IDENTIFICATION AND FUNCTIONAL CHARACTERIZATION OF P210 BCR/ABL SUBSTRATE

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The broad long term goal of this project is to understand, on a molecular level, how the p210 bcr/abl protein tyrosine kinase activity ultimately causes a myeloid expansion in chronic phase CML. In order to achieve this objective, it becomes essential to: (a) Identify and characterize the relevant p210bcr/abl substrates, and their interactions, in primary primitive chronic phase CML blasts by immunoprecipitation and immunoblotting with available antibodies to known proteins or by protein purification, sequencing and cloning of, an generation of antibodies to, potential novel proteins; (b) Ascertain the biological functions of the relevant substrates. Ascertain whether the constitutive tyrosine phosphorylation of a substrate in primary primitive chronic phase CML blasts is aberrant or untimely by examining primary primitive and maturing normal subpopulations; (d) Determine the growth factor signal transduction pathways in which the relevant substrates may be involved. The specific aims of the project are: Aim 1. To further identify and characterize the relevant p210 bcr/abl substrates and signal transduction pathways affected in primary primitive chronic phase CML blasts: (1a) Characterization of other known relevant substrates; (1c) Further identification of relevant substrates; (id) Identification of P-tyr proteins in kit ligand pathway in primary primitive normal blasts. Aim 2. To isolate, sequence and characterize p56; (2a) cDNA cloning and sequencing; (2b) Antibody production; (2c) Functional analyses. Aim 3. To analyze the structure and function of p155, a protein whose tyrosine phosphorylation is elevated in primary CML blasts (3a) Further characterization of p155 in primary chronic phase CML blasts and p210 bcr/abl expressing cell lines; (3b) Purification of p155; (3c) Cloning and sequencing p p155 (ed) Functional analyses.