title: Platelet-derived growth factor (PDGF) regulates Slingshot phosphatase activity via Nox1-dependent auto-dephosphorylation of serine 834 in vascular smooth muscle cells.
abstract: Migration of vascular smooth muscle cells (VSMCs) contributes to vascular pathology. PDGF induces VSMC migration by a Nox1-based NADPH oxidase mediated mechanism. We have previously shown that PDGF-induced migration in VSMCs requires Slingshot-1L (SSH1L) phosphatase activity. In the present work, the mechanism of SSH1L activation by PDGF is further investigated. We identified a 14-3-3 consensus binding motif encompassing Ser-834 in SSH1L that is constitutively phosphorylated. PDGF induces SSH1L auto-dephosphorylation at Ser-834 in wild type (wt), but not in Nox1(-/y) cells. A SSH1L-S834A phospho-deficient mutant has significantly lower binding capacity for 14-3-3 when compared with the phospho-mimetic SSH1L-S834D mutant, and acts as a constitutively active phosphatase, lacking of PDGF-mediated regulation. Given that Nox1 produces reactive oxygen species, we evaluated their participation in this SSH1L activation mechanism. We found that H(2)O(2) activates SSH1L and this is accompanied by SSH1L/14-3-3 complex disruption and 14-3-3 oxidation in wt, but not in Nox1(-/y) cells. Together, these data demonstrate that PDGF activates SSH1L in VSMC by a mechanism that involves Nox1-mediated oxidation of 14-3-3 and Ser-834 SSH1L auto-dephosphorylation.
authors.affiliation: Department of Medicine, Division of Cardiology, Emory University, Atlanta, Georgia 30322.
authors.forename: Mithunan
authors.initials: M
authors.lastname: Maheswaranathan
authors.affiliation: Department of Medicine, Division of Cardiology, Emory University, Atlanta, Georgia 30322.
authors.forename: Hope K A
authors.initials: HKA
authors.lastname: Gole
authors.affiliation: Department of Medicine, Division of Cardiology, Emory University, Atlanta, Georgia 30322.
authors.forename: Isabel
authors.initials: I
authors.lastname: Fernandez
authors.affiliation: Department of Medicine, Division of Cardiology, Emory University, Atlanta, Georgia 30322.
authors.forename: Bernard
authors.initials: B
authors.lastname: Lassègue
authors.affiliation: Department of Medicine, Division of Cardiology, Emory University, Atlanta, Georgia 30322.
authors.forename: Kathy K
authors.initials: KK
authors.lastname: Griendling
authors.affiliation: Department of Medicine, Division of Cardiology, Emory University, Atlanta, Georgia 30322. Electronic address: asanmartin@emory.edu.
authors.forename: Alejandra
authors.initials: A
authors.lastname: San Martín
mesh_terms: D048948:14-3-3 Proteins / Q000235:genetics / Q000378:metabolism; D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D000818:Animals; D004789:Enzyme Activation / Q000502:physiology; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D009131:Muscle, Smooth, Vascular / Q000201:enzymology*; D020125:Mutation, Missense; D032389:Myocytes, Smooth Muscle / Q000201:enzymology*; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D000074624:NADPH Oxidase 1; D010084:Oxidation-Reduction; D010749:Phosphoprotein Phosphatases / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000502:physiology; D010982:Platelet-Derived Growth Factor / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000235:genetics / Q000378:metabolism
chemical_list: D048948:14-3-3 Proteins; D010982:Platelet-Derived Growth Factor; D012694:Serine; D009247:NADH, NADPH Oxidoreductases; D000074624:NADPH Oxidase 1; C000614711:NOX1 protein, mouse; D010749:Phosphoprotein Phosphatases; C529575:SSH1 protein, mouse