The broad long term goal of the project is to characterize the functional role of GAT62, a protein identified as being constitutive tyrosine phosphorylated in chronic phase progenitor cells from human chronic myelogenous leukemia (CML) patients. CML is a clonal disorder of the hematopoietic stem cell characterized by the Philadelphia chromosome in which the c-abl proto-oncogene becomes linked to the bcr gene on chromosome 22, resulting in a chimeric protein termed p210 bcr/abl with deregulated tyrosine kinase activity. There is significant evidence implying that the elevated tyrosine kinase activity of bcr-abl is responsible for the proliferative and maturational abnormalities evident in the chronic phase of CML. However, the intracellular signaling cascades perturbed by bcr/abl remain to be defined. This project examines the potential role of GAT62 as mediator of p210 bcr/abl signal transduction.
The specific aims are: 1) To determine whether p210 bcr/abl is the protein kinase responsible for the tyrosine phosphorylation on GAT62. 2) To map the domain(s) of GAT62 and p210 bcr/abl required for interaction. 3) To characterize the relevance of GAP-p210 bcr/abl interaction. 4) To identify and characterize proteins that associate with GAT62. 5) To define the subcellular localization of constitutive tyrosine phosphorylatd GAT62. 6) To examine the involvement of GAT62 in P210 bcr/abl mediated transformation. The project presents biochemical and molecular/cell biological approaches to study the functional role of GAT62 in CML. In vitro binding studies and phosphopeptide mapping will be performed to address whether GAT62 is a direct substrate of p210 bcr/ab1 and deletion/site directed mutagenesis will be employed for domain mapping. The importance of the GAT62/GAP interaction will be defined by performing GTPase activating protein assays, measuring the Ra-bound GDP/GTP ration and defining the subcellular localization of tyrosine phosphorylated GAT62 and GAP. The latter will be examined using two approaches, standard subcellular frationation techniques and immunochemistry. The biological relevance of GAT62 will be assessed using two assays, an in vitro assay with rat-1 fibroblasts and an in vivo assay with primary bone marrow cells. The health-relatedness of this project is that by defining the function of GAT62, as potential substrate of p210 bcr-abl, new insights may be provided into the molecular mechanisms underlying CML and new potential targets for therapeutic intervention by uncovered.
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