Protein Interactions of the Germ Cell Kinase Haspin

描述(申请人提供)：Haspin是在真核生物中发现的一组独特的新丝氨酸/苏氨酸激酶的典型成员。Haspin mRNA在雄性生殖细胞中的表达最高，而在增殖体细胞亚群中的表达水平要低得多。这种蛋白质位于圆形和细长精子细胞的细胞核中，当被转移到培养细胞中时，它位于一个尚未确定的核亚室中，可能是核仁。在单倍体精子细胞中，Haspin蛋白水平似乎在核仁溶解和失活时达到峰值。Haspin的过度表达会导致细胞周期停滞。因此，haspin很可能参与了单倍体生殖细胞减数分裂的停止。几乎可以肯定的是，haspin的调控涉及蛋白质-蛋白质相互作用和磷酸化事件，这两种活动通常由细胞过程的关键调节器使用。因此，haspin结合蛋白的鉴定是了解其作用机制的关键。利用酵母双杂交筛选人睾丸cDNA文库，我们已经鉴定了潜在的haspin结合蛋白。其中两个候选(PAR4和最深)与haspin的N-末端结合，而另一个(Will Be)与C-末端的激酶结构域相互作用。PAR4(前列腺凋亡反应蛋白-4，也称为PRKC凋亡WT1调节因子，PAWR)使细胞对凋亡敏感，是WT1的调节因子，与蛋白激酶Dik/ZIP Kinase和PKcheeta具有功能相互作用。Deepest(也称为Spag5、Astrin或hMAP126)是一种最近被描述的蛋白质，主要在粗线期精母细胞和圆形精子细胞中表达，但也可以结合到有丝分裂纺锤体中。Will Be是一种以前没有特征的蛋白质，其组织分布与haspin相似。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。这些对这种耐人寻味的激酶和其他调节蛋白之间新的相互作用的研究应该为控制精子发生过程中从减数分裂到精子细胞分化的转变提供重要的见解。

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.