Phosphatase Slingshot Research Articles

Nox1‐mediated activation of Slingshot phosphatase in VSMC

PDGF stimulates vascular smooth muscle cell (VSMC) migration by a NADPH oxidase 1 (Nox1)‐derived, H2O2 dependent mechanism. Migration is partially controlled by cofilin, an actin binding protein activated by Slingshot‐1L (SSH1L) phosphatase. We previously showed that SSH1L is activated and required for PDGF‐mediated cofilin activation and migration in VSMCs, but the mechanism of SSH1L activation is unclear. 14‐3‐3 proteins are one potential regulatory mechanism, and bind to partners that contain phosphorylated consensus motifs. We hypothesized that PDGF disrupts a SSH1L/14‐3‐3 inhibitory complex through Nox1/H2O2‐mediated SSH1L dephosphorylation in a 14‐3‐3 binding motif. We found that SSH1L is Ser‐phosphorylated at a 14‐3‐3 binding motif (probably S834) and co‐immunoprecipitates with 14‐3‐3‐β and ‐γ. In cells transfected with 14‐3‐3K49E DN, which fails to recognize phosphorylated partners, SSH1L is activated (94±6 vs. 45±1 A.U.p=0.01), and PDGF has no further effect (44.6±1.4 vs. 40.2±10.8 A.U.). Interestingly, PDGF reduces phospho‐SSH1L and 14‐3‐3 binding in wild type (29±11 vs. 100±8 A.U.p=0.01) but not in Nox1 knockout cells (101±18 vs. 86±14 A.U.), and the SSH1L‐S834A mutant is constitutively active. Our work shows that PDGF induces SSH1L/14‐3‐3 complex disruption and SSH1L activation via its Nox1‐dependent dephosphorylation in a 14‐3‐3 binding motif.

Regulation of AMPA receptor channels and synaptic plasticity by cofilin phosphatase Slingshot in cortical neurons

Regulation of AMPA receptor channels and synaptic plasticity by cofilin phosphatase Slingshot in cortical neurons

Protein Kinase D Regulates Cell Migration by Direct Phosphorylation of the Cofilin Phosphatase Slingshot 1 Like

Protein kinase D (PKD) has been identified as a negative regulator of epithelial cell migration; however, its molecular substrates and downstream signaling pathways that mediate this activity have remained elusive. In this study, we provide evidence that the cofilin phosphatase slingshot 1 like (SSH1L), an important regulator of the complex actin remodeling machinery, is a novel in vivo PKD substrate. PKD-mediated phosphorylation of serines 937 and 978 regulates SSH1L subcellular localization by binding of 14-3-3 proteins and thus impacts the control of local cofilin activation and actin remodeling during cell migration. In line with this, we show that the loss of PKD decreases cofilin phosphorylation, induces a more spread cell morphology, and stimulates chemotactic migration of breast cancer cells in an SSHL1-dependent fashion. Our data thus identify PKD as a central regulator of the cofilin signaling network via direct phosphorylation and regulation of SSH1L.

14-3-3 ζ/τ heterodimers regulate Slingshot activity in migrating keratinocytes

Defining the pathways required for keratinocyte cell migration is important for understanding mechanisms of wound healing and tumor cell metastasis. We have recently identified an α6β4 integrin-Rac1 signaling pathway via which the phosphatase Slingshot (SSH) activates/dephosphorylates cofilin, thereby determining keratinocyte migration behavior. Here, we assayed the role of 14-3-3 isoforms in regulating the activity of SSH1. Using amino or carboxy terminal domains of 14-3-3 ζ, we demonstrate that in keratinocytes 14-3-3 ζ/ τ heterodimers bind SSH1, in the absence of Rac1 signaling. This interaction leads to an inhibition of SSH1 activity, as measured by an increase in phosphorylated cofilin levels. Overexpression of the carboxy terminal domain of 14-3-3 ζ acts as a dominant negative and inhibits the interaction between 14-3-3 τ and SSH1. These results implicate 14-3-3 ζ/ τ heterodimers as key regulators of SSH1 activity in keratinocytes and suggest they play a role in cytoskeleton remodeling during cell migration.

LIM Kinase regulation of cofilin activity mediates PDGF‐induced VSMC migration

Platelet derived growth factor (PDGF) is a pro‐migratory factor released in response to vascular injury. Vascular smooth muscle cells (VSMCs) mediate this response and contribute significantly to neointimal formation, thus delineating the molecular mechanisms of VSMC migration is critical to understanding the vascular repair process. Signaling cascades activated by PDGF ultimately converge upon the actin cytoskeleton which is a key regulator of migration. Recent studies report that cofilin, which upon activation depolymerizes actin and it's phosphatase Slingshot (SSH) are essential to PDGF‐induced VSMC migration. In addition to SSH, cofilin activity is also regulated by LIM‐kinase, which attenuates its activity. Thus, in these studies we examined the role of LIMK in PDGF‐induced VSMC migration. Immunoblotting with phosphospecific antibodies confirmed that LIMK activity was increased in a time dependent manner in response to PDGF. Transfection of VSMCs cells with LIMK siRNA attenuated PDGF induced migration by 70% vs. control VSMCs. The knockdown of LIMK expression by siRNA also decreased basal phosphorylation of cofilin and potentiated the cofilin response to PDGF‐induced activation. As PDGF‐induced LIMK regulation appears to mediate VSMC migration, these studies show the importance of further examining the SSH/LIMK balance in mediating cytoskeletal events associated with cofilin activity.

INTERACTIONS BETWEEN HUMAN SLINGSHOT PHOSPHATASE 2 AND PHOSPHO-COFILIN: A MOLECULAR DYNAMICS STUDY

Human slingshot phosphatase 2 (SSH2) is one of the dual specificity protein tyrosine phosphatases, which can activate cofilin substrate by binding its phosphorylation state. Because the interaction model of SSH2 and phospho-cofilin (P-cofilin) was unknown, we obtained the complex through macromolecular docking method. The molecular dynamics studies were used to investigate the complex dynamics in an aqueous solution. To understand the binding specificity, the free energy was calculated with the molecular mechanics Poisson–Boltzmann surface area (MM/PBSA) approach and the interaction mode in active site was analyzed. The results indicated that the interaction of the P-loop of SSH2 with phosphoserine of human P-cofilin was stabilized by molecular mechanics energy and nonpolar solvation energy components, while polar solvation energy and the entropic contributions were unfavorable for the combination of the two proteins. In addition, the electrostatic contributions were negative for the formation of the complex on the whole, but seen from the active local, the Coulomb interaction between the phosphoserine and the P-loop residues could play an important role in determining substrate specificity.

Dopamine D4Receptors Regulate AMPA Receptor Trafficking and Glutamatergic Transmission in GABAergic Interneurons of Prefrontal Cortex

GABAergic interneurons in prefrontal cortex (PFC) play a critical role in cortical circuits by providing feedforward and feedback inhibition and synchronizing neuronal activity. Impairments in GABAergic inhibition to PFC pyramidal neurons have been implicated in the abnormal neural synchrony and working memory disturbances in schizophrenia. The dopamine D(4) receptor, which is strongly linked to neuropsychiatric disorders, such as attention deficit-hyperactivity disorder (ADHD) and schizophrenia, is highly expressed in PFC GABAergic interneurons, while the physiological role of D(4) in these interneurons is largely unknown. In this study, we found that D(4) activation caused a persistent suppression of AMPAR-mediated synaptic transmission in PFC interneurons. This effect of D(4) receptors on AMPAR-EPSC was via a mechanism dependent on actin/myosin V motor-based transport of AMPA receptors, which was regulated by cofilin, a major actin depolymerizing factor. Moreover, we demonstrated that the major cofilin-specific phosphatase Slingshot, which was activated by calcineurin downstream of D(4) signaling, was required for the D(4) regulation of glutamatergic transmission. Thus, D(4) receptors, by using the unique calcineurin/Slingshot/cofilin signaling mechanism, regulate actin dynamics and AMPAR trafficking in PFC GABAergic interneurons. It provides a potential mechanism for D(4) receptors to control the excitatory synaptic strength in local-circuit neurons and GABAergic inhibition in the PFC network, which may underlie the role of D(4) receptors in normal cognitive processes and mental disorders.

Chronophin Mediates an ATP-Sensing Mechanism for Cofilin Dephosphorylation and Neuronal Cofilin-Actin Rod Formation

Actin and its key regulatory component, cofilin, are found together in large rod-shaped assemblies in neurons subjected to energy stress. Such inclusions are also enriched in Alzheimer's disease brain, and appear in transgenic models of neurodegeneration. Neuronal insults, such as energy loss and/or oxidative stress, result in rapid dephosphorylation of the cellular cofilin pool prior to its assembly into rod-shaped inclusions. Although these events implicate a role for phosphatases in cofilin rod formation, a mechanism linking energy stress, phosphocofilin turnover, and subsequent rod assembly has been elusive. We demonstrate the ATP-sensitive interaction of the cofilin phosphatase chronophin (CIN) with the chaperone hsp90 to form a biosensor that mediates cofilin/actin rod formation. Our results suggest a model whereby attenuated interactions between CIN and hsp90 during ATP depletion enhance CIN-dependent cofilin dephosphorylation and consequent rod assembly, thereby providing a mechanism for the formation of pathological actin/cofilin aggregates during neurodegenerative energy flux.

Cell Adhesion-dependent Cofilin Serine 3 Phosphorylation by the Integrin-linked Kinase·c-Src Complex

Integrin-linked kinase (ILK) is involved in signal transduction by integrin-mediated cell adhesion that leads to dynamic actin reorganization. Actin (de)polymerization is regulated by cofilin, the Ser(3) phosphorylation (pS(3)cofilin) of which inhibits its actin-severing activity. To determine how ILK regulates pS(3)cofilin, we examined the effects of ILK on pS(3)cofilin using normal RIE1 cells. Compared with suspended cells, fibronectin-adherent cells showed enhanced pS(3)cofilin, depending on ILK expression and c-Src activity. The ILK-mediated pS(3)cofilin in RIE1 cells did not involve Rho-associated kinase, LIM kinase, or testicular protein kinases, which are known to be upstream of cofilin. The kinase domain of ILK, including proline-rich regions, appeared to interact physically with the Src homology 3 domain of c-Src. In vitro kinase assay revealed that ILK immunoprecipitates phosphorylated the recombinant glutathione S-transferase-cofilin, which was abolished by c-Src inhibition. Interestingly, epidermal growth factor treatment abolished the ILK effects, indicating that the linkage from ILK to cofilin is biologically responsive to extracellular cues. Altogether, this study provides evidence for a new signaling connection from ILK to cofilin for dynamic actin polymerization during cell adhesion, depending on the activity of ILK-associated c-Src.

Dual Regulation of Cofilin Activity by LIM Kinase and Slingshot-1L Phosphatase Controls Platelet-Derived Growth Factor–Induced Migration of Human Aortic Smooth Muscle Cells

Platelet-derived growth factor (PDGF) plays a central role in vascular healing, atherosclerosis, and restenosis, partly by stimulating vascular smooth muscle cell (VSMC) migration. Migration requires rapid turnover of actin filaments, which is partially controlled by cofilin. Although cofilin is negatively regulated by Ser3 phosphorylation, the upstream signaling pathways have not been defined, nor has its role in VSMC migration been studied. We hypothesized that PDGF-induced migration of VSMCs involves cofilin activation and that this is regulated by the serine kinase LIM kinase (LIMK) and the novel phosphatase Slingshot (SSH)1L. In human VSMCs, stimulation with PDGF increased G-actin incorporation into the actin cytoskeleton. PDGF transiently activated the cofilin kinase, LIMK, with a peak at 5 minutes. However, cofilin was dephosphorylated between 5 and 45 minutes, with a maximum of 43+/-5% dephosphorylation at 30 minutes, suggesting that PDGF also activates a cofilin phosphatase. We found that VSMCs express SSH1L, which is induced and activated (564+/-73 versus 1021+/-141 picomoles of PO(4); P=0.015) by PDGF. Of importance, small interfering RNA directed against SSH1L blocked cofilin dephosphorylation and decreased migration (528+/-33 versus 318+/-25 cells/field; P<0.01). Taken together, our results suggest that PDGF participates in actin dynamics by dual regulation of cofilin activity via LIMK and SSH1L.

Phosphorylated Cofilin Not Inactive After All

Cofilin is an actin-depolymerizing factor, and this activity is inhibited when the protein is phosphorylated by LIM-kinase or TES-kinase. In an attempt to identify the mechanism by which the muscarinic acetylcholine receptor activated phospholipase D (PLD) downstream of the Rho effector Rho-kinase, Han et al . found that in the fibroblast cell line HEK293 and the neuroblastoma cell line N1E-115, PLD1 was not directly phosphorylated by Rho-kinase, but instead carbachol-stimulated PLD1 activation was inhibited by expression of a kinase-deficient LIM-kinase 1, which is a target for Rho-kinase. However, PLD1 was not a substrate for LIM-kinase 1 either, suggesting another step in the path from the receptor to PLD1. As cofilin is a known substrate for LIM-kinase 1, the authors overexpressed cofilin or a nonphosphorylatable mutant S3A cofilin and showed that carbachol-mediated stimulation of PLD1 was enhanced in the presence of excess wild-type cofilin and inhibited by S3A cofilin. RNAi-mediated depletion of cofilin also reduced carbachol-mediated activation of PLD1, consistent with a role for endogenous cofilin. Expression of a phosphorylation-mimic mutant of cofilin S3D also enhanced carbachol-stimulated PLD1 activation, whereas overexpression of the cofilin phosphatase slingshot inhibited PLD activation. Pull-down assays, as well as colocalization assays and coimmunoprecipitation assays with transfected cells, showed a direct interaction between PLD1 and phosphorylated, but not unphosphorylated, cofilin. If the interaction between cofilin and PLD1 was disrupted by expressing a fragment of PLD1 (F-3 fragment) responsible for mediating the interaction, then carbachol-mediated PLD1 activation was inhibited. Carbachol stimulation of stress fiber formation was also inhibited by the F-3 fragment. Carbachol stimulated the transient phosphorylation of cofilin and increased the interaction between the cofilin and PLD1 in coimmunoprecipitation assays. Phosphorylated cofilin stimulated PLD1 activity in vitro. Thus, phosphorylated cofilin is not an inactive entity but rather interacts with partners other than actin, such as PLD1. L. Han, M. B. Stope, M. López de Jesús, P. A. Oude Weernink, M. Urban, T. Wieland, D. Rosskopf, K. Mizuno, K. H. Jakobs, M. Schmidt, Direct stimulation of receptor-controlled phospholipase D1 by phospho-cofilin. EMBO J. 26 , 4189-4202 (2007). [PubMed]

Direct stimulation of receptor-controlled phospholipase D1 by phospho-cofilin

The activity state of cofilin, which controls actin dynamics, is driven by a phosphorylation-dephosphorylation cycle. Phosphorylation of cofilin by LIM-kinases results in its inactivation, a process supported by 14-3-3zeta and reversed by dephosphorylation by slingshot phosphatases. Here we report on a novel cellular function for the phosphorylation-dephosphorylation cycle of cofilin. We demonstrate that muscarinic receptor-mediated stimulation of phospholipase D1 (PLD1) is controlled by LIM-kinase, slingshot phosphatase as well as 14-3-3zeta, and requires phosphorylatable cofilin. Cofilin directly and specifically interacts with PLD1 and upon phosphorylation by LIM-kinase1, stimulates PLD1 activity, an effect mimicked by phosphorylation-mimic cofilin mutants. The interaction of cofilin with PLD1 is under receptor control and encompasses a PLD1-specific fragment (aa 585-712). Expression of this fragment suppresses receptor-induced cofilin-PLD1 interaction as well as PLD stimulation and actin stress fiber formation. These data indicate that till now designated inactive phospho-cofilin exhibits an active cellular function, and suggest that phospho-cofilin by its stimulatory effect on PLD1 may control a large variety of cellular functions.

Control of Actin Assembly Dynamics in Cell Motility

Control of Actin Assembly Dynamics in Cell Motility

Converting Attraction to Repulsion

During nervous system development, axonal growth cones respond to attractive and repulsive cues that help them navigate to their targets. Wen et al . discovered that, whereas embryonic Xenopus spinal axons in an in vitro culture system were attracted to a gradient of bone morphogenetic protein 7 (BMP7) presented 4 to 8 hours after being placed in culture, BMP7 repelled the same axons after they had been cultured overnight. The type II BMP receptor (BMPRII) mediated both attractive and repulsive responses, and overexpression of a shortened form of BMPRII [which lacks a region that mediates interactions with LIM kinase I (LIMKI) but is dispensable for activation of Smad signaling] abolished BMP7-induced turning. A 32-amino-acid peptide that competitively inhibits LIMKI abolished attractive turning to BMP7, as did a dominant-negative LIMKI mutant, whereas neither affected repulsive turning. In neurons cultured overnight that overexpressed a dominant-negative mutant of the phosphatase Slingshot (SSH), however, the response to BMP7 was converted from repulsion to attraction. A combination of live imaging of the actin cytoskeleton, immunofluorescence analysis of the phosphorylation of XAC [ Xenopus actin-depolymerizing factor (ADF)/cofilin], and expression of wild-type and mutant forms of XAC revealed that LIMKI and SSH regulated growth cone turning through phosphorylation of XAC. BMP7 elicited an increase in growth cone calcium concentration in neurons cultured overnight but not in those cultured for 6 hours. Manipulations designed to block calcium signaling failed to affect attractive turning in 4- to 8-hour cultures, whereas in overnight cultures these treatments abolished the BMP-dependent decrease in XAC phosphorylation and converted repulsive to attractive turning, as did pharmacological inhibition of calcineurin. The transient receptor potential channel TRPC1 was more abundant in overnight cultures than in 4- to 8-hour cultures, and expression of a dominant-negative TRPC1 mutant abolished BMP7-dependent repulsion in overnight cultures, whereas overexpression of wild-type TRPC1 elicited BMP7-dependent repulsion in 4- to 8-hour cultures. Thus, opposing actions of LIMKI and SSH on XAC phosphorylation appear to regulate the actin cytoskeleton and thereby growth cone turning, with calcium influx through TRPC1 switching attraction to repulsion by shifting the balance toward SSH. Z. Wen, L. Han, J. R. Bamburg, S. Shim, G.-l. Ming, J. Q. Zheng, BMP gradients steer nerve growth cones by a balancing act of LIM kinase and Slingshot phosphatase on ADF/cofilin. J. Cell Biol. 178 , 107-119 (2007). [Abstract] [Full Text]

BMP gradients steer nerve growth cones by a balancing act of LIM kinase and Slingshot phosphatase on ADF/cofilin

Bone morphogenic proteins (BMPs) are involved in axon pathfinding, but how they guide growth cones remains elusive. In this study, we report that a BMP7 gradient elicits bidirectional turning responses from nerve growth cones by acting through LIM kinase (LIMK) and Slingshot (SSH) phosphatase to regulate actin-depolymerizing factor (ADF)/cofilin-mediated actin dynamics. Xenopus laevis growth cones from 4–8-h cultured neurons are attracted to BMP7 gradients but become repelled by BMP7 after overnight culture. The attraction and repulsion are mediated by LIMK and SSH, respectively, which oppositely regulate the phosphorylation-dependent asymmetric activity of ADF/cofilin to control the actin dynamics and growth cone steering. The attraction to repulsion switching requires the expression of a transient receptor potential (TRP) channel TRPC1 and involves Ca2+ signaling through calcineurin phosphatase for SSH activation and growth cone repulsion. Together, we show that spatial regulation of ADF/cofilin activity controls the directional responses of the growth cone to BMP7, and Ca2+ influx through TRPC tilts the LIMK-SSH balance toward SSH-mediated repulsion.

Coronin 1B Coordinates Arp2/3 Complex and Cofilin Activities at the Leading Edge

Actin filament formation and turnover within the treadmilling actin filament array at the leading edge of migrating cells are interdependent and coupled, but the mechanisms coordinating these two activities are not understood. We report that Coronin 1B interacts simultaneously with Arp2/3 complex and Slingshot (SSH1L) phosphatase, two regulators of actin filament formation and turnover, respectively. Coronin 1B inhibits filament nucleation by Arp2/3 complex and this inhibition is attenuated by phosphorylation of Coronin 1B at Serine 2, a site targeted by SSH1L. Coronin 1B also directs SSH1L to lamellipodia where SSH1L likely regulates Cofilin activity via dephosphorylation. Accordingly, depleting Coronin 1B increases phospho-Cofilin levels, and alters lamellipodial dynamics and actin filament architecture at the leading edge. We conclude that Coronin 1B's coordination of filament formation by Arp2/3 complex and filament turnover by Cofilin is required for effective lamellipodial protrusion and cell migration.

The Cdi/TESK1 kinase is required for Sevenless signaling and epithelial organization in theDrosophilaeye

How cellular behaviors such as cell-to-cell communication, epithelial organization and cell shape reorganization are coordinated during development is poorly understood. The developing Drosophila eye offers an ideal model system to study these processes. Localized actin polymerization is required to constrict the apical surface of epithelial cells of the eye imaginal disc to maintain the refined arrangement of the developing ommatidia. The identity of each photoreceptor cell within the epithelium is determined by cell-to-cell contacts involving signal transduction events. The R7 photoreceptor cell requires the activity of the Sevenless RTK to adopt a proper cell fate. We performed an EP screen for negative regulators of this inductive process, and we identified the serine/threonine kinase Center divider (cdi) as a suppressor of the phenotype caused by an activated Sevenless receptor. Cdi is homologous to the human testis-specific kinase 1 (TESK1), a member of the LIM kinases involved in cytoskeleton control through ADF/cofilin phosphorylation. We have analyzed the effects of gain- and loss-of-function of cdi and found alterations in actin organization and in the adherens junctions proteins DE-cadherin and beta-catenin, as well as in Sevenless apical localization. Interference with the function of the ADF/cofilin phosphatase Slingshot (ssh), which antagonizes Cdi, also results in a suppression of signaling triggered by the Sevenless RTK. Our results reveal a critical interplay between the localization of molecules involved in epithelial organization and signal transduction.

The phosphatase slingshot modulates the dynamics of endothelial cell protrusions

The phosphatase slingshot modulates the dynamics of endothelial cell protrusions

Myelin-Associated Inhibitors Regulate Cofilin Phosphorylation and Neuronal Inhibition through LIM Kinase and Slingshot Phosphatase

Myelin-associated inhibitors (MAIs) signal through a tripartate receptor complex on neurons to limit axon regeneration in the CNS. Inhibitory influences ultimately converge on the cytoskeleton to mediate growth cone collapse and neurite outgrowth inhibition. Rho GTPase and its downstream effector Rho kinase are key signaling intermediates in response to MAIs; however, the links between Rho and the actin cytoskeleton have not been fully defined. We found that Nogo-66, a potent inhibitory fragment of Nogo-A, signals through LIM (LIM is an acronym of the three gene products Lin-11, Isl-1, and Mec-3) kinase and Slingshot (SSH) phosphatase to regulate the phosphorylation profile of the actin depolymerization factor cofilin. Blockade of LIMK1 activation and subsequent cofilin phosphorylation circumvents myelin-dependent inhibition in chick dorsal root ganglion neurons, suggesting that phosphorylation and inactivation of cofilin is critical for neuronal inhibitory responses. Subsequent activation of SSH1 phosphatase mediates cofilin dephosphorylation and reactivation. Overexpression of SSH1 does not mimic the neurite outgrowth inhibitory effects of myelin, suggesting an alternative role in MAI inhibition. We speculate that SSH-mediated persistent cofilin activation may be responsible for maintaining an inhibited neuronal phenotype in response to myelin inhibitors.

Spatial and temporal regulation of cofilin activity by LIM kinase and Slingshot is critical for directional cell migration

Cofilin mediates lamellipodium extension and polarized cell migration by accelerating actin filament dynamics at the leading edge of migrating cells. Cofilin is inactivated by LIM kinase (LIMK)–1-mediated phosphorylation and is reactivated by cofilin phosphatase Slingshot (SSH)-1L. In this study, we show that cofilin activity is temporally and spatially regulated by LIMK1 and SSH1L in chemokine-stimulated Jurkat T cells. The knockdown of LIMK1 suppressed chemokine-induced lamellipodium formation and cell migration, whereas SSH1L knockdown produced and retained multiple lamellipodial protrusions around the cell after cell stimulation and impaired directional cell migration. Our results indicate that LIMK1 is required for cell migration by stimulating lamellipodium formation in the initial stages of cell response and that SSH1L is crucially involved in directional cell migration by restricting the membrane protrusion to one direction and locally stimulating cofilin activity in the lamellipodium in the front of the migrating cell. We propose that LIMK1- and SSH1L-mediated spatiotemporal regulation of cofilin activity is critical for chemokine-induced polarized lamellipodium formation and directional cell movement.