The protein products of a number of retroviral oncogenes and growth factor receptors are protein-tyrosine kinases. A key question is how the signal produced by tyrosine kinase activity is transduced into changes in cellular biochemistry leading to the transformed state. We have studied the phosphorylation of serine residues of ribosomal protein S6 as one biochemical event amenable to analysis and one which is stimulated by many protein tyrosine-kinases. We have isolated the stimulated S6 kinase activity from insulin-treated oocytes and purified it to homogeneity. It is not a substrate for purified tyrosine kinases indicating there must be one or more intermediates between the S6 serine kinase and the protein tyrosine kinase. This possibility will be evaluated by using monoclonal antibodies against phosphotyrosine that specifically immunoprecipitate phosphotyrosyl containing protein. The only identified phosphatase that can dephosphorylate S6 in vitro is an enzyme known as protein phosphatase-1 (PrP-1). This enzyme has been reported by others to be phosphorylated on tyrosine residues and inactivated by several protein-tyrosine kinases in vitro. We have confirmed this finding in our work, and this raises the possibility phosphatase inactivation is in part responsible for increased S6 phosphorylation in response to oncogenes and growth factors. This possibility will be explored in the next year by evaluating whether PrP-1 can be isolated as a phosphotyrosyl-containing protein from oocytes injected with protein-tyrosine kinases. By examining both S6 kinases and phosphatases, the complete regulation of this pathway of signal transduction will be evaluated.
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