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Cofilin plays an essential role in actin filament dynamics and membrane protrusion in motile cells. Cofilin is inactivated by phosphorylation at Ser-3 by LIM kinase and reactivated by dephosphorylation by cofilin-phosphatase Slingshot (SSH). Although cofilin is dephosphorylated in response to various extracellular stimuli, signaling pathways regulating SSH activation and cofilin dephosphorylation have remained to be elucidated. Here we show that insulin stimulates the phosphatase activity of Slingshot-IL (SSH1L) and cofilin dephosphorylation in cultured cells, in a manner dependent on phosphoinositide 3-kinase (PI3K) activity. Consistent with this, the level of Ser-3-phosphorylated cofilin is increased in PTEN (phosphatase and tensin homolog deleted in chromosome 10)-overexpressing cells and decreased in PTEN-deficient cells. Insulin induced the accumulation of SSH1L and active Akt (a downstream effector of PI3K), together with a PI3K product phosphatidylinositol 3,4,5-trisphosphate, onto membrane protrusions. Cofilin, but not Ser-3-phosphorylated coillin, accumulated in membrane protrusions in insulinstimulated cells, indicating that cofilin is dephosphorylated in these areas. Finally, suppression of SSHIL expression by RNA interference abolished insulin-induced cofilin dephosphorylation and the membrane protrusion. These findings suggest that SSHIL is activated downstream of PI3K and plays a critical role in insulin-induced membrane protrusion by dephosphorylating and activating cofilin. [ABSTRACT FROM AUTHOR]

Examines the role of stress-activated protein kinase/c-Jun NH[sub 2]-terminal kinase (SAPK/JNK) in stress signaling. Generation of mkk7 mouse embryonic stem (ES) cells in addition to sek1 cells; Comparison of the two kinases in terms of the activation and phosphorylation of JNK; Generation of MKK7-deficient ES cells.