Validation of a testis specific serine/threonine kinase [TSSK] family and the substrate of TSSK1 & 2, TSKS, as contraceptive targets.
A family of testis specific serine/threonine kinases, TSSK1-4 and SSTK, in addition to the substrate of TSSK1 & 2, TSKS, have been studied during the past several years in our laboratory. This paper will provide a general background on these kinases through review of pertinent literature and then will summarize data from our laboratory germane to evaluating these kinases as candidate targets for future development of small molecule kinase inhibitors that may serve to regulate male fertility. Bio-informatic and structural analyses of human TSSK1-4 and SSTK indicate that these kinases constitute a unique subfamily belonging to the AMPK branch on the human kinome tree. Expression studies showed that all five kinases and the TSKS substrate are testis abundant, if not strictly testis specific, indicating that tissue specific contraceptive targeting is possible. In situ hybridization further confirmed that mouse TSSK2, SSTK and TSKS are post-meiotic in their expression patterns, a finding that makes them possible targets of reversible contraceptive intervention by preserving spermatogonia and spermatocytes. Our laboratory detected TSSK2, TSKS and SSTK proteins in mature spermatozoa for the first time. TSKS was localized to the centrioles of human spermatozoa, while TSSK2 was observed in the sperm neck, equatorial segment and mid-piece of the sperm tail, and SSTK was localized in the equatorial segment. The interaction and binding between human TSSK2 and TSKS was confirmed by several methods: this substrate and enzyme interaction offers a particularly interesting opportunity for drug design. In vitro kinase assay showed phosphorylation of TSKS by TSSK2. The TSKS phosphopeptide, HGLSPATPIQGCSGPPGS*PEEPPR, was identified by IMAC-LC-FTMS, with serine 285 being phosphorylated (representend by asterisk). These results provide a rationale for high-throughput screening of inhibitors for TSKS phosphorylation and further studies of members of this kinase family as targets for both male contraception and intra-vaginal spermicides.
Xu
B
B
Center for Research in Contraceptive and Reproductive Health (CRCRH), Department of Cell Biology, University of Virginia, Charlottesville, VA 22908, USA.
Hao
Z
Z
Jha
K N
KN
Digilio
L
L
Urekar
C
C
Kim
Y H
YH
Pulido
S
S
Flickinger
C J
CJ
Herr
J C
JC
D000818:Animals; D003272:Contraceptive Agents, Male / Q000494:pharmacology*; D003598:Cytoskeletal Proteins; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D008297:Male; D010750:Phosphoproteins; D017346:Protein Serine-Threonine Kinases / Q000032:analysis / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D013091:Spermatogenesis / Q000502:physiology*; D013737:Testis / Q000201:enzymology*
D003272:Contraceptive Agents, Male; D003598:Cytoskeletal Proteins; D004791:Enzyme Inhibitors; D010750:Phosphoproteins; C432423:TSKS protein, human; C497813:TSSK1A protein, human; C497812:TSSK2 protein, human; D017346:Protein Serine-Threonine Kinases