The association of protein tyrosine phosphorylation with the regulation of the growth properties of cells has justifiably generated considerable interest in the identification of protein-tyrosine kinases that specifically interact with growth regulating receptors. Recently, much of this interest has been directed toward lymphocytes and the antigen receptors found on their cell surfaces. B and T lymphocytes express higher levels of protein-tyrosine kinase activity than are found in most other nontransformed cells and, as important mediators of cellular and humoral immune responses, they interact with a variety of external factors that influence their growth and differentiation. Thus, lymphocytes provide opportunities for the study of new mechanisms of signal transduction that are coupled to changes in tyrosine phosphorylation. The long-term objective of our research has been to understand on a molecular basis the role of protein-tyrosine kinases in regulating cell growth in lymphocytes. To understand the role of tyrosine phosphorylation in the response to lymphocytes to regulators of growth and differentiation, it is important to first understand the identities and characteristics of the kinases that are expressed in specific lymphoid cell types. this proposal focuses on the characterization of a novel protein-tyrosine kinase, PTK72, which is part of a signal transduction complex associated with the B lymphocyte antigen receptor. Proposed studies are focused on: 1) characterization of the association of PTK72 with the antigen receptor and its regulation by receptor crosslinking; 2) characterization of the expression and cellular distribution of PTK72 and its role in activation in other cell types; and 3) isolation and sequence analysis of the cDNA for PTK72. The methodologies that we propose to use include: 1) cellular assays of lymphocyte activation using cultured lymphocyte subpopulations and transformed lymphoid cell lines, 2) immunoprecipitations and immune-complex kinase assays, 3) ATP:peptide and ATP:protein phosphotransferase assays, 4) cloning and expression of cDNAs for lymphocyte protein-tyrosine kinases, 5) Western and Northern blot analyses, 6) identification and use of specific protein-tyrosine kinase inhibitors, 7) immunofluorescence localization, 6) antibody production, and 7) biosynthetic labeling of protein kinases. It is our contention that a greater understanding of the biochemical pathways that regulate normal cell growth and development is necessary to begin to define the lesions within this pathway that can lead to malignant growth. It is likely that the molecular mechanisms of signal transduction in B lymphocytes will serve as a prototype for receptor signaling pathways in a variety of cell types.
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