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Report |
Address correspondence to K. Mizuno, Dept. of Biomolecular Sciences, Graduate School of Life Sciences, Tohoku University, Aoba-ku, Sendai, Miyagi 980-8578, Japan. Tel.: 81-22-217-6676. Fax: 81-22-217-6678. email: kmizuno{at}biology.tohoku.ac.jp
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Abstract |
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Key Words: LIM-kinase; cell polarity; 14-3-3; actin filaments; MCF-7
Abbreviations used in this paper: Lat-A, latrunculin-A; LIMK, LIM-kinase; NRG, neuregulin-1ß; P-cofilin, Ser-3phosphorylated cofilin; pS, phospho-Ser; SSH1L, Slingshot-1L.
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Introduction |
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Cofilin is inactivated by phosphorylation at Ser-3 by LIM-kinase (LIMK; Arber et al., 1998; Yang et al., 1998). Suppression of cofilin activity by LIMK overexpression abolished lamellipodium formation and polarized cell migration, which implicates cofilin in cell polarity formation and migration (Zebda et al., 2000; Dawe et al., 2003). Slingshot-1L (SSH1L) is a member of a Slingshot phosphatase family that dephosphorylates and reactivates an inactive Ser-3phosphorylated cofilin (P-cofilin; Niwa et al., 2002). Previous studies showed that cofilin is dephosphorylated in response to various external stimuli that alter cell motility and morphology (Moon and Drubin, 1995). However, mechanisms that regulate SSH activity and cofilin dephosphorylation in response to external cues are poorly understood. Here, we provide evidence that SSH1L is locally activated in lamellipodia by associating with actin filaments and 14-3-3 proteins negatively regulate this activation by sequestering it in the cytoplasm.
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Results and discussion |
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Pretreatment of cells with latrunculin-A (Lat-A), an inhibitor of actin assembly, blocked NRG-induced lamellipodium formation and SSH1L accumulation (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200401136/DC1). Lat-A increased the basal level of P-cofilin in MCF-7 cells and inhibited NRG-induced cofilin dephosphorylation (Fig. 1 F), which indicates that actin filament assembly is required for cofilin dephosphorylation. NRG induced Rac activation but this was not affected by Lat-A treatment (Fig. 1 F). Expression of SSH1L induced cofilin dephosphorylation in MCF-7 cells, but treatment of the cells with Lat-A suppressed SSH1L-induced cofilin dephosphorylation (Fig. 1 G), which suggests that cofilin-phosphatase activity of SSH1L depends on actin filament assembly.
As SSH1L has the potential to bind to F-actin (Niwa et al., 2002), we examined if actin filaments would directly control SSH1L activity. In vitro cofilin-phosphatase assays revealed that SSH1L activity was remarkably enhanced in the presence of F-actin, but not G-actin (Fig. 2 A). F-actin alone had no apparent effect. As reported previously (Ohta et al., 2003), full-length SSH1L and an NH2-terminal NP fragment have the potential to dephosphorylate P-cofilin, but a COOH-terminal PC fragment did not do so (Fig. 2 B). Addition of F-actin enhanced cofilin-phosphatase activity of full-length SSH1L, but not either the activity of NP or PC (Fig. 2 B). Kinetic analysis revealed that F-actin increased 10-fold the cofilin-phosphatase activity of full-length SSH1L, whereas it had no apparent effect on the activity of NP (Fig. 2 C). Because NP scarcely binds to F-actin (Ohta et al., 2003), SSH1L is likely activated by F-actin binding to the COOH-terminal region.
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Next, we examined the effects of 14-3-3 expression on the P-cofilin level in cells. Overexpression of 14-3-3
increased
1.2-fold the cellular P-cofilin level (Fig. 3 F), which could be explained by 14-3-3
inhibition of the cofilin-phosphatase activity of SSH1L in the cells. Expression of SSH1L(WT) or SSH1L(2SA) in COS cells resulted in a decrease in the cellular P-cofilin level. Coexpression of 14-3-3
suppressed the cofilin dephosphorylation caused by SSH1L(WT) but not that by SSH1L(2SA) (Fig. 3 F), which strongly suggests that 14-3-3
increased the cellular P-cofilin level by binding to and inhibiting SSH1L. Recently, it was reported that 14-3-3
interacts with P-cofilin and thereby protects P-cofilin from dephosphorylation (Gohla and Bokoch, 2002). However, we were unable to detect the specific interaction between P-cofilin (or cofilin) and 14-3-3
(or ß or
isoform) in our assay system (Fig. S5, available at http://www.jcb.org/cgi/content/full/jcb.200401136/DC1). LIMK1 was also reported to bind to 14-3-3
(Birkenfeld et al., 2003), but we detected no interaction between LIMK1 and 14-3-3ß under the conditions in which SSH1L and TESK1 (another cofilin kinase reported to bind to 14-3-3ß; Toshima et al., 2001b) tightly bound to 14-3-3ß (Fig. S5).
SSH1L accumulates in lamellipodia after NRG stimulation (Fig. 1 D). To examine the role of 14-3-3 for SSH1L localization, MCF-7 cells were coexpressed with 14-3-3 and CFP-SSH1L. Expression of 14-3-3
significantly suppressed NRG-induced lamellipodium formation and SSH1L accumulation to the lamellipodium (compare Fig. 4 A with Fig. 1 D). When a 2SA mutant was expressed, it colocalized with F-actin before and after NRG stimulation and accumulated in lamellipodia after NRG stimulation (Fig. 4 B). Coexpression of 14-3-3
had no apparent effect on SSH1L(2SA) accumulation in lamellipodia (Fig. 4 C). These results suggest that 14-3-3
suppresses SSH1L translocation to the lamellipodium, in a manner dependent on Ser-937/978 phosphorylation. We also examined the effects of 14-3-3
on the level and localization of cofilin/P-cofilin in MCF-7 cells (Fig. 4 D). Expression of 14-3-3
suppressed both cofilin accumulation in the lamellipodium and reduction in the P-cofilin level after NRG stimulation. Thus, 14-3-3 seems to inhibit SSH1L translocation to the lamellipodium and thereby suppress cofilin dephosphorylation in this region.
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During cell migration, the cell is polarized to form an F-actinrich lamellipodial extension in the direction of cell movement. In our model, once the F-actinrich structure is established in the front of migrating cells, SSH1L is recruited and activated in this region and supports local activation of cofilin in the lamellipodium. Given the essential role of cofilin in actin filament turnover, spatially restricted activation of SSH1L by F-actin will be an important mechanism for maintaining and extending lamellipodia in the front of the cell and sustaining polarized cell migration.
In addition, LIMK1 was also activated by NRG stimulation (Fig. S7, available at http://www.jcb.org/cgi/content/full/jcb.200401136/DC1), as reported previously (Vadlamudi et al., 2002). Previous studies showed that LIMK1 is required for lamellipodium formation and cell migration (Yang et al., 1998; Nishita et al., 2002). LIMK1 may play a role in the establishment of lamellipodia by transiently inactivating cofilin and inducing actin polymerization. LIMK1 may also contribute to stimulate actin turnover in lamellipodia by releasing free actin and cofilin from an actincofilin complex, which is produced from the pointed end of actin filaments by the action of cofilin (Rosenblatt and Mitchison, 1998). Because the released P-cofilin can be reused after dephosphorylation by SSH1L, coordinated activation of LIMK1 and SSH1L can accelerate the recycling of cofilin and thereby promote actin turnover. In contrast to the accumulation of SSH1L in lamellipodia after NRG stimulation, LIMK1 localizes diffusely in the cytoplasm. Therefore, the spatially distinct distribution of LIMK1 and SSH1L can generate the polarized pattern of cofilin activation and may contribute to polarized cell migration.
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Materials and methods |
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Cell culture and staining
MCF-7, COS-7, and HEK293T cells were maintained in DME supplemented with 10% FCS. Cells were transfected with expression plasmids using FuGENE6 (Roche). Serum-starved MCF-7 cells were treated with 50 ng/ml NRG (Genzyme) or 0.5 µM Lat-A (Molecular Probes). For staining, cells were fixed with 4% formaldehyde and incubated with 100% methanol. After blocking with 5 mg/ml BSA, cells were stained with anticofilin or antiP-cofilin antibody. Rhodamine phalloidin was used to stain F-actin. Fluorescent images were obtained using a confocal microscope (model LSM510; Carl Zeiss MicroImaging, Inc.).
In vitro phosphatase assay
Cofilin-(His)6 expressed in Vero cells and purified with Ni-NTA agarose was used as a substrate. SSH1L was immunoprecipitated and incubated for 1 h at 30°C with 100 ng cofilin-(His)6 in 20 µl of lysis buffer containing 0.01% BSA (Niwa et al., 2002). Reaction mixtures were run on SDS-PAGE and P-cofilin was analyzed by immunoblotting with antiP-cofilin antibody or staining with Pro-Q Diamond phosphoprotein gel stain kit (Molecular Probes), and cofilin was analyzed by Coomassie brilliant blue staining. To examine the effects of F-actin, purified rabbit muscle actin was polymerized in F-buffer (Niwa et al., 2002). G-actin was prepared in G-buffer (2 mM Tris-Cl, pH 8.0, 1 mM DTT, 0.2 mM ATP, and 0.2 mM CaCl2). SSH1L was incubated with 5 µg F- or G-actin and 100 ng cofilin-(His)6 in 20 µl of lysis buffer. To examine the effect of 14-3-3, GST-14-3-3
expressed in Escherichia coli was used after thrombin cleavage.
In vitro kinase assay
MCF-7 cells were stimulated with NRG and LIMK1 was immunoprecipitated with anti-LIMK1 antibody and subjected to in vitro kinase reaction as described previously (Nishita et al., 2002).
Purification of SSH1L-binding proteins
Three 100-mm plates of COS-7 cells were transfected with (Myc+His)-SSH1L. Lysates were precleared with Sepharose-4B and then precipitated with Ni-NTA agarose. The pellets were washed, run on SDS-PAGE, and stained by silver. To identify SSH1L-binding proteins, the proteins resolved on SDS-PAGE were transferred onto a PVDF membrane and stained with Colloidal gold (Bio-Rad Laboratories). The protein bands were cut out, digested with lysyl endopeptidase, and analyzed by mass spectrometry.
In vitro pull-down assay
In vitro pull-down assay was done as described previously (Toshima et al., 2001b). Lysates of COS-7 cells expressing Myc-SSH1L or its mutants were incubated with GST or GST-14-3-3ß and glutathione Sepharose. After centrifugation, the pellets were subjected to SDS-PAGE and analyzed by immunoblotting with an anti-Myc antibody.
Subcellular fractionation
MCF-7 cells treated with or without NRG were extracted for 3 min on ice with 1% Triton X-100 in PEM buffer (100 mM Pipes, pH 6.8, 1 mM EGTA, and 1 mM MgCl2) containing 2 µM phalloidin, 10 mM NaF, 1 mM Na3VO4, and 10 µg/ml leupeptin (Svitkina and Borisy, 1999). The soluble fraction was removed, then the residual insoluble fraction was washed once with PEM buffer and scraped with SDS-sample buffer. Equal amounts of soluble and insoluble fractions were subjected to SDS-PAGE and analyzed by immunoblotting.
Online supplemental material
Video 1 shows the time-lapse phase-contrast microscopy of MCF-7 cell shape changes for 38 min after NRG stimulation. Fig. S1 shows kinetics of actin filament reorganization after NRG stimulation. Fig. S2 shows that Lat-A inhibits NRG-induced lamellipod formation and SSH1L accumulation. Fig. S3 shows the interaction of SSH1L14-3-3ß in the yeast two-hybrid system. Fig. S4 shows the inhibition of the SSH1L14-3-3ß interaction by phosphatase treatment. Fig. S5 shows that 14-3-3 proteins interact with SSH1L and TESK1, but not with cofilin, P-cofilin, or LIMK1. Fig. S6 shows the specificity of an anti-pS978 antibody. Fig. S7 shows NRG-induced activation of LIMK1. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200401136/DC1.
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Acknowledgments |
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This work was supported by a grant for creative scientific research from the Japan Society of the Promotion of Science to K. Mizuno and a grant from Core Research for Evolution Science and Technology, Japan Science and Technology Corporation to T. Uemura.
Submitted: 27 January 2004
Accepted: 12 April 2004
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