The purpose of the proposed research is to characterize and to determine the physiological significance of a phosphorylation system (endogenous protein kinase and substrate) detected in the outer plasma membrane of 3T3 cells. The activity is correlated with cell growth in 3T3 cells: it is greater in growing cells than in quiescent ones; it decreases as cell density increases; it is 5-10 fold greater in SV40-3T3 cells than in normal 3T3. Stimulation of quiescent cells with serum brings about a 2-4 fold increase in incorporation of phosphate as does also treatment with small amounts of insulin, prostaglandin E1 or F2alpha. The increase in phosphorylation appears to be a G1 event. Inhibitors of RNA or protein synthesis block the increase in phosphorylation as well as passage through G1. Hydroxyurea, which blocks at the G1/S boundary, does not prevent the increase in phosphorylation. The endogenous activity is not stimulated with cyclic-AMP. Like other cyclic-AMPindependent kinases, GTP, as well as ATP, can be used as a phosphate donor. In contrast, the soluble, cytoplasmic kinases from the cells, which are cyclic-AMP dependent, do not use GTP. On the basis of autoradiographs of SDS gels of phosphorylated cells, GTP and ATP appear to label the same proteins. Phorphorylation with GTP appears similar to that with ATP in all other respects tested. We plan to use this differential incorporation of GTP and ATP to distinguish soluble kinases from membrane bound. We plan to isolate membranes from 3T3 and SV40-3T3 cells and characterize their phosphorylation activity. We will test membranes from quiescent and stimulated cells. Immunoprecipitation of membrane proteins will be done with antibody made against intact 3T3 cells. Selective membrane extraction procedures will also be used to try to separate enzymes and substrates. We also plan a series of experiments utilizing double labeling of cells with 32P and 33P. We will compare changes in phosphorylation in the same cells in various stages of the growth cycle.