Molecular and functional analysis of Pctk1

Pctk1 is a member of the cyclin dependent kinases (CDK). These serinehreonine kinases consist of a catalytic and a regulatory subunit, the cyclin, and are best known for their function in cell cycle regulation. In contrast to these well characterised kinases, Pctk1 is only poorly understood and neither a cyclin partner nor a substrate have been identified so far. In order to define the function of this enigmatic kinase, we have deleted the Pctk1 gene in the mouse and found that lack of Pctk1 causes male infertility. Histological analysis of the testis has revealed that Pctk1 is involved in the terminal steps of spermatogenesis, which are required for the production of motile spermatozoa. These results demonstrate that Pctk1 is an essential gene and further prove that sperm morphogenesis is genetically controlled. The genetic basis of human male infertility, which affects 5-7% of couples and often involves defects in the latest steps in spermatogenesis, is not well understood. Thus, Pctk1 deficient mice serve as an important model system for male infertility. In this project we propose to dissect the pathogenesis of infertility due to Pctk1 deficiency by performing a histological and ultrastructural analysis of testis sections and spermatids obtained from Pctk1 deficient mice. In addition, since we now know that Pctk1 is essential for spermatogenesis, we will use testis proteins to search for Pctk1 activators and substrates in order to define the biochemical function of this kinase. By defining the mode of activation as well as substrates, in combination with analysis of the subcellular localisation of Pctk1 in spermatids, we hope to be able to elucidate the function of this kinase in the testis. The results obtained by these experiments may then help to elucidate the poorly understood mechanisms that regulate the process of sperm morphogenesis, which involves dramatic cytoplasmic changes that are controlled by posttranslational protein modification. In addition to deciphering the role of Pctk1 in spermatogenesis, these analyses might lead to the discovery of new means for the diagnosis and, eventually, treatment of male infertility. In addition, since Pctk1 only effects the terminal steps in spermatogenesis, Pctk1 inhibitors might serve as reversible inhibitors of spermatogenesis, i.e. male contraceptives.

In this project we identified the mode of activation, regulation and function of cyclin dependent kinase (CDK) 16, previously known as PCTAIRE kinase 1 (PCTK1). By using biochemical and cell biological techniques, we found CDK16 to be activated by binding to Cyclin Y and to function as a serine/threonine protein kinase. This interaction was first established using in vitro reconstitution assays and later confirmed by the analysis of CDK16 complexes purified from murine brain and testis, in which both proteins are most highly expressed and in which CDK16 is active. To define the function of CDK16 in vivo, we analyzed mice lacking the Cdk16 gene. Consistent with the high expression of CDK16 in spermatids, targeted deletion of the Cdk16 gene in mice resulted in male infertility. Sperm isolated from Cdk16 knockout male mice exhibited reduced motility and displayed many structural defects, demonstrating that CDK16 function is required for the generation of functional spermatozoa. Analysis of a large cohort of infertile men led to the identification of a mutation in the CDK16 gene (R477P) suggesting that mutations in this gene might also underlie inborn errors of human male fertility. By studying the mechanism how Cyclin Y activates CDK16, we discovered novel interaction domains between the two proteins and found that their interaction is controlled by the phosphorylation state of CDK16. Based on these results we retrieved CDK16 related sequences from various genome databases and could show that only animals but not plants or unicellular eukaryotes contain CDK16-related kinases, suggesting that this protein kinase co-evolved with the formation of a central nervous system. Vertebrates contain 3-5 CDK16 related sequences, most of which are co-expressed in neurons and are likely to carry out similar functions, explaining why CDK16 knockout mice do not display an obvious neuronal phenotype. These findings expand our knowledge how cyclins are able to activate CDKs and provide the basis for a more detailed structural analysis of the CDK16 enzyme complex as well as the development of specific chemical inhibitors that might be useful as reversible inhibitors of spermatogenesis for contraception in males.Purified active recombinant CyclinY-CDK16 was found to display high enzymatic activity in vitro, allowing us to use is to identify potential substrates of this protein kinase. We performed in vitro kinase assays by analyzing over 700 peptides using in vitro phosphorylation assays, which allowed us to derive a novel consensus phosphorylation motif for CyclinY-CDK16. This motif, SPPGSPSPP (displayed in the single letter abbreviation code for amino acid sequences) lacks a positively charged residue, suggesting that the substrate spectrum for CDK16 is very different to that of other CDKs. In addition, candidate substrate screens were performed using testis extracts as well as a panel of more than 6500 arrayed recombinant human proteins to compile a CDK16 candidate kinase substrate list.