Molecular dissection of the mechanisms of substrate recognition and F-actin-mediated activation of cofilin-phosphatase Slingshot-1.
Slingshot-1 (SSH1), a member of a dual-specificity protein phosphatase family, regulates actin dynamics by dephosphorylating and reactivating cofilin, an actin-depolymerizing factor. SSH1 has the SSH family-specific, N-terminal, noncatalytic (SSH-N) domain, consisting of the A and B subdomains. SSH1 is activated by binding to actin filaments. In this study, we examined the mechanisms of SSH1 substrate recognition of phospho-cofilin (P-cofilin) and SSH1 activation by F-actin. We found that P-cofilin binds to a phosphatase-inactive mutant, SSH1(CS), in which the catalytic Cys-393 is replaced by Ser. Using a series of deletion mutants, we provided evidence that both the phosphatase (P) domain and the adjacent B domain are indispensable for P-cofilin binding of SSH1(CS) and cofilin-phosphatase activity of SSH1. In contrast, the A domain is required for the F-actin-mediated activation of SSH1, but not for P-cofilin binding or basal cofilin-phosphatase activity. The P domain alone is sufficient for the phosphatase activity toward p-nitrophenyl phosphate (pNPP), indicating that the SSH-N domain is not essential for the basal phosphatase activity of SSH1. Addition of F-actin increased the cofilin-phosphatase activity of SSH1 more than 1200-fold, but the pNPP-phosphatase activity only 2.2-fold, which suggests that F-actin principally affects the cofilin-specific phosphatase activity of SSH1. When expressed in cultured cells, SSH1, but not its mutant deleted of SSH-N, accumulated in the rear of the lamellipodium. Together, these findings suggest that the conserved SSH-N domain plays critical roles in P-cofilin recognition, F-actin-mediated activation, and subcellular localization of SSH1.
Kurita
Souichi
S
Department of Biomolecular Sciences, Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi 980-8578, Japan.
Watanabe
Yosuke
Y
Gunji
Emi
E
Ohashi
Kazumasa
K
Mizuno
Kensaku
K
D000199:Actins / Q000737:chemistry*; D002384:Catalysis; D051338:Cofilin 1 / Q000737:chemistry* / Q000235:genetics; D003545:Cysteine / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation; D006801:Humans; D008840:Microfilament Proteins / Q000737:chemistry; D010749:Phosphoprotein Phosphatases / Q000737:chemistry / Q000235:genetics* / Q000502:physiology*; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011554:Pseudopodia / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D013379:Substrate Specificity
D000199:Actins; D051338:Cofilin 1; D008840:Microfilament Proteins; C072061:coronin proteins; D012694:Serine; D010749:Phosphoprotein Phosphatases; C484329:SSH1 protein, human; D010744:Phosphoric Monoester Hydrolases; D003545:Cysteine