Xu B, Hao Z, Jha K N, Digilio L, Urekar C, Kim Y H, Pulido S, Flickinger C J, Herr J C
Center for Research in Contraceptive and Reproductive Health (CRCRH), Department of Cell Biology, University of Virginia, Charlottesville, VA 22908, USA.
Soc Reprod Fertil Suppl. 2007;63:87-101.
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.
在过去几年里，我们实验室对一个睾丸特异性丝氨酸/苏氨酸激酶家族，即TSSK1 - 4和SSTK，以及TSSK1和2的底物TSKS进行了研究。本文将通过回顾相关文献，提供这些激酶的一般背景信息，然后总结我们实验室的数据，这些数据与评估这些激酶作为未来小分子激酶抑制剂开发的候选靶点相关，这些抑制剂可能用于调节男性生育能力。对人类TSSK1 - 4和SSTK的生物信息学和结构分析表明，这些激酶构成了人类激酶组树上属于AMPK分支的一个独特亚家族。表达研究表明，所有这五种激酶和TSKS底物在睾丸中含量丰富，即便并非严格的睾丸特异性，这表明进行组织特异性避孕靶向是可行的。原位杂交进一步证实，小鼠TSSK2、SSTK和TSKS在减数分裂后的表达模式，这一发现使得它们有可能成为通过保留精原细胞和精母细胞进行可逆性避孕干预的靶点。我们实验室首次在成熟精子中检测到了TSSK2、TSKS和SSTK蛋白。TSKS定位于人类精子的中心粒，而TSSK2则在精子颈部、赤道段和精子尾部的中段被观察到，SSTK定位于赤道段。通过多种方法证实了人类TSSK2与TSKS之间的相互作用和结合：这种底物与酶的相互作用为药物设计提供了一个特别有趣的机会。体外激酶分析表明TSSK2可使TSKS磷酸化。通过IMAC - LC - FTMS鉴定出TSKS磷酸肽HGLSPATPIQGCSGPPGS*PEEPPR，其中丝氨酸285被磷酸化（用星号表示）。这些结果为高通量筛选TSKS磷酸化抑制剂以及进一步研究这个激酶家族的成员作为男性避孕和阴道内杀精剂的靶点提供了理论依据。
