Chronic myelogenous leukemia (CML) arises from a genetic abnormality, the Philadelphia chromosome, that consists of reciprocal translocation of portions of human chromosomes 9 and 22. The fusion protein that results from this somatic cell translocation, called p210bcr/abl, is a protein kinase with abnormally high tyrosine kinase activity, a property shared with the v-abl viral oncogene product. The apparent abnormal level of tyrosine kinase activity of p210 is correlated with the occurrence of the disease in both chronic and acute stages, ultimately leading to abnormal numbers of myeloid cells. The overall goal of this project is to understand how the phosphorylation of the p210bcr/abl fusion protein relates to its functions, and how these phosphorylations and functions are altered in myeloid cells expressing p210bcr/abl compared to those on c-ABL or BCR proteins in normal myeloid cells. The initial goal is to identify and quantitate sites on the proteins that are phosphorylated in vivo, as they are found in human cell lines of myeloid lineage expressing p210bcr/abl. The functional relevance of these phosphorylations will be probed through mutagenesis of the corresponding sites on plasmids, followed by expression f the mutant proteins. The identity of the protein kinases involved in the functionally relevant phosphorylation events will be evaluated through substrate specificity analysis, stable complex formulation, and cell division cycle dependence.
The specific aims of this project are: 1. To determine the sites and extent of phosphorylations on the p210bcr/abl fusion protein compared to those on the BCR and c-ABL proteins in cells of myeloid lineage. 2. To analyze the function of the phosphorylation sites by examining the effects of mutations in those sites in c-abl and bcr/abl. 3. To identify the protein kinases responsible for the functional phosphorylations on BCR, c-ABL and p210bcr/abl. Regulation of p210bcr/abl activities, including DNA binding, tyrosine kinase, localization, and interactions with other proteins, appear to be regulated by phosphorylation. Understanding the regulation of the activities of the p210bcr/abl protein relative to that of the normal c- Abl and BCR proteins will give insight into the molecular basis of chronic myelogenous leukemia. Further, these findings may suggest targets for new chemo-therapeutic approaches to controlling CML.
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