LIM kinase 1 and Cell Cycle Regulation

描述(申请人提供)：在理解细胞骨架调节蛋白表达的变化如何导致细胞周期异常进展导致非整倍体、中心体增殖和细胞增殖改变方面存在空白。我们的长期目标是更好地了解G1/S期进展的各个步骤的潜在机制，以及作为肌动蛋白和微管细胞骨架调节蛋白的LIM激酶1是如何参与G1到S期进展的，因为LIM激酶1的表达变化导致了短暂的G1/S停滞、G2/M进展延迟和中心体增殖。这一特殊应用的目的是确定G1进展的初始事件，如早期G1调节蛋白的表达，G1细胞周期蛋白/CDKs的激活和CDK抑制剂的失活是如何通过蛋白质磷酸化和去磷酸化来调节的，以及LIM激酶1如何参与这些过程；同时，确定LIMK1的表达变化如何导致G1细胞向S期的缓慢进展。中心假说是，LIMK1通过改变G1期和S期CDKs的激活和抑制因子的定位和功能，负向调节细胞从G1期到S期的进程。这项研究的基本原理是，了解G1/S期进展的基本机制有可能转化为更好地理解LIMK1在上皮细胞癌中异常表达的发病机制。因此，拟议的研究与NIH使命中与发展基础知识相关的部分相关，这可能有助于减轻人类癌症的负担。在强大的初步数据的指导下，这一假说将被两个特定的目的所验证：1)检测G0同步表达LIMK1的RWPE1细胞中G1/S期调控蛋白的表达、定位和LIMK1的激活状态，以响应生长因子的刺激；2)阐明G1/S期调控蛋白通过形成复合体而相互作用，导致细胞周期蛋白/CDKs在有丝分裂信号作用下的功能激活或失活。在特定目的1下，我们将使用LIMK1过表达的方法，利用稳定的细胞系来确定LIMK1的表达、磷酸化和催化功能对G1期进展的特异性影响，以及G1期和S期激酶及其抑制物的表达。在第二个目标下，将使用激酶分析来检测G1期和S期CDKs的激活及其激活剂和抑制剂的稳定性。这种方法是创新的，因为它利用了我们小组的一项新观察，获得了关于G1到S相变机制的更多信息。这项拟议的研究具有重要意义，因为它有望促进和扩大对激酶如何影响G1/S期进程以及这些激酶的异常表达如何干扰正常细胞周期进程的理解。公共卫生相关性：拟议的研究是一个重要的和正在研究的领域，调节G1/S期和LIM激酶1的功能，对理解LIM激酶1在癌症中异常表达所导致的发病机制具有潜在的适用性。LIM激酶1通过蛋白-蛋白相互作用和磷酸化来调节细胞功能，并通过肌动蛋白细胞骨架的重塑来维持细胞形态。这项拟议的研究与公共健康相关，因为它调查了细胞周期进展的基本机制，该机制在癌症发展过程中经常变得异常。

DESCRIPTION (provided by applicant): There is a gap in understanding how variation in expression of cytoskeleton modulatory proteins leads to abnormal progression of cell cycle resulting in aneuploidy, centrosomal multiplication and altered cell proliferation. The long-term goal is to understand better the underlying mechanism of individual steps of G1/S phase progression and how LIM kinase 1, an actin and microtubule cytoskeleton modulatory protein, is involved in G1 to S phase progression as variation in expression of LIM kinase 1 induced transient G1/S arrest, delayed progression of G2/M and centrosome multiplication. The objective of this particular application is to identify how initial events of G1 progression such as expression of early G1 regulatory proteins, activation of G1 Cyclin/Cdks and inactivation of Cdk inhibitors is regulated through protein phosphorylation and dephosphorylation, and how LIM kinase 1 participates in these processes; also, to determine how variation in expression of LIMK1 leads to sluggish progression of G1 cells to the S phase. The central hypothesis is that LIMK1 negatively regulates progression of cells through G1 to S phase through altered localization and functions of activators and inhibitors of G1 and S phase Cdks. The rationale for the proposed research is that understanding the fundamental mechanism of G1/S phase progression has the potential to translate into better understanding the pathogenesis of abnormal expression of LIMK1 in epithelial cell cancers. Thus, the proposed research is relevant to that part of NIH's mission that pertains to developing fundamental knowledge that will potentially help to reduce the burdens of human cancers. Guided by strong preliminary data, this hypothesis will be tested by two specific aims: 1) Examine the expression, localization of G1/S phase regulatory proteins and activation status of LIMK1 in G0 synchronized RWPE1 cells expressing LIMK1 in response to growth factor stimulation and 2) Elucidate interactions of G1/S phase regulatory proteins through complex formation leading to functional activation or inactivation of Cyclin/Cdks in response to mitogenic signals. Under specific aim 1 we will use the approach of overexpression of LIMK1 and utilize stable cell lines to determine specific effects of expression, phosphorylation and catalytic function of LIMK1 on progression of G1 phase, and expression of G1 and S phase kinases and their inhibitors. Under the second aim, activation of G1 and S phase Cdks and stability of their activator and inhibitors will be examined using kinase assays. This approach is innovative because it capitalizes on a new observation from our group to obtain additional information on the mechanism of G1 to S phase transition. The proposed research is significant, because it is expected to advance and expand the understanding of how kinases influence G1/S phase progression and how aberrant expression of these kinases interferes with normal cell cycle progression. PUBLIC HEALTH RELEVANCE: The proposed studies are of an important and under investigated area of regulation of G1/S phase and function of LIM kinase 1 that has potential applicability to understanding the pathogenesis caused by abnormal expression of LIM kinase 1 in cancer. LIM kinase 1 regulates cellular functions through protein-protein interaction and phosphorylation, and maintains cell shape through remodeling of actin cytoskeleton. The proposed research has relevance to public health, because it investigates a fundamental mechanism of cell cycle progression, which often becomes aberrant during development of cancer.