Insulin receptor substrate-4 binds to slingshot-1 phosphatase and promotes cofilin dephosphorylation

Yuta Homma, Shin Ichiro Kanno, Kazutaka Sasaki, Michiru Nishita, Akira Yasui, Tomoichiro Asano, Kazumasa Ohashi, Kensaku Mizuno

Research output: Contribution to journalArticlepeer-review

15 Citations (Scopus)

Abstract

Cofilin plays an essential role in cell migration and morphogenesis by enhancing actin filament dynamics via its actin filament- severing activity. Slingshot-1 (SSH1) is a protein phosphatase that plays a crucial role in regulating actin dynamics by dephosphorylating and reactivating cofilin. In this study, we identified insulin receptor substrate (IRS)-4 as a novel SSH1- binding protein. Co-precipitation assays revealed the direct endogenous binding of IRS4 to SSH1. IRS4, but not IRS1 or IRS2, was bound to SSH1. IRS4 was bound to SSH1 mainly through the unique region (amino acids 335-400) adjacent to the C terminus of the phosphotyrosine-binding domain of IRS4. The N-terminal A, B, and phosphatase domains of SSH1 were bound to IRS4 independently. Whereas in vitro phosphatase assays revealed that IRS4 does not directly affect the cofilin phosphatase activity of SSH1, knockdown of IRS4 increased cofilin phosphorylation in cultured cells. Knockdown of IRS4 decreased phosphatidylinositol 3-kinase (PI3K) activity, and treatment with an inhibitor of PI3K increased cofilin phosphorylation. Akt preferentially phosphorylated SSH1 at Thr-826, but expression of a non-phosphorylatable T826A mutant of SSH1 did not affect insulin-induced cofilin dephosphorylation, and an inhibitor of Akt did not increase cofilin phosphorylation. These results suggest that IRS4 promotes cofilin dephosphorylation through sequential activation of PI3K and SSH1 but not through Akt. In addition, IRS4 co-localized with SSH1 in F-actin-rich membrane protrusions in insulin-stimulated cells, which suggests that the association of IRS4 with SSH1 contributes to localized activation of cofilin in membrane protrusions.

Original languageEnglish
Pages (from-to)26302-26313
Number of pages12
JournalJournal of Biological Chemistry
Volume289
Issue number38
DOIs
Publication statusPublished - 2014 Sep 19

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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