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• W2023985493 abstract "RasGRPs constitute a new group of diacylglycerol-dependent GDP/GTP exchange factors that activate Ras subfamily GTPases. Despite a common structure, Ras-GRPs diverge in their GTPase specificity, subcellular distribution, and downstream biological effects. The more divergent family member is RasGRP2, a Rap1-specific exchange factor with low affinity toward diacylglycerol. The regulation of RasGRP2 during signal transduction has remained elusive up to now. In this report, we show that the subcellular localization of Ras-GRP2 is highly dependent on actin dynamics. Thus, the induction of F-actin by cytoskeletal regulators such as Vav, Vav2, Dbl, and Rac1 leads to the shift of RasGRP2 from the cytosol to membrane ruffles and its co-localization with F-actin. Treatment of cells with cytoskeletal disrupting drugs abolishes this effect, leading to an abnormal localization of RasGRP2 in cytoplasmic clusters of actin. The use of Rac1 effector mutants indicates that the RasGRP2 translocation is linked exclusively to actin polymerization and is independent of other pathways such as p21-activated kinase JNK, or superoxide production. Biochemical experiments demonstrate that the translocation of RasGRP2 to membrane ruffles is mediated by the direct association of this protein with F-actin, a property contained within its 150 first amino acids. Finally, we show that the RasGRP2/F-actin interaction promotes the regionalized activation of Rap1 in juxtamembrane areas of the cell. These results reveal a novel function of the actin cytoskeleton in mediating the spatial activation of Ras subfamily GTPases through the selective recruitment of GDP/GTP exchange factors. RasGRPs constitute a new group of diacylglycerol-dependent GDP/GTP exchange factors that activate Ras subfamily GTPases. Despite a common structure, Ras-GRPs diverge in their GTPase specificity, subcellular distribution, and downstream biological effects. The more divergent family member is RasGRP2, a Rap1-specific exchange factor with low affinity toward diacylglycerol. The regulation of RasGRP2 during signal transduction has remained elusive up to now. In this report, we show that the subcellular localization of Ras-GRP2 is highly dependent on actin dynamics. Thus, the induction of F-actin by cytoskeletal regulators such as Vav, Vav2, Dbl, and Rac1 leads to the shift of RasGRP2 from the cytosol to membrane ruffles and its co-localization with F-actin. Treatment of cells with cytoskeletal disrupting drugs abolishes this effect, leading to an abnormal localization of RasGRP2 in cytoplasmic clusters of actin. The use of Rac1 effector mutants indicates that the RasGRP2 translocation is linked exclusively to actin polymerization and is independent of other pathways such as p21-activated kinase JNK, or superoxide production. Biochemical experiments demonstrate that the translocation of RasGRP2 to membrane ruffles is mediated by the direct association of this protein with F-actin, a property contained within its 150 first amino acids. Finally, we show that the RasGRP2/F-actin interaction promotes the regionalized activation of Rap1 in juxtamembrane areas of the cell. These results reveal a novel function of the actin cytoskeleton in mediating the spatial activation of Ras subfamily GTPases through the selective recruitment of GDP/GTP exchange factors. One frequent event in the signaling pathways engaged by most extracellular factors is the activation of GTPases of the Ras subfamily (1Pronk G.J. Bos J.L. Biochim. Biophys. Acta. 1994; 1198: 131-147PubMed Google Scholar). This group of GTP-binding proteins is composed of Ras (K-RasA, K-RasB, N-Ras, and H-Ras), R-Ras (R-Ras, TC21/R-Ras2, and M-Ras/R-Ras3), Rap (Rap1A, Rap1B, Rap2A, and Rap2B), and Ral (RalA and RalB) proteins (2Ehrhardt A. Ehrhardt G.R. Guo X. Schrader J.W. Exp. Hematol. 2002; 30: 1089-1106Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). These GTPases share a common tridimensional structure and a regulatory cycle strictly dependent on the binding of guanosine nucleotides (1Pronk G.J. Bos J.L. Biochim. Biophys. Acta. 1994; 1198: 131-147PubMed Google Scholar, 3Lowy D.R. Willumsen B.M. Annu. Rev. Biochem. 1993; 62: 851-891Crossref PubMed Scopus (1122) Google Scholar). In nonactivated cells, these GTPases are found in an inactive, GDP-bound state. The stimulation of receptors by their respective ligands leads to the release of GDP and the incorporation of GTP molecules. This exchange of nucleotides induces a conformational change within the switch I and switch II of the GTPases that converts them in optimal docking sites for effector molecules such as c-Raf family proteins, Ral GDP dissociation stimulator (RalGDS), 1The abbreviations used are: RalGDS, Ral GDP dissociation stimulator; CFP, cyan fluorescent protein; DAG, diacylglycerol; DH, Dbl homology domain; EGFP, enhanced green fluorescent protein; FRET, fluorescence resonance energy transfer; GAP, GTPase-activating protein; GEF, guanosine nucleotide exchange factor; GRF, guanosine nucleotide-releasing factor; GRP, GDP-releasing protein; GST, glutathione S-transferase; HA, hemagglutinin; JNK, c-Jun N-terminal kinase; PAGE, polyacrylamide gel electrophoresis; PAK, p21-activated kinase; PH, pleckstrin homology domain; RBD, Ras binding domain; SH, Src homology; Sos, son-of-sevenless; YFP, yellow fluorescent protein; ZF, zinc finger. 1The abbreviations used are: RalGDS, Ral GDP dissociation stimulator; CFP, cyan fluorescent protein; DAG, diacylglycerol; DH, Dbl homology domain; EGFP, enhanced green fluorescent protein; FRET, fluorescence resonance energy transfer; GAP, GTPase-activating protein; GEF, guanosine nucleotide exchange factor; GRF, guanosine nucleotide-releasing factor; GRP, GDP-releasing protein; GST, glutathione S-transferase; HA, hemagglutinin; JNK, c-Jun N-terminal kinase; PAGE, polyacrylamide gel electrophoresis; PAK, p21-activated kinase; PH, pleckstrin homology domain; RBD, Ras binding domain; SH, Src homology; Sos, son-of-sevenless; YFP, yellow fluorescent protein; ZF, zinc finger. and phosphatidylinositol 3-kinase (1Pronk G.J. Bos J.L. Biochim. Biophys. Acta. 1994; 1198: 131-147PubMed Google Scholar, 4Marshall M.S. FASEB J. 1995; 9: 1311-1318Crossref PubMed Scopus (271) Google Scholar). The concomitant activation of these effector proteins by either post-translational modification (i.e. phosphorylation) or changes in their subcellular localization initiate the stimulation of several signaling cascades that determine, among other biological responses, the proliferative, developmental, and/or motility status of the cell that has received the extracellular stimulus (1Pronk G.J. Bos J.L. Biochim. Biophys. Acta. 1994; 1198: 131-147PubMed Google Scholar, 4Marshall M.S. FASEB J. 1995; 9: 1311-1318Crossref PubMed Scopus (271) Google Scholar). The GTPase signal is subsequently shut off at the end of the stimulation period by the hydrolysis of the bound GTP, a step mediated by GTPase-activating proteins (GAPs) (1Pronk G.J. Bos J.L. Biochim. Biophys. Acta. 1994; 1198: 131-147PubMed Google Scholar, 3Lowy D.R. Willumsen B.M. Annu. Rev. Biochem. 1993; 62: 851-891Crossref PubMed Scopus (1122) Google Scholar). The importance of this cycle is underscored by the observation that point mutations affecting either the GTP hydrolysis or the GDP/GTP exchange originate GTPase proteins with high oncogenic potential (3Lowy D.R. Willumsen B.M. Annu. Rev. Biochem. 1993; 62: 851-891Crossref PubMed Scopus (1122) Google Scholar).Because of this activation/deactivation cycle, the main regulatory step in the modulation of the activity of these GTPases is the stimulus-dependent triggering of GDP/GTP exchange. This exchange is catalyzed by enzymes known indistinctly as guanosine nucleotide exchange factors (GEFs), guanosine nucleotide-releasing factors (GRFs), GDP-releasing proteins (GRPs), or GDP dissociation stimulators (5Quilliam L.A. Rebhun J.F. Castro A.F. Prog. Nucleic Acids Res. Mol. Biol. 2002; 71: 391-444Crossref PubMed Google Scholar). GEF families described so far for Ras subfamily proteins include Sos proteins (Sos1 and Sos2), Ras GRFs (RasGRF1 and RasGRF2), cyclic nucleotide Ras GEF (CNRasGEF), and Ras GRPs (RasGRP1, RasGRP3, RasGRP4, and the N-myristoylatable and nonmyristoylatable RasGRP2 versions generated by the differential splicing of the rasgrp2 gene) (5Quilliam L.A. Rebhun J.F. Castro A.F. Prog. Nucleic Acids Res. Mol. Biol. 2002; 71: 391-444Crossref PubMed Google Scholar). In addition to these families, there are other GEFs specific only for Rap proteins, such as C3G and Epac. Although these GEFs are highly divergent from a structural point of view, they have in common the presence of a Cdc25 domain responsible for stimulating nucleotide exchange on either Rap and/or Ras GTP hydrolases (5Quilliam L.A. Rebhun J.F. Castro A.F. Prog. Nucleic Acids Res. Mol. Biol. 2002; 71: 391-444Crossref PubMed Google Scholar). Sos and RasGRF proteins also contain a Dbl homology domain involved in the activation of Rho/Rac GTPases (5Quilliam L.A. Rebhun J.F. Castro A.F. Prog. Nucleic Acids Res. Mol. Biol. 2002; 71: 391-444Crossref PubMed Google Scholar). According to this, several reports have indicated that these GEFs can promote the activation of Rac1 (6Nimnual A.S. Yatsula B.A. Bar-Sagi D. Science. 1998; 279: 560-563Crossref PubMed Scopus (387) Google Scholar, 7Fan W.T. Koch C.A. de Hoog C.L. Fam N.P. Moran M.F. Curr. Biol. 1998; 8: 935-938Abstract Full Text Full Text PDF PubMed Google Scholar).Most GEF families have become specialized in the activation of Ras subfamily proteins in specific signaling contexts. Sos and C3G proteins are involved in connecting protein-tyrosine kinases with the stimulation of Ras subfamily GTPases (5Quilliam L.A. Rebhun J.F. Castro A.F. Prog. Nucleic Acids Res. Mol. Biol. 2002; 71: 391-444Crossref PubMed Google Scholar). Ras-GRF, CNRasGEF, and Epac proteins are coupled to heterotrimeric G-proteins (5Quilliam L.A. Rebhun J.F. Castro A.F. Prog. Nucleic Acids Res. Mol. Biol. 2002; 71: 391-444Crossref PubMed Google Scholar). In contrast to those GEFs, the different members of the RasGRP family are more difficult to assign to a common framework of biological functions and regulatory mechanisms. This is because these exchange factors diverge significantly in a number of biochemical, cellular, and biological properties. At the biochemical level, RasGRPs work catalytically on an overlapping, but not identical, subset of GTPases. Thus, RasGRP1 activates the exchange activity of TC21, M-Ras, and Ras proteins. Instead, it is inactive on Rap GTPases (8Ohba Y. Mochizuki N. Yamashita S. Chan A.M. Schrader J.W. Hattori S. Nagashima K. Matsuda M. J. Biol. Chem. 2000; 275: 20020-20026Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 9Ebinu J.O. Bottorff D.A. Chan E.Y. Stang S.L. Dunn R.J. Stone J.C. Science. 1998; 280: 1082-1086Crossref PubMed Scopus (545) Google Scholar, 10Tognon C.E. Kirk H.E. Passmore L.A. Whitehead I.P. Der C.J. Kay R.J. Mol. Cell. Biol. 1998; 18: 6995-7008Crossref PubMed Scopus (203) Google Scholar, 11Ebinu J.O. Stang S.L. Teixeira C. Bottorff D.A. Hooton J. Blumberg P.M. Barry M. Bleakley R.C. Ostergaard H.L. Stone J.C. Blood. 2000; 95: 3199-3203Crossref PubMed Google Scholar). RasGRP2 acts mostly on Rap1 and, with lower affinity, on other Ras proteins except H-Ras (8Ohba Y. Mochizuki N. Yamashita S. Chan A.M. Schrader J.W. Hattori S. Nagashima K. Matsuda M. J. Biol. Chem. 2000; 275: 20020-20026Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 12Kawasaki H. Springett G.M. Toki S. Canales J.J. Harlan P. Blumenstiel J.P. Chen E.J. Bany I.A. Mochizuki N. Ashbacher A. Matsuda M. Housman D.E. Graybiel A.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13278-13283Crossref PubMed Scopus (309) Google Scholar). Interestingly, the catalytic specificity of RasGRP2 appears to be different in function of the splice variant used (13Clyde-Smith J. Silins G. Gartside M. Grimmond S. Etheridge M. Apolloni A. Hayward N. Hancock J.F. J. Biol. Chem. 2000; 275: 32260-32267Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). RasGRP4 activates Ras but not Rap proteins (14Reuther G.W. Lambert Q.T. Rebhun J.F. Caligiuri M.A. Quilliam L.A. Der C.J. J. Biol. Chem. 2002; 277: 30508-30514Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). RasGRP3 is the more promiscuous member, being active on the majority of Ras subfamily GTPases tested so far (8Ohba Y. Mochizuki N. Yamashita S. Chan A.M. Schrader J.W. Hattori S. Nagashima K. Matsuda M. J. Biol. Chem. 2000; 275: 20020-20026Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 15Yamashita S. Mochizuki N. Ohba Y. Tobiume M. Okada Y. Sawa H. Nagashima K. Matsuda M. J. Biol. Chem. 2000; 275: 25488-25493Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). As a probable consequence of this biochemical heterogeneity, RasGRPs exhibit different biological properties, including differences in the activation of extracellular-regulated kinases (inhibited by RasGRP2 and stimulated by the other RasGRPs), the stimulation of JNK (stimulated preferentially by RasGRP1 and -3), the induction of neuronal differentiation (where RasGRP2 is inactive), and the promotion of anchorage-independent growth (induced by Ras-GRP1, -3, and -4) (12Kawasaki H. Springett G.M. Toki S. Canales J.J. Harlan P. Blumenstiel J.P. Chen E.J. Bany I.A. Mochizuki N. Ashbacher A. Matsuda M. Housman D.E. Graybiel A.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13278-13283Crossref PubMed Scopus (309) Google Scholar, 14Reuther G.W. Lambert Q.T. Rebhun J.F. Caligiuri M.A. Quilliam L.A. Der C.J. J. Biol. Chem. 2002; 277: 30508-30514Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 15Yamashita S. Mochizuki N. Ohba Y. Tobiume M. Okada Y. Sawa H. Nagashima K. Matsuda M. J. Biol. Chem. 2000; 275: 25488-25493Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). RasGRP proteins are also highly divergent in their organ and tissue distribution (12Kawasaki H. Springett G.M. Toki S. Canales J.J. Harlan P. Blumenstiel J.P. Chen E.J. Bany I.A. Mochizuki N. Ashbacher A. Matsuda M. Housman D.E. Graybiel A.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13278-13283Crossref PubMed Scopus (309) Google Scholar, 14Reuther G.W. Lambert Q.T. Rebhun J.F. Caligiuri M.A. Quilliam L.A. Der C.J. J. Biol. Chem. 2002; 277: 30508-30514Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 15Yamashita S. Mochizuki N. Ohba Y. Tobiume M. Okada Y. Sawa H. Nagashima K. Matsuda M. J. Biol. Chem. 2000; 275: 25488-25493Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 16Li L. Yang Y. Stevens R. Mol. Immunol. 2002; 38: 1283Crossref PubMed Scopus (14) Google Scholar, 17Pierret P. Mechawar N. Vallee A. Patel J. Priestley J.V. Dunn R.J. Dower N.A. Stone J.C. Richardson P.M. Neuroscience. 2002; 111: 83-94Crossref PubMed Scopus (9) Google Scholar, 18Pierret P. Vallee A. Mechawar N. Dower N.A. Stone J.C. Richardson P.M. Dunn R.J. Neuroscience. 2001; 108: 381-390Crossref PubMed Scopus (10) Google Scholar, 19Toki S. Kawasaki H. Tashiro N. Housman D.E. Graybiel A.M. J. Comp. Neurol. 2001; 437: 398-407Crossref PubMed Scopus (26) Google Scholar, 20Pierret P. Dunn R.J. Djordjevic B. Stone J.C. Richardson P.M. J. Neurocytol. 2000; 29: 485-497Crossref PubMed Scopus (12) Google Scholar). Finally, and perhaps more intriguingly from a regulatory point of view, RasGRPs show a different susceptibility toward upstream signals. The activation of RasGRP1, -3, and -4 is mediated via the phospholipase C-γ-dependent generation of diacylglycerol (DAG) (9Ebinu J.O. Bottorff D.A. Chan E.Y. Stang S.L. Dunn R.J. Stone J.C. Science. 1998; 280: 1082-1086Crossref PubMed Scopus (545) Google Scholar, 21Rong S.B. Enyedy I.J. Qiao L. Zhao L. Ma D. Pearce L.L. Lorenzo P.S. Stone J.C. Blumberg P.M. Wang S. Kozikowski A.P. J. Med. Chem. 2002; 45: 853-860Crossref PubMed Scopus (21) Google Scholar, 22Lorenzo P.S. Kung J.W. Bottorff D.A. Garfield S.H. Stone J.C. Blumberg P.M. Cancer Res. 2001; 61: 943-949PubMed Google Scholar, 23Lorenzo P.S. Beheshti M. Pettit G.R. Stone J.C. Blumberg P.M. Mol. Pharmacol. 2000; 57: 840-846PubMed Google Scholar). This second messenger binds to a C-terminal ZF region present in those GEFs (see Fig. 5), making it possible the translocation of RasGRPs to membranes and their subsequent association with the target GTPases (5Quilliam L.A. Rebhun J.F. Castro A.F. Prog. Nucleic Acids Res. Mol. Biol. 2002; 71: 391-444Crossref PubMed Google Scholar, 21Rong S.B. Enyedy I.J. Qiao L. Zhao L. Ma D. Pearce L.L. Lorenzo P.S. Stone J.C. Blumberg P.M. Wang S. Kozikowski A.P. J. Med. Chem. 2002; 45: 853-860Crossref PubMed Scopus (21) Google Scholar). In contrast, RasGRP2 shows a very poor response to DAG and, as a consequence, it does not undergo the characteristic rapid translocation of other RasGRPs to the plasma membrane and endomembranes when cells are treated with DAG agonists (13Clyde-Smith J. Silins G. Gartside M. Grimmond S. Etheridge M. Apolloni A. Hayward N. Hancock J.F. J. Biol. Chem. 2000; 275: 32260-32267Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 24Caloca M.J. Zugaza J.L. Bustelo X.R. J. Biol. Chem. 2003; 278: 33465-33473Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). The divergence among the RasGRP ZF regions also conditions the subcellular localization of these proteins. Thus, RasGRP1 and -3 localize preferentially in both the endoplasmic reticulum and Golgi apparatus in exponentially growing cells (24Caloca M.J. Zugaza J.L. Bustelo X.R. J. Biol. Chem. 2003; 278: 33465-33473Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). In contrast, RasGRP2 is found in the cytoplasm or in both the cytosol and the plasma membrane depending on the splice variant analyzed (13Clyde-Smith J. Silins G. Gartside M. Grimmond S. Etheridge M. Apolloni A. Hayward N. Hancock J.F. J. Biol. Chem. 2000; 275: 32260-32267Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 24Caloca M.J. Zugaza J.L. Bustelo X.R. J. Biol. Chem. 2003; 278: 33465-33473Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). The above observations indicate that RasGRP2 is the most divergent member of the family, in terms of substrate specificity, subcellular localization, and regulation by upstream signals.The different behavior or RasGRP2 led us to look for regulatory signals that could modulate its activity during cell stimulation. During our work with RasGRPs (24Caloca M.J. Zugaza J.L. Bustelo X.R. J. Biol. Chem. 2003; 278: 33465-33473Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar), we observed that the nonmyristoylatable RasGRP2 isoform could be found in exponentially growing cells in membrane areas rich in F-actin. Moreover, and unlike RasGRP1 and -3, the treatment of quiescent cells with either epidermal growth factor or phorbol esters did not induce the effective translocation of this protein toward the plasma membrane or the Golgi apparatus. Instead, a small enrichment of RasGRP2 in peripheral membrane ruffles was observed. These observations led us to investigate whether this idiosyncratic member of the RasGRP family could be regulated by signals promoting actin polymerization and cytoskeletal change. In this report, we show that different cytoskeletal regulators such as Vav, Vav2, Dbl, and Rac1 can promote the effective translocation of RasGRP2 toward juxtamembrane areas and membrane ruffles. Moreover, we demonstrate that this effect is because of the intrinsic property of RasGRP2 of associating with polymerized filaments of actin via its N-terminal domain.MATERIALS AND METHODSPlasmids—Mammalian expression vectors encoding FLAG-tagged RasGRP2 (pCXN2-RasGRP2), EGFP-tagged RasGRP2 (pMJC39), and EGFP-tagged Rac1 (pNM32) have been described previously (24Caloca M.J. Zugaza J.L. Bustelo X.R. J. Biol. Chem. 2003; 278: 33465-33473Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). The mammalian expression vector encoding the EGFP-Rac1G12V+ΔIns mutant (pMJC8) was generated by ligating into the pEGFP-C1 vector a fragment containing the rac1G12V+ΔIns cDNA obtained by PCR from the pCGT-Rac1G12V+ΔIns plasmid (provided by Dr. D. Bar-Sagi, State University of New York, Stony Brook, NY) (25Joneson T. Bar-Sagi D. J. Biol. Chem. 1998; 273: 17991-17994Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). Deletion mutants of Ras-GRP2 were generated by PCR using pCXN2-RasGRP2 as template and subcloned into either the pCEFL-FLAG or the pGEX-4T3 vectors for expression in eukaryotes and prokaryotes, respectively. pcDNA3-Vav (Δ1-186) (pMJC10) and pcDNA3-Vav (Δ1-66) were generated by PCR using the pJC11 plasmid as DNA template (26Schuebel K.E. Movilla N. Rosa J.L. Bustelo X.R. EMBO J. 1998; 17: 6608-6621Crossref PubMed Scopus (223) Google Scholar). Expression vectors for Vav point mutants were generated by site-directed mutagenesis (QuikChange, Stratagene). The oligonucleotides used for the generation of RasGRP2 and Vav mutants are available upon request. The expression vector for the Vav (Δ1-186+Δ608-845) mutant (pNM103) has been described previously (26Schuebel K.E. Movilla N. Rosa J.L. Bustelo X.R. EMBO J. 1998; 17: 6608-6621Crossref PubMed Scopus (223) Google Scholar, 27Zugaza J.L. Lopez-Lago M.A. Caloca M.J. Dosil M. Movilla N. Bustelo X.R. J. Biol. Chem. 2002; 277: 45377-45392Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). The expression vector for the dbl oncogene (pcDNA-oncDBL) was obtained from Dr. Silvio Gutkind (National Institutes of Health, Bethesda, MD) (28Coso O.A. Chiariello M. Yu J.C. Teramoto H. Crespo P. Xu N. Miki T. Gutkind J.S. Cell. 1995; 81: 1137-1146Abstract Full Text PDF PubMed Scopus (1559) Google Scholar). The expression plasmid encoding the HA-tagged Sos1 (pCEFL-KZ-HA-Sos1-isof1) was provided by Dr. J. M. Rojas (Instituto Carlos III, Madrid, Spain) (29Jorge R. Zarich N. Oliva J.L. Azanedo M. Martinez N. de la Cruz X. Rojas J.M. J. Biol. Chem. 2002; 277: 44171-44179Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). An expression plasmid encoding a FLAG-tagged version of RasGRF2 (pCEFL-FLAG-RasGRF2) was obtained from Dr. M. F. Moran (University of Toronto, Toronto, Canada) (30Fam N.P. Fan W.T. Wang Z. Zhang L.J. Chen H. Moran M.F. Mol. Cell. Biol. 1997; 17: 1396-1406Crossref PubMed Scopus (133) Google Scholar). A plasmid encoding a FLAG-tagged version of lbc oncogene product (pSRNeo-Lbc) was a gift from Dr. D. Toksoz (Tufts University, Boston, MA) (31Toksoz D. Williams D.A. Oncogene. 1994; 9: 621-628PubMed Google Scholar). pRaichu-Rap1-404X was given to us by Dr. M. Matsuda (Research Institute for Microbial Diseases, Osaka University, Osaka, Japan) (32Mochizuki N. Yamashita S. Kurokawa K. Ohba Y. Nagai T. Miyawaki A. Matsuda M. Nature. 2001; 411: 1065-1068Crossref PubMed Scopus (494) Google Scholar). pEGFP vectors were purchased from Clontech. A bacterial expression vector encoding the GST fused to the Ras binding domain of RalGDS (amino acid residues 701-851) (pGEX-RalGDS-RBD) was obtained from Dr. P. Crespo (University of Cantabria/Instituto de Investigaciones Biomédicas, Santander, Cantabria, Spain).Tissue Culture and DNA Transfections—COS1 and Jurkat cells were cultured at 37 °C and a humidified 5% CO2 atmosphere. COS1 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% calf serum plus 100 units·ml-1 penicillin and streptomycin. Jurkat cells were grown in RPMI medium supplemented with 10% fetal bovine serum plus 100 units·ml-1 penicillin and streptomycin. All tissue culture reagents were obtained from Invitrogen. For ectopic protein expression, COS1 cells were transfected with the appropriate plasmids using a liposomal carrier (FuGENE6, Roche Molecular Biochemicals) according to the instructions from the manufacturer. For standard immunofluorescence studies, COS1 cells were transfected with 1 μg of each mammalian expression vector. For Rap1 GTP pull-down experiments, cells were transfected with 0.1 and 1 μg of vectors encoding RasGRP2 and Vav (Δ1-186), respectively. For fluorescence resonance energy transfer (FRET) analyses, COS1 cells were transfected with 0.5 μg of pRaichu-Rap1-404X, 0.5 μg of the vector encoding EGFP-Vav (Δ1-186), and 1 μg of the plasmid containing FLAG-tagged RasGRP2 in the appropriate combinations. Jurkat T cells were transfected by electroporation with 10 μg of each indicated plasmid. Electroporations were performed in Opti-MEM medium (Invitrogen) with a Gene Pulser II apparatus (Bio-Rad) using 0.4-cm gap electroporation cuvettes with settings of 280 V and 1200 microfarads. In all cases, transfections were supplemented with empty vector to normalize the total amount of plasmid DNA used in each transfection.F-actin Co-sedimentation Assays—Actin co-sedimentation assays were performed as reported (33Scita G. Tenca P. Areces L.B. Tocchetti A. Frittoli E. Giardina G. Ponzanelli I. Sini P. Innocenti M. Di Fiore P.P. J. Cell Biol. 2001; 154: 1031-1044Crossref PubMed Scopus (102) Google Scholar). Briefly, F-actin was obtained by incubating G-actin (Sigma) in F-buffer (5 mm Tris-HCl (pH 7.8), 1 mm ATP, 0.5 mm dithiothreitol, 0.2 mm CaCl2, 0.2 mm MgCl2, 100 mm KCl) for 60 min at 37 °C. GST-RasGRP2 (amino acids 1-150) was purified by standard GST fusion protein purification protocols. Purified proteins were incubated with various amounts of F-actin in F-buffer in a total volume of 100 μl at 37 °C for 45 min and then ultracentrifuged for 60 min at 100,000 × g in a TLA-100 rotor (Beckman). After the centrifugation, supernatants (100 μl) were taken off and diluted 1:1 with SDS-PAGE sample buffer. Pellets were resuspended in 100 μl of SDS-PAGE sample buffer. Aliquots of the supernatants (30 μl) and pellets (15 μl) were denatured by boiling, separated by SDS-PAGE, transferred onto nitrocellulose filters, and immunoblotted with antibodies to GST (Santa Cruz Biotechnology, 1:1,000 dilution) and actin (Sigma, 1:5,000 dilution). Immunoreactive bands were visualized using a chemiluminescence detection system (ECL, Amersham Biosciences).GST Pull-down Assays—For Rap1 activity assays, COS1 cells were lysed in a buffer containing 50 mm Tris-HCl (pH 7.6), 150 mm NaCl, 10 mm MgCl2, 1% IGEPAL (Sigma), 100 μm Na3VO4 (Sigma), 1 mm NaF (Sigma), and a mixture of protease inhibitors (Cømplete, Roche Molecular Biochemicals). Lysates were centrifuged at 14,000 × g for 10 min at 4 °C. The resulting supernatants were incubated for 60 min at 4 °C with 10 μg of GST protein fused to the Ras/Rap binding domain of RalGDS immobilized onto glutathione-Sepharose beads (Amersham Biosciences). After incubation, beads were collected and washed three times with lysis buffer. Proteins were then eluted from the beads using Laemmli's sample buffer, separated electrophoretically, and analyzed by immunoblotting using anti-Rap1 antibodies (Santa Cruz Biotechnology). Pull-down assays conducted with GST proteins containing either the N-terminal domain (residues 1-150) of RasGRP2 or the Rac1 binding domain of PAK1 were performed as described above. When appropriate, the proteins transferred to the nitrocellulose filter were visualized by staining with Ponceau solution (Sigma), followed by three washes with water.Immunofluorescence Analysis—COS1 cells were grown onto glass coverslips and transfected as indicated above. 24-48 h after transfection, cells were washed, fixed with formaldehyde (Sigma), and subjected to immunostaining. Immunological reagents used were anti-FLAG M2 (Sigma), anti-HA (Covance), and anti-Dbl (Santa Cruz Biotechnology). Cy3- and Cy2-labeled secondary antibodies were obtained from Jackson Immunolaboratories. Rhodamine-phalloidin and cytochalasin D were obtained from Molecular Probes and Sigma, respectively. For FRET imaging, COS-1 cells were grown on polylysine-coated glasses and transfected. 24 h after transfection, cells were serum-starved for 4 h, washed, fixed, and analyzed by confocal microscopy. Capture of FRET images was performed as described previously (32Mochizuki N. Yamashita S. Kurokawa K. Ohba Y. Nagai T. Miyawaki A. Matsuda M. Nature. 2001; 411: 1065-1068Crossref PubMed Scopus (494) Google Scholar). Immunofluorescent signals were registered using a laser scanning confocal microscope (LSM510, Zeiss). Imports of confocal images were made using the LSM510 software (Zeiss). Final processing of images was done with the Adobe PhotoShop (Adobe Systems) program.Integrin Activation—30 h after electroporation, Jurkat cells were purified away from cellular debri by centrifugation onto Ficoll-Hypaque medium gradients (Sigma), resuspended in RPMI 1640 supplemented with 10% fetal calf serum (Invitrogen), and allowed to settle for 30 min onto 12-mm coverslips coated with 50 μg/ml poly-l-Lys (Sigma). Cells were then fixed in ice-cold 4% formaldehyde for 10 min and permeabilized by an incubation of 5 min with 0.5% Triton X-100 in phosphate-buffered saline solution. Detection of doubly transfected cells was carried out by microscopic detection of EGFP fusion proteins and staining with biotinylated anti-FLAG (M2 clone) and Alexa350-conjugated streptavidin. To detect activation of β1 integrins, we performed staining experiments with the HUTS-21 monoclonal antibody (34Luque A. Gomez M. Puzon W. Takada Y. Sanchez-Madrid F. Cabanas C. J. Biol. Chem. 1996; 271: 11067-11075Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar), whereas total β1 integrin was detected with the TS2/16 monoclonal antibody (34Luque A. Gomez M. Puzon W. Takada Y. Sanchez-Madrid F. Cabanas C. J. Biol. Chem. 1996; 271: 11067-11075Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar). In both cases, Alexa568-conjugated goat anti-mouse IgGs was employed. Quantification was performed by direct" @default.
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• W2023985493 title "F-actin-dependent Translocation of the Rap1 GDP/GTP Exchange Factor RasGRP2" @default.
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