Activation of Stat1α by Phosphatase Inhibitor Vanadate in Glomerular Mesangial Cells: Involvement of Tyrosine and Serine Phosphorylation

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Abstract
Vanadate is an insulinomimetic agent that has potent inhibitory effect on tyrosine phosphatases. We have recently demonstrated that low concentration of vanadate stimulates phosphotyrosine-dependent signal transduction pathways leading to gene expression and DNA synthesis in mesangial cells. To further examine the mechanisms by which vanadate activates mesangial cell, we studied its effect on signal transducer and activators of transcription (STAT). Incubation of lysates from vanadate-stimulated mesangial cells with a specific high affinity sis-inducible DNA element (SIE) resulted in the formation of protein-DNA complex. Supershift analysis using monoclonal antibody against STAT1 alpha showed its exclusive presence in the DNA-protein complex. Incubation of cell lysate with antiphosphotyrosine antibody or with excess phosphotyrosine caused decrease in binding of STAT1 alpha to SIE probe indicating that tyrosine phosphorylation and dimerization of this transcription factor are necessary for its activation. Immunoprecipitation followed by immunecomplex kinase assay showed increased tyrosine kinase activity of Janus kinase 2 (JAK2) in vanadate-treated mesangial cells. The addition of a monoclonal antiphosphoserine antibody to lysates from vanadate-treated mesangial cells results in supershift of protein-DNA complex indicating the presence of serine phosphorylated STAT1 alpha in this complex. Treatment of lystates from vanadated-stimulated mesangial cells with serine phosphatase PP2A causes inhibition of DNA-protein interaction. Collectively, our data indicate that at least one mechanism of activation of mesangial cells during vanadate treatment is increased activation of STAT1 alpha by both tyrosine and serine phosphorylation.