Activation of STAT1 alpha by phosphatase inhibitor vanadate in glomerular mesangial cells: Involvement of tyrosine and serine phosphorylation

Vanadate is an insulinomimetic agent that has potent inhibitory effect on t yrosine phosphatases. We have recently demonstrated that low concentration of vanadate stimulates phosphotyrosine-dependent signal transduction pathwa ys leading to gene expression and DNA synthesis in mesangial cells. To furt her examine the mechanisms by which vanadate activates mesangial cell, we s tudied its effect on signal transducer and activators of transcription (STA T). Incubation of lysates from vanadate-stimulated mesangial cells with a s pecific high affinity sis-inducible DNA element (SIE) resulted in the forma tion of protein-DNA complex. Supershift analysis using monoclonal antibody against STAT1 alpha showed its exclusive presence in the DNA-protein comple x. Incubation of cell lysate with antiphosphotyrosine antibody or with exce ss phosphotyrosine caused decrease in binding of STAT1 alpha to SIE probe i ndicating that tyrosine phosphorylation and dimerization of this transcript ion factor are necessary for its activation. Immunoprecipitation followed b y immunecomplex kinase assay showed increased tyrosine kinase activity of J anus kinase 2 (JAK2) in vanadate-treated mesangial cells. The addition of a monoclonal antiphosphoserine antibody to lysates from vanadate-treated mes angial cells results in supershift of protein-DNA complex indicating the pr esence of serine phosphorylated STAT1 alpha in this complex. Treatment of l ystates from vanadated-stimulated mesangial cells with serine phosphatase P P2A causes inhibition of DNA-protein interaction. Collectively, our data in dicate that at least one mechanism of activation of mesangial cells during vanadate treatment is increased activation of STAT la by both tyrosine and serine phosphorylation.