Cofilin phosphorylation and actin cytoskeletal dynamics regulated by rho- and Cdc42-activated LIM-kinase 2.
(33/2130)
The rapid turnover of actin filaments and the tertiary meshwork formation are regulated by a variety of actin-binding proteins. Protein phosphorylation of cofilin, an actin-binding protein that depolymerizes actin filaments, suppresses its function. Thus, cofilin is a terminal effector of signaling cascades that evokes actin cytoskeletal rearrangement. When wild-type LIMK2 and kinase-dead LIMK2 (LIMK2/KD) were respectively expressed in cells, LIMK2, but not LIMK2/KD, phosphorylated cofilin and induced formation of stress fibers and focal complexes. LIMK2 activity toward cofilin phosphorylation was stimulated by coexpression of activated Rho and Cdc42, but not Rac. Importantly, expression of activated Rho and Cdc42, respectively, induced stress fibers and filopodia, whereas both Rho- induced stress fibers and Cdc42-induced filopodia were abrogated by the coexpression of LIMK2/KD. In contrast, the coexpression of LIMK2/KD with the activated Rac did not affect Rac-induced lamellipodia formation. These results indicate that LIMK2 plays a crucial role both in Rho- and Cdc42-induced actin cytoskeletal reorganization, at least in part by inhibiting the functions of cofilin. Together with recent findings that LIMK1 participates in Rac-induced lamellipodia formation, LIMK1 and LIMK2 function under control of distinct Rho subfamily GTPases and are essential regulators in the Rho subfamilies-induced actin cytoskeletal reorganization. (+info)
Rho-kinase-mediated pathway induces enhanced myosin light chain phosphorylations in a swine model of coronary artery spasm.
(34/2130)
OBJECTIVE: We recently demonstrated in our swine model of coronary artery spasm that enhanced myosin light chain (MLC) phosphorylations (both MLC mono- and diphosphorylations) play a central role in the pathogenesis of the spasm. However, the molecular mechanism for and the phosphorylation sites for the enhanced MLC phosphorylations were unknown. In the present study, we addressed these points using hydroxyfasudil, a novel inhibitor of protein kinases, which we found preferentially inhibits Rho-kinase. METHODS: The specificity of the inhibitory effects of hydroxyfasudil on Rho-kinase, MLCK, MRCK beta and PKC were examined by kinase assay in vitro. The left porcine coronary artery was chronically treated with interleukin-1 beta (IL-1 beta, 2.5 micrograms). Two weeks after the operation, coronary artery vasomotion was examined both in vivo and in vitro. MLC phosphorylations were examined by Western blot analysis and the sites for the phosphorylations by anti-phosphorylated MLC antibodies that identified the monophosphorylation site as Ser19 and diphophorylation sites as Ser19/Thr18 of MLC. RESULTS: Inhibitory effects of hydroxyfasudil was at least 100 times more potent for Rho-kinase as compared with other protein kinases tested. Intracoronary serotonin (10 micrograms/kg) caused coronary hyperconstriction at the IL-1 beta-treated site in vivo, which was dose-dependently inhibited by hydroxyfasudil (p < 0.01). The coronary segment taken from the spastic site also showed hypercontractions to serotonin in vitro, which were again dose-dependently inhibited by hydroxyfasudil (p < 0.01). Western blot analysis showed that MLC monophosphorylation was significantly greater in the spastic segment than in the control segment, while MLC diphosphorylation was noted only at the spastic segment (p < 0.01). The sites for the mono- and diphosphorylated MLC were identified as the monophosphorylated site Ser19 and diphosphorylated sites Ser19/Thr18 of MLC, respectively. Both types of MLC phosphorylations at the spastic segment were markedly inhibited by hydroxyfasudil (p < 0.01). CONCLUSION: These results indicate that hydroxyfasudil-sensitive Rho-kinase-mediated pathway appears to mediate the enhanced MLC phosphorylations (on Ser19 and Ser19/Thr18 residues) and plays a central role in the pathogenesis of coronary artery spasm. (+info)
Inhibition of Rho-associated kinase blocks agonist-induced Ca2+ sensitization of myosin phosphorylation and force in guinea-pig ileum.
(35/2130)
Ca2+ sensitization of smooth muscle contraction involves the small GTPase RhoA, inhibition of myosin light chain phosphatase (MLCP) and enhanced myosin regulatory light chain (LC20) phosphorylation. A potential effector of RhoA is Rho-associated kinase (ROK). The role of ROK in Ca2+ sensitization was investigated in guinea-pig ileum. Contraction of permeabilized muscle strips induced by GTPgammaS at pCa 6.5 was inhibited by the kinase inhibitors Y-27632, HA1077 and H-7 with IC50 values that correlated with the known Ki values for inhibition of ROK. GTPgammaS also increased LC20 phosphorylation and this was prevented by HA1077. Contraction and LC20 phosphorylation elicited at pCa 5.75 were, however, unaffected by HA1077. Pre-treatment of intact tissue strips with HA1077 abolished the tonic component of carbachol-induced contraction and the sustained elevation of LC20 phosphorylation, but had no effect on the transient or sustained increase in [Ca2+]i induced by carbachol. LC20 phosphorylation and contraction dynamics suggest that the ROK-mediated increase in LC20 phosphorylation is due to MLCP inhibition, not myosin light chain kinase activation. In the absence of Ca2+, GTPgammaS stimulated 35S incorporation from [35S]ATPgammaS into the myosin targeting subunit of MLCP (MYPT). The enhanced thiophosphorylation was inhibited by HA1077. No thiophosphorylation of LC20 was detected. These results indicate that ROK mediates agonist-induced increases in myosin phosphorylation and force by inhibiting MLCP activity through phosphorylation of MYPT. Under Ca2+-free conditions, ROK does not appear to phosphorylate LC20 in situ, in contrast to its ability to phosphorylate myosin in vitro. In particular, ROK activation is essential for the tonic phase of agonist-induced contraction. (+info)
Signal transduction by G-proteins, rho-kinase and protein phosphatase to smooth muscle and non-muscle myosin II.
(36/2130)
We here review mechanisms that can regulate the activity of myosin II, in smooth muscle and non-muscle cells, by modulating the Ca2+ sensitivity of myosin regulatory light chain (RLC) phosphorylation. The major mechanism of Ca2+ sensitization of smooth muscle contraction and non-muscle cell motility is through inhibition of the smooth muscle myosin phosphatase (MLCP) that dephosphorylates the RLC in smooth muscle and non-muscle. The active, GTP-bound form of the small GTPase RhoA activates a serine/threonine kinase, Rho-kinase, that phosphorylates the regulatory subunit of MLCP and inhibits phosphatase activity. G-protein-coupled release of arachidonic acid may also contribute to inhibition of MLCP acting, at least in part, through the Rho/Rho-kinase pathway. Protein kinase C(s) activated by phorbol esters and diacylglycerol can also inhibit MLCP by phosphorylating and thereby activating CPI-17, an inhibitor of its catalytic subunit; this mechanism is independent of the Rho/Rho-kinase pathway and plays only a minor, transient role in the G-protein-coupled mechanism of Ca2+ sensitization. Ca2+ sensitization by the Rho/Rho-kinase pathway contributes to the tonic phase of agonist-induced contraction in smooth muscle, and abnormally increased activation of myosin II by this mechanism is thought to play a role in diseases such as high blood pressure and cancer cell metastasis. (+info)
Involvement of Rho-kinase in angiotensin II-induced hypertrophy of rat vascular smooth muscle cells.
(37/2130)
Angiotensin II (Ang II) is now believed to play a critical role in the pathogenesis of hypertrophy and/or hyperplasia of vascular smooth muscle cells (VSMCs). Several G(i)- and G(q)-coupled receptors, including the Ang II type 1 (AT(1)) receptor, activate Rho and Rho-associated kinase in Swiss 3T3 cells and cardiac myocytes. However, little is known about the role of Rho-kinase in Ang II-induced vascular hypertrophy in VSMCs. In the present study, we explored the role of Rho and Rho-kinase in Ang II-induced protein synthesis in VSMCs. In unstimulated cells, RhoA was observed predominantly in the cytosolic fraction, but it was translocated in part to the particulate fraction in response to Ang II (100 nmol/L). This effect was completely blocked by the AT(1) receptor blocker candesartan but not by the Ang II type 2 (AT(2)) receptor antagonist PD123319. Botulinum C(3) exoenzyme, which inactivated RhoA, attenuated Ang II-induced [(3)H]leucine incorporation. The specific Rho-kinase inhibitor, Y-27632, dose-dependently abolished Ang II-induced protein synthesis and also suppressed Ang II-induced c-fos mRNA expression. On the other hand, Y-27632 had no effect on Ang II-stimulated phosphorylation of p70 S6 kinase and extracellular signal-regulated kinase 1/2, which are reported to be involved in Ang II-induced protein synthesis, nor had it any effect on the Ang II-induced phosphorylation of PHAS-I, a heat- and acid-stable eIF-4E-binding protein. The phosphorylation of PHAS-I is regulating for translation initiation. These observations suggest that the Rho, Rho-kinase, and c-fos pathways may play a role in Ang II-induced hypertrophic changes of VSMCs through a novel pathway. (+info)
In smooth muscle, a Rho-regulated system of myosin phosphatase exists; however, it has yet to be established whether Rho kinase, one of the downstream effectors of Rho, mediates the regulation of myosin phosphatase activity in vivo. In the present study, we demonstrate in permeabilized vascular smooth muscle cells (SMCs) that the vasodilator 1-(5-isoquinolinesulfonyl)-homopiperazine (HA-1077), which we show to be a potent inhibitor of Rho kinase, dose dependently inhibits Rho-mediated enhancement of Ca(2+)-induced 20-kDa myosin light chain (MLC(20)) phosphorylation due to abrogating Rho-mediated inhibition of MLC(20) dephosphorylation. By an immune complex phosphatase assay, we found that guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) stimulation of permeabilized SMCs caused a decrease in myosin phosphatase activity with an increase in the extent of phosphorylation of the 130-kDa myosin-binding regulatory subunit (MBS) of myosin phosphatase in a Rho-dependent manner. HA-1077 abolished both of the Rho-mediated events. Moreover, we observed that the pleckstrin homology/cystein-rich domain protein of Rho kinase, a dominant negative inhibitor of Rho kinase, inhibited GTPgammaS-induced phosphorylation of MBS. These results provide direct in vivo evidence that Rho kinase mediates inhibition of myosin phosphatase activity with resultant enhancement of MLC(20) phosphorylation in smooth muscle and reveal the usefulness of HA-1077 as a Rho kinase inhibitor. (+info)
Mildly oxidised low density lipoprotein induces platelet shape change via Rho-kinase-dependent phosphorylation of myosin light chain and moesin.
(39/2130)
Oxidised low density lipoprotein (LDL) plays an important role in the pathogenesis of atherosclerosis. Here we demonstrate that mildly oxidised (mox) LDL engages the GTPase Rho and its effector molecule p160 Rho-kinase to induce phosphorylation of myosin light chain and of moesin leading to platelet shape change. Pretreatment of platelets with the selective Rho inhibitor C3-transferase from Clostridium botulinum or with the Rho-kinase inhibitor Y-27632 blocked mox-LDL-induced myosin light chain phosphorylation, moesin phosphorylation and shape change. Mox-LDL did not induce an increase in cytosolic Ca(2+) during shape change. We propose that Rho/Rho-kinase inhibition could be a strategy for prevention of the pathologic platelet activation during atherogenesis. (+info)
Rho-associated kinase ROCK activates LIM-kinase 1 by phosphorylation at threonine 508 within the activation loop.
(40/2130)
LIM-kinase 1 (LIMK1) phosphorylates cofilin, an actin-depolymerizing factor, and regulates actin cytoskeletal reorganization. LIMK1 is activated by the small GTPase Rho and its downstream protein kinase ROCK. We now report the site of phosphorylation of LIMK1 by ROCK. In vitro kinase reaction revealed that the active forms of ROCK phosphorylated LIMK1 on the threonine residue and markedly increased its cofilin-phosphorylating activity. A LIMK1 mutant (T508A) with replacement of Thr-508 within the activation loop of the kinase domain by alanine was neither phosphorylated nor activated by ROCK. Replacement of Thr-508 by serine changed the ROCK-catalyzed phosphorylation residue from threonine to serine. A LIMK1 mutant with replacement of Thr-508 by two glutamates increased the kinase activity about 2-fold but was not further activated by ROCK. In addition, wild-type LIMK1, but not its T508A mutant, was activated by co-expression with ROCK in cultured cells. These results suggest that ROCK activates LIMK1 in vitro and in vivo by phosphorylation at Thr-508. Together with the recent finding that PAK1, a downstream effector of Rac, also activates LIMK1 by phosphorylation at Thr-508, these results suggest that activation of LIMK1 is one of the common targets for Rho and Rac to reorganize the actin cytoskeleton. (+info)