slingshot: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References
Gene name - slingshot

Synonyms -

Cytological map position - 96B10-11

Function - phosphatase

Keywords - cytoskeleton, bristles, actin dynamics, Twinstar/cofilin

Symbol - ssh

FlyBase ID: FBgn0029157

Genetic map position -

Classification - SSH family

Cellular location - cytoplasmic



NCBI links: Precomputed BLAST | Entrez Gene | UniGene
BIOLOGICAL OVERVIEW

The ADF (actin-depolymerizing factor)/cofilin family, represented by Twinstar in Drosophila, is a stimulus-responsive mediator of actin dynamics. In contrast to the mechanisms of inactivation of ADF/cofilin by kinases such as LIM-kinase 1 (LIMK1: see Drosophila LIM-kinase1), much less is known about its reactivation through dephosphorylation. Slingshot (Ssh), a family of phosphatases that have the property of F actin binding, is described. In Drosophila, loss of ssh function dramatically increases levels of both F actin and phospho-cofilin (P cofilin) and disorganizes epidermal cell morphogenesis. In mammalian cells, human Ssh homologs (hSSHs) suppress LIMK1-induced actin reorganization. Furthermore, Ssh and the hSSHs dephosphorylate P cofilin in cultured cells and in cell-free assays. These results strongly suggest that the SSH family plays a pivotal role in actin dynamics by reactivating ADF/cofilin in vivo (Niwa, 2002).

Regulation of the dynamics of the actin cytoskeleton is fundamental in the construction and remodeling of a variety of polarized subcellular structures. The reorganization of the actin cytoskeleton is controlled at multiple levels. When focused on elongation of actin filaments, profilin (chickadee in Drosophila) promotes the elongation at barbed ends, and capping protein (CP: capping protein beta in Drosophila) stabilizes the barbed ends; a member of the ADF (actin-depolymerizing factor)/cofilin family accelerates depolymerization at pointed ends and severs long actin filaments (Niwa, 2002 and references therein).

Among actin binding proteins, ADF/cofilin is the most-characterized stimulus-responsive mediator of actin dynamics. In response to insulin and lysophosphatidic acid, LIM-kinases (LIMKs), activated by effectors of Rho family GTPases, phosphorylate ADF/cofilin specifically at Ser-3, and thereby inhibit filament-severing and monomer binding activities of ADF/cofilin proteins. Testicular protein kinases (TESKs) also phosphorylate Ser-3 of ADF/cofilin and inhibit its activity, although upstream pathways of TESKs activation appear to be separate from that of LIMKs. ADF/cofilin undergoes rapid dephosphorylation as well, in response to several extracellular stimuli, which could result from downregulation of the kinases, upregulation of a phosphatase(s), or both. In contrast to characterization of the kinases, however, much less is known about the phosphatases that reactivate ADF/cofilin (Niwa, 2002 and references therein).

Actin dynamics in vivo have been studied genetically in several systems, including Drosophila. Bristles and hairs on appendages are actin-based protrusions exposed on body surfaces; therefore, they are easily scored landmarks for isolating mutations that cause malformations. In fact, loci reported to be involved in such malformations include chickadee (chic) encoding Profilin, capping protein beta (cpb) encoding the subunit of CP, and twinstar (tsr) encoding ADF/cofilin. This study reports another locus, which has been named slingshot (ssh) after the bifurcation phenotypes of the bristles and hairs in these mutants. The ssh gene encodes a phosphatase that is conserved among several animal species. Loss of ssh function dramatically increases the level of actin filaments, which is similar to a phenotype of tsr mutant cells (Baum, 2000; Baum, 2001). In cultured mammalian cells, such a strong enhancement of actin polymerization is induced by overproduction of LIMKs or TESKs (Niwa, 2002 and references therein).

One hypothesis would be that Ssh and these kinases share the same substrate, ADF/cofilin, regulate its phosphorylation level, and consequently control its actin-depolymerizing activity. To pursue this possibility, multiple approaches were undertaken. Evidence is presented that loss of ssh function in Drosophila increases the level of phospho-cofilin (P cofilin) and that ssh genetically interacts with the Drosophila LIMK gene in actin-based cell morphogenesis. In cultured cells, expression of either of two human Ssh homologs (hSSHs) with LIMK1 or TESK1 suppresses actin reorganization induced by these kinases; and Ssh or hSSHs expression reduces the level of P cofilin. Finally, Ssh and the hSSHs dephosphorylate P cofilin as judged from the results of cell-free assays. These results suggest that the SSH family plays a critical role in controlling actin dynamics, presumably through dephosphorylating and thus reactivating cofilin in cellular and developmental contexts (Niwa, 2002).

In the course of genetic screening for loci that affect bristle number and/or morphology, a focus was placed on l(3)01207, which has one copy of PZ inserted into CG6238. Lethality of l(3)01207 was due to loss of CG6238 function, as shown by the facts that the lethality was recovered by remobilization of the P element and by CG6238 cDNA expression using a widely expressed GAL4 driver, daughterless (da)-GAL4. Through subsequent analysis, it was found that adult escapers of hypomorphic alleles had bifurcated and twisted bristles. This locus was renamed slingshot (ssh) (Niwa, 2002).

To study exactly how actin reorganization is affected by ssh mutations, ssh mutant clones were stained with dye-conjugated phalliodin. Loss of ssh function causes a dramatic increase in the level of F actin in a cell-autonomous manner. This phenotype is found in mutant clones in larval imaginal discs, pupal wings, and follicle epithelia of the egg chamber. Along the apico-basal cell axis, overaccumulation of actin filaments was not necessarily restricted to the apical subcellular region; basolateral positions were also brightly stained. At 30-36 hr after puparium formation (h APF) at 25°C, each epidermal cell in the wing localizes an assembly of actin bundles to its distal-most vertex, producing a single prehair in the wild-type. Prehairs of the mutant cells were much more intensely stained with dye-conjugated phalliodin than those of normal cells, indicating that individual mutant hairs contained more actin filaments. ssh null cells seem to show no obvious defect in proliferation or cell-cell adhesion when compared with cells in wild-type twin spots (Niwa, 2002).

A dramatic increase in the F actin level has also been reported in twinstar (tsr)/cofilin clones (Baum, 2000; Baum, 2001); in cultured mammalian cells, excessive actin polymerization is caused by overproduction of LIMKs or TESKs (Arber, 1998; Yang, 1998; Toshima, 2001a, 2001b). Therefore, the possibility was examined that those kinases and the SSH family share the same substrate, ADF/cofilin, and thus control the level of phospho-ADF/cofilin in vivo (Niwa, 2002).

Whether loss of ssh function increases the level of phosphorylated Drosophila cofilin (P Dcofilin) in fly tissues was examined. For this purpose, binding and specificity of anti-P cofilin antibody to P Dcofilin was first characterized. Anti-P cofilin antibody was originally made against a phosphopeptide of mammalian cofilin (Toshima, 2001a). When ssh1-11 clones were analyzed by staining or Western blotting with this anti-P cofilin antibody, the endogenous P Dcofilin level in ssh/ssh mutant cells was significantly elevated compared with that in the surrounding ssh/+ and +/+ wild-type cells, whereas the total amount of cofilin was not altered. This result is consistent with the hypothesis that a physiological expression level of Ssh is required for dephosphorylating P Dcofilin in vivo (Niwa, 2002).

Loss of ssh function and expression of Drosophila LIMK (DLIMK) exerts synergistic effects on wing hair morphogenesis and on the levels of F actin and P Dcofilin. Double-stranded RNA (dsRNA) of ssh sequences was expressed in dorsal cells of the wing. This phenocopied a hypomorphic ssh condition through a phenomenon called RNA interference (RNAi). DLIMK production on its own, whether the wild-type form (wt) or a kinase-inactive form (KI), does not give detectable phenotypes under the experimental conditions employed. The weak RNAi phenotype of ssh is strongly enhanced by expression of DLIMK-wt, but DLIMK-KI does not show this enhancement. Similarly, drastic increases in F actin and P Dcofilin levels result from coexpression of the dsRNA and DLIMK-wt. In those cells, unusually thick actin bundles are observed when images are collected with a low gain. Cells on ventral surfaces are not affected, where the transgene was silent (Niwa, 2002).

In addition to experiments using Drosophila, the approach of transfecting mammalian cell lines with hSSH plasmids was taken to test the following hypothesis: if hSSH dephosphorylates P cofilin, coexpression of hSSH together with LIMKs or TESKs is expected to counterbalance the promotion effect of those kinases on actin polymerization. To evaluate the degree of actin polymerization, F actin patterns were classified into three categories. In control cells expressing GFP alone, strong F actin signals were seen infrequently and most of the cells were scored as class 3, whereas LIMK1 or TESK1 expression made class 1 (strongest F actin signals) and class 2 (intermediate signal) predominant. Coexpression of hSSH-1L(wt) or hSSH-2(wt) with either kinase restored the cells to class 3 category; the CS phosphatase mutant forms, however, failed to counteract the kinases. These results support that the hSSH phosphatase activity inhibits LIMK1- or TESK1-dependent actin reorganization (Niwa, 2002).

The above coexpression results could be explained by the hypothesis that P cofilin is a direct target of the hSSHs; however, another interpretation is also possible. The kinase activity of LIMK1 is enhanced by phosphorylation at Thr-508 (Ohashi, 2000); therefore, the hSSHs might dephosphorylate LIMK1 and make it less active. This second hypothesis is less likely; a phosphomimetic form of LIMK1, LIMK1(T508EE), which is no longer phosphorylated at the residue of 508, induces actin polymerization. Nevertheless, hSSH-1L is still able to counteract LIMK1(T508EE) since it is LIMK1(wt) (Niwa, 2002).

Coexpression of hSSH-1L(CS) with LIMK1 evokes robust assembly of actin filaments in a way that is hardly induced by LIMK1 expression alone. When compared to F actin patterns in LIMK1-expressing cells, it looked as if almost all actin filaments in the coexpressing cell are assembled to build a limited number of thick bundles. These F-actin patterns were counted as class 2. These unusual structures were also induced by expressing hSSH-1L(CS) alone, but not at all by expressing the wild-type form. This abnormal assembly of the filaments is reminiscent of the consequence of coexpression of ssh-dsRNA and DLIMK in Drosophila epidermal cells, suggesting the possibility that hSSH-1L(CS) overexpression exerts a dominant-negative effect. hSSH-2(CS) also tends to induce actin reorganization, as shown by a slight expansion of the class 2 population when compared with the effect of its wild-type expression; however, unlike hSSH-1L(CS), hSSH-2(CS) can not generate the thick actin bundles (Niwa, 2002).

A straightforward prediction of the above results would be that expression of hSSH-1L(wt), hSSH-2(wt), or Ssh(wt) reduces the P ADF/cofilin level in cells. This was indeed found to be the case. Expression of hSSH-1L, hSSH-2, or Ssh causes a prominent decrease in the level of P cofilin or P Dcofilin; in contrast, hSSH-3(wt) expression is ineffective. Neither of the two CS forms nor MKP-5, which shows a limited sequence similarity to the catalytic domain of the SSH family, is able to decrease the P cofilin level. Complementary experiments have indicated that hSSH-1L is unable to dephosphorylate p38alpha and JNK2, which are efficiently inactivated by MKP-5. These results suggest that the SSH family displays distinct substrate specificity from the MKP family (Niwa, 2002).

Whether hSSH-1L, hSSH-2, and/or Ssh dephosphorylate P cofilin was directly assayed in cell-free systems. The hSSHs and cofilin were expressed in COS-7 cells; Ssh and Dcofilin were expressed in S2 cells. The immunoprecipitated enzymes and the purified substrates, which contain phosphorylated forms, were subject to in vitro reactions. Each wild-type form of the hSSHs or Ssh dephosphorylated P cofilin, but the CS forms do not, supporting the notion that cofilin is a substrate of hSSH-1L, hSSH-2, and Ssh. In the cell-free assay, 90% of P cofilin was dephosphorylated by Ssh after 15 min reaction (Niwa, 2002).

Under the experimental conditions used, hSSH-2 was reproducibly less active than hSSH-1L. When comparable amounts of hSSH-1L(wt) and hSSH-2(wt) were reacted with the same amount of P cofilin, over 99% of the substrate was dephosphorylated by hSSH-1L(wt), whereas only 60% was by hSSH-2(wt). ADF (also called destrin) is a close relative of cofilin in the vertebrate ADF/cofilin family and is also phosphorylated at Ser-3 by LIMKs and TESKs (Amano, 2001; Toshima, 2001a); hSSH-1L dephosphorylates phospho-ADF as well (Niwa, 2002).

In a transfection experiment, it was found that hSSH-1L(CS) predominantly colocalizes with F actin. This result prompted an investigation to see whether hSSHs and Ssh have the property of binding F actin. Tagged hSSHs and Ssh were incubated with actin filaments and centrifuged to examine whether each of them cosediment with F actin or not. Both wild-type and CS forms of hSSH-1L and Ssh preferentially cosedimented with actin filaments, whereas hSSH-2(wt), hSSH-2(CS), and hSSH-3(wt) exhibit weaker binding capabilities. A fraction of hSSH-3(CS) precipitates in the absence of actin filaments; thus, whether hSSH-3(CS) binds F actin or not was difficult to address with this method. Consistent with the result of the cosedimentation assay, Ssh(wt) and Ssh(CS) appear to colocalize with cortical actin in wing epithelia at 30 h APF (Niwa, 2002).

These studies using the whole animal and cell lines have demonstrated that Ssh and two human homologs (hSSH-1L and -2) prevent excessive actin polymerization and these SSH family members reduce the P cofilin level in cells. These studies have also shown in cell-free assays that Ssh and the hSSHs dephosphorylate cofilin efficiently and bind to actin filaments. All of these results are consistent with the hypothesis that Ssh and the hSSHs control reorganization of actin cytoskeleton by reactivating cofilin. Both the cofilin-phosphatase activity and the F actin binding ability of hSSH-2 are weaker than those of hSSH-1L under the experimental conditions used. Extensive domain-swapping between hSSH-1L and -2 may reveal whether the two activities are related to one another (Niwa, 2002).

In genomes of S. cerevisiae, C. elegans, and Arabidopsis, appear to have no ortholog of genes encoding LIMK, TESK, and Ssh, although cofilin in all of these species has a Ser-3 or equivalent serine residue. This is suggestive of a model in which the actin-depolymerizing activity of cofilin in those species may be regulated in vivo by a set of enzymes structurally distinct from LIMK, TESK, and Ssh. Alternatively, the activity may not be regulated by serine phosphorylation, but by other means such as PIP2 binding or intracellular pH changes. The latter possibility is supported in S. cerevisiae and D. discoideum by the report that P cofilin is not detected in vegetatively growing cells (Niwa, 2002).

The analysis of ssh mutants shows that a critical role of Ssh is in the building of wing hair, bristle, and arista, presumably through reactivating P cofilin. These cellular extensions in insects share a number of structural features with those found throughout the animal kingdom, such as the brush border of intestinal epithelial cell. Formation of all those protrusions on apical cell surfaces requires packing of parallel filaments through actin-bundling proteins. Therefore, thickening and/or splitting phenotypes in ssh mutants are most likely due to overaccumulated filaments that were not arranged in a normal array. Ssh does not appear to be required for cell proliferation or viability, which provides a contrast to an effect of loss of a generic enzyme PP1 or PP2A. All these results are consistent with the hypothesis that cofilin is the major substrate of Ssh and that Ssh does not display broad substrate specificity (Niwa, 2002).

It is worth noting that mutations of some other loci cause phenotypes similar to or reminiscent of those of ssh and twinstar (tsr)/cofilin. In act up (acu)/caplet (capt) clones in imaginal discs and follicle cells, the level of actin filaments is elevated as in ssh clones, and bristle malformation occurs. acu/capt encodes cyclase-associated protein (CAP), and CAP limits filament formation catalyzed by Enabled at apical cell junctions. The splitting or branching of wing hair, bristle, and arista is caused by loss of tricornered (trc) or furry (fry). The Trc protein is an evolutionally conserved kinase; Fry is also conserved among species, but its biochemical function is unknown. It would be intriguing to explore whether trc or fry phenotypes are associated with altered levels of actin filaments and P cofilin (Niwa, 2002).

The current studies did not focus on pursuing whether or not Ssh regulates cofilin functions in cell behaviors other than epithelial cell morphogenesis. For example, one of the important functions of ADF/cofilin is the dynamic regulation of the cortical ring at the cleavage furrow to complete cytokinesis (Nagaoka, 1995; Abe, 1996). During ana/telophase in tsr mutants, aberrantly large actin-based structures appear at the site of contractile ring formation and fail to disassemble at the end of telophase (Gunsalus, 1995). The apparent dispensability of Ssh in mitosis in the wing could be explained by the possibility that only a small fraction of active cofilin molecules are required for dynamics of the contractile ring and/or that a cofilin-phosphatase(s) other than Ssh is responsible for the progression of cytokinesis (Niwa, 2002).

In response to extracellular stimuli, some cells are known to undergo rapid dephosphorylation of ADF/cofilin (Moon, 1995); all of these stimuli result in cytoskeletal reorganization, possibly by way of reactivation of ADF/cofilin. During semaphorin-3A (Sema-3A)-induced growth cone collapse, a rapid increase and subsequent decrease in P cofilin occurs in growth cones within 5 min after exposure to Sema-3A (Aizawa, 2001). This supports a mechanism of local and transient regulation of phosphorylation-dephosphorylation cycling. In some systems, stimulation of cortical actin dynamics occurs without a net change in the ratio of P to non-P ADF/cofilin. Instead, what may be crucial is the turnover rate of the P ADF/cofilin pools or differential distributions of phosphorylation and dephosphorylation spots within cells (Meberg, 1998; Yang, 1998). The studies reported here raise an obvious question to be addressed, that is, whether the Ssh family is involved in the above stimuli-driven actin reorganizations. It is thus necessary to explore whether the activity of the SSH family is regulated in response to the stimuli, and if so, to determine the components of such regulatory mechanisms (Niwa, 2002).


GENE STRUCTURE

cDNA clone length - 4467

Bases in 5' UTR - 459

Exons - 8

Bases in 3' UTR - 870


PROTEIN STRUCTURE

Amino Acids - 1045

Structural Domains

The Ssh protein has a phosphatase domain whose amino acid sequences are distantly related to those of the family of MAP kinase phosphatases (MKPs). The protein made from the cDNA construct comigrates with endogenous Ssh, supporting that the cDNA spanned the entire open reading frame. Three human homologs of ssh were identified in the draft sequences, and analysis of transcripts suggests that at least six different polypeptides would be produced from these three loci. Two of the human proteins (hSSHs), hSSH-1L and hSSH2, are enzymatically active when reacted with an artificial substrate, p-nitrophenyl phosphate (pNPP). For unknown reasons, pNPP-hydrolyzing activity of Ssh could not be detected; nevertheless, Ssh dephosphorylates P cofilin, which is considered to be a bona fide substrate. hSSH3 did not exhibit any activity toward pNPP; neither did its expression reduce the level of P cofilin in COS-7 cells (Niwa, 2002).

Besides the catalytic domain, there are two other domains conserved between Ssh and hSSHs that do not have known motifs and are unique to the SSH family. Both Ssh and hSSH-1L have long C tails of 530 and 609 residues, respectively, although their sequences are not very similar to each other. EST sequences of ascidian, sea urchin, and zebrafish were found that correspond to domains A and/or B. However, neither domain A nor B was found in predicted proteins in Saccharomuces cerevisae, Caenorhabditis elegans, or Arabidopsis under standard conditions for searching databases, suggesting that those species may not have ssh orthologs (Niwa, 2002).


EVOLUTIONARY HOMOLOGS

Cofilin, a key regulator of actin filament dynamics, is inactivated by phosphorylation at Ser-3 by LIM-kinases and is reactivated by dephosphorylation by a family of protein phosphatases, termed Slingshot (SSH). Two novel isoforms of SSHs, termed SSH-2L and SSH-3L have been identified, and they were characterized in comparison with the previously reported SSH-1L. SSH-1L and SSH-2L, but not SSH-3L, bind tightly to and co-localize with actin filaments. When expressed in cultured cells, SSH-1L, SSH-2L and SSH-3L decrease the level of Ser-3-phosphorylated cofilin (P-cofilin) in cells and suppressed LIM-kinase-induced actin reorganization, although SSH-3L was less effective than SSH-1L and SSH-2L. In cell-free assays, SSH-1L and SSH-2L efficiently dephosphorylated P-cofilin, whereas SSH-3L did so only weakly. Using deleted mutants of SSH-1L and SSH-2L, it was found that the N-terminal and C-terminal extracatalytic regions are critical for cofilin-phosphatase and F-actin-binding activities, respectively. In situ hybridization analyses revealed characteristic patterns of expression of each of the mouse Ssh genes in both neuronal and non-neuronal tissues; in particular, expression of Ssh-3 in epithelial tissues is evident. It is concluded that SSH-1L, SSH-2L and SSH-3L have the potential to dephosphorylate P-cofilin, but subcellular distribution, F-actin-binding activity, specific phosphatase activity and expression patterns significantly differ, which suggests that they have related but distinct functions in various cellular and developmental events (Ohta, 2003).

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). 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 rescues 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 (Kaji, 2003).

Growth cone motility and morphology are based on actin-filament dynamics. Cofilin plays an essential role for the rapid turnover of actin filaments by severing and depolymerizing them. The activity of cofilin is repressed by phosphorylation at Ser3 by LIM kinase and is reactivated by dephosphorylation by phosphatases, termed Slingshot (SSH). The roles of cofilin, LIMK, and SSH were investigated in growth cone motility and morphology and in neurite extension by expressing fluorescence protein-labeled cofilin, LIMK1, SSH1, or their mutants in chick dorsal root ganglion (DRG) neurons and then monitoring live images of growth cones by time-lapse video fluorescence microscopy. The expression of LIMK1 remarkably represses growth cone motility and neurite extension, whereas the expression of SSH1 or a nonphosphorylatable S3A mutant of cofilin enhances these events. The fan-like shape of growth cones is disorganized by the expression of any of these proteins. The repressive effects on growth cone behavior by LIMK1 expression are significantly rescued by the coexpression of S3A-cofilin or SSH1. These findings suggest that LIMK1 and SSH1 play critical roles in controlling growth cone motility and morphology and neurite extension by regulating the activity of cofilin and may be involved in signaling pathways that regulate stimulus-induced growth cone guidance. Using various mutants of cofilin, evidence was obtained that the actin-filament-severing activity of cofilin is critical for growth cone motility and neurite extension (Endo, 2003).

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. Cofilin-phosphatase activity of SSH1L is shown to increase 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. Evidence is provided 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 induces 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 also that 14-3-3 proteins negatively regulate SSH1L activity by sequestering it in the cytoplasm (Nagata-ohashi, 2004).

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. This study shows 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 increases in PTEN-overexpressing cells and decreased in PTEN-deficient cells. Insulin induced the accumulation of SSH1L and active Akt, together with a PI3K product phosphatidylinositol 3,4,5-trisphosphate, onto membrane protrusions. Cofilin, but not Ser-3-phosphorylated cofilin, accumulates in membrane protrusions in insulin-stimulated cells, indicating that cofilin is dephosphorylated in these areas. Finally, suppression of SSH1L expression by RNA interference abolishes 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 (Nishita, 2004).

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. Cofilin activity is temporally and spatially regulated by LIMK1 and SSH1L in chemokine-stimulated Jurkat T cells. The knockdown of LIMK1 suppresses chemokine-induced lamellipodium formation and cell migration, whereas SSH1L knockdown produces and retaines multiple lamellipodial protrusions around the cell after cell stimulation and impaired directional cell migration. These 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. It is proposed that LIMK1- and SSH1L-mediated spatiotemporal regulation of cofilin activity is critical for chemokine-induced polarized lamellipodium formation and directional cell movement (Nishita, 2005).

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. 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. The phosphatase activity of purified SSH-1L is F-actin dependent and is negatively regulated via phosphorylation by PAK4. 14-3-3zeta binds to and phosphorylates slingshot, decreases the amount of slingshot that co-sediments with F-actin, but does not alter slingshot activity. A novel ADF/cofilin phosphoregulatory complex is defined and a new mechanism is suggested for the regulation of ADF/cofilin activity in mediating changes to the actin cytoskeleton (Soosairajah, 2005).

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. This study 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, it is suggested that Ca2+-induced cofilin dephosphorylation is mediated by calcineurin-dependent activation of SSH1L (Wang, 2005).

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. Nogo-66, a potent inhibitory fragment of Nogo-A, signals through LIM 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. It is speculated that SSH-mediated persistent cofilin activation may be responsible for maintaining an inhibited neuronal phenotype in response to myelin inhibitors (Hsieh, 2006).

Slingshot and gastrulation in Xenopus

ADF/cofilin is a phosphorylation-regulated protein essential for actin filament dynamics in cells. Two cDNAs were cloned encoding Xenopus ADF/cofilin (XAC)-specific phosphatase, slingshot (XSSH), one of which contains an extra 15 nucleotides in a coding sequence of the other, possibly generated by alternative splicing. Whole mount in situ hybridization showed XSSH transcripts in the blastopore lip and sensorial ectoderm at stage 11, and subsequently localized to developing brain, branchial arches, developing retina, otic vesicle, cement gland, and spinal chord in neurula to tailbud embryos. Immunostaining of animal-vegetal sections of gastrula embryos demonstrated that both XAC and XSSH proteins are predominant in ectodermal and involuting mesodermal cells. Microinjection of either a wild type (thus induces overexpression) or a phosphatase-defective mutant (functions as dominantly negative form) resulted in defects in gastrulation, and often generated the spina bifida phenotype with reduced head structures. Interestingly, the ratio of phosphorylated XAC to dephosphorylated XAC markedly increases from the early gastrula stage (stage 10.5), although the amount of XSSH protein markedly increases from this stage. These results suggest that gastrulation movement requires ADF/cofilin activity through dynamic regulation of its phosphorylation state (Tanaka, 2005a).

ADF/cofilin is a key regulator for actin dynamics during cytokinesis. Its activity is suppressed by phosphorylation and reactivated by dephosphorylation. Little is known, however, about regulatory mechanisms of ADF/cofilin function during formation of contractile ring actin filaments. Using Xenopus cycling extracts, it was found that ADF/cofilin is dephosphorylated at prophase and telophase. In addition, constitutively active Rho GTPase induces dephosphorylation of ADF/cofilin in the egg extracts. This dephosphorylation is inhibited by Na3VO4 but not by other conventional phosphatase-inhibitors. A Xenopus homologue of Slingshot phosphatase (XSSH) was cloned, and antibody was raised specific for the catalytic domain of XSSH. This inhibitory antibody significantly suppresses the Rho-induced dephosphorylation of ADF/cofilin in extracts, suggesting that the dephosphorylation at telophase is dependent on XSSH. XSSH binds to actin filaments with a dissociation constant of 0.4 microM, and the ADF/cofilin phosphatase activity is increased in the presence of F-actin. When latrunculin A, a G-actin-sequestering drug, was added to extracts, both Rho-induced actin polymerization and dephosphorylation of ADF/cofilin were markedly inhibited. Jasplakinolide, an actin-stabilizing drug, alone induced actin polymerization in the extracts and led to dephosphorylation of ADF/cofilin. These results suggest that Rho-induced dephosphorylation of ADF/cofilin is dependent on the XSSH activation that is caused by increase in the amount of F-actin induced by Rho signaling. XSSH colocalized with both actin filaments and ADF/cofilin in the actin patches formed on the surface of the early cleavage furrow. Injection of inhibitory antibody blocked cleavage of blastomeres. Thus, XSSH may reorganize actin filaments through dephosphorylation and reactivation of ADF/cofilin at early stage of contractile ring formation (Tanaka, 2005b).

Cofilin activity downstream of Pak1 regulates cell protrusion efficiency by organizing lamellipodium and lamella actin networks

Protrusion of the leading edge of migrating epithelial cells requires precise regulation of two actin filament (F-actin) networks, the lamellipodium and the lamella. Cofilin is a downstream target of Rho GTPase signaling that promotes F-actin cycling through its F-actin-nucleating, -severing, and -depolymerizing activity. However, its function in modulating lamellipodium and lamella dynamics, and the implications of these dynamics for protrusion efficiency, has been unclear. Using quantitative fluorescent speckle microscopy, immunofluorescence, and electron microscopy, this study established that the Rac1/Pak1/LIMK1 signaling pathway controls cofilin activity within the lamellipodium. Enhancement of cofilin activity accelerates F-actin turnover and retrograde flow, resulting in widening of the lamellipodium. This is accompanied by increased spatial overlap of the lamellipodium and lamella networks and reduced cell-edge protrusion efficiency. It is proposed that cofilin functions as a regulator of cell protrusion by modulating the spatial interaction of the lamellipodium and lamella in response to upstream signals (Delorme, 2007).

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. This study reports 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, this study shows 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 (Wen, 2007).

Phosphorylation of Ser 402 impedes phosphatase activity of slingshot 1

By using mass spectrometry, Ser 402 has been identified 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. It was further demonstrated 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. This study has thus identified a universal mechanism by which PKD controls SSH1 phosphatase activity (Barisic, 2011).

PKD inhibits directed cell migration through phosphorylation of SSH1 at Ser 937 and 978 (Eiseler, 2009). The co-expression of constitutive active (ca) PKD1 with SSH1 wild type significantly decreased cell migration in comparison to SSH1 wild type alone. However, this was partly rescued by the SSH1 S402A mutant, indicating that PKD-controlled phosphorylation of this site substantially contributes to the regulation of SSH1-dependent directed cell migration. The sequences surrounding Ser 402 are highly conserved across species, suggesting that the mechanism of negative regulation through phosphorylation at Ser 402 could be universal. Drosophila was used as a model organism for further in vivo studies. Here, the C-terminus of SSH lacks the two PKD phospho-sites, whereas the previously identified phosphorylation site at Ser 477 (Ser 402 in mammals) is conserved. Similarly to the human PKD1ca protein, Drosophila PKDca-GFP displayed constitutive activity. In in vitro kinase assays, Drosophila PKDca was able to directly phosphorylate a truncated Drosophila SSH-GST-fusion protein comprising the amino acids 461-671. These results show that Drosophila SSH is a PKD substrate in vitro. Loss of Drosophila SSH results in an accumulation of both P-cofilin and F-actin. If PKD is a negative regulator of SSH in Drosophila, an accumulation of P-cofilin and of F-actin would be expected, when active PKD is expressed in Drosophila tissues. Thus cell clones overexpressing PKDca-GFP were induced that were surrounded by wild-type tissue in developing wing and eye imaginal anlagen. There was a significant accumulation of F-actin and P-cofilin that was detectable in cells overexpressing the active PKD protein, compared with the neighbouring wild-type sister cells. Both F-actin and P-cofilin were also enriched in cells that lack the SSH protein. These data indicate that PKD negatively regulates SSH activity in Drosophila tissue, probably by phosphorylation of Ser 477 (Barisic, 2011).


slingshot: Developmental Biology | Effects of Mutation | References

date revised: 20 April 2012

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