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• W2004638925 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. 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. guanine nucleotide exchange factor GTPase-activating protein protein kinase A glutathioneS-transferase hemagglutinin-1 nerve growth factor isobutylmethylxanthine N-[2-hydroxy-1, 1-bis(hydroxymethyl)ethyl]glycine The Ras GTPases are timed molecular switches that cycle between GDP- and GTP-bound forms to control pathways of cellular growth and differentiation (1Macara I.G. Lounsbury K.M. Richards S.A. McKiernan C. Bar-Sagi D. FASEB J. 1996; 10: 625-630Crossref PubMed Scopus (211) Google Scholar). In addition to the well established roles for Ras in the proliferation of normal and malignant cells (2Barbacid M. Annu. Rev. Biochem. 1987; 56: 779-827Crossref PubMed Scopus (3778) Google Scholar), there is increasing evidence that Ras also regulates critical functions in terminally differentiated cells such as neurons (3Rosen L.B. Ginty D.D. Weber M.J. Greenberg M.E. Neuron. 1994; 12: 1207-1221Abstract Full Text PDF PubMed Scopus (599) Google Scholar). The GTPase cycle is controlled by guanine nucleotide exchange factors (GEFs)1 and GTPase-activating proteins (GAPs) (4Boguski M.S. McCormick F. Nature. 1993; 366: 643-654Crossref PubMed Scopus (1760) Google Scholar), with the balance between the effective GEF and GAP activities determining the activation state of Ras because it is the GTP-bound form that activates downstream effector pathways. The principal control in many instances may be the activation process through the activity or subcellular localization of the GEF, as there is increasing evidence that these Ras activators are highly regulated (5Quilliam L.A. Rebhun J.F. Castro A.F. Prog. Nucleic Acids Res. Mol. Biol. 2002; 71: 391-444Crossref PubMed Google Scholar). In other cases, such as type 1 neurofibromatosis, where there is loss of the Ras-GAP activity of the protein neurofibromin (6DeClue J.E. Cohen B.D. Lowy D.R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9914-9918Crossref PubMed Scopus (176) Google Scholar), physiological control is shown to require the deactivation step. Indeed, oncogenic mutations in Ras that block the GAP-catalyzed deactivation of Ras from the GTP- to GDP-bound states (7Trahey M. McCormick F. Science. 1987; 238: 542-545Crossref PubMed Scopus (838) Google Scholar), emphasize the importance of this switch mechanism. The Ras-GRF1 exchange factor (8Shou C. Farnsworth C.L. Neel B.G. Feig L.A. Nature. 1992; 358: 351-354Crossref PubMed Scopus (290) Google Scholar), which is also termed CDC25Mm (9Cen H. Papageorge A.G. Vass W.C. Zhang K.E. Lowy D.R. Mol. Cell. Biol. 1993; 13: 7718-7724Crossref PubMed Scopus (46) Google Scholar, 10Martegani E. Vanoni M. Zippel R. Coccetti P. Brambilla R. Ferrari C. Sturani E. Alberghina L. EMBO J. 1992; 11: 2151-2157Crossref PubMed Scopus (189) Google Scholar), is highly expressed in neurons of the central nervous system (11Wei W. Schreiber S.S. Baudry M. Tocco G. Broek D. Brain Res. Mol. Brain Res. 1993; 19: 339-344Crossref PubMed Scopus (27) Google Scholar, 12Ferrari C. Zippel R. Martegani E. Gnesutta N. Carrera V. Sturani E. Exp. Cell Res. 1994; 210: 353-357Crossref PubMed Scopus (30) Google Scholar, 13Zippel R. Gnesutta N. Matus-Leibovitch N. Mancinelli E. Saya D. Vogel Z. Sturani E. Renata Z. Nerina G. Noa M.L. Enzo M. Daniella S. Zvi V. Emmapaola S. Brain Res. Mol. Brain Res. 1997; 48: 140-144Crossref PubMed Scopus (62) Google Scholar). Ras-GRF1 is expressed predominantly at synapses (14Sturani E. Abbondio A. Branduardi P. Ferrari C. Zippel R. Martegani E. Vanoni M. Denis-Donini S. Exp. Cell Res. 1997; 235: 117-123Crossref PubMed Scopus (56) Google Scholar), a neuronal subcellular distribution it shares with the Ras-GAP termed SynGap (15Chen H.J. Rojas-Soto M. Oguni A. Kennedy M.B. Neuron. 1998; 20: 895-904Abstract Full Text Full Text PDF PubMed Scopus (483) Google Scholar). This colocalization suggests that the proteins may reciprocally regulate Ras at synapses. Interestingly, Ras (16Zhu J.J. Qin Y. Zhao M. Van Aelst L. Malinow R. Cell. 2002; 110: 443-455Abstract Full Text Full Text PDF PubMed Scopus (634) Google Scholar) and the Ras effector mitogen-activated protein kinase (17Bailey C.H. Bartsch D. Kandel E.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13445-13452Crossref PubMed Scopus (610) Google Scholar, 18Patterson S.L. Pittenger C. Morozov A. Martin K.C. Scanlin H. Drake C. Kandel E.R. Neuron. 2001; 32: 123-140Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar) have been implicated in the control of synaptic plasticity, which is the proposed cellular corollary of memory storage, whereas mice that lack Ras-GRF1 have defects in memory (19Brambilla R. Gnesutta N. Minichiello L. White G. Roylance A.J. Herron C.E. Ramsey M. Wolfer D.P. Cestari V. Rossi-Arnaud C. Grant S.G. Chapman P.F. Lipp H.P. Sturani E. Klein R. Nature. 1997; 390: 281-286Crossref PubMed Scopus (399) Google Scholar, 20Giese K.P. Friedman E. Telliez J.B. Fedorov N.B. Wines M. Feig L.A. Silva A.J. Neuropharmacology. 2001; 41: 791-800Crossref PubMed Scopus (124) Google Scholar). Thus, Ras-GRF1 may participate in the regulation of synaptic plasticity in the central nervous system. Ras-GRF1 couples heterotrimeric G proteins (21Shou C. Wurmser A. Suen K.L. Barbacid M. Feig L.A. Ling K. Oncogene. 1995; 10: 1887-1893PubMed Google Scholar, 22Zippel R. Orecchia S. Sturani E. Martegani E. Oncogene. 1996; 12: 2697-2703PubMed Google Scholar, 23Mattingly R.R. Macara I.G. Nature. 1996; 382: 268-272Crossref PubMed Scopus (156) Google Scholar, 24Mattingly R.R. Saini V. Macara I.G. Cell. Signal. 1999; 11: 603-610Crossref PubMed Scopus (22) Google Scholar) and calcium signals (8Shou C. Farnsworth C.L. Neel B.G. Feig L.A. Nature. 1992; 358: 351-354Crossref PubMed Scopus (290) Google Scholar, 25Buchsbaum R. Telliez J.B. Goonesekera S. Feig L.A. Mol. Cell. Biol. 1996; 16: 4888-4896Crossref PubMed Scopus (91) Google Scholar) to the activation of Ras. The activation of Ras-GRF1 by G protein-coupled receptors is closely associated with an increase in its phosphorylation at certain serine residues (24Mattingly R.R. Saini V. Macara I.G. Cell. Signal. 1999; 11: 603-610Crossref PubMed Scopus (22) Google Scholar), with the first of these to be identified being Ser916 (in the mouse sequence, equivalent to Ser898 in the rat sequence) (26Mattingly R.R. J. Biol. Chem. 1999; 274: 37379-37384Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Phosphorylation of Ser916/898, which is an in vivo and in vitro substrate for protein kinase A (PKA), is necessary for full activation of the Ras-GEF activity of Ras-GRF1 (26Mattingly R.R. J. Biol. Chem. 1999; 274: 37379-37384Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). The current study confirms that Ser916/898 is a physiologically relevant site of regulated phosphorylation in the endogenous Ras-GRF1 exchange factor that is expressed in rat forebrain. Localization of this regulatory phosphorylation event to the apical dendrites of prefrontal pyramidal cells suggests that Ras signaling is activated at these loci. Constructs in the pKH3 mammalian expression vector (27Mattingly R.R. Sorisky A. Brann M.R. Macara I.G. Mol. Cell. Biol. 1994; 14: 7943-7952Crossref PubMed Scopus (84) Google Scholar) that encode murine Ras-GRF1, Ras-GRF1ΔN, Ras-GRF1Δ900, and the S916A mutant have previously been described (23Mattingly R.R. Macara I.G. Nature. 1996; 382: 268-272Crossref PubMed Scopus (156) Google Scholar, 26Mattingly R.R. J. Biol. Chem. 1999; 274: 37379-37384Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Ras-GRF1Δ976 was prepared by PCR using a 5′-primer with a BamHI restriction site from the template pKH3Ras-GRF1ΔN and verified by sequencing. Mammalian expression vectors for rat Ras-GRF1 (8Shou C. Farnsworth C.L. Neel B.G. Feig L.A. Nature. 1992; 358: 351-354Crossref PubMed Scopus (290) Google Scholar), Sos1 (28Egan S.E. Giddings B.W. Brooks M.W. Buday L. Sizeland A.M. Weinberg R.A. Nature. 1993; 363: 45-51Crossref PubMed Scopus (1010) Google Scholar), Myc-tagged H-Ras (29Jones M.K. Jackson J.H. J. Biol. Chem. 1998; 273: 1782-1787Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), and muscarinic receptor subtypes 1 and 2 (30Bonner T.I. Buckley N.J. Young A.C. Brann M.R. Science. 1987; 237: 527-532Crossref PubMed Scopus (1220) Google Scholar) were generously provided by Profs. L .A. Feig, M. Czech, J. Jackson, and M. R. Brann, respectively. COS-7 cells were transfected by calcium phosphate coprecipitation (27Mattingly R.R. Sorisky A. Brann M.R. Macara I.G. Mol. Cell. Biol. 1994; 14: 7943-7952Crossref PubMed Scopus (84) Google Scholar), and PC12 cells by electroporation (31McKiernan C.J. Stabila P.F. Macara I.G. Mol. Cell. Biol. 1996; 16: 4985-4995Crossref PubMed Scopus (53) Google Scholar). Expression of recombinant wild-type Ras-GRF1-(900–983) and Ras-GRF1-(900–983) with the S916A mutation as fusion proteins with glutathione S-transferase (GST) has been previously described (26Mattingly R.R. J. Biol. Chem. 1999; 274: 37379-37384Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). GST-Ras-GRF1-(632–1262) (equivalent to GST-GRF1ΔN) was expressed from the vector pGEX.GRF1-(632–1262), which was constructed by subcloning the BamHI/EcoRI insert from pKH3GRF1ΔN (23Mattingly R.R. Macara I.G. Nature. 1996; 382: 268-272Crossref PubMed Scopus (156) Google Scholar) into pGEX-2T. Forebrain slices cut from postnatal day 15 rat brains were labeled in six-well tissue culture plates in 1 ml/well phosphate-free Dulbecco's modified Eagle's medium (Invitrogen) with 2 mCi of [32P]orthophosphate (ICN, Costa Mesa, CA) for 2 h in a 37 °C incubator that was supplied with 95% O2 and 5% CO2. The slices were lysed, and Ras-GRF1 was immunoprecipitated using antibody sc-224 (Santa Cruz Biotechnology, Santa Cruz, CA) as described (23Mattingly R.R. Macara I.G. Nature. 1996; 382: 268-272Crossref PubMed Scopus (156) Google Scholar). Immunoprecipitated labeled Ras-GRF1 was processed for Western blotting and digestion with cyanogen bromide as described (26Mattingly R.R. J. Biol. Chem. 1999; 274: 37379-37384Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Antibodies were developed in rabbits immunized with a 13-residue phosphopeptide that represents the region flanking Ser916 of mouse Ras-GRF1. This sequence is highly conserved (with only three conservative substitutions) in rat Ras-GRF1 (8Shou C. Farnsworth C.L. Neel B.G. Feig L.A. Nature. 1992; 358: 351-354Crossref PubMed Scopus (290) Google Scholar). The specificity of the antibodies was tested, and affinity-purified antibody 2152 was characterized as a selective agent for recognition of both mouse and rat Ras-GRF1 only when phosphorylated at the relevant serine (see below). Western blots were developed by enhanced chemiluminescent detection (32Mattingly R.R. Felczak A. Chen C.C. McCabe Jr., M.J. Rosenspire A.J. Toxicol. Appl. Pharmacol. 2001; 176: 162-168Crossref PubMed Scopus (31) Google Scholar). Dual labeling indirect confocal immunofluorescence of transfected PC12 cells was performed as previously described (33Lounsbury K.M. Richards S.A. Carey K.L. Macara I.G. J. Biol. Chem. 1996; 271: 32834-32841Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). For neurite outgrowth experiments, the primary antibodies used were anti-Myc monoclonal antibody 9e10 (1:1000 dilution; Sigma) for detection of cells expressing Myc-H-Ras and anti-hemagglutinin-1 (HA1) polyclonal antibody Y-11 (1:150 dilution; Santa Cruz Biotechnology) for detection of cells expressing HA13-Ras-GRF1. The secondary antibodies used were Cy3-coupled anti-mouse antibody (1:300 dilution; Jackson Laboratories, Bar Harbor, ME) and Oregon Green-coupled anti-rabbit antibody (1:300 dilution; Molecular Probes, Inc., Eugene, OR). Pictures were taken with a ×40 water immersion lens on a Zeiss LSM310 microscope. Neurite outgrowth was quantified as previously described (34Yang H. Xiao Z.-c. Becker B. Hillenbrand R. Rougon G. Schachner M. J. Neurosci. Res. 1999; 55: 687-701Crossref PubMed Scopus (30) Google Scholar). To determine phosphorylation of Ras-GRF1, the procedure was similar, except that the primary antibodies used were anti-HA1 monoclonal antibody 12CA5 (1:500 dilution) for detection of HA13-Ras-GRF1 and polyclonal antibody 2152 (1:300 dilution) for detection of Ras-GRF1 phosphorylated at Ser916/898, and pictures were taken with a ×63 oil immersion lens. Forebrain slices of postnatal day 15 rat brain were fixed in 4% paraformaldehyde in phosphate-buffered saline and incubated in 50% sucrose/phosphate-buffered saline overnight at 4 °C. The slices were then permeabilized for 10 min with methanol that had been precooled to −20 °C; rinsed with phosphate-buffered saline; and blocked with phosphate-buffered saline containing 2% bovine serum albumen, 5% goat serum, and 0.25% Triton X-100 for 4 h at room temperature. Indirect confocal immunofluorescence was carried out using the following primary polyclonal antibodies: antibody 2152 (anti-Ras-GRF1 Ser(P)916/898; 1:200 dilution); antibody sc-224 (anti-Ras-GRF1; 1:200 dilution); or, as a negative control, antibody sc-863 (anti-Ras-GRF1 antibody that is competent for Western blotting, but does not recognize the protein in immunoprecipitation or immunofluorescence protocols; 1:200 dilution). The secondary antibody used was Oregon Green-coupled anti-rabbit immunoglobulin (1:150 dilution). Pictures were taken with an Olympus Fluoview laser scanning confocal microscope using a ×20 objective. GST-GRF1-(900–983) (wild-type and mutant S916A) and GST-GRF1-(632–1262) were reacted with PKA (Sigma) as described (26Mattingly R.R. J. Biol. Chem. 1999; 274: 37379-37384Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). It is now clear that there are multiple GEFs that can serve to activate Ras proteins, including those in the Sos, Ras-GRF1/CDC25Mm, Ras-guanyl nucleotide-releasing protein, and other families (5Quilliam L.A. Rebhun J.F. Castro A.F. Prog. Nucleic Acids Res. Mol. Biol. 2002; 71: 391-444Crossref PubMed Google Scholar). Much work has been performed on the ubiquitous Sos exchange factors that activate Ras in response to stimulation of tyrosine kinase-mediated signals (28Egan S.E. Giddings B.W. Brooks M.W. Buday L. Sizeland A.M. Weinberg R.A. Nature. 1993; 363: 45-51Crossref PubMed Scopus (1010) Google Scholar, 35Corbalan-Garcia S. Margarit S.M. Galron D. Yang S.S. Bar-Sagi D. Mol. Cell. Biol. 1998; 18: 880-886Crossref PubMed Scopus (83) Google Scholar, 36Innocenti M. Tenca P. Frittoli E. Faretta M. Tocchetti A. Di Fiore P.P. Scita G. J. Cell Biol. 2002; 156: 125-136Crossref PubMed Scopus (146) Google Scholar). In contrast, Ras-GRF1 is expressed predominantly in the neurons of the central nervous system (11Wei W. Schreiber S.S. Baudry M. Tocco G. Broek D. Brain Res. Mol. Brain Res. 1993; 19: 339-344Crossref PubMed Scopus (27) Google Scholar, 12Ferrari C. Zippel R. Martegani E. Gnesutta N. Carrera V. Sturani E. Exp. Cell Res. 1994; 210: 353-357Crossref PubMed Scopus (30) Google Scholar, 13Zippel R. Gnesutta N. Matus-Leibovitch N. Mancinelli E. Saya D. Vogel Z. Sturani E. Renata Z. Nerina G. Noa M.L. Enzo M. Daniella S. Zvi V. Emmapaola S. Brain Res. Mol. Brain Res. 1997; 48: 140-144Crossref PubMed Scopus (62) Google Scholar) and activates Ras in response to G protein-coupled and calcium signals (8Shou C. Farnsworth C.L. Neel B.G. Feig L.A. Nature. 1992; 358: 351-354Crossref PubMed Scopus (290) Google Scholar, 21Shou C. Wurmser A. Suen K.L. Barbacid M. Feig L.A. Ling K. Oncogene. 1995; 10: 1887-1893PubMed Google Scholar, 22Zippel R. Orecchia S. Sturani E. Martegani E. Oncogene. 1996; 12: 2697-2703PubMed Google Scholar, 23Mattingly R.R. Macara I.G. Nature. 1996; 382: 268-272Crossref PubMed Scopus (156) Google Scholar, 24Mattingly R.R. Saini V. Macara I.G. Cell. Signal. 1999; 11: 603-610Crossref PubMed Scopus (22) Google Scholar). To examine the relative expression levels of Sos and Ras-GRF1 in rat brain, a quantitative Western blot protocol was developed. Ras-GRF1 and Sos1 were expressed in COS-7 cells with identical triple-HA1 epitope tags at their N termini. The lysates from these transfections were standardized by Western blotting with anti-HA1 monoclonal antibody 12CA5. The standardized HA13-Ras-GRF1 and HA13-Sos1 lysates were then used to establish the relative sensitivities of two polyclonal antibodies directed against the Ras-GRF1 and Sos proteins (Fig.1). The polyclonal antibodies detected significantly more Ras-GRF1 than Sos in lysates of rat brain forebrain, even though the sensitivity of the anti-Sos antibody is greater. Correcting for the sensitivity of the antibodies, we found that there was 11-fold more Ras-GRF1 than Sos in rat forebrain slices. Ras-GRF1 is regulated by a complex of mechanisms that include regulated phosphorylation of serine residues (24Mattingly R.R. Saini V. Macara I.G. Cell. Signal. 1999; 11: 603-610Crossref PubMed Scopus (22) Google Scholar). Phosphorylation of Ser916, which is an in vivo substrate for PKA, has been shown to be necessary in model systems for full activation of the Ras-GEF activity of Ras-GRF1 by muscarinic receptors (26Mattingly R.R. J. Biol. Chem. 1999; 274: 37379-37384Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). To test whether phosphorylation of this residue also occurs in endogenous Ras-GRF1, we metabolically labeled rat forebrain slices with [32P]orthophosphate, stimulated the slices with forskolin to activate PKA, and immunoprecipitated Ras-GRF1. Ras-GRF1 was then cleaved at methionine residues using cyanogen bromide, and the fragments were separated by peptide PAGE (Fig.2). Phosphorylation of mouse Ras-GRF1 at Ser916 produces a 32P-labeled fragment with an apparent mobility of ∼6.5 kDa (26Mattingly R.R. J. Biol. Chem. 1999; 274: 37379-37384Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). The positions of the methionines that flank this phosphorylation site are conserved between mouse and rat. Transfection of COS-7 cells with rat Ras-GRF1 demonstrated that the equivalent phosphorylation (at Ser898) was revealed in a similarly sized cyanogen bromide cleavage product. Stimulation of forebrain slices of rat brain with forskolin induced the appearance of32P in the equivalent fragment of endogenous Ras-GRF1, confirming that Ser898 is a site of regulated phosphorylation in the brain. We have previously shown that phosphorylation of Ras-GRF1 at Ser916 is necessary for maximal activation of Ras-GEF activity in a biochemical assay with recombinant Ras substrate (26Mattingly R.R. J. Biol. Chem. 1999; 274: 37379-37384Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Because we have shown here that this phosphorylation event occurred in the endogenous exchange factor in the brain, we investigated whether this phosphorylation plays a critical role in Ras activation in a neuronal context. We used the well established model of neurite extension from PC12 cells in response to Ras activation (37Bar-Sagi D. Feramisco J.R. Cell. 1985; 42: 841-848Abstract Full Text PDF PubMed Scopus (569) Google Scholar). The results show that expression of either the C-terminal half of Ras-GRF1 (Ras-GRF1ΔN) or a Myc-tagged H-Ras protein alone did not induce neurite outgrowth (Fig. 3). It has previously been demonstrated that wild-type Ras proteins (those without a constitutively activating, oncogenic mutation) are not sufficient to induce neurite outgrowth (37Bar-Sagi D. Feramisco J.R. Cell. 1985; 42: 841-848Abstract Full Text PDF PubMed Scopus (569) Google Scholar), and this result further suggests that the Ras-GRF1ΔN protein is not able to activate the endogenous Ras protein in PC12 cells to induce neurite extension. Because H-Ras is the preferred in situ substrate for Ras-GRF1 (29Jones M.K. Jackson J.H. J. Biol. Chem. 1998; 273: 1782-1787Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), we assayed for neurite extension in cotransfections of Ras-GRF1ΔN and wild-type H-Ras and found robust outgrowth. Deletion of the Ras-GRF1 construct to leave the C-terminal third (Ras-GRF1Δ900), although removing the Ras exchange motif sequence that is common to Ras-GEFs (5Quilliam L.A. Rebhun J.F. Castro A.F. Prog. Nucleic Acids Res. Mol. Biol. 2002; 71: 391-444Crossref PubMed Google Scholar), did not reduce the H-Ras-dependent induction of neurite outgrowth. Further deletion to leave the C-terminal quarter of Ras-GRF1 (Ras-GRF1Δ976) did, however, significantly reduce neurite extension. This deletion leaves intact the CDC25 Ras-GEF domain that is necessary for biochemical activity (38Coccetti P. Monzani E. Alberghina L. Casella L. Martegani E. Biochim. Biophys. Acta. 1998; 1383: 292-300Crossref PubMed Scopus (2) Google Scholar) and so reveals a stimulatory function within residues 900–975 of Ras-GRF1. The same reduction in neurite extension was produced by point mutation of the Ser916phosphorylation site to alanine (Fig. 3). The appearance of the32P-labeled fragment of Ras-GRF1 following cyanogen bromide cleavage provides an assay for the regulated phosphorylation of Ser916/898. This assay is time-consuming and provides poor sensitivity, however. To develop a more sensitive and powerful assay approach, antibodies that selectively recognize the Ser(P)916/898 form of Ras-GRF1 were developed. To confirm the selectivity of antibody 2152, it was tested against recombinant Ras-GRF1 proteins that were subjected to phosphorylation by PKA (Fig.4). The data show that antibody 2152 exhibited great selectivity for recognition of Ras-GRF1 only when it was phosphorylated, with detectable signal produced from as little as 10 ng of Ras-GRF1 Ser(P)916, whereas unphosphorylated Ras-GRF1 was only detected when microgram quantities were present. Note that PKA treatment of the S916A mutant protein did not produce any reactivity to antibody 2152. To test whether antibody 2152 recognizes both mouse and rat Ras-GRF1 expressed in mammalian cells, it was tested against whole cell lysates of COS-7 cells that had been transfected to express Ras-GRF1 and then treated with various agonists (Fig. 5). The data show that antibody 2152 reacted with a single band that was present only in the cells transfected to express Ras-GRF1 and that this band had the same mobility as that recognized by an independent anti-Ras-GRF1 antibody (sc-863). Furthermore, it is clear that the recognition of the mouse and rat Ras-GRF1 proteins was similar and that all recognition was absolutely dependent on phosphorylation at Ser916/898, as no reactivity occurred in cells expressing Ras-GRF1(S916A). Both forskolin and serum (the latter to a lesser extent, which is clearer when the Western blots were given prolonged exposure) stimulated an increase in the phosphorylation of Ras-GRF1 at Ser916/898. Interestingly, treatment of the cells with thapsigargin, which increases cytosolic calcium levels (39Mattingly R.R. Garrison J.C. FEBS Lett. 1992; 296: 225-230Crossref PubMed Scopus (18) Google Scholar), failed to increase phosphorylation of Ras-GRF1 at Ser916/898 above the basal level. This result supports previous observations on transfected fibroblasts that increases in calcium do not stimulate the phosphorylation of Ras-GRF1 (40Mattingly R.R. In Vitro Mol. Toxicol. 1998; 11: 57-62Google Scholar), although it has been reported that Ras-GRF1 is an in vitro substrate for calmodulin-dependent kinase II (14Sturani E. Abbondio A. Branduardi P. Ferrari C. Zippel R. Martegani E. Vanoni M. Denis-Donini S. Exp. Cell Res. 1997; 235: 117-123Crossref PubMed Scopus (56) Google Scholar). To investigate the phosphorylation of Ras-GRF1 in a neuronal context, we coexpressed Ras-GRF1 with muscarinic receptors in PC12 cells. Western blotting of PC12 cell lysates (Fig. 6) demonstrated that activation of muscarinic receptors by carbachol, stimulation of PKA by forskolin, and activation of endogenous Trk receptors by nerve growth factor (NGF) all increased the phosphorylation of Ras-GRF1 at Ser916. To extend these results, either HA13-Ras-GRF1 or its S916A mutant was coexpressed in PC12 cells with muscarinic receptors, and the cells were stimulated and then fixed and processed for indirect confocal immunofluorescence (Fig.7). It is clear that antibody 2152 reactivity was again dependent on phosphorylation at Ser916, as no reactivity was seen in untransfected cells, in transfected cells that were not stimulated, or in stimulated cells that were transfected with the Ras-GRF1(S916A) mutant. In agreement with the Western blot results, carbachol, forskolin, and NGF all induced the phosphorylation of Ras-GRF1 at Ser916.Figure 7Regulated phosphorylation of Ras-GRF1 at Ser916 in PC12 cells as revealed by indirect confocal immunofluorescence. PC12 cells were cotransfected with pKH3Ras-GRF1 (left panels) or pKH3Ras-GRF1(S916A) (right panels) plus expression vectors for human muscarinic receptor subtypes 1 and 2. The cells were deprived of serum overnight and then stimulated with 100 μm carbachol, with 100 μm IBMX plus 20 μm forskolin, or with 10 ng/ml NGF. The cells were fixed and processed for indirect confocal immunofluorescence using red detection for the HA1 epitope tags at the N termini of the Ras-GRF1 constructs and green detection for antibody 2152 (raised against Ras-GRF1 Ser(P)916(Ras-GRF1phospho916)). The fluorescence images are presented overlaid with a phase-contrast image of the cells that reveals the presence of untransfected control cells. Note that green fluorescence due to antibody 2152 reactivity was found only when wild-type Ras-GRF1 (Ras-GRF1,wt) was present (thus colocalized with red fluorescence and appears yellow) and only in cells that had been stimulated with agonists that induce Ser916phosphorylation. Data shown are representative of five independent experiments. Ras-GRF1,916A, Ras-GRF1(S916A).View Large Image Figure ViewerDownload (PPT) To investigate whether endogenous Ras-GRF1 is regulated by phosphorylation in a manner similar to that defined in the model systems, antibody 2152 was used to assess phosphorylation of Ras-GRF1 in rat forebrain slices. It was difficult to detect any reactivity to antibody 2152 in lysates of brain slices by Western blotting, perhaps suggesting that only a subset of Ras-GRF1 proteins (perhaps, in particular, neurons) is phosphorylated at Ser898. When Ras-GRF1 was first immunoprecipitated from the lysate using an antibod" @default.
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• W2004638925 date "2003-04-01" @default.
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• W2004638925 title "Phosphorylation of the Ras-GRF1 Exchange Factor at Ser916/898 Reveals Activation of Ras Signaling in the Cerebral Cortex" @default.
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