| Title : Signaling through transforming G protein-coupled receptors in NIH 3T3 cells involves c-Raf activation. Evidence for a protein kinase C-independent pathway - Crespo_1994_J.Biol.Chem_269_21103 |
| Author(s) : Crespo P , Xu N , Daniotti JL , Troppmair J , Rapp UR , Gutkind JS |
| Ref : Journal of Biological Chemistry , 269 :21103 , 1994 |
| Abstract : We have studied the role of Raf-1 in mitogenesis and cellular transformation induced by G protein-coupled receptors in NIH 3T3 cells transfected with the human m1 muscarinic receptor. We have observed that in m1-expressing NIH 3T3 cells, the cholinergic agonist carbachol induces a dose- and time-dependent shift in the electrophoretic mobility of p72Raf-1, equivalent to that observed when using phorbol esters or platelet-derived growth factor as stimulants. Phosphoamino acid analysis of slower mobility forms of p72Raf-1 revealed both phosphoserine and phosphothreonine. Carbachol potently induced c-Raf activity as judged by its in vitro phosphorylating activity using MEK as a substrate. However, induction of Raf-1 kinase activity by carbachol occurred much earlier than changes in its electrophoretic mobility. Raf-1 kinase activation followed a kinetic similar to that exhibited by an epitope-tagged ERK2 protein when coexpressed in the same cells. Conventional protein kinase C (PKC) inactivation by means of sustained phorbol ester treatment or by a new nontoxic PKC-specific inhibitor, GF 109203X, abolished p72Raf-1 mobility shift induced by carbachol or by phorbol esters. However, c-Raf and ERK2 enzymatic activity in response to carbachol 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 m1 receptors. In contrast, cotransfection of NIH 3T3 cells with the Raf-1 dominant negative mutant Raf-301 (K375W) drastically decreased the transforming ability of m1 receptors. Thus, our findings implicate Raf-1 activation in transformation by G protein-coupled receptors. In addition, our data suggest that activation of p72Raf-1 and ERK2 by G protein-coupled receptors involves PKC-independent pathways. |
| ESTHER : Crespo_1994_J.Biol.Chem_269_21103 |
| PubMedSearch : Crespo_1994_J.Biol.Chem_269_21103 |
| PubMedID: 8063729 |
Crespo P, Xu N, Daniotti JL, Troppmair J, Rapp UR, Gutkind JS (1994)
Signaling through transforming G protein-coupled receptors in NIH 3T3 cells involves c-Raf activation. Evidence for a protein kinase C-independent pathway
Journal of Biological Chemistry
269 :21103
Crespo P, Xu N, Daniotti JL, Troppmair J, Rapp UR, Gutkind JS (1994)
Journal of Biological Chemistry
269 :21103