REGULATION OF CD45 ACTIVITY BY PHOSPHORYLATION

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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.