Actin filaments-stabilizing and -bundling activities of cofilin-phosphatase Slingshot-1.
Slingshot-1 (SSH1) is known to regulate actin filament dynamics by dephosphorylating and activating cofilin, an actin-depolymerizing factor. SSH1 binds to filamentous (F-) actin through its multiple F-actin-binding sites and its cofilin-phosphatase activity is enhanced by binding to F-actin. In this study, we demonstrate that SSH1 has F-actin-stabilizing and -bundling activities. In vitro actin depolymerization assays revealed that SSH1 suppressed spontaneous and cofilin-induced actin depolymerization in a dose-dependent manner. SSH1 inhibited F-actin binding and severing activities of cofilin. Low-speed centrifugation assays combined with fluorescence and electron microscopic analysis revealed that SSH1 has F-actin-bundling activity, independently of its cofilin-phosphatase activity. Deletion of N- or C-terminal regions of SSH1 significantly reduced its F-actin-stabilizing and -bundling activities, indicating that both regions are critical for these functions. As SSH1 does not form a homodimer, it probably bundles F-actin through its multiple F-actin-binding sites. Knockdown of SSH1 expression by RNA interference significantly suppressed stress fiber formation in C2C12 myoblast cells, indicating a role for SSH1 in stress fiber formation or stabilization in cells. SSH1 thus has the potential to regulate actin filament dynamics and organization in cells via F-actin-stabilizing and -bundling activities, in addition to its ability to dephosphorylate cofilin.
Kurita
Souichi
S
Department of Biomolecular Sciences, Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi 980-8578, Japan.
Gunji
Emi
E
Ohashi
Kazumasa
K
Mizuno
Kensaku
K
D000199:Actins / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001704:Biopolymers / Q000737:chemistry / Q000378:metabolism; D002460:Cell Line; D051340:Cofilin 2 / Q000378:metabolism*; D019281:Dimerization; D004355:Drug Stability; D051379:Mice; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020836:Protein Structure, Quaternary; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D022502:Stress Fibers / Q000378:metabolism
D000199:Actins; D001704:Biopolymers; D051340:Cofilin 2; D034741:RNA, Small Interfering; D010749:Phosphoprotein Phosphatases