Protein Interactions of the Germ Cell Kinase Haspin

描述（由申请人提供）：Haspin是在整个真核生物中发现的一组独特的新丝氨酸/苏氨酸激酶的原型成员。Haspin mRNA表达在雄性生殖细胞中最高，在增殖体细胞亚群中水平低得多。当转染到培养细胞中时，该蛋白位于圆形和细长精子细胞的细胞核中以及尚未确定的核亚室中，可能是核仁。Haspin蛋白水平在单倍体精子细胞中出现峰值，此时核仁发生溶解和失活。haspin的过度表达导致细胞周期停滞。因此，haspin可能参与了单倍体生殖细胞减数分裂的停止。haspin的调节几乎肯定涉及蛋白质-蛋白质相互作用和磷酸化事件，这些活动通常由细胞过程的关键调节剂使用。因此，识别haspin结合蛋白对于理解其作用机制至关重要。利用酵母双杂交筛选的人睾丸cDNA文库，我们已经确定了潜在的haspin结合蛋白。这些候选物中的两个（Par 4和DEEPEST）与haspin的N-末端结合，而另一个（Willbe）与C-末端激酶结构域相互作用。Par 4（前列腺凋亡反应蛋白-4，也称为PRKC凋亡WT 1调节因子，PAWR）使细胞对凋亡敏感，是WT 1的调节因子，并与蛋白激酶DIk/ZIP激酶和PKCzeta具有功能性相互作用。DEEPEST（也称为Spag 5、Astrin或hMAP 126）是最近描述的主要在粗线期精母细胞和圆形精子细胞中表达的蛋白质，但也可以并入有丝分裂纺锤体中。Willbe是一种以前未表征的蛋白质，具有与haspin相似的组织分布。我们将在哺乳动物细胞中证实这些相互作用。为了进一步了解这些蛋白质的功能，我们将确定haspin及其结合伙伴的亚核定位。这些有趣的激酶和其他调节蛋白之间的新的相互作用的研究应该提供重要的见解控制从减数分裂到精子细胞分化在精子发生过程中的过渡。

DESCRIPTION (provided by applicant): Haspin is the prototypical member of a distinctive new group of serine/threonine kinases found throughout the eukaryotes. Haspin mRNA expression is highest in male germ cells and present at much lower levels in subsets of proliferating somatic cells. The protein is located in the nucleus of round and elongated spermatids and in a yet undefined nuclear sub-compartment, possibly the nucleolus, when transfected into cultured cells. Haspin protein levels appear to peak in haploid spermatids at a time when nucleoli undergo dissolution and inactivation. Over expression of haspin causes cell cycle arrest. It is therefore likely that haspin is involved in the cessation of meiosis in haploid germ cells. Regulation by and of haspin almost certainly involves both protein-protein interactions and phosphorylation events, activities commonly employed by critical regulators of cellular processes. Therefore, identification of haspin binding proteins will be crucial to understand its mechanism of action. Using a yeast-two hybrid screen of a human testis cDNA library, we have identified potential haspin binding proteins. Two of these candidates (Par4 and DEEPEST) bind to the N-terminus of haspin, while the other (Willbe) interacts with the C-terminal kinase domain. Par4 (prostate apoptosis response protein-4, also known as PRKC apoptosis WT1 regulator, PAWR) sensitizes cells to apoptosis, is a regulator of WT1 and has functional interactions with the protein kinases DIk/ZIP kinase and PKCzeta. DEEPEST (also known as Spag5, Astrin or hMAP126) is a recently described protein that is expressed predominantly in pachytene spermatocytes and round spermatids, but can also be incorporated into the mitotic spindle. Willbe is a previously uncharacterized protein with a similar tissue distribution to haspin. We will confirm these interactions in mammalian cells. To provide additional insight into the function of these proteins, we will determine the sub-nuclear localization of haspin and its binding partners. These studies of novel interactions between this intriguing kinase and other regulatory proteins should provide important insights into the control of the transition from meiosis to spermatid differentiation during spermatogenesis.