Kueng P, Nikolova Z, Djonov V, Hemphill A, Rohrbach V, Boehlen D, Zuercher G, Andres A C, Ziemiecki A
Department of Clinical Research, University of Berne, CH-3004 Berne, Switzerland.
J Cell Biol. 1997 Dec 29;139(7):1851-9. doi: 10.1083/jcb.139.7.1851.
The molecular mechanisms regulating the spectacular cytodifferentiation observed during spermiogenesis are poorly understood. We have recently identified a murine testis-specific serine kinase (tssk) 1, constituting a novel subfamily of serine/threonine kinases. Using low stringency screening we have isolated and molecularly characterized a second closely related family member, tssk 2, which is probably the orthologue of the human DGS-G gene. Expression of tssk 1 and tssk 2 was limited to the testis of sexually mature males. Immunohistochemical staining localized both kinases to the cytoplasm of late spermatids and to structures resembling residual bodies. tssk 1 and tssk 2 were absent in released sperms in the lumen of the seminiferous tubules and the epididymis, demonstrating a tight window of expression restricted to the last stages of spermatid maturation. In vitro kinase assays of immunoprecipitates containing either tssk 1 or tssk 2 revealed no autophosphorylation of the kinases, however, they led to serine phosphorylation of a coprecipitating protein of approximately 65 kD. A search for interacting proteins using the yeast two-hybrid system with tssk 1 and tssk 2 cDNA as baits and a prey cDNA library from mouse testis, led to the isolation of a novel cDNA, interacting specifically with both tssk 1 and tssk 2, and encoding the coprecipitated 65-kD protein phosphorylated by both kinases. Interestingly, expression of the interacting clone was also testis specific and paralleled the developmental expression observed for the kinases themselves. These results represent the first demonstration of the involvement of a distinct kinase family, the tssk serine/threonine kinases, together with a substrate in the cytodifferentiation of late spermatids to sperms.
精子发生过程中所观察到的显著细胞分化的分子调控机制仍知之甚少。我们最近鉴定出一种小鼠睾丸特异性丝氨酸激酶（tssk）1，它构成了丝氨酸/苏氨酸激酶的一个新亚家族。利用低严谨度筛选，我们分离并对第二个密切相关的家族成员tssk 2进行了分子特征分析，它可能是人类DGS - G基因的直系同源物。tssk 1和tssk 2的表达仅限于性成熟雄性的睾丸。免疫组织化学染色将这两种激酶定位于晚期精子细胞的细胞质以及类似残余小体的结构中。在生精小管和附睾管腔中释放的精子中不存在tssk 1和tssk 2，这表明其表达严格局限于精子细胞成熟的最后阶段。对含有tssk 1或tssk 2的免疫沉淀物进行的体外激酶分析显示，这些激酶没有自磷酸化，然而，它们导致了一种共沉淀的约65 kD蛋白质的丝氨酸磷酸化。利用酵母双杂交系统，以tssk 1和tssk 2的cDNA为诱饵，从小鼠睾丸的猎物cDNA文库中寻找相互作用蛋白，从而分离出一个新的cDNA，它与tssk 1和tssk 2都特异性相互作用，并编码这两种激酶磷酸化的共沉淀65 kD蛋白质。有趣的是，相互作用克隆的表达也是睾丸特异性的，并且与所观察到的激酶自身的发育表达平行。这些结果首次证明了一个独特的激酶家族，即tssk丝氨酸/苏氨酸激酶，以及一种底物参与了晚期精子细胞向精子的细胞分化过程。
