Phosphorylation of serine 916 of Ras-GRF1 contributes to the activation of exchange factor activity by muscarinic receptors.
The Ras-GRF1 exchange factor is strongly implicated in the control of neuronal Ras. The activity of Ras-GRF1 is regulated by increases in intracellular calcium and the release of Gbetagamma subunits from heterotrimeric G-proteins. Increases in Ras-GRF1 activity toward Ras that are stimulated by receptors coupled to G-proteins are associated with enhanced phosphorylation of Ras-GRF1 on one or more serine residues. Co-expression of Ras-GRF1 with subtype 1 human muscarinic receptors in COS-7 cells allowed mapping of a carbachol-stimulated phosphorylation site to a region composed of residues 916-976. Site-directed mutagenesis replaced each of the serine residues within this region with alanine and demonstrated that serine 916 is a major site of in vivo phosphorylation of Ras-GRF1 in both COS-7 cells and NIH-3T3 fibroblasts. Serine 916 was a substrate for protein kinase A both in vivo and in vitro, suggesting a novel link between the cAMP and Ras signaling systems. Carbachol-dependent phosphorylation of serine 916 occurred through a protein kinase A-independent pathway, however. Full-length Ras-GRF1 that contains an alanine 916 mutation was only partially activated by carbachol, suggesting that phosphorylation at residue 916 is necessary for full activation. Phosphorylation of serine 916 in response to forskolin treatment did not, however, increase the activity of Ras-GRF1, indicating that it is not sufficient for activation.
Mattingly
R R
RR
Department of Pharmacology, Wayne State University, Program in Molecular Biology and Genetics, Barbara Ann Karmanos Cancer Institute, Detroit, Michigan 48201, USA. r.mattingly@wayne.edu
D016475:3T3 Cells; D000818:Animals; D019556:COS Cells; D005576:Colforsin / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D006801:Humans; D051379:Mice; D010766:Phosphorylation; D011976:Receptors, Muscarinic / Q000502:physiology*; D012694:Serine / Q000378:metabolism*; D013329:Structure-Activity Relationship; D020666:ras-GRF1 / Q000378:metabolism*
D011976:Receptors, Muscarinic; D020666:ras-GRF1; D005576:Colforsin; D012694:Serine; D017868:Cyclic AMP-Dependent Protein Kinases