Examining the role of Cbk1/NDR kinase in regulating mRNA localization

说明（由申请人提供）：NDR激酶是与多种癌症有关的肿瘤抑制因子，包括软组织肉瘤、B和T细胞淋巴瘤、胰腺癌、前列腺癌和卵巢癌。NDR突变导致包括神经元在内的多种细胞的细胞生长和极化形态发生缺陷。尽管NDR激酶的重要性，但它们影响细胞形态发生和癌症进展的机制知之甚少，从而限制了NDR介导的癌症的诊断和治疗的发展。我们最近的数据支持了S.酿酒酵母NDR 1/2直系同源物Cbk 1在调节mRNA极性和翻译中的作用，在细胞极性、不对称细胞中起决定性作用的过程， 分裂发育和形态发生具体来说，我们已经表明，Cbk 1磷酸化的核糖核蛋白（RNP）Ssd 1和促进极性定位的一个子集的Ssd 1-mRNA复合物，这表明NDR激酶促进细胞极性通过指导mRNA-RNP复合物的网站本地化的蛋白质表达。相反，Cbk 1抑制或细胞应激重定向Ssd 1-mRNA复合物的P体和应激颗粒，从而抑制mRNA的翻译，并导致细胞形态和裂解缺陷。此外，Ssd 1招聘的P机构被取消Sit 4/PP 6磷酸酶删除，这表明Sit 4/PP 6是一个NDR-反对磷酸酶。最后，初步数据表明，人类hnRNP A1和A2/B1作为功能性Ssd 1直系同源物。基于这些数据，我们假设NDR激酶通过增强mRNA-RNP与mRNA转运蛋白的相互作用并同时抑制mRNA-RNP向翻译抑制P体/应激颗粒的募集，在促进细胞极性方面发挥普遍作用。由于mRNA极性和局部翻译对细胞极性、发育和细胞运动性至关重要，这些数据可能有助于解释NDR突变如何引起神经元和其他细胞的形态发生缺陷。此外，在极化生长和应激反应中，调节mRNA-RNP复合物主动转运的调控机制尚不清楚。该提案的主要目的是揭示NDR激酶和反作用磷酸酶途径如何通过RNP调节来控制细胞生长和发育。在目标1中，我们确定如何Cbk 1促进Ssd 1介导的不对称mRNA定位，在目标2中，我们将确定如何Sit 4磷酸酶调节Ssd 1招聘到P机构，在目标3中，我们测试的假设，NDR激酶调节细胞极性通过hnRNPA 1和A2/B1。预计这些研究将提供深入了解NDR和抵消途径如何调节hnRNP依赖的mRNA定位的机制。因此，这些研究可能揭示了NDR激酶在细胞极性、神经元形态发生和肿瘤抑制活性中的关键机制。

DESCRIPTION (provided by applicant): NDR kinases are tumor suppressors implicated in a variety of cancers, including soft tissue sarcomas, B and T cell lymphomas, pancreatic, prostate and ovarian cancers. NDR mutations cause cell growth and polarized morphogenesis defects in a variety of cells, including neurons. Despite the importance of NDR kinases, the mechanisms by which they influence cellular morphogenesis and cancer progresion are poorly understood, thus limiting the development of diagnostics and therapeutics for NDR-mediated cancers. Our recent data support a novel role for the S. cerevisiae NDR1/2 orthologue Cbk1 in regulating mRNA polarity and translation, processes that play defining roles in cell polarity, asymmetric cell division, development, and morphogenesis. Specifically, we have shown that Cbk1 phosphorylates the ribonucleoprotein (RNP) Ssd1 and promotes the polar localization of a subset of Ssd1-mRNA complexes, suggesting that NDR kinases promote cell polarity by directing mRNA-RNP complexes to sites of localized protein expression. In contrast, Cbk1 inhibition or cellular stress redirect Ssd1-mRNA complexes to P bodies and stress granules, thereby repressing mRNA translation and causing cell morphology and lysis defects. Moreover, Ssd1 recruitment to the P bodies is abolished by Sit4/PP6 phosphatase deletion, suggesting that Sit4/PP6 is an NDR-opposing phosphatase. Finally, preliminary data suggest that human hnRNP A1 and A2/B1 serve as functional Ssd1 orthologues. Based on these data, we hypothesize that NDR kinases play a universal role in promoting cell polarity by enhancing mRNA-RNP interactions with mRNA transport proteins and concurrently inhibiting mRNA-RNP recruitment to translational repressing P bodies/stress granules. Because mRNA polarity and localized translation are critical for cell polarity, development and cell motility these data may help explain how NDR mutations give rise to morphogenesis defects in neurons and other cells. Moreover, the regulatory mechanisms for modulating active transport of mRNA-RNP complexes during polarized growth and in response to stress are unknown. The main objective of this proposal is to reveal how NDR kinases and a counteracting phosphatase pathway control cell growth and development via RNP regulation. In Aim 1 we determine how Cbk1 promotes Ssd1- mediated asymmetric mRNA localization, in Aim 2 we will determine how Sit4 phosphatase regulates Ssd1 recruitment to P bodies and in Aim 3 we test the hypothesis that NDR kinases regulate cell polarity via hnRNP A1 and A2/B1. It is expected that these studies will provide mechanistic insight into how NDR and counteracting pathways regulate hnRNP-dependent mRNA localization. As such, these studies may reveal a key mechanism for NDR kinases in cell polarity, neuronal morphogenesis and tumor suppressor activity.