The long-term objective of this research is to understand the molecular basis of how oncogene products interfere with the normal regulation of intracellular signal transduction pathways. These studies focus on the mechanism of regulating 40S ribosomal protein S6 protein kinase activity in cells incubated with growth factors or phorbol esters or in cells expressing the Rous sarcoma virus transforming gene product, pp60v-src. The key to this understanding will require continued purification of the S6 kinase for characterization as well as production of antibodies. Classical biochemical approaches for enzyme purification will continue to be used. Strategies for producing polyclonal and monoclonal antibodies with limited quantities of antigen will be modified from existing protocols. Antibodies will be used to immunoprecipitate biosynthetically-labeled S6 kinase from culture cells, to identify src- and growth-regulated post- translational modifications, to screen expression libraries for S6 kinase cDNA, to perform immuno-cytological studies and to improve purification protocols if required. In parallel with S6 kinase purification, pharmacological agents will be used to study possible interactions of S6 protein kinase with other potential signal transducers such as protein kinase C, cAMP-dependent protein kinase, Ca2+/calmodulin-dependent protein kinases and G- proteins at a biochemical and cell biological level. Additional S6 protein kinase substrates will be identified by a combination of in vitro protein phosphorylation and two-dimensional gel analysis of phosphoproteins in 32Pi-labeled cells in which S6 protein kinase activity has been stimulated. The pharmacological studies and cell growth variants will be particularly useful in the latter approach as the S6 kinase can be stimulated under conditions not affecting the activity of other signal transducing proteins. Finally, the growth-stimulated phosphorylation of nuclear proteins and the enzymes responsible for these modifications will be studied. Interest will focus on nuclear matrix or DNA-binding phosphoproteins potentially involved in regulating gene expression. Gel retardation studies will be used to identify 32-Pi- labeled proteins which bind to the control elements of growth- regulated genes. These studies will strengthen our understanding of the molecular basis of communication within cells and will provide a biochemical means for elucidating the molecular mechanism of src-induced oncogenesis.
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