Bardgette J, Abboud H E, Choudhury G G
Department of Medicine, University of Texas Health Science Center at San Antonio 78284, USA.
J Recept Signal Transduct Res. 1999 Sep;19(5):865-84. doi: 10.3109/10799899909042878.
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.
钒酸盐是一种具有胰岛素样作用的物质，对酪氨酸磷酸酶有强大的抑制作用。我们最近证明，低浓度的钒酸盐能刺激磷酸酪氨酸依赖性信号转导通路，从而导致系膜细胞中的基因表达和DNA合成。为了进一步研究钒酸盐激活系膜细胞的机制，我们研究了其对信号转导子和转录激活子（STAT）的影响。用特异性高亲和力的sis诱导性DNA元件（SIE）孵育钒酸盐刺激的系膜细胞裂解物，会形成蛋白质-DNA复合物。使用抗STAT1α单克隆抗体进行的超迁移分析表明，它在DNA-蛋白质复合物中单独存在。用抗磷酸酪氨酸抗体或过量的磷酸酪氨酸孵育细胞裂解物，会导致STAT1α与SIE探针的结合减少，这表明该转录因子的酪氨酸磷酸化和二聚化对其激活是必要的。免疫沉淀后进行免疫复合物激酶分析表明，在钒酸盐处理的系膜细胞中，Janus激酶2（JAK2）的酪氨酸激酶活性增加。向钒酸盐处理的系膜细胞裂解物中添加单克隆抗磷酸丝氨酸抗体，会导致蛋白质-DNA复合物超迁移，表明该复合物中存在丝氨酸磷酸化的STAT1α。用丝氨酸磷酸酶PP2A处理钒酸盐刺激的系膜细胞裂解物，会抑制DNA-蛋白质相互作用。总的来说，我们的数据表明，钒酸盐处理期间系膜细胞激活的至少一种机制是通过酪氨酸和丝氨酸磷酸化增加STAT1α的激活。
