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.

Actin cytoskeletal reorganization is essential for numerous cell activities, including migration, morphological change, and vesicle transport. The actin-depolymerizing factor/cofilin family proteins are key regulators for actin filament dynamics and reorganization, with potential to bind to actin monomers and filaments and stimulate depolymerization and severance of actin filaments (1)(2)(3)(4). These activities of actin-depolymerizing factor/cofilin (henceforth referred to as cofilin) are negatively regulated by phosphorylation at Ser-3 and reactivated by dephosphorylation (5,6). Cofilin undergoes phosphorylation and dephosphorylation in response to various extracellular stimuli that trigger changes in the actin cytoskeleton (1)(2)(3)(4). Given the essential role of cofilin in actin filament dynamics, it is important to elucidate signal transduction mechanisms that regulate cofilin phosphorylation and dephosphorylation in order to bet-ter understand stimulus-induced actin cytoskeletal remodeling and the cell activities related to it.
As for phosphatases responsible for cofilin dephosphorylation, genetic and biochemical analyses have led to identification of a Slingshot (SSH) 1 family of protein phosphatases (SSH in Drosophila and SSH1L, SSH2L, and SSH3L in mammals), which can specifically dephosphorylate and reactivate an inactive Ser-3-phosphorylated cofilin (P-cofilin), both in vitro and in vivo (16 -19). Cofilin dephosphorylation is induced in response to various extracellular stimuli in many different cell types (1-4, 20 -30). Although multiple signaling pathways, including those involving Ca 2ϩ , cAMP, and phosphoinositide 3-kinase, have been proposed for stimulus-induced cofilin dephosphorylation (20 -28), neither downstream signaling pathways nor the roles of SSHs in stimulus-induced cofilin dephosphorylation have been determined. We recently showed that SSH1L is activated downstream of phosphoinositide 3-kinase in insulinstimulated cells, but the signaling pathway linking phosphoinositide 3-kinase and SSH1L has remained unknown (29). We also showed that the cofilin-phosphatase activity of SSH1L is increased by association with actin filaments and suppressed by 14-3-3 proteins (30). However, the mechanisms regulating SSH1L activity through F-actin and 14-3-3 proteins are not well understood.
In the present study, we investigated the signaling mechanism of Ca 2ϩ signal-induced cofilin dephosphorylation. Previous studies reported that elevation of the intracellular Ca 2ϩ concentration stimulates cofilin dephosphorylation in a variety of cell types (20 -24), and this dephosphorylation is blocked by inhibitors of calcineurin, a Ca 2ϩ /calmodulin-dependent protein phosphatase (also called protein phosphatase 2B), thus indicating that calcineurin is involved in Ca 2ϩ -induced cofilin dephosphorylation (23,31). However, it remained to be determined whether or not calcineurin directly dephosphorylates cofilin or merely mediates cofilin dephosphorylation in cells through ac-tivation of a cofilin-specific phosphatase, such as SSH1L. Here we now provide evidence that Ca 2ϩ -induced cofilin dephosphorylation is mediated by SSH1L via calcineurin.
Cell Culture and Transfection-293T and HeLa cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. 293T and HeLa cells were transfected with expression plasmids using FuGENE 6 transfection reagent (Roche Applied Science) and Lipofectamine (Invitrogen), respectively. Cells were used for assays after being in culture for 30 -44 h.
In Vitro Phosphatase Assay of SSH1L-Cofilin-His 6 expressed in Vero cells was purified and used as a substrate for in vitro phosphatase reactions. 293T or HeLa cells were lysed in phosphatase buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 5% glycerol, 1 mM dithiothreitol, and 10 g/ml leupeptin). SSH1L was immunoprecipitated with anti-SSH1L antibody and incubated with purified cofilin-His 6 for 2 h at 30°C. Reaction mixtures were analyzed by immunoblotting using antibodies to cofilin, P-cofilin, and SSH1L. To examine the effect of calcineurin on SSH1L activity in cell-free assays, lysates of 293T cells were immunoprecipitated with anti-SSH1L antibody or control IgG. The precipitates were incubated for 1 h at 30°C with 7.5 units/l recombinant active calcineurin A␣ containing calcineurin B subunit, 14 g/ml calmodulin, and 6.3 g/ml cofilin-His 6 in 20 l of reaction buffer (50 mM HEPES, pH 7.4, 0.1 mM CaCl 2 , 0.5 mM EDTA, 3 mM MgCl 2 , 100 mM NaCl, 1 mM dithiothreitol, 0.5 mg/ml bovine serum albumin, and 1 g/ml leupeptin) and then further incubated for 30 min at 30°C in reaction buffer containing 0.25 mg/ml F-actin. Reaction mixtures were separated by SDS-PAGE and analyzed by immunoblotting using antibodies to cofilin, P-cofilin, and SSH1L.
Dephosphorylation of SSH1L by Calcineurin in Cell-free Assays-Lysates of 293T cells transfected with plasmids for Myc-SSH1L were immunoprecipitated with anti-Myc antibody or control IgG. The precipitates were incubated with recombinant active calcineurin A␣ and B and calmodulin, as described above. Phosphatase inhibitor (5 mM pyrophosphate) was preincubated with active calcineurin for 15 min at 4°C. Reaction mixtures were run on SDS-PAGE, and the amounts of phosphorylated SSH1L and total SSH1L were analyzed by staining the gel with Pro-Q Diamond phosphoprotein gel stain kit (Molecular Probes) and Coomassie Brilliant Blue, respectively.
Cell Staining-HeLa cells were fixed with 4% formaldehyde in phosphate-buffered saline for 20 min and permeabilized with 100% methanol for 5 min. After blocking with 1% bovine serum albumin in phosphatebuffered saline, cells were incubated with 3F10 rat anti-HA monoclonal antibody and rabbit anti-P-cofilin antibodies, followed by incubation with fluorescein isothiocyanate-conjugated anti-rat IgG antibody and rhodamine-conjugated anti-rabbit IgG antibody. Coverslips were washed with phosphate-buffered saline and mounted on glass slides. Images were obtained using a Leica DMLB fluorescence microscope.

RESULTS
Ca 2ϩ Ionophore Stimulates Cofilin Dephosphorylation and SSH1L Activation-It was reported that cofilin undergoes dephosphorylation in response to extracellular stimuli that increase the intracellular Ca 2ϩ concentration (20 -24). To explore signaling pathways of Ca 2ϩ -induced cofilin dephosphorylation, we first examined the effects of the Ca 2ϩ ionophore A23187 on levels of cofilin phosphorylation in cultured cells. Serumstarved 293T or HeLa cells were exposed to 5 M A23187 for different time periods, and cell lysates were analyzed by immunoblotting with an anti-P-cofilin antibody that specifically recognizes P-cofilin and an anti-cofilin antibody that recognizes both phosphorylated and non-phosphorylated forms of cofilin (10). Stimulation of 293T or HeLa cells with A23187 significantly decreased the level of P-cofilin in a time-dependent manner, without affecting the total cofilin level (Fig. 1A). A23187-induced cofilin dephosphorylation was also observed in WI-38 human lung fibroblasts (data not shown). These findings suggest that the increase in intracellular Ca 2ϩ concentrations induces cofilin dephosphorylation in various types of cells.
We next analyzed changes in cofilin-phosphatase activity of endogenous SSH1L after stimulating the cells with A23187. Serum-starved 293T or HeLa cells were incubated with or without 5 M A23187 for 10 min, cell lysates were prepared, and SSH1L was immunoprecipitated with an anti-SSH1L antibody. The immunoprecipitates were subjected to an in vitro phosphatase assay, using a recombinant phosphorylated cofilin-His 6 substrate. Cofilin-phosphatase activity was measured by the decrease in P-cofilin immunoreactivity. In both 293T and HeLa cells, the cofilin-phosphatase activity of SSH1L significantly increased after A23187 stimulation (Fig. 1B). These results suggest that Ca 2ϩ signaling induces both SSH1L activation and cofilin dephosphorylation.
Ca 2ϩ -mobilizing Agents Induce Cofilin Dephosphorylation and SSH1L Activation-Extracellular ATP and histamine are known to elevate the intracellular Ca 2ϩ concentration by mobilizing Ca 2ϩ from internal stores in 293T and HeLa cells, respectively (34). We asked whether these Ca 2ϩ -mobilizing agents would induce cofilin dephosphorylation and SSH1L activation in these cells. The level of P-cofilin significantly decreased after exposure of 293T cells to 50 M ATP ( Fig. 2A, top panel). In vitro phosphatase assay showed that the cofilin-phosphatase activity of SSH1L in 293T cells increased after ATP stimulation ( Fig. 2A, bottom  panel). In a similar manner, stimulation of HeLa cells with 10 mM histamine induced both cofilin dephosphorylation and SSH1L activation (Fig. 2B). Therefore extracellular Ca 2ϩ -mobilizing agents do have the potential to induce cofilin dephosphorylation and SSH1L activation.
Calcineurin Inhibitors Block Ca 2ϩ -induced Cofilin Dephosphorylation and SSH1L Activation-A23187-induced cofilin dephosphorylation was reported to be blocked by calcineurin inhibitors, which suggested that calcineurin is involved in the Ca 2ϩ -induced cofilin dephosphorylation (23). However, it is unclear whether or not calcineurin directly dephosphorylates cofilin. We examined the effects of calcineurin inhibitors on A23187-or histamine-induced cofilin dephosphorylation and SSH1L activation in HeLa cells. When HeLa cells were preincubated with calcineurin inhibitors (10 M cyclosporin A, 50 nM FK506, or 100 nM cypermethrin) and then treated with 5 M A23187 for 10 min, A23187-induced cofilin dephosphorylation was almost completely blocked in the presence of any one of these calcineurin inhibitors (Fig. 3A, top panel). Interestingly, A23187-induced SSH1L activation was also suppressed by calcineurin inhibitors (Fig. 3A, bottom panel), which indicates that calcineurin is required for the Ca 2ϩ -induced SSH1L activation. Both histamine-induced cofilin dephosphorylation and SSH1L activation in HeLa cells were inhibited by pretreatment of the cells with FK506 (Fig. 3B). These findings suggest that Ca 2ϩ -induced cofilin dephosphorylation and SSH1L activation are dependent on calcineurin activity and that calcineurin functions upstream of SSH1L. In addition, pretreatment of cells with 1 M okadaic acid (an inhibitor of protein phosphatases type 1 and 2A) had no apparent effect on histamine-induced cofilin dephosphorylation and SSH1L activation (Fig. 3C), which suggests that neither protein phosphatase type 1 nor 2A is involved in Ca 2ϩ -induced cofilin dephosphorylation and SSH1L activation.
Expression of Dominant-negative Calcineurin Suppresses A23187-induced Cofilin Dephosphorylation and SSH1L Activation-To further examine the role of calcineurin in Ca 2ϩ -induced cofilin dephosphorylation and SSH1L activation, we overexpressed either HA-tagged wild-type calcineurin A (CnA(WT)) or its dominant-negative form (⌬CnA(H160Q)) in HeLa cells and analyzed changes in the levels of P-cofilin and SSH1L activity before and after A23187 treatment. Expression of CnA(WT) alone had no apparent effect, unless cells had been exposed to agents that increased the intracellular Ca 2ϩ concentration (32). A23187-induced cofilin dephosphorylation and SSH1L activation were suppressed in cells expressing ⌬CnA(H160Q), but not in cells expressing CnA(WT) (Fig. 4). These data further indicate that calcineurin activity is required for Ca 2ϩ -induced SSH1L activation and cofilin dephosphorylation.
Phosphatase-inactive SSH1L Suppresses A23187-induced Cofilin Dephosphorylation-To examine the role of SSH1L in Ca 2ϩ -induced cofilin dephosphorylation, we overexpressed either SSH1L(WT) or SSH1L(CS) in HeLa cells and analyzed changes in P-cofilin levels before and after A23187 stimulation. In cells expressing SSH1L(WT), the level of P-cofilin decreased significantly even before A23187 stimulation and declined further after stimulation (Fig. 5A). In contrast, in cells expressing SSH1L(CS), a phosphatase-inactive Slingshot-1L mutant in which catalytic Cys is replaced by Ser, A23187-induced cofilin dephosphorylation was significantly suppressed (Fig. 5A). Similar results were obtained when CnA(WT) was co-expressed with either SSH1L(WT) or SSH1L(CS) in HeLa cells. A23187induced cofilin dephosphorylation in CnA(WT)-expressing cells was stimulated by co-expression of SSH1L(WT) and inhibited by SSH1L(CS) (Fig. 5B, compare lanes 3, 6, and 9). These findings suggest that the phosphatase activity of SSH1L is FIG. 1. Calcium ionophore A23187 induces cofilin dephosphorylation and SSH1L activation. A, calcium ionophore-induced cofilin dephosphorylation. Serum-starved 293T (left panels) or HeLa (right panels) cells were stimulated with 5 M A23187 for the indicated times. Cell lysates were immunoblotted with anti-Pcofilin and anti-cofilin antibodies. Bottom panels show the relative P-cofilin levels, with the value in non-treated cells taken as 100%. B, calcium ionophore-induced SSH1L activation. Serum-starved 293T or HeLa cells were stimulated with 5 M A23187 for 10 min. Cell lysates were immunoprecipitated (IP) with anti-SSH1L antibody or control IgG and subjected to an in vitro phosphatase assay, using cofilin-His 6 as a substrate. Reaction mixtures were analyzed by immunoblotting with anti-P-cofilin, anti-cofilin, and anti-SSH1L antibodies. Bottom panels show the relative P-cofilin levels after the in vitro cofilin-phosphatase assay, with the value obtained from control IgG precipitates taken as 100%.

FIG. 2. Cofilin dephosphorylation and SSH1L activation in ATP-stimulated 293T cells and histamine-stimulated HeLa cells.
A, ATP-induced cofilin dephosphorylation and SSH1L activation in 293T cells. Serum-starved 293T cells were stimulated with 50 M ATP for the indicated times. Cell lysates were analyzed by immunoblotting with antibodies to P-cofilin and cofilin (top panel). SSH1L was immunoprecipitated and subjected to in vitro phosphatase assay, as described in Fig. 1B (bottom panel). B, histamine-induced cofilin dephosphorylation and SSH1L activation in HeLa cells. Serum-starved HeLa cells were stimulated with 10 mM histamine for the indicated times. Changes in the levels of P-cofilin (top panel) and SSH1L activity (bottom panel) were measured in cells as described in A.
critical for Ca 2ϩ /calcineurin-induced cofilin dephosphorylation and that SSH1L(CS) acts as a dominant-negative mutant.
Suppression of SSH1L Expression by siRNA Inhibits A23187-induced Cofilin Dephosphorylation-To confirm that SSH1L mediates Ca 2ϩ /calcineurin-induced cofilin dephosphorylation, expression of endogenous SSH1L in HeLa cells was suppressed by transfection of a pSUPER siRNA expression plasmid, pSUPER-SSH1L, which directs the synthesis of an siRNA targeting human SSH1L (29). An empty pSUPER vector and siRNA plasmid mutated in the SSH1L target sequence, pSUPER-SSH1L(mt), were transfected as control experiments. Immunoblot analysis revealed that transfection with pSUPER-SSH1L significantly reduced the level of endogenous SSH1L expression, whereas control vector or mutated siRNA plasmid did not suppress it (Fig. 6A). HeLa cells were co-transfected with CnA(WT) and siRNA plasmids, and changes in P-cofilin levels in cells were analyzed before and after A23187 stimulation. In cells transfected with control pSUPER vector or pSU-PER-SSH1L(mt), A23187 stimulation induced cofilin dephosphorylation (Fig. 6B, lanes 3 and 9). In contrast, in cells transfected with pSUPER-SSH1L, A23187-induced cofilin dephosphorylation was significantly inhibited (Fig. 6B, lane 6). These observations further suggest that SSH1L plays a critical role in Ca 2ϩ /calcineurin-induced cofilin dephosphorylation.  Fig. 2. B, inhibition of histamineinduced cofilin dephosphorylation and SSH1L activation by FK506. HeLa cells were preincubated with or without 50 nM FK506 and then stimulated with 10 mM histamine for 10 min. Changes in the levels of P-cofilin and SSH1L activity in cells were analyzed as described in Fig.  2. C, effects of okadaic acid on P-cofilin levels and SSH1L activity. HeLa cells were treated with 1 M okadaic acid for 10 min and then stimulated with 10 mM histamine for 10 min. Changes in the levels of P-cofilin and SSH1L activity were analyzed as described in Fig. 2. induced cofilin dephosphorylation by staining cells with an anti-P-cofilin antibody. HeLa cells transfected with plasmids coding for HA-tagged constitutively active calcineurin A and calcineurin B (⌬CnA/B) with or without pSUPER-SSH1L siRNA plasmids were cultured for 44 h and then co-stained with anti-HA and anti-P-cofilin antibodies. As shown in the left panels of Fig. 7, the P-cofilin level significantly decreased in cells expressing ⌬CnA/B (indicated by arrowheads), compared with the levels in neighboring non-expressing cells. In contrast, the P-cofilin levels in cells co-transfected with ⌬CnA/B and pSUPER-SSH1L were similar to those in surrounding cells (Fig. 7, right panels), which indicates that SSH1L is required for ⌬CnA/B-induced cofilin dephosphorylation. These observations strongly suggest that calcineurin induces cofilin dephosphorylation via SSH1L activation.
Calcineurin Dephosphorylates and Activates SSH1L in Cellfree Assays-We next examined whether calcineurin has the potential to dephosphorylate and activate SSH1L in cell-free assays. Lysates of 293T cells transfected with plasmids for Myc-SSH1L were immunoprecipitated with anti-Myc antibody or control IgG, the precipitates were incubated in the presence or absence of recombinant active calcineurin, and the levels of SSH1L phosphorylation were analyzed (Fig. 8A). The amounts of phosphorylated SSH1L (P-SSH1L) and total SSH1L were measured by Pro-Q Diamond phosphoprotein staining and Coomassie Brilliant Blue staining, respectively. Pro-Q Diamond staining revealed that Myc-SSH1L was phosphorylated in 293T cells (Fig.  8A, lane 3) and dephosphorylated in vitro by treatment with active calcineurin (Fig. 8A, lane 5). Calcineurin-catalyzed dephosphorylation was blocked by pretreatment of calcineurin with pyrophosphate, an inhibitor of phosphatases including calcineurin (Fig. 8A, lane 6). These results suggest that SSH1L is a phosphoprotein in cultured cells and that calcineurin has the potential to dephosphorylate SSH1L in cell-free assays. The slight increase in the P-SSH1L level by pyrophosphate may be due to the inhibitory effect on SSH1L autodephosphorylation (Fig. 8A, lane 4). We also examined the effect of active calcineurin on the cofilin-phosphatase activity of SSH1L in cell-free assays. SSH1L purified from 293T cells by immunoprecipitation was incubated with or without recombinant active calcineurin and subjected to the in vitro cofilin-phosphatase assay (Fig. 8B). Incubation of SSH1L with calcineurin significantly increased the cofilin-phosphatase activity of SSH1L (Fig. 8B, lanes 3 and 4). In control experiments, calcineurin alone had no apparent effect on the P-cofilin level (Fig. 8B, lane 2). These results suggest that calcineurin has the potential to stimulate the cofilin-phosphatase activity of SSH1L, but not to catalyze cofilin dephosphorylation itself, in cell-free assays. These data, taken together with the data of Fig. 8A, indicate that calcineurin appears to activate SSH1L by dephosphorylation. DISCUSSION Cofilin plays an essential role in regulating actin filament dynamics. Its actin-depolymerizing and -severing activities are inhibited by phosphorylation at Ser-3 by LIM kinases and testicular protein kinases and reactivated by dephosphorylation by SSH family phosphatases (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). Although a variety of extracellular stimuli induce cofilin dephosphorylation, the signaling pathways that lead to cofilin dephosphorylation have not been well understood. In the present study, we provided evidence that Ca 2ϩ signal-induced cofilin dephosphorylation is mediated by consecutive activation of two protein phosphatases, calcineurin and SSH1L. We showed that Ca 2ϩ ionophore and Ca 2ϩ -mobilizing agents induced SSH1L activation and cofilin dephosphorylation, both of which were suppressed by inhibition of calcineurin. These data indicate that Ca 2ϩ -induced SSH1L activation and cofilin dephosphorylation are mediated by calcineurin. Importantly, knockdown of endogenous SSH1L expression by siRNA abolished Ca 2ϩ -or calcineurininduced cofilin dephosphorylation. Furthermore, calcineurin dephosphorylated SSH1L and stimulated cofilin-phosphatase activity of SSH1L in cell-free assays, but it had no apparent activity to catalyze cofilin dephosphorylation directly. Taken together, these findings suggest that SSH1L is essential for Ca 2ϩ -induced cofilin dephosphorylation as a downstream effector of calcineurin and that calcineurin stimulates cofilin dephosphorylation apparently only through SSH1L activation. Thus, we propose a novel signaling cascade of Ca 2ϩ -calcineurin-SSH1L-cofilin for Ca 2ϩ signal-induced cofilin dephosphorylation and activation.
The precise mechanism of calcineurin-mediated SSH1L activation is still unclear. It is important to identify the residue(s) of SSH1L that is phosphorylated in resting cells but becomes dephosphorylated by calcineurin in response to stimulation of the Ca 2ϩ -induced signaling pathway. SSH1L activity is inhibited by association with 14-3-3 proteins, in a manner dependent on the phosphorylation of SSH1L serine residues (30), and it is possible that calcineurin stimulates SSH1L activity by dephosphorylating the serine residues that are involved in 14-3-3 binding. In the case of Ca 2ϩ -induced apoptosis, calcineurin-mediated dephosphorylation of BAD causes its dissociation from 14-3-3 and translocation to mitochondria to inhibit Bcl-xL (35). Additional studies are necessary to determine whether calcineurin induces SSH1L activation through a similar mechanism.
Ca 2ϩ is a versatile intracellular signal mediator that can regulate many different cellular processes, including those related to actin cytoskeletal rearrangement. Ca 2ϩ -mediated cofilin dephosphorylation has been observed in various cell types and is thought to contribute to various cell responses, such as thrombininduced platelet aggregation (20), chemotactic peptide-induced neutrophil migration (28), and nicotine-induced noradrenalin secretion by adrenal chromaffin cells (24). Because cofilin appears to play an essential role in actin filament dynamics and remodeling, Ca 2ϩ -induced SSH1L activation and cofilin dephosphorylation/activation must represent an important signaling pathway supporting stimulus-induced actin cytoskeletal changes and related cell responses. In neurons, intracellular Ca 2ϩ signals play important roles in regulating growth cone motility and guidance (36). Local Ca 2ϩ signals can cause growth cone steering responses, with higher Ca 2ϩ signals mediating attraction and lower Ca 2ϩ signals mediating repulsion (37). Based on findings that both cofilin and SSH1L stimulate growth cone motility and extension (18,38), local activation of the Ca 2ϩ -SSH1L-cofilin signaling pathway appears to stimulate the directional steering of growth cones. In addition, Ca 2ϩ and calcineurin are known to regulate neuronal plasticity and memory formation in the central nervous system (39). Modulation of actin cytoskeletal dynamics in dendritic spines by cofilin phosphorylation and dephosphorylation contributes to spine structure, persistence of long-term potentiation, and synaptic plasticity (40,41). Thus, Ca 2ϩ /calcineurin-induced SSH1L activation may be one of the mechanisms by which Ca 2ϩ /calcineurin regulates neuronal actin cytoskeletal dynamics and hence modulates synaptic function and neuronal plasticity.
Various signaling pathways are involved in stimulus-induced cofilin dephosphorylation. In insulin-stimulated cells, phosphoinositide 3-kinase and its product, phosphatidylinositol-3,4,5-trisphosphate, are involved in SSH1L activation and cofilin dephosphorylation (29). In other types of cells, cAMP and protein kinase C signals stimulate cofilin dephosphorylation (1,2,(21)(22)(23). In contrast to the Ca 2ϩ signal, cAMP-or protein kinase C signal-induced cofilin dephosphorylation is insensitive to calcineurin or calmodulin inhibitors (23,28), which indicates the existence of calcineurin-independent signaling pathways for cofilin dephosphorylation. Additional studies are required to determine whether SSH1L, other members of a SSH family (e.g. SSH2L and SSH3L), or other more general FIG. 8. Calcineurin dephosphorylates and activates SSH1L in vitro. A, dephosphorylation of SSH1L by calcineurin. Lysates of 293T cells expressing Myc-SSH1L were immunoprecipitated with anti-Myc antibody or control IgG. The precipitates were incubated with recombinant active calcineurin (active Cn) and/or pyrophosphate (Cn inhibitor), as indicated. Reaction mixtures were run on SDS-PAGE, and phospho-SSH1L (P-SSH1L) and total SSH1L were measured by Pro-Q Diamond and Coomassie Brilliant Blue (CBB) staining, respectively. Bottom panel shows the relative phospho-SSH1L levels, with the value obtained from non-treated SSH1L taken as 100%. Results are shown as the means Ϯ S.D. of three independent experiments. B, activation of SSH1L by calcineurin. Lysates of 293T cells were immunoprecipitated with anti-SSH1L antibody or control IgG. The precipitates were incubated with cofilin-His 6 substrate in the presence (ϩ) or absence (Ϫ) of recombinant active calcineurin (active Cn), as indicated. Reaction mixtures were analyzed by immunoblotting with anti-P-cofilin, anti-cofilin, and anti-SSH1L antibodies. Bottom panel shows the relative P-cofilin levels after the in vitro cofilin-phosphatase assay, with the value obtained from control IgG precipitates in the absence of calcineurin taken as 100%. Results are shown as the means Ϯ S.D. of three independent experiments. classes of protein phosphatases, such as protein phosphatase type 1 and 2A (27), are involved in cAMP-and protein kinase C-induced cofilin dephosphorylation. Indeed, cofilin dephosphorylation/activation appears to be one of the important and convergent points in a cell signaling network through which a variety of extracellular stimuli regulate actin cytoskeletal dynamics and organization.