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 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.

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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 (Fernandez- Medarde and Santos, 2011). "
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    • "Rasgrf1 is most highly expressed in central nervous system neurons (Sturani et al. 1997; Zippel et al. 2000), with lower expression levels in other somatic tissues (Font de Mora et al. 2003; Plass et al. 1996). Its product, RasGRF1 protein, is a guanine nucleotide exchange factor for Ras and Rac (Cen et al. 1993; Innocenti et al. 1999), activating these G-proteins in response to cellular calcium influx (Farnsworth et al. 1995) or serine phosphorylation (Mattingly et al. 1999; Yang et al. 2003) in pathways downstream of muscarinic receptor activity (Mattingly & Macara 1996), heterotrimeric G-protein subunit dissociation (Kiyono et al. 2000; Shou et al. 1995), and neurotrophin binding to TrkA, TrkB, and TrkC receptors (MacDonald et al. 1999; Robinson et al. 2005). "
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    ABSTRACT: Rasgrf1 is genomically imprinted; only the paternally inherited allele is expressed in the neonatal mouse brain until weaning, at which time expression becomes biallelic. Whereas Rasgrf1 has been implicated in learning and memory via knockout studies in adult mice, the effect of its normal imprinted expression on these phenotypes has not yet been examined. Neonatal mice with experimentally manipulated patterns of imprinted Rasgrf1 expression were assessed on an associative olfactory task. Neonates lacking the normally expressed wild-type paternal allele exhibited significant impairment in olfactory associative memory. Adult animals in which neonatal imprinting had been manipulated were also behaviorally assessed; while neonatal imprinting significantly affects body weight even into adulthood, no learning and memory phenotype attributable to imprinting was observed in adults. Additional analyses of neonates showed imprinted Rasgrf1 transcript selective to olfactory bulb even in mice that were null for Rasgrf1 in the rest of the brain and showed that Rasgrf1 affects Ras and Rac activation in the brain. Taken together, these results indicate that Rasgrf1 expression from the wild-type paternal allele contributes to learning and memory in neonatal mice.
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    • "Recent studies have documented the functional contribution of Ras signaling pathways to various neural processes , including control of synaptic plasticity and connectivity (Arendt et al., 2004; Seeger et al., 2004), management of neuronal excitability (Tonini et al., 2001) or neuronal polarity and building of neuronal cytoarchitecture (Arendt et al., 2004; Yoshimura et al., 2006). In the same neuronal context, it is known that Ras-GRF1 may become phosphorylated in response to Ca 2ϩ , PKA, LPA/heterotrimeric G proteins and NGF stimulation (Mattingly and Macara, 1996; Mattingly et al., 1999; Yang et al., 2003; Norum et al., 2005; Robinson et al., 2005; Kesavapany et al., 2006), thus driving subsequent Ras activation. Ras- GRF1 is also known to couple NMDA glutamate receptors to Rac–p38, Ras–ERK and Ras–CREB pathways (Krapivinsky et al., 2003; Tian et al., 2004; Li et al., 2006a). "
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