REGULATION OF CD45 ACTIVITY BY PHOSPHORYLATION

描述(改编自调查摘要)：CD45是一种跨膜 受体类型蛋白酪氨酸磷酸酶(PTP)高表达于 几乎所有的造血系细胞。CD45的表达是 对抗原活化以及T细胞和T细胞的分化至关重要 B淋巴细胞。CD45研究的一个关键优势是 修改CD45的实验可以用相关的功能分析来测量 -即抗原激活。调查人员最近报告了这种使用 新的方法来识别在体内，自然发生的 CD45的磷酸化位点。这项研究的假设是 CD45的磷酸化调节CD45的功能和活性，并且 CD45磷酸化与其在抗原介导中的作用 激活。调查员计划实现这些目标如下。这个 CD45在抗原活化细胞中的磷酸化位点将被确定 通过放射性标记细胞，然后分离和分析CD45 免疫沉淀、肽图谱、高效液相色谱和MALDI-MS。这个 上面确定的关键磷酸化短链在CD45 PTP活性中的作用将 通过磷酸化位点的突变来确定，然后 酶活性和底物专一性的测定。最后， CD45的磷酸化位点突变形式重建细胞周期的能力 CD45阴性细胞的信号反应性将被确定。这些 研究对于理解磷酸化事件很重要 调节T细胞对抗原信号的接受性。

DESCRIPTION (Adapted from Investigator's abstract): CD45 is a transmembrane receptor-type protein tyrosine phosphatase (PTP) that is highly expressed on virtually all cells of the hematopoietic lineage. Expression of CD45 is essential for antigen activation as well as for the differentiation of T and B lymphocytes. A key advantage to the study of CD45 is that the outcome of experiments modifying CD45 can be measured with a relevant functional assay - i.e., antigen activation. The investigator has recently reported the use of novel methods to identify the in vivo, naturally occurring phosphorylation sites of CD45. The hypothesis of this study is that the phosphorylation of CD45 regulates the function and the activity of CD45, and to relate CD45 phosphorylation to its function in antigen mediated activation. The investigator plans to address these goals as follows. The phosphorylation sites of CD45 in antigen activated cells will be identified by radiolabeling cells followed by isolation and analysis of CD45 by immunoprecipitation, peptide mapping, HPLC and MALDI-mass spectrometry. The role of key phosphorylation sties identified above in CD45 PTP activity will be determined by mutagenesis of phosphorylation sites followed by determination of enzyme activity and substrate specificity. Finally, the ability of phosphorylation site-mutant forms of CD45 to reconstitute the signaling responsiveness of CD45 negative cells will be determined. These studies are important to the understanding of phosphorylation events which regulate the receptivity of T cells to antigenic signals.