CD45 is a receptor-type protein tyrosine phosphatase (PTP) which is ubiquitous and abundant in hematopoietic cells. The overall objective of this study is to delineate the role of CD45 phosphorylation in the regulation of cell division in normal and transformed cells. We previously reported that CD45 PTP activity is elevated in lymphocytes during G1 and G2/M. The temporal relationship of this elevation with the phosphorylation events occurring in the cell cycle suggests an important role for CD45 in the regulation of the phosphorylation state of membrane signaling elements. This study is designed to determine whether CD45 becomes activated during the cell cycle. The role of CD45 will be studied by separation of CTLL-2 cells into cell cycle stages by centrifugal elutriation, followed by detailed analysis of CD45 phosphorylation and PTP activity. The kinetics of cell cycle progression will be compared in CD45 positive and negative cells. The detailed phosphorylation sites of CD45 in cell cycle phases have been determined by phosphoamino acid analysis, by phosphopeptide mapping and by MALDI-MS. This approach has already led to the identification of six sites. Key phosphorylation sites are being modified by site directed mutagenesis to confirm their role in CD45 activity. Finally, the proteins and protein kinases which associate with CD45 cells will be identified. These studies are important to the understanding of phosphorylation events which regulate the cell cycle and the receptivity of cells to external signals, and will contribute to the understanding of the deregulation of the cell cycle often associated with neoplastic events and cancer.
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