SIGNALING THROUGH TRANSFORMING G-PROTEIN-COUPLED RECEPTORS IN NIH 3T3CELLS INVOLVES C-RAF ACTIVATION - EVIDENCE FOR A PROTEIN KINASE-C-INDEPENDENT PATHWAY

We have studied the role of Raf-1 in mitogenesis and cellular transfor mation induced by G protein-coupled receptors in NIH 3T3 cells transfe cted with the human m1 muscarinic receptor. We have observed that in m 1- expressing NIH 3T3 cells, the cholinergic agonist carbachol induces a dose- and time-dependent shift in the electrophoretic mobility of p 72(Raf-1), equivalent to that observed when using phorbol esters or pl atelet-derived growth factor as stimulants. Phosphoamino acid analysis of slower mobility forms of p72(Raf-1) revealed both phosphoserine an d phosphothreonine. Carbachol potently induced c-Raf activity as judge d by its in vitro phosphorylating activity using MEK as a substrate. H owever, induction of Raf-1 kinase activity by carbachol occurred much earlier than changes in its electrophoretic mobility. Raf-1 kinase act ivation followed a kinetic similar to that exhibited by an epitope-tag ged ERK2 protein when coexpressed in the same cells. Conventional prot ein kinase C (PKC) inactivation by means of sustained phorbol ester tr eatment or by a new nontoxic PKC-specific inhibitor, GF 109203X, aboli shed p72(Raf-1) mobility shift induced by carbachol or by phorbol este rs. However, c-Raf and ERK2 enzymatic activity in response to carbacho l was at least 50-80% PKC-independent. Furthermore, inhibition of PKC failed to affect DNA synthesis or focus formation induced by carbachol in cells expressing mi receptors. In contrast, cotransfection of NIH 3T3 cells with the Raf-1 dominant negative mutant Raf-301 (K375W) dras tically decreased the transforming ability of mi receptors. Thus, our findings implicate Raf-1 activation in transformation by G protein-cou pled receptors. In addition, our data suggest that activation of p72(R af-1) and ERK2 by G protein-coupled receptors involves PKC-independent pathways.