LIM kinase 1 and Cell Cycle Regulation

描述（由申请人提供）：在理解细胞骨架调节蛋白表达的变化如何导致细胞周期的异常进展方面存在差距，从而导致非整倍性、中心体增殖和细胞增殖改变。长期目标是更好地理解G1/S期进展的各个步骤的潜在机制，以及LIM激酶1（肌动蛋白和微管细胞骨架调节蛋白）如何参与G1至S期进展，因为LIM激酶1表达的变化诱导短暂的G1/S停滞，G2/M的延迟进展和中心体增殖。该特定申请的目的是鉴定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/Cdk的活化和Cdk抑制剂的失活如何通过蛋白磷酸化和去磷酸化来调节，以及LIM激酶1如何参与这些过程;也确定LIMK 1表达的变化如何导致G1细胞缓慢进展到S期。中心假设是LIMK 1通过改变G1和S期Cdks的激活剂和抑制剂的定位和功能来负调节细胞从G1期到S期的进展。这项研究的基本原理是，理解G1/S期进展的基本机制有可能转化为更好地理解上皮细胞癌中LIMK 1异常表达的发病机制。因此，拟议的研究与NIH的使命的一部分有关，该使命涉及开发可能有助于减轻人类癌症负担的基础知识。在强有力的初步数据的指导下，这一假设将通过两个具体目标进行检验：1）检查表达式，G1/S期调节蛋白的定位和LIMK 1在响应生长因子刺激的表达LIMK 1的G 0同步化RWPE 1细胞中的活化状态，和2）阐明G1/S期调节蛋白通过复合物形成的相互作用，导致Cyclin/Cyclin激酶的功能活化或失活。Cdks对促有丝分裂信号的反应。在具体目标1下，我们将使用LIMK 1过表达的方法，并利用稳定的细胞系来确定LIMK 1的表达、磷酸化和催化功能对G1期进展以及G1和S期激酶及其抑制剂的表达的具体影响。在第二个目标下，G1和S期Cdks的活化及其活化剂和抑制剂的稳定性将使用激酶测定来检查。这种方法是创新的，因为它利用了我们小组的一个新的观察，以获得更多的信息G1到S相变的机制。这项研究意义重大，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：这些研究是LIM激酶1的G1/S期调控和功能研究的一个重要领域，对理解LIM激酶1异常表达引起的癌症发病机制具有潜在的应用价值。LIM激酶1通过蛋白质-蛋白质相互作用和磷酸化调节细胞功能，并通过肌动蛋白细胞骨架的重塑维持细胞形状。这项拟议的研究与公共卫生相关，因为它研究了细胞周期进展的基本机制，而细胞周期进展在癌症发展过程中往往会变得异常。

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.