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.
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ABSTRACT: We have observed dephosphorylation of the soluble, 48 kDa insulin receptor tyrosine kinase domain following its tyrosine autophosphorylation. Dephosphorylation was associated with generation of inorganic phosphate, thereby making catalysis by reversal of the kinase reaction unlikely. The kinase domain preparations could not be shown to contain detectable, contaminating protein tyrosine phosphatase activity. In addition, dephosphorylation was insensitive to protein phosphatase inhibitors. However, it was blocked by the kinase inhibitor staurosporine. These results are consistent with insulin receptor kinase domain auto-dephosphorylation via catalysis involving the kinase itself. These findings raise the possibility of a novel mechanism for termination of the insulin receptor signal.Biochemical and Biophysical Research Communications 01/1993; 189(3):1457-63. · 2.48 Impact Factor