LIM Kinase 1 Coordinates Microtubule Stability and Actin Polymerization in Human Endothelial Cells

Matvey Gorovoy,Jiaxin Niu,Ora Bernard,Jasmina Profirovic,Richard Minshall,Radu Neamu,Tatyana Voyno-Yasenetskaya

doi:10.1074/jbc.m502921200

Abstract

Microtubule (MT) destabilization promotes the formation of actin stress fibers and enhances the contractility of cells; however, the mechanism involved in the coordinated regulation of MTs and the actin cytoskeleton is poorly understood. LIM kinase 1 (LIMK1) regulates actin polymerization by phosphorylating the actin depolymerization factor, cofilin. Here we report that LIMK1 is also involved in the MT destabilization. In endothelial cells endogenous LIMK1 co-localizes with MTs and forms a complex with tubulin via the PDZ domain. MT destabilization induced by thrombin or nocodazole resulted in a decrease of LIMK1 colocalization with MTs. Overexpression of wild type LIMK1 resulted in MT destabilization, whereas the kinase-dead mutant of LIMK1 (KD) did not affect MT stability. Importantly, down-regulation of endogenous LIMK1 by small interference RNA resulted in abrogation of the thrombin-induced MTs destabilization and the inhibition of thrombin-induced actin polymerization. Expression of Rho kinase 2, which phosphorylates and activates LIMK1, dramatically decreases the interaction of LIMK1 with tubulin but increases its interaction with actin. Interestingly, expression of KD-LIMK1 or small interference RNA-LIMK1 prevents thrombin-induced microtubule destabilization and F-actin formation, suggesting that LIMK1 activity is required for thrombin-induced modulation of microtubule destabilization and actin polymerization. Our findings indicate that LIMK1 may coordinate microtubules and actin cytoskeleton.

Highlights

Microtubules, polymers of ␣- and ␤-tubulins, are key component of the cytoskeleton and are involved in multiple cellular processes such as migration, mitosis, protein, and organelle transport [1, 2]
To determine whether a certain pool of LIM kinase 1 (LIMK1) protein is associated with microtubule cytoskeleton, we have adapted a novel fixation approach that allows the extraction of cytosolic proteins, including monomeric tubulin, from living cells, preserving only polymeric cytoskeletal structures [17]
LIMK1 Interacts with Tubulin—We have demonstrated here that in endothelial cells LIMK1 is colocalized with MTs and can form a complex with tubulin via its PDZ domain

Summary

Introduction

Microtubules, polymers of ␣- and ␤-tubulins, are key component of the cytoskeleton and are involved in multiple cellular processes such as migration, mitosis, protein, and organelle transport [1, 2] Microtubule dynamics and their spatial arrangements are affected by a number of signaling molecules. The activity of LIMK1 is regulated by the members of the Rho-GTPase family members, Rho, Rac, and Cdc (8, 14 –15) through the activation of their effectors serine/threonine kinases, Rho kinase (ROCK) and the p21-activated kinases (PAK) PAK1 and PAK4, respectively. These kinases phosphorylate LIMK1 at Thr-508 located in the activation loop of the kinase domain, resulting in its activation. We show that LIMK1 induces microtubule destabilization and actin stress fiber formation that requires the kinase activity of LIMK1

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Results

Conclusion