Identification of Ribosome-binding Protein p34 as an Intracellular Protein That Binds Acidic Fibroblast Growth Factor
2002; Elsevier BV; Volume: 277; Issue: 26 Linguagem: Inglês
10.1074/jbc.m112193200
ISSN1083-351X
AutoresCamilla Skiple Skjerpen, Jørgen Wesche, Sjur Olsnes,
Tópico(s)Galectins and Cancer Biology
ResumoWith the aim of identifying new intracellular binding partners for acidic fibroblast growth factor (aFGF), proteins from U2OS human osteosarcoma cells were adsorbed to immobilized aFGF. One of the adsorbed proteins is a member of the leucine-rich repeat protein family termed ribosome-binding protein p34 (p34). This protein has previously been localized to endoplasmic reticulum membranes and is thought to span the membrane with the N terminus on the cytosolic side. Confocal microscopy of cells transfected with Myc-p34 confirmed the endoplasmic reticulum localization, and Northern blotting determined p34 mRNA to be present in a multitude of different tissues. Cross-linking experiments indicated that the protein is present in the cell as a dimer. In vitro translated p34 was found to interact with maltose-binding protein-aFGF through its cytosolic coiled-coil domain. The interaction between aFGF and p34 was further characterized by surface plasmon resonance, giving aK D of 1.4 ± 0.3 μm. Even though p34 interacted with mitogenic aFGF, it bound poorly to the non-mitogenic aFGF(K132E) mutant, indicating a possible involvement of p34 in intracellular signaling by aFGF. With the aim of identifying new intracellular binding partners for acidic fibroblast growth factor (aFGF), proteins from U2OS human osteosarcoma cells were adsorbed to immobilized aFGF. One of the adsorbed proteins is a member of the leucine-rich repeat protein family termed ribosome-binding protein p34 (p34). This protein has previously been localized to endoplasmic reticulum membranes and is thought to span the membrane with the N terminus on the cytosolic side. Confocal microscopy of cells transfected with Myc-p34 confirmed the endoplasmic reticulum localization, and Northern blotting determined p34 mRNA to be present in a multitude of different tissues. Cross-linking experiments indicated that the protein is present in the cell as a dimer. In vitro translated p34 was found to interact with maltose-binding protein-aFGF through its cytosolic coiled-coil domain. The interaction between aFGF and p34 was further characterized by surface plasmon resonance, giving aK D of 1.4 ± 0.3 μm. Even though p34 interacted with mitogenic aFGF, it bound poorly to the non-mitogenic aFGF(K132E) mutant, indicating a possible involvement of p34 in intracellular signaling by aFGF. acidic fibroblast growth factor basic fibroblast growth factor mitogen-activated protein kinase aFGF intracellular binding protein FGF-binding protein-1 phosphate-buffered saline maltose-binding protein disuccinimidyl suberate glutathione S-transferase green fluorescent protein dithiothreitol matrix-assisted laser desorption ionization time-of-flight mass spectrometry endoplasmic reticulum Acidic fibroblast growth factor (aFGF)1 belongs to the large family of FGF growth factors. It is involved in cellular processes such as stimulation of DNA synthesis and cell proliferation as well as differentiation and cell migration (1Burgess W.H. Maciag T. Annu. Rev. Biochem. 1989; 58: 575-606Crossref PubMed Google Scholar, 2Wiedlocha A. Falnes P.Ø. Madshus I.H. Sandvig K. Olsnes S. Cell. 1994; 76: 1039-1051Abstract Full Text PDF PubMed Scopus (218) Google Scholar, 3Basilico C. Moscatelli D. Adv. Cancer Res. 1992; 59: 115-165Crossref PubMed Scopus (1049) Google Scholar, 4Szebenyi G. Fallon J.F. Int. Rev. Cytol. 1999; 185: 45-106Crossref PubMed Google Scholar, 5Mason I.J. Cell. 1994; 78: 547-552Abstract Full Text PDF PubMed Scopus (525) Google Scholar). In vivo, aFGF has been shown to play a role in mesoderm induction; angiogenesis; wound healing; and development of the nervous, skeletal, and vascular systems (3Basilico C. Moscatelli D. Adv. Cancer Res. 1992; 59: 115-165Crossref PubMed Scopus (1049) Google Scholar). During the last decade, an increasing amount of evidence has suggested that both aFGF and basic FGF (bFGF) do not conform to the paradigm that protein growth factors act only through cell-surface receptors. Both aFGF and bFGF have been found in the cytosol and nucleus of different cell types. aFGF has been shown to enter NIH/3T3, BALB/c 3T3, and human umbilical vein endothelial cells as well as U2OS cells that were stably transfected with high affinity FGF receptor-4 (2Wiedlocha A. Falnes P.Ø. Madshus I.H. Sandvig K. Olsnes S. Cell. 1994; 76: 1039-1051Abstract Full Text PDF PubMed Scopus (218) Google Scholar, 6Imamura T. Oka S. Tanahashi T. Okita Y. Exp. Cell Res. 1994; 215: 363-372Crossref PubMed Scopus (73) Google Scholar, 7Wiedlocha A. Falnes P.Ø. Rapak A. Klingenberg O. Muñoz R. Olsnes S. J. Biol. Chem. 1995; 270: 30680-30685Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 8Wiedlocha A. Falnes P.Ø. Rapak A. Muñoz R. Klingenberg O. Olsnes S. Mol. Cell. Biol. 1996; 16: 270-280Crossref PubMed Scopus (96) Google Scholar), whereas bFGF has been found to enter the nucleus from the cytosol of adult bovine aortic endothelial cells in a cell cycle-dependent manner (9Baldin V. Roman A.M. Bosc-Bierne I. Amalric F. Bouche G. EMBO J. 1990; 9: 1511-1517Crossref PubMed Scopus (303) Google Scholar). aFGF signaling through cell-surface receptors is sufficient to induce tyrosine phosphorylation of the receptors and concomitant activation of the MAPK cascade (2Wiedlocha A. Falnes P.Ø. Madshus I.H. Sandvig K. Olsnes S. Cell. 1994; 76: 1039-1051Abstract Full Text PDF PubMed Scopus (218) Google Scholar, 8Wiedlocha A. Falnes P.Ø. Rapak A. Muñoz R. Klingenberg O. Olsnes S. Mol. Cell. Biol. 1996; 16: 270-280Crossref PubMed Scopus (96) Google Scholar). Furthermore, translocation of aFGF to the nucleus is a sufficient signal to stimulate DNA synthesis. However, both these signals are necessary to stimulate cell proliferation, at least in certain cells (8Wiedlocha A. Falnes P.Ø. Rapak A. Muñoz R. Klingenberg O. Olsnes S. Mol. Cell. Biol. 1996; 16: 270-280Crossref PubMed Scopus (96) Google Scholar). Similarly, bFGF located in the nucleus can activate transcription of ribosomal genes, and its nuclear accumulation is associated with cell proliferation (1Burgess W.H. Maciag T. Annu. Rev. Biochem. 1989; 58: 575-606Crossref PubMed Google Scholar,9Baldin V. Roman A.M. Bosc-Bierne I. Amalric F. Bouche G. EMBO J. 1990; 9: 1511-1517Crossref PubMed Scopus (303) Google Scholar, 10Bouche G. Gas N. Prats H. Baldin V. Tauber J.P. Teissie J. Amalric F. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 6770-6774Crossref PubMed Scopus (389) Google Scholar, 11Amalric F. Bouche G. Bonnet H. Brethenou P. Roman A.M. Truchet I. Quarto N. Biochem. Pharmacol. 1994; 47: 111-115Crossref PubMed Scopus (95) Google Scholar). An aFGF mutant in which lysine 132 has been replaced by glutamic acid has been reported to possess greatly reduced mitogenic activity (12Burgess W.H. Shaheen A.M. Ravera M. Jaye M. Donohue P.J. Winkles J.A. J. Cell Biol. 1990; 111: 2129-2138Crossref PubMed Scopus (91) Google Scholar). Despite this, it retains the ability to bind to heparin, has normal receptor-binding activity, and is capable of stimulating the tyrosine kinase activity of the receptor and expression of proto-oncogenes (12Burgess W.H. Shaheen A.M. Ravera M. Jaye M. Donohue P.J. Winkles J.A. J. Cell Biol. 1990; 111: 2129-2138Crossref PubMed Scopus (91) Google Scholar,13Burgess W.H. Friesel R. Winkles J.A. Mol. Reprod. Dev. 1994; 39: 56-60Crossref PubMed Scopus (15) Google Scholar). In addition, both wild-type and mutant aFGFs were found in the nucleus following transfection of NIH/3T3 cells, even though only wild-type aFGF was found to induce a transformed phenotype (13Burgess W.H. Friesel R. Winkles J.A. Mol. Reprod. Dev. 1994; 39: 56-60Crossref PubMed Scopus (15) Google Scholar). A number of mutations in aFGF with less dramatic effects were described by Klingenberg et al. (14Klingenberg O. Wiedlocha A. Olsnes S. J. Biol. Chem. 1999; 274: 18081-18086Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). They altered amino acids close to or in an exposed loop containing a phosphorylation site recognized by protein kinase C. Although all the mutants could bind to specific FGF receptors, activate the MAPK cascade, and be translocated to the nucleus, the mutations affected to varying extent the ability to stimulate DNA synthesis and cell proliferation. As a result of the accumulating evidence of an intracellular role for both aFGF and bFGF, several groups have attempted to identify cellular proteins interacting with these two growth factors. aFGF has been shown to interact with aFGF intracellular binding protein (FIBP) (15Kolpakova E. Wiedlocha A. Stenmark H. Klingenberg O. Falnes P.Ø. Olsnes S. Biochem. J. 1998; 336: 213-222Crossref PubMed Scopus (45) Google Scholar), mortalin (16Mizukoshi E. Suzuki M. Loupatov A. Uruno T. Hayashi H. Misono T. Kaul S.C. Wadhwa R. Imamura T. Biochem. J. 1999; 343: 461-466Crossref PubMed Scopus (80) Google Scholar), and a secreted protein named FGF-binding protein-1 (FGF-BP1) (17Tassi E., Al Attar A. Aigner A. Swift M.R. McDonnell K. Karavanov A. Wellstein A. J. Biol. Chem. 2001; 276: 40247-40253Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). During secretion, aFGF also forms complexes with synaptotagmin-1 and the calcium-binding protein S100A13 (18LaVallee T.M. Tarantini F. Gamble S. Carreira C.M. Jackson A. Maciag T. J. Biol. Chem. 1998; 273: 22217-22223Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 19Carreira C.M. LaVallee T.M. Tarantini F. Jackson A. Lathrop J.T. Hampton B. Burgess W.H. Maciag T. J. Biol. Chem. 1998; 273: 22224-22231Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 20Tarantini F. LaVallee T. Jackson A. Gamble S. Carreira C.M. Garfinkel S. Burgess W.H. Maciag T. J. Biol. Chem. 1998; 273: 22209-22216Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). bFGF has been shown to interact with CK2 (21Bonnet H. Filhol O. Truchet I. Brethenou P. Cochet C. Amalric F. Bouche G. J. Biol. Chem. 1996; 271: 24781-24787Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar) and with FGF-BP1 (17Tassi E., Al Attar A. Aigner A. Swift M.R. McDonnell K. Karavanov A. Wellstein A. J. Biol. Chem. 2001; 276: 40247-40253Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar); and recently, we also found that aFGF interacts with both the α- and β-subunits of protein kinase CK2. 2C. S. Skjerpen, J. Wesche, and S. Olsnes, manuscript in preparation.2C. S. Skjerpen, J. Wesche, and S. Olsnes, manuscript in preparation. Furthermore, bFGF was reported to associate with platelet-derived growth factor-BB (22Russo K. Ragone R. Facchiano A.M. Capogrossi M.C. Facchiano A. J. Biol. Chem. 2002; 277: 1284-1291Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar), the nuclear protein FGF-2-interacting factor (23Van den Berghe B.L. Laurell H. Huez I. Zanibellato C. Prats H. Bugler B. Mol. Endocrinol. 2000; 14: 1709-1724Crossref PubMed Scopus (62) Google Scholar), and the ribosomal proteins L6 and s19 (24Shen B. Arese M. Gualandris A. Rifkin D.B. Biochem. Biophys. Res. Commun. 1998; 252: 524-528Crossref PubMed Scopus (27) Google Scholar, 25Soulet F., Al Saati T. Roga S. Amalric F. Bouche G. Biochem. Biophys. Res. Commun. 2001; 289: 591-596Crossref PubMed Scopus (54) Google Scholar). Despite the accumulating data for both aFGF and bFGF acting inside cells and for interaction with cytosolic and nuclear proteins, the role of these interactions with respect to the intracellular trafficking and functioning of aFGF remains largely unexplained. In an attempt to elucidate the intracellular role of aFGF, we precipitated proteins that bind to aFGF. We identified by precipitation and mass spectrometry two new proteins that bind to aFGF. One was protein kinase CK2, a constitutively active serine/threonine kinase (26Guerra B. Issinger O.G. Electrophoresis. 1999; 20: 391-408Crossref PubMed Scopus (363) Google Scholar, 27Guerra B. Boldyreff B. Sarno S. Cesaro L. Issinger O.G. Pinna L.A. Pharmacol. Ther. 1999; 82: 303-313Crossref PubMed Scopus (85) Google Scholar, 28Allende J.E. Allende C.C. FASEB J. 1995; 9: 313-323Crossref PubMed Scopus (584) Google Scholar) previously found to interact with bFGF (21Bonnet H. Filhol O. Truchet I. Brethenou P. Cochet C. Amalric F. Bouche G. J. Biol. Chem. 1996; 271: 24781-24787Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 29Bailly K. Soulet F. Leroy D. Amalric F. Bouche G. FASEB J. 2000; 14: 333-344Crossref PubMed Scopus (69) Google Scholar). The other one was a protein with an apparent molecular mass of 35 kDa. In this work, we report the identification of this aFGF-interacting protein as ribosome-binding protein p34. p34 is located in the endoplasmic reticulum (30Ohsumi T. Ichimura T. Sugano H. Omata S. Isobe T. Kuwano R. Biochem. J. 1993; 294: 465-472Crossref PubMed Scopus (23) Google Scholar). It is a protein that contains a leucine-rich repeat domain and a coiled-coil domain (both presumably located in the cytosol) as well as a transmembrane domain close to the C-terminal tail (30Ohsumi T. Ichimura T. Sugano H. Omata S. Isobe T. Kuwano R. Biochem. J. 1993; 294: 465-472Crossref PubMed Scopus (23) Google Scholar). We found that although p34 binds to mitogenic aFGF, it does not bind to the non-mitogenic K132E mutant. Phosphate-buffered saline (PBS) contained 140 mm NaCl and 10 mmNa2HPO4 (pH 7.2); lysis buffer contained 100 mm NaCl, 10 mm Na2HPO4(pH 7.2), 1% Triton X-100, and 1 mm EDTA. Protein A-Sepharose CL-4B, CNBr-activated Sepharose, glutathione-Sepharose, heparin-Sepharose, [35S]methionine, and [33P]dCTP were from Amersham Biosciences (Uppsala, Sweden). Restriction endonucleases and amylose resin were from New England Biolabs Inc. (Beverly, MA). The Dynabeads mRNA DIRECT kit was from Dynal (Oslo, Norway). Coomassie Brilliant Blue G and reduced glutathione were from Sigma. Anti-c-Myc antibody 9E10 was from American Type Culture Collection (Manassas, VA). Anti-calreticulin antibody was from Stressgen Biotechnologies Corp. (Victoria, British Colombia, Canada). Anti-MBP-FIBP antibody was obtained from Dr. Elona Kolpakova (Institute for Cancer Research, Oslo). The secondary antibodies (horseradish peroxidase-conjugated IgGs, lissamine rhodamine-labeled anti-mouse IgG, and fluorescein isothiocyanate-labeled anti-rabbit IgG) were from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). SuperSignal chemiluminescent substrate and disuccinimidyl suberate (DSS) were from Pierce. FuGENE 6 reagent and Complete protease inhibitor mixture were from Roche Molecular Biochemicals. RNasin, T7 RNA polymerase, nuclease-treated rabbit reticulocyte lysate, and canine pancreatic microsomal membranes were from Promega. Bradford reagent and recombinant GST were from Bio-Rad. The multiple-tissue Northern blot (human 12-lane MTNTM blot), ExpressHybTMhybridization solution, and the probe for human β-actin were from CLONTECH (Palo Alto, CA). Isopropyl-β-d-thiogalactopyranoside was from Saween Biotech (Malmö, Sweden). U2OS and COS-1 cells were propagated in Dulbecco's modified essential medium with 10% (v/v) fetal calf serum in a 5% CO2 atmosphere at 37 °C. Transient expression of the p34 protein or GFP-aFGF was achieved by transiently transfecting COS-1 cells with pcDNA3-Myc-p34 or pEGFP-aFGF using the FuGENE 6 transfection agent according to the manufacturer's recommendations. Cells were used for experiments 20–24 h after transfection. All plasmid constructs containing MBP fusions of aFGF, aFGF mutants, bFGF, and FIBP have been described previously (14Klingenberg O. Wiedlocha A. Olsnes S. J. Biol. Chem. 1999; 274: 18081-18086Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar,15Kolpakova E. Wiedlocha A. Stenmark H. Klingenberg O. Falnes P.Ø. Olsnes S. Biochem. J. 1998; 336: 213-222Crossref PubMed Scopus (45) Google Scholar), except for the MBP-aFGF(S113A) mutant, which was made using polymerase chain reaction-directed mutagenesis with the MBP-aFGF plasmid as template. p34 was obtained from a U2OS cDNA library made from U2OS cells using the Dynabeads mRNA DIRECT kit following the manufacturer's recommendations, followed by reverse transcription. The forward and reverse primers used were 5′-GCCGCATGCGAATTCCATGACCAAGGCCGGTAGCAAG-3′ and 5′-GCCGGTCTAGACTCGAGTCACTGCTGAGAGTCGGTCTG-3′, respectively. cDNA coding for p34 was cloned into theEcoRI/XhoI site of both pGEX-6P-1 (AmershamBiosciences) and pcDNA3-Myc (constructed by Dr. Camilla Raiborg (Institute for Cancer Research) by inserting the Myc epitope into theHindIII/XhoI sites of pcDNA3 (Invitrogen)). pMal-p34 was made by inserting the cDNA for p34 into the BamHI/SalI site of pMal-C2 (New England Biolabs Inc.). All the constructs or parts of the p34 gene were made by PCR. These partial genes were then cloned into theEcoRI/XhoI sites of pcDNA3-Myc. pEGFP-aFGF was made by inserting the cDNA for aFGF into the multiple cloning site of the pEGFP vector (CLONTECH). The plasmid pRc/CMV-HA-CK2α was a gift from Dr. D. W. Litchfield (Manitoba Institute of Cell Biology, Winnipeg, Canada) (31Penner C.G. Wang Z. Litchfield D.W. J. Cell. Biochem. 1997; 64: 525-537Crossref PubMed Scopus (70) Google Scholar). The construct pcDNA-CK2β was made by subcloning CK2β from plasmid pCMVES-CK2β (a gift from Dr. Götz, University of the Saarland, Saarland, Germany) (32Gotz C. Kartarius S. Scholtes P. Montenarh M. Biochem. Biophys. Res. Commun. 2000; 268: 882-885Crossref PubMed Scopus (18) Google Scholar) into theBamHI/SalI sites of pGEX-5X-3 using PCR. Expression of MBP fusion proteins in Escherichia coli DH5α was induced for 2 h by addition of 0.3 mm isopropyl-β-d-thiogalactopyranoside. The cells were harvested and frozen at −20 °C. The cell pellet was resuspended in 25 ml of column buffer A (20 mm Tris (pH 7.5), 200 mm NaCl, 1 mm DTT, and 1 mm EDTA,) with one tablet of Complete protease inhibitor mixture, sonicated to disrupt the cells, and centrifuged at 12,100 × g for 20 min at 4 °C. The supernatant was diluted 1:1 with column buffer A, loaded onto a column packed with amylose resin, and washed with 200 ml of column buffer A. The fusion proteins were eluted with 10 mm maltose in column buffer A, and the protein concentration was estimated using the Bradford assay (33Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211946) Google Scholar) and by SDS-PAGE. Expression of fusion proteins with GST was induced as described for MBP fusion proteins. The cell pellet was resuspended in 20 ml of column buffer B (20 mm Tris (pH 7.5) and 150 mm NaCl) with one tablet of Complete protease inhibitor mixture and sonicated. Triton X-100 was added to 1%, and the lysate was rotated for 30 min at 4 °C before being centrifuged at 12,100 × g for 20 min at 4 °C. The supernatant was diluted 1:1 with column buffer B, and 1 ml of prewashed glutathione-Sepharose was added. The mixture was rotated for 30 min at room temperature and loaded onto a column. The proteins were washed with 30 ml of column buffer B and eluted with 10 mm reduced glutathione in column buffer B (pH 8.0). Recombinant aFGF was produced in E. coli BL21. Expression of aFGF was induced for 2 h with 3 mmisopropyl-β-d-thiogalactopyranoside; the cells were harvested; and the cell pellet was frozen at −20 °C. Bacterial pellets were resuspended in 20 ml of column buffer C (20 mmTris (pH 7.4), 0.5 m NaCl, 10 mm DTT, and 1 mm EDTA) with one tablet of Complete protease inhibitor mixture and sonicated. After centrifugation at 12,100 ×g for 20 min at 4 °C, the supernatant was diluted 1:1 with column buffer C and incubated for 2 h at 4 °C with prewashed heparin-Sepharose (2.5 g). The Sepharose slurry was applied to a column and washed first with column buffer C, followed by column buffer C containing 0.7 m NaCl. The protein was eluted with 2 m NaCl in column buffer C. For precipitation purposes, MBP fusion proteins were bound to either CNBr-activated Sepharose or protein A-Sepharose. Binding to protein A-Sepharose was via an antibody against MBP-FIBP, whereas the MBP fusion proteins were coupled to CNBr-activated Sepharose by incubating 1 ml (0.2–2 mg) of protein solution in PBS with 0.5 ml of prewashed CNBr-activated Sepharose. The reaction was quenched by incubation for another hour with 100 mm glycine before washing repeatedly with high salt and high and low pH buffers. Subconfluent U2OS cells were labeled overnight with [35S]methionine/cysteine, washed with PBS, and lysed on ice for 20 min in lysis buffer (100 mm NaCl, 10 mm Na2HPO4 (pH 7.2), 1% Triton X-100, and 1 mm EDTA) with 10 mm DTT and Complete protease inhibitor mixture. The cells were collected with a cell scraper and centrifuged at 3020 × g for 10 min at 4 °C. The supernatant was diluted 1:1 with PBS and incubated for 2 h at 4 °C with CNBr-activated Sepharose without additional bound protein. The precipitation mixture was centrifuged at 3020 × g for 5 min at 4 °C, and the supernatant was incubated for another 2 h at 4 °C with Sepharose-bound MBP-interferon-γ (control). After another centrifugation, the supernatant was incubated with Sepharose-bound MBP-aFGF for 2.5 h at 4 °C. The beads were then washed four times with a 1:1 mixture of lysis buffer and PBS, and the bound proteins were eluted with 2 m NaCl in PBS on ice for 15 min. Proteins were precipitated with 5% trichloroacetic acid on ice for 1 h, and the pellet was extracted three times with ether. The proteins were analyzed by SDS-PAGE (12% (w/v) gel), followed by staining with Coomassie Brilliant Blue G; and the dried gel was subjected to autoradiography. Defined bands were excised from the gel and subjected to in-gel trypsin treatment, followed by either MALDI mass spectrometry alone or MALDI mass spectrometry and internal sequencing. The protein sequence data were obtained at the Rockefeller University Protein/DNA Technology Center (New York, NY) (34Fernandez J. Gharahdaghi F. Mische S.M. Electrophoresis. 1998; 19: 1036-1045Crossref PubMed Scopus (162) Google Scholar, 35Fernandez J. Andrews L. Mische S.M. Anal. Biochem. 1994; 218: 112-117Crossref PubMed Scopus (143) Google Scholar). Transiently transfected COS-1 cells were washed with PBS and lysed on ice in lysis buffer containing 10 mm DTT and Complete protease inhibitor mixture. The lysate was centrifuged at 20,800 × g for 3 min at 4 °C. The supernatant was diluted 1:1 with PBS and incubated with Sepharose-bound MBP fusion protein for 1 h at 4 °C. Precipitates were collected by centrifugation and washed three times with a 1:1 mixture of PBS and lysis buffer before SDS sample buffer was added to elute the proteins. The samples were subsequently subjected to SDS-PAGE, followed by transfer to a polyvinylidene difluoride membrane. The membrane was blocked with 5% nonfat dry milk powder in washing buffer (PBS with 0.1% Tween 20) and incubated with mouse anti-c-Myc antibody 9E10, and proteins were visualized after incubation with a horseradish peroxidase-conjugated secondary antibody and SuperSignal chemiluminescent substrate. [35S]Methionine-labeled p34 and its domains as well as CK2α and CK2β were produced in a rabbit reticulocyte lysate system as described previously (2Wiedlocha A. Falnes P.Ø. Madshus I.H. Sandvig K. Olsnes S. Cell. 1994; 76: 1039-1051Abstract Full Text PDF PubMed Scopus (218) Google Scholar). The template vectors were pcDNA3-Myc-p34, pRc/CMV-HA-CK2α, and pcDNA3-CK2β, respectively. In short, the plasmid was linearized downstream of the coding sequence and transcribed for 60 min in a 20-μl reaction mixture using T7 RNA polymerase. The mRNA was precipitated with ethanol, dissolved in 10 μl of H2O containing 10 mm DTT and 0.2 units/μl RNasin, and subsequently translated for 60 min in a nuclease-treated rabbit reticulocyte lysate in the presence of [35S]methionine. The translation mixture was dialyzed against dialysis buffer (20 mm Hepes (pH 7.0), 140 mm NaCl, and 20 mm CaCl2) to remove free [35S]methionine. Fifteen μg of MBP fusion protein bound to protein A-Sepharose beads was added together with 1–20 μl of in vitro translated p34, one of the p34 domain proteins, or hemagglutinin-tagged CK2α or CK2β to a 1:1 mixture of lysis buffer and PBS with 5 mm DTT. The mixture was incubated for 90 min at 4 °C and washed three times with the same buffer or with the same buffer with additional NaCl. The bound proteins were eluted with SDS sample buffer and subjected to SDS-PAGE, followed by fluorography. In the competition experiments, binding was performed in the presence of the indicated amounts of unlabeled recombinant protein. The equilibrium dissociation constant (K D ) for the binding between aFGF and p34 was determined using a BIAcore X (BIAcore AB, Uppsala) at 25 °C. GST-p34 was coupled to a CM5 sensor chip (BIAcore) using the GST kit for fusion capture (BIAcore). Anti-GST antibody was covalently linked to the carboxylated dextran matrix of the CM5 sensor chip according to the manual (GST kit for fusion capture). Thirty μl of GST-p34 (5 μg/ml) was then loaded onto the sensor chip by injection at a flow rate of 5 μl/ml. The reference cell was coated similarly with recombinant GST. Injections of recombinant aFGF in buffer containing 10 mm Hepes (pH 7.3), 0.15 m NaCl, 3 mm EDTA, and 0.25 mg/ml carboxylmethyl-dextran were carried out at a flow rate of 30 μl/min, and sensorgrams were recorded. The surface was regenerated between each measurement with 10 mm glycine (pH 2.2). The sensorgrams were analyzed using the BIAevaluation Version 3.0 software. The means ± S.D. were calculated based on four experiments. The human multiple-tissue Northern blot containing 1 μg of poly(A+) RNA/lane was probed with a [33P]dCTP-labeled, random-primed DNA probe using the 560-bp 5′-terminal fragment of p34, the 300-bp 5′-terminal fragment of aFGF, or a probe for human β-actin. The blot was hybridized overnight in ExpressHybTM hybridization solution and washed for 40 min at room temperature with 2× SSC (150 mm NaCl and 15 mm Na3 citrate (pH 7.0)) and 0.05% SDS, twice for 40 min at 50 °C with 0.1× SSC and 0.1% SDS, and finally for 1 h at 50 °C with 0.5× SSC and 0.2% SDS. Membranes were exposed using a phosphorimaging screen and scanned. After hybridization of the membrane with a p34 probe, the membrane was stripped, rehybridized with an aFGF probe, and finally stripped and hybridized with a probe for human β-actin. COS-1 cells were seeded on sterile coverslips and transiently transfected with pcDNA3-Myc-p34 alone or in combination with pEGFP-aFGF using FuGENE 6 transfection agent. Twenty-four h after transfection, the cells were washed three times with PBS and fixed in 3% paraformaldehyde in PBS for 15 min at room temperature. The cells were washed with PBS, and autofluorescence was quenched by incubation in 50 mm NH4Cl in PBS for 10 min at room temperature. After another wash, the cells were permeabilized with 0.5% Triton X-100 in PBS for 4 min at room temperature, washed, and incubated for 20 min at room temperature with the appropriate primary antibody (anti-calreticulin and/or anti-c-Myc) diluted in PBS, 0.1% Tween 20, and 5% nonfat dry milk powder. The cells were washed and incubated for 20 min at room temperature with the secondary antibody and then washed a final time and mounted in Mowiol. Immunofluorescence images were taken using a Leica confocal microscope and processed using Adobe Photoshop Version 5.0. Antibodies against MBP-p34 were raised in rabbits and purified by affinity chromatography on an Affi-Gel-10 column with covalently bound MBP-p34. The antibodies were eluted with 100 mm glycine (pH 2.8), and the pH was immediately neutralized with 3 m Tris-HCl (pH 8.8). U2OS cells grown in Dulbecco's modified essential medium were either permeabilized with digitonin (40 μg/ml) or left untreated for 10 min at room temperature and then washed once with PBS. DSS was added to PBS to a final concentration of 2 mm, and the cells were kept on ice for 2 h in PBS with or without DSS. The reaction was quenched with 20 mm Tris, and the cells were kept on ice for an additional 15 min. The cells were then washed three times with PBS, scraped off, and centrifuged (20,800 × g, 3 min, 4 °C), and the pellet was resuspended in SDS sample buffer. The samples were analyzed by SDS-PAGE, followed by Western blotting with an antibody against MBP-p34. In a screen for proteins of the U2OS human osteosarcoma cell line that bind to MBP-aFGF, a promising candidate was a protein with an apparent molecular mass of 35 kDa (Fig. 1). U2OS cells were chosen because they are of human origin, which would simplify the identification process, and because they were used in our previous work (2Wiedlocha A. Falnes P.Ø. Madshus I.H. Sandvig K. Olsnes S. Cell. 1994; 76: 1039-1051Abstract Full Text PDF PubMed Scopus (218) Google Scholar, 8Wiedlocha A. Falnes P.Ø. Rapak A. Muñoz R. Klingenberg O. Olsnes S. Mol. Cell. Biol. 1996; 16: 270-280Crossref PubMed Scopus (96) Google Scholar, 36Klingenberg O. Wiedlocha A. Citores L. Olsnes S. J. Biol. Chem. 2000; 275: 11972-11980Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). We chose to work with MBP fusion proteins because the MBP fusion (as opposed to the GST fusion) with aFGF was as potent as wild-type aFGF in binding to and activating FGF receptors (data not shown). In addition to the 35-kDa band, specific bands with molecular masses of ∼28 (later identified as the regulatory subunit of protein kinase CK2), 43, and 17 kDa could be seen on the gels. Also some minor, but apparently specific bands with molecular masses of 29, 40, and 72 kDa could be seen in most of the experiments (data not shown). These bands could represent the catalytic subunit of protein kinase CK2 (p43), FGF-BP1 (p29), FIBP (p40), and mortalin (p72). Both FIBP and mortalin have been previously shown to bind to aFGF (15Kolpakova E. Wiedlocha A. Stenmark H. Klingenberg O. Falnes P.Ø. Olsnes S. Biochem. J. 1998; 336: 213-222Crossref PubMed Scopus (45) Google Schol
Referência(s)