The broad, long-term objectives of the project are to identify and characterize protein tyrosine phosphatases (PTPases) as potential antagonists of the aberrant tyrosine phosphorylation associated with chronic myelogenous leukemia (CML). CML is a clonal disorder of the haematopoietic stem cell characterized by the Philadelphia chromosome, in which the c-Abl proto-oncogene becomes linked to the bcr gene on chromosome 22. This results in the generation of a fusion protein termed p210 bcr/abl, the kinase activity of which is enhanced relative to c-Abl, producing abnormal patterns of tyrosine phosphorylation in CML cells. Much research effort has focussed on the p210 bcr/abl kinase and the role of tyrosine phosphorylation in the disease. However protein phosphorylation in vivo is a reversible process in which the net phosphorylation state of a particular substrate depends upon the balance between the competing action of kinases and phosphatases. This project examines the potential involvement of PTPases in CML.
The specific aims are: 1) To characterize the role of PTP1B in controlling Abl functions in terms of: a) the physical association between PTP1B and c-Abl p210 bcr/abl, b) the effect of association of the enzyme activity of each component, c) the biological consequence of association and the ability of PTP1B to antagonize Abl. 2) To characterize modifications of PTP1B that may play a role in enhancing the stability of the PTP1B protein in p210 bcr/abl expressing derivatives of Mo7 cells. 3) To characterize a novel ser/Thr kinase that is recovered in immunoprecipitates of PTP1B. 4) To characterize novel PTPases in CML model systems and in human patient samples. The project presents chemical and molecular/cell biological approaches to studying PTPases in CML. The interaction between PTP1B and Abl will be defined by deletion/site directed mutagenesis. The effects of each component on the activity of the other will be determine in enzymatic assays. The biological consequences will be ascertained using retroviral expressions systems, for example to examine the potential of PTPases such as PTP1B to counter bcr/abl function. Covalent modifications, particularly phosphorylation, of PTP1B will be characterized by direct sequencing and comparative phosphopeptide mapping of in vitro and in vivo phosphorylated protein. Potential roles for such modification in determining the half-life of PTP1B and in controlling protein:protein interactions will be studied by a variety of techniques including analysis of immunoprecipitates of the PTPase from labelled cells. The grant also proposes reverse transcriptase-PCR based strategies to identify novel PTPases in CML model systems and patient samples to establish differences in PTPase expression that correlate with the presence of p210 bcr/abl. The health-relatedness of the project is that through the identification of PTPases that antagonize the activity of bcr/abl new insights will be provided into the molecular mechanisms underlying CML and new potential targets for therapeutic intervention will be uncovered.
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