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.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 T 细胞的激活和增殖受到造血酪氨酸磷酸酶 (HePTP)（一种 38 kDa I 类非受体蛋白酪氨酸磷酸酶）的负调节。 HePTP 通过使激活环中的 Tyr185 去磷酸化，使细胞外信号调节激酶 2 (Erk2)（一种丝裂原激活蛋白激酶 (MAPK)）失活。除了其 C 端 PTP 催化结构域外，HePTP 还包含一个短的 N 端激酶相互作用基序 (KIM)，这对于 Erk2 结合至关重要。我们研究的目的是通过解析以下晶体结构来了解 HePTP 对 Erk2 去磷酸化的底物特异性的分子基础：1) HePTP 的 PTP 催化结构域与对应于 Erk2 双重磷酸化激活环的肽结合， 2) 全长 HePTP 与全长双磷酸化 Erk2 蛋白结合。为了选择性地填充这些瞬时复合物，我们生成了多个 HePTP 构建体，其中它们的关键催化残基已突变为丝氨酸或丙氨酸。酶活性显着降低但底物亲和力未改变的那些突变体充当底物捕获突变体。我们确定突变的 Cys270（HePTP 的催化半胱氨酸）比野生型 HePTP 的磷酸酶活性降低了几个数量级。因此，Cys270 突变体可能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 Erk2 肽和 HePTP Cys270 突变体的混合物中获得了晶体。因为之前报道 WT-HePTP 仅在含有磷酸盐或磷酸盐类似物的条件下结晶，所以我们相信我们的 HePTP Cys270 突变体晶体含有结合在其活性位点的 Erk2 肽。