Rho plays a key role in TGF-beta1-induced cytoskeletal rearrangement in human retinal pigment epithelium

Proliferative vitroretinopathy (PVR) is caused by retinal pigment epithelial (RPE) cell proliferation and transformation into fibrotic cells that produce extracellular matrix (ECM) components. Transforming growth factor beta1 (TGF-beta1) is known to play an important role in PVR pathogenesis. To determine how TGF-beta1 mediates the pathogenic changes in RPE cells, we characterized the effects of TGF-beta1 on the morphology, ECM accumulation, and stress fiber formation of ARPE-19 cells, a human RPE cell line. We then elucidated the signaling pathways that mediate these effects. Serum-starved ARPE-19 cells were incubated with 10 ng/ml TGF-beta1 and their morphological changes were examined by phase-contrast microscopy. Actin reorganization was examined by immunochemistry and confocal microscopy. Protein phosphorylation was analyzed by Western blot analysis. TGF-beta1 treatment induced cytoskeleton reorganization, alpha-SMA expression, increased the phosphorylation of ERK, Smad2/3, and AKT, and activated RhoA and Rac1. Cytoskeletal rearrangement was prevented by pretreatment with a Rho inhibitor and by expression of a dominant negative form of Rho. TGF-beta1 also increased LIM kinase and cofilin phosphorylation and the Rho inhibitor blocked this effect. We propose that TGF-beta1 induces human RPE cells to undergo cytoskeletal actin rearrangement via Rho GTPase-dependent pathways that modulate LIM kinase and cofilin activity. This inhibits actin depolymerization and induces the cytoskeletal rearrangements in RPE cells that result in the characteristic features of PVR.     

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.     

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.     

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.     

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.     

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.     

Suppression of the invasive capacity of rat ascites hepatoma cells by knockdown of Slingshot or LIM kinase

Actin cytoskeletal reorganization is essential for tumor cell migration, adhesion, and invasion. Cofilin and actin-depolymerizing factor (ADF) act as key regulators of actin cytoskeletal dynamics by stimulating depolymerization and severing of actin filaments. Cofilin/ADF are inactivated by phosphorylation of Ser-3 by LIM kinase-1 (LIMK1) and reactivated by dephosphorylation by Slingshot-1 (SSH1) and -2 (SSH2) protein phosphatases. In this study, we examined the roles of cofilin/ADF, LIMK1, and SSH1/SSH2 in tumor cell invasion, using an in vitro transcellular migration assay. In this assay, rat ascites hepatoma (MM1) cells were overlaid on a primary-cultured rat mesothelial cell monolayer and the number of cell foci that transmigrated underneath the monolayer in the presence of lysophosphatidic acid (LPA) was counted. The knockdown of cofilin/ADF, LIMK1, or SSH1/SSH2 expression by small interfering RNAs (siRNAs) significantly decreased the LPA-induced transcellular migration of MM1 cells and their motility in two-dimensional culture. Knockdown of LIMK1 also suppressed fibronectin-mediated cell attachment and focal adhesion formation. Our results suggest that both LIMK1-mediated phosphorylation and SSH1/SSH2-mediated dephosphorylation of cofilin/ADF are critical for the migration and invasion of tumor cells and that LIMK1 is involved in the transcellular migration of tumor cells by enhancing both adhesion and motility of the cells.     

Mitosis-dependent phosphorylation and activation of LIM-kinase 1.

LIM-kinases (LIMK1 and LIMK2) regulate actin cytoskeletal reorganization through phosphorylation of cofilin, an actin-depolymerizing factor of actin filaments. Here, we describe a detailed analysis of the cell-cycle-dependent activity of endogenous LIMK1. When HeLa cells were synchronized at prometaphase by nocodazole-treatment, LIMK1 was hyperphosphorylated, and its activity toward cofilin phosphorylation was markedly increased. During cell cycle progression, LIMK1 activity was low in interphase but reached a maximal level during mitosis. Activation of LIMK1 during mitosis was abrogated by roscovitine, a specific inhibitor of cyclin-dependent kinases (CDKs), suggesting that activation of CDKs directly or indirectly participates in LIMK1 activation. These results strongly suggest that LIMK1 may play an important role in the cell cycle progression through regulation of actin cytoskeletal rearrangements.     

Selective amino acid restriction differentially affects the motility and directionality of DU145 and PC3 prostate cancer cells

We previously found that selective restriction of amino acids inhibits invasion of two androgen-independent human prostate cancer cell lines, DU145 and PC3. Here we show that the restriction of tyrosine (Tyr) and phenylalanine (Phe), methionine (Met) or glutamine (Gln) modulates the activity of G proteins and affects the balance between two actin-binding proteins, cofilin and profilin, in these two cell lines. Selective amino acid restriction differentially reduces G protein binding to GTP in DU145 cells. Tyr/Phe deprivation reduces the amount of Rho-GTP and Rac1-GTP. Met deprivation reduces the amount of Ras-GTP and Rho-GTP, and Gln deprivation decreases Ras-GTP, Rac-GTP, and Cdc42-GTP. Restriction of these amino acids increases the amount of profilin, cofilin and phosphorylation of cofilin-Ser(3). Increased PAK1 expression and phosphorylation of PAK1-Thr(423), and Ser(199/204) are consistent with the increased phosphorylation of LIMK1-Thr(508). In PC3 cells, Tyr/Phe or Gln deprivation reduces the amount of Ras-GTP, and all of the examined amino acid restrictions reduce the amount of profilin. PAK1, LIMK1 and cofilin are not significantly altered. These data reveal that specific amino acid deprivation differentially affects actin dynamics in DU145 and PC3. Modulation on Rho, Rac, PAK1, and LIMK1 likely alter the balance between cofilin and profilin in DU145 cells. In contrast, profilin is inhibited in PC3 cells. These effects modulate directionality and motility to inhibit invasion.     

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.     

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.     

Regulation of cofilin phosphorylation and asymmetry in collective cell migration during morphogenesis

During Drosophila oogenesis, two actin dynamics regulators, cofilin and Rac, are required for the collective migration of a coherent cluster of cells called border cells. Cell culture data have shown that Rac and cofilin are both essential for lamellipodium formation, but Rac signaling results in phosphorylation and hence inactivation of cofilin. So it remains unclear whether cofilin phosphorylation plays a promoting or inhibitory role during cell migration. We show here that cofilin is required for F-actin turnover and lamellipodial protrusion in the border cells. Interestingly, reducing the dosage of cofilin by half or expressing a phospho-mimetic mutant form, S3E, partially rescues the migration and protrusion defects of Rac-deficient border cells. Moreover, cofilin exhibits moderate accumulation in border cells at the migratory front of the cluster, whereas phospho-cofilin has a robust and uniform distribution pattern in all the outer border cells. Blocking or overactivating Rac signaling in border cells greatly reduces or increases cofilin phosphorylation, respectively, and each abolishes cell migration. Furthermore, Rac may signal through Pak and LIMK to result in uniform phosphorylation of cofilin in all the outer border cells, whereas the guidance receptor Pvr (PDGF/VEGF receptor) mediates the asymmetric localization of cofilin in the cluster but does not affect its phosphorylation. Our study provides one of the first models of how cofilin functions and is regulated in the collective migration of a group of cells in vivo.     

HIV-1 Tat activates dual Nox pathways leading to independent activation of ERK and JNK MAP kinases

Human immunodeficiency virus, type 1 Tat is known to exert pleiotropic effects on the vascular endothelium through mitogen-activated protein (MAP) kinases, although the signaling pathways leading to MAP kinase activation are incompletely understood. We focused on proximal pathways potentially governing downstream MAP kinase activity by Tat. Within 2 min, Tat activated both Ras and Rho GTPases in endothelial cells, leading to ERK phosphorylation by 10 min. Notably, Rac1 was necessary for downstream activation of RhoA and both Rac1 and RhoA acted upstream of the Ras/ERK cassette. Antioxidants and the oxidase inhibitor diphenylene iodonium blocked ERK phosphorylation, but specific interference with the canonical Nox2 oxidase had no effect on ERK. Instead, knock down of the novel oxidase Nox4 completely suppressed Tat-dependent Ras and ERK activation downstream of Rac1 and RhoA. Conversely, interference with Rac1, PAK1, and Nox2 blocked JNK phosphorylation, whereas RhoA(N19) and Nox4 knock down did not. Further, knock down of Nox2, but not Nox4, blocked Tat-induced cytoskeletal rearrangement, whereas knock down of Nox4, but not Nox2, blocked Tat-dependent proliferation. Rac1, therefore, bifurcates Tat signaling, leading to concurrent but separate Nox4-dependent Ras/ERK activation, and Nox2-dependent JNK activation. Tat signaling, therefore, provides an example of Nox-specific differential control of MAP kinase pathways.     

Molecular mechanism of cofilin dephosphorylation by ouabain

We previously reported that phosphorylated cofilin-triosephosphate isomerase (TPI) complex interacts with Na,K-ATPase and enhances the pump activity through the phosphorylation of cofilin via Rho-mediated signaling pathway. In this study, we tested the hypothesis that the dephosphorylation of cofilin may be induced through Na,K-ATPase inhibition by ouabain. The phosphorylation level of cofilin by ouabain which decreases in a time- and dose-dependent manner in various human cell lines, remains unchanged by pretreatment with Src inhibitor, PP2; epidermal growth factor receptor (EGFR) inhibitor, AG1478; Raf-1 kinase (Raf) inhibitor, GW5074; and ERK kinase (MEK) inhibitor, PD98059, and by transfection of Ras dominant negative mutant (RasN17). This suggests that ouabain dephosphorylates cofilin through the Src/EGFR/Ras/Raf/MEK pathway. Ouabain activates Ras/Raf/MEK pathway, but down-regulates Rho kinase (ROCK)/LIM kinase (LIMK)/cofilin pathway, implying that there may be a cross-talk by ouabain between the Ras/Raf/MEK and the ROCK/LIMK/cofilin pathways. Immunofluorescence and flow cytometry suggest that ouabain-induced active form of cofilin may be involved in cytoskeletal reorganization and cell volume regulation. Thus, these findings demonstrate a new molecular mechanism for the dephosphorylation of cofilin through the inhibition of Na,K-ATPase by ouabain.     

LIM-kinase1

LIM-kinase1 (LIMK1) is a serine-only protein kinase that contains LIM and PDZ protein-protein interaction domains which is highly expressed in neurons. Overexpression of LIMK1 in cultured cells results in accumulation of filamentous (F-) actin. LIMK1 phosphorylates cofilin, an actin depolymerisation factor, which is then unable to bind and depolymerise F-actin. Rac-GTP enhances phosphorylation of LIMK1 and cofilin, which leads to accumulation of F-actin, while Rac-GDP and PMA reduce these effects. LIMK1 is therefore a key component of a signal transduction network that connects extracellular stimuli to changes in cytoskeletal structure. Control of cell morphology and mobility via LIMK1 activity may provide novel approaches to cancer therapy.     

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.     

Different activity regulation and subcellular localization of LIMK1 and LIMK2 during cell cycle transition

LIM kinases (LIMK1 and LIMK2) regulate actin cytoskeletal reorganization through phosphorylating and inactivating cofilin, an actin-depolymerizing factor of actin filaments. Here, we describe a detailed analysis of the cell-cycle-dependent activity of LIMK2, and a subcellular localization of LIMK1 and LIMK2. The activity of LIMK2, distinct from LIMK1, toward cofilin phosphorylation did not change in the normal cell division cycle. In contrast, LIMK2 was hyperphosphorylated and its activity was markedly increased when HeLa cells were synchronized at mitosis with nocodazole treatment. Immunofluorescence analysis showed that LIMK1 was localized at cell-cell adhesion sites in interphase and prophase, redistributed to the spindle poles during prometaphase to anaphase, and accumulated at the cleavage furrow in telophase. In contrast, LIMK2 was diffusely localized in the cytoplasm during interphase, redistributed to the mitotic spindle, and finally to the spindle midzone during anaphase to telophase. These findings suggest that LIMK2 is activated in response to microtubule disruption, and that LIMK1 and LIMK2 may play different roles in regulating for the mitotic spindle organization, chromosome segregation, and cytokinesis during the cell division cycle.     

A role for cofilin and LIM kinase in Listeria-induced phagocytosis

The pathogenic bacterium Listeria monocytogenes is able to invade nonphagocytic cells, an essential feature for its pathogenicity. This induced phagocytosis process requires tightly regulated steps of actin polymerization and depolymerization. Here, we investigated how interactions of the invasion protein InlB with mammalian cells control the cytoskeleton during Listeria internalization. By fluorescence microscopy and transfection experiments, we show that the actin-nucleating Arp2/3 complex, the GTPase Rac, LIM kinase (LIMK), and cofilin are key proteins in InlB-induced phagocytosis. Overexpression of LIMK1, which has been shown to phosphorylate and inactivate cofilin, induces accumulation of F-actin beneath entering particles and inhibits internalization. Conversely, inhibition of LIMK's activity by expressing a dominant negative construct, LIMK1(-), or expression of the constitutively active S3A cofilin mutant induces loss of actin filaments at the phagocytic cup and also inhibits phagocytosis. Interestingly, those constructs similarly affect other actin-based phenomenons, such as InlB-induced membrane ruffling or Listeria comet tail formations. Thus, our data provide evidence for a control of phagocytosis by both activation and deactivation of cofilin. We propose a model in which cofilin is involved in the formation and disruption of the phagocytic cup as a result of its local progressive enrichment.     

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.