Specific Activation of LIM kinase 2 via Phosphorylation of Threonine 505 by ROCK, a Rho-dependent Protein Kinase

Abstract

LIM-kinase 1 (LIMK1) and LIM-kinase 2 (LIMK2) regulate actin cytoskeletal reorganization via cofilin phosphorylation downstream of distinct Rho family GTPases. We report our findings that ROCK, a downstream protein kinase of Rho, specifically activates LIMK2 but not LIMK1 downstream of RhoA. LIMK1 and LIMK2 activities toward cofilin phosphorylation were stimulated by co-expression with the active form of ROCK (ROCK-⌬3), whereas full-length ROCK selectively activates LIMK2 but not LIMK1. Activation of LIMK2 by RhoA was inhibited by Y-27632, a specific inhibitor of ROCK, but Rac1-mediated activation of LIMK1 was not. ROCK directly phosphorylated the threonine 505 residue within the activation segment of LIMK2 and markedly stimulated LIMK2 activity. A LIMK2 mutant with replacement of threonine 505 by valine abolished LIMK2 activities for cofilin phosphorylation and actin cytoskeletal changes, whereas replacement by glutamate enhanced the protein kinase activity and stress fiber formation by LIMK2. These results indicate that ROCK directly phosphorylates threonine 505 and activates LIMK2 downstream of RhoA and that this phosphorylation is essential for LIMK2 to induce actin cytoskeletal reorganization. Together with the finding that LIMK1 is regulated by Pak1, LIMK1 and LIMK2 are regulated by different protein kinases downstream of distinct Rho family GTPases.

The cofilin pathway in breast cancer invasion and metastasis

“…In mammals, two different LIMK isoforms, LIMK1 and 2, have been described that can phosphorylate cofilin downstream of Rho-family GTPases 20,64,65 . LIMKs can be activated upstream by Pak 66 as well as ROCK 65,67 and MRCK (myotonic dystrophy kinase-related CDC42-binding kinase) 68 .…”

Rho kinase in the regulation of cell death and survival

“…Most of the ROCK substrates are cellular proteins associated with the regulation of actin cytoskeleton. Besides MLC and MYPT1, this important subset of ROCK substrates include CPI-17 [63], calponin [58], LIM kinases [80,99,132], ezrin/radixin/moesin (ERM) [83], adducin [37], sodium-hydrogen exchanger (NHE1) [137] and ZIP kinase [43].…”

Scaffold mediated regulation of MAPK signaling and cytoskeletal dynamics: A perspective

“…Signaling through its effectors Rho kinase (ROCK) and mDia, Rho stimulates actin stress fiber formation by stimulating the monomeric actin binding protein profilin, and stabilizes stress fibers by inhibiting the activity of the actin depolymerizing protein, cofilin [35][36][37][38][39][40]. Bundling of actin filaments with myosin to form stress fibers, itself stimulated by ROCK and myosin light chain kinase, stimulates cell contraction and stabilization of focal adhesions [41][42][43][44], points of integrin-extracellular matrix contact between the ventral surface of the cell and the underlying substrate.…”

“…Rho-ROCK signaling influences contraction through effects on both the myosin and actin: ROCK elevates myosin light chain phosphorylation by inhibiting myosin phosphatase and/or by directly phosphorylating myosin light chain (MLC) [41][42][43]. ROCK also phosphorylates and activates LIM kinase [64], which in turn phosphorylates and inactivates the actin severing protein, cofilin [36,65].…”

“…While Rac entrains PAK for this function [66,67], Cdc42 signals through MRCK [36,65]. Finally, the recently discovered TESKs can directly phosphorylate cofilin [68], perhaps in response to Rac signaling [69] (Fig.…”