HEMATOPOIETIC TYROSINE PHOSPHATASE

造血酪氨酸磷酸酶

• 批准号: 8170599
• 负责人: Rebecca Page
• 金额: $ 0.56万
• 依托单位: BROOKHAVEN SCIENCE ASSOC-BROOKHAVEN LAB
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8170599
• 关键词: Active Sites; Affinity; Alanine; Binding; C-terminal; Catalytic Domain; Complex; Computer Retrieval of Information on Scientific Projects Database; Cysteine; Funding; Grant; Hematopoietic; Institution; Length; Mitogen Activated Protein Kinase 1; Mitogen-Activated Protein Kinases; Molecular; Mutate; N-terminal; Peptides; Phosphoric Monoester Hydrolases; Phosphotransferases; Protein Dephosphorylation; Protein Tyrosine Phosphatase; Proteins; Reporting; Research; Research Personnel; Resources; Serine; Source; Structure; Substrate Specificity; T-Cell Proliferation; United States National Institutes of Health; analog; base; human PTPRT protein; inorganic phosphate; mutant

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The activation and proliferation of T cells is negatively regulated by hematopoietic tyrosine phosphatase (HePTP), a 38 kDa class I non-receptor protein tyrosine phosphatase. HePTP inactivates the extracellular signal-regulated kinase 2 (Erk2), a mitogen-activated protein kinase (MAPK), by dephosphorylating Tyr185 in its activation loop. In addition to its C-terminal PTP catalytic domain, HePTP contains a short N-terminal kinase interaction motif (KIM), which is essential for Erk2 binding. The objectives of our research are to understand the molecular basis of substrate specificity of HePTP for Erk2 dephosphorylation by solving the crystal structures of the following: 1) the PTP catalytic domain of HePTP bound to a peptide corresponding to the dually phosphorylated activation loop of Erk2, and 2) full-length HePTP bound to full-length, dually phosphorylated Erk2 protein. In order to selectively populate these transient complexes, we have generated multiple constructs of HePTP in which their key catalytic residues have been mutated to serine or alanine. Those mutants with substantially reduced enzymatic activity but unaltered substrate affinity function as substrate-trapping mutants. We determined that mutating Cys270, the catalytic cysteine of HePTP, reduces phosphatase activity by several orders of magnitude than that of wild-type HePTP. Thus the Cys270 mutant may potentially serve as a substrate-trapping mutant. We obtained crystals from a mixture of the Erk2 peptide and the HePTP Cys270 mutant in conditions lacking both phosphate and phosphate analogs. Because WT-HePTP has previously been reported to crystallize only in conditions containing either phosphate or phosphate analogs, we believe that our crystals of the HePTP Cys270 mutant contain the Erk2 peptide bound at its active site.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 T细胞的活化和增殖受造血酪氨酸磷酸酶（HePTP）负调节，HePTP是38 kDa的I类非受体蛋白酪氨酸磷酸酶。HePTP通过使其激活环中的Tyr 185去磷酸化来使细胞外信号调节激酶2（Erk 2）失活，Erk 2是一种促分裂原活化蛋白激酶（MAPK）。除了其C-末端PTP催化结构域之外，HePTP还含有短的N-末端激酶相互作用基序（KIM），其对于Erk 2结合是必需的。我们的研究目的是通过解决以下晶体结构来理解HePTP对Erk 2去磷酸化的底物特异性的分子基础：1）HePTP的PTP催化结构域结合到对应于Erk 2的双磷酸化活化环的肽，以及2）全长HePTP结合到全长双磷酸化Erk 2蛋白。为了选择性地填充这些瞬时复合物，我们已经产生了HePTP的多个构建体，其中它们的关键催化残基已经突变为丝氨酸或丙氨酸。那些酶活性显著降低但底物亲和力不变的突变体作为底物捕获突变体发挥功能。我们确定，突变Cys 270，催化半胱氨酸的HePTP，降低磷酸酶活性的几个数量级比野生型HePTP。因此，Cys 270突变体可能潜在地充当底物捕获突变体。我们在缺乏磷酸盐和磷酸盐类似物的条件下从Erk 2肽和HePTP Cys 270突变体的混合物中获得晶体。因为WT-HePTP先前已被报道仅在含有磷酸盐或磷酸盐类似物的条件下结晶，我们相信我们的HePTP Cys 270突变体晶体含有结合在其活性位点的Erk 2肽。