Activation of cytosolic Slingshot-1 phosphatase by gelsolin-generated soluble actin filaments

https://doi.org/10.1016/j.bbrc.2014.10.108Get rights and content

Highlights

  • Slingshot-1 binds to gelsolin via soluble actin filaments in the cytosol.

  • Gelsolin promotes the solubilization of actin filaments and Slingshot-1 in cells.

  • Slingshot-1 is activated by gelsolin-generated soluble actin filaments.

  • Gelsolin enhances neuregulin-induced cofilin dephosphorylation.

Abstract

Slingshot-1 (SSH1) is a protein phosphatase that dephosphorylates and activates cofilin, an actin-severing and -disassembling protein. SSH1 is bound to and activated by F-actin, but not G-actin. SSH1 is accumulated in the F-actin-rich lamellipodium but is also diffusely distributed in the cytoplasm. It remains unknown whether SSH1 is activated by soluble (low-level polymerized) actin filaments in the cytoplasm. In this study, we show that SSH1 binds to gelsolin via actin filaments in the cytosolic fraction. Gelsolin promoted solubilization of actin filaments and SSH1 in cell-free assays and in cultured cells. SSH1 was activated by gelsolin-generated soluble actin filaments. Furthermore, gelsolin enhanced cofilin dephosphorylation in neuregulin-stimulated cells. Our results suggest that cytosolic SSH1 forms a complex with gelsolin via soluble actin filaments and is activated by gelsolin-generated soluble actin filaments and that gelsolin promotes stimulus-induced cofilin dephosphorylation through increasing soluble actin filaments, which support SSH1 activation in the cytoplasm.

Introduction

Regulation of actin filament dynamics is essential for cell migration and morphogenesis. Cofilin plays a fundamental role in actin filament dynamics and remodeling by severing and depolymerizing actin filaments [1], [2]. Cofilin is inactivated by phosphorylation at Ser-3 by LIMKs and TESKs [3], [4], [5] and is reactivated by dephosphorylation by Slingshot (SSH) family protein phosphatases, which comprise SSH1, SSH2, and SSH3 in mammals [6], [7], [8]. SSH1 binds to F-actin, and the cofilin-phosphatase activity of SSH1 is markedly enhanced by its binding to F-actin [6], [7], [8], [9], [10], [11], [12]. Upon stimulation with growth factors or chemokines, SSH1 accumulates into F-actin-rich lamellipodia at the front of migrating cells [9], [13]. These observations suggest that SSH1 plays a critical role in cell migration by activating cofilin and thereby promoting actin filament turnover in lamellipodia [9], [13]. On the other hand, a considerable proportion of SSH1 is diffusely distributed in the cytoplasm. However, it remains unknown whether cytosolic SSH1 binds to and is activated by soluble (low-level polymerized) actin filaments.

Gelsolin (GSN) and its closely related protein scinderin (SCIN) are Ca2+-dependent actin-regulatory proteins that stimulate severing of actin filaments, capping of the plus ends of actin filaments, and nucleation of actin assembly [14], [15]. The severing and capping activities of GSN produce GSN-capped short actin filaments [14]. Thus, GSN plays important roles in various cell functions, including cell migration and morphogenesis, through the regulation of actin dynamics [14], [15], [16].

In this study, we show that SSH1 co-precipitates GSN and SCIN through actin filaments. We provide evidence that SSH1 forms a protein complex with GSN and soluble actin filaments in the cytosolic fraction and is activated by GSN-generated soluble actin filaments. Our results suggest that GSN plays a role in cofilin dephosphorylation through soluble actin filament-induced SSH1 activation.

Section snippets

Plasmid construction

cDNAs encoding human GSN and SCIN were PCR-amplified from the MegaMan human transcriptome library (Agilent Technologies). The cDNAs were subcloned into FPC1-Myc (GE Healthcare), pGEX (GE Healthcare), and pEYFP-C1 (Clontech) expression vectors. Plasmids for SSH1 and its mutants were constructed as described previously [12].

Cell culture, transfection, and staining

293T cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum. MCF-7 cells were cultured in Eagle’s minimum essential medium

GSN co-precipitates and co-localizes with SSH1

To examine the mechanisms regulating SSH1 activity, we searched for SSH1-binding proteins using co-precipitation assay. 293T cell lines stably expressing FLAG-tagged SSH1 were established and the cell lysates were immunoprecipitated with an anti-FLAG antibody. The co-precipitated proteins were separated by SDS–PAGE and analyzed by mass spectrometry (Fig. 1A). Actin and several actin-binding proteins, including GSN, SCIN, filamin-A, and the α1 and α2 subunits of capping protein (CapZα1 and

Discussion

The cofilin-phosphatase activity of SSH1 is dramatically increased by its binding to F-actin [9], [12]. However, it is unknown whether cytosolic SSH1 is activated by soluble (low-level polymerized) actin filaments in the cytoplasm, similar to SSH1 bound to insoluble (highly polymerized) cytoskeletal actin filaments. In this study, we showed that SSH1 forms a protein complex with GSN and soluble actin filaments in the cytosolic fraction of cultured cells. GSN promoted the solubilization of actin

Acknowledgments

We thank Drs. K. Ohashi and A. Yasui for helpful comments. This work was supported by Grants for Scientific Research (24370051 and 26102505 to K.M.) from the Ministry of Education, Culture, Science, Sports, and Technology of Japan.

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