HEMATOPOIETIC TYROSINE PHOSPHATASE

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 T细胞的激活和增殖受造血酪氨酸磷酸酶(HePTP)的负调控，HePTP是一种38 kDa的非受体蛋白酪氨酸磷酸酶。HePTP通过去磷酸化激活环中的Tyr185来灭活细胞外信号调节激酶2(ERK2)，ERK2是一种丝裂原激活的蛋白激酶(MAPK)。除了其C端的PTP催化结构域外，HePTP还含有一个短的N端激酶相互作用基序(KIM)，这是ERK2结合所必需的。本研究的目的是通过解决以下晶体结构来了解HePTP对ERK2去磷酸化的底物专一性的分子基础：1)HePTP的PTP催化结构域与ERK2的双磷酸化激活环相对应的多肽结合；2)全长HePTP与全长、双磷酸化的ERK2蛋白结合。为了选择性地填充这些瞬时络合物，我们产生了多个HePTP结构，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没有改变的突变体的功能是底物捕获突变体。我们确定突变的Cys270，HePTP的催化半胱氨酸，比野生型HePTP降低磷酸酶活性几个数量级。因此，Cys270突变体有可能成为底物捕捉突变体。在缺乏磷酸和磷酸类似物的条件下，我们从ERK2肽和HePTP Cys270突变体的混合物中获得了晶体。由于WT-HePTP先前已被报道仅在含有磷酸或磷酸类似物的条件下结晶，我们相信我们的HePTP Cys270突变体的晶体含有结合在其活性部位的ERK2肽。

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.