The protein products of a number of retroviral oncogenes and growth factor receptors are protein tyrosine kinases. The 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 that is stimulated by virtually all known protein tyrosine kinases. We have stimulated S6 kinase activity from insulin-treated oocytes, purified it to homogeneity, and shown it to be a substrate for activation by phosphorylation by another kinase known as microtubule-associated protein, or MAP kinase. MAP kinase itself is activated by phosphorylation on tyrosine and threonine residues by yet another kinase in the pathway known as MAP kinase activator. A major objective of this grant is to purify the activator to homogeneity, clone the gene for it, and determine the mechanisms of activation of the activator by insulin and other agents which work through - activation of tyrosine kinase activity. Other studies will evaluate oncogene and proto-oncogene interactions in the pathway of signalling that leads to activation of MAP kinase and S6 kinase. In particular, there appear to be important interactions between the v-src oncogene, which encodes a tyrosine kinase, the Harvey Ras oncogene which encodes a GTP binding protein, and the c- mos proto- oncogene which encodes a serine/threonine protein kinase activity. Each of these agents is capable of eliciting the activation of MAP kinase and S6 kinase by pathways that are either dependent or independent of one another. Using the Xenopus oocyte and egg system, as well as extracts from these cells, the action of these oncogenes will be studied singly and in combination in terms of the activation of MAP kinase and S6 kinase enzymes. Finally, the substrate specificity of the S6 kinase enzyme itself will be evaluated using site-directed mutagenesis to mutate particular domains of the enzyme involved in structure/function relationships. The enzyme is unusual in having two complete kinase domains, and the main focus of this work will be mutation of residues in one of the other kinase domain that may lead to changes in protein kinase activity and ability to be activated by MAP kinase. By continuing to characterize each step of the signalling pathway leading to S6 phosphorylation and working backwards up the pathway towards the tyrosine kinase activity of the insulin receptor, the complete regulation of this pathway of signal transduction should be elucidated.
Showing the most recent 10 out of 41 publications
