Cell death of prostate cancer cells by specific amino acid restriction depends on alterations of glucose metabolism

Selective amino acid restriction targets mitochondria resulting in DU145 and PC3 prostate cancer cell death. This study shows that restriction of tyrosine and phenylalanine (Tyr/Phe), glutamine (Gln), or methionine (Met) differentially modulates glucose metabolism, glycogen synthase kinase 3β (GSK3β), p53, and pyruvate dehydrogenase (PDH) in these two cell lines. In DU145 cells, Gln and Met restriction increase glucose consumption, but Tyr/Phe restriction does not. Addition of glucose to culture media diminishes cell death induced by Tyr/Phe‐restriction. Addition of pyruvate reduces cell death due to Tyr/Phe and Gln restriction. Tyr/Phe, Gln and Met restriction increase phosphorylation of GSK3β‐Ser⁹, phosphorylation of p53‐Ser¹⁵ and reduce the mitochondrial localization of PDH. Addition of glucose or pyruvate to cultures significantly reverses the alterations in GSK3β, p53 and PDH induced by amino acid restriction. In p53‐null PC3 cells, Tyr/Phe, Gln and Met restriction decreases glucose consumption, reduces phosphorylation of Akt‐Ser⁴⁷³, and increases phosphorylation of GSK3β‐Ser⁹. Addition of pyruvate or glucose reduces death of Met‐restricted cells. Addition of glucose increases phosphorylation of Akt‐Ser⁴⁷³ in amino acid‐restricted cells reduces phosphorylation of GSK3β‐Ser⁹ in Tyr/Phe and Gln restricted cells and increases phosphorylation of GSK3β‐Ser⁹ in Met restricted cells. Addition of pyruvate reduces phosphorylation of GSK3β‐Ser⁹ in all amino acid‐restricted cells. In summary, cell death induced by specific amino acid restriction is dependent on or closely related to the modulation of glucose metabolism. GSK3β (DU145 and PC3) and p53 (DU145) are crucial switches connecting metabolism and these signaling molecules to cell survival during amino acid restriction. J. Cell. Physiol. 224: 491–500, 2010. © 2010 Wiley‐Liss, Inc.     

Selective amino acid restriction targets mitochondria to induce apoptosis of androgen-independent prostate cancer cells

Relative specific amino acid dependency is one of the metabolic abnormalities of cancer cells, and restriction of specific amino acids induces apoptosis of prostate cancer cells. This study shows that restriction of tyrosine and phenylalanine (Tyr/Phe), glutamine (Gln), or methionine (Met), modulates Raf and Akt survival pathways and affects the function of mitochondria in DU145 and PC3, in vitro. These three restrictions inhibit energy production (ATP synthesis) and induce generation of reactive oxygen species (ROS). Restriction of Tyr/Phe or Met in DU145 and Met in PC3 reduces mitochondrial membrane potential (DeltaPsim) and induces caspase-dependent and -independent apoptosis. In DU145, Tyr/Phe or Met restriction reduces activity of Akt, mitochondrial distribution of phosphorylated Raf and apoptosis inducing factor (AIF), and increases mitochondrial distribution of Bak. Mitochondrial Bcl-XL is increased in Tyr/Phe-restricted but decreased in Met-restricted cells. Under Tyr/Phe or Met restriction, reduced mitochondrial Raf does not inactivate the pro-apoptotic function of Bak. Tyr/Phe restriction also inhibits Bcl-2 and Met restriction inhibits Bcl-XL in mitochondria. These comprehensive actions damage the integrity of the mitochondria and induce apoptosis of DU145. In PC3, apoptosis induced by Met restriction was not associated with alterations in intracellular distribution of Raf, Bcl-2 family proteins, or AIF. All of the amino acid restrictions inhibited Akt activity in this cell line. We conclude that specific amino acid restriction differentially interferes with homeostasis/balance between the Raf and Akt survival pathways and with the interaction of Raf and Bcl-2 family proteins in mitochondria to induce apoptosis of DU145 and PC3 cells.     

Nischarin inhibits LIM kinase to regulate cofilin phosphorylation and cell invasion

Nischarin is a novel protein that regulates cell migration by inhibiting p21-activated kinase (PAK). LIM kinase (LIMK) is a downstream effector of PAK, and it is known to play an important role in cell invasion. Here we show that nischarin also associates with LIMK to inhibit LIMK activation, cofilin phosphorylation, and LIMK-mediated invasion of breast cancer cells, suggesting that nischarin regulates cell invasion by negative modulation of the LIMK/cofilin pathway. The amino terminus of nischarin binds to the PDZ and kinase domains of LIMK. Although LIMK activation enhances the interaction with nischarin, only phosphorylation of threonine 508 of LIMK is crucial for the interaction. Inhibition of endogenous nischarin expression by RNA interference stimulates breast cancer cell invasion. Also, nischarin small interfering RNA (siRNA) enhances cofilin phosphorylation. In addition, knock-down of nischarin showed branched projection actin structures. Collectively these data indicate that nischarin siRNA may enhance random migration, resulting in stimulation of invasion.     

Interplay between components of a novel LIM kinase-slingshot phosphatase complex regulates cofilin

Slingshot (SSH) phosphatases and LIM kinases (LIMK) regulate actin dynamics via a reversible phosphorylation (inactivation) of serine 3 in actin-depolymerizing factor (ADF) and cofilin. Here we demonstrate that a multi-protein complex consisting of SSH-1L, LIMK1, actin, and the scaffolding protein, 14-3-3zeta, is involved, along with the kinase, PAK4, in the regulation of ADF/cofilin activity. Endogenous LIMK1 and SSH-1L interact in vitro and co-localize in vivo, and this interaction results in dephosphorylation and downregulation of LIMK1 activity. We also show that the phosphatase activity of purified SSH-1L is F-actin dependent and is negatively regulated via phosphorylation by PAK4. 14-3-3zeta binds to phosphorylated slingshot, decreases the amount of slingshot that co-sediments with F-actin, but does not alter slingshot activity. Here we define a novel ADF/cofilin phosphoregulatory complex and suggest a new mechanism for the regulation of ADF/cofilin activity in mediating changes to the actin cytoskeleton.     

A PAK4-LIMK1 pathway drives prostate cancer cell migration downstream of HGF

Hepatocyte growth factor (HGF) is associated with tumour progression and increases the invasiveness of prostate carcinoma cells. Cell migration and invasion requires reorganisation of the actin cytoskeleton; processes mediated by the Rho family GTPases. p21 activated kinase 4 (PAK4), an effector of the Rho family protein Cdc42, is activated downstream of HGF. We report here the novel finding that in prostate cancer cells PAK4 binds to and phosphorylates LIM kinase 1 (LIMK1) in an HGF-dependent manner. We show for the first time that variations in the level of PAK4 expression change the level of cofilin phosphorylation in cells, a change we correlate with LIMK1 activity, cell morphology and migratory behaviour. We identify for the first time a direct and localised interaction between PAK4 and LIMK1 within cells using FRET: FLIM. Moreover we show here that HGF mediates this interaction which is concentrated in small foci at the cell periphery. PAK4 and LIMK1 act synergistically to increase cell migration speed, whilst a reduction in PAK4 expression decreases cell speed. It is well established that unphosphorylated (active) cofilin is a required to drive cell migration. Our results support a model whereby HGF-stimulated cell migration also requires a cofilin phosphorylation step that is mediated by PAK4.     

LIM kinase and slingshot are critical for neurite extension

Cofilin and its closely related protein, actin-depolymerizing factor (ADF), are key regulators of actin cytoskeleton dynamics that have been implicated in growth cone motility and neurite extension. Cofilin/ADF are inactivated by LIM kinase (LIMK)-catalyzed phosphorylation and reactivated by Slingshot (SSH)-catalyzed dephosphorylation. Here we examined the roles of cofilin/ADF, LIMKs (LIMK1 and LIMK2), and SSHs (SSH1 and SSH2) in nerve growth factor (NGF)-induced neurite extension. Knockdown of cofilin/ADF by RNA interference almost completely inhibited NGF-induced neurite extension from PC12 cells, and double knockdown of SSH1/SSH2 significantly suppressed both NGF-induced cofilin/ADF dephosphorylation and neurite extension from PC12 cells, thus indicating that cofilin/ADF and their activating phosphatases SSH1/SSH2 are critical for neurite extension. Interestingly, NGF stimulated the activities of both LIMK1 and LIMK2 in PC12 cells, and suppression of LIMK1/LIMK2 expression or activity significantly reduced NGF-induced neurite extension from PC12 cells or chick dorsal root ganglion (DRG) neurons. Inhibition of LIMK1/LIMK2 activity reduced actin filament assembly in the peripheral region of the growth cone of chick DRG neurons. These results suggest that proper regulation of cofilin/ADF activities through control of phosphorylation by LIMKs and SSHs is critical for neurite extension and that LIMKs regulate actin filament assembly at the tip of the growth cone.     

Cytoskeletal changes regulated by the PAK4 serine/threonine kinase are mediated by LIM kinase 1 and cofilin.

PAK4 is the most recently identified member of the PAK family of serine/threonine kinases. PAK4 differs from other members of the PAK family in sequence and in many of its functions. Previously, we have shown that an important function of this kinase is to mediate the induction of filopodia in response to the Rho GTPase Cdc42. Here we show that PAK4 also regulates the activity of the protein kinase LIM kinase 1 (LIMK1). PAK4 was shown to interact specifically with LIMK1 in binding assays. Immune complex kinase assays revealed that both wild-type and constitutively active PAK4 phosphorylated LIMK1 even more strongly than PAK1, and activated PAK4 stimulated LIMK1's ability to phosphorylate cofilin. Immunofluorescence experiments revealed that PAK4 and LIMK1 cooperate to induce cytoskeletal changes in C2C12 cells. Furthermore, dominant negative LIMK1 and a mutant cofilin inhibited the specific cytoskeletal and cell shape changes that were induced in response to a recently characterized constitutively activated PAK4 mutant.     

PAK1 and PAK2 have different roles in HGF-induced morphological responses

Hepatocyte growth factor (HGF) stimulates dissociation of epithelial cells (scattering) and cell migration. Several Rho GTPases are required for HGF-induced scattering. PAK1 and PAK2 are members of the p21-activated kinase (PAK) family of serine/threonine kinases, and are activated by the Rho GTPases Rac and Cdc42. Here we investigate the contributions of PAK1 and PAK2 to HGF-induced motile response. HGF stimulates phosphorylation of PAK1 and PAK2. Knockdown of PAK1 inhibits HGF-stimulated migration and loss of cell–cell junctions in DU145 prostate carcinoma cells, whereas knockdown of PAK2 enhances loss of cell–cell junctions and increases lamellipodium extension but does not affect migration speed. On the other hand, in PC3 prostate carcinoma cells, which lack cell–cell junctions, knockdown of PAK1 or PAK2 reduces HGF-stimulated migration. PAK2 knockdown increases phosphorylation of PAK1, indicating that PAK2 provides a negative feedback on PAK1. We hypothesise that PAK2 acts in part via PAK1 to regulate HGF-induced scattering.     

DGCR6L,a novel PAK4 interaction protein,regulates PAK4-mediated migration of human gastric cancer cell via LIMK1

Overexpression, genetic amplification and mutations of p21-activated kinase 4 (PAK4) were found in a variety of human cancers. PAK4 regulated actin cytoskeleton reorganization by phosphorylating LIMK1 and promoted cancer cells migration. Using yeast two-hybrid screen, we identified a novel PAK4 binding protein, DGCR6L, which was associated with cancer cell metastasis. We confirmed PAK4 binding to the DGCR6L specifically by GST pull-down assay, and found an association between endogenous PAK4 and DGCR6L by immunoprecipitation in mammalian cells. Furthermore, L115 of DGCR6L was the critical amino acid to bind 466–572aa in the very C-terminus of PAK4. Importantly, DGCR6L was required for the formation of PAK4-DGCR6L-β-actin complex. Overexpressed DGCR6L promoted migration of AGS cells mediated by PAK4, whereas knock-down of DGCR6L markedly inhibited the migration of those cells. Moreover, DGCR6L (L115V), which did not bind to PAK4, lost the ability to promote AGS cells migration. DGCR6L colocalized with PAK4 or F-actin and enhanced the phosphorylation level of LIMK1 and cofilin in a dose dependent manner. Taken together, our results demonstrated that DGCR6L, a novel PAK4 interacting protein, regulated PAK4-mediated migration of human gastric cancer cells via LIMK1.     

Phosphorylated LIM Kinases Colocalize with Gamma-Tubulin in Centrosomes During Early Stages of Mitosis

LIM kinases (LIMK1 and LIMK2) are LIM domain containing serine/threonine kinases that modulate reorganization of actin cytoskeleton through inactivating phosphorylation of cofilin. The Rho family of small GTPases regulates the catalytic activity of LIMK1 and LIMK2 through activating phosphorylation by ROCK or by p21 kinase. Recent studies have suggested that LIMK1 could play a role in modulation of cellular growth by alteration of the cell cycle in breast and prostate tumor cells; however, the direct mitogenic effects of LIMK1 in these tumor cells is yet to be elucidated. Via immunofluorescence, in this study, we show that phosphorylated LIM kinases (pLIMK1/2) are colocalized with γ-tubulin in the centrosomes during the early mitotic phases of human breast and prostate cancer cells (MDA-MB-231 and DU145); apparent colocalization begins in the centrosomes in prophase. As shown by both bright field (MDA-MB-231) and fluorescent immunohistochemistry (MDA-MB-231 and DU145), pLIMK1/2 does not localize to centrosomes during interphase. By bright field immunohistochemistry, the largest area of the centrosome that is stained with pLIMK1/2 occurs at anaphase. In early telophase, reduced staining of pLIMK1/2 at the spindle poles and concomitant accumulation of pLIMK1/2 at the cleavage furrow begins to occur. In late telophase, loss of staining of pLIMK1/2 and of colocalization with γ-tubulin occurs at the poles and pLIMK1/2 became further concentrated at the junction between the two daughter cells. Co-immunoprecipitation studies indicated that γ-tubulin associates with phosphorylated LIMK1 and LIMK2 but not with dephosphorylated LIMK1 or LIMK2. The results suggest that activated LIMK1/2 may associate with γ-tubulin and play a role in mitotic spindle assembly.     

Protein kinase D regulates cofilin activity through p21-activated kinase 4

Dynamic reorganization of the actin cytoskeleton at the leading edge is required for directed cell migration. Cofilin, a small actin-binding protein with F-actin severing activities, is a key enzyme initiating such actin remodeling processes. Cofilin activity is tightly regulated by phosphorylation and dephosphorylation events that are mediated by LIM kinase (LIMK) and the phosphatase slingshot (SSH), respectively. Protein kinase D (PKD) is a serine/threonine kinase that inhibits actin-driven directed cell migration by phosphorylation and inactivation of SSH. Here, we show that PKD can also regulate LIMK through direct phosphorylation and activation of its upstream kinase p21-activated kinase 4 (PAK4). Therefore, active PKD increases the net amount of phosphorylated inactive cofilin in cells through both pathways. The regulation of cofilin activity at multiple levels may explain the inhibitory effects of PKD on barbed end formation as well as on directed cell migration.     

Mitosis-specific activation of LIM motif-containing protein kinase and roles of cofilin phosphorylation and dephosphorylation in mitosis

Actin filament dynamics play a critical role in mitosis and cytokinesis. LIM motif-containing protein kinase 1 (LIMK1) regulates actin reorganization by phosphorylating and inactivating cofilin, an actin-depolymerizing and -severing protein. To examine the role of LIMK1 and cofilin during the cell cycle, we measured cell cycle-associated changes in the kinase activity of LIMK1 and in the level of cofilin phosphorylation. Using synchronized HeLa cells, we found that LIMK1 became hyperphosphorylated and activated in prometaphase and metaphase, then gradually returned to the basal level as cells entered into telophase and cytokinesis. Although Rho-associated kinase and p21-activated protein kinase phosphorylate and activate LIMK1, they are not likely to be involved in mitosis-specific activation and phosphorylation of LIMK1. Immunoblot and immunofluorescence analyses using an anti-phosphocofilin-specific antibody revealed that the level of cofilin phosphorylation, similar to levels of LIMK1 activity, increased during prometaphase and metaphase then gradually declined in telophase and cytokinesis. Ectopic expression of LIMK1 increased the level of cofilin phosphorylation throughout the cell cycle and induced the formation of multinucleate cells. These results suggest that LIMK1 is involved principally in control of mitosis-specific cofilin phosphorylation and that dephosphorylation and reactivation of cofilin at later stages of mitosis play a critical role in cytokinesis of mammalian cells.     

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.     

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.     

Stromal Cell-Derived Factor 1α Activates LIM Kinase 1 and Induces Cofilin Phosphorylation for T-Cell Chemotaxis

Stromal cell-derived factor 1 alpha (SDF-1alpha), the ligand for G-protein-coupled receptor CXCR4, is a chemotactic factor for T lymphocytes. LIM kinase 1 (LIMK1) phosphorylates cofilin, an actin-depolymerizing and -severing protein, at Ser-3 and regulates actin reorganization. We investigated the role of cofilin phosphorylation by LIMK1 in SDF-1alpha-induced chemotaxis of T lymphocytes. SDF-1alpha significantly induced the activation of LIMK1 in Jurkat human leukemic T cells and peripheral blood lymphocytes. SDF-1alpha also induced cofilin phosphorylation, actin reorganization, and activation of small GTPases, Rho, Rac, and Cdc42, in Jurkat cells. Pretreatment with pertussis toxin inhibited SDF-1alpha-induced LIMK1 activation, thus indicating that Gi protein is involved in LIMK1 activation. Expression of dominant negative Rac (DN-Rac), but not DN-Rho or DN-Cdc42, blocked SDF-1alpha-induced activation of LIMK1, which means that SDF-1alpha-induced LIMK1 activation is mediated by Rac but not by Rho or Cdc42. We used a cell-permeable peptide (S3 peptide) that contains the phosphorylation site (Ser-3) of cofilin to inhibit the cellular function of LIMK1. S3 peptide inhibited the kinase activity of LIMK1 in vitro. Treatment of Jurkat cells with S3 peptide inhibited the SDF-1alpha-induced cofilin phosphorylation, actin reorganization, and chemotactic response of Jurkat cells. These results suggest that the phosphorylation of cofilin by LIMK1 plays a critical role in the SDF-1alpha-induced chemotactic response of T lymphocytes.     

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.     

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.     

Spatial and temporal control of cofilin activity is required for directional sensing during chemotaxis

BACKGROUND: Previous work has led to the hypothesis that cofilin severing, as regulated by PLC, is involved in chemotactic sensing. We have tested this hypothesis by investigating whether activation of endogenous cofilin is spatially and temporally linked to sensing an EGF point source in carcinoma cells. RESULTS: We demonstrate that inhibition of endogenous cofilin activity with either siRNA or overexpression of LIMK suppresses directional sensing in carcinoma cells. LIMK siRNA knockdown, which suppresses cofilin phosphorylation, and microinjection of S3C cofilin, a cofilin mutant that is constitutively active and not phosphorylated by LIMK, also inhibits directional sensing and chemotaxis. These results indicate that phosphorylation of cofilin by LIMK, in addition to cofilin activity, is required for chemotaxis. Cofilin activity concentrates rapidly at the newly formed leading edge facing the gradient, whereas cofilin phosphorylation increases throughout the cell. Quantification of these results indicates that the amplification of asymmetric actin polymerization required for protrusion toward the EGF gradient occurs at the level of cofilin but not at the level of PLC activation by EGFR. CONCLUSIONS: These results indicate that local activation of cofilin by PLC and its global inactivation by LIMK phosphorylation combine to generate the local asymmetry of actin polymerization required for chemotaxis.     

Cofilin phosphorylation and actin cytoskeletal dynamics regulated by rho- and Cdc42-activated LIM-kinase 2

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.     

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.