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

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 thrombininduced 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.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. Conversely, changes in microtubule dynamics modulate intracellular signal transduction (for review, see Ref. 1).The actin cytoskeleton undergoes rearrangement under the control of various actin binding, capping, nucleating, and severing proteins, which are intimately involved in regulating the contractile status of the cells (3). Actin dynamics is regulated via transduction of extracellular signals to intracellular events primarily through the members of the Rho family of small GTPases. Rho is known to induce stress fiber formation, whereas Cdc42 and Rac are involved in formation of lamellipodia and filopodia, respectively (4).Microtubule disassembly promotes the formation of actin stress fibers and enhances the contractility of cells (5). Agents such as nocodazole or vinblastine that disrupt microtubules induce rapid assembly of actin filaments and focal adhesions (6), whereas microtubule stabilization with taxol attenuated these effects. Regulation of the actin cytoskeleton by microtubules requires the Rho GTPases (for review, see Ref. 7). However, the mechanisms involved in the coordinated regulation of microtubules and the actin cytoskeleton remain poorly understood. LIMK1 1 is a serine/threonine kinase that regulates a...

Trp-Asp (WD) Repeat Domain 1 Is Essential for Mouse Peri-implantation Development and Regulates Cofilin Phosphorylation

“…In addition, LIMK1 is concentrated to the microtubules in Wdr1 KO MEF cells and the disassembly of microtubules can partly rescue the phosphorylated Cofilin in these cells. Previous reports show that the PDZ domain is a cytoskeleton binding domain and the PDZ domain can localize LIMK1 to the microtubules (21,37,38). Thus, these results suggest that WDR1 might inhibit the binding of LIMK1 to microtubules and release LIMK1 to the cytoplasm to catalyze Cofilin phosphorylation, therefore regulating the binding of F-actin to Cofilin.…”

“…Previous reports showed that LIMK1 activity was inhibited by binding with BMP-RII (bone morphogenetic proteins receptor II) or microtubules (21,37,38). Because the LIMK1 antibody did not work well for immunostaining, LIMK1 with the Myc tag was overexpressed in control and Wdr1 KO MEF cells to examine whether WDR1 regulates LIMK1 by changing its localization.…”

LIM Kinase 1 and Cofilin Regulate Actin Filament Population Required for Dynamin-dependent Apical Carrier Fission from theTrans-Golgi Network

“…Another major challenge is to identify the mechanisms that couple the actin-dependent fission mechanisms described here with our recent observation that kinesin 5B is essential for generation and transport of p75 carrier vesicles from Golgi to PM (Jaulin et al, 2007). In this regard, it is interesting to mention a recent report that postulates a role for LIMK1 in coordinating MT and actin cytoskeleton function (Gorovoy et al, 2005).…”

LIM-kinase 2, a regulator of actin dynamics, is involved in mitotic spindle integrity and sensitivity to microtubule-destabilizing drugs

“…The mechanism(s) of how LIMK2 regulates microtubule-targeted drug action is not yet known. Earlier studies have shown that LIMK1 regulates microtubule stability (Gorovoy et al, 2005) through phosphorylation of p25 also known as tubulin polymerization promoting protein (Acevedo et al, 2007). Phosphorylation of p25 by LIMK1 inhibits its ability to polymerize tubulin resulting in microtubule disassembly.…”