The aim is to carry out further studies on the mechanism of ATP-stimulated mitogenesis in mammalian cells and to elucidate the role of cyclic-AMP (CAMP) when extracellular ATP synergizes with other growth factors. A major mitogenic pathway for ATP in 3T3 cells involves receptor binding, arachidonic acid release, prostaglandin E2 formation and stimulation of adenylyl cyclase. Pharmacological evidence suggests that elevation in cAMP levels is obligatory for ATP-dependent mitogenesis. Surprisingly, ATP synergizes with another potent mitogen, lysophosphatidic acid, which inhibits adenylyl cyclase. Such findings raise questions about the role of cAMP changes in DNA synthesis in certain situations and how mitogens with opposite effects can synergize. The strategy will be to construct 3T3 mutants that are blocked in cellular processes mediated by cAMP-dependent protein kinase. Expression vectors from Drs. G.S. McKnight and M. Gottesman are being used. If mutant studies confirm an essential function for cAMP in ATP-dependent mitogenesis, kinetic analysis of CAMP levels in the presence of ATP, alone and in combination with other mitogens, will be carried out. Kinetic studies of protein phosphorylation patterns will also be performed. In addition, comparison of phosphorylation patterns of wild type and 3T3 mutants, using 2D gels, may indicate that certain proteins whose phosphorylation depends on cAMP are absent or greatly reduced in the mutant cells. If, unexpectedly, genetic studies indicate that elevation of intracellular cAMP does not play a role in ATP-stimulated mitogenesis, then a search for an unidentified mitogenic metabolite of arachidonic acid will be carried out. Growth factors with which extracellular ATP synergizes will be examined using the same mutants. Recent reports describe constitutive activation of adenylyl cyclase by certain oncogenes. Accordingly, a better understanding of how cAMP regulates growth will help in efforts to control growth of cancer cells.
