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

Abstract

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

Figure 1.

NRG stimulates cofilin dephosphorylation in a manner dependent on Rac, SSH1L, and actin assembly. (A) NRG induces cofilin dephosphorylation. MCF-7 cells were stimulated with NRG. Cell lysates were immunoblotted with antibodies against P-cofilin and cofilin. Relative P-cofilin levels are shown as means ± SEM of triplicate experiments. (B and C) Expression of RacN17 or SSH1L(CS) inhibits NRG-induced cofilin dephosphorylation. MCF-7 cells transfected with HA-RacN17 (B) or Myc-SSH1L(CS) (C) were stimulated with NRG for 20 min and the P-cofilin levels were analyzed as in A. (D) SSH1L accumulates in NRG-induced lamellipodia. MCF-7 cells expressing CFP-SSH1L were stimulated with NRG for 15 min and stained with rhodamine phalloidin for F-actin. Bar, 20 μm. (E) Cofilin, but not P-cofilin, accumulates in NRG-induced lamellipodia. MCF-7 cells were stimulated with NRG for 15 min and stained with antibodies against cofilin or P-cofilin. Bar, 20 μm. (F) Lat-A inhibits NRG-induced cofilin dephosphorylation. MCF-7 cells were treated with Lat-A for 10 min, then stimulated with NRG for 20 min, and the P-cofilin levels were analyzed. Active GTP-bound form of Rac was analyzed using pull-down assay with GST-PBD (p21-binding domain of PAK1; Nishita et al., 2002). (G) Lat-A inhibits SSH1L- induced cofilin dephosphorylation. MCF-7 cells transfected with Myc-SSH1L(WT) were treated with Lat-A for 30 min and the P-cofilin levels were analyzed as in A.

Figure 2.

F-actin activates the cofilin-phosphatase activity of SSH1L. (A) F-actin activates SSH1L. Myc-SSH1L expressed in COS cells was precipitated with anti-Myc antibody and subjected to in vitro phosphatase assay, using cofilin-(His)₆ as a substrate, with or without F-actin (F) or G-actin (G). P-cofilin and total cofilin were measured by Pro-Q and Coomassie brilliant blue (CBB) staining, respectively. The bottom panel indicates the P-cofilin levels, as means ± SD of triplicate experiments. (B) F-actin activates SSH1L(WT), but not its NP or PC fragment. The conserved regions between a SSH family are indicated as A, B, P (phosphatase), and S domains. Cofilin-phosphatase activities of Myc-SSH1L(WT) and its NP and PC fragments with or without F- or G-actin were analyzed as in A. (C) Kinetic analyses of cofilin-phosphatase activity of WT and NP mutant of SSH1L with or without F-actin. Top panels show the P-cofilin levels at indicated times after incubation with WT or NP mutant of SSH1L, measured by Pro-Q staining. Bottom panels indicate the time courses of P-cofilin dephosphorylation as means ± SD of triplicate experiments.

Figure 3.

14-3-3 proteins bind to and inhibit SSH1L, dependent on Ser-937 and Ser-978 phosphorylation. (A) Purification of 14-3-3 proteins as SSH1L-interacting proteins. Lysates of COS cells expressing WT or CS mutant of (Myc+His)-SSH1L were precipitated with Ni-NTA agarose, run on SDS-PAGE, and stained by silver. Right panel shows similar experiments done for the indicated mutants of (Myc+His)-SSH1L. (B) The COOH-terminal region of SSH1L is required for binding to 14-3-3β. WT and deletion mutants of Myc-SSH1L were expressed in COS cells and subjected to in vitro pull-down assay with GST or GST-14-3-3β. (C) SSH1L interacts with 14-3-3β, dependent on Ser-937 and Ser-978 phosphorylation. WT and point mutants of Myc-SSH1L expressed in COS cells were subjected to in vitro pull-down assay. (D) 14-3-3γ inhibits SSH1L, but not its 2SA mutant, in cell-free assay. Myc-SSH1L or its 2SA mutant expressed in 293T cells were precipitated with an anti-Myc antibody and subjected to in vitro phosphatase assay, using cofilin-(His)₆ as a substrate, with or without recombinant (Rec.) 14-3-3γ. Reaction mixtures were analyzed by blotting with anti–P-cofilin antibody. It is noted that SSH1L(WT), but not its 2SA mutant, coprecipitated endogenous (Endo.) 14-3-3 proteins (bottom). (E) 14-3-3γ protects SSH1L from F-actin– induced activation. Endogenous SSH1L in MCF-7 cells was precipitated with anti-SSH1L antibody or GST-14-3-3γ and subjected to in vitro phosphatase assay, as in Fig. 2 A. (F) Expression of 14-3-3γ increases the cellular P-cofilin level and suppresses SSH1L-induced cofilin dephosphorylation. Myc-14-3-3γ was expressed alone or with WT or 2SA mutant of Myc-SSH1L in COS cells, and lysates were analyzed by immunoblotting with indicated antibodies. The bottom panel indicates the relative levels of P-cofilin as means ± SD of triplicate experiments.

Figure 4.

14-3-3γ inhibits NRG-induced accumulation of SSH1L and cofilin in lamellipodia. (A–C) 14-3-3γ inhibits NRG-induced accumulation of SSH1L, but not its 2SA mutant, in lamellipodia. MCF-7 cells expressing CFP-SSH1L(WT) and HA-14-3-3γ (A), CFP-SSH1L(2SA) alone (B), or CFP-SSH1L(2SA) and HA-14-3-3γ (C), were stimulated with NRG for 15 min and stained with rhodamine phalloidin for F-actin. (D) 14-3-3γ inhibits NRG-induced cofilin dephosphorylation and accumulation in lamellipodia. MCF-7 cells transfected with HA-14-3-3γ were stimulated with NRG for 15 min and stained with anticofilin or anti–P-cofilin antibody. Arrowheads and arrows indicate 14-3-3γ–expressing and nonexpressing cells, respectively. Bar, 20 μm.

Figure 5.

NRG induces translocation of SSH1L into the Triton X-100–insoluble cytoskeletal fraction and dephosphorylation at Ser-978. (A) NRG induces translocation of SSH1L to the Triton X-100–insoluble fraction. MCF-7 cells with or without pretreatment of Lat-A were exposed to NRG for 10 min and cell lysates were fractionated into Triton X-100–soluble (S) and –insoluble (I) fractions. Each fraction was run on SDS-PAGE and blotted using anti-SSH1L and anti–β-actin antibodies. Bottom panels indicate the ratio of the amounts of SSH1L and β-actin in the insoluble fraction, as means ± SD of triplicate experiments. (B) 14-3-3γ suppresses basal and NRG-induced translocation of SSH1L to the insoluble fraction. MCF-7 cells transfected with Myc-SSH1L(WT) or Myc-SSH1L(2SA) alone or cotransfected with 14-3-3γ were stimulated with NRG and cell lysates were fractionated and analyzed as in A. (C) NRG induces Ser-978 dephosphorylation of SSH1L. Lysates of MCF-7 cells before and after NRG stimulation were immunoprecipitated with anti-SSH1L antibody and blotted with anti-SSH1L and anti-pS978 antibodies. (D) A proposed model of the NRG-induced SSH1L activation and cofilin dephosphorylation.