Characterization of SKAP/Kinastrin isoforms
SKAP (Small Kinetochore-Associated Protein/Kinastrin) is a multifunctional protein involved in mitotic regulation, apoptosis and cell migration. Additionally, it is classified as an oncogene in human skin cancers. Despite being associated with important cellular processes, mechanisms underlying SKAPs roles in these processes are not yet fully understood. SKAP is predicted to be expressed in different isoforms, however, studies reported in the literature did not differentiate between them. In this work I studied the expression profile and functional differences between SKAP isoforms in human and mouse. Various human tissues and cells of different origin were analyzed by RT-PCR, Western blotting and immunocytochemistry. Newly generated anti-SKAP monoclonal antibodies revealed for the first time that human SKAP exists in two protein isoforms: SKAP16 represents the ubiquitously expressed isoform whereas SKAP1 showed only testis/sperm-specific expression. Similarly, in mouse SKAP1 is expressed in testes at 4 weeks postnatally, after completion of the first wave of spermatogenesis when elongated spermatids can be detected in the testes. Further biochemical analyses aimed at identifying novel SKAP1-specific interaction partners. Pull-down and co-immunoprecipitation experiments discovered that Pontin, Reptin, β-catenin and TIP60 bind to SKAP1. Particularly interesting is the interaction between SKAP1 and Pontin, a member of AAA+ ATPases. Co-localization of SKAP1 and Pontin in the flagellar region of human sperm suggests a function for SKAP1-Pontin interaction in sperm motility. Additionally, homo-oligomerization of both SKAP1 and SKAP16 was revealed by FRET and FCCS, showing that SKAP proteins in vivo function as homomeric complexes. Finally, qPCR analyses showed a significant overexpression of SKAP in colon cancer compared to healthy colon tissue, suggesting that SKAP indeed is a potential oncogene.