The Interaction of Src and RACK1 Is Enhanced by Activation of Protein Kinase C and Tyrosine Phosphorylation of RACK1
2001; Elsevier BV; Volume: 276; Issue: 23 Linguagem: Inglês
10.1074/jbc.m101375200
ISSN1083-351X
AutoresBetty Chang, Meiling Chiang, Christine A. Cartwright,
Tópico(s)PI3K/AKT/mTOR signaling in cancer
ResumoRACK1 is an intracellular receptor for the serine/ threonine protein kinase C. Previously, we demonstrated that RACK1 also interacts with the Src protein-tyrosine kinase. RACK1, via its association with these protein kinases, may play a key role in signal transduction. To further characterize the Src-RACK1 interaction and to analyze mechanisms by which cross-talk occurs between the two RACK1-linked signaling kinases, we identified sites on Src and RACK1 that mediate their binding, and factors that regulate their interaction. We found that the interaction of Src and RACK1 is mediated, in part, by the SH2 domain of Src and by phosphotyrosines in the sixth WD repeat of RACK1, and is enhanced by serum or platelet-derived growth factor stimulation, protein kinase C activation, and tyrosine phosphorylation of RACK1. To the best of our knowledge, this is the first report of tyrosine phosphorylation of a member of the WD repeat family of proteins. We think that tyrosine phosphorylation of these proteins is an important mechanism of signal transduction in cells. RACK1 is an intracellular receptor for the serine/ threonine protein kinase C. Previously, we demonstrated that RACK1 also interacts with the Src protein-tyrosine kinase. RACK1, via its association with these protein kinases, may play a key role in signal transduction. To further characterize the Src-RACK1 interaction and to analyze mechanisms by which cross-talk occurs between the two RACK1-linked signaling kinases, we identified sites on Src and RACK1 that mediate their binding, and factors that regulate their interaction. We found that the interaction of Src and RACK1 is mediated, in part, by the SH2 domain of Src and by phosphotyrosines in the sixth WD repeat of RACK1, and is enhanced by serum or platelet-derived growth factor stimulation, protein kinase C activation, and tyrosine phosphorylation of RACK1. To the best of our knowledge, this is the first report of tyrosine phosphorylation of a member of the WD repeat family of proteins. We think that tyrosine phosphorylation of these proteins is an important mechanism of signal transduction in cells. receptor for activated protein kinase C protein kinase C Src homology 2 Src homology 3 tryptophan-aspartic acid platelet-derived growth factor platelet-derived growth factor receptor Dulbecco's modified Eagle medium monoclonal antibody Tris-buffered saline phosphate-buffered saline fetal bovine serum glutathioneS-transferase phorbol-12-myristate-13-acetate bovine serum albumin hemagglutinin fluorescein isothiocyanate polyacrylamide gel electrophoresis GTPase-activating protein CHinese hamster ovary radioimmunoprecipitation assay phospholipase C high performance liquid chromatography The Src family of intracellular protein-tyrosine kinases participates in diverse signaling pathways that regulate cell growth, differentiation, adhesion, and architecture (reviewed in Ref. 1Brown M.T. Cooper J.A. Biochim. Biophys. Acta. 1996; 128: 121-149Google Scholar). Identification of Src-binding proteins has led to better understanding of Src regulation and has provided clues about the function of Src in normal and transformed cells. For example, characterization of the interaction between Src and polyoma middle T antigen led to discovery of a fundamental mechanism by which the cellular Src protein is converted to a transforming protein (by dephosphorylation at Tyr-527) and defined the requirement of Src for polyoma transformation (2Courtneidge S.A. Smith A.E. Nature. 1983; 303: 435-439Crossref PubMed Scopus (269) Google Scholar, 3Bolen J.B. Thiele C.J. Israel M.A. Yonemoto W. Lipsich L.A. Brugge J.S. Cell. 1984; 38: 767-777Abstract Full Text PDF PubMed Scopus (145) Google Scholar, 4Kmiecik T.E. Shalloway D. Cell. 1987; 49: 65-73Abstract Full Text PDF PubMed Scopus (411) Google Scholar, 5Piwnica-Worms H. Saunders K.B. Roberts T.M. Smith A.E. Cheng S.H. Cell. 1987; 49: 75-824Abstract Full Text PDF PubMed Scopus (313) Google Scholar, 6Cartwright C.A. Eckhart W. Simon S. Kaplan P.L. Cell. 1987; 49: 83-91Abstract Full Text PDF PubMed Scopus (228) Google Scholar, 7Reynolds A.B. Vila J. Lansing T.J. Potts W.M. Weber M.J. Parsons J.T. EMBO J. 1987; 6: 2359-2364Crossref PubMed Scopus (89) Google Scholar). Thus, characterization of a single Src-binding protein contributed substantially to our understanding of both RNA and DNA tumor biology. Recently, using the unique domain/SH2/SH3 domain of Src as bait, and a human lung fibroblast cDNA library as prey, we identified RACK1, a known intracellular receptor for activatedCkinase (RACK),1 as a Src-binding protein (8Chang B.Y. Conroy K.B. Machleder E.M. Cartwright C.A. Mol. Cell. Biol. 1998; 18: 3245-3256Crossref PubMed Google Scholar). We found that overexpression of RACK1 inhibited the specific activity of Src tyrosine kinases (as measured in vitro) and the growth of NIH 3T3 cells. RACK1 exerted its effect on growth, in part, by prolonging the G0/G1phase of the cell cycle. RACK1 was the first of a group of proteins (collectively called RACKs) to be identified and characterized by Mochly-Rosen and co-workers (reviewed in Refs. 9Mochly-Rosen D. Science. 1995; 268: 247-251Crossref PubMed Scopus (834) Google Scholar, 10Mochly-Rosen D. Kauvar L.M. Adv. Pharmacol. 1998; 44: 91-145Crossref PubMed Scopus (89) Google Scholar, 11Mochly-Rosen D. Gordon A.S. FASEB J. 1998; 12: 35-42Crossref PubMed Scopus (510) Google Scholar). RACK1 has sequence homology with the β subunit of heterotrimeric G proteins. RACK1 and Gβ are both members of an ancient family of regulatory proteins made up of highly conserved repeating units usually ending with Trp-Asp (WD) (reviewed in Refs. 12Neer E.J. Schmidt C.J. Nambudripad R. Smith T.F. Nature. 1994; 371: 297-300Crossref PubMed Scopus (1292) Google Scholarand 13Neer E.J. Smith T.F. Cell. 1996; 84: 175-178Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). WD repeat proteins are functionally diverse, although all seem to be regulatory and few are enzymes. The WD repeats in RACK1 are conserved from Chlamydomonas to human (reviewed in Ref. 13Neer E.J. Smith T.F. Cell. 1996; 84: 175-178Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). Thus, the function of RACK1 was probably fixed before the evolutionary divergence of plants and animals. Protein kinase C (PKC) is a family of serine/threonine kinases whose activity depends upon phospholipid, diacylglycerol, and in some case on calcium (reviewed in Refs. 9Mochly-Rosen D. Science. 1995; 268: 247-251Crossref PubMed Scopus (834) Google Scholar, 10Mochly-Rosen D. Kauvar L.M. Adv. Pharmacol. 1998; 44: 91-145Crossref PubMed Scopus (89) Google Scholar, 11Mochly-Rosen D. Gordon A.S. FASEB J. 1998; 12: 35-42Crossref PubMed Scopus (510) Google Scholar and 14Newton A.C. Curr. Opin. Cell Biol. 1997; 9: 161-167Crossref PubMed Scopus (851) Google Scholar). Upon stimulation with tumor promoter phorbol esters or hormones that increase intracellular concentrations of diacylglycerol, PKCs become activated and translocate to new subcellular sites where they phosphorylate isozyme-specific substrates. Individual, activated, PKC isozymes are translocated to distinct compartments, suggesting that they mediate distinct cellular functions (9Mochly-Rosen D. Science. 1995; 268: 247-251Crossref PubMed Scopus (834) Google Scholar, 10Mochly-Rosen D. Kauvar L.M. Adv. Pharmacol. 1998; 44: 91-145Crossref PubMed Scopus (89) Google Scholar, 11Mochly-Rosen D. Gordon A.S. FASEB J. 1998; 12: 35-42Crossref PubMed Scopus (510) Google Scholar, 15Ron D. Jiang Z. Yao L. Diamond I. Gordon A. J. Biol. Chem. 1999; 274: 27039-27046Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). RACKs interact only with activated forms of PKCs, suggesting that PKC binding to RACK occurs after cell stimulation, to localize the active enzyme to the RACK site (reviewed in Refs. 9Mochly-Rosen D. Science. 1995; 268: 247-251Crossref PubMed Scopus (834) Google Scholar, 10Mochly-Rosen D. Kauvar L.M. Adv. Pharmacol. 1998; 44: 91-145Crossref PubMed Scopus (89) Google Scholar, 11Mochly-Rosen D. Gordon A.S. FASEB J. 1998; 12: 35-42Crossref PubMed Scopus (510) Google Scholar). Moreover, there are isozyme-specific RACKs, which presumably anchor each PKC isozyme close to its physiologic substrate. For example, in cardiac myocytes RACK1 is specific for βIIPKC, whereas RACK2 is specific for εPKC (15Ron D. Jiang Z. Yao L. Diamond I. Gordon A. J. Biol. Chem. 1999; 274: 27039-27046Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 16Ron D. Chen C.H. Caldwell J. Jamieson L. Orr E. Mochly-Rosen D. Proc. Natl. Acad. Sci., U. S. A. 1994; 91: 839-843Crossref PubMed Scopus (646) Google Scholar, 17Csukai M. Chen C.H. De Matteis M.A. Mochly-Rosen D. J. Biol. Chem. 1997; 272: 29200-29206Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). Thus, it appears that the specificity of PKC function may be determined, in part, by the different locations of isozyme-specific RACKs. The observation that RACK1 interacts with two, distinct, cytoplasmic protein kinases raises interesting questions about the role of RACK1 in orchestrating the intersection of tyrosine and serine/threonine kinase signaling pathways. The purpose of this study was to further characterize the Src-RACK1 interaction and to begin to analyze the mechanism by which cross-talk occurs between two RACK1-linked signaling protein kinases. We found that the interaction of Src and RACK1 is mediated by the SH2 domain of Src and phosphotyrosines in the sixth WD repeat of RACK1, and is enhanced by serum, PDGF stimulation, PKC activation, and tyrosine phosphorylation of RACK1. NIH 3T3 cells overexpressing the β-platelet-derived growth factor (PDGF) receptor (a gift from Sara Courtneidge, Sugen, San Francisco, CA; Ref. 18Twamley-Stein G.M. Pepperkok R. Ansorge W. Courtneidge S.A. Proc. Natl. Acad. Sci., U. S. A. 1993; 90: 7696-7700Crossref PubMed Scopus (295) Google Scholar) or wild-type or Y527F chicken c-Src (6Cartwright C.A. Eckhart W. Simon S. Kaplan P.L. Cell. 1987; 49: 83-91Abstract Full Text PDF PubMed Scopus (228) Google Scholar) were cultured in Dulbecco's modified Eagle's medium (DMEM) (Mediatech, Herndon, VA) supplemented with 10% calf serum (Sigma), and maintained in G418 (200 μg/ml) (Life Technologies, Inc.). CHO cells (American Type Culture Collection (ATCC), Rockville, MD) were cultured in Ham's F-12 medium (Mediatech) supplemented with 10% fetal bovine serum (FBS) (Sigma). HeLa cells (ATCC) were cultured in DMEM supplemented with 10% FBS. pGEX-3X plasmids containing the SH2 domain(s) of phospholipase Cγ1 (PLCγ), Shp-2, Shp-1, p85, Abl, Grb2, rasGAP, Csk, or Shc were gifts from Lewis Cantley (Harvard University, Boston, MA) (19Zhou S. Shoelson S.E. Chaudhuri M. Gish G. Pawson T. Haser W.G. King F. Roberts T. Ratnofsky S. Lechleider R.J. Neel B.G. Birge R.B. Fajardo J.E. Chou M.M. Hanafusa H. Schaffhausen B. Cantley L.C. Cell. 1993; 72: 767-778Abstract Full Text PDF PubMed Scopus (2383) Google Scholar). pGEXsrc-SH2 and pGEX-RACK1 were constructed as described (8Chang B.Y. Conroy K.B. Machleder E.M. Cartwright C.A. Mol. Cell. Biol. 1998; 18: 3245-3256Crossref PubMed Google Scholar). pGEX plasmids were used to generate GST-fusion proteins. Plasmids encoding wild-type (pM5H) or mutant dl155–157 (pM155) chicken c-src were gifts from Sarah Parsons (University of Virginia, Charlottesville, VA; Ref. 20Moyers J.S. Bouton A.B. Parsons S.J. Mol. Cell. Biol. 1993; 13: 2391-2400Crossref PubMed Google Scholar). Src inserts from the plasmids were subcloned into pcDNA3 (Invitrogen, La Jolla, CA) to create pcDNA3 wild-type or dl155 c-src. pcDNA3-HA-RACK1 was generated as described (8Chang B.Y. Conroy K.B. Machleder E.M. Cartwright C.A. Mol. Cell. Biol. 1998; 18: 3245-3256Crossref PubMed Google Scholar). pcDNA3 plasmids were used for transient protein expression assays. pGEMsrc, a gift from Tony Hunter (Salk Institute, La Jolla, CA), was used to generate in vitro translated Src (8Chang B.Y. Conroy K.B. Machleder E.M. Cartwright C.A. Mol. Cell. Biol. 1998; 18: 3245-3256Crossref PubMed Google Scholar). For Src antibodies, 1) monoclonal antibody (mAb) 327 (21Lipsich L.A. Lewis A.J. Brugge J.S. J. Virol. 1983; 48: 352-360Crossref PubMed Google Scholar) was used (unless otherwise stated) for immunoprecipitation and immunoblot analyses; 2) anti-peptide antibody N16, which recognizes the unique domain of Src (Santa Cruz Laboratories, Santa Cruz, CA), was used for immunoprecipitation; and 3) anti-peptide antibody R7, which recognizes the C terminus of Src (8Chang B.Y. Conroy K.B. Machleder E.M. Cartwright C.A. Mol. Cell. Biol. 1998; 18: 3245-3256Crossref PubMed Google Scholar) was used for immunoprecipitation. RACK1 mAb (Transduction Laboratories, Lexington, KY; Ref. 7Reynolds A.B. Vila J. Lansing T.J. Potts W.M. Weber M.J. Parsons J.T. EMBO J. 1987; 6: 2359-2364Crossref PubMed Scopus (89) Google Scholar) was used for immunoblot analyses. Anti-phosphotyrosine mAb PY20 (Transduction Laboratories; Ref. 22Glenny J.R. Zokas L. Kamps M.P. J. Immunol. Meth. 1988; 109: 277-285Crossref PubMed Scopus (171) Google Scholar) were used for immunoprecipitation and immunoblot analyses. Polyclonal anti-GST was a gift from Anson Lowe (Stanford University, Stanford, CA). Cultures ofEscherichia coli DH5α containing various pGEX-SH2 or pGEX RACK1 plasmids were induced with 0.1 mmisopropyl-β-d-thiogalactopyranoside (United States Biochemical Corp., Cleveland, OH) for 3 h at 30 °C as described (8Chang B.Y. Conroy K.B. Machleder E.M. Cartwright C.A. Mol. Cell. Biol. 1998; 18: 3245-3256Crossref PubMed Google Scholar). Bacteria were harvested, resuspended in Tris-buffered saline (TBS) containing 1% Triton X-100 and 100 mm EDTA and sonicated. After centrifugation at 12,000 × g for 10 min to remove debris, the supernatant was incubated with glutathione-agarose beads (Sigma) for 2 h at 4 °C with agitation. Beads were washed three times with TBS. GST fusion proteins were eluted by the addition of 100 mm Tris, pH 8.0, 120 mm NaCl, and 20 mm glutathione and dialyzed four times against TBS. Purified GST fusion proteins (1–5 μg) were incubated with cell lysates or radiolabeled, in vitro translated Src for 3 h at 4 °C as described (8Chang B.Y. Conroy K.B. Machleder E.M. Cartwright C.A. Mol. Cell. Biol. 1998; 18: 3245-3256Crossref PubMed Google Scholar). Protein complexes were collected with the addition of 30 μl of glutathione beads, washed four times in buffer containing 0.5% Nonidet P-40, 20 mm Tris, pH 8.0, 100 mm sodium chloride (NaCl), and 1 mm EDTA, and boiled in sodium dodecyl sulfate (SDS) sample buffer. Proteins were resolved by SDS-PAGE and detected by fluorography (see below) or subjected to immunoblot analysis and detected by enhanced chemiluminescence (ECL) (Amersham Pharmacia Biotech), according to the manufacturer's protocol. CHO cells were transfected with pcDNA3, pcDNA3-HA-RACK1, or pcDNA3-HA-RACK1 together with pcDNA3c-src, using LipofectAMINE (Life Technologies, Inc.) according to the manufacturer's protocol and as described (8Chang B.Y. Conroy K.B. Machleder E.M. Cartwright C.A. Mol. Cell. Biol. 1998; 18: 3245-3256Crossref PubMed Google Scholar). Briefly, 2 × 105 cells were seeded in six-well plates in Ham's F-12 medium containing 10% FBS. 24 h later, transfections were performed using 0.5–1 μg of plasmid DNA and 10 μl of LipofectAMINE in serum-free media. 5 h later, cells were placed in fresh media containing 10% FBS. Cells were lysed 48 h after transfection. Cells were washed three times with ice-cold TBS and lysed in modified RIPA buffer (0.1% SDS, 1% Nonidet P-40, 1% sodium deoxycholate, 150 mm NaCl, 10 mm sodium phosphate, pH 7.0, 100 μm sodium vanadate, 50 mm sodium fluoride, 50 μm leupeptin, 1% aprotinin, 2 mm EDTA, and 1 mm dithiothreitol) (6Cartwright C.A. Eckhart W. Simon S. Kaplan P.L. Cell. 1987; 49: 83-91Abstract Full Text PDF PubMed Scopus (228) Google Scholar, 8Chang B.Y. Conroy K.B. Machleder E.M. Cartwright C.A. Mol. Cell. Biol. 1998; 18: 3245-3256Crossref PubMed Google Scholar, 23Cartwright C.A. Hutchinson M. Eckhart W. Mol. Cell. Biol. 1985; 5: 2647-2652Crossref PubMed Scopus (26) Google Scholar, 24Cartwright C.A. Kaplan P.L. Cooper J.A. Hunter T. Eckhart W. Mol. Cell. Biol. 1986; 6: 1562-1570Crossref PubMed Scopus (71) Google Scholar, 25Cartwright C.A. Meisler M.A. Eckhart W. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 558-562Crossref PubMed Scopus (281) Google Scholar, 26Park J. Cartwright C.A. Mol. Cell. Biol. 1995; 15: 2374-2382Crossref PubMed Scopus (51) Google Scholar, 27Walter A.O. Peng Z.Y. Cartwright C.A. Oncogene. 1999; 18: 1911-1920Crossref PubMed Scopus (64) Google Scholar). Lysates were centrifuged at 14,000 × gfor 1 h at 4 °C. Protein concentrations were measured by the BCA protein assay (Pierce), and samples were standardized to equal amounts of total cellular protein (6Cartwright C.A. Eckhart W. Simon S. Kaplan P.L. Cell. 1987; 49: 83-91Abstract Full Text PDF PubMed Scopus (228) Google Scholar, 8Chang B.Y. Conroy K.B. Machleder E.M. Cartwright C.A. Mol. Cell. Biol. 1998; 18: 3245-3256Crossref PubMed Google Scholar, 23Cartwright C.A. Hutchinson M. Eckhart W. Mol. Cell. Biol. 1985; 5: 2647-2652Crossref PubMed Scopus (26) Google Scholar, 24Cartwright C.A. Kaplan P.L. Cooper J.A. Hunter T. Eckhart W. Mol. Cell. Biol. 1986; 6: 1562-1570Crossref PubMed Scopus (71) Google Scholar, 25Cartwright C.A. Meisler M.A. Eckhart W. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 558-562Crossref PubMed Scopus (281) Google Scholar, 26Park J. Cartwright C.A. Mol. Cell. Biol. 1995; 15: 2374-2382Crossref PubMed Scopus (51) Google Scholar, 27Walter A.O. Peng Z.Y. Cartwright C.A. Oncogene. 1999; 18: 1911-1920Crossref PubMed Scopus (64) Google Scholar). Lysates were incubated for 3 h at 4 °C with excess antibody (1 μg of mAb 327 or PY20, or anti-peptide N16 or R7) and protein complexes were collected with the addition of 30 μl of protein A/G-Sepharose beads (Amersham Pharmacia Biotech). Protein kinase assays were performed by incubating mAb 327 immunoprecipitates (of Y527F Src-overexpressing NIH 3T3 cell lysates) for 10 min at 30 °C in 30 μl of kinase buffer containing 50 mm piperazine-N,N′-bis (2-ethanesulfonic acid), pH 7.0, 10 mm manganese chloride, 10 mm dithiothreitol, 1 μg of GST-RACK1 or GST, and, in some cases, 1 mm ATP (6Cartwright C.A. Eckhart W. Simon S. Kaplan P.L. Cell. 1987; 49: 83-91Abstract Full Text PDF PubMed Scopus (228) Google Scholar, 8Chang B.Y. Conroy K.B. Machleder E.M. Cartwright C.A. Mol. Cell. Biol. 1998; 18: 3245-3256Crossref PubMed Google Scholar, 23Cartwright C.A. Hutchinson M. Eckhart W. Mol. Cell. Biol. 1985; 5: 2647-2652Crossref PubMed Scopus (26) Google Scholar, 24Cartwright C.A. Kaplan P.L. Cooper J.A. Hunter T. Eckhart W. Mol. Cell. Biol. 1986; 6: 1562-1570Crossref PubMed Scopus (71) Google Scholar, 25Cartwright C.A. Meisler M.A. Eckhart W. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 558-562Crossref PubMed Scopus (281) Google Scholar, 26Park J. Cartwright C.A. Mol. Cell. Biol. 1995; 15: 2374-2382Crossref PubMed Scopus (51) Google Scholar, 27Walter A.O. Peng Z.Y. Cartwright C.A. Oncogene. 1999; 18: 1911-1920Crossref PubMed Scopus (64) Google Scholar). Phosphorylated proteins were detected by immunoblot analysis with anti-phosphotyrosine PY20 or by binding to radiolabeled, in vitro translated Src (see below). Src or PY20 immunoprecipitates were resolved on 10% SDS-polyacrylamide gels (acrylamide-bisacrylamide, 29:0.8). Proteins were transferred to polyvinylidene difluoride membranes (Immobilon-P™; Millipore, Bedford, MA) in transfer buffer (25 mm Tris-HCl, pH 7.4, 192 mm glycine, and 15% methanol) using a Trans-Blot apparatus (Bio-Rad) for 2 h at 60 V (6Cartwright C.A. Eckhart W. Simon S. Kaplan P.L. Cell. 1987; 49: 83-91Abstract Full Text PDF PubMed Scopus (228) Google Scholar, 8Chang B.Y. Conroy K.B. Machleder E.M. Cartwright C.A. Mol. Cell. Biol. 1998; 18: 3245-3256Crossref PubMed Google Scholar, 23Cartwright C.A. Hutchinson M. Eckhart W. Mol. Cell. Biol. 1985; 5: 2647-2652Crossref PubMed Scopus (26) Google Scholar, 24Cartwright C.A. Kaplan P.L. Cooper J.A. Hunter T. Eckhart W. Mol. Cell. Biol. 1986; 6: 1562-1570Crossref PubMed Scopus (71) Google Scholar, 25Cartwright C.A. Meisler M.A. Eckhart W. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 558-562Crossref PubMed Scopus (281) Google Scholar, 26Park J. Cartwright C.A. Mol. Cell. Biol. 1995; 15: 2374-2382Crossref PubMed Scopus (51) Google Scholar, 27Walter A.O. Peng Z.Y. Cartwright C.A. Oncogene. 1999; 18: 1911-1920Crossref PubMed Scopus (64) Google Scholar). Protein binding sites on the membranes were blocked by incubating membranes overnight in TNT buffer (10 mm Tris-HCl, pH 7.5, 100 mm sodium chloride, 0.1% (v/v) Tween 20 (Sigma)) containing 3% nonfat, powdered milk (blocking buffer). Membranes were incubated with mAb RACK1 (0.08 mg/ml), affinity-purified mAb 327 ascites (2 μg/ml), mAb PY20 (0.08 mg/ml), or polyclonal anti-GST (2 mg/ml) for 1 h, washed in TNT buffer with changes every 5 min for 30 min, and incubated with horseradish peroxidase-conjugated donkey anti-mouse IgM (Zymed Laboratories Inc., San Francisco, CA) for RACK1 blots, goat anti-mouse IgG (Bio-Rad) for mAb 327 or PY20 blots, or goat anti-rabbit IgG (Bio-Rad) for anti-GST blots (6Cartwright C.A. Eckhart W. Simon S. Kaplan P.L. Cell. 1987; 49: 83-91Abstract Full Text PDF PubMed Scopus (228) Google Scholar, 8Chang B.Y. Conroy K.B. Machleder E.M. Cartwright C.A. Mol. Cell. Biol. 1998; 18: 3245-3256Crossref PubMed Google Scholar, 23Cartwright C.A. Hutchinson M. Eckhart W. Mol. Cell. Biol. 1985; 5: 2647-2652Crossref PubMed Scopus (26) Google Scholar, 24Cartwright C.A. Kaplan P.L. Cooper J.A. Hunter T. Eckhart W. Mol. Cell. Biol. 1986; 6: 1562-1570Crossref PubMed Scopus (71) Google Scholar, 25Cartwright C.A. Meisler M.A. Eckhart W. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 558-562Crossref PubMed Scopus (281) Google Scholar, 26Park J. Cartwright C.A. Mol. Cell. Biol. 1995; 15: 2374-2382Crossref PubMed Scopus (51) Google Scholar, 27Walter A.O. Peng Z.Y. Cartwright C.A. Oncogene. 1999; 18: 1911-1920Crossref PubMed Scopus (64) Google Scholar). Proteins were detected by ECL (see above). pGEMsrc (2 μg) was transcribed and translated in vitro using a TnT coupled rabbit reticulocyte lysate system (Promega, Madison, WI), as instructed by the manufacturer and as described (8Chang B.Y. Conroy K.B. Machleder E.M. Cartwright C.A. Mol. Cell. Biol. 1998; 18: 3245-3256Crossref PubMed Google Scholar). In vitro translated products labeled with Pro-Mix[35S] (70% l-[35S]methionine and 30%l-[35S]cysteine; >1,000 Ci/mmol; Amersham Pharmacia Biotech) were diluted (1:100) in buffer (50 mmTris, pH 7.5, 150 mm NaCl, and 0.2% Nonidet P-40) and incubated with 1 μg of purified GST or GST-RACK1 for 3 h at 4 °C as described above. 1/20 of the unbound translation reaction product was loaded directly on the gel as a marker for in vitro translated Src. Gels were treated with Fluoro-Hance (Research Products International Corp., Mount Prospect, IL), and radiolabeled proteins were detected by fluorography. Cell lysates, GST fusion proteins, or immunoprecipitates were incubated in phosphatase buffer (50 mm Tris-HCl, pH 8.5, 0.1 mm EDTA) with or without the addition of purified calf intestinal alkaline phosphatase (50 units) (Promega) for 30 min at room temperature. The reaction was stopped by heating the mixture to 75 °C for 15 min (27Walter A.O. Peng Z.Y. Cartwright C.A. Oncogene. 1999; 18: 1911-1920Crossref PubMed Scopus (64) Google Scholar, 28Liu X. Brodeur S.R. Gish G. Songyang Z. Cantley L.C. Laudano A.P. Pawson T. Oncogene. 1993; 8: 1119-1126PubMed Google Scholar, 29Hardie, D. G. (ed) (1993) Protein Phosphorylation: A Practical Approach, pp.231-249, IRL Press/Oxford University Press, OxfordGoogle Scholar, 30Cobb B.S. Schaller M.D. Leu T.H. Parsons J.T. Mol. Cell. Biol. 1994; 14: 147-155Crossref PubMed Scopus (484) Google Scholar). Tyrosine-phosphorylated (or identical unphosphorylated) peptides corresponding to the sequence surrounding each of the 6 tyrosines of RACK1 were synthesized by the Protein Structure Core Facility of the Digestive Disease Center, Stanford University (Director, Gary Schoolnik), on an automated Milligen Peptide Synthesizer using FMOC-Novasyn KA resin (NovaBioChem, San Diego, CA) (31Cooper J.A. Esch F.S. Taylor S.S. Hunter T. J. Biol. Chem. 1984; 259: 7835-7841Abstract Full Text PDF PubMed Google Scholar, 32Ottinger E.A. Botfield M.C. Shoelson S.E. J. Biol. Chem. 1998; 273: 729-735Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 33Liao J. Lowthert L.A. Ku N.O. Fernandez R. Omary M.B. J. Cell Biol. 1995; 131: 1291-1301Crossref PubMed Scopus (73) Google Scholar). Phosphotyrosine was incorporated as Fmoc (N-(9-fluorenyl)methoxycarbonyl)-Tyr(PO3H2)-OH. The crude peptides were purified using reverse phase high performance liquid chromatography (HPLC) (3.9 × 300-mm C18 column) and a linear gradient containing 0.05% trifluoroacetic acid in 15–65% acetonitrile. The purity of the HPLC-purified products was confirmed using mass spectrometry. The purified phosphopeptides (Tyr-52, TRDETNY(PO4)GIPQ; Tyr-140, TLGVCKY(PO4)TVQD; Tyr-195, HIGHTGY(PO4)LNTV; Tyr-228, NEGKHLY(PO4)TLD; Tyr-246, CFSPNRY(PO4)WLCA; Tyr-302, QTLFAGY(PO4)TDNL), or the identical unphosphorylated peptides, were used for peptide competition assays. CHO cells were treated with phorbol-12-myristate-13-acetate (PMA) (Life Technologies, Inc.) (10 ng/ml) at 37 °C for 10 min prior to lysis in RIPA buffer. Lysate containing 200 μg of total cellular protein was incubated with peptide (100 μm) and GST-Src-SH2 (500 nm) or GST for 1 h at 4 °C (8Chang B.Y. Conroy K.B. Machleder E.M. Cartwright C.A. Mol. Cell. Biol. 1998; 18: 3245-3256Crossref PubMed Google Scholar, 34Alonso G. Koegl M. Mazurenko N. Courtneidge S.A. J. Biol. Chem. 1995; 270: 9840-9848Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). Glutathione-agarose beads (30 μl) were added, and the mixture was incubated with gentle rocking for 2 h at at 4 °C. Proteins were eluted from the beads, resolved by SDS-PAGE, and subjected to immunoblot analysis with anti-RACK1, as described above. Oligonucleotide-directed mutagenesis was used to substitute phenylalanine for tyrosine at residues 52, 140, 194, 228, 246, or 302 of RACK1, utilizing the Transformer site-directed mutagenesis kit according to the manufacturer's protocol (CLONTECH, Palo Alto, CA) and the following oligonucleotides: Y52F oligo, GATGAGACCAACTTTGGAATTCCA; Y140F oligo, GGTGTGTGCAAATTCACTGTCCAG; Y195F oligo, CACACAGGCTTTCTGAACACGGTG; Y228F oligo, GGCAAACACCTTTTCACGCTAGAT; Y246F oligo, CCTAACCGCTTCTGGCTGTGTGCT; Y302F oligo, CTGTTTGCTGGCTTCACGGACAAC. The sequence of each RACK1 mutant was confirmed by automated DNA sequencing (Protein and Nucleic Acid Facility, Stanford University, Stanford, CA). Mutant RACK1 genes were inserted into pcDNA3 to create pcDNA3-HA-RACK1(Y52F, Y140F, Y195F, Y228F, Y246F, or Y302F) as described (8Chang B.Y. Conroy K.B. Machleder E.M. Cartwright C.A. Mol. Cell. Biol. 1998; 18: 3245-3256Crossref PubMed Google Scholar). CHO cells were transfected with pcDNA3 plasmids as described above. 18 h later after transfection, cells were placed in fresh media containing 0.5% FBS. 24 h later, cells were treated for various time periods with PMA, which was dissolved in dimethyl sulfoxide (Me2SO) and used at a concentration of 10 ng/ml (35Emkey R. Kahn C.R. J. Biol. Chem. 1997; 272: 31172-31181Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 36Rodriguez-Fernandez J.L. Rozengurt E. J. Biol. Chem. 1996; 271: 27895-27901Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). In some cases, cells were pre-treated with a PKC inhibitor: GF109203X, chelerythrine, or calphostin C (each was dissolved in Me2SO and used at a concentration of 0.1 μm) (Calbiochem, La Jolla, CA) for 30 min prior to PMA stimulation. Control cells were treated with Me2SO alone. NIH 3T3 cells that were stably overexpressing the PDGFR were maintained in 0.5% serum for 48 h before treatment with PDGF-BB (Sigma) (10 ng/ml) for various time periods. For immunoblot analysis with anti-phosphotyrosine PY20, cells were treated with 100 μm sodium vanadate (Sigma) for 30 min prior to harvesting (8Chang B.Y. Conroy K.B. Machleder E.M. Cartwright C.A. Mol. Cell. Biol. 1998; 18: 3245-3256Crossref PubMed Google Scholar). Cells were harvested by trypsinization, collected by centrifugation, and lysed in SDS sample buffer. NIH 3T3 cells stably overexpressing c-Src were grown subconfluently on coverslips in DMEM supplemented with 10% FBS for 24 - 48 h and then in fresh media containing 0.5% FBS for 72 h. Cells were treated with PMA (10 ng/ml) or Me2SO for various time periods prior to fixation in 3.7% paraformaldehyde in phosphate-buffered saline (PBS) for 20 min at room temperature and permeabilization in 0.4% Triton X-100/PBS for 20 min at room temperature (37Jou T.S. Nelson W.J. J. Cell Biol. 1998; 142: 85-100Crossref PubMed Scopus (259) Google Scholar). Nonspecific sites were blocked with 10% goat serum and 1% bovine serum albumin (BSA) in PBS containing 50 mm NH4Cl, for 1 h. Cells were incubated with primary antibodies, anti-RACK1 (1:500) and anti-Src (mAb 327) (1:100) in PBS containing 5% goat serum and 0.2% BSA for 1 h. After washing three times in PBS containing 0.2% BSA, cells were incubated with secondary antibodies, FITC-conjugated goat anti-mouse IgG (Jackson Immunoresearch Laboratories, West Grove, PA) and lissamine-rhodamine conjugated goat anti-mouse IgM (Jackson Immunoresearch), which were diluted 1:100 in PBS containing 5% goat serum and 0.2% BSA, for 40 min in the dark. Coverslips were washed three times in PBS containing 0.2% BSA and a fourth time in PBS containing bisbenzamidine. Src and RACK1 immunostaining were visualized with FITC and Texas Red filters, respectiv
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