Regulation of STAT Activity by Tyrosine Phosphatases

描述(申请人提供)：细胞因子通过酪氨酸磷酸化激活潜伏的细胞质转录因子STAT(信号转导和转录激活因子)家族。酪氨酸磷酸化的STATs转位到细胞核，在那里它们与DNA结合以激活转录。STAT的活性依赖于酪氨酸磷酸化，它在细胞核中的失活是由一种以前未知的蛋白质酪氨酸磷酸酶完成的。结构性STAT的激活与癌症有关。利用生物化学和遗传学相结合的方法，我们现在已经确定TC45是一种蛋白磷酸酶，可以使细胞核中的STAT1失活。这项建议的总体目标是研究STAT酪氨酸去磷酸化的分子机制、调节和特异性。这些研究可能会增强我们利用细胞因子设计合理治疗策略的能力。 为了研究STAT去磷酸化的分子机制，我们将通过体外和体内相互作用试验来检验TC45是否优先去磷酸化STAT1二聚体或单体。参与TC45识别STAT1的蛋白质结构域将通过突变分析和遗传重组实验进行检测。为了研究STAT去磷酸化的调控，我们将研究STAT1 DNA结合活性以及STAT1相互作用的蛋白是否可能调节STAT1去磷酸化。将分析细胞因子在转录或转录后水平对TC45的可能调节。对耐酪氨酸去磷酸化的突变体STAT1进行检测，以确定其在调节STAT1去磷酸化过程中可能存在的缺陷。为了研究STAT去磷酸化的特异性，我们将进一步研究TC45介导的STAT去磷酸化在不同类型的TC45缺失细胞中的特异性。我们将通过生化分析和结构域交换分析来验证TC45对STATS具有内在底物特异性的假设。其他统计数据的去磷酸化将被检查。

DESCRIPTION (provided by applicant): Cytokines activate the STAT (signal transducer and activator of transcription) family of latent cytoplasmic transcription factors by tyrosine phosphorylation. Tyrosine phosphorylated STATs translocate into the nucleus where they bind to DNA to activate transcription. The activity of STAT is dependent on tyrosine phosphorylation and its inactivation in the nucleus is accomplished by a previously unknown protein tyrosine phosphatase. Constitutive STAT activation has been associated with cancer. Using combined biochemical and genetic approaches, we have now identified TC45 as a protein phosphatase that can inactivate Stat1 in the nucleus. The overall goal of this proposal is to study the molecular mechanism, the regulation, and the specificity of STAT tyrosine dephosphorylation. These studies may enhance our ability to design rational therapeutic strategies employing cytokines. To study the molecular mechanism of STAT dephosphorylation, we will test if TC45 preferentially dephosphorylates Stat1 dimer or monomers by in vitro and in vivo interaction assays. Protein domains involved in the recognition of Stat1 by TC45 will be examined by mutational analysis and genetic reconstitution experiments. To study the regulation of STAT dephosphorylation, we will investigate if Stat1 DNA binding activity as well as Stat1-interacting proteins may regulate Stat1 dephosphorylation. The possible regulation of TC45 by cytokines at transcriptional or post-transcriptional levels will be analyzed. A mutant Stat1 resistant to tyrosine dephosphorylation will be examined for possible defects in the regulation of Stat1 dephosphorylation. To study the specificity of STAT dephosphorylation, we will further characterize the specificity of TC45-mediated STAT dephosphorylation in different types of TC45-null cells. We will test the hypothesis that TC45 has intrinsic substrate specificity toward STATs by biochemical assays and domain-swapping analysis. The dephosphorylation of other STATs will be examined.