Phosphorylation of the Ras-GRF1 exchange factor at Ser916/898 reveals activation of Ras signaling in the cerebral cortex

J Biol Chem. 2003 Apr 11;278(15):13278-85. doi: 10.1074/jbc.M209805200. Epub 2003 Jan 21.


The Ras-GRF1 exchange factor, which is regulated by increases in intracellular calcium and the release of G beta gamma subunits from heterotrimeric G proteins, plays a critical role in the activation of neuronal Ras. Activation of G protein-coupled receptors stimulates an increase in the phosphorylation of Ras-GRF1 at certain serine residues. The first of these sites to be identified, Ser(916) in the mouse sequence (equivalent to Ser(898) in the rat sequence), is required for full activation of the Ras exchange factor activity of Ras-GRF1 by muscarinic receptors. We demonstrate here that Ras-GRF1 is highly expressed in rat brain compared with the Sos exchange factor and that there is an increase in incorporation of (32)P into Ser(898) of brain Ras-GRF1 following activation of protein kinase A. Phosphorylation of Ras-GRF1 at Ser(916) is also required for maximal induction of Ras-dependent neurite outgrowth in PC12 cells. A novel antibody (termed 2152) that selectively recognizes Ras-GRF1 when it is phosphorylated at Ser(916/898) confirmed the regulated phosphorylation of Ras-GRF1 by Western blotting in both model systems of transfected COS-7 and PC12 cells and also of the endogenous protein in rat forebrain slices. Indirect confocal immunofluorescence of transfected PC12 cells using antibody 2152 demonstrated reactivity only under conditions in which Ras-GRF1 was phosphorylated at Ser(916/898). Confocal immunofluorescence of cortical slices of rat brain revealed widespread and selective phosphorylation of Ras-GRF1 at Ser(898). In the prefrontal cortex, there was striking phosphorylation of Ras-GRF1 in the dendritic tree, supporting a role for Ras activation and signal transduction in neurotransmission in this area.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Binding Sites
  • COS Cells
  • Cerebral Cortex / metabolism
  • Chlorocebus aethiops
  • Colforsin / pharmacology
  • In Vitro Techniques
  • Peptide Fragments / chemistry
  • Phosphopeptides / chemistry
  • Phosphorylation
  • Phosphoserine / metabolism
  • Prosencephalon / metabolism*
  • Rats
  • Recombinant Proteins / metabolism
  • Serine*
  • Thapsigargin / pharmacology
  • Transfection
  • ras Guanine Nucleotide Exchange Factors / metabolism*
  • ras-GRF1 / chemistry
  • ras-GRF1 / metabolism*


  • Peptide Fragments
  • Phosphopeptides
  • Recombinant Proteins
  • ras Guanine Nucleotide Exchange Factors
  • ras-GRF1
  • Phosphoserine
  • Colforsin
  • Serine
  • Thapsigargin