Testis specific serine-threonine kinases : regulation and their role in disease

Abstract

The phosphorylation of proteins catalyzed by the protein kinases is one of the most important post-translational modifications found in the living cells and is involved in the control, either directly or indirectly, of all processes occurring in the cell. A major challenge in the signal transduction field is to define sequence, structural and mechanistic features responsible for the substrate selectivity, regulation and cellular function of individual protein kinases. We took part in this challenge and studied the family of Testis Specific Serine-threonine Kinases (TSSK) that comprises four members up to date. These kinases are expressed exclusively in testis during spermatogenesis and therefore they are the potential candidates that could be critical for fertilization. The main aim of this thesis was to give more insight into the biochemical properties and possible function(s) of TSSK3. Chapter 2 describes the analysis of biochemical properties, substrate specificity and in vitro activation of one of TSSK family members, TSSK3. In vitro TSSK3 exhibited the ability to autophosphorylate and to phosphorylate test substrates providing the evidence that TSSK3 is a genuine kinase. Sequence comparison indicated the existence within the TSSK3 catalytic domain of a so-called 'T-loop' a structure present in the AGC-family of protein kinases. To test if this T-loop is engaged in TSSK3 regulation, we mutated the critical threonine within the T-loop to alanine (T168A) and this resulted in an inactive TSSK3 kinase. Furthermore in vitro Thr-168 is phosphorylated by the T-loop kinase phosphoinositide-dependent protein kinase-1 (PDK1) and phosphorylation by PDK1 increased in vitro TSSK3 kinase activity, suggesting that TSSK3 can be regulated in the same way as AGC-kinase family members. Furthermore, analysis of a range of peptide sequences defines the peptide sequence, RRSSSY, as an efficient and specific substrate for TSSK3. In Chapter 3, we searched for TSSK3 potential regulatory interacting proteins by a yeast two-hybrid approach. We identified, a novel, widely expressed protein, RUSC2 and we showed that it binds to but is not a substrate for TSSK3. We also observed it to be phosphorylated by Jun NH2-terminal kinase (JNK). Next, we demonstrated that RUSC2 transiently interacts with JNK kinases after oxidative stress treatment and that TSSK3 appears in this stress-induced complex as well. Furthermore we showed that this interaction is abolished by inhibitory mutant of MKK4. In this chapter we pointed out the possible connection between RUSC2, TSSK3 and oxidative stress induced JNK signaling pathway. Chapter 4 describes our search for mutations in two other genes of the TSSK family, TSSK1 and 2, in patients with infertility. We found, single nucleotide polymorphism (SNP) in the TSSK2 gene, which changed threonine residue, located in the C-terminal regulatory domain of TSSK2, to methionine (T280M) that may be correlated with spermatogenic failure. Thus, TSSK2 might be a candidate gene for molecular marker for genetic diagnosis of male infertility. In Chapter 5, a model for RUSC2 assembly with TSSK3, JNK and H-Ras is proposed and the possible directions of future studies on TSSK family and their role in signal transduction are discussed