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

Yang, Haiyan; Cooley, Desma; Legakis, Julie E.; Ge, Qingyuan; Andrade, Rodrigo; Mattingly, Raymond R.

doi:10.1074/jbc.m209805200

Cited by 37 publications

(45 citation statements)

References 66 publications

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“…Previous studies have suggested that the Rem domain of RasGRF1 is regulatory in function, containing phosphorylation sites and PEST motifs (25)(26)(27)(28). The level of activity we observe for the isolated Cdc25 domain of RasGRF1, although significantly greater than the intrinsic rate of nucleotide release from Ras, is much lower than the maximum rate observed for allosterically activated Sos cat .…”

Section: Discussioncontrasting

confidence: 43%

A Ras-induced conformational switch in the Ras activator Son of sevenless

Freedman

Sondermann

Friedland

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

133

152

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The Ras-specific guanine nucleotide-exchange factors Son of sevenless (Sos) and Ras guanine nucleotide-releasing factor 1 (RasGRF1) transduce extracellular stimuli into Ras activation by catalyzing the exchange of Ras-bound GDP for GTP. A truncated form of RasGRF1 containing only the core catalytic Cdc25 domain is sufficient for stimulating Ras nucleotide exchange, whereas the isolated Cdc25 domain of Sos is inactive. At a site distal to the catalytic site, nucleotide-bound Ras binds to Sos, making contacts with the Cdc25 domain and with a Ras exchanger motif (Rem) domain. This allosteric Ras binding stimulates nucleotide exchange by Sos, but the mechanism by which this stimulation occurs has not been defined. We present a crystal structure of the Rem and Cdc25 domains of Sos determined at 2.0-Å resolution in the absence of Ras. Differences between this structure and that of Sos bound to two Ras molecules show that allosteric activation of Sos by Ras occurs through a rotation of the Rem domain that is coupled to a rotation of a helical hairpin at the Sos catalytic site. This motion relieves steric occlusion of the catalytic site, allowing substrate Ras binding and nucleotide exchange. A structure of the isolated RasGRF1 Cdc25 domain determined at 2.2-Å resolution, combined with computational analyses, suggests that the Cdc25 domain of RasGRF1 is able to maintain an active conformation in isolation because the helical hairpin has strengthened interactions with the Cdc25 domain core. These results indicate that RasGRF1 lacks the allosteric activation switch that is crucial for Sos activity.Cdc25 ͉ nucleotide-exchange factor ͉ crystal structure ͉ Ras exchanger motif (Rem) domain ͉ Ras guanine nucleotide-releasing factor 1

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Section: Discussioncontrasting

confidence: 43%

A Ras-induced conformational switch in the Ras activator Son of sevenless

Freedman

Sondermann

Friedland

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

133

152

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“…Plasmids encoding the same inserts with enhanced green fluorescent protein (GFP) replacing the HA1 epitope tags were prepared by subcloning BamHI/EcoRI fragments into the pRK7sGFP vector (Carey et al, 1996). A plasmid encoding the ⌬N mutant fused to glutathione S-transferase (GST) has previously been described (Yang et al, 2003). Similar constructs expressing the ⌬C mutant or Rac1 were prepared by subcloning the relevant BamHI/EcoRI fragments into pGEX-2T.…”

Section: Plasmidsmentioning

confidence: 99%

“…Transfected PC12 cells were fixed and processed for confocal indirect and GFP fluorescence broadly as previously described (Yang et al, 2003), with the following modifications: The primary antibodies used were anti-Myc monoclonal antibody (mAb) 9e10 (1:1000 dilution; Sigma) for detection of cells expressing Myc-tagged Ras or Rac proteins, anti-Ras-GRF1 polyclonal antibody sc224 (1:500 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) to detect untagged Ras-GRF1, anti-Flag mAb M2 (1:200 dilution; Sigma) to detect Flag-tagged MEK1.K97M, and either anti-hemagglutinin-1 (HA1) polyclonal antibody Y-11 (1:150 dilution; Santa Cruz) or 12CA5 mAb (1:500) for detection of cells expressing HA1 3 -tagged proteins. For detection of neurofilaments, mAb RT97 (NICHD Developmental Studies Hybridoma Bank, Iowa City, IA) was used at a 1:100 dilution.…”

Section: Confocal Immunofluorescencementioning

confidence: 99%

“…Pathways that stimulate the Ras exchange activity of Ras-GRF1 have been found to be those from G protein-coupled receptors (Shou et al, 1995;Mattingly and Macara, 1996;Zippel et al, 1996), which may require the release of G protein ␤␥ subunits (Mattingly and Macara, 1996), and via direct interactions between Ras-GRF1 and the TrkA nerve growth factor (NGF) receptor (MacDonald et al, 1999;Robinson et al, 2005), and Ras-GRF1 and the NR2B NMDA receptor (Krapivinsky et al, 2003). G protein-coupled agonists, NGF, and NMDA/glycine have all been demonstrated to increase phosphorylation of endogenous Ras-GRF1 at Serine-916 or its equivalent (e.g., Ser-898 in the rat sequence and Ser-927 in the human sequence; Mattingly, 1999;Yang et al, 2003;Norum et al, 2005;Schmitt et al, 2005).The exchange factor activity toward Rac1 is conferred by the DH/PH domains in the N-terminal half of Ras-GRF1. This activity is also stimulated by overexpression of G protein ␤␥ subunits (Kiyono et al, 1999) or tyrosine phosphorylation by Src (Kiyono et al, 2000b) and is likely to be regulated by G protein-coupled agonists such as lysophosphatidic acid (LPA;Innocenti et al, 1999).…”

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confidence: 99%

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The Ras-GRF1 Exchange Factor Coordinates Activation of H-Ras and Rac1 to Control Neuronal Morphology

Yang

Mattingly

2006

MBoC

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The Ras-GRF1 exchange factor has regulated guanine nucleotide exchange factor (GEF) activity for H-Ras and Rac1 through separate domains. Both H-Ras and Rac1 activation have been linked to synaptic plasticity and thus could contribute to the function of Ras-GRF1 in neuronal signal transduction pathways that underlie learning and memory. We defined the effects of Ras-GRF1 and truncation mutants that include only one of its GEF activities on the morphology of PC12 phaeochromocytoma cells. Ras-GRF1 required coexpression of H-Ras to induce morphological effects. Ras-GRF1 plus H-Ras induced a novel, expanded morphology in PC12 cells, which was characterized by a 10-fold increase in soma size and by neurite extension. A truncation mutant of Ras-GRF1 that included the Ras GEF domain, GRF⌬N, plus H-Ras produced neurite extensions, but did not expand the soma. This neurite extension was blocked by inhibition of MAP kinase activation, but was independent of dominant-negative Rac1 or RhoA. A truncation mutant of Ras-GRF1 that included the Rac GEF domains, GRF⌬C, produced the expanded phenotype in cotransfections with H-Ras. Cell expansion was inhibited by wortmannin or dominant-negative forms of Rac1 or Akt. GRF⌬C binds H-Ras.GTP in both pulldown assays from bacterial lysates and by coimmunoprecipitation from HEK293 cells. These results suggest that coordinated activation of H-Ras and Rac1 by Ras-GRF1 may be a significant controller of neuronal cell size. INTRODUCTIONThe Ras superfamily of GTPases are regulated switches that control many intracellular pathways. The Ras family, which includes H-, K-, and N-Ras and other closely related isoforms, has been particularly associated with the control of proliferation in cells such as fibroblasts and epithelia (Lowy and Willumsen, 1986). This action is thought to be of particular relevance to the common involvement of activated Ras in human cancer (Barbacid, 1987), which can occur by mutational activation (Taparowsky et al., 1982), by inappropriate activation of other elements in the Ras activation pathway, such as the overexpression or aberrant stimulation of growth factor receptors (Malaney and Daly, 2001), or by loss of a deactivating GTPase-activating protein (GAP), such as in type 1 neurofibromatosis (DeClue et al., 1991). Ras proteins are also, however, highly involved in the function of terminally differentiated cells such as neurons of the CNS (Weeber et al., 2002). The Rho family small GTPases, which include Rac1 and many other members, have multiple cellular functions during both cellular differentiation (Beqaj et al., 2002;Sordella et al., 2003) and in the mature phenotype, including regulation of the cytoskeleton and cellular morphology, and coupling to transcription factor pathways (Aznar and Lacal, 2001). There is increasing evidence that the functions of Ras and Rho family small GTPases can be coordinated to produce regulation of cellular phenotypes, with most models suggesting that Ras activation occurs before the activation of Rho proteins (Sarner et al., 2000;Men...

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“…79 Moreover, phospho-specific antibodies to this site in GRF1 demonstrated that this site does get phosphorylated in the dendrites of cortical neurons in the brain, implying that this phenomenon plays some role in synaptic plasticity. 80 Interestingly, this site on GRF1 was shown to become phosphorylated in response to signals that induce LTP in the CA1 region of the hippocampus. 81 This event was shown to involve CamKI, although PKA was likely the enzyme that directly phosphorylates GRF1.…”

Section: Pka and Camkimentioning

confidence: 99%

Regulation of Neuronal Function by Ras-GRF Exchange Factors

Feig

2011

Genes & Cancer

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Ras-GRF1 (GRF1) and Ras-GRF2 (GRF2) constitute a family of guanine nucleotide exchange factors (GEFs). The main isoforms, p140-GRF1 and p135-GRF2, have 2 GEF domains that give them the capacity to activate both Ras and Rac GTPases in response to signals from a variety of neurotransmitter receptors. GRF1 and GRF2 proteins are found predominantly in adult neurons of the central nervous system, although they can also be detected in a limited number of other tissues. p140-GRF1 and p135-GRF2 contain calcium/calmodulin-binding IQ domains that allow them to act as calcium sensors to mediate the actions of NMDA-type and calcium-permeable AMPA-type glutamate receptors. p140-GRF1 also mediates the action of dopamine receptors that signal through cAMP. Although p140-GRF1 and p135-GRF2 have similar functional domains, studies of GRF knockout mice show that they can play strikingly different roles in regulating MAP kinase family members, neuronal synaptic plasticity, specific forms of learning and memory, and behavioral responses to psychoactive drugs. In addition, the function of GRF proteins may vary in different regions of the brain. Alternative splice variants yielding smaller GRF1 gene isoforms with fewer functional domains also exist; however, their distinct roles in neurons have not been revealed. Continuing studies of these proteins should yield important insights into the biochemical basis of brain function as well as novel concepts to explain how complex signal transduction proteins, like Ras-GRFs, integrate multiple upstream signals into specific downstream outputs to control brain function.

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

Cited by 37 publications

References 66 publications

A Ras-induced conformational switch in the Ras activator Son of sevenless

A Ras-induced conformational switch in the Ras activator Son of sevenless

The Ras-GRF1 Exchange Factor Coordinates Activation of H-Ras and Rac1 to Control Neuronal Morphology

Regulation of Neuronal Function by Ras-GRF Exchange Factors

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