Protein Interactions of the Germ Cell Kinase Haspin

生殖细胞激酶 Haspin 的蛋白质相互作用

• 批准号: 6595864
• 负责人: JONATHAN M HIGGINS
• 金额: $ 8.65万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-01 至 2005-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6595864
• 关键词: HeLa cells; SDS polyacrylamide gel electrophoresis; autoradiography; binding proteins; cell differentiation; cell growth regulation; cell line; enzyme activity; fluorescence microscopy; immunoprecipitation; nucleic acid sequence; phosphorylation; polymerase chain reaction; protein localization; protein protein interaction; serine threonine protein kinase; sperm; spermatogenesis; transfection; western blottings; yeast two hybrid system

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.

描述（由申请人提供）：Haspin是整个真核生物中发现的独特新丝氨酸/苏氨酸激酶的典型成员。 HASPIN mRNA表达在男性生殖细胞中最高，并且在增殖体细胞的子集中的水平低得多。该蛋白质位于圆形和伸长的精子核中，当转染到培养的细胞中时，蛋白质位于尚未定义的核子室（可能是核仁）中。在核仁发生溶解和灭活的时候，HASPIN蛋白水平似乎在单倍体精子中达到峰值。 haspin的表达导致细胞周期停滞。因此，Haspin可能参与单倍生殖细胞中减数分裂的停止。 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，这是细胞过程关键调节剂通常采用的活动。因此，识别haspin结合蛋白对于了解其作用机理至关重要。使用人类睾丸cDNA文库的酵母 - 两种杂交筛选，我们确定了潜在的HASPIN结合蛋白。这些候选者中的两个（PAR4和最深）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。 PAR4（前列腺凋亡反应蛋白-4，也称为PRKC凋亡WT1调节剂PAWR，PAWR）将细胞敏感到凋亡，是WT1的调节剂，与蛋白激酶DIK/ZIP激酶和PKCZETA具有功能相互作用。最深的（也称为Spag5，Astrin或HMAP126）是一种最近描述的蛋白质，主要在Pachytene的精子细胞和圆形精子中表达，但也可以掺入有丝分裂的纺锤体中。 Willbe是一种先前未表征的蛋白质，其组织分布与Haspin相似。我们将在哺乳动物细胞中确认这些相互作用。为了提供对这些蛋白质功能的更多见解，我们将确定Haspin及其结合伙伴的亚核定位。这些有趣的激酶与其他调节蛋白之间的新型相互作用的研究应为控制精子发生过程中从减数分裂到精子分化的过渡提供重要见解。