Hao Zhonglin, Jha Kula N, Kim Young-Hwan, Vemuganti Soumya, Westbrook V Anne, Chertihin Olga, Markgraf Karin, Flickinger Charles J, Coppola Michael, Herr John C, Visconti Pablo E
Center for Research in Contraception and Reproductive Health (CRCRH), Department of Cell Biology, University of Virginia, Charlottesville, VA 22908, USA.
Mol Hum Reprod. 2004 Jun;10(6):433-44. doi: 10.1093/molehr/gah052. Epub 2004 Mar 25.
Two members of the human testis-specific serine/threonine (Ser/Thr) kinase family, TSSK 1 and TSSK 2, were cloned and sequenced from a human testis adaptor-ligated cDNA library using a PCR strategy. Within the cDNA, open reading frames (ORF) were defined encoding proteins of 367 and 358 amino acids respectively, as well as conserved kinase domains typical of the superfamily of Ser/Thr kinases. Both genes were intronless and mapped to chromosomes 5 and 22 respectively. The human and mouse homologues of TSSK 1 and TSSK 2, together with TSSK 3 and SSTK/FKSG82, constitute a kinase subfamily closely related to the calmodulin kinases and SNF/nim 1 kinase subfamilies. Similar to the mouse, tissue expression by northern and dot blot analysis revealed that human TSSK 1 and 2 messages are expressed exclusively in the testis. However, mRNA for these kinases can be detected in other tissues using real-time PCR. In addition, TSKS, the human homologue of a putative substrate of TSSK 1 and 2, was cloned. TSKS had an ORF of 592 amino acids and was also expressed exclusively in the testis as demonstrated by northern and dot blot analyses; however, lower levels of expression in other tissues were detected using real-time PCR. Human TSSK 2 and TSKS interacted in a yeast two-hybrid system and also co-immunoprecipitated after in vitro translation. TSSK 2 expressed in yeast and bacteria was able to autophosphorylate and also phosphorylated recombinant TSKS in vitro. Antibodies against recombinant TSSK 2 demonstrated that a member of the TSSK family was present in human testis and localized to the equatorial segment of ejaculated human sperm. In contrast, TSKS was only found in the testis. The finding of a TSSK family member in mature sperm suggests that this family of kinases might play a role in sperm function.
利用聚合酶链式反应（PCR）策略，从人睾丸衔接子连接的cDNA文库中克隆并测序了人睾丸特异性丝氨酸/苏氨酸（Ser/Thr）激酶家族的两个成员，即TSSK 1和TSSK 2。在cDNA中，分别定义了编码367和358个氨基酸的开放阅读框（ORF），以及Ser/Thr激酶超家族典型的保守激酶结构域。这两个基因均无内含子，分别定位于5号和22号染色体。TSSK 1和TSSK 2的人和小鼠同源物，连同TSSK 3和SSTK/FKSG82，构成了一个与钙调蛋白激酶和SNF/nim 1激酶亚家族密切相关的激酶亚家族。与小鼠相似，通过Northern印迹和斑点印迹分析的组织表达显示，人TSSK 1和2的信使核糖核酸（mRNA）仅在睾丸中表达。然而，使用实时聚合酶链反应（PCR）可在其他组织中检测到这些激酶的mRNA。此外，还克隆了TSSK 1和2假定底物的人同源物TSKS。TSKS有一个592个氨基酸的开放阅读框，Northern印迹和斑点印迹分析表明其也仅在睾丸中表达；然而，使用实时聚合酶链反应（PCR）检测到其在其他组织中的表达水平较低。人TSSK 2和TSKS在酵母双杂交系统中相互作用，体外翻译后也能进行共免疫沉淀。在酵母和细菌中表达的TSSK 2能够自身磷酸化，并且在体外也能磷酸化重组TSKS。针对重组TSSK 2的抗体表明，TSSK家族的一个成员存在于人睾丸中，并定位于射出的人类精子的赤道段。相比之下，TSKS仅在睾丸中发现。在成熟精子中发现TSSK家族成员表明，该激酶家族可能在精子功能中发挥作用。
