Cloning And Function Of The Testis-specific Protein Kinase Tssk4

Post-translational modifications of proteins through the phosphorylation of serine, threonine, and tyrosine residues by protein kinases play a role in many cellular processes such as transduction of extracellular signals, intracellular transport, and cell cycle progression. Considering the integral role that protein kinases play in the control of cellular mechanisms and signal transduction, it is not surprising that several protein kinases have been shown to be involved in spermatogenesis. Nevertheless, only a few kinases have been characterized, whose expression is restricted to either germ cells or to the testis.In this study, the cDNA sequences of several testis-specific serine/threonine kinases, TSSK1 ,TSSK2,TSSK3 were used as information probes to make homology screen, and we found a novel homologous gene. We isolated it from the human testis cDNA library. This cDNA has an open reading frame consisting of 984 bases that encode a protein of 328 amino acids, which consists of a serine/threonine protein kinase domain. Sequence alignment revealed that this protein has higher homology to the TSSK1, TSSK2, TSSK3, so maybe it is the fourth member of this family, named TSSK4. We got the mouse homologous gene tssk4 by the same way. Analysis of tissue expression pattern revealed that the gene also expresses exclusively in testis, immunohistochemical localization of tssk4 revealed it is mainly expressed in haploid round spermatids of mouse testis.In order to learn more information about the physiological function of TSSK4, we began our research with searching the proteins that interact with the TSSK4 via yeast two-hybrid system, isolating the cAMP responsive element binding protein (CREB) from human testis cDNA library. In vitro experiments including GST pulldown and phosphorylation show that TSSK4 can not only interact with CREB, but also phosphorylate it. To further confirm the physiological function of the process that TSSK4 phosphorylate CREB, we examined the regulation function of TSSK4 in Hek 293T cells using a CRE reporter system, and found that TSSK4 can enhance CRE (cAMP responsive element) -dependent transcription. These results suggested that this kinase may be involved in spermatogenesis by phosphorylating CREB and then stimulate CRE-dependent gene transcription.