Protein-tyrosine kinases have been broadly implicated in the control of both normal and neoplastic cell proliferation. For example, the binding of many polypeptide growth factors to their cellular receptors results in the rapid activation of a receptor- associated protein-tyrosine kinase that phosphorylates the receptor itself, as well as a number of cellular proteins. Furthermore, the ability of many oncogenic retroviruses to transform cells in dependent on their encoding and expressing a constitutively active protein-tyrosine kinase. As an initial step towards our ultimate goal of understanding the mechanisms whereby tyrosine phosphorylation regulates cell growth, we recently produced a monoclonal antibody that has high affinity and great specificity for phosphotyrosyl-proteins. Making extensive use of this antibody, the proposed studies seek to identify cellular functions related to cell proliferation that are controlled by protein- tyrosine kinases and their cellular substrates. These studies seek to : (i) identify cellular responses to growth factors and to oncogenic proteins that are perturbed by antibodies to phosphotyrosine; (ii) identify cellular functions influenced by individual phosphotyrosyl-proteins by inserting them into quiescent, non-transformed cells; (iii) prepare antibodies to three cellular phosphotyrosyl-proteins that appear to be involved in tyrosine-kinase responses and to use the antibodies to examine the expression, distribution and subcellular location of these three proteins as well as to examine and perturb their functions in intact cells. A main approach in all these aims involves the insertion of antibodies (or phosphotyrosyl proteins) into cells by osmotic lysis of pinocytic vesicles.
