Identification of multiple actin-binding sites in cofilin-phosphatase Slingshot-1L

Slingshot-1L (SSH1L) is a phosphatase that specifically dephosphorylates and activates cofilin, an actin-severing and -depolymerizing protein. SSH1L binds to and is activated by F-actin in vitro, and co-localizes with F-actin in cultured cells. We examined the F-actin-binding activity, F-actin-mediated phosphatase activation, and subcellular distribution of various mutants of SSH1L. We identified three sites involved in F-actin binding of SSH1L: Trp-458 close to the C-terminus of the phosphatase domain, an LHK motif in the N-terminal region, and an LKR motif in the C-terminal region. These sites play unique roles in the control of subcellular localization and F-actin-mediated activation of SSH1L.     

A pathway of neuregulin-induced activation of cofilin-phosphatase Slingshot and cofilin in lamellipodia

Cofilin mediates lamellipodium extension and polarized cell migration by stimulating actin filament dynamics at the leading edge of migrating cells. Cofilin is inactivated by phosphorylation at Ser-3 and reactivated by cofilin-phosphatase Slingshot-1L (SSH1L). Little is known of signaling mechanisms of cofilin activation and how this activation is spatially regulated. Here, we show that cofilin-phosphatase activity of SSH1L increases approximately 10-fold by association with actin filaments, which indicates that actin assembly at the leading edge per se triggers local activation of SSH1L and thereby stimulates cofilin-mediated actin turnover in lamellipodia. We also provide evidence that 14-3-3 proteins inhibit SSH1L activity, dependent on the phosphorylation of Ser-937 and Ser-978 of SSH1L. Stimulation of cells with neuregulin-1beta induced Ser-978 dephosphorylation, translocation of SSH1L onto F-actin-rich lamellipodia, and cofilin dephosphorylation. These findings suggest that SSH1L is locally activated by translocation to and association with F-actin in lamellipodia in response to neuregulin-1beta and 14-3-3 proteins negatively regulate SSH1L activity by sequestering it in the cytoplasm.     

The Cofilin Phosphatase Slingshot Homolog 1 (SSH1) Links NOD1 Signaling to Actin Remodeling

NOD1 is an intracellular pathogen recognition receptor that contributes to anti-bacterial innate immune responses, adaptive immunity and tissue homeostasis. NOD1-induced signaling relies on actin remodeling, however, the details of the connection of NOD1 and the actin cytoskeleton remained elusive. Here, we identified in a druggable-genome wide siRNA screen the cofilin phosphatase SSH1 as a specific and essential component of the NOD1 pathway. We show that depletion of SSH1 impaired pathogen induced NOD1 signaling evident from diminished NF-κB activation and cytokine release. Chemical inhibition of actin polymerization using cytochalasin D rescued the loss of SSH1. We further demonstrate that NOD1 directly interacted with SSH1 at F-actin rich sites. Finally, we show that enhanced cofilin activity is intimately linked to NOD1 signaling. Our data thus provide evidence that NOD1 requires the SSH1/cofilin network for signaling and to detect bacterial induced changes in actin dynamics leading to NF-κB activation and innate immune responses.     

Insulin Receptor Substrate-4 Binds to Slingshot-1 Phosphatase and Promotes Cofilin Dephosphorylation

Cofilin plays an essential role in cell migration and morphogenesis by enhancing actin filament dynamics via its actin filament-severing activity. Slingshot-1 (SSH1) is a protein phosphatase that plays a crucial role in regulating actin dynamics by dephosphorylating and reactivating cofilin. In this study, we identified insulin receptor substrate (IRS)-4 as a novel SSH1-binding protein. Co-precipitation assays revealed the direct endogenous binding of IRS4 to SSH1. IRS4, but not IRS1 or IRS2, was bound to SSH1. IRS4 was bound to SSH1 mainly through the unique region (amino acids 335–400) adjacent to the C terminus of the phosphotyrosine-binding domain of IRS4. The N-terminal A, B, and phosphatase domains of SSH1 were bound to IRS4 independently. Whereas in vitro phosphatase assays revealed that IRS4 does not directly affect the cofilin phosphatase activity of SSH1, knockdown of IRS4 increased cofilin phosphorylation in cultured cells. Knockdown of IRS4 decreased phosphatidylinositol 3-kinase (PI3K) activity, and treatment with an inhibitor of PI3K increased cofilin phosphorylation. Akt preferentially phosphorylated SSH1 at Thr-826, but expression of a non-phosphorylatable T826A mutant of SSH1 did not affect insulin-induced cofilin dephosphorylation, and an inhibitor of Akt did not increase cofilin phosphorylation. These results suggest that IRS4 promotes cofilin dephosphorylation through sequential activation of PI3K and SSH1 but not through Akt. In addition, IRS4 co-localized with SSH1 in F-actin-rich membrane protrusions in insulin-stimulated cells, which suggests that the association of IRS4 with SSH1 contributes to localized activation of cofilin in membrane protrusions.     

Phosphoinositide 3-kinase-mediated activation of cofilin phosphatase Slingshot and its role for insulin-induced membrane protrusion

Cofilin plays an essential role in actin filament dynamics and membrane protrusion in motile cells. Cofilin is inactivated by phosphorylation at Ser-3 by LIM kinase and reactivated by dephosphorylation by cofilin-phosphatase Slingshot (SSH). Although cofilin is dephosphorylated in response to various extracellular stimuli, signaling pathways regulating SSH activation and cofilin dephosphorylation have remained to be elucidated. Here we show that insulin stimulates the phosphatase activity of Slingshot-1L (SSH1L) and cofilin dephosphorylation in cultured cells, in a manner dependent on phosphoinositide 3-kinase (PI3K) activity. Consistent with this, the level of Ser-3-phosphorylated cofilin is increased in PTEN (phosphatase and tensin homolog deleted in chromosome 10)-overexpressing cells and decreased in PTEN-deficient cells. Insulin induced the accumulation of SSH1L and active Akt (a downstream effector of PI3K), together with a PI3K product phosphatidylinositol 3,4,5-trisphosphate, onto membrane protrusions. Cofilin, but not Ser-3-phosphorylated cofilin, accumulated in membrane protrusions in insulin-stimulated cells, indicating that cofilin is dephosphorylated in these areas. Finally, suppression of SSH1L expression by RNA interference abolished insulin-induced cofilin dephosphorylation and the membrane protrusion. These findings suggest that SSH1L is activated downstream of PI3K and plays a critical role in insulin-induced membrane protrusion by dephosphorylating and activating cofilin.     

Mitotic phosphorylation rescues Abl from F-actin-mediated inhibition

We have previously shown that F-actin exerts a negative effect on Abl tyrosine kinase activity. This inhibition results from a direct association of F-actin with the C terminus of Abl and accounts, in part, for the loss of Abl activity in detached fibroblasts. We report here that Abl from mitotic cells or cells treated with the protein phosphatase inhibitor okadaic acid remains active when detached from the extracellular matrix. Aspartic acid substitution of Thr(566), which is phosphorylated in mitotic or okadaic acid-treated cells, is sufficient to abolish F-actin-mediated inhibition and to maintain Abl activity despite cell detachment. A recent crystal structure of the Abl N-terminal region has revealed autoinhibitory interactions among the Src homology 3 (SH3), SH2, and kinase domains. We found that deletion of the SH2 domain also abolished the negative effect of F-actin on kinase activity. Immediately following the kinase domain in Abl is a proline-rich linker (PRL) that binds to several SH3 adaptor proteins. Interestingly, binding of the Crk N-terminal SH3 domain to the PRL also disrupted F-actin-mediated inhibition of Abl kinase. These results suggest that F-actin may reinforce the autoinhibitory interactions to regulate Abl kinase and that inhibition can be relieved through phosphorylation and/or protein interactions with the Abl PRL region.     

Activation of cytosolic Slingshot-1 phosphatase by gelsolin-generated soluble actin filaments

Slingshot-1 (SSH1) is a protein phosphatase that dephosphorylates and activates cofilin, an actin-severing and -disassembling protein. SSH1 is bound to and activated by F-actin, but not G-actin. SSH1 is accumulated in the F-actin-rich lamellipodium but is also diffusely distributed in the cytoplasm. It remains unknown whether SSH1 is activated by soluble (low-level polymerized) actin filaments in the cytoplasm. In this study, we show that SSH1 binds to gelsolin via actin filaments in the cytosolic fraction. Gelsolin promoted solubilization of actin filaments and SSH1 in cell-free assays and in cultured cells. SSH1 was activated by gelsolin-generated soluble actin filaments. Furthermore, gelsolin enhanced cofilin dephosphorylation in neuregulin-stimulated cells. Our results suggest that cytosolic SSH1 forms a complex with gelsolin via soluble actin filaments and is activated by gelsolin-generated soluble actin filaments and that gelsolin promotes stimulus-induced cofilin dephosphorylation through increasing soluble actin filaments, which support SSH1 activation in the cytoplasm.     

The coiled-coil domain is required for HS1 to bind to F-actin and activate Arp2/3 complex

HS1 (hematopoietic lineage cell-specific protein 1), a substrate of protein tyrosine kinases in lymphocytes, binds to F-actin, and promotes Arp2/3 complex-mediated actin polymerization. However, the mechanism for the interaction between HS1 and F-actin has not yet been fully characterized. HS1 contains 3.5 tandem repeats, a coiled-coil region, and an SH3 domain at the C terminus. Unlike cortactin, which is closely related to HS1 and requires absolutely the repeat domain for F-actin binding, an HS1 mutant with deletion of the repeat domain maintains a significant F-actin binding activity. On the other hand, deletion of the coiled-coil region abolished the ability of HS1 to bind to actin filaments and to activate the Arp2/3 complex for actin nucleation and actin branching. Furthermore, a peptide containing the coiled-coil sequence only was sufficient for F-actin binding. Within cells overexpressing green fluorescent protein-tagged HS1 proteins, wild type HS1 co-localizes with cortical F-actin at the cell leading edge, whereas mutants with deletion of either the coiled-coil region or the repeat domain diffuse in the cytoplasm. Immunoprecipitation analysis reveals that the coiled-coil deletion mutant binds poorly to F-actin, whereas the mutant without the repeat domain fails to bind to both Arp2/3 complex and F-actin. These data suggest that the HS1 coiled-coil region acts synergistically with the repeat domain in the modulation of the Arp2/3 complex-mediated actin polymerization.     

Phosphorylation of Ser 402 impedes phosphatase activity of slingshot 1

By using mass spectrometry, we have identified Ser 402 as a new phosphorylation site within the catalytic domain of human slingshot 1 (SSH1). Phosphorylation at this site inhibits substrate binding and, thus, phosphatase activity in vitro, resulting in enrichment of phosphorylated cofilin in monolayer cell culture. We further demonstrate that protein kinase D (PKD) is upstream from Ser 402 phosphorylation. Accordingly, expression of active PKD in Drosophila phenotypically mimics the loss of SSH activity by inducing accumulation of phosphorylated cofilin and filamentous actin. We thus identify a universal mechanism by which PKD controls SSH1 phosphatase activity.     

Calcium signal-induced cofilin dephosphorylation is mediated by Slingshot via calcineurin

Cofilin, an essential regulator of actin filament dynamics, is inactivated by phosphorylation at Ser-3 and reactivated by dephosphorylation. Although cofilin undergoes dephosphorylation in response to extracellular stimuli that elevate intracellular Ca2+ concentrations, signaling mechanisms mediating Ca2+-induced cofilin dephosphorylation have remained unknown. We investigated the role of Slingshot (SSH) 1L, a member of a SSH family of protein phosphatases, in mediating Ca2+-induced cofilin dephosphorylation. The Ca2+ ionophore A23187 and Ca2+-mobilizing agonists, ATP and histamine, induced SSH1L activation and cofilin dephosphorylation in cultured cells. A23187- or histamine-induced SSH1L activation and cofilin dephosphorylation were blocked by calcineurin inhibitors or a dominant-negative form of calcineurin, indicating that calcineurin mediates Ca2+-induced SSH1L activation and cofilin dephosphorylation. Importantly, knockdown of SSH1L expression by RNA interference abolished A23187- or calcineurin-induced cofilin dephosphorylation. Furthermore, calcineurin dephosphorylated SSH1L and increased the cofilin-phosphatase activity of SSH1L in cell-free assays. Based on these findings, we suggest that Ca2+-induced cofilin dephosphorylation is mediated by calcineurin-dependent activation of SSH1L.     

Calcium-binding protein 1 of Entamoeba histolytica transiently associates with phagocytic cups in a calcium-independent manner

EhCaBP1, a calcium-binding protein of the parasite Entamoeba histolytica, is known to participate in cellular processes involving actin filaments. This may be due to its direct interaction with actin. In order to understand the kinetics of EhCaBP1 in such processes, its movement was studied in living cells expressing GFP-EhCaBP1. The results showed that EhCaBP1 accumulated at phagocytic cups and pseudopods transiently. The time taken for appearance and disappearance of EhCaBP1 was found to be around 12 s. Site-directed mutagenesis was used to generate an EhCaBP1 mutant with reduced Ca(2+)- and G-actin binding ability without any defect in its ability to bind F-actin. The overexpression of this mutant EhCaBP1 in the E. histolytica trophozoites resulted in the impairment of erythrophagocytosis, uptake of bacterial cells, killing of target cells but not fluid-phase pinocytosis. However, the mutant protein was still found to transiently localize with F-actin at the phagocytic cups and pseudopods. The mutant protein displayed reduced ability to activate endogenous kinase(s) suggesting that phagosome formation may require Ca(2+)-EhCaBP1 transducing downstream signalling but initiation of phagocytosis may be independent of its intrinsic ability to bind Ca(2+). The results suggest a dynamic association of EhCaBP1 with F-actin-mediated processes.     

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.     

Platelet-derived growth factor (PDGF) regulates Slingshot phosphatase activity via Nox1-dependent auto-dephosphorylation of serine 834 in vascular smooth muscle cells

Migration of vascular smooth muscle cells (VSMCs) contributes to vascular pathology. PDGF induces VSMC migration by a Nox1-based NADPH oxidase mediated mechanism. We have previously shown that PDGF-induced migration in VSMCs requires Slingshot-1L (SSH1L) phosphatase activity. In the present work, the mechanism of SSH1L activation by PDGF is further investigated. We identified a 14-3-3 consensus binding motif encompassing Ser-834 in SSH1L that is constitutively phosphorylated. PDGF induces SSH1L auto-dephosphorylation at Ser-834 in wild type (wt), but not in Nox1(-/y) cells. A SSH1L-S834A phospho-deficient mutant has significantly lower binding capacity for 14-3-3 when compared with the phospho-mimetic SSH1L-S834D mutant, and acts as a constitutively active phosphatase, lacking of PDGF-mediated regulation. Given that Nox1 produces reactive oxygen species, we evaluated their participation in this SSH1L activation mechanism. We found that H(2)O(2) activates SSH1L and this is accompanied by SSH1L/14-3-3 complex disruption and 14-3-3 oxidation in wt, but not in Nox1(-/y) cells. Together, these data demonstrate that PDGF activates SSH1L in VSMC by a mechanism that involves Nox1-mediated oxidation of 14-3-3 and Ser-834 SSH1L auto-dephosphorylation.     

GPCR-mediated PLCβγ/PKCβ/PKD signaling pathway regulates the cofilin phosphatase slingshot 2 in neutrophil chemotaxis

Chemotaxis requires precisely coordinated polymerization and depolymerization of the actin cytoskeleton at leading fronts of migrating cells. However, GPCR activation-controlled F-actin depolymerization remains largely elusive. Here, we reveal a novel signaling pathway, including Gαi, PLC, PKCβ, protein kinase D (PKD), and SSH2, in control of cofilin phosphorylation and actin cytoskeletal reorganization, which is essential for neutrophil chemotaxis. We show that PKD is essential for neutrophil chemotaxis and that GPCR-mediated PKD activation depends on PLC/PKC signaling. More importantly, we discover that GPCR activation recruits/activates PLCγ2 in a PI3K-dependent manner. We further verify that PKCβ specifically interacts with PKD1 and is required for chemotaxis. Finally, we identify slingshot 2 (SSH2), a phosphatase of cofilin (actin depolymerization factor), as a target of PKD1 that regulates cofilin phosphorylation and remodeling of the actin cytoskeleton during neutrophil chemotaxis.     

Cell cycle-associated changes in Slingshot phosphatase activity and roles in cytokinesis in animal cells

During cytokinesis the actomyosin-based contractile ring is formed at the equator, constricted, and then disassembled prior to cell abscission. Cofilin stimulates actin filament disassembly and is implicated in the regulation of contractile ring dynamics. However, little is known about the mechanism controlling cofilin activity during cytokinesis. Cofilin is inactivated by phosphorylation on Ser-3 by LIM-kinase-1 (LIMK1) and is reactivated by a protein phosphatase Slingshot-1 (SSH1). Here we show that the phosphatase activity of SSH1 decreases in the early stages of mitosis and is elevated in telophase and cytokinesis in HeLa cells, a time course correlating with that of cofilin dephosphorylation. SSH1 co-localizes with F-actin and accumulates onto the cleavage furrow and the midbody. Expression of a phosphatase-inactive SSH1 induces aberrant accumulation of F-actin and phospho-cofilin near the midbody in the final stage of cytokinesis and frequently leads to the regression of the cleavage furrow and the formation of multinucleate cells. Co-expression of cofilin rescued the inhibitory effect of phosphatase-inactive SSH1 on cytokinesis. These results suggest that SSH1 plays a critical role in cytokinesis by dephosphorylating and reactivating cofilin in later stages of mitosis.     

Neuregulin Mediates F-actin-driven Cell Migration through Inhibition of Protein Kinase D1 via Rac1 Protein

Neuregulin (NRG; heregulin) is overexpressed in ∼30% of breast cancers and mediates various processes involved in tumor progression, including tumor cell migration and invasion. Here, we show that NRG mediates its effects on tumor cell migration via PKD1. Downstream of RhoA, PKD1 can prevent directed cell migration through phosphorylation of its substrate SSH1L. NRG exerts its inhibitory effects on PKD1 through Rac1/NADPH oxidase, leading to decreased PKD1 activation loop phosphorylation and decreased activity toward SSH1L. The consequence of PKD1 inhibition by NRG is decreased binding of 14-3-3 to SSH1L, localization of SSH1L to F-actin at the leading edge, and increased cofilin activity, resulting in increased reorganization of the actin cytoskeleton and cell motility. Our data provide a mechanism through which the Rho GTPase Rac1 cross-talks with PKD1 signaling pathways to facilitate directed cell migration.     

Regulation of TGF-beta1/MAPK-mediated PAI-1 gene expression by the actin cytoskeleton in human mesangial cells

The importance of transforming growth factor-beta1 (TGF-beta1) in plasminogen activator inhibitor-1 (PAI-1) gene expression has been established, but the precise intracellular mechanisms are not fully understood. Our hypothesis is that the actin cytoskeleton is involved in TGF-beta1/MAPK-mediated PAI-1 expression in human mesangial cells. Examination of the distributions of actin filaments (F-actin), alpha-actinin, extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) by immunofluorescence and immunoprecipitation revealed that ERK and JNK associate with alpha-actinin along F-actin and that TGF-beta1 stimulation promote the dissociation of ERK and JNK with F-actin. Disassembly of the actin cytoskeleton inhibited phosphorylation of ERK and JNK and modulated PAI-1 expression and promoter activity under both basal and TGF-beta1-stimulated conditions. Stabilizing actin prevented dephosphorylation of ERK and JNK. ERK and JNK inhibitors and overexpressed dominant negative mutants antagonized the ability of TGF-beta1 to increase PAI-1 expression and promoter activity. Disassembly of F-actin also inhibited AP-1 DNA binding activity as determined by electrophoretic mobility shift assay using AP-1 consensus oligonucleotides derived from human PAI-1 promoter. F-actin stabilization prevented loss of AP-1 DNA binding activity. Therefore, changes in actin cytoskeleton modulate the ability of TGF-beta1 to stimulate PAI-1 expression through a mechanism dependent on the activation of MAPK/AP-1 pathways.     

Identification of a Novel Actin-Binding Domain within the Rho Guanine Nucleotide Exchange Factor TEM4

Spatio-temporal activation of Rho GTPases is essential for their function in a variety of biological processes and is achieved in part by regulating the localization of their activators, the Rho guanine nucleotide exchange factors (RhoGEFs). In this study, we provide the first characterization of the full-length protein encoded by RhoGEF TEM4 and delineate its domain structure, catalytic activity, and subcellular localization. First, we determined that TEM4 can stimulate guanine nucleotide exchange on RhoA and the related RhoB and RhoC isoforms. Second, we determined that TEM4, like other Dbl RhoGEFs, contains a functional pleckstrin homology (PH) domain immediately C-terminal to the catalytic Dbl homology (DH) domain. Third, using immunofluorescence analysis, we showed that TEM4 localizes to the actin cytoskeleton through sequences in the N-terminus of TEM4 independently of the DH/PH domains. Using site-directed mutagenesis and deletion analysis, we identified a minimal region between residues 81 and 135 that binds directly to F-actin and has an ～90-fold higher affinity for ATP-loaded F-actin. Finally, we demonstrated that a single point mutation (R130D) within full-length TEM4 abolishes actin binding and localization of TEM4 to the actin cytoskeleton, as well as dampens the in vivo activity of TEM4 towards RhoC. Taken together, our data demonstrate that TEM4 contains a novel actin binding domain and binding to actin is essential for TEM4 subcellular localization and activity. The unique subcellular localization of TEM4 suggests a spatially-restricted activity and expands the diversity of mechanisms by which RhoGEF function can be regulated.     

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.