Article

Phosphorylation of the Ras-GRF1 Exchange Factor at Ser916/898 Reveals Activation of Ras Signaling in the Cerebral Cortex

Department of Pharmacology, Wayne State University, Detroit, Michigan 48201, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 05/2003; 278(15):13278-85. DOI: 10.1074/jbc.M209805200
Source: PubMed
ABSTRACT
The Ras-GRF1 exchange factor, which is regulated by increases in intracellular calcium and the release of Gβγ 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,
Ser916 in the mouse sequence (equivalent to Ser898 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 32P into Ser898 of brain Ras-GRF1 following activation of protein kinase A. Phosphorylation of Ras-GRF1 at Ser916 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 Ser916/898 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 Ser916/898. Confocal immunofluorescence of cortical slices of rat brain revealed widespread and selective phosphorylation of Ras-GRF1
at Ser898. 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.

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    • "Ras-GRF1 is highly expressed in certain CNS neurons [120, 121] , and is particularly enriched in postsynaptic densities [122], where it can bind directly to the NMDA NR2B receptor and mediate its activation of ERK [123]. Ras-GRF1 can also integrate signals from the modulatory G protein-coupled receptors that also control learning and memory [11,124125126. Mice deficient in Ras-GRF1 have learning deficits that include defects in hippocampal-dependent contextual memories [127, 128] and sensory memory formation [129]. "
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    • "Our results link the activation of GRF1 by alcohol to the cAMP/PKA pathway. Specifically, alcohol exposure resulted in increased phosphorylation of GRF1 on a PKA site (serine 916), a post-translational modification that enhances the intrinsic GDP to GTP exchange activity of GRF1 (Mattingly, 1999; Yang et al., 2003). Although the increase in GRF1 activity can influence the function of other members of the p21Ras family, several studies suggest that GRF1 is the specific exchange factor for H-Ras but not N-Ras or K-Ras ( Medarde and Santos, 2011). "
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    • "RasGrf1 has been shown in other studies to be an activator of both Ras and Rac in transfected HEK cells. Specifically, in HEK and Cos-7 cells, RasGrf1 activates Ras downstream of the non-receptor tyrosine kinase ACK1, cAMP-activated PKA, calcium, and GPCR activity (Mattingly 1999; Kiyono et al. 2000a; Yang et al. 2003). Conversely, RasGrf1 activates Rac downstream of the non-receptor tyrosine kinase Src (Kiyono et al. 2000b ). "
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