The association of protein-tyrosine kinase activity with growth factor receptors and oncogene products suggests an important role for these enzymes in the regulation of cellular proliferation. We feel that useful model systems are needed for studying protein-tyrosine kinases that are under normal cellular control. The system we have chosen is lymphocytes, whose normal response to external stimuli is to proliferate and differentiate. It is our hypothesis that protein-tyrosine kinases play important roles in the regulation of these responses. To test this hypothesis we have taken both biochemical and cellular approaches. Our biochemical approach has included the identification of a novel 40,000 dalton, cytosolic, protein-tyrosine kinase. This enzyme appears to be detectable in lymphoctye homogenates only after the removal of an endogenous inhibitor. We have purified this enzyme to near homogeneity from calf thymus using a number of chromatographic procedures. We are proposing to study its physical properties, substrate specificity, distribution, regulation and role in lymphocyte function. We have also identified a T cell-specific, membrane-associated, protein-tyrosine kinase that is down-regulated in response to mitogens or tumor-promoting phorbol esters. We have proposed, in this study, to elucidate the mechanisms that trigger the loss of this kinase from the membrane and examine the consequences of this event on its activity and location within the cell. To determine if the down-regulation of a membrane-associated protein-tyrosine kinase is a general mechanism of lymphocyte activation, we have proposed also to examine the effects of mitogens on B lymphocyte enzymes. We are also interested in how the various protein-tyrosine kinases in lymphocytes interact with one another and with other protein kinases present in the cell. The methodologies that we propose to use include: 1) enzyme purification and characterization, 2) ATP:peptide phosphotransferase assays, 3) cell culture of lymphocyte subpopulations and transformed lymphoid cell lines, 4) subcellular fractionation and detection of endogenous protein kinases, 5) antibody production, and 6) 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.
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