Characterization of a Novel Interaction between ELMO1 and ERM Proteins
2005; Elsevier BV; Volume: 281; Issue: 9 Linguagem: Inglês
10.1074/jbc.m510647200
ISSN1083-351X
AutoresCynthia Grimsley, Mingjian Lu, Lisa B. Haney, Jason M. Kinchen, Kodi S. Ravichandran,
Tópico(s)Galectins and Cancer Biology
ResumoERMs are closely related proteins involved in cell migration, cell adhesion, maintenance of cell shape, and formation of microvilli through their ability to cross-link the plasma membrane with the actin cytoskeleton. ELMO proteins are also known to regulate actin cytoskeleton reorganization through activation of the small GTPbinding protein Rac via the ELMO-Dock180 complex. Here we showed that ERM proteins associate directly with ELMO1 as purified recombinant proteins in vitro and at endogenous levels in intact cells. We mapped ERM binding on ELMO1 to the N-terminal 280 amino acids, which overlaps with the region required for binding to the GTPase RhoG, but is distinct from the C-terminal Dock180 binding region. Consistent with this, ELMO1 could simultaneously bind both radixin and Dock180, although radixin did not alter Rac activation via the Dock180-ELMO complex. Most interestingly, radixin binding did not affect ELMO binding to active RhoG and a trimeric complex of active RhoG-ELMO-radixin could be detected. Moreover, the three proteins colocalized at the plasma membrane. Finally, in contrast to most other ERM-binding proteins, ELMO1 binding occurred independently of the state of radixin C-terminal phosphorylation, suggesting an ELMO1 interaction with both the active and inactive forms of ERM proteins and implying a possible role of ELMO in localizing or retaining ERM proteins in certain cellular sites. Together these data suggest that ELMO1-mediated cytoskeletal changes may be coordinated with ERM protein crosslinking activity during dynamic cellular functions. ERMs are closely related proteins involved in cell migration, cell adhesion, maintenance of cell shape, and formation of microvilli through their ability to cross-link the plasma membrane with the actin cytoskeleton. ELMO proteins are also known to regulate actin cytoskeleton reorganization through activation of the small GTPbinding protein Rac via the ELMO-Dock180 complex. Here we showed that ERM proteins associate directly with ELMO1 as purified recombinant proteins in vitro and at endogenous levels in intact cells. We mapped ERM binding on ELMO1 to the N-terminal 280 amino acids, which overlaps with the region required for binding to the GTPase RhoG, but is distinct from the C-terminal Dock180 binding region. Consistent with this, ELMO1 could simultaneously bind both radixin and Dock180, although radixin did not alter Rac activation via the Dock180-ELMO complex. Most interestingly, radixin binding did not affect ELMO binding to active RhoG and a trimeric complex of active RhoG-ELMO-radixin could be detected. Moreover, the three proteins colocalized at the plasma membrane. Finally, in contrast to most other ERM-binding proteins, ELMO1 binding occurred independently of the state of radixin C-terminal phosphorylation, suggesting an ELMO1 interaction with both the active and inactive forms of ERM proteins and implying a possible role of ELMO in localizing or retaining ERM proteins in certain cellular sites. Together these data suggest that ELMO1-mediated cytoskeletal changes may be coordinated with ERM protein crosslinking activity during dynamic cellular functions. In eukaryotic cells, rearrangements of the cortical actin cytoskeleton provide the molecular basis for changes in cell shape, motility, adhesion, and division. Organized signaling complexes at the cell membrane are thought to coordinate actin assembly with membrane association during these processes. However, the exact molecular nature of these signaling complexes is not well understood. The highly conserved ERM (ezrin/radixin/moesin) protein family has been implicated in embryonic development, formation of microvilli, cell motility, formation of membrane ruffles, and formation of cell-cell/cellmatrix adhesion sites through their ability to cross-link the actin cytoskeleton to the plasma membrane (for reviews see Refs. 1Bretscher A. Edwards K. Fehon R.G. Nat. Rev. Mol. Cell Biol. 2002; 3: 586-599Crossref PubMed Scopus (1137) Google Scholar, 2Mangeat P. Roy C. Martin M. Trends Cell Biol. 1999; 9: 187-192Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar, 3Tsukita S. Yonemura S. J. Biol. Chem. 1999; 274: 34507-34510Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar). Each ERM protein contains an ∼300 residue FERM (band 4.1 and ERM) domain at its N terminus followed by an extended α-helical structure (∼160 amino acids) and a highly conserved C-terminal region (∼90 amino acids). An actin filament-binding domain site is located within the last 34 amino acids of this C-terminal region (4Pestonjamasp K. Amieva M.R. Strassel C.P. Nauseef W.M. Furthmayr H. Luna E.J. Mol. Biol. Cell. 1995; 6: 247-259Crossref PubMed Scopus (154) Google Scholar, 5Turunen O. Wahlstrom T. Vaheri A. J. Cell Biol. 1994; 126: 1445-1453Crossref PubMed Scopus (348) Google Scholar). The FERM domain promotes targeting to the plasma membrane via several transmembrane receptors and membrane-associated proteins. Transmembrane binding partners for ERM proteins identified so far include CD44, CD43, NEP, syndecan-2, and I-CAM-1, -2, and -3 (6Tsukita S. Oishi K. Sato N. Sagara J. Kawai A. J. Cell Biol. 1994; 126: 391-401Crossref PubMed Scopus (685) Google Scholar, 7Serrador J.M. Alonso-Lebrero J.L. del Pozo M.A. Furthmayr H. Schwartz- Albiez R. Calvo J. Lozano F. Sanchez-Madrid F. J. Cell Biol. 1997; 138: 1409-1423Crossref PubMed Scopus (202) Google Scholar, 8Granes F. Urena J.M. Rocamora N. 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Sidor A. J. Biol. Chem. 1998; 273: 25856-25863Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). ERM proteins also interact with PtdIns(4Pestonjamasp K. Amieva M.R. Strassel C.P. Nauseef W.M. Furthmayr H. Luna E.J. Mol. Biol. Cell. 1995; 6: 247-259Crossref PubMed Scopus (154) Google Scholar,5Turunen O. Wahlstrom T. Vaheri A. J. Cell Biol. 1994; 126: 1445-1453Crossref PubMed Scopus (348) Google Scholar)P2 3The abbreviations used are: PtdIns(4Pestonjamasp K. Amieva M.R. Strassel C.P. Nauseef W.M. Furthmayr H. Luna E.J. Mol. Biol. Cell. 1995; 6: 247-259Crossref PubMed Scopus (154) Google Scholar,5Turunen O. Wahlstrom T. Vaheri A. J. Cell Biol. 1994; 126: 1445-1453Crossref PubMed Scopus (348) Google Scholar)P2, phosphatidylinositol 4,5-biphosphate; GEF, guanine nucleotide exchange factor; MOPS, 4-morpholinepropanesulfonic acid; BSA, bovine serum albumin; HA, hemagglutinin; GST, glutathione S-transferase; GFP, green fluorescent protein; YFP, yellow fluorescent protein; FAK, focal adhesion kinase. at the plasma membrane as well as certain signaling proteins, including RhoGDI, Dbl, PALS1, N-WASP, the p85 subunit of phosphatidylinositol 3-kinase, and hamartin via the N-terminal FERM domain (15Lamb R.F. Roy C. Diefenbach T.J. Vinters H.V. Johnson M.W. Jay D.G. Hall A. Nat. Cell Biol. 2000; 2: 281-287Crossref PubMed Scopus (278) Google Scholar, 16Niggli V. Andreoli C. Roy C. Mangeat P. FEBS Lett. 1995; 376: 172-176Crossref PubMed Scopus (165) Google Scholar, 17Cao X. Ding X. Guo Z. Zhou R. Wang F. Long F. Wu F. Bi F. Wang Q. Fan D. Forte J.G. Teng M. Yao X. J. Biol. 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Cell Sci. 1992; 103: 131-143PubMed Google Scholar, 22Franck Z. Gary R. Bretscher A. J. Cell Sci. 1993; 105: 219-231Crossref PubMed Google Scholar). Biochemical and structural studies suggest that native full-length ERM proteins exist predominantly in a dormant state, by virtue of an intramolecular and/or intermolecular interaction between the N-terminal FERM domain and the C-terminal tail (23Magendantz M. Henry M.D. Lander A. Solomon F. J. Biol. Chem. 1995; 270: 25324-25327Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 24Gary R. Bretscher A. Mol. Biol. Cell. 1995; 6: 1061-1075Crossref PubMed Scopus (377) Google Scholar, 25Ishikawa H. Tamura A. Matsui T. Sasaki H. Hakoshima T. Tsukita S. J. Mol. Biol. 2001; 310: 973-978Crossref PubMed Scopus (29) Google Scholar, 26Pearson M.A. Reczek D. Bretscher A. Karplus P.A. Cell. 2000; 101: 259-270Abstract Full Text Full Text PDF PubMed Scopus (492) Google Scholar). In this auto-inhibited conformation, the C-terminal F-actin-binding site and N-terminal protein interaction sites are masked. The dormant forms of ERM proteins have no reported binding partners except for the regulatory subunit of protein kinase A (27Dransfield D.T. Bradford A.J. Smith J. Martin M. Roy C. Mangeat P.H. Goldenring J.R. EMBO J. 1997; 16: 35-43Crossref PubMed Scopus (268) Google Scholar). Therefore, to allow N-terminal and C-terminal binding to the plasma membrane and F-actin, respectively, an activation mechanism that opens the molecular structure is required. Two types of distinct signals have been proposed to generate and/or maintain the active state of ERM proteins. The first involves the phosphorylation of a C-terminal threonine residue that is conserved in all three ERM proteins (T564 in radixin, T567 in ezrin, and T558 in moesin). Phosphorylated ERM proteins are concentrated in actin-rich membrane structures in a variety of cells and tissues, whereas total ERM proteins are distributed in both the cytoplasm and plasma membranes (28Hayashi K. Yonemura S. Matsui T. Tsukita S. J. Cell Sci. 1999; 112: 1149-1158Crossref PubMed Google Scholar, 29Oshiro N. Fukata Y. Kaibuchi K. J. Biol. Chem. 1998; 273: 34663-34666Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 30Yonemura S. Matsui T. Tsukita S. J. Cell Sci. 2002; 115: 2569-2580Crossref PubMed Google Scholar). Phosphorylation of this threonine residue, or engineered phosphomimetic mutation of this residue (e.g. T564E radixin), reduces the affinity of the C-terminal tail for the FERM domain in all ERM proteins (31Huang L. Wong T.Y. Lin R.C. Furthmayr H. J. Biol. Chem. 1999; 274: 12803-12810Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 32Gautreau A. Louvard D. Arpin M. J. Cell Biol. 2000; 150: 193-203Crossref PubMed Scopus (236) Google Scholar), induces cytoskeletal changes (29Oshiro N. Fukata Y. Kaibuchi K. J. Biol. Chem. 1998; 273: 34663-34666Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 32Gautreau A. Louvard D. Arpin M. J. Cell Biol. 2000; 150: 193-203Crossref PubMed Scopus (236) Google Scholar), and promotes stronger binding to actin filaments in vitro (31Huang L. Wong T.Y. Lin R.C. Furthmayr H. J. Biol. Chem. 1999; 274: 12803-12810Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 33Nakamura F. Huang L. Pestonjamasp K. Luna E.J. Furthmayr H. Mol. Biol. Cell. 1999; 10: 2669-2685Crossref PubMed Scopus (126) Google Scholar). Conversely, nonphosphorylatable Thr → Ala mutants associate poorly with the actin cytoskeleton and act as dominant negative inhibitors of wild type ERM proteins (29Oshiro N. Fukata Y. Kaibuchi K. J. Biol. 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Cell Biol. 1994; 126: 1445-1453Crossref PubMed Scopus (348) Google Scholar)P2 binding enhances ERM binding to membrane proteins and actin filaments (9Heiska L. Alfthan K. Gronholm M. Vilja P. Vaheri A. Carpen O. J. Biol. Chem. 1998; 273: 21893-21900Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar, 31Huang L. Wong T.Y. Lin R.C. Furthmayr H. J. Biol. Chem. 1999; 274: 12803-12810Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 33Nakamura F. Huang L. Pestonjamasp K. Luna E.J. Furthmayr H. Mol. Biol. Cell. 1999; 10: 2669-2685Crossref PubMed Scopus (126) Google Scholar, 35Hirao M. Sato N. Kondo T. Yonemura S. Monden M. Sasaki T. Takai Y. Tsukita S. J. Cell Biol. 1996; 135: 37-51Crossref PubMed Scopus (512) Google Scholar). The ability of ERM proteins to function in such a highly regulated fashion, and their association with certain signaling molecules, strongly suggests that ERM proteins might also help organize signaling complexes that serve to regulate cytoskeletal assembly. Members of the evolutionarily conserved family of ELMO (engulfment and cell motility) proteins have been shown to regulate actin cytoskeleton reorganization and formation of membrane protrusions through an interaction with the protein Dock180 (36Brugnera E. Haney L. Grimsley C. Lu M. Walk S.F. Tosello-Trampont A.C. Macara I.G. Madhani H. Fink G.R. Ravichandran K.S. Nat. Cell Biol. 2002; 4: 574-582Crossref PubMed Scopus (481) Google Scholar, 37Gumienny T.L. Brugnera E. Tosello-Trampont A.C. Kinchen J.M. Haney L.B. Nishiwaki K. Walk S.F. Nemergut M.E. Macara I.G. Francis R. Schedl T. Qin Y. Van Aelst L. Hengartner M.O. Ravichandran K.S. Cell. 2001; 107: 27-41Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar, 38Katoh H. Negishi M. Nature. 2003; 424: 461-464Crossref PubMed Scopus (297) Google Scholar). The Dock180-ELMO complex functions as a guanine nucleotide exchange factor (GEF) specific for Rac, which mediates actin cytoskeletal reorganization and lamellipodia formation through several downstream effectors (39Bishop A.L. Hall A. Biochem. J. 2000; 348: 241-255Crossref PubMed Scopus (1682) Google Scholar). ELMO and Dock180 have also been found to localize to actinrich polarized membrane ruffles in cells (37Gumienny T.L. Brugnera E. Tosello-Trampont A.C. Kinchen J.M. Haney L.B. Nishiwaki K. Walk S.F. Nemergut M.E. Macara I.G. Francis R. Schedl T. Qin Y. Van Aelst L. Hengartner M.O. Ravichandran K.S. Cell. 2001; 107: 27-41Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar, 40Grimsley C.M. Kinchen J.M. Tosello-Trampont A.C. Brugnera E. Haney L.B. Lu M. Chen Q. Klingele D. Hengartner M.O. Ravichandran K.S. J. Biol. Chem. 2004; 279: 6087-6097Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). One mode of targeting of ELMO to the membrane occurs via ELMO binding to the activated form of another small GTPase, RhoG. The simultaneous interaction of ELMO with active RhoG and Dock180 (via the N- and C-terminal regions of ELMO, respectively) serves as an evolutionarily conserved mechanism for RhoG-dependent Rac activation leading to cell migration and neuronal outgrowth (38Katoh H. Negishi M. Nature. 2003; 424: 461-464Crossref PubMed Scopus (297) Google Scholar). Here we report that ELMO1 is a direct physiological binding partner for ERM proteins. An interaction of ELMO1 with radixin was observed both in vitro with recombinant proteins and at endogenous protein levels. Most interestingly, the interaction of ELMO1 with radixin appears to be distinct from other ERM-binding proteins, in that ELMO1 associated strongly with the closed, dormant form of the molecule as well as the open, active form of the molecule. Moreover, ELMO1 displayed only weak association with the isolated FERM domain of radixin, in contrast to other known ERM-binding proteins. Therefore, ELMO1 appears to be a novel ERM-binding protein, and the ELMO1-ERM interaction could have important implications for coordinated regulation of actin cytoskeleton reorganization during dynamic cellular functions. Antibodies and Reagents—Purified rabbit polyclonal anti-ELMO1 was generated in our laboratory and has been described previously (37Gumienny T.L. Brugnera E. Tosello-Trampont A.C. Kinchen J.M. Haney L.B. Nishiwaki K. Walk S.F. Nemergut M.E. Macara I.G. Francis R. Schedl T. Qin Y. Van Aelst L. Hengartner M.O. Ravichandran K.S. Cell. 2001; 107: 27-41Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar). Mouse monoclonal anti-GFP (clone B2), mouse monoclonal anti-HA (clone F7), goat polyclonal anti-Dock180 (C19), rabbit polyclonal anti-GST (clone Z5), mouse monoclonal anti-actin (clone C-2), and horseradish peroxidase-conjugated donkey anti-goat IgG antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). FLAG peptide and mouse monoclonal anti-FLAG (clone M2) were from Sigma. Mouse monoclonal anti-Myc (9E10) and mouse monoclonal anti-Rac (clone 23A8) were from Upstate Biotechnology, Inc. (Lake Placid, NY). Mouse monoclonal anti-radixin/moesin antibody was from BD Transduction Laboratories. Horseradish peroxidase-conjugated anti-mouse and antirabbit secondary antibodies were from Amersham Biosciences. All immunoblots were developed using enhanced chemiluminescence (Pierce). G-actin purified from muscle was a generous gift from Dorothy Schafer (University of Virginia). Plasmids—All mutant constructs generated were sequenced to confirm the fidelity and presence of the appropriate mutations. PEBB-ELMO1-GFP and pEBB-ELMO1-FLAG have been described previously (37Gumienny T.L. Brugnera E. Tosello-Trampont A.C. Kinchen J.M. Haney L.B. Nishiwaki K. Walk S.F. Nemergut M.E. Macara I.G. Francis R. Schedl T. Qin Y. Van Aelst L. Hengartner M.O. Ravichandran K.S. Cell. 2001; 107: 27-41Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar). The T280, T558, and Δ531 mutants of ELMO1 were generated by a PCR-based approach and were sequenced to confirm the appropriate mutations. The GST-ELMO1, GST-ARM1, GST-ARM2, GST-T115, and GST-T558 mutants of ELMO1 were described previously (41Debakker C.D. Haney L.B. Kinchen J.M. Grimsley C. Lu M. Klingele D. Hsu P.K. Chou B.K. Cheng L.C. Blangy A. Sondek J. Hengartner M.O. Wu Y.C. Ravichandran K.S. Curr. Biol. 2004; 14: 2208-2216Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). Fulllength pCXN2-FLAG-Dock180, was a generous gift from Dr. Michiyuki Matsuda (42Hasegawa H. Kiyokawa E. Tanaka S. Nagashima K. Gotoh N. Shibuya M. Kurata T. Matsuda M. Mol. Cell. Biol. 1996; 16: 1770-1776Crossref PubMed Scopus (294) Google Scholar). pEF-radixin-HA, pEF-radixin-T564A-HA, and pEF-radixin-T564E-HA were the generous gifts of Dr. Sachilo Tsukita (Kyoto University). PGEX-2T-radixin was kindly provided by Dr. Christian Roy (Universite Montpellier II). We generated the pGEX-2T-radixin-T564A and pGEX-2T-radixin-T564E mutants by replacing wild type pGEX-2T-radixin with the corresponding fragments of pEF-radixin-T564A-HA and pEF-radixin-T564E-HA. pEF-radixin-FERM (residues 1-310) was generated by a PCR-based approach using pEF-radixin as a template. PGEX-2T-ezrin was a gift of Dr. Monique Arpin (UMR144 CNRS/Institut Curie). PRK-moesin-HA was a gift of Dr. David Brautigan (University of Virginia). pEF-ezrin-HA and pEF-moesin-HA were generated by a PCR-based approach using pGEX-2T-ezrin and pRK-moesin-YFP as a template. The plasmid encoding HA-Tiam1 (C1199) was provided by Dr. John Collard (Netherlands Cancer Institute). The FLAG-tagged Rac1Q61L and GFP-FAK plasmids were kindly provided from Dr. Tom Parsons (University of Virginia). pEGFPC3-RhoGN17 and pEGFPC3-RhoGL61 for mammalian expression were kindly provided by Dr. Ann Blangy (Centre de Recherches en Biochimie Macromoleculaire; Montpellier, France) (43Blangy A. Vignal E. Schmidt S. Debant A. Gauthier-Rouviere C. Fort P. J. Cell Sci. 2000; 113: 729-739Crossref PubMed Google Scholar). Yeast Two-hybrid Screen—The mouse embryonic cDNA library was kindly provided by Dr. Ian Macara (University of Virginia). The cDNAs were cloned into the NotI site of the pVP16 vector. The yeast strain used in the two-hybrid screen was HF7C, with His, Trp, and Leu as selection markers. Yeast cells were transformed using the LiAc-based transformation protocol. Before the library screen, the full-length ELMO1 was tested on a -Trp, -His SCM plate, containing 5 mm 3-amino-1,2,4-triazole, and showed no transcriptional activation. When the ELMO1-Dock180 interaction was tested as a positive control, yeast cells transformed with pGBT10-ELMO1 and pVP16-Dock1-161 grew well on a -Trp, -Leu, -His SCM plate containing 10 mm 3-amino-1,2,4-triazole. The pGBT10-ELMO1-transformed yeast cells were then transformed with library plasmids and spread on -Trp, -Leu SCM plates, and ∼3 million colonies were screened. The yeasts grown out of the double dropout plates were replicated onto selection SCM plates (-Trp, -Leu, -His, containing 10 mm 3-amino-1,2,4-triazole). The yeast cells were allowed to grow into single colonies. After further restreaking on selective plates, the plasmid mixtures were then isolated from each clone after culture of the yeast in 10 ml of selective SCM medium. The library plasmids were rescued by transforming the individual plasmids mixture into KC8 Escherichia coli. Plasmids isolated from transformed KC8 were re-transformed into HF7C yeast cells with either the pGBT10-ELMO1 plasmid or the pGBT10 vector to confirm the specific interaction. Once this interaction was confirmed, the pVP16 library vectors derived from the two-hybrid screen were sequenced to identify the inserts. Transfection, Immunoprecipitations, and Immunoblotting—293T and HeLa cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal calf serum and 1% penicillin/streptomycin/glutamine. The J774 macrophage cell line was cultured in RPMI 1640 medium supplemented with 10% fetal calf serum, 10 mm HEPES, pH 7.4, 0.05 μm β-mercaptoethanol, 4.5 g/liter glucose, and antibiotics. 293T cells were transiently transfected by the calcium phosphate method, and HeLa cells were transiently transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. In all experiments, carrier DNA was added to keep equal plasmid concentration between different samples. Lysis, immunoprecipitation, and immunoblotting were performed as described previously (36Brugnera E. Haney L. Grimsley C. Lu M. Walk S.F. Tosello-Trampont A.C. Macara I.G. Madhani H. Fink G.R. Ravichandran K.S. Nat. Cell Biol. 2002; 4: 574-582Crossref PubMed Scopus (481) Google Scholar, 37Gumienny T.L. Brugnera E. Tosello-Trampont A.C. Kinchen J.M. Haney L.B. Nishiwaki K. Walk S.F. Nemergut M.E. Macara I.G. Francis R. Schedl T. Qin Y. Van Aelst L. Hengartner M.O. Ravichandran K.S. Cell. 2001; 107: 27-41Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar). Briefly, 293T cells were transiently transfected in 10-cm dishes with ELMO1 (3 μg), radixin (3 μg), Dock180 (10 μg), or other (3 μg) plasmids as indicated. For immunoprecipitation using the FLAG tag, 20 μl of anti-FLAG M2 antibody (Sigma) directly coupled to agarose was used per transfection condition. For ELMO1 immunoprecipitation, anti-ELMO1 antibody was incubated with protein A-Sepharose (Santa Cruz Biotechnology) for 1 h followed by three washes with lysis buffer. For HA immunoprecipitation, 15 μl of anti-HA 12CA5 antibody (Santa Cruz Biotechnology) directly coupled to agarose was used per transfection condition. For GST precipitation, 20 μl of glutathione-Sepharose beads was used per condition. Cells were then harvested, lysed, and incubated with the beads for 1-2 h. Beads were then washed four times, and precipitation of proteins was assessed by SDS-PAGE and immunoblotting. Purification of Recombinant Proteins from Bacteria—BL21 transformants were inoculated into 3 ml of LB medium containing 100 μg/ml ampicillin and incubated overnight at 37 °C. The culture was then diluted 1:100 into 250 ml of LB medium containing 100 μg/ml ampicillin and 2% (v/v) of ethanol and incubated for 1 h at 37°C. One mm of isopropyl 1-thio-β-d-galactopyranoside was then added, and the culture was incubated for 5 h. Bacteria were then collected by centrifugation, and the pellet was stored overnight at -80 °C. The pellet was then resuspended in 8 ml of lysis buffer (100 mm Tris, pH 7.6, 100 mm NaCl, 1 mm EDTA, 1 mg/ml lysozyme, 1 mm dithiothreitol, 10 μg/ml pepstatin, 10 μg/ml aprotinin, 10 μg/ml leupeptin) for 20 min. The suspension was then sonicated three times for 15 s each on ice. Eight mg of sodium deoxycholate in lysis buffer was then added and incubated for 20 min at room temperature. Then 25 μl of 10 mg/ml DNase was added and incubated for 20 min at room temperature. Proteins were then collected by centrifugation and incubated with 300 μl of glutathione-Sepharose beads for 2 h at 4°C on Nutator. The beads were then washed eight times in washing buffer (50 mm Tris, pH 7.6, 1% (v/v) Nonidet P-40, 150 mm NaCl, 10% (v/v) glycerol, 1 mm dithiothreitol, 10 μg/ml pepstatin, 10 μg/ml aprotinin, 10 μg/ml leupeptin). The beads were then resuspended in an equal volume of washing buffer containing 20% (v/v) glycerol and stored at -80 °C. Proteins were quantitated by SDS-PAGE followed by Coomassie staining. Cleavage of GST from ELMO1 and ERM Proteins—GST was cleaved from fusion proteins, where indicated, using a thrombin cleavage kit (Novagen). GST fusion proteins bound to glutathione-Sepharose beads were incubated with 1 μl of thrombin and 100 μl of cleavage buffer in a 1-ml total volume overnight at 4 °C on Nutator. Cleaved proteins were then collected by brief centrifugation and stored at -80 °C. The cleavage appeared virtually complete as determined by SDS-PAGE and staining with Coomassie Blue. Purification and Elution of FLAG-ELMO1 Proteins—293T cells were transfected in 10-cm dishes, one dish per condition of purified FLAG-ELMO1. Twenty four to 48 h later, cells were lysed, and FLAG-ELMO1 was immunoprecipitated and washed. FLAG-ELMO1 was then eluted using FLAG peptide (Sigma) according to the manufacturer's instructions. In Vivo Rac GTP-loading Assay—Bacterially produced GST-CRIB proteins bound to glutathione-Sepharose beads were incubated with lysates from 293T cells transfected with the indicated plasmids for 1 h at 4 °C. The beads were then washed, and the levels of Rac-GTP present in the lysates were analyzed by SDS-PAGE and immunoblotting for Rac (40Grimsley C.M. Kinchen J.M. Tosello-Trampont A.C. Brugnera E. Haney L.B. Lu M. Chen Q. Klingele D. Hengartner M.O. Ravichandran K.S. J. Biol. Chem. 2004; 279: 6087-6097Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). In Vitro GEF Assay—The radioactivity-based in vitro GEF assay was performed as described previously (36Brugnera E. Haney L. Grimsley C. Lu M. Walk S.F. Tosello-Trampont A.C. Macara I.G. Madhani H. Fink G.R. Ravichandran K.S. Nat. Cell Biol. 2002; 4: 574-582Crossref PubMed Scopus (481) Google Scholar). 293T cells in 10-cm dishes were transfected with the indicated plasmids, and the cell lysates were immunoprecipitated with anti-FLAG antibody. Precipitated proteins were then eluted with FLAG peptide following the manufacturer's instructions into 120 μl. The eluted proteins were quantitated via Western blotting. Levels of Dock180 were kept constant, and conditions were analyzed for Rac GEF activity as follows. 5 μg of bacterially expressed and purified Rac was loaded with 50 μCi of [α-32P]GTP (3000 Ci/mmol) in 40 mm MOPS, pH 7.1, 1 mm EDTA, 1 mg/ml BSA, and 0.3 mm unlabeled GTP for 20 min on ice. 10 mm MgCl2 was then added and incubated on ice for an additional 10 min. 250 ng of [32P]GTP-loaded Rac was added with 2 μl of eluted proteins (for wild type Dock180, volume for other samples was adjusted based on the concentration of eluted Dock180) in reaction buffer (25 mm MOPS, pH 7.1, 6.25 mm MgCl2, 0.6 mm NaH2PO4, 0.5 mg/ml BSA, 1.25 mm unlabeled GDP) in a final volume of 100 ml. After 15 min at 30 °C, 50 ml of the exchange reaction was subjected to nitrocellulose filter binding followed by scintillation counting. The presence of GEF activity was revealed by loss of radioactivity bound to Rac (due to the exchange reaction). [32P]GTP binding to Rac, in the control conditions with precipitates from untransfe
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