LIM kinase 1 and Cell Cycle Regulation

Chakrabarti, Ratna

Abstract

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. ? ? ?