Involvement of PI3K??in the Regulation of Erythroid
Differentiation of K562 Cells
Dissertation
zur Erlangung des akademischen Grades
doctor rerum naturalium
vorgelegt dem Rat der Biologisch-Pharmazeutischen Fakultät
der Friedrich-Schiller-Universität Jena
VON YUANTAO LIU
AUS BEIJING, CHINA
Gutachter:
1. Prof. Dr. Reinhard Wetzker
2. PD Dr. Frank-D. Böhmer
3. Prof. Dr. Bernd Nürnberg
Tag des Rigorosums: 04, June, 2003
Datum der Verteidigung: 23, July, 2003
Contents
1. INTRODUCTION___________________________________________ 1
1.1 BACKGROUND--------------------------------------------------------------------------------------------------------1
1.2 PHOSPHOINOSITIDE 3-KINASES -------------------------------------------------------------------------------------1
1.2.1 Class I PI3Ks ---------------------------------------------------------------------------------------------------3
1.2.2 Class II PI3Ks--------------------------------------------------------------------------------------------------9
1.2.3 Class III PI3Ks----------------------------------------------------------------------------------------------- 10
1.3 SIGNALLING PROTEINS DOWNSTREAM OF PI3KS--------------------------------------------------------------- 10
1.4 PI3KS AND CELL DIFFERENTIATION------------------------------------------------------------------------------ 14
1.4.1 Involvement of PI3Ks in hematopoiesis------------------------------------------------------------------ 14
1.4.2 Involvement of PI3Ks in differentiation of non-hematopoietic cells ------------------------------- 18
1.5 AIM OF THE WORK ------------------------------------------------------------------------------------------------- 20
2. MATERTIALS AND METHODS_____________________________ 21
2.1 MATERIALS --------------------------------------------------------------------------------------------------------- 21
2.1.1 Cells------------------------------------------------------------------------------------------------------------ 21
2.1.2 Bacterial strains (E. coli) ----------------------------------------------------------------------------------- 21
2.1.3 Plasmids------------------------------------------------------------------------------------------------------- 21
2.1.4 Primers for RT-PCR ---------------------------------------------------------------------------------------- 22
2.1.5 Antibodies----------------------------------------------------------------------------------------------------- 22
2.1.6 Medium and substances for cell culture ----------------------------------------------------------------- 23
2.1.7 Materials for silver staining-------------------------------------------------------------------------------- 24
2.1.8 Other reagents and materials ------------------------------------------------------------------------------ 24
2.1.9 Buffers and Solutions --------------------------------------------------------------------------------------- 25
2.2 METHODS ----------------------------------------------------------------------------------------------------------- 27
2.2.1 Plasmid preparation----------------------------------------------------------------------------------------- 27
2.2.2 Isolation of CD34+ progenitors from peripheral blood ------------------------------------------------ 28
2.2.3 Culture of CD34+ cells and induction of differentiation----------------------------------------------- 28
2.2.4 Flow cytometry analysis ------------------------------------------------------------------------------------ 28
2.2.5 K562 cell culture and induction of differentiation ----------------------------------------------------- 29
2.2.6 Assessment of erythroid differentiation of K562 cells-------------------------------------------------- 29
2.2.7 Stable transfection of K562 cells -------------------------------------------------------------------------- 30
2.2.8 Western Blot -------------------------------------------------------------------------------------------------- 30
2.2.9 Purification of polyclonal anti-PI3K ??antibody--------------------------------------------------------- 31
2.2.10 Coupling anti-PI3k ??monoclonal antibody to protein G- Sepharose------------------------------- 32
2.2.11 Immunoprecipitation -------------------------------------------------------------------------------------- 33
2.2.12 PI3K ??Protein Kinase activity assay--------------------------------------------------------------------- 33
2.2.13 Silver staining----------------------------------------------------------------------------------------------- 33
2.2.14 RNA isolation and RT-PCR ------------------------------------------------------------------------------ 34
3. RESULTS ________________________________________________ 36
3.1 INVOLVEMENT OF PI3K??IN THE ERYTHROID DIFFERENTIATION OF HEMATOPOIETIC STEM CELLS ------- 36
3.2 INVOLVEMENT OF PI3K??IN THE ERYTHROID DIFFERENTIATION OF K562 CELLS--------------------------- 38
3.3 INFLUENCE OF SUPPRESSING ENDOGENOUS PI3K??EXPRESSION ON THE ERYTHROID DIFFERENTIATION OF
K562 CELLS ------------------------------------------------------------------------------------------------------------- 42
3.4 INFLUENCE OF OVEREXPRESSION OF PI3K??ON THE ERYTHROID DIFFERENTIATION OF K562 CELLS----- 45
3.5 RELATIONSHIP OF PI3K??KINASE ACTIVITIES TO THE ERYTHROID DIFFERENTIATION OF K562 CELLS --- 46
3.6 EXPRESSION OF P101 IN K562 CELLS --------------------------------------------------------------------------- 49
3.7 INFLUENCE OF PI3K??ON MAPK ACTIVITY IN K562 CELLS -------------------------------------------------- 50
3.8 DOWNSTREAM TARGETS FOR PI3K??PROTEIN KINASE ACTIVITY --------------------------------------------- 54
4 DISCUSSION ______________________________________________ 58
4.1 PI3K??IS A NEGATIVE REGULATOR OF THE ERYTHROID DIFFERENTIATION OF K562 CELLS --------------- 58
4.2 PI3K??NEGATIVELY REGULATES THE ERYTHROID DIFFERENTIATION PRESUMABLY VIA ITS PROTEIN
KINASE ACTIVITY ------------------------------------------------------------------------------------------------------- 60
4.3 THE EFFECT OF PI3K??ON THE ERYTHROID DIFFERENTIATION IS INDEPENDENT OF P101 ------------------ 62
4.4 THE EFFECT OF PI3K??ON THE ERYTHROID DIFFERENTIATION IS NOT MEDIATED BY MAPK-------------- 62
4.5 ATTEMPTS TO IDENTIFY THE DOWNSTREAM TARGETS FOR PI3K??PROTEIN KINASE ACTIVITY ----------- 63
4.6 CONCLUSION ------------------------------------------------------------------------------------------------------- 65
4.7 OUTLOOK ----------------------------------------------------------------------------------------------------------- 65
LITERATURE ______________________________________________ 67
SUMMARY _________________________________________________ 81
ZUSAMMENFASSUNG ______________________________________ 83
APPENDIX _________________________________________________ 85
ABBREVIATIONS -------------------------------------------------------------------------------------------------------- 85
ACKNOWLEDGEMENTS------------------------------------------------------------------------------------------------- 87
LEBENSLAUF ------------------------------------------------------------------------------------------------------------ 88
SELBSTÄNDIGKEITSERKLÄRUNG ------------------------------------------------------------------------------------- 89
ERKLÄRUNG ZUR BEWERBUNG --------------------------------------------------------------------------------------- 90
1.Introduction
1
1. INTRODUCTION
1.1 Background
Differentiation is a basic developmental process by which a cell or a number of cells that
lack specific functions proliferate for a defined period and change their pattern of gene
expression to produce specified cell types that perform highly specialised biochemical
functions. Embryogenesis is the most striking form of differentiation, during which all
specialised functions of the adult arise from a single fertilised egg.
During normal differentiation, each step in the “proliferation-execute genetic
differentiation program-stop proliferation” cascade is under stringent control from both
extracellular and intracellular stimuli. For example, in haematopoietic tissue, stromal cells
produce numerous cytokines, growth factors, and cell adhesion molecules, supporting the
proliferation of hematopoietic progenitors and specifying their direction of differentiation.
In the nucleus of differentiating hematopoietic progenitors, both lineage-restricted and
ubiquitous transcription factors play critical roles in governing a highly organised gene
expression program. Currently, one of the major tasks in cell differentiation study is to
define the signalling events that link the extracellular stimuli to specific gene(s)
expression.
It is known that the transfer of extracellular stimuli to their nuclear targets is mediated by
various intracellular messengers or signalling pathways. Phosphoinositide 3-kinases
(PI3Ks), are a family of ubiquitous enzymes that have been shown to be important
mediators of intracellular signalling in mammalian cells. Previous work has demonstrated
PI3Ks are important regulators of mitogenesis, cell survival, metabolic control, and
cytoskeletal rearrangements. More recently, the role of PI3Ks in differentiation has begun
to be revealed.
1.2 Phosphoinositide 3-kinases
Phosphoinositide 3-kinases (PI3Ks) are a large family of ubiquitous lipid kinases. To date,
eight members of this family have been cloned and characterised in mammalian cells. A
common feature of these enzymes is there ability to catalyse the phosphorylation on the 3-
hydroxyl of the inositol ring of phosphoinositide (PI) and its derivatives. These enzymes
differ in structure, and have distinct mode of regulation and in vitro lipid substrate
1.Introduction
2
specificity. On the basis of these differences, they can be divided into three classes
(Vanhaesebroeck, B et al., 1997).
Fig.1 Reactions catalysed by PI3K and related enzymes for the synthesis and hydrolysis of
phosphoinositides. The basic structure of PtdIns and its numbering system of the inositol ring are
presented in the upper of this figure.
Diacylglycerol
Inositol-1,4,5-trisphosphate
PI(3,4)-P2 PI(3,4,5)P3
PI(4,5)P2 PI(4)P
PI(3)P
PtdIns
PtdIns-4-K
PI3K PTEN
SHIP
PtdIns-5-K
5-phosphatase
PtdIns-4-K
4-phosphatase Phospholipase C
PI3K PTEN PI3K
O- O= O
O
1
2 3
OH
OH
4
5
6
OH
OH
OH
Fatty Acid-COO
Fatty Acid -COO
Phophatidylinositol(PtdIns)
1.Introduction
3
1.2.1 Class I PI3Ks
Among the PI3K family, the Class I PI3Ks are the most intensively investigated and best
characterised. In vitro they utilise phosphatidylinositol (PtdIns), phosphatidylinositol 4-
phosphate (PtdIns(4)P), phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) as substrates
to produce PtdIns(3)P, PtdIns(3, 4)P2 and PtdIns(3,4,5)P3 respectively. In vivo, their
preferential substrate is PtdIns(4,5)P2. Class I PI3Ks comprise four isoforms, namely,
PI3K?, ?, ?, and ?. Based on the difference in mode of regulation and adaptor association,
they are further subdivided into class IA and class IB PI3Ks (Vanhaesebroeck, B et al.,
1997)
Class IA PI3Ks
The class IA PI3Ks are heterodimers consisting of a p110 catalytic subunit and an
adaptor/regulatory subunit. In mammals, three isoforms of catalytic subunits have been
identified, termed p110??(Hiles, ID et al., 1992), p110??(Hu, P et al., 1993), and p110
??(Vanhaesebroeck, B et al., 1997). They are encoded by three separate genes, with
molecular weights of about 110-120 kDa. p110??and p110??are widely expressed in
mammalian tissues, in contrast p110??shows a restricted distribution and is mainly found in
leukocytes (Vanhaesebroeck, B et al., 1997). Despite the differences in tissue distribution,
these catalytic subunits are quite similar in structure (Fig.2). A striking structural feature of
these proteins is characterised by the presence of an N-terminal p85-binding domain. In the
C-terminus, they also share a high degree of homology in the catalytic domain, which
contains the binding sites for ATP and the PI3K inhibitor wortmannin. In addition, they
also share a Ras–binding domain located in the N-terminal part, a C2 domain , and a
phosphoinositide kinase domain (PIK). The C2 domain is conserved in all PI3Ks and is
thought to mediate the interaction with the phospholipid membrane. The PIK domain is
conserved in all lipid kinase enzymes, and is thought to mediate protein-protein
interactions (Walker, EH et al., 1999).
1.Introduction
4
Fig.2 structural features of mammalian PI3Ks
The catalytic subunits are constitutively associated with an adaptor protein. There are three
genes that encode at least five different adaptor proteins (p85?, p55?, p50?, p85?, and
p55?) (for an overview, see Okkenhaug, K, 2001). p85??and p85?, encoded by different
genes, represent the full-length versions of the regulatory subunits. These two proteins are
similar in structure. Each contains a SH3 domain, a bcr homology (BH) flanked by two
proline-rich domains, two SH2 domains separated by an inter SH2 domain, which contains
the p110 binding region. The two SH2 domains can bind specific phosphorylated tyrosine
residues in activated receptor proteins or other tyrosine phosphorylated protein adaptors
and thus to bring the p110 catalytic subunit to the membrane in proximity to its substrate.
The SH3 domain mediates the interactions between p85 and proteins with proline-rich
sequences, such as SOS (son of sevenless) and Cbl. These interactions create an alternative
signal mechanism for receptors to recruit p110 to the membrane (Kivens, WJ et al., 1998).
The BH domain of p85 is homologous to the Rho family GAPs. Some evidence has been
presented that this domain can bind Cdc42, an interaction that may contribute to the
activation of p110 (Jimenez, C et al., 2000, Vanhaesebroeck, B et al., 2001). Among the
shorter versions of the regulatory subunits, p55 ??and p50??are splicing variants derived
from the same gene as p85??(Fruman, DA et al., 1996), whereas p55??is encoded by a
different gene. These short forms of regulatory subunits share the common SH2 domains
Adaptor-binding Ras-binding kinase core domain PIK C2
Class Structural features
p110?, ?,?
p110?
PI3K-C2 ?,?,?
Vps34p
analogues
I
II
III
Isoforms
1.Introduction
5
and inter SH2 domain structure with p85??and p85???but lack the N-terminal half
containing the SH3 domain, N-terminal proline-rich domain, and BH domain. Due to these
structural differences, the longer p85 subunits have been shown to possess additional
functions to that of the shorter forms. The main function of the regulatory subunits is to
bring the p110 catalytic subunits to the membrane, where the catalytic subunit
phosphorylates its lipid substrates. However, in the unstimulated state, a regulatory subunit
plays a role in maintaining the thermal stability of the p110 catalytic subunit but also
keeps it in low activity state (Yu, J et al., 1998 ). Intriguingly, there is increasing evidence
to suggest that the regulatory subunits in fact can exert some effects independently of the
catalytic subunit. For instance, p85??was shown to independently stimulate signalling
pathways involved in actin cytoskeletal rearrangements (Jimenez, C et al., 2000,
Okkenhaug, K et al., 2001).
In response to different stimuli, Class IA PI3Ks can be activated in various ways (for an
overview, see {Wymann, MP et al., 1998}). One type of activation involves binding of p85
to the tyrosine phosphorylated sites of the activated receptor tyrosine kinases. For instance,
PI3K binds to the PDGF receptor through the SH2 domain of p85 (Panayotou, G et al.,
1992). Nonreceptor tyrosine kinases, such as src-family or JAK kinases, are also
implicated in the activation of class I PI3Ks through the interactions with B- and T-cell
antigen receptors, cytokine receptors, and several costimulatory molecules (such as CD28,
CD2, CD19). In addition, class IA PI3Ks can also be activated by the small GTPase Ras
(Downward, J., 1997; Rodriguez-Viciana, P., 1994, 1996). Under some circumstances,
p110??can be activated by G-protein coupled receptors (Kurosu, H et al., 1997, Yart, A et
al., 2002).
There is evidence that different activation mechanisms could recruit distinct PI3K
complexes to distinct intracellular locations, which lead to specificity in signal transduction
(Wang, Q et al., 1998). For instance, insulin–induced glucose transport in adipocytes is
mediated by p110??but not p110?, despite the fact that both of them are present and can
equally well associate with the adaptor subunits (Wang, Q et al., 1998). This concept is
further reinforced by the observation that p110??can mediate insulin and lysophosphatidic
acid (LPA) induced mitogenesis in fibroblasts, whereas the platelet derived growth factor
(PDGF)-induced mitogenesis in these cells apparently does not require the activation of
p110??(Roche, S et al., 1998).
In addition to their function as lipid kinases, PI3Ks have an intrinsic protein-serine kinase
activity. Initial studies showed that p110?-mediated phosphorylation on p85??adaptor
1.Introduction
6
reduces the lipid kinase activity of the heterodimer in vitro (Dhand, R et al., 1994). PI3K?
has been shown to undergo autophosphorylation both in vitro and in vivo
(Vanhaesebroeck, B et al., 1999), and the in vivo autophosphorylation induced by activated
receptors (such as CD28 ) leads to a concomitant decrease in associated lipid kinase
activity. Similar to p110?, p110??can also phosphorylate p85 adapator, albeit to a lower
degree than does p110??(Beeton, CA et al., 2000). Despite the finding that class IA PI3Ks
are able to phosphorylate insulin receptor substrate-1 (IRS-1) in vitro (Vanhaesebroeck, B
et al., 1999), the physiological protein kinase targets have not been identified.
Nevertheless, in vitro protein kinase activity is a distinguishing feature among the PI3K
isoforms and may contribute to isoform-specific functions.
Class IB
Up to date, only one isoform of class IB PI3K has been identified. Initial studies in
neutrophils (Stephens, L et al., 1994) and platelets (Thomason, PV et al., 1994) revealed
that the accumulation of PI(3,4,5)P3 occurs in response to activation of heterotrimeric G
protein-linked receptors, implying the existence of a PI3K isoform different from classIA
PI3Ks. Later on, by screening of the human bone marrow cDNA library, a novel PI3K
isoform was cloned and characterised in our lab, namely, PI3K??(Stoyanov, B et al.,
1995). In structure, PI3K??is similar to that of class IA p110 proteins, but its N-terminal
part lacks the binding domain for the p85 adaptor family. Instead of binding p85 family
protein, Stephens et al. found the porcine version of human PI3K??is complexed with a
novel protein, termed p101 (Stephens, L et al., 1997). This protein shows no sequence
homology to any known protein. There is some evidence showing that the lipid kinase
activity of PI3K??is positively regulated by p101. Stephens et al, demonstrated PI3K???
when expressed in SF9 cells or COS-7 cells, exhibits a significant basal activity that is only
slightly stimulated by G???dimers, but when co-expressed with p101, the PI3K??activity is
stimulated to a much greater extent by G???dimers. In agreement with the above
observation, Krugmann et al, generated a panel of catalytically active PI3K??fragments
using a deletion approach, and demonstrated that those fragments incapable of binding
p101 are insensitive to G???-stimulation (Krugmann, S et al., 1999). Thus, according to
these studies, the function of p101 seems to increase the sensitivity of PI3K??to G???-
stimulation. However, there are some other observations arguing against an indispensable
role of p101 in G???stimulation of PI3K??by showing that PI3K?, in the absence of p101,
1.Introduction
7
can be substantially activated by G-proteins (Stoyanov, B et al., 1995; Leopoldt, D et al.,
1998 ). In another study, it was demonstrated that purified PI3K??from SF9 cells has kinase
activity towards several 3-phosphoinositides, addition of p101 increases its specificity for
PI(4,5)P2. Thus the authors proposed that p101 might be responsible for the lipid substrate
selectivity of PI3K??in its G???-stimulated state (Maier, U et al., 1999). Up to date , the
precise molecular basis of p101 in the regulation of PI3K??activity is unclear.
In contrast to classIA PI3Ks which signal downstream of tyrosine kinases, PI3K??is mainly
activated by agonists of G-protein coupled receptors (GPCR), including the bacterial
chemotatic peptide formyl-Met-Leu-Phe (fMLP), complement factor 5, interleukin-8.
GPCR agonists lead to the activation of heterotrimeric G????proteins by exchanging the
GDP molecule bound to the G??subunit for GTP, thus releasing the G???heterodimer
(Gilman, AG, 1987). The mechanism whereby PI3K??is activated by GPCR is not
completely defined, but likely involves direct binding of G???to the PI3K??/p101
heterodimers. Direct binding of G???to PI3K??is evidenced by co-purification of G???with
a GST-PI3K??fusion protein (Leopoldt, D et al., 1998). Involvement of p101 in binding to
G???is supported by a deletion analysis of p101 which indicates the G???stimulated
activity of PI3K??requires the N-terminus of p101 and correlates with the tightness of
binding of p101 to PI3K??(Krugmann, S et al.,1999). In any case, G???seems to play a
important role in the activation of PI3K??(Stoyanov, B et al., 1995, Murga, C et al., 1998),
and possibly by localising PI3K??to the membrane (Leopoldt, D et al., 1998; Murga, C et
al., 1998) where its lipid substrates reside. In support of this hypothesis, a membranetargeted
PI3K??mutant was shown to constitutively produce PIP3 (Bondeva, T et al., 1998).
It should be noted that, only specific G???dimers can activate PI3K?. G?5?2, for instance,
does not activate PI3K??(Maier, U et al., 2000)
To date, PI3K??has been implicated in several signalling pathways. In COS-7 cells, PI3K?
was shown to mediate a G???-dependent pathway leading to mitogen-activated protein
kinase (MAPK) stimulation, and possibly through the activation of a tyrosine kinase-Shc-
Grb2-Sos-Ras dependent pathway (Lopez-Ilasaca, M et al., 1997). Nevertheless, the
putative tyrosine kinase activated downstream of PI3K??in this study remains to be
identified. In contrast, it was reported that PI3K??can mediate a Ras-independent
activation on MAPK in CHO cells treated with lysophosphatidic acid (LPA) , an agonist of
GPCRs (Takeda, H et al., 1999). The authors showed that PI3K??activation leads to the
stimulation of MEK through PKC?, a downstream target of PI(3,4,5)P3. In addition to
1.Introduction
8
MAPK, PI3K??also mediates G??-dependent activation of Jun kinase (JNK) in COS-7 cells
(Lopez-Ilasaca, M et al. 1998). In endothelial cells, shear stress leads to both MAPK and
JNK activation (Go, YM et al., 1998). However, in this study, the authors have
demonstrated that PI3K??only mediates the JNK, but not the MAPK activation. Thus, it
seems PI3K??can participate in different signalling pathways depending on the cell type
and stimulus.
It should be noted, in addition to its lipid kinase activity, PI3K??was also demonstrated to
possess a serine kinase activity. Similar to PI3K??(Vanhaesebroeck, B et al., 1999?, PI3K?
is able to autophosphorylate in vitro (Stoyanova, S et al., 1997). Furthermore, it was
demonstrated that PI3K??can transphosphorylate the adaptor protein p101 and the mitogenactivated
protein kinase kinase 1 (MEK-1) in vitro (Bondev, A et al., 1999). The lipid and
protein kinase activities of PI3K??could be involved in distinct cellular processes. Using a
panel of engineered PI3K??constructs, Bondeva et al. demonstrated that the lipid kinase
activity is needed for the lipid production and the subsequent activation of PKB, whereas
the protein kinase activity is responsible for MAPK activation (Bondeva, T et al., 1998).
Furthermore, it was also found that the membrane-targeted form of PI3K??constitutively
produces PI(3,4,5)P3 and activates PKB but interferes with the MAPK activation,
suggesting the lipid and protein kinase activities of PI3K??are related to its cellular
localisation. Although PI3K??protein kinase activity was shown to be sufficient to activate
MAPK in this transient transfection system, its direct target protein has not been firmly
established. Importantly, further work is required to assess whether this protein kinase
activity is also operative under more physiological conditions.
PI3K??appears to be present only in mammals where it shows a more restricted tissue
distribution than class IA PI3Ks. It is predominantly expressed in cells of hematopoietic
origin (Ho, LK et al., 1997). Accordingly, multiple roles have been revealed for PI3K??in
the regulation of blood cell functions. Studies of mice lacking PI3K??showed decreased
migration of neutrophils and macrophages in response to chemokines in vitro and in vivo
(Hirsch, E et al., 2000; Sasaki, T et al., 2000). As a consequence, the mice had reduced
inflammatory responses to bacteria. In neutrophils, the chemokine-induced oxidative bursts
were impaired (Sasaki, T et al., 2000). In addition, the T cell mediated inflammatory and
humoral immune responses were also compromised in PI3K??deficient mice. (Sasaki, T et
al., 2000).
1.Introduction
9
1.2.2 Class II PI3Ks
Class II PI3Ks are a recently identified subclass of PI3K family. They are large molecules
about twice the size (170-220 kDa) of type I PI3Ks. So far, three isoforms of class II
PI3Ks, PI3K-C2??(Domin, J et al.,1997), ??(Arcaro, A et al., 1998), ??(Ono, F et al.,
1998??have been identified in mammals. These enzymes share high similarity in structure.
However they have different pattern in tissue distribution. The PI3K-C2??and PI3KC2?
are ubiquitously expressed, in contrast PI3K-C2??is mainly found in the liver. The
striking feature of these proteins in structure is characterised by the presence of C-terminal
C2 domain which is lacking in class I PI3Ks. The C2 domain was originally defined as the
second of four conserved regions within mammalian protein kinase C (PKC) which confers
a Ca2+ sensitive phospholipid binding (Kaibuchi, K et al., 1989). Whereas the C2-like
domain of class II PI3Ks has been shown to bind in vitro to phospholipids in a Ca2+-
independent manner (MacDougall, LK et al., 1995). The deletion of C2 domain increased
the in vitro lipid kinase activity of PI3K-C2??(Arcaro, A et al., 1998) toward PI. It is
possible that the C2 domain competes with the catalytic domain for binding to
phosphotidylinositol substrate, thus behaving as a negative regulator of lipid activity of the
enzyme. The catalytic domain is also distinct from that of the class I enzyme. A feature of
this domain is its ability to utilise Ca2+ -ATP for the in vitro lipid kinase activity (Arcaro,
A et al., 1998). The N-terminal regions of class II PI3Ks are the most divergent among
these isoforms, and show no homology to those of class I PI3Ks. Although a Ras-binding
fold exists in this regions, class II PI3Ks have not been found to associate with Ras
(Arcaro, A et al., 1998). In contrast to class I PI3Ks which are mainly cytosolic, class II
PI3Ks show a predominant membrane localisation. In vitro, they preferentially utilise
PtdIns, PtdIns(4)P, but can, albeit poorly phosphorylate PtdIns(4,5)P2. (Domin, J et
al.,1997, Arcaro, A et al., 1998). However, it is still unclear which lipids they produce in
vivo. Insulin (Brown, RA et al., 1999), EGF (Wheeler, M, et al., 2001), and integrin
(Zhang, J et al., 1998) have been shown to be able to activate class II PI3Ks in in vitro lipid
activity assay system. However, the signalling events linking the above stimuli to the
activation of these PI3Ks are unclear. Besides, the physiological relevance of these
enzymes still remains to be established. Only a recent study has shown that PI3K-C2??is
involved in cell growth of a human small cell lung carcinoma (SCLC) cell line in response
to SCF stimulation (Arcaro, A et al., 2002).
1.Introduction
10
1.2.3 Class III PI3Ks
To date, a single class III PI3K catalytic subunit has been identified in mammals (Volinia,
S et al., 1995) which is the homolog of the yeast vesicular-protein-sorting protein Vps34p
(Schu, PV et al., 1993). The only substrate for this enzyme is PtdIns, both in vivo and in
vitro (Volinia, S et al., 1995). This catalytic subunit exists in association with a Ser/Thr
protein kinase (Vps15 in yeast, p150 in human). Vps15 has been shown to be essential for
Vps34p localisation and activation (Stack. JH et al., 1995), and a similar role is defined for
p150 in mammals (Panaretou, C et al., 1997). The major cellular function of class III
PI3Ks has been implicated in vacuolar protein sorting in yeast and membrane trafficking
processes in mammalian.
1.3 Signalling proteins downstream of PI3Ks
Upon activation in response to various of stimuli, PI3Ks phosphorylate inositol lipids at the
D-3 position of the inositol ring to generate the 3-phosphoinositides PI(3)P, PI(3,4)P2,
PI(3,4,5)P3 in vitro. These lipids in turn serve as second messengers and function in signal
transduction by interacting with 3-phosphoinositide-binding modules present in a broad
variety of proteins. Specifically, certain FYVE domains bind to PI(3)P, whereas certain
pleckstrin homology domains bind PI(3,4)P2 and PI(3,4,5)P3.
FYVE domains were named after four of the proteins in which they are first identified:
Fab1p ,YOTB, Vac1p and Early endosome antigen 1 (EEA1). They are cysteine-rich zincfinger-
like motifs that specifically bind PI(3)P but not PI(3,4)P2 , PI(3,4,5)P3 The FYVE
domain-PI(3)P interactions are shown to be essential for vesicular trafficking (Corvera, S
et al., 1998).
PH domains are structurally conserved modules of approximately 100 amino acids that was
shown to be involved in protein-protein interaction and binding to PI(4,5)P2 (Lemmon, M.
A et al., 1997). Now it is clear that PH-domains bind preferentially to PI(3,4)P2 and
PI(3,4,5)P3 over other inositol lipids (Salim, K et al., 1996). To date, PH domain has been
found to be present in numerous proteins. These proteins include serine/threonine kinase
Akt and 3-phosphoinositide-dependent kinase-1(PDK1), certain guanine nucleotide
exchange factors (GEFs), certain tyrosine kinases and phospholipase-C(PLC)??.
The serine/threonine kinase Akt is one of the well-established targets of PI3K (Kandel, ES
et al., Hay, 1999). Akt, also termed PKB, is the cellular homologue of the transforming
viral oncogene v-Akt and bears significant homology to PKA and PKC (Bellacosa, A et
1.Introduction
11
al., 1993). Akt contains an N-terminal PH domain, a central kinase domain with an
activation-loop, a Thr308 phosphorylation site, and a conserved regulatory serine
phosphorylation site Ser473 near the C terminus. The activation of Akt entails a complex
series of events involving additional proteins. First, PI3Ks generated lipid production
PI(3,4)P2 and PI(3,4,5)P3 recruit Akt to the plasma membrane through their affinity for the
PH domain of Akt (Burgering, BM et al., 1995), where it is phosphorylated on Thr308 by
another PH domain containing protein, PDK1 (Anderson, KE et al., 1998).
Phosphorylation of Thr308 is a prerequisite for kinase activation, but phosphorylation of
Ser 473 is required for full activation of Akt kinase. The mechanism by which the Ser 473
is activated is incompletely understood. Although the existence of a kinase that targets Ser
473, termed PDK-2, has been postulated (Anderson, KE et al., 1998), the identification for
this kinase has not been successful.
The activation of Akt has been shown to mediate many cellular functions by subsequent
phosphorylation of a host of other proteins. One of the major functions of Akt is to
promote growth factor-mediated cell survival and to block programmed cell death or
apoptosis. It was shown that Akt phosphorylates the proapoptotic factor, BAD, on a serine
residue and prevents its binding to Bcl-2 family members Bcl-2 and Bcl-XL, thus releasing
them for a survival response (Datta, SR et al., 1997; Brunnet, A et al., 2001).
Subsequently, it was reported that Akt-induced phosphorylation of a death protease called
caspase-9 prevents apoptosis by inhibiting the protease activity directly (Cardone, MH et
al., 1998). In addition, Akt can interfere with cell death through transcription-based
mechanisms. For instance, Akt induces the degradation of I?B by promoting IKK??activity
and subsequently increase the activity of nuclear factor-?B (NF-?B) leading to increased
synthesis of antiapoptotic proteins, such as c-myc and Bcl-XL (Lee, HH et al., 1999;
Romashkova, JA et al., 1999). It was also shown that Akt promotes cell survival by
preventing the expression of proapoptotic factors such as Fas ligand through the
phosphorylation and inactivation of the Forkhead transcription factor (Brunet, A et al.,
1999).
Akt can also influence protein synthesis. Forkhead transcription factors, cAMP-response
element binding protein (CREB), E2F, and NF?B have been shown to be targets for Akt
(Brunet, A et al., 1999). Akt modulates the activity of multiple factors that are required for
mRNA translation. Akt can induce initiation of translation through phosphorylation and
inactivition the repressor of translation 4E-BP (eIF-4E binding protein), and subsequent
activation of elongation/initiation factor eIF-4E (Gingras, AC et al., 1998; Miron, M et al.,
1.Introduction
12
2001). The mammalian target of rapamycin (mTOR) has been shown to be directly
phosphorylated by Akt, which in turn mediates activation of the elongation factor eEF2
(Wang, X et al., 2001). In addition to Akt, PDK1 may also contribute to protein synthesis.
PDK1 is reported to activate p70S6 kinase (Pullen, N et al., 1998). The p70S6 kinase can
regulate mRNA translation through the phosphorylation of 40S ribosomal protein S6
(Ballou, LM et al., 1991).
The cellular function of Akt is also involved in the regulation of cell–cycle entry. Cyclin D
1 is a substrate of glycogen synthase kinase 3 (GSK3), which when phosphorylated by
GSK3, is targeted for degradation (Diehl, JA et al., 1998). However, phosphorylation of
GSK3 by Akt results in the inactivation of GSK3, which allows cyclin D1 to accumulate.
In addition, Akt can phosphorylate and inactivate Forkhead transcription factors. The
inactivation of these transcription factors leads to the decreased transcription of cyclindependent
kinase (CDK) inhibitor, p27kip1 (Medema, RH et al., 2000, Schmidt, M et al.,
2002 ). Thus, Akt might have several inputs in control of the cell cycle.
Some of the metabolic actions of insulin are also mediated by Akt. It enhances glucose
uptake in insulin-response tissues by affecting the glucose transportors GLUT1, GLUT3
and GLUT4. Akt induces the expression of GLUT1, GLUT3 (Berthel, A et al., 1999;
Hajduch, E et al. 1998) and the tanslocation of GLUT4 (Kohn, AD et al., 1996). It also
activates glycogen synthesis through the inactivation of GSK3., which in turn leads to the
activation of glycogen synthase (Cross, DA et al., 1997). In addition, Akt is shown to
directly activate 6-phosphofucto-2-kinase (PFK2), which is an important regulator of
glycolysis (Deprez, J et al., 1997).
The PH-domain of the Bruton’s tyrosine kinase (Btk) is also a downstream target of class I
PI3Ks. Btk is a member of Tec kinases, a family of nonrecptor tyrosine kinases. The PH
domain of Btk has been found to bind selectively to PI(3,4,5)P3 (Rameh, LE et al., 1997)
and is thought to mediate the translocation of Btk to the plasma membranes where it is
activated by Src family kinases or PDK1-related enzymes. Once activated, Btk-related
kinases can phosphorylate and activate PLC?. Btk activation is involved in B-cell
development and mutations in its PH-domain have been found to be responsible for Xlinked
immunodeficiency in mice (Xid) and X-linked agamma-globulinaemia in humans
(Fukuda, M et al., 1996).
1.Introduction
13
Fig.3 Main signalling pathways downstream of PI3Ks
PKC isoforms have also been shown to be the downstream targets of PI3Ks. However, the
precise mechanism underlying the PKC activation by PI3Ks is unclear. Several studies
have provided evidence that some of the atypical (???) and novel PKC(?) isoforms, as well
as the PKC related kinase (PRK) can bind to and are directly activated by PI(3,4)P2 and/or
PI(3,4,5)P3 (Nakanishi, H et al., 1993; Palmer, R.H et al., 1995; Toker, A et al., 1994).
Alternatively, it is proposed the activation of PKCs is controlled by PDK1, based on the
motility
Rac
Cdc42
GEFs
PI(3,4)P??PI(3,4,5)P3
PDK1 Akt
p70S6K
BAD,
Capase9
FKHR
survival
NF-kB
GSK3
Cyclin D
cell cycle entry protein synnthesis
PKC ?
?
GLUTs, PFK1
Glucogen
synthase
metabolism
PI3Ks
1.Introduction
14
observation that PDK1 can phosphorylate critical residues in the activation loop of several
PKC isoforms (Le Good , JA et al., 1998).
PH domains are also present in many guanine nucleotide exchanging factors (GEFs)
specific for the Rho family of GTPases, which include Rho, Rac and Cdc42. PI(3,4)P2 and
PI(3,4,5)P3 have been shown to bind to the PH-domain of the exchange factor VAV
(Palmby, TR et al, 2002). The binding of PI(3,4,5)P3 is thought to cause an allosteric
activation of its exchange activity towards the small GTPases Rac and CDC42. Both
GTPases are involved in regulation of cytoskeletal rearrangement and cell migration
(Rameh, LE et al., 1999).
The PI3K signalling is negatively regulated by at least two types of phosphatases. The Srchomology
2(SH2)-containing phosphatases (SHIP1 and SHIP2) convert PI(3,4,5)P3 to
PI(3,4)P2 by dephosphorylating the 5 position of the inositol ring. PTEN, another kind of
phosphatase, dephosphorylates the 3 position of PI(3,4,5)P3 and PI(3,4)P2 . Loss of PTEN
or function has been found in a large fraction of human cancers, indicating that the
uncontrolled PI3K signalling contributes to tumor formation ( for an overview, see
{Cantley, LC, 2002}).
1.4 PI3Ks and cell differentiation
1.4.1 Involvement of PI3Ks in hematopoiesis
Cell differentiation plays an important role in the developmental biology. An excellent
model system in differentiation study is the hematopoietic system. The formation of
different types of blood cells, hematopoiesis, is essential for the development and survival
of an individual. During normal hematopoiesis, a small pool of pluripotent, hematopoietic
stem cells gives rise to several blood cell types through a process of simultaneous lineage
commitment, cell proliferation, and differentiation (Fig 4).
1.Introduction
15
Fig.4 Scheme of hematopoiesis
This process is, at least in part, regulated by a complex network of hematopoietic growth
factors (for an overview, see {Lotem, J et al., 2002}). Interleukin (IL)-3, also known as
multi-colony-stimulating factor (multiple-CSF), stimulates the proliferation of
multipotential progenitors as well as various lineage-committed progenitors, granulocyte
colony-stimulating factor (G-CSF) also stimulates early progenitors in addition to
granulocytes and macrophges. In contrast, erythropoietin (EPO), thrombopoietin (TPO),
granulocyte colony-stimulating factor (G-CSF), and IL-5 predominantly stimulate
erythrocytes, megakaryocytes, granulocytes and eosinophils, respectively (for an overview,
see {Miyajima, A et al., 1999}). In addition, other cytokines such as interleukin (IL)-6 and
stem cell factor are essential for the expansion of the primitive stem cell and the
proliferation of various progenitors (Rappold, I et al., 1999). It should be noted multiple
cytokines usually exhibits overlapping functions, because the receptors of these cytokines
share some common structures. Currently, how these growth factors elicit varied
developmental responses leading to different blood cell lineages has been only partially
understood.
NEUTROPHILS
MONOCYTES
PLETELETS
EOSINOPHILS
THYMUS
G0 phase
stem cell
S phase
stem cell
Lymphoid
progenitor
Pre B
stem cell
Thymocyte
CFU-GM
BFU-E CFU-E
Meg-blast CFU-Meg
IL-3
GM-CSF
SCF
IL-6
IL-3
IL-3
GM-CSF
IL-6
IL-3
GM-CSF
SCF
IL-12
IL-3
IL-3
IL-2, IL-4, IL-7
IL-4, IL-7
GM-CSF,M-CSF
GM-CSF,IL-5
IL-3, SCF
G-CSF
TPO
EPO
RED CELLS
BASOPHILS
B CELLS
T CELLS
1.Introduction
16
Once binding to their receptors, these cytokines are able to activate distinct intracellular
signalling pathways. Among these well established signalling pathways involved in
hematopoiesis are the Ras/Raf/MAPK pathway, and the Janus kinases (JAKs)/signal
transducers and activators of transcription (STATs) pathway ( for an overview, see
{Miyajima, A et al., 1999}). Recently, several lines of evidence have shown that PI3Ks are
also involved in the process of hematopoiesis.
The direct evidence implicating PI3K in hematopoiesis was derived from gene targeting
studies. The class IA p85??regulatory subunit of PI3K has been knocked out by two
independent groups (Fruman, DA et al., 1999; Suzuki, H et al., 1999). One strategy has led
to the elimination of all the splice variants (Fruman, DA et al., 1999), whereas another
eliminated the long (p85??) splice variant, but allowing for the expression of the shorter
p50??and p55??isoforms (Suzuki, H et al., 1999). Both lines of p85 knockout mice have
impaired pre-B cell development, reduced numbers of mature B cells in the spleen. These
results suggest class IA PI3Ks play a role in the development of B cells. More recently, a
defective B-cell development was also revealed in mice lacking the PI3K??(Jou, ST et al.,
2002). It was shown that the percentages of pro-B cells (CD43+ B220+) were similar for
wild type and PI3K??deficient bone marrow. However, the more mature B-cell
subpopulation of B220+ CD43- cells was significantly reduced in the bone marrow of
PI3K??knockout mice. Taken together, these results point to an essential and nonredundant
and stage-specific role for PI3K??in the development and differentiation of B cells.
In addition to B cells, recent studies have implicated PI3Ks in erythropoiesis, the process
by which erythrocytes are formed. The process of erythropoiesis can be divided into a
number of discrete parts: recruitment and commitment of pluripotent hematopoeitic stem
cells to erythroid progenitor cells including erythroid burst-forming unit (BFU-E) and
erythroid colony-forming unit (CFU-E), commitment of CFU-E to erythroblasts in the
presence of EPO, and enucleation of the mature erythroblasts and their release into the
bloodstream (Perry, C et al., 2002). Glycophorin A(GPA) is a marker for early erythroid
differentiation, which has been most commonly used to define erythroid progenitors
(Chasis, JA et al., 1992). Myklebust et al. have demonstrated that the formation of GPApositive
cells from CD34+ stem cells were significantly inhibited by LY294002, the
specific PI3K inhibitor (Myklebust, JH et al., 2002). In another study, it was shown that
LY294002 not only inhibited the proliferation of erythroid progenitor cells (CFU-E), but
also induced apoptosis of these cells as well (Haseyama Y et al., 1999 ).
1.Introduction
17
Although the above data provide evidence that PI3Ks play an essential role in the earlier
stages of erythroid differentiation, the isoform-specific role of PI3Ks in this process has
not been well established. Recently, Kubota et al. have shown that the antisense
p85??oligonucleotide inhibited the EPO-induced formation of CFU-E from normal human
hematopoietic progenitor cells, implicating the class IA PI3Ks in erythropoiesis (Kubota,
Y et al., 2001). It is proposed the binding of p85??through its SH2 domain to the tyrosinephosphorylated
EPO receptor is an essential step for the EPO induced erythroid
differentiation signals. However, this in vitro result was not confirmed in p85?-deficient
mice which show defects in B cell development but not in erythropoiesis (Fruman, DA et
al., 1999; Suzuki, H et al., 1999). The authors suggested other molecules of PI3K family or
other signalling molecules may compensate for the defect of p85??function in
erythropoiesis during the early development of p85?-deficient mice. However, the precise
reason for this discrepancy is still unclear.
The importance of PI3Ks in the regulation of erythropoiesis is also highlighted by the
study on the cell transformation processes leading to erythroleukemia. PU.1 is a
hematopoietic-specific Ets transcription factor, promoting differentiation of lymphoid and
myeloid lineages. PU.1 dysregulated activation in erythroid progenitors can induce
erythroleukemia in mice (Moreau-Gachelin, F et al., 1996). Barnache et al. showed that the
overexpression of PU.1 in murine proerythroblasts induces their differentiation arrest and
hyperproliferation, and the transformation ability of PU.1 is, at least in part, due to the
subsequent activation of PI3Ks (Barnache, S et al., 2001). Thus the dysregulated activation
of PI3Ks seems to interfere with the terminal erythroid differentiation. Paradoxically,
Bavelloni et al. have demonstrated the dimethyl sulfoxide (DMSO) induced terminal
differentiation of murine erythroleukemia cells was inhibited by LY294002 (Bavellooni, A
et al., 2000). Thus the exact role of PI3Ks in the terminal differentiation of erythrocyte is
unknown.
In addition to the above data, PI3Ks have been shown to be involved in other lineage
differentiation processes. In this regard, the neutrophilic differentiation of HL60 cells
induced by G-CSF was enhanced by wortmannin in a dose-dependent manner (Kanayasu-
Toyoda, T et al., 2002). In another study, Baier et al. have demonstrated that PI3K??plays
an essential and nonredundant role in the differentiation of myelomonocytic U973 cells,
despite in PI3K??deficient mice the myeloid differentiation seems to be normal (Bayer, R
et al., 1999; Hirsch, E et al., 2000; Sasaki T et al., 2000). It should be noted that a targeted
gene disruption in vivo does not simply indicate a functional defect of the targeted
1.Introduction
18
molecule, but provide a sum of alternative mechanisms by which other factors adapt to the
unusual circumstances. The action of compensatory or redundant pathways may obscure
the authentic in vivo role of the targeted gene.
1.4.2 Involvement of PI3Ks in differentiation of non-hematopoietic cells
The involvement of PI3Ks in differentiation is not only restricted to cells of hematopoietic
origin, several lines of evidence have implicated PI3Ks in the differentiation processes in
other cell types.
Rat pheochromocytoma PC12 cells are a model system for the differentiation of neuronal
cells. After stimulation with nerve growth factor (NGF), neurite-specific genes are induced
and neurite outgrowth occurs (Halegoua, S et al., 1991). It was shown that PI3K is
activated in PC12 cells after NGF treatment, and inhibition of PI3K by the PI3K specific
inhibitor wortmannin abrogated the NGF induced neurite outgrowth of these cells (Kimura,
K et al., 1994). In addition, a dominant-negative mutant of p85 was also shown to have a
similar inhibitory effect on neurite outgrowth as with wortmannin (Jackson, TR et al.,
1996). Conversely, in the absence of NGF, neurite outgrowth could be partially induced by
the microinjection of a constitutively active mutant of PI3K, and this effect of PI3K
seemed to be due to the subsequent activation of the Rac-JNK pathway (Kita, Y et al.,
1998).
A similar role for PI3K was also revealed in the myogenic differentiation. Chicken embryo
myoblasts (CEM) are the precursors of chicken skeletal-muscle cells. CEM undergo
myogenic differentiation in vitro. The oncogene p3k, coding for a homolog of the catalytic
subunit p110??of PI3K with constitutive activity, was shown to strongly enhance the
myogenic differentiation in cultures of CEM. This effect was abrogated by the use of PI3K
inhibitor LY294002 (Jiang, BH et al., 1998). Another study on the mechanism by which
insulin-like growth factors induce myogenesis in cultures of rat skeletal-muscle myoblast
cell line L6E9 also identified PI3K as an essential component in this process (Kaliman, P
et al., 1998).
While the above findings point to a positive role of PI3K in the regulation of cellular
differentiation, in some other studies PI3K was shown to exert an opposite effect. A recent
study by Ptasznik et al. has demonstrated that blockade of the basal level of PI3K activity
in primary cultured human fetal pancreatic cells resulted in a robust activation of
spontaneous endocrine differentiation. Thus, the primary role of PI3K in these cells seems
to keep them in an undifferentiated state (Ptasznik, A et al., 1997). Similarly, a negative
1.Introduction
19
role of PI3K in differentiation was also demonstrated in human colon cancer cells, HT29
and Caco-2 (Wang, D et al., 2001). In this study the authors showed the PI3K inhibitor
wortmannin significantly enhanced the sodium butyrate induced differentiation of these
cells in a time- and dose-dependent fashion, and the effect of wortmannin was mimicked
by overexpressing the dominant negative mutant of p85. Interestingly, overexpression of
PTEN also augmented the inductive effect of sodium butyrate on the differentiation of
these cells, which suggests a contributory role of the lipid products of PI3K in intestinal
cell differentiation.
In summary, available data suggest PI3Ks are important regulators for cellular
differentiation in mammalian cells. A major change in the future work is to define the
nonredundant role for each of the PI3K isoforms in a specific differentiation process.
Furthermore, studies directed at downstream signalling elements coupled to PI3Ks should
be of great value, as will the investigation of the transcription factors by which PI3Ks are
linked to tissue-specific gene regulation.
1.Introduction
20
1.5 Aim of the work
Phosphoinositide 3-kinase (PI3K) is a ubiquitous enzyme that has been shown to be an
important mediator of various intracellular signalling pathways. In mammals, complexity
arises by the expression of multiple PI3K isoforms which differ in structure, substrate
specificity, binding partner and mode of regulation. Despite of this, much progress has
been made in defining the role of PI3Ks in many aspects of cellular processes , such as cell
proliferation, cell migration, cell cycle entry and survival. However, our knowlege about
the role of PI3Ks in cell differentiation is relatively limited. Several lines of evidence have
indicated PI3Ks can be positive or negative regulators of cell differentiation in different
cell types. PI3K?, the only isoform of class IB PI3K is predominantly expressed in cells of
hematopoietic origin. It was previously implicated in the retinoic acid induced
differentiation of U937 cell line. This prompted us to make further investigations using
human peripheral blood stem cells. These cells were induced to differentiate into several
lineages by using different combinations of growth factors. The preliminary results showed
a downregulation of PI3K??expression is specifically associated with erythropoietininduced
erythroid differentiation, indicating a possible involvement of PI3K??in this
process. The aim of this work was to explore the role of PI3K??in erythroid differentiation
in more details using K562 cell line as a model system. As PI3K??possesses both lipid
kinase and protein kinase activities, we intend to make use of a panel of engineered
PI3K??mutants to address which kinase activity is involved in this process. Furthermore,
immunoprecipitation will be used to study the potential protein-protein interactions in
attempts to define the downstream targets of PI3K?.
2. Materials and Methods
21
2. MATERTIALS AND METHODS
2.1 Materials
2.1.1 Cells
??CD34+ stem cells
Isolated from peripheral blood of healthy adult donors.
??K562 Cell line
Human chronic myeloid leukemia in blast crisis, established from the pleural effusion of a
53-year-old woman with CML in blast crisis in 1970 (Lozzio and Lozzio 1975); The cells
produce haemoglobin. They carry the Philadelphia chromosome with b3-a2 fusion gene.
This cell line was purchased from DSM, Braunschweig, Germany.
2.1.2 Bacterial strains (E. coli)
Escherischia coli XL1-Blue (Stratagene, La Jolla, CA, USA ) , used for plasmid
amplification
2.1.3 Plasmids
??pcDNA3 empty vector (Invitrogen, GmbH, Karlsruhe,Germany)
??pcDNA3-PI3K??-wild type
Encoding the full length PI3K?
??pcDNA3-PI3K??-K832R
Mutant of PI3K??with mutation in wortmannin binding site, catalytically inactive
??pcDNA3-PI3K??-FRAP
Swap mutant between PI3K??and mammalian TOR (FRAP)
??pcDNA3-PI3K??CAAX
Membrane associated form of PI3K??by adding into C-terminus of PI3K??sequence of
Raf/K-Ras-CAAX for membrane localisation
??pcDNA3-PI3K??-antisense
Generated by inverting the orientation of the wild-type PI3K??full-length cDNA into
pcDNA3.
2. Materials and Methods
22
??pcDNA3-GFP-PI3K?
A cDNA of PI3K??subcloned in pEGFP-N1 Plasmid (Clontech, Heidelberg, Germany)
2.1.4 Primers for RT-PCR
??Primers used for an amplification of human p101:
forward primer: 5' gacatgcacggaggaccgcatccag 3'
reverse primer: 5' acagcaaggaactcggcctg3'
??Primers used for an amplification of human ?-actin:
forward primer: 5' ccgtaccactggcatcgtgat 3'
reverse primer: 5' catactcctgcttgctgatc 3'
2.1.5 Antibodies
Antibodies for Western blot and Immunoprecipitation
??Anti-PI3K??monoclonal antibody, directed against the N-terminal fragment of PI3K??,
used for Western blot (FSU facilities)
??Anti–PI3K??monoclonal antibody, clone 641, used for Immunoprecipitation
(Eurogentech facilities, Berlin, Germany)
??Anti–PI3K??polyclonal antibody, directed against the peptide SOLPESFRVPYDPGN
located in the C-terminal fragment of PI3K??, used for immunoprecipitatation (FSU
facilities)
??Anti–PI3K??polyclonal antibody (s-19) (Santa Cruz Biotechnology, Inc., Heidelberg,
Germany)
??Anti-p85 polyclonal antibody (Upstate biotechnology, Hamburg, Germany)
??Anti–HA monoclonal antibody (BabCO, Richmond, California, USA)
??Anti-phospho-Akt (S473) polyclonal antibody (New England Biolabs, Frankfurt,
Germany)
??Anti-Akt1 polyclonal antibody (Pharmingen, Heidelberg, Germany)
??Anti-phospho-p42/44 MAPK monoclonal antibody (New England Biolabs, Frankfurt,
Germany)
2. Materials and Methods
23
??Anti-pan-ERK monoclonal antibody (BD Transduction Laboratories, Heidelberg,
Germany)
??Anti-vinculin mouse monoclonal antibody (Serotec LTD,UK)
??Horseradish-peroxidase-conjugated goat-anti-mouse IgG (Santa Cruz Biotechnology,
Inc, Heidelberg, Germany)
??Horseradish-peroxidase-conjugated goat-anti-rabbit IgG (Santa Cruz Biotechnology,
Inc, Heidelberg, Germany)
Antibodies for immunocytochemistry and FACS analysis
??Anti-human hemoglobinA polyclonal antibody (DAKO, Diagnostika GmbH, Hamburg,
Germany)
??Anti-CD34-PC5 (phycoerythrin)monoclonal antibody (BD Transduction Laboratories,
Heidelberg, Germany)
??Isotype control for IgG1-PC5 ( Beckman /Coulter, Germany)
??Anti-CD61-FITC (fluoresceinisothiocyanate) monoclonal antibody (DAKO,
Diagnostika GmbH, Hamburg, Germany)
??Anti-Glycophorin A-PE monoclonal antibody (DAKO, Diagnostika GmbH, Hamburg ,
Germany)
??Isotype control antibodies IgG1-FITC, PE (DAKO,Diagnostika GmbH, Hamburg,
Germany)
??Streptavindin-Biotin-AP-conjugated anti-rabbit secondary antibody (DAKO Alkalinephosphatase-
kit 5005)
2.1.6 Medium and substances for cell culture
??RPMI1640 (BIOCHEM AG, Berlin, Germany)
??Iscoves modified Dulbecco medium (IMDM) (BIOCHROM KG , Berlin, Germany)
??Fetal calf serum (Life Technologies GmbH, Eggenstein, Germany )
??Recombinant human erythropoietin (EPO) (Gilag GmbH, Fresenius AG, Germany)
??Recombinant human thrombopoietin (TPO) (TEBU GmbH, Frankfurt, Germany)
??Recombinant human IL6 (Roche Diagnostics GmbH, Mannheim, Germany)
??Recombinant human granulocyte colony-stimulating factor (C-GSF) (AMGEN GmbH,
Munich, Germany)
??Recombinant human stem cell factor (SCF) (AMGEN, Thousand Oaks, CA, USA)
2. Materials and Methods
24
??N-Acetyl-L-alanyl-L-glutamin (Biochrom KG, Berlin, Germany)
??Iron saturated transferrin (Sigma, Munich, Germany)
??Antibiotics: 100 U/ml penicillin/ 0.1 mg/ml streptomycin (Sigma, Munich, Germany)
??Cytosine arabinoside (Amersham Pharmacia Biotech, Freiburg, Germany)
2.1.7 Materials for silver staining
??Acetic acid glacial (Carl Roth GmbH & Co KG, Karlsruhe, Germany)
??Formaldehyde (37 %, w/v) ( Carl Roth GmbH & Co KG, Karlsruhe, Germany)
??Methanol (Carl Roth GmbH & Co KG, Karlsruhe, Germany)
??Silver nitrate (Carl Roth GmbH & Co KG, Karlsruhe, Germany)
??Sodium carbonate (anhydrous) (Carl Roth GmbH & Co KG, Karlsruhe, Germany)
??Sodium thiosulfate (Carl Roth GmbH & Co KG, Karlsruhe, Germany)
2.1.8 Other reagents and materials
??Acrylamide, Rotiphorese Gel 30 (30 % Acrylamide / 0,8 % Bisacrylamide) (Carl Roth
GmbH & Co KG, Karlsruhe, Germany)
??Actigel ALD Superflow kit (Sterogene, supplied in 20% Ethenal, 0.02%Sodium azide).
??[?32P]ATP, 3000 Ci/mmol (NEN Life Science, Brussels, Belgium)
??Benzidine dihydrochloride (Sigma, Munich, Germany)
??BioMax MR-1 Film (Amersham, Freiburg, Germany)
??Bradford-reagent (Sigma, Munich, Germany)
??CliniMACS separation system (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany)
??Dimethyl pimelimidate (Sigama, USA )
??Ethanolamine (Merck, frankfurt, germany)
??ECL-Kit (NEN Life Science, Brussels,Belgium)
??Gel drying film (Promega, Heidelberg, Germany)
??Geneticin (G418) (Invitrogen, Karlsruhe, Germany )
??Glycerol (Carl Roth GmbH & Co KG, Karlsruhe, Germany)
??LipofectAMINE PLUSTM Reagent (Invitrogen, Karlsruhe, Germany)
??LY 294002 (Calbiochem Novabiochem, Bad Soden, Germany)
??NP-40 (Serva, Heidelberg, Germany)
??PD 98059 (Calbiochem Novabiochem, Bad Soden, Germany)
??Peptone (Becton Dickinson, Heidelberg,Germany)
2. Materials and Methods
25
??Protease inhibitors (Sigma, Munich, Germany)
??Protein A-Sepharose (Amersham Pharmacia Biotech, Freiburg, Germany)
??Protein G-Sepharose (Amersham Pharmacia Biotech, Freiburg, Germany)
??Polyvinylidene fluoride (PVDF)-membrane (Millipore, Eschborn,Germany)
??Qiagen plasmid-purification kit (QIAGEN GmbH, Hilden, Germany)
??Qiagen RNeasy Mini Kit (QIAGEN GmbH, Hilden, Gernany)
??Qiagen One-step RT-PCR Kit (QIAGEN GmbH, Hilden, Gernany)
??Sodium borate (Carl Roth GmbH & Co KG, Karlsruhe, Germany)
??Sodiumdodecylsulfate (SDS) (Carl Roth GmbH & Co KG, Karlsruhe, Germany)
??Sterile filters, 0.22 µm (Millipore Corporation, Eschborn,Germany)
??Triton X-100 (Carl Roth GmbH & Co KG, Karlsruhe, Germany)
??Tween 20 (Serva, Heidelberg, Germany)
??Yeast extract (Serva, Heidelberg, Germany)
2.1.9 Buffers and Solutions
All buffers and solutions were prepared with deionized water. Specifications in percentage
are in weight per volume for dry substances and volume per volume for liquids.
Phosphate-buffered saline (PBS)
Na2HPO4?7 H2O 4.3 mM
KH2PO4 1.4 mM
NaCl 137 mM
KCl 2.7 mM
TAE(1x)
0.04M Tris-acetate
0.01 M EDTA
Buffers for bacteria work
5 x KCM-buffer
KCl 500 mM
CaCl2 150 mM
MgCl2 250 mM
1?LB-medium
Bacto-Trypton (Peptone) 10 g
yeast extract 5 g
NaCl 10 g
fill up to 1L with H2O
for LB-agar , add 15g of agar
TSB
2?LB 75 ml
DMSO 7.5 ml
1M MgCl2 1.5 ml
1M MgSO4 1.5 ml
PEG4000 15 g
Add to 150ml with H2O and Steriled with
0.22 µm filter
2. Materials and Methods
26
Buffers for Western blot
Lysis buffer
20 mM HEPES (pH 7.5),
150 mM NaCl
1 0mM EGTA
1 % NP40
10 µg/ml leupeptin?
1 µg/ml pepstatin A?
1 µg/ml apportion?
1 mM benzamidine?
10 mM Pefabloc?
0.1 mM PMSF?
1 mM sodium-orthovanadate?
10 mM ?-glycerolphosphate?
10 mM NaF?
1 mM DTT?
(??added freshly just before use)
5 x Loading buffer
50 % glycerol
500 mM Tris(pH: 6.8)
5 mM EDTA
10 % SDS
5% ?-mercaptoethanol
0.1 % bromophenol blue
SDS-PAGE electrophoresis buffer
25 mM Tris
250 mM Glycine
0.1 % SDS
Semi-dry transfer buffer
TrisBase 48 mM
Glycine 39 mM
SDS 0.037 %
Methanol 20 %
TBS-Tween washing buffer(pH,7.5)
TrisHCl pH 7.5 20 mM
NaCl 137 mM
0.1 % Tween-20
Membrane Strip-Buffer
Tris-HCl pH6.8 62 mM
?-Mercaptoethanol 100 mM
SDS 2 %
Coomassie-stain solution
Coomassie G250 (for membranes)
Coomassie R250 (for gels) 2 g/L
Acetic acid 10 %
Methanol 50 %
Destaining solution for Coomassiestained
gel
acetic acid 10 %
Methanol 25 %
PI3K??protein kinase reaction buffer
12.5 mM MOPS pH 7.5
7.5 mM MgCl2
12.5 mM ?-glycerolphosphate
10 mM NaF?
1 mM Sodium-orthovanadate?
(??added freshly just before use)
2. Materials and Methods
27
Silver staining solutions
??Fixation solution
Methanol:Acetic acid: Water 50:5:45(v/v/v)
??Sensitizing solution
Sodium Thiosulfate 0.02 %
??Staining Solution
Silver nitrate 0.1 %
??Developing solution
Formaldehyde 0.04 in 2 % sodium carbonate
??Stop solution
Acetic acid 1 %
2.2 Methods
2.2.1 Plasmid preparation
Competent bacteria preparation
2ml LB-medium (without ampicillin) were inoculated with E.coli XL1-Blue and grown
over night at 37°C. 1 ml of this culture was transferred to 150 ml fresh LB-medium
without ampicillin. The culture was incubated on a shaker for 2-4 hours at 37°C until
OD600 reaches 0.6. Then the cells were pelleted by centrifugation of the culture for 10 min
at 5000rpm, and resuspended in 15 ml TSB. This suspension was then incubated on ice for
10 minutes, aliquoted and stored at -80°C.
Transformation of Competent bacteria and plasmid preparation
100 µl competent E.coli XL-1 blue cells were thawed on ice and mixed with a suspension
containing 80 µl H2O, 20 µl 5 x KCM-buffer and 0.1-10 µg plasmid DNA. The cells were
then incubated on ice for 20-40 min and afterwards subjected to a heat shock at 42°C for
45 seconds. The bacteria were cultured in 1ml LB medium without ampicillin on a shaker
for 1 hour at 37°C. Then the cells were pelleted by centrifugation for 10 min at 5000 rpm,
and plated on a LB-agar plate containing ampicillin (100 µg/ml) and grown at 37°C over
2. Materials and Methods
28
night. From the plates, single colonies were picked and used to inoculate 2-5 ml LBmedium
as a starter culture for plasmid preparation. Plasmid DNA was purified from
bacterial cultures using Qiagen plasmid-purification kits following the manufacturer’s
instructions.
2.2.2 Isolation of CD34+ progenitors from peripheral blood
The peripheral blood stem cells (PBSC) were collected on day 4, 5 and 6 after G-CSF (10
?g/kg body weight) treatment from healthy donors after informed consent by means of a
Cobe spectra cell separator. The CD34+ positive cells were isolated by positive selection
using anti-CD34 antibody (GBEND/10) and magnetic cell sorting system for clinical use
according to the manufacturer’s instructions.
2.2.3 Culture of CD34+ cells and induction of differentiation
CD34+ cells were cultured in Iscoves modified Dulbecco medium (IMDM), supplemented
with 30% pre-tested fetal calf serum (FCS), 12 ?l/ml N-Acetyl-L-alanyl-L-glutamine, 8
?l/ml iron saturated transferrin (27 mg/ml in IMDM), 10-6 M Mercaptoethanol, 100 U/ml
Penicillin/100 ?g/ml streptomycin.10 ng/ml SCF, 100 ng/ml IL6 at 37°C in a humidified
atmosphere containing 5% CO2. After expansion for 5 days, cell differentiation was
induced by addition of 50ng/mL TPO or 4 U/ml EPO to the above culture medium in the
presence of SCF/IL6 for the indicated time periods .
2.2.4 Flow cytometry analysis
Cells were washed once in PBS containing 2 % human serum albumin (PBS-A), and
incubated with antibodies directed against the following antigens CD34, Glycophorin A, or
CD61 for 30 minutes applied at the recommended concentrations. Control stainings were
performed with isotype-matched control antibodies under the same conditions. After the
staining, cells were washed once and resuspended in PBS-A. Fluorescence was analysed
on a coulter EPICS profile II flow cytometer. Data acquisition and analysis were
performed by “Flow mate” list mode software (DAKO).
2. Materials and Methods
29
2.2.5 K562 cell culture and induction of differentiation
K562 Cells were cultured in RPMI 1640 medium containing 10% fetal calf serum.
Cultures were maintained at 37°C with 5% of CO2. The cultured cells were passed every
three days, seeding at a density of about 2 ??105 cells/ml. Cell counts were determined
using the trypan-blue dye exclusion test.
For the induction of erythroid differentiation, exponentially growing K562 cells were
seeded at a density of 2 ??105 /ml. A single-dose of freshly-prepared Ara-C was added at
the seeding time. Cells were cultured for indicated time without changing the medium.
2.2.6 Assessment of erythroid differentiation of K562 cells
Benzidine staining
Benzidine staining was performed as described previously (Belhacene, et al. 1998).
Briefly, 50 microliters of cell suspension (0.5-1x106 cells/ml) in PBS were mixed with
10µl of freshly- prepared benzidine reagent (0.6 % H2O2, 0.5 % acetic acid, and 0.2 %
benzidine dihydrochloride) and incubated at 37oC for 5 min. The percentage of benzidinepositive
cells (blue) was determined by light microscopic examination of at least 200 cells
per sample. Each experiment was performed in triplicate, and results were averaged.
Immunocytochemistry for the detection of Haemoglobin A
Cells were washed twice in PBS and fixed with 4 % paraformaldehyde at 4°C for 30min.
After 1 wash with PBS, cells were distributed on to slides by cytospin, and post-fixed with
pre-cold (-30°C) acetone at 4°C for 2 minutes. Following 3 washes with PBS, cells were
incubated with rabbit-anti-haemoglobin A antibody (1:2000, in PBS containing 2 % human
serum albumin diluted) overnight at 4°C in a humid chamber. After 3 washes with PBS,
cells were subsequently incubated with Streptavindin-Biotin-AP-conjugated anti-rabbit
secondary antibody (DAKO Alkaline-phosphatase-kit 5005) for 30minutes. After 3 washes
in PBS, the positive staining was visualised by incubated with the chromogen-substrate for
Alkaline-phosphatase (accompanied with the kit) until a sufficient degree of staining is
obtained. Finally, the slides were counterstained with hematoxilin for 3 minutes, and
covered with glycerolgelatin.
2. Materials and Methods
30
2.2.7 Stable transfection of K562 cells
Exponentially growing cells were transfected using LipofectAMINE PLUSTM Reagent
1. Pre-complex the DNA with PLUS Reagent: dilute 2-4 µg plasmid DNA into 250 µl
serum-free medium, then add 5µl PLUS Reagent, mix gently and incubate at room
temperature for 15 minutes.
2. Dilute 10µl LipefectAMINE Reagent into 250 µl serum-free medium in a second tube,
and mix gently .
3. Combine the pre-complexed DNA and diluted 10µl LipefectAMINE Reagent; mix and
incubate for 15 minutes at room temperature.
4. Add the above complex to a 60-mm tissue culture plate containing about 0. 5-1?106
cells in 1.5 ml serum-free medium.
5. After 3 hours incubation, the DNA containing medium was replaced by normal
medium containing 10% FCS.
As negative controls, cells were mock-transfected without the addition of plasmid DNA.
48 hours after transfection, the cells were seeded in RPMI1640 medium supplemented with
1 mg/ml G418 for selection. Two to 3 weeks later, when mock-transfected cells died, cells
were pooled and maintained in medium with 0. 4 mg/ml G418.
2.2.8 Western Blot
Preparation of protein extracts
Cells were pelleted by centrifugation at 1000 rpm for 4 minutes and washed twice with
PBS, then lysed in an appropriate ice-cold lysis buffer depending on the subsequent
sample preparation. The lysates were vortexed and kept on ice for 15 minutes. Cellular
debris was removed by centrifugation at 14,000 rpm for 20 minutes at 4°C. The
supernatant was then used for the assay.
Western blotting
Proteins were resolved by electrophoresis using appropriate percent sodium dodecyl sulfate
polyacrylamide (SDS-PAGE) gel according to the molecular weight of the proteins of
interest. After that proteins were transferred onto PVDF membrane using semi-dry transfer
conditions. In semi-dry blotting, the gel and the membrane were sandwiched between
2. Materials and Methods
31
buffer-soaked 3MM Whatmann papers. The PVDF-membrane was pre-wetted in methanol
for 10 seconds before it was submerged in transfer-buffer for 15 minutes to allow for
equilibration. Proteins were transferred at a constant current of 2.5 mA/cm2 for one hour.
Thereafter, the membrane was blocked with TBS-Tween buffer containing 5 % dry milk or
1 % BSA for 30 minutes at room temperature. Following blocking, the membrane was
incubated with primary antibody diluted in TBS-Tween buffer containing 1 % dry milk or
1%BSA for 1 hour at room temperature. Phosphospecific antibodies were incubated with
the membrane over night (16 hours) at 4°C. The antibodies were diluted at concentrations
as recommended by the manufacturers. The membrane was then washed three times with
TBS-Tween, and, subsequently incubated for 1 hour at room temperature with appropriate
HRP-conjugated secondary antibody diluted in TBS-Tween containing 1% dry milk. After
three washes in TBS-Tween, the membrane was submerged for 1 minute in ECL solution,
wrapped in Saran foil and exposed to Kodak Biomax films. Blots can be stripped by
incubating the membrane in stripping buffer for 30 minutes at 50°C to remove the bound
antibodies, and re-probed with other antibodies.
2.2.9 Purification of polyclonal anti-PI3K ??antibody
Polyclonal antibody against PI3K??used for immunoprecipitation was purified from the
immunized rabbit serum using Actigel ALD Superflow kit (supplied in 20% Ethanol, 0.02
% sodium azide).
??Preparation for the following buffers
1. 0.1 M Phosphate buffer , pH 7.4
77.4 ml buffer A (0.1 M Na2HPO4)
+ 22.6 ml buffer B (0.1 M NaH2PO4)
2. 0.015 M Phosphate buffer, pH 7.4
3. 0.5 M NaCl, pH 7.2
4. 20 mM Glycine, pH 2.5
5. 1 M Tris, pH 8.0
??Peptide- coupling to Actigel ALD Superflow resin
1. Take 1 ml of suction-tried resin, wash the resin with 5 ml of 0.1 M Phosphate buffer
for three times on a Buchner funnel, and pack the resin in a column.
2. Materials and Methods
32
2. Mix 100 µl of peptide (about 1mg) which was used for raising the PI3K??antibody with
100µl of ALD-coupling solution (1 M NaCNBH3) and 800 µl of 0.1 M Phosphate
buffer.
3. Apply the mixture to the column and let it stand overnight at 4°C.
??Purification of the antibody
1. Wash the column with 10 ml of 0.1 M Phosphate buffer for equilibration.
Pass 10-15 ml of rabbit serum containing the anti-PI3K??antibody through the column.
2. wash the column with 20 ml of 0.015 M Phosphate buffer (pH 7.2), followed by 20 ml
of 0.5 M NaCl (pH 7.2).
3. Elute the column with 20 mM Glycine (pH 2.5). Collect the eluate in 1.5 ml conical
tubes containing 50 µl of 1 M Tris (pH 8.0). Mix each tube gently to bring the pH to
neutral.
4. Determine the antibody concentration in each fractions by Bradford method and store
the antibody in 50 % glycerol with 0.02 % sodium azide.
2.2.10 Coupling anti-PI3k ??monoclonal antibody to protein G- Sepharose
1. Take 2 ml of tissue-culture supernatant containing about 10-20 µg of monoclonal
antibody, mix with 20 ?l of protein G beads (prior to use, washed three times with 10
mM Tris (pH 8.0) to remove the PBS/ethanol solution the resin is stored in) at room
temperature for 1hour with gentle shaking.
2. Wash the beads twice with 10 volumes of 0.2 M sodium borate (pH 9.0) by
centrifugation at 5000 rpm for 2 minutes.
3. Resuspend the beads in 10 volumes of 0.2 M sodium borate (pH 9.0), add dimethyl
pimelimidate to bring the final concentation to 20 mM.
4. Incubate for 30 minutes at room temperature with gentle mixing.
5. Stop the reaction by washing the beads once in 0.2 M ethanolamine (pH 8.0) and then
incubate at room temperature for half a hour in 0.2 M ethanolamine (pH 8.0) with
gentle mixing.
6. After the final wash, resuspend the beads in PBS with 0.01 Merthiolate.
2. Materials and Methods
33
2.2.11 Immunoprecipitation
For immunoprecipitation, 1x10 8 of cells were pelleted and washed twice with PBS, then
lysed in 3 ml of ice-cold lysis buffer. The soluble material was incubated with about 10 µg
of antibodies for 2 hours at 4°C. Immune complex was then incubated with 30 µl of protein
G or protein A-Sepharose for 1 hour at 4°C (prior to use, the Sepharose was washed three
times with lysis buffer to remove the PBS/ethanol solution the resin is stored in).
Alternatively, for special purpose, cell lysate was incubated directly with monoclonal
antibody pre-coupled protein G beads for 1 hour at 4°C. After that the antigen-antibodyprotein
A/G complex was collected by centrifugation at 3000 rpm for 2 minutes.
Immunoprecipitate was washed three times with lysis buffer. Pellet was then processed for
in vitro protein kinase assay or resuspended in electrophoresis sample buffer for
subsequent immunoblotting analysis.
2.2.12 PI3K ??Protein Kinase activity assay
Cells were pelleted, lysed and subjected to anti-PI3K??immunoprecipitation.
immunoprecipitates were washed three times with lysis buffer and once with protein kinase
buffer. Pellets were then resuspented in 50 µl of protein kinase buffer, incubated in the
presence or absence of 10 µM LY 294002 for 30 minutes at 30°C. The reactions were
initiated by adding 20 µm ATP and 10 µCi ??32P?ATP to the protein kinase buffer. After
30-minute incubation at 37°C on a thermomixer, reactions were stopped by adding 12.5 µl
of 5 ??sample buffer. Samples were denatured at 95°C for 10 minutes and resolved on 8%
SDS-PAGE gel. After silver or Coomassie staining, the gel was dried on a vacuum geldryer.
The phosphorylated proteins were revealed by exposure of the gels to BioMax MR-1
Films.
2.2.13 Silver staining
Silver staining was performed as described by Shevchenk (Shevchenko, et al. 1996) with
some modifications
1. After electrophoresis, fix the gel in fixation solution for 20 minutes.
2. Wash the gel with 50% methanol for 10 minutes, and with water for 30 minutes on a
shaking platform.
2. Materials and Methods
34
3. Incubate the gel in sensitizing sulotion for 1 minute. Discard solution and quickly rinse
the gel with two changes of water ( 1 minute each).
4. Incubate the gel in chilled 0.1% silver nitrate for 20 minutes at 4°C.
5. Discard the silver nitrate solution and quickly rinse the gel twice with water (1 minute
each).
6. Incubate the gel in developing solution for 1-10 minutes until a sufficient degree of
staining is obtained.
7. Stop the staining by discarding the developing solution and replace with 1% acetic acid
, and dry the gel with Gel-drying film.
2.2.14 RNA isolation and RT-PCR
RNA isolation
Total RNA was isolated using the Qiagen RNeasy Mini Kit according to the
manufacturer’s instructions. Briefly, cells were lysed in lysis buffer containing the highly
denaturing agent guanidine isothiocyanate, which immediately inactivates RNAses to
ensure the isolation of intact RNA. To shear genomic DNA and to reduce the viscosity, the
lysate was passed through a 20 -G needle (Ø 0.9 mm) 5 to 10 times for homogenization.
Add 1 volume of 70 % ethanol to the homogenized lysate to adjust conditions for the
binding of RNA to the RNeasy column. Apply the lysate on the RNeasy column and
centrifuge for 15 seconds at 12000 rpm to allow for the binding of RNA to the column
membrane. After removing contaminants with washing buffer, the RNA bound on the
column was eluted with 30 ?l of RNase free water. Measure the RNA concentration and
store it at -20°C.
RT-PCR
RT-PCRs was performed using the One-step RT-PCR Kit from Qiagen. The following
conditions was used for each reaction:
??Reaction mixture for one reaction
RNA template 1 ?g
5x reaction buffer 10 ?l
2. Materials and Methods
35
Q-solution 10 ?l
forward primer 50 pmol
reverse primer 50 pmol
dNTP 2 ?l
RT-PCR enzyme 2 ?l
ddH2O to 50 ?l final volume
Thermal-cycler conditions
Reverse transcription: 30 minutes 50°C
Initial PCR activation: 15 minutes 95°C
3-Step cycling
Denaturation: 45 seconds 94°C
Annealing: 1 minute 56°C
Elongation: 1 minute 72°C
Number of cycles: 30 cycles
Final elongation 5 minutes 72°C
The products of the amplified PCR fragments were electrophoreticaly separated on 1.5 %
agarose gel in 1x TAE containing 0.5 µg/ml of ethidium bromide at 90 V. Gels were
illuminated with UV light for visualisation.
3. results
36
3. RESULTS
3.1 Involvement of PI3K??in the erythroid differentiation of hematopoietic stem cells
Previous work from our lab has indicated that PI3K??plays a role in ATRA (all-trans
retinoic acid)-induced U937 cell differentiation (Baier, R et al., 1999). However, it is
unclear whether PI3K??is also involved in other lineage–specific differentiation processes.
To address this issue, we first analysed the profile of PI3K??protein expression during the
differentiation processes of human CD34+ progenitor cells. CD34+ cells are able to
differentiate in vitro into different hematopoietic lineage’s depending on the cytokine
combination given. In this study, the CD34+ cells were isolated from human peripheral
blood. Before induced to differentiate, the cells were pre-cultured in the presence of
SCF/IL6 for 5 days for expansion. Thereafter the day 5 cells were induced to differentiate
by using different combination of growth factors.
For the induction of erythroid differentiation, day 5 cells were grown in medium
containing SCF/IL6, and cultured in the presence of 4 U/ml of EPO for the indicated
periods. At each time points, expression of glycophorin A, which is one specific marker for
erythroid differentiation, was analysed by flow cytometry using direct
immunofluorescence. As shown in Fig.5, glycophorin A-positive cells increased from 0.5
% at day 0 to 15 %, 38 % or 69 % after 4, 6, or 8 days in the presence of EPO.
For the induction of megakaryocytic differentiation, day 5 cells were grown in medium
containing SCF/IL6, and cultured in the presence of 50 ng/ml of TPO for the indicated
periods. At each time points, cells were collected and subjected to flow cytometric analysis
of the cell surface expression of CD61, a marker for megakaryocytic differentiation. As
shown in Fig. 6, CD61+ cells increased from 1.4 % at day 0 to 9 %, 12 % or 30 % after 4,
6, or 8 days.
3. results
37
80
70
60
50
40
30
20
10
0
EPO(-)
EPO(+)
0 4 6 8 (days)
Glycophorin A -positive cells (%)
Fig.5 Erythroid differentiation of CD34+ stem cells induced by EPO. CD 34+ stem cells were
pre-cultured in the presense of SCF/IL6 for 5 days for expansion. Day 5 cells were cultured in the
presense of SCF/IL6, or in combination with EPO for the indicated time periods. The erythroid
differentiation was determined by flow cytometric analysis for the cell suface expression of
glycophorin A. Results represent the Mean±SEM of triplicate experiments.
0 4 6 8 (days)
CD61-positive cells (%
TPO(-)
TPO(+)
50
40
30
20
10
0
Fig.6 Megakaryocytic differentiation of CD34+ stem cells induced by TPO. CD34+ stem cells
were pre-cultured in the presense of SCF/IL6 for 5 days for expansion. Day 5 cells were cultured
in the presense of SCF/IL6, or in combination of TPO for the indicated time periods. The
megakaryocytic differentiation was determined by flow cytometric analysis for the CD61
expression. Results represent the Mean±SEM of triplicate experiments.
3. results
38
In parallel with the flow cytometric analysis, cell samples were taken at each time points of
the induction period, and frozen at –80°C. Finally, cells were lysed, and the expression of
PI3K??was evaluated by Western blot. As shown in Fig.7, the PI3K??expression was
significantly downregulated after EPO treatment. In contrast, TPO treatment did not
significantly change the PI3K??expression. These results indicate an inverse correlation
exists between the PI3K??protein level and the erythroid differentiation.
Fig.7 EPO downregulates PI3K??expression in CD34+ stem cells. CD34+ stem cells were precultured
in the presence of SCF/IL6 for 5 days for expansion. Thereafter these cells were cultured
in the presence of SCF/IL6, or in combination of EPO or TPO. Whole cell extracts from the
indicated time points were subjected to immunoblot analysis with the anti-PI3K??antibody. Equal
loading was monitored by immunoblotting with an antibody against pan-ERK.
3.2 Involvement of PI3K??in the erythroid differentiation of K562 cells
Because of the difficulties in the isolation and maintenance of adequate numbers of CD34+
stem cells, we have used the K562 cell line as a model system to explore the possible
functional significance of PI3K??in erythroid differentiation. The K562 is a
erythroleukemia cell line. These cells are pluripotent in that they are able to differentiate
along both erythroid and megakaryocytic lineages in response to a variety of differentiation
inducers (Leary, JF et al., 1987).
In order to clarify if PI3K??is down-regulated in K562 cells upon induced erythroid
differentiation, we therefore analysed the PI3K??expression before and after Ara-c
treatment, because Ara-C is known to be a potent inducer of erythroid differentiation in
PI3K?
0 4 6 8 4 6 8 4 6 8 (Days)
EPO(+) TPO(+) EPO/TPO(-)
pan-ERK
3. results
39
K562 cells (Rowley, PT et al., 1981; Yamada, H et al., 1998). For the induction of
erythroid differentiation, exponentially growing K562 cells were cultured in the presence
of increasing concentrations of Ara-C from 0.1µg/ml to 50 µg/ml for 4 days. After that, the
erythroid differentiation was determined by the benzidine staining for haemoglobin. As
shown in Fig.8, at doses less than 10 µg/ml, Ara-C induced the erythroid differentiation in
a dose-dependent manner. At the concentration of 5µg/ml, Ara-C almost achieved the
maximum inductive effect, resulting in about 33 % of benzidine positive cells, this
concentration was therefore used in subsequent experiments. In the absence of Ara-C,
about 3% cells were benzidine positive, indicative of a minor spontaneous differentiation
of K562 cells under normal culture conditions.
Fig.8 Dose-dependent erythroid differentiation in K562 cells induced by Ara-C. Exponentially
growing K562 cells were seeded at a density of 2 ??105 cells /ml, and cultured in the absence or in
the presence of increasing concentrations of Ara-C as indicated. After 4 days, erythroid
differentiation was determined by benzidine staining. Results represent the Mean±SEM of triplicate
experiments.
After Ara-C treatment, cells were lysed and analysed by Western blot to detect the
expression of PI3K?. As shown in Fig.9A, Ara-C treatment significantly reduces the PI3K?
expression in a dose-dependent manner, further confirming that the PI3K??expression is
inversely correlated with the erythroid differentiation.
In order to know whether PI3K??is a specific isoform of PI3Ks which is associated with
erythroid differentiation, the expression of PI3K?, and its adaptor protein p85 was also
Benzidine-positive cells (%)
0
10
20
30
40
50
0 0.1 1 5 10 50 (?g/ml)
3. results
40
analysed by Western blot upon Ara-C treatment. In contrast to PI3K?, the expression of
PI3K??and p85 was not obviously affected by Ara-C (Fig. 9B).
Fig.9 Selective downregulation of PI3K??expression during erythroid differentiation of K562
cells. Exponentially growing K562 cells were cultured in the absence or in the presence of 5µg/ml
of Ara-C for 4 days. Immunoblots with antibodies against PI3K?, PI3K?, and p85 were performed.
Equal loading of protein was monitored by immunoblottings with anti-vinculin antibody.
To further reveal the relationship of Ara-C induced decrease in PI3K??expression to the
erythroid differentiation, a time-course study was performed. Exponentially growing K562
cells were left untreated or treated with 5 µg/ml of Ara-C from 24 to 72 hours. Erythroid
differentiation was determined every 24 hours in triplicate using benzidine staining. At
each time points, part of the cells was aliquoted, and frozen at –80°C. At the end of
treatment, the PI3K??expression was determined by Western blot. As shown in Fig.10, the
PI3K??expression was significantly decreased 72 hours post Ara-C treatment,
concomitantly, an increase in benzidine-positive cells was also observed at this time point
(Fig.11).
A)
Ara-C(?g/ml ) - 0.1 1 5 10 50
PI3K?
vinculin
B)
Ara-C(?g/ml ) - 0.1 1 5 10 50
PI3K?
p85
vinculin
3. results
41
Fig.10 Time-dependent downregulation of PI3K??expression during erythroid differentiation
of K562 cells. Exponentially growing K562 cells were cultured in the absence or in the presence
of 5µg/ml of Ara-C for indicated time points. At each time point, cells were collected, and the cell
lysates were subjected to Western blotting using anti-PI3K??antibody. Equal loading of protein was
monitored by immunoblotting with anti-vinculin antibody.
Fig.11 Time-dependent erythroid differentiation in K562 cells induced by Ara-C.
Exponentially growing K562 cells were seeded at a density of 2 ??105 cells /ml, and cultured in the
absence or in the presence of 5?g/ml Ara-C for the indicated time periods. Erythroid differentiation
was determined by benzidine staining. Results represent the Mean±SEM of triplicate experiments.
Benzidine-positive cells (%)
30
20
10
0
40
0 1 2 3 (days)
Ara-C(5?g/ml) - + - + - +
24h 48h 72h
PI3K?
vinculin
3. results
42
3.3 Influence of suppressing endogenous PI3K??expression on the erythroid
differentiation of K562 cells
Because the level of PI3K??decreases as the cells undergo differentiation, it is important to
ascertain whether the differentiation event itself requires the downregulation of this
protein. To address this issue, an antisense strategy was used in the present study. A
plasmid encoding full-length PI3K??in antisense orientation was used. K562 cells was
stably transfected with this plasmid. As negative controls, cells were stably transfected
with an empty vector. The endogenous PI3K??expression was analysed after transfection
by western blot. As shown in Fig.12A, cells stably expressing the antisense plasmid
displayed a reduced PI3K??expression in comparison to the cells transfected with an empty
vector, confirming that the expression of an antisense cDNA partially inhibits the
endogenous PI3K??expression.
Fig.12A Overexpression of PI3K??antisense cDNA reduces endogenous PI3K??expression.
K562 cells were stably tansfected with PI3K??antisense plasmid, or an empty vector (Vector) as
described in “Materials and Methods”. Protein levels of PI3K??expression were detected by
Western blot using anti-PI3K??antibody. Equal loading of protein was monitored by
immunoblotting with anti-vinculin antibody .
In order to confirm the inhibition of this construct on protein expression is specific for
PI3K?, the protein level of PI3K??was also examined in parallel. As shown in Fig.12B,
overexpression of this antisense construct did affect the endogenous PI3K??expression.
Vector Antisense
vinculin
PI3K?
3. results
43
Fig.12B Overexpression of PI3K??antisense cDNA does not affect endogenous PI3K?
expression. Lystes form cells stably tansfected with PI3K??antisense plasmid, or an empty vector
were subjected to immunoblot analysis with the anti-PI3K??antibody. Equal loading was monitored
by immunoblotting with an antibody against vinculin.
Subsequently, these stably transfected cells were analysed for their ability to produce
haemoglobin, which was detected by benzidine staining and immunocytochemistry,
respectively. Fig.13 shows the results of benzidine staining. Without Ara-C induction,
about 3% of the cells transfected with an empty vector were benzidine-positive, which is
similar to that of non-transfected parental cells (as shown in Fig.8). In contrast, the cells
expressing the antisense of PI3K??displayed spontaneous differentiation of about 13%.
Furthermore, after induction with 5 µg/ml of Ara-C for 4 days, the antisense transfected
cells exhibited a significantly higher percentage of benzidine-positive cells than that of
control cells tranfected with an empty vector (53% vs 27%).
Ara-C(-), pcDNA3 Ara-C(+), pcDNNA3
Ara-C(+), Antisense
A)
Ara-C(-), Antisense
Vector Antisense
PI3K?
vinculin
3. results
44
The above results were further confirmed by immunocytochemistry using a specific
antibody against haemoglobin A (Fig.14A). Compared with benzidine staining, this
method seems to be less sensitive, as the percentages of positive cells were shown to be
proportionally reduced (Fig.14B). Therefore, benzidine staining method was used in
subsequent experiments. Taken together, these results indicate that suppressing the
expression of PI3K??potentiates the spontaneous and promotes the Ara-C induced erythroid
differentiation of K562 cells
Benzidine-positive cells(%)
B)
Ara-C(-) Ara-C(+)
10
0
20
30
40
50
60
70
vector
antisense
Ara-C(-), pcDNA3
Ara-C(-), Antisense
Ara-C(+), pcDNA3
Ara-C(+), Antisense
A)
Fig.13 Evaluation of erythroid differentiation by benzidine staining. Exponentially growing
K562 cells were cultured in the absence or in the presence of 5 µg/ml of Ara-C for 4 days.
Erythroid differentiation was then detected by benzidine staining. A) representative pictures for
benzidine staining (original magnification, 20 x). B) Quantitative presentation of benzidinepositive
cells. Results represent the Mean±SEM of triplicate experiments.
3. results
45
3.4 Influence of overexpression of PI3K??on the erythroid differentiation of K562 cells
To gain further insight into the role of PI3K??in erythroid differentiation, K562 cells were
stably transfected with wild type PI3K??or a dominant negative PI3K?-KR mutant. The
expression of these proteins was detected by Western blot as shown in Fig.15.
Fig.15 Protein expression in K562 cells stably transfected with PI3K??constructs. K562 cells
were stably tansfected with wild type PI3K??(PI3K?-WT), PI3K?-KR mutant, or empty vector
alone as described in “Materials and Methods”. Protein levels of PI3K??expression were detected
by Western blot using anti-PI3K??antibody. Equal loading of protein was monitored by
immunoblotting with anti-vinculin antibody.
Vector PI3K?-WT PI3K?-KR
PI3K?
vinculin
HbA-positive cells(%)
0
10
20
30
40
50
60
70
Ara-c(-) Ara-c(+)
B)
vector
antisense
Fig.14 Evaluation of erythroid differentiation by immunocytochemistry. Exponentially
growing K562 cells were cultured in the absence or in the presence of 5 µg/ml of Ara-C for 4 days.
Erythroid differentiation was then detected by staining with an anti-hemoglobin A antibody A)
Hemoglobin-containing cells appear red (original magnification, 20 x). B) Quantitative
presentation of hemoglobin A-positive cells. Results represent the Mean±SEM of triplicate
experiments.
3. results
46
Subsequently, erythroid differentiation of these cells was evaluated. Exponentially growing
cells were induced to differentiation by the addition of 5 µg/ml of Ara-C. 4 days post
induction, erythroid differentiation was determined by benzidine staining. Fig.16 shows
that the Ara-C induced erythroid differentiation was significantly inhibited in cells
transfected with the wild type PI3K??in comparison to the vector controls. whereas Ara-C
induced erythroid differentiation of cells transfected with PI3K?-KR mutant was
significantly enhanced. Furthermore, cells transfected with PI3K?-KR mutant showed an
obvious increase (about 10%) in spontaneous erythroid differentiation. These results
indicate that PI3K??is a negative regulator for erythroid differentiation, and this function of
PI3K??is related to its kinase activity .
Fig.16 Erythroid differentiation of stable transfectans overexpressing wild type PI3K??and
PI3K?-KR mutant. Exponentially growing K562 cells stably transfected wild type PI3K??and
PI3K?-KR mutant were cultured in the absence or presence of Ara-C (5 µg/ml) for 4 days.
Erythroid differentiation was then estimated by benzidine staining. Data are expressed as
percentage of benzidine-positive cells. Results represent the Mean±SEM of triplicate experiments.
3.5 Relationship of PI3K??kinase activities to the erythroid differentiation of K562
cells
As PI3K??possesses both lipid an protein kinase activity, we therefore tried to figure out
which activity is responsible for the observed inhibitory effect of PI3K??on the erythroid
differentiation. For this purpose, we took the advantage of the PI3K?-FRAP mutant, which
only retains the protein kinase activity, but lacks lipid kinase activity (Bondeva, T et al.,
Benzidine-positive cells (%)
80
70
60
50
40
30
20
10
0
Vector WT KR
Ara-C(-)
Ara-C(+)
3. results
47
1998). K562 cells were stably transfected with PI3K?-FRAP mutant. The expression of
this protein was confirmed by Western blot as shown in Fig.17.
Erythroid differentiation ability of these cells was estimated subsequently. It was observed
that erythroid differentiation of PI3K?-FRAP expressing cells in response to Ara-C
induction was significantly reduced as compared to the control cells transfected with an
empty vector (Fig.18). This result indicates the protein kinase activity of PI3K??is inversely
correlated with the erythroid differentiation.
Fig.17 Overexpression of PI3K?-FRAP mutant. K562 cells were stably tansfected with wild
type PI3K?-FRAP mutant, or empty vector alone as described in "Materials and Methods". Protein
expression of PI3K??was detected by Western blot using anti-PI3K??antibody. Equal loading of
protein was monitored by immunoblotting with anti-vinculin antibody.
Fig.18 Inhibitory effect of PI3K?-FRAP on erythroid differention. Exponentially growing cells
stably transfected with PI3K?-FRAP mutant, or empty vector alone were cultured in the absence or
presence of Ara-C (5 µg/ml) for 4 days. Erythroid differentiation was then detected by benzidine
staining. Data are expressed as percentages of benzidine-positive cells. Results represent the
Mean±SEM of triplicate experiments.
PI3K?-FRAP Vector
PI3K?
Vinculin
Benzidine-positive cells (%)
Vector FRAP
Ara-C(-)
Ara-C(+)
10
30
0
20
40
50
3. results
48
In order to define the potential role of the lipid kinase activity of PI3K??in erythroid
differentiation, the correlation between the lipid kinase activity of PI3K??and the erythroid
differentiation in cells overexpressing PI3K??or PI3K??functional mutants was analysed.
For convenience, activation of Akt, an effector enzyme downstream of PI3K (Franke , TF
et al., 1995) was used as a readout for the evaluation of the lipid kinase activity.
Exponentially growing cells under normal culture conditions were collected, and lysed
directly in electrophoresis sample buffer. The Akt phosphorylation was detected by
western blot using an antibody which specifically recognises the phosphorylated form of
Akt (ser 473). As expected, overexpression of PI3K?-FRAP mutant did not affect the basal
level of Akt phosphorylation (Fig.19). Interestingly, overexpression of the dominant
negative PI3K?-KR mutant failed to inhibit the Akt phosphorylation, despite this mutant
led to spontaneous erythroid differentiation. This suggests the effect of this mutant on
erythroid differentiation is not caused by modulating the endogenous PI3K??lipid kinase
activity. Intriguingly, overxepression of the wild type PI3K??was unable to activate Akt.
As a positive control, overexpression of a membrane targeted PI3K??(PI3K?-CAAX)
resulted in an elevated Akt phosphorylation. Taken together, these results indicate the lipid
kinase activity of PI3K??is not correlated with the spontaneous erythroid differentiation.
Fig.19 Effects of overexpressing PI3K??and PI3K??mutants on basal Akt phospophorylation.
Exponentially growing cells stably tansfected with wild type PI3K??PI3K?-KR mutant, PI3K?-
FRAP mutant, PI3K?-CAAX mutant, or empty vector were collected, lysed directly in
electrophoresis sample buffer and analysed by anti-phospho-Akt (serine 473), and anti-PI3K?
immunoblotting. Equal loading of protein was monitored by immunoblotting with anti-Akt1
antibody using the same-stripped membrane.
PI3K?
Vector
PI3K?-WT
PI3K?-KR
PI3K?-FRAP
p-Akt
Akt1
PI3K?-CAAX
3. results
49
Furthermore, in order to know whether the lipid kinase activity of PI3K??is directly related
to Ara-C induced erythroid differentiation, the effect of Ara-C on Akt phosphorylation was
analysed. Exponentially growing cells cultured under normal conditions were subjected to
Ara-C treatment from 30 minutes to 24 hours. As shown in Fig.20, Ara-C treatment did not
significantly affect the Akt phosphorylation during the period observed .
Fig.20 Effect of Ara-C treatment on Akt phosphorylation. Exponentially growing K562 cells
were cultured in the absence or in the presence of 5 µg/ml of Ara-C for the indicated time periods.
At each time point, cells were then collected, lysed directly in electrophoresis sample buffer, and
analysed by anti-phospho-Akt immunoblotting. Equal loading of protein was monitored by
immunoblotting with anti-Akt1 antibody using the same stripped membrane.
3.6 Expression of p101 in K562 cells
The activity of PI3K??has been shown to be regulated by its adaptor protein p101
(Stephens, et al., 1997). In this study, the expression p101 was also examined. Because of
the lack of an antibody against p101, a Reverse Transciption Polymerase Chain Reaction
(RT-PCR) was used to detect the messenger RNA of p101. Gene specific primers were
designed against human p101 sequence. The size of the product expected was
approximately 516 base pairs. ?-actin primers were used to amplify a product of
approximately 600 base pairs which was used as an internal control. Cells cultured under
normal conditions were collected and total RNA was extracted using the Qiagen Mini Kit.
RT-PCR was then performed using the one-step RT-PCR Kit from Qiagen as described
under “Materials and Methods”. To our surprise, no amplified p101 cDNA was detected in
Akt1
p-Akt
- +
30 min
Ara-C - +
1h
- +
2h
- +
4h
- +
8h
- +
24h
3. results
50
K562 cells (Fig. 21). This result suggested that the function of PI3K??in K562 cells could
be independent of p101.
Fig.21 Detection of p101 expression in K562 and U937 cells by RT-PCR. p101 specific RTPCR
was performed using total RNA of K562 cells (lane 1). The total RNA of U937 cells was used
as a positive control (lane 2). Reaction in the absence of the template RNA was used as a negative
control (lane 3). The products of the amplified PCR fragments were electrophoreticaly separated on
1.5 % agarose gel containing 0.5 µg/ml of ethidium bromide, and visualised under UV light. The ?-
actin product was used as an internal control for the amount of total RNA used in the RT-PCR
reactions.
3.7 Influence of PI3K??on MAPK activity in K562 cells
The PI3K??protein kinase activity has been shown to be sufficient to activate MAPK in
COS-7 cells (Bondeva et al., 1998). Previous studies have also implicated MAPK pathway
in erythroid differentiation of K562 cells (Racke, FK et al .,1997; Shelly, C et al., 1998;
Witt, O et al., 2000). Therefore, we ask whether the observed effect of PI3K??on erythroid
differentiation is associated with the MAPK signalling pathway. The activity of MAPK
was evaluated by Western blot using an antibody that specifically recognises the activated,
phosphorylated form of the ERK p42/44 MAP kinase.
0.6 Kb
0.4 Kb
?-actin p101
1 2 3 1 2 3
p101
3. results
51
First, we detected the basal level of MAPK activity and determined whether the basal level
of MAPK activity could be affected by overexpressing PI3K??and its functional mutants.
As shown in Fig.22, under normal culture conditions, K562 cell exhibits a detectable basal
level of MAPK activation, which was inhibited by the MEK inhibitor PD98059. Consistent
with previous study (Herrera, R et al., 1998; Kang. C., 1998), the inhibition of basal
MAPK activity led to an increase in spontaneous erythroid differentiation (Fig.25).
Fig.22 Inhibition of basal MAPK activity by MEK inhibitor. Exponentially growing K562 cells
were cultured in the absence or in the presence of 20 µM PD98059 or an equivalent volume of
DMSO for the indicated time periods. At each time point, cells were collected, lysed directly in
electrophoresis sample buffer and analysed by anti-phospho-ERK immunoblotting. Equal loading
of protein was monitored by immunoblotting with anti-pan-ERK antibody using the same stripped
membrane.
However, we found that the basal level of MAPK activity was not affected by the
overexepression of PI3K??and the functional mutants (Fig.23)
PD98059
- + - + - + - + - +
1h 2h 4h 8h 24 h
p-ERK
pan-ERK
3. results
52
Fig.23 Effect of overexpression of PI3K??or its functional mutants on the basal leve of MAPK
activity. Exponentially growing K562 cells stably transfected with wild type PI3K??(PI3K?-WT),
PI3K??KR mutant (PI3K?-KR), PI3K??FRAP mutant (PI3K?-FRAP), or empty vector were lysed
directly in electrophoresis sample buffer and analysed by anti-phospho-ERK immunoblotting.
Equal loading of protein was monitored by immunoblotting with anti-pan-ERK antibody using the
same stripped membrane.
Next, we have investigated the possible contribution of PI3K??to MAPK activity linked to
Ara-c treatment. It was found that MAPK was activated 2 hours after Ara-C treatment,
which lasted at least up to 24 hours. This stimulation was completely abrogated by
PD98059 (Fig.24). Interestingly, inhibition of MAPK activity by PD98059 enhanced the
Ara-C induced erythroid differentiation (Fig.25), pointing to a negative role for MAPK in
Ara-C induced erythroid differentiation. We therefore compared the MAPK activities in
cells stably transfected with PI3K??and its functional mutants upon Ara-C treatment. Cells
were cultured in full medium in the presence of 5 µg/ml of Ara-C for 24 hours. After that,
cells were lysed directly in electrophoresis sample buffer and analysed by anti-phospho-
ERK immunoblotting. As shown in Fig.26, overexpression of wild type PI3K??or the
PI3K?-FRAP mutant which are assumed to possess protein kinase activity did not increase
the Ara-C induced ERK phosphorylation over that of the empty vector control.
Furthermore the Ara-C induced ERK phosphorylation was not inhibited by overexpression
of PI3K?-KR mutant. Thus, the MAPK pathway seems to be acting rather in parallel than
downstream of PI3K??in K562 cells.
Vector
PI3k?-WT
PI3k?-KR
PI3k?-FRAP
PI3K?
p-ERK
pan-ERK
3. results
53
Fig.24 Effect of Ara-C on MAPK activity. Exponentially growing K562 cells were cultured in
the absence or in the presence of 5µg/ml Ara-C with 20µM PD98059 or an equivalent volume of
DMSO for the indicated time periods, PD98059 and DMSO were added 1hour before Ara-C
treatment. Cells were then collected, lysed directly in electrophoresis sample buffer and analysed
by anti-phospho-ERK immunoblotting. Equal loading of protein was monitored by immunoblotting
with anti-pan-ERK antibody using the same stripped membrane.
Ara-C - + + - + + - + + - + +
PD58059 - - + - - + - - + - - +
p-ERK
pan-ERK
2h 4h 8h 24h
Benzidine positive cells (%)
10
0
20
30
40
50
60
70
Ara-C(-) Ara-C(+)
PD(-)
PD(+)
Fig. 25 Inhibition of MAPK activity enhances erythroid differentiation of K562 cells.Cells were
incubated in the absence or in the presence of 5 µg/ml of Ara-C with a single dose of 10 µM
PD98059 or an equivalent of DMSO for 4 days. PD98059 was added 1 hour before Ara-C
treatment. The erythroid differentiation was determined by benzidine staining. Results are shown as
the Mean??SEM of triplicate experiments.
3. results
54
3.8 Downstream targets for PI3K??protein kinase activity
Our results demonstrated that both the PI3K??wild type and PI3K?-FRAP are capable of
inhibiting the Ara-C induced erythroid differentiation of K562 cells. As the common
feature of these proteins is their shared protein kinase activity, we therefore tried to address
the question: what is the target(s) of this type of activity? At the first step, we performed
anti-PI3K??immunoprecipitation experiments using two different types of antibodies to
detect the binding partners for PI3K?. The polyclonal antibody is directed against the cterminal
peptide of the PI3K?, a monoclonal antibody is mapped to recognize the Nterminal
part of the protein. To increase the amount of PI3K?, a GFP-fusion PI3K??was
transiently expressed in K562 cells. Immunoprecipitations were performed using 106 to 108
of cells for each sample. After extensive washing, the immunocomplexes were separated
on 8% SDS-PAGE gel and stained with silver for the detection of the associated proteins.
As shown in Fig.27, both antibodies were able to precipitate GFP-PI3K??(approximately
158 kDa) and the endogenous PI3K??(approximately 120kDa). Together with PI3K?,
several other bands were co-precipitated. These proteins are absent in the
immunoprecipitate when the blocking peptide (blocking the PI3K??binding sites in the
Fig. 26 Effect of overexpression of PI3K??or its functional mutants on Ara-C induced MAPK
activity. Exponentially growing cells stably tansfected with wild type PI3K??(WT), PI3K?-KR
mutant (KR), PI3K?-FRAP mutant (FRAP), or an empty vector (vector) were treated with 5 µg/ml
of Ara-C for 24 hours. Immunoblotting was performed with anti-phospho-ERK, anti-pan-ERK
and anti-PI3K??antibodies. Equal loading of protein was monitored by immunoblotting with antipan-
ERK antibody using the same stripped membrane.
PI3K?
p-ERK
pan-ERK
Vector
- + + + +
Vector WT FRAP KR
Ara-C
3. results
55
polyclonal antibody) was used prior to immunoprecipitaion, which was used as a negative
control.
Fig.27 Detection of PI3K??associated proteins. 1x108 cells stably expressing GFP-PI3K??were
lysed and the cleared lysates were used for PI3K??immunoprecipitation using either monoclonal or
polyclonal antibody. As a negative control, anti-PI3K??polyclonal antibody was preincubated with
the blocking peptide (20 µg/1µg antibody) before addition to the cell lysate. After extensive
washing, precipitated proteins were separated on 8 % SDS-PAGE and revealed by silver staining.
It should be pointed out that different proteins were detected in complex with PI3K?
depending on the antibody used for immunoprecipitation. The monoclonal antibody pulled
down mainly three proteins with molecular weight of about 100 kDa, 80 kDa, and 70 kDa,
respectively. In addition to these proteins, the polyclonal antibody precipitated two
additional bands with molecular weight approximately 200 kDa and 110 kDa. We have
Blocking peptide
+ Pab
Pab IP Mab
110kD
200kD
158kD
212kD
97.2 kD
116kD
66.4 kD
42.7 kD
55.6 kD
36.5 kD
70kD
80kD
100kD
PI3K?
GFP-PI3K?
3. results
56
attempted to identify these binding partners of PI3K?, however we were unable to pull
down sufficient amount of protein(s).
In subsequent experiments, we tried to clarify: are their targets for PI3K??protein kinase
activity present in the co-precipitated proteins? For this purpose, immunoprecipitations
were performed using the monoclonal antibody. The immunocomplexes were further
subjected to in vitro kinase assay using ???-32P?ATP to check if any of the associated
proteins can be phosphorylated by PI3K??in vitro. The reactions for in vitro protein kinase
assay were carried out for 30 minutes in the absence or in the presence of the PI3Ks
inhibitor LY294002 (10 ?M). After that, proteins were separated on SDS-PAGE gel. The
gel was then stained with Coomassie, dried and exposed to film. As shown in Fig.28, the
precipitated proteins were visualised on the Coomassie stained gel (right panel), the left
panel displayed the phosphorylation pattern of these proteins. PI3K??exhibits intrinsic
protein kinase activity, as evidenced by its autophosphorylation. As expected, the
autophosphorylation of PI3K??was inhibited by LY294002. In addition to PI3K?, the
phosphorylation of two other proteins with molecular weight of approximately 70 kDa and
50 kDa was also sensitive to LY294002.
3. results
57
Fig.28 In vitro PI3K??protein kinase assay. 1x108 non-transfected K562 cells for each sample
were lysed and the cleared lysates were subjected to immunoprecipitation using anti-PI3K?
monoclonal antibody which was covalently-coupled to protein G beads. An isotype-matched
monoclonal anti-HA antibody was used as a negative control. After the immunoprecipitation, the
immunoprecipitates were subjected to in vitro protein kinase assay as described under ‘Material
and Methods’. After that , the proteins present in the immunoprecipitates were separated by 8 %
SDS-PAGE followed by Coomassie staining (right panel). The phosphorylation of PI3K??and the
associated proteins were visualised by exposure to the dried gel to film (left panel).
In this manner, we were able to demonstrate that PI3K??is involved in a complex of
proteins in K562 cells, and some of the associated proteins show to be the direct targets for
its intrinsic protein kinase activity under in vitro conditions. Further investigations are
needed to identify these proteins and to clarify the physiological relevance of these
interactions for the erythroid differentiation process.
PI3K?
IP: ??-PI3K???-Ha
- + -
70Kd
50Kd
LY294002
212Kd
IP: ??-PI3K???-Ha
- + -
158Kd
116Kd
97.2Kd
42.7Kd
66.4Kd
55.6Kd
36.5Kd
26.6Kd
4 Disscussion
58
4 DISCUSSION
Hematopoiesis, the differentiation of hematopoietic stem cells and progenitors into various
lineages, is essential for the development and survival of a normal individual. New blood
cells belonging to different cell lineages are formed from stem cells during embryogenesis
and throughout the lifetime of the adult to replace cells that have completed their life span.
Abnormalities in the normal developmental program for blood cell formation result in
hematological diseases including leukemia. Understanding the cellular and molecular
controls of normal blood development will make it possible to answer questions about the
origin and treatment of these diseases.
Cell differentiation can be regulated at several levels. One level is the regulation of
intracellular programs that are controlled by intracellular signal messengers. As important
mediators of intracellular signalling pathways in mammalian cells, PI3Ks have been
implicated in cell differentiation. Depending on cell type, both negative and positive roles
for PI3Ks in differentiation have been revealed (Kimura, K et al., 1994; Ptasznik, A et al.,
1997). However, little is known about the role of PI3Ks in hematopoietic stem cell
differentiation.
In the present study, the role of PI3Ks in hematopoiesis was investigated using both
normal hematopoietic stem cells and K562 cell line. By using various approaches, we
revealed a nonredundant role for PI3K??as a negative regulator of the erythroid
differentiation in K562 cells. Furthermore, we demonstrated that PI3K??performs this
function presumably via its protein kinase activity.
4.1 PI3K??is a negative regulator of the erythroid differentiation of K562 cells
PI3K??is preferentially expressed in cells of hematopoietic origin. Previous work in our lab
has shown that a specific up-regulation of PI3K??isoform precedes ATRA (all-trans
retinoic acid)-induced cell differentiation in U937, a myeloid leukemia cell line (Baier, R
et al., 1999). This provides the first evidence that PI3K??could be involved in
hematopoietic cell differentiation, and raises the question whether PI3K??contributes to any
other lineage specific differentiation process.
To answer this question, we screened the expression profile of PI3K??protein during the
differentiation of CD34+ progenitor cells derived from human peripheral blood. It was
observed that PI3K??expression is significantly downregulated after induction of the
CD34+ cells with EPO, which predominantly led to the development of erythroid
4 Disscussion
59
differentiation. In contrast, the PI3K??expression was not obviously affected when these
cells were induced to megakaryocytic differentiation by TPO (Fig. 7), despite
megakaryocytic and erythroid are closely linked lineage’s (Debili, N et al., 1996). These
preliminary results provide the first clue that PI3K??is associated with erythroid
differentiation.
Due to the difficulties in the isolation and maintenance of adequate numbers of
hematopoietic stem cells, we therefore made further investigation using K562 cells. K562
is a human erythroleukemia cell line. Similar to hematopoietic stem cells, these cells are
pluripotent in that they are able to differentiate along a megakaryocytic, erythroid, or, to a
lesser extent, monocytic lineage (Lozzio, CB et al., 1975). Erythrroid differentiation of
K562 cells can be induced by a number of chemical agents including cytosine arabinoside
(Ara-C), hemin, and butyric acid (Leary, JF et al, 1987). Importantly, in vitro
differentiation of these cells recapitulates many aspects of the normal erythroid terminal
differentiation programs, including haemoglobin synthesis and proliferation arrest. Thus,
this cell line is a useful model for in vitro differentiation studies.
To document that PI3K??is also downregulated in K562 cells upon induced erythroid
differentiation as it is in induced erythroid differentiation of normal stem cells, K562 cells
were induced to undergo erythroid differentiation by Ara-C. As shown in Fig.8, after
treatment with Ara-C for 4 days, K562 cells were partially induced to erythroid
differentiation, as evidenced by the haemoglobin synthesis. This process was accompanied
by significant decrease in PI3K??expression. In contrast to PI3K?, the expression of
PI3K??and its adaptor protein p85 was not significantly affected. Together with the results
from CD34+ cells, these findings indicate that PI3K??is a specific isoform of PI3Ks that is
involved in erythroid differentiation.
To define the potential role of PI3K??in erythroid differentiation, it is important to ascertain
whether this process can be affected by suppressing or increasing the PI3K??expression
level. To achieve the specific suppression of PI3K??expression, an antisense strategy was
used. The decrease in endogenous PI3K?, by stable expression of PI3K??antisense cDNA
in K562 cells increased spontaneous and Ara-C induced erythroid differentiation.
Conversely, overexpression of the wild type PI3K??blocked the effect of Ara-C on
erythroid differentiation. These results indicate PI3K??is a negative regulator of erythroid
differentiation in K562 cells. Furthermore, as overexpression of a dominant negative
4 Disscussion
60
PI3K??mutant displayed an effect opposite to that of the wild type PI3K???we conclude that
the observed inhibitory effect of PI3K??seems to be associated with its kinase activity.
In contrast to our results, previous studies have suggested the PI3Ks seems to play a
positive role in erythropoiesis. In this regard, Myklebust, et al. have shown the PI3K
inhibitor LY294002 suppresses the EPO-induced formation of glycophorin A positive
erythroid cells from CD34+ progenitors (Myklebust, JH et al., 2002). In addition, the
terminal differentiation of murine erythroleukemia cells induced by dimethylsulfoxyde
(DMSO) was also shown to be blocked by LY294002 (Bavelloni, A et al., 2000). A
possible explanation for this apparent discrepancy could be due to the use of PI3K
inhibitors in the above studies, which unselectively inhibit the activity of all PI3K
isoforms. It is possible that other isoforms of PI3Ks may play a permissive role for the
erythroid differentiation process during which the inhibition of PI3K??activity is needed. In
consistent with this hypothesis, it is reported that inhibition of the overall PI3Ks activity by
LY29002 induced erythroid precursor cells to undergo apoptosis (Haseyama,Y et al.,
1999).
4.2 PI3K??negatively regulates the erythroid differentiation presumably via its
protein kinase activity
PI3Ks are dual specificity enzymes. In addition to their function as lipid kinases, PI3Ks
have an intrinsic protein kinase activity. The protein kinase activity of PI3Ks was initially
discovered by the finding that p110??can phosphorylate the p85??regulatory subunit,
which results in a reduced lipid kinase activity of the heterodimer in vitro (Dhand, R et al.,
1994). Thereafter, protein kinase activity of other class IA PI3K isoforms has been
revealed. PI3K??has been shown to undergo autophosphorylation both in vitro and in vivo
(Vanhaesebroeck, B, 1999), and the in vivo autophosphorylation induced by activated
receptors (such as CD28) leads to a concomitant decrease in associated lipid kinase
activity.
In addition to class IA PI3Ks, PI3K???the only class IB isoform of PI3K, also possesses an
intrinsic protein kinase activity. Similar to PI3K?, PI3K??is able to autophosphorylate in
vitro (Stoyanova, S et al., 1997). Furthermore, it has been demonstrated that PI3K??is able
to transphosphorylate the adaptor protein p101 and the mitogen-activated protein kinase
kinase 1 (MEK-1) in vitro (Bondev, A et al., 1999). To define the individual function
4 Disscussion
61
played by the lipid kinase and protein kinase of PI3K?, the use of PI3K inhibitor is not
suitable, because they apparently interfere with both activities. To circumvent this
situation, Bondeva et al. generated a PI3K??mutant construct, termed PI3K?-FRAP, by
replacing a sequence within the conserved catalytic core of PI3K??with the corresponding
sequence from FRAP (a member of target of rapamycin family without assigned lipid
kinase activity) (Bondeva, T et al., 1998). The resultant PI3K?-FRAP protein does not have
lipid kinase but still retain protein kinase activity. Taking advantage of this mutant, in the
present study, we overexpressed PI3K?-FRAP in K562 cells, and its effect on erythroid
differentiation was observed. Our results showed that the overexpression of PI3K?-FRAP
inhibits the Ara-C induced erythroid differentiation of K562 cells as effectively as the wide
type PI3K??(Fig.16, 18). Because the common feature of these two proteins is the shared
protein kinase activity, thus the observed inhibitory effect of PI3K??on erythroid
differentiation in K562 cells is presumably due to its protein kinase activity.
To address whether the lipid kinase activity of PI3K??is uncoupled from the erythroid
differentiation process, we first compared the lipid kinase activities in cells stably
transfected with PI3K??and its functional mutants. For convenience, the activation of Akt,
an effector enzyme downstream of PI3K (Franke, TF et al., 1995) was used as a readout of
lipid kinase activity. As expected, overxepression of PI3K?-FRAP mutant did not activate
Akt because this mutant does not have a lipid kinase activity. Intriguingly, even
overexpressing a wild type PI3K??was not sufficient to increase the Akt phosphorylation
under conditions used in this work. Furthermore, the basal level of Akt phosphorylation
was not inhibited by overexpressing the dominant negative PI3K?-KR mutant. Thus,
despite cells stably transfected with PI3K??and its functional mutants exhibited different
potentials for erythroid differentiation, these different potentials were not correlated with
their lipid kinase activities. In addition, treatment of K562 cells with Ara-C from 30
minutes up to 24h did not obviously affect Akt phosphorylation, suggesting that Ara-C has
no direct effect on the lipid kinase activity of PI3Ks. Taken together, these results indicate
the lipid kinase activity does not account for the inhibitory effect of PI3K??on the erythroid
differentiation in K562 cells.
In contrast to our results, ATRA increased the overall PI3K lipid products which was
apparently correlated with the upregulation of P13K??expression in U937 cells (Baier, R et
al., 1999). In this study, the authors suggested the elevated lipid kinase activity resulted in
activation of MAPK, which is supposed to be responsible for ATRA-induced
4 Disscussion
62
differentiation in these cells. It is possible that PI3K??may function either as lipid kinase or
protein kinase to regulate distinct cellular responses depending on the cell type and the
cellular context.
4.3 The effect of PI3K??on the erythroid differentiation is independent of p101
Class I PI3Ks are all heterodimers containing a 110-kDa catalytic subunit and a regulatory
subunit. The class IA members p110 ?, ?, and ??are associated with a p85 family
regulatory subunit. The primary function of p85 is to recruit the catalytic subunits to the
plasma membrane, where the catalytic subunit phosphorylates its lipid substrates. In
addition, p85 also stabilises the catalytic subunits by protecting them from degradation
(Yu, J et al, 1998). PI3K??does not interact with a p85 family adaptor. Instead, PI3K??has
been shown to be associated with a p101 regulatory subunit which has no homology with
any known protein (Stephens, L et al., 1997). Up to date, the functional significance of
p101 remains elusive.
In present study, the expression of p101 was examined by a method of RT-PCR.
Surprisingly, no p101 expression could be detected in K562 cells (Fig 21). The precise
reason for the absence of p101 in K562 cells is unknown. Nevertheless, this suggests the
observed effect of PI3K??on erythroid differentiation of K562 cells is independent of p101.
Previously, Stephens et al. showed that, in the presence of p101, the G???mediated lipid
kinase activity of PI3K??was significantly enhanced (Stephens, L et al., 1997). Recently, it
was demonstrated the membrane recruitment of PI3K??mediated by p101 is essential for
the activation of PI3K??in response to G???stimulation (Brock, C et al., 2002). Consistent
with this notion, we found overexpression of a membrane-targeted form of PI3K??mutant
(PI3K?-CAAX) resulted in an elevated Akt phosphorylation, whereas overexpression of
the wild type PI3K??did not (Fig. 19). It is tempting to speculate, the absence of p101 in
K562 cells may provide a cellular context which predisposes PI3K??to function as a protein
kinase in the cytosol.
4.4 The effect of PI3K??on the erythroid differentiation is not mediated by MAPK
As our results support the inhibitory effect of PI3K??on erythroid differentiation could be
related to its protein kinase activity, it raises the question: what is the signalling evens that
act downstream of this protein kinase activity? A possible candidate is the ERK/MAPK
4 Disscussion
63
pathway. Previous work in our lab has demonstrated that the protein kinase activity of
PI3K??is sufficient to activate MAPK in COS-7 cells (Bondeva, T et al., 1998).
Furthermore, available data also implicate ERK/MAPK in erythroid differentiation. For
example, it has been shown the TPA-induced sustained activation of MAPK not only
commits these cells to megakaryocytic differentiation, but also suppresses the transcription
of haemoglobin, a marker of erythroid differentiation (Lumelsky, NL et al., 1991).
In present study, we demonstrated that Ara-C treatment resulted in ERK phosphorylation
(Fig. 24). The specific MEK inhibitor PD98059 completely abrogated Ara-C induced ERK
phosphorylation, and consequently enhanced erythroid differentiation (Fig. 25).
Furthermore, inhibition of the basal level of MAPK activity by PD98059 increased the
spontaneous erythroid differentiation (Fig. 25). Consistent with the previous study (Shelly,
C et al., 1998), our results confirm MAPK could function as a negative regulator of
erythroid differentiation of K562 cells. Therefore it is tempting to speculate that the
inhibitory effect of PI3K??on erythroid differentiation might be mediated by MAPK.
Next, we tested whether the protein kinase of PI3K??contributes to the baseline and Ara-C
induced MAPK activation. The activity of MAPK was evaluated in Western blots using an
antibody that specifically recognises the activated, phosphorylated form of the ERK p42/44
MAP kinase. As shown in Fig. 23 and Fig. 26, the baseline and Ara-C -induced ERK
phosphorylation was neither increased by overexpressing PI3K??wide type or PI3K?-
FRAP, nor inhibited by the overexpressing the dominant negative PI3K?-KR mutant.
These results indicate that the protein kinase activity of PI3K??does not contribute to the
MAPK activation in this system. Thus the ERK/MAPK pathway seems to act in parallel
rather than downstream of PI3K??to convey an inhibitory effect on erythroid
differentiation.
4.5 Attempts to identify the downstream targets for PI3K??protein kinase activity
As PI3K??fails to activate MAPK in K562 cells, it excludes MEK1 as the substrate for
PI3K??protein kinase activity in this system, though MEK1 has been previously shown to
be phosphorylated by PI3K??in vitro (Bondava, T et al., 1998). This prompted us to search
for the direct target (or targets) of PI3K??protein kinase activity. For this purpose,
immunoprecipitation using a PI3K??specific antibody and subsequent in vitro protein
kinase assay were performed. Using the monoclonal antibody, we have detected several
4 Disscussion
64
proteins that were co-precipitated with PI3K??(Fig. 28). These proteins are believed to be
constitutively associated with PI3K??because they were not present in the
immunoprecipitate using a control HA-antibody that failed to pull down PI3K?. When the
anti-PI3K??immunoprecipitate was subjected to in vitro kinase assay, we found two
proteins with molecular weights of about 70 kDa, and 50 kDa were phosphorylated.
Importantly, the phosphorylation of these two proteins was abrogated by the PI3K inhibitor
LY294002, strongly suggesting the phosphorylation of these proteins are catalysed by
PI3K?. Thus these two proteins could be direct downstream substrates for PI3K??protein
kinase activity. Due to the limited sensitivity of immunoprecipitation method, currently we
can not exclude the existence of other substrates for PI3K??protein kinase activity in this
system. Actually we have detected additional proteins present in anti-PI3K?
Immunoprecipitate using polyclonal antibody (Fig. 27). Because the polyclonal antibody
apparently inhibits the intrinsic activity of PI3K?, it is currently unknown whether these
proteins can be phosphorylated by PI3K??or not. Nevertheless, these results confirm the
existence of potential substrates downstream of PI3K??protein kinase activity in K562 cells
The molecular events that links the phosphorylation of the downstream proteins of PI3K?
protein activity to erythroid differentiation is currently unknown. In fact, erythroid
differentiation is a complex and finely-tuned multi-step process. It is now widely accepted
that this process in controlled by two groups of functionally antagonistic transcription
factors (Perry, C et al., 2002). The normal erythroid differentiation requires not only the
activation of differentiation-promoting factors, but also the concomitant suppression of
differentiation-inhibiting factors as well (Nerlov, C et al., 2000). In this regard, PU.1, a
member of Ets family of transcription factors, has been shown to be an inhibiting factor for
erythroid differentiation (Rekhtman, N et al, 1999). Repression of PU.1 function is a
critical step for erythroid differentiation, because overexpression of PU.1 in erythroblast
blocks the terminal erythroid cell differentiation (Deljado, MD et al., 1998). Interestingly,
Rekhtman et al. have demonstrated that ectopic expression of PU.1 in erythroid cells
blocks their maturation, while normal erythroid differentiation of these cells can be
restored by overexpression of GATA-1, an erythroid differentiation-promoting
transcription factor (Rekhtman, N et al., 1999). As the activities of transcription factors are
to be regulated by phosphorylation, it is possible that the protein kinase activity of PI3K?
may exert its effect through a mechanism of triggering a phosphorylation cascade which
ultimately affects the activities of these transcription factors.
4 Disscussion
65
Based on our results, a model for PI3K??mediated signalling mechanism could be depicted
as following: without induction, PI3K??may constitutively bind with and phosphorylate
some downstream proteins via its protein kinase activity. After phosphorylated by PI3K?,
these proteins may trigger a signalling pathway which conveys an inhibitory effect on
erythroid differentiation and keep the cells in undifferentiated state. After induction, the
expression of PI3K??is downregulated and its inhibitory effect is removed, thus allowing
for the terminal differentiation of the cells.
4.6 Conclusion
In the present study, we established a specific role for PI3K??as a negative regulator of
erythroid differentiation of K562 cells. Interestingly, it has been recently shown that the
inhibition of PI3K activity in primary cultured fetal pancreatic cells results in a robust
activation of endocrine differentiation (Ptasznik, A et al., 1997). The blockade of PI3K was
also shown to enhance the neutrophilic differentiation in HL60 cells (Kanayasu-Toyoda, T
et al, 2002). Despite the isoform-specific role of PI3Ks was not clarified in these studies, at
least these data indicate the negative regulation of cellular differentiation by PI3Ks may be
a general phenomenon. Nevertheless, other authors have previously shown a positive
involvement of PI3Ks in PC12 pheochromocytoma (Kimura, K et al., 1994), SH-SY5Y
human neuroblastoma (Lopez-Carballo, G et al., 2002) and U937 (Baier, R et al., 1999)
cell lines differentiation. Together, all available data suggest that PI3Ks may play dual
roles as both positive and negative regulators of cellular differentiation depending on cell
type.
Up to date, almost all the established cellular functions of PI3K??are believed to be related
to its lipid products. In the present study, we demonstrated PI3K??plays a negative role in
erythroid differentiation of K562 cells, and the function of PI3K??in this process is
mediated by its protein kinase activity. To our knowledge, this is the first time that a
physiological relevance for PI3K??protein kinase activity has been established.
4.7 Outlook
In the present study, we clearly demonstrate that PI3K??plays a negative role in the
regulation of erythroid differentiation in K562 cells. However, in mice lacking functional
PI3K??(PI3K??-/- mice) (Hirsch, E et al., 2000; Sasaki, T et al., 2000), the erythropoiesis
4 Disscussion
66
seems not to be obviously affected, as evidenced by the normal count of erythrocytes in the
peripheral blood. Presently, the precise reason for this is unknown. One possibility is that
PI3K??may affect erythropoiesis in a differentiation-stage dependent manner. In the present
study, PI3K??was shown to affect the haemoglobin synthesis, suggesting an involvement of
PI3K??in the later phase of erythroid differentiation. It is possible PI3K??may only
facilitate the terminal differentiation of the committed erythroid precursor cells, but has no
effect on the generation of these erythroid precursor cells from the multipotent progenitors.
To test this hypothesis, a close examination of the kinetics of erythroid differentiation of
hematopoietic stem cells from PI3K??-/- mice will be helpful. On the other hand, K562 is a
leukemia cell line, and it is well known that the arrest of differentiation is a common
feature of tumor cells. Thus a more speculative explanation is that the observed inhibitory
effect of PI3K??on erythroid differentiation in K562 cells may reflect an intrinsic property
of PI3K??in leukemogenesis. To test this concept, future investigation is needed to
determine if the PI3K??protein kinase activity can produce the same inhibitory effect on the
erythroid differentiation of normal blood stem cells. Furthermore, in the present study, we
detected two proteins that are constitutively associated with PI3K??and phosphorylated by
PI3K??in vitro, implying they are direct targets for PI3K??protein kinase activity. Thus,
these proteins could be the possible candidates that mediate the inhibitory effect of PI3K?
on erythroid differentiation in vivo. Future work is needed to identify these proteins and
elucidate how the phosphorylation of these proteins by PI3K??affects the molecular evens
linked to erythroid lineage–specific gene(s) expression.
5. Literature
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6. Summary
81
SUMMARY
Cell differentiation is a complex, multi-step process that can be regulated at several levels.
One level is the regulation of intracellular programs that are controlled by intracellular
signal messengers. As important mediators of intracellular signaling, PI3Ks have been
implicated in cell differentiation. Depending on cell types, both positive and negative roles
for PI3Ks have been revealed. PI3K??is the only isoform of class IB PI3Ks, which is
preferentially expressed in cells of hematopoietic origin.
In the present study, the role of PI3K??in erythroid differentiation was investigated. PI3K?
expression was found to be rapidly downregulated in human hematopoietic stem cells
when these cells were induced to undergo erythroid differentiation by EPO. This finding
was further corroborated in K562 cells. K562 is an erythroleukemia cell line, which is able
to differentiate along the erythroid lineage in response to a variety of chemical agents. It
was observed that PI3K??expression was downregulated along erythroid differentiation of
K562 cells induced by Ara-C, a potent differentiation inducer. In contrast, the expression
of PI3K??and its adaptor protein p85 was not significantly affected by Ara-C treatment,
suggesting that PI3K??is a specific PI3K isoform related to the erythroid differentiation
process.
In subsequent experiments, a negative role of PI3K??in erythroid differentiation was
established by using antisense and overexpression strategies. It was found that antisense
inhibition of the endogenous PI3K??expression increased the spontaneous and Ara-C
induced erythroid differentiation, whereas overexpression of wild type PI3K??attenuated
the inductive effect of Ara-C.
As overexpression of the dominant negative PI3K?-KR mutant displayed an effect opposite
to that of the wild type PI3K?, we conclude that observed effect of PI3K??on eythroid
differentiation is linked its kinase activities. PI3K??possesses both lipid kinase activity and
protein kinase activity. To distinguish which activity is responsible for the inhibiton of
erythroid differentiation, the effect of the PI3K?-FRAP mutant, which possesses protein
kinase activity, but lacks lipid kinase activity, was evaluated. It was observed that
overexpression of this mutant inhibited the Ara-C induced erythroid differentiation. Thus,
the inhibitory effect of PI3K??on erythroid differentiation is presumably due to its protein
kinase activity. This concept was further substantiated by the following findings: (i).
overexpression of the dominant negative PI3K?-KR did not affect the basal endogenous
PI3K??lipid kinase activity, despite overexpression of this mutant increased the
6. Summary
82
spontaneous erythroid differentiation. (ii). Ara-C has no direct effect on lipid kinase
activity of PI3Ks. Thus the lipid activity of PI3K??seems to be uncoupled from the
erythroid differentiation process.
The activity of PI3K??has been shown to be subject to the regulation of its adaptor protein
p101. In the present study, the expression of p101 was examined by the method of RTPCR.
No detectable p101 expression was found at mRNA level in K562 cells. Thus, the
observed effect of PI3K??on erythroid differentiation is independent of p101.
As the PI3K??protein kinase activity has been previously shown to be sufficient to activate
MAPK, a possible involvement of MAPK in mediating the effect of PI3K??in this system
was investigated. Overexpressing PI3K??wild-type or PI3K?-FRAP did not increase the
basal or Ara-C induced MAPK activity over the vector controls. In addition, the basal and
Ara-C induced MAPK activity was not inhibited by the overexpression of the dominant
negative PI3K?-KR mutant. These results indicate that the protein kinase activity of
PI3K??does not contribute to the MAPK activation in this system.
To search for the direct target (or targets) for PI3K??protein kinase activity, anti-PI3K?
immunoprecipitation and subsequent in vitro kinase assay was performed. Several proteins
were coprecipitated with P13K?. In vitro, the phosphorylation of two of these proteins with
molecular weight of approximately 70 kDa and 50 kDa was abrogated by the PI3Ks
inhibitor LY294002, suggesting these two proteins are downstream substrates for the
PI3K??protein kinase activity in vitro. These two proteins are the possible candidates that
mediate the inhibitory effect of PI3K??on erythroid differentiation in vivo.
To conclude, in the present study, we revealed a nonredundant role of PI3K??as a negative
regulator for erythroid differentiation in K562 cells. This is the first time that a role of the
protein kinase activity of PI3K??in a physiological condition has been established. The
identification of these substrates for PI3K??protein kinase activity and the elucidation of
their downstream signalling events linked to the erythroid-specific gene(s) expression are
the aim of our future investigations.
7. Zusammenfassung
83
ZUSAMMENFASSUNG
Zelldifferenzierung ist ein komplexer, mehrstufiger Prozess, der auf verschiedenen Ebenen
reguliert werden kann. Eine Ebene ist die Regulation von intrazellulären Programmen, die
durch intrazelluläre Signal-Botenstoffe kontrolliert werden. Für PI3-Kinasen als wichtige
Mediatoren der intrazellulärer Signaltransduktion wird eine Beteiligung an der
Zelldifferenzierung angenommen. Abhängig vom Zelltyp konnte für PI3-Kinasen sowohl
positiver als auch negativer Einfluß gezeigt werden. PI3K??ist die einzige Isoform der
Klasse 1B PI3-Kinasen, welche bevorzugt in hämatopoietischen Zellen expremiert wird.
In der vorliegenden Arbeit wurde die Rolle der PI3K??bei der erythroiden Differenzierung
untersucht. Eine schnelle Absenkung der PI3K??Expression nach Induktion der erythroiden
Differenzierung durch EPO konnte gezeigt werden. Diese Beobachtung konnte in K562
Zellen weiter bestätigt werden. K562 ist eine erythroleukämische Zelllinie, welche in der
Lage ist, in Reaktion auf eine Vielzahl an chemischen Agenzien die erythroide
Differenzierung zu durchlaufen. Es wurde beobachtet, dass die PI3K??Expression während
der Ara-C - induzierten erythroiden Differenzierung von K562 Zellen herab reguliert wird.
Im Gegensatz dazu, wird die Expression von PI3K??und deren Adaptorprotein p85 durch
Ara-C Behandlung nicht signifikant beeinflußt. Dies ist ein Hinweis darauf, dass die
Funktion der PI3K??bei der erythroiden Differenzierung Isoform-spezifisch ist.
In folgenden Experimenten wurde durch Verwendung von Antisense - und
Überexpressions - Strategien eine negative Rolle der PI3K??während der erythroiden
Differenzierung etabliert. Es wurde gezeigt, dass die Unterdrückung der endogenen PI3K?
Expression durch Antisense-RNA zu einer Erhöhungen sowohl der spontanen als auch der
Ara-C induzierten erythroiden Differenzierung führt, während die Überexpression von
Wildtyp-PI3K??den induzierenden Effekt von Ara-C abschwächt.
Da Überexpression der dominant negativen PI3K?-KR Mutante verglichen mit Wildtyp-
PI3K??den gegenteiligen Effekt hat, schließen wir, dass für den PI3K??- Effekt deren
Kinaseaktivitäten wesentlich sind. PI3K??besitzt sowohl Lipid - als auch
Proteinkinaseaktivität. Um zu unterscheiden, welche von beiden für die Inhibierung der
erythroiden Differenzierung verantwortlich ist, wurde der Effekt der PI3K?-FRAP
Mutante, welche nur Proteinkinaseaktivität besitzt, untersucht. Es wurde beobachtet, dass
Überexpression dieser Mutante die Ara-C induzierte erythroide Differenzierung inhibiert.
Demnach ist für den inhibitorischen Effekt der PI3K??anscheinend deren
Proteinkinaseaktivität nötig. Diese Annahme wurde durch die folgenden Beobachtungen
7. Zusammenfassung
84
untermauert: 1. Überexpression der dominant negativen PI3K?-KR Mutante ist ohne
Einfluß auf die basale endogene PI3K??Lipidkinaseaktivität, obwohl die Überexpression
dieser Mutante die spontane erythroide Differenzierung erhöht, 2. Ara-C hat keinen
direkten Effekt auf die Lipidkinaseaktivität von PI3-Kinasen. Die Lipidkinaseaktivität
scheint also unabhängig von der erythroiden Differenzierung zu sein.
Für PI3K??wurde gezeigt, dass ihre Aktivität der Regulation durch das Adaptorprotein
p101 unterliegt. In der vorliegenden Arbeit wurde die Expression von p101 mittels RTPCR
untersucht. Auf der Ebene der mRNA wurde in K562 Zellen keine detektierbare p101
Expression gefunden. Der beobachtete Effekt der PI3K??auf die erythroide Differenzierung
ist demnach unabhängig von p101.
Da kürzlich beschrieben wurde, dass die Proteinkinaseaktivität der PI3K??für die
Aktivierung der MAPK ausreichend ist, wurde die mögliche Beteiligung der MAPK an der
Vermittlung der PI3K??Effekte in diesem System untersucht. Überexpression der PI3K?
WT oder der PI3K?-FRAP erhöht weder die basale noch die Ara-C induzierte MAPK
Aktivität über das Kontrollniveau. Die basale und die Ara-C induzierte MAPK Aktivierung
wird auch nicht durch Überexpression der dominant negativen PI3K?-KR Mutante
inhibiert. Diese Ergebnisse weisen darauf hin, dass die Proteinkinaseaktivität der PI3K??in
diesem System nicht an der Aktivierung der MAPK beteiligt ist.
Um das direkte Substrat (oder Substrate) der PI3K??Proteinkinaseaktivität zu identifizieren,
wurden anti-PI3K?-Immunpräzipitationen, gefolgt von einem in vitro Kinaseassay,
durchgeführt. Mehrere Proteine werden mit PI3K??copräzipitiert. In vitro wird die
Phosphorylierung von zwei dieser Proteine mit einem Molekulargewicht von etwa 50 und
70 kDa durch den PI3K-Inhibitor LY294002 verhindert. Dies weist darauf hin, dass diese
beiden Proteine in vitro nachgeschaltete Substrate der Proteinkinaseaktivität der PI3K?
sind. Diese beiden Proteine sind mögliche Kandidaten für die Vermittlung der
inhibitorischen Effekte der PI3K??auf die erythroide Differenzierung in vivo.
In der vorliegenden Arbeit beschreiben wir die Rolle von PI3K??als negativer Regulator
der erythroiden Differenzierung in K562 Zellen. Erstmalig wurden Hinweise für eine
physiologische Funktion der PI3K??Proteinkinaseaktivität gefunden. Die Identifizierung
der gefundenen Substrate der PI3K??Proteinkinaseaktivität und die Aufdeckung der
nachgeschalteten Signalwege, die zur Expression von erythroid-spezifischen Genen führen,
sind das Ziel weiterer Untersuchungen.
8. Appendix
85
APPENDIX
Abbreviations
Ara-C cytosine arabinoside
ATP adenosine 5’triphosphate
ATRA all-trans retinoic acid
BCR breakpoint cluster region
Btk Bruton’s tyrosine kinase
C-GSF granulocyte colony-stimulating factor
DTT dithiothreitol
ECL enhanced chemiluminescence
EDTA ethylenediamine-tetraacetic acid
EGF epidermal growth factor
EGTA ethylene-bis (oxyethylenenitrilo) tetraacetic acid
EGFR epidermal growth factor receptor
EPO erythropoietin
ERK extracellular signal regulated kinase
FCS fetal calf serum
FITC fluorescein isothiocyanate
FRAP FKPB12 and rapamycin-associated protein
GAP GTPase activating protein
GDP guanosine di-phosphate
GEF guanidine-nucleotide exchange factor
GPCR G-protein coupled receptor
GST glutathione-S-transferase
GTP guanosine tri-phosphate
HEPES (N- (2-hydroxyethyl) piperazine-N´-(2-ethanesulfonic acid))
IL6 interleukin 6
IRS insulin receptor substrate
JNK c-Jun N-terminal kinase
Jun oncogene of the avian sarcoma virus 17 (ju-nana, japanese:17)
LPA lysophosphatidic acid
MAPK mitogen activated protein kinase
MEK MAPK/ERK kinase
8. Appendix
86
MOPS 3-(N-morpholino)-propanesulfonic acid
PBS phosphate buffered saline
PCR polymerase chain reaction
PD98059 AMF, 2’-amino-3’-methoxy-flavone
PDK phosphotidylinositol dependent kinase
PE phycoerythrin
PH pleckstrin homology
PLC phospholipase C
PKB protein kinase B
PKC protein kinase C
PMSF phenylmethylsulfonyl fluoride
PtdIns phosphatidylinositol
PTEN phosphatase and tensin homologue deleted on chromosome 10
SCF stem cell factor
SDS-PAGE sodium dodecyl sulfate-polcralamine gel electrophoresis
SH2 src homology 2
TOR target of rapamycin
TPA tetradecanoylphorbol 13-acetate
TPO thrombopoietin
8. Appendix
87
Acknowledgements
I would like to express my deepest gratitude to professor Reinhard Wetzker for providing
me the opportunity to work in this lab, and for his continual support and help during my
Ph.D study.
I am grateful to professor Felix Zintl for his kind support for my study.
I would like to sincerely thank Dr. Boehmer Frank for his kind help and valuable advice.
I thank Dr. Tzvetanka Bondeva, and Angelika Mueller for their close guidance to my
experiments and critical reading of the manuscript and helpful discussions while I am
writing this dissertation.
Thanks also go to Rico Pusch for translating the “summary” of this dissertation into
German.
I thank Dr. Ralf Haefer for his kind help with FACS analysis.
I am grateful to all members of the lab for their friendship and kind help during my study.
Special thanks to my family for their continual encouragement and understanding.
8. Appendix
88
Lebenslauf
Name: Yuantao Liu
Geburtsdatum: 07.09.1965
Geburtsort: ShanDong, V.R.China
Familienstand: verheiratet
Nationalität: chinesisch
Ausbildung:
09/1984-08/1989: Studium an der ShanDong Medical University, ShanDong,
V.R.China, Abschlu??als ‘Bachelor of Medicine’.
09/1989-08/1994: Klinische Tätigkeit im ‘Beijing Tongren Hospital’.
09/1994-07/1997: Studium an der ‘Capital University of Medical Sciences’, Beijing,
V.R.China, Abschlu??als ‘Master of Medicine’ mit dem Thema:
‘Effect and mechanism of low molecular weight heparin on
diabetic nephropathy in rats’.
09/1997-07/1999: Klinische Tätigkeit im ‘Beijing Tongren Hospital’.
seit 09/1999: Promotionsstudent in der Arbeitsgruppe Molekulare Zellbiologie,
Friedrich-Schiller-Universität.
Jena, den 17.03.2003
Yuantao Liu
8. Appendix
89
Selbständigkeitserklärung
Ich erkläre, dass ich die vorliegende Arbeit selbständig und nur unter Verwendung der
angegebenen Hilfsmittel und Literatur angefertigt habe.
Jena, den 17.03.2003
Yuantao Liu
8. Appendix
90
Erklärung zur Bewerbung
Hiermit erkläre ich, dass ich mich mit der vorliegenden Arbeit an keiner anderen
Hochschule um den akademischen Grad Doctor rerum naturalium beworben habe und dass
ich weder früher noch gegenwärtig die Eröffnung eines Verfahrens zum Erwerb des o.g.
akademischen Grades an einer anderen Hochschule beantragt habe.
Jena, den 17.03.2003
Yuantao Liu

