Rho-associated kinase ROCK activates LIM-kinase 1 by phosphorylation at threonine 508 within the activation loop

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.     

Specific activation of LIM kinase 2 via phosphorylation of threonine 505 by ROCK, a Rho-dependent protein kinase

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-Delta3), 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.     

Effects of Hippocampal LIMK Inhibition on Memory Acquisition,Consolidation, Retrieval,Reconsolidation, and Extinction

Long-lasting changes in dendritic spines provide a physical correlate for memory formation and persistence. LIM kinase (LIMK) plays a critical role in orchestrating dendritic actin dynamics during memory processing, since it is the convergent downstream target of both the Rac1/PAK and RhoA/ROCK pathways that in turn induce cofilin phosphorylation and prevent depolymerization of actin filaments. Here, using a potent LIMK inhibitor (BMS-5), we investigated the role of LIMK activity in the dorsal hippocampus during contextual fear memory in rats. We first found that post-training administration of BMS-5 impaired memory consolidation in a dose-dependent manner. Inhibiting LIMK before training also disrupted memory acquisition. We then demonstrated that hippocampal LIMK activity seems to be critical for memory retrieval and reconsolidation, since both processes were impaired by BMS-5 treatment. Contextual fear memory extinction, however, was not sensitive to the same treatment. In conclusion, our findings demonstrate that hippocampal LIMK activity plays an important role in memory acquisition, consolidation, retrieval, and reconsolidation during contextual fear conditioning.     

Protein kinase C-related kinase and ROCK are required for thrombin-induced endothelial cell permeability downstream from Galpha12/13 and Galpha11/q

Increase in vascular permeability occurs under many physiological conditions such as wound repair, inflammation, and thrombotic reactions and is central in diverse human pathologies, including tumor-induced angiogenesis, ocular diseases, and septic shock. Thrombin is a pro-coagulant serine protease, which causes the local loss of endothelial barrier integrity thereby enabling the rapid extravasation of plasma proteins and the local formation of fibrin-containing clots. Available information suggests that thrombin induces endothelial permeability by promoting actomyosin contractility through the Rho/ROCK signaling pathway. Here we took advantage of pharmacological inhibitors, knockdown approaches, and the emerging knowledge on how permeability factors affect endothelial junctions to investigate in detail the mechanism underlying thrombin-induced endothelial permeability. We show that thrombin signals through PAR-1 and its coupled G proteins Galpha(12/13) and Galpha(11/q) to induce RhoA activation and intracellular calcium elevation, and that these events are interrelated. In turn, this leads to the stimulation of ROCK, which causes actin stress-fiber formation. However, this alone is not sufficient to account for thrombin-induced permeability. Instead, we found that protein kinase C-related kinase, a Rho-dependent serine/threonine kinase, is activated in endothelial cells upon thrombin stimulation and that its expression is required for endothelial permeability and the remodeling of cell-extracellular matrix and cell-cell adhesions. Our results demonstrate that the signal initiated by thrombin bifurcates at the level of RhoA to promote changes in the cytoskeletal architecture through ROCK, and the remodeling of focal adhesion components through protein kinase C-related kinase. Ultimately, both pathways converge to cause cell-cell junction disruption and provoke vascular leakage.     

LIM kinase 1 coordinates microtubule stability and actin polymerization in human endothelial cells

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.     

LIM-kinase 2 and cofilin phosphorylation mediate actin cytoskeleton reorganization induced by transforming growth factor-beta

Reorganization of the actin cytoskeleton in response to growth factor signaling, such as transforming growth factor beta (TGF-beta), controls cell adhesion, motility, and growth of diverse cell types. In Swiss3T3 fibroblasts, a widely used model for studies of actin reorganization, TGF-beta1 induced rapid actin polymerization into stress fibers and concomitantly activated RhoA and RhoB small GTPases. Consequently, dominant-negative RhoA and RhoB mutants blocked TGF-beta1-induced actin reorganization. Because Rho GTPases are known to regulate the activity of LIM-kinases (LIMK), we found that TGF-beta1 induced LIMK2 phosphorylation with similar kinetics to Rho activation. Cofilin and LIMK2 co-precipitated and cofilin became phosphorylated in response to TGF-beta1, whereas RNA interference against LIMK2 blocked formation of new stress fibers by TGF-beta1. Because the kinase ROCK1 links Rho GTPases to LIMK2, we found that inhibiting ROCK1 activity blocked completely TGF-beta1-induced LIMK2/cofilin phosphorylation and downstream stress fiber formation. We then tested whether the canonical TGF-beta receptor/Smad pathway mediates regulation of the above effectors and actin reorganization. Adenoviruses expressing constitutively activated TGF-beta type I receptor led to robust actin reorganization and Rho activation, whereas the constitutively activated TGF-beta type I receptor with mutated Smad docking sites (L45 loop) did not affect either actin organization or Rho activity. In line with this, ectopic expression of the inhibitory Smad7 inhibited TGF-beta1-induced Rho activation and cytoskeletal reorganization. Our data define a novel pathway emanating from the TGF-beta type I receptor and leading to regulation of actin assembly, via the kinase LIMK2.     

MAPKAPK-2-mediated LIM-kinase activation is critical for VEGF-induced actin remodeling and cell migration

Vascular endothelial growth factor-A (VEGF-A) induces actin reorganization and migration of endothelial cells through a p38 mitogen-activated protein kinase (MAPK) pathway. LIM-kinase 1 (LIMK1) induces actin remodeling by phosphorylating and inactivating cofilin, an actin-depolymerizing factor. In this study, we demonstrate that activation of LIMK1 by MAPKAPK-2 (MK2; a downstream kinase of p38 MAPK) represents a novel signaling pathway in VEGF-A-induced cell migration. VEGF-A induced LIMK1 activation and cofilin phosphorylation, and this was inhibited by the p38 MAPK inhibitor SB203580. Although p38 phosphorylated LIMK1 at Ser-310, it failed to activate LIMK1 directly; however, MK2 activated LIMK1 by phosphorylation at Ser-323. Expression of a Ser-323-non-phosphorylatable mutant of LIMK1 suppressed VEGF-A-induced stress fiber formation and cell migration; however, expression of a Ser-323-phosphorylation-mimic mutant enhanced these processes. Knockdown of MK2 by siRNA suppressed VEGF-A-induced LIMK1 activation, stress fiber formation, and cell migration. Expression of kinase-dead LIMK1 suppressed VEGF-A-induced tubule formation. These findings suggest that MK2-mediated LIMK1 phosphorylation/activation plays an essential role in VEGF-A-induced actin reorganization, migration, and tubule formation of endothelial cells.     

Activation of LIM kinases by myotonic dystrophy kinase-related Cdc42-binding kinase alpha

LIM kinases (LIMK1 and LIMK2) regulate actin cytoskeletal reorganization through cofilin phosphorylation downstream of distinct Rho family GTPases. Pak1 and ROCK, respectively, activate LIMK1 and LIMK2 downstream of Rac and Rho; however, an effector protein kinase for LIMKs downstream of Cdc42 remains to be defined. We now report evidence that LIMK1 and LIMK2 activities toward cofilin phosphorylation are stimulated in cells by the co-expression of myotonic dystrophy kinase-related Cdc42-binding kinase alpha (MRCKalpha), an effector protein kinase of Cdc42. In vitro, MRCKalpha phosphorylated the protein kinase domain of LIM kinases, and the site in LIMK2 phosphorylated by MRCKalpha proved to be threonine 505 within the activation segment. Expression of MRCKalpha induced phosphorylation of actin depolymerizing factor (ADF)/cofilin in cells, whereas MRCKalpha-induced ADF/cofilin phosphorylation was inhibited by the co-expression with the protein kinase-deficient form of LIM kinases. These results indicate that MRCKalpha phosphorylates and activates LIM kinases downstream of Cdc42, which in turn regulates the actin cytoskeletal reorganization through the phosphorylation and inactivation of ADF/cofilin.     

C. elegans LIN-18 is a Ryk ortholog and functions in parallel to LIN-17/Frizzled in Wnt signaling

Wnt proteins are intercellular signals that regulate various aspects of animal development. In Caenorhabditis elegans, mutations in lin-17, a Frizzled-class Wnt receptor, and in lin-18 affect cell fate patterning in the P7.p vulval lineage. We found that lin-18 encodes a member of the Ryk/Derailed family of tyrosine kinase-related receptors, recently found to function as Wnt receptors. Members of this family have nonactive kinase domains. The LIN-18 kinase domain is dispensable for LIN-18 function, while the Wnt binding WIF domain is required. We also found that Wnt proteins LIN-44, MOM-2, and CWN-2 redundantly regulate P7.p patterning. Genetic interactions indicate that LIN-17 and LIN-18 function independently of each other in parallel pathways, and different ligands display different receptor specificities. Thus, two independent Wnt signaling pathways, one employing a Ryk receptor and the other a Frizzled receptor, function in parallel to regulate cell fate patterning in the C. elegans vulva.     

ROCK2 is a major regulator of axonal degeneration,neuronal death and axonal regeneration in the CNS

The Rho/ROCK/LIMK pathway is central for the mediation of repulsive environmental signals in the central nervous system. Several studies using pharmacological Rho-associated protein kinase (ROCK) inhibitors have shown positive effects on neurite regeneration and suggest additional pro-survival effects in neurons. However, as none of these drugs is completely target specific, it remains unclear how these effects are mediated and whether ROCK is really the most relevant target of the pathway. To answer these questions, we generated adeno-associated viral vectors to specifically downregulate ROCK2 and LIM domain kinase (LIMK)-1 in rat retinal ganglion cells (RGCs) in vitro and in vivo. We show here that specific knockdown of ROCK2 and LIMK1 equally enhanced neurite outgrowth of RGCs on inhibitory substrates and both induced substantial neuronal regeneration over distances of more than 5 mm after rat optic nerve crush (ONC) in vivo. However, only knockdown of ROCK2 but not LIMK1 increased survival of RGCs after optic nerve axotomy. Moreover, knockdown of ROCK2 attenuated axonal degeneration of the proximal axon after ONC assessed by in vivo live imaging. Mechanistically, we demonstrate here that knockdown of ROCK2 resulted in decreased intraneuronal activity of calpain and caspase 3, whereas levels of pAkt and collapsin response mediator protein 2 and autophagic flux were increased. Taken together, our data characterize ROCK2 as a specific therapeutic target in neurodegenerative diseases and demonstrate new downstream effects of ROCK2 including axonal degeneration, apoptosis and autophagy.     

LIMK1 depletion enhances fasudil-dependent inhibition of urethral fibroblast proliferation and migration

LIM kinase 1 (LIMK1) is an important regulator of the cell cytoskeleton. This study aimed to examine the role of LIMK1 in mediating the effects of the Rho kinase (ROCK) inhibitor fasudil. In vitro cultures of urethral fibroblasts were divided into LIMK1 knockdown (LIMK1 KD) and LIMK1 control (LIMK1 NC) experimental groups. Each group was incubated with fasudil (50 μmol/L) with or without transforming growth factor β1 (10 ng/mL) for 24 hours. Wound healing and Transwell assays were performed to determine cell migration. Flow cytometry was used to determine apoptosis. LIMK1, collagen I, collagen III, phospho-myosin light chain (p-MLC), alpha smooth muscle actin (α-SMA), and phospho-Cofilin (p-Cofilin) expression was examined by Western blot analysis. The expression of LIMK1 was further validated in human urethral scar tissues. Transwell and wound healing assays revealed that the cells of the LIMK1 KD group exhibited significantly attenuated migration, when compared with those of the LIMK1 NC group ( P < 0.05). Cell migration was also attenuated in the LIMK1 KD group treated with fasudil ( P < 0.05). Flow cytometry analysis revealed that apoptosis was higher in the LIMK1 KD group than that in LIMK1 NC group ( P < 0.05). Apoptosis was also enhanced in the LIMK1 KD group treated with fasudil ( P < 0.05). Western blot analysis demonstrated that LIMK1, collagen I, collagen III, p-MLC, α-SMA, and p-Cofilin expression was significantly attenuated in both the fasudil-treated and untreated LIMK1 KD groups ( P < 0.05). LIMK1 was positively expressed in human urethral scar tissues while it was negatively expressed in normal urethra tissues. In conclusion, loss of LIMK1 expression inhibits the Rho/ROCK pathway-dependent proliferation and migration via downregulation of collagen I, collagen III, p-Cofilin, and α-SMA. LIMK1 loss can also enhance the inhibitory effects of fasudil on the proliferation and migration of urethral fibroblasts.     

LIM kinase 1 modulates cortical actin and CXCR4 cycling and is activated by HIV-1 to initiate viral infection

Almost all viral pathogens utilize a cytoskeleton for their entry and intracellular transport. In HIV-1 infection, binding of the virus to blood resting CD4 T cells initiates a temporal course of cortical actin polymerization and depolymerization, a process mimicking the chemotactic response initiated from chemokine receptors. The actin depolymerization has been suggested to promote viral intracellular migration through cofilin-mediated actin treadmilling. However, the role of the virus-mediated actin polymerization in HIV infection is unknown, and the signaling molecules involved remain unidentified. Here we describe a pathogenic mechanism for triggering early actin polymerization through HIV-1 envelope-mediated transient activation of the LIM domain kinase (LIMK), a protein that phosphorylates cofilin. We demonstrate that HIV-mediated LIMK activation is through gp120-triggered transient activation of the Rack-PAK-LIMK pathway, and that knockdown of LIMK through siRNA decreases filamentous actin, increases CXCR4 trafficking, and diminishes viral DNA synthesis. These results suggest that HIV-mediated early actin polymerization may directly regulate the CXCR4 receptor during viral entry and is involved in viral DNA synthesis. Furthermore, we also demonstrate that in resting CD4 T cells, actin polymerization can be triggered through transient treatment with a pharmacological agent, okadaic acid, that activates LIMK and promotes HIV latent infection of resting CD4 T cells. Taken together, our results suggest that HIV hijacks LIMK to control the cortical actin dynamics for the initiation of viral infection of CD4 T cells.     

Regulation of the actin cytoskeleton by rho kinase controls antigen presentation by CD1d

CD1d molecules are MHC class I-like molecules that present lipid Ags to NKT cells. Although we have previously shown that several different cell signaling molecules can play a role in the control of Ag presentation by CD1d, a defined mechanism by which a cell signaling pathway regulates CD1d function has been unclear. In the current study, we have found that the Rho kinases, Rho-associated, coiled-coil containing protein kinase (ROCK)1 and ROCK2, negatively regulate both human and mouse CD1d-mediated Ag presentation. Inhibition of ROCK pharmacologically, through specific ROCK1 and ROCK2 short hairpin RNA, or by using dendritic cells generated from ROCK1-deficient mice all resulted in enhanced CD1d-mediated Ag presentation compared with controls. ROCK regulates the actin cytoskeleton by phosphorylating LIM kinase, which, in turn, phosphorylates cofilin, prohibiting actin fiber depolymerization. Treatment of APCs with the actin filament depolymerizing agent, cytochalasin D, as well as knockdown of LIM kinase by short hairpin RNA, resulted in enhanced Ag presentation to NKT cells by CD1d, consistent with our ROCK inhibition data. Therefore, our overall results reveal a model whereby CD1d-mediated Ag presentation is negatively regulated by ROCK via its effects on the actin cytoskeleton.     

Inhibition of nuclear import of LIMK2 in endothelial cells by protein kinase C-dependent phosphorylation at Ser-283

LIM kinases (LIMKs) are mainly in the cytoplasm and regulate actin dynamics through cofilin phosphorylation. Recently, it has been reported that nuclear localization of LIMKs can mediate suppression of cyclin D1 expression. Using immunofluorescence monitoring of enhanced green fluorescent protein-tagged LIMK2 in combination with photobleaching techniques and leptomycin B treatment, we demonstrate that LIMK2 shuttles between the cytoplasm and the nucleus in endothelial cells. Sequence analysis predicted two PKC phosphorylation sites in LIMK2 but not in LIMK1. One site at Ser-283 is present between the PDZ and the kinase domain, and the other site at Thr-494 is within the kinase domain. Activation of PKC by phorbol ester treatment of endothelial cells stimulated LIMK2 phosphorylation at Ser-283 and inhibited nuclear import of LIMK2 and the PDZ kinase construct of LIMK2 (amino acids 142-638) but not of LIMK1. The PKC-delta isoform phosphorylated LIMK2 at Ser-283 in vitro. Mutational analysis indicated that LIMK2 phosphorylation at Ser-283 but not Thr-494 was functional. Serum stimulation of endothelial cells also inhibited nuclear import of PDZK-LIMK2 by protein kinase C-dependent phosphorylation of Ser-283. Our study shows that phorbol ester and serum stimulation of endothelial cells inhibit nuclear import of LIMK2 but not LIMK1. This effect was dependent on PKC-delta-mediated phosphorylation of Ser-283. Since phorbol ester enhanced cyclin D1 expression and subsequent G1-to-S-phase transition of endothelial cells, we suggest that the PKC-mediated exclusion of LIMK2 from the nucleus might be a mechanism to relieve suppression of cyclin D1 expression by LIMK2.     

LIM kinase and Diaphanous cooperate to regulate serum response factor and actin dynamics

The small GTPase RhoA controls activity of serum response factor (SRF) by inducing changes in actin dynamics. We show that in PC12 cells, activation of SRF after serum stimulation is RhoA dependent, requiring both actin polymerization and the Rho kinase (ROCK)-LIM kinase (LIMK)-cofilin signaling pathway, previously shown to control F-actin turnover. Activation of SRF by overexpression of wild-type LIMK or ROCK-insensitive LIMK mutants also requires functional RhoA, indicating that a second RhoA-dependent signal is involved. This is provided by the RhoA effector mDia: dominant interfering mDia1 derivatives inhibit both serum- and LIMK-induced SRF activation and reduce the ability of LIMK to induce F-actin accumulation. These results demonstrate a role for LIMK in SRF activation, and functional cooperation between RhoA-controlled LIMK and mDia effector pathways.     

ROCK1 and LIMK2 interact in spread but not blebbing cancer cells

Cancer cells migrating within a 3D microenvironment are able to adopt either a mesenchymal or amoeboid mode of migration. Amoeboid migration is characterised by membrane blebbing that is dependent on the Rho effectors, ROCK1/2. We identify LIMK2 as the preferred substrate for ROCK1 but find that LIMK2 did not induce membrane blebbing, suggesting that a LIMK2 pathway is not involved in amoeboid-mode migration. In support of this hypothesis, novel FRET data demonstrate a direct interaction between ROCK1 and LIMK2 in polarised but not blebbing cells. Our results point to a specific role for the ROCK1:LIMK2 pathway in mesenchymal-mode migration.     

Cytoplasmic p21Cip1 is involved in Ras-induced inhibition of the ROCK/LIMK/cofilin pathway

Accumulating evidence suggests that p21(Cip1) located in the cytoplasm might play a role in promoting transformation and tumor progression. Here we show that oncogenic H-RasV12 contributes to the loss of actin stress fibers by inducing cytoplasmic localization of p21(Cip1), which uncouples Rho-GTP from stress fiber formation by inhibiting Rho kinase (ROCK). Concomitant with the loss of stress fibers in Ras-transformed cells, there is a decrease in the phosphorylation level of cofilin, which is indicative of a compromised ROCK/LIMK/cofilin pathway. Inhibition of MEK in Ras-transformed NIH3T3 results in restoration of actin stress fibers accompanied by a loss of cytoplasmic p21(Cip1), and increased phosphorylation of cofilin. Ectopic expression of cytoplasmic but not nuclear p21(Cip1) in Ras-transformed cells was effective in preventing stress fibers from being restored upon MEK inhibition and inhibited phosphorylation of cofilin. p21(Cip1) was also found to form a complex with ROCK in Ras-transformed cells in vivo. Furthermore, inhibition of the PI 3-kinase pathway resulted in loss of p21(Cip1) expression accompanied by restoration of phosphocofilin, which was not accompanied by stress fiber formation. These results suggest that restoration of cofilin phosphorylation in Ras-transformed cells is necessary but not sufficient for stress fiber formation. Our findings define a novel mechanism for coupling cytoplasmic p21(Cip1) to the control of actin polymerization by compromising the Rho/ROCK/LIMK/cofilin pathway by oncogenic Ras. These studies suggest that localization of p21(Cip1) to the cytoplasm in transformed cells contributes to pathways that favor not only cell proliferation, but also cell motility thereby contributing to invasion and metastasis.