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Nox1‐mediated activation of Slingshot phosphatase in VSMC
Author(s) -
Martin Alejandra San,
Maheswaranathan Mithunan,
Griendling Kathy K
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.lb185
Subject(s) - nox1 , dephosphorylation , phosphorylation , microbiology and biotechnology , cofilin , chemistry , vascular smooth muscle , phosphatase , platelet derived growth factor receptor , nadph oxidase , transfection , biology , biochemistry , growth factor , receptor , cell , cytoskeleton , actin cytoskeleton , reactive oxygen species , endocrinology , smooth muscle , gene
PDGF stimulates vascular smooth muscle cell (VSMC) migration by a NADPH oxidase 1 (Nox1)‐derived, H 2 O 2 dependent mechanism. Migration is partially controlled by cofilin, an actin binding protein activated by Slingshot‐1L (SSH1L) phosphatase. We previously showed that SSH1L is activated and required for PDGF‐mediated cofilin activation and migration in VSMCs, but the mechanism of SSH1L activation is unclear. 14‐3‐3 proteins are one potential regulatory mechanism, and bind to partners that contain phosphorylated consensus motifs. We hypothesized that PDGF disrupts a SSH1L/14‐3‐3 inhibitory complex through Nox1/H 2 O 2 ‐mediated SSH1L dephosphorylation in a 14‐3‐3 binding motif. We found that SSH1L is Ser‐phosphorylated at a 14‐3‐3 binding motif (probably S834) and co‐immunoprecipitates with 14‐3‐3‐β and ‐γ. In cells transfected with 14‐3‐3K49E DN, which fails to recognize phosphorylated partners, SSH1L is activated (94±6 vs. 45±1 A.U.p=0.01), and PDGF has no further effect (44.6±1.4 vs. 40.2±10.8 A.U.). Interestingly, PDGF reduces phospho‐SSH1L and 14‐3‐3 binding in wild type (29±11 vs. 100±8 A.U.p=0.01) but not in Nox1 knockout cells (101±18 vs. 86±14 A.U.), and the SSH1L‐S834A mutant is constitutively active. Our work shows that PDGF induces SSH1L/14‐3‐3 complex disruption and SSH1L activation via its Nox1‐dependent dephosphorylation in a 14‐3‐3 binding motif.