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• W2035555613 abstract "The Zn2+- and Ca2+-binding S100B protein is implicated in multiple intracellular and extracellular regulatory events. In glial cells, a relationship exists between cytoplasmic S100B accumulation and cell morphological changes. We have identified the IQGAP1 protein as the major cytoplasmic S100B target protein in different rat and human glial cell lines in the presence of Zn2+ and Ca2+. Zn2+ binding to S100B is sufficient to promote interaction with IQGAP1. IQ motifs on IQGAP1 represent the minimal interaction sites for S100B. We also provide evidence that, in human astrocytoma cell lines, S100B co-localizes with IQGAP1 at the polarized leading edge and areas of membrane ruffling and that both proteins relocate in a Ca2+-dependent manner within newly formed vesicle-like structures. Our data identify IQGAP1 as a potential target protein of S100B during processes of dynamic rearrangement of cell membrane morphology. They also reveal an additional cellular function for IQGAP1 associated with Zn2+/Ca2+-dependent relocation of S100B. The Zn2+- and Ca2+-binding S100B protein is implicated in multiple intracellular and extracellular regulatory events. In glial cells, a relationship exists between cytoplasmic S100B accumulation and cell morphological changes. We have identified the IQGAP1 protein as the major cytoplasmic S100B target protein in different rat and human glial cell lines in the presence of Zn2+ and Ca2+. Zn2+ binding to S100B is sufficient to promote interaction with IQGAP1. IQ motifs on IQGAP1 represent the minimal interaction sites for S100B. We also provide evidence that, in human astrocytoma cell lines, S100B co-localizes with IQGAP1 at the polarized leading edge and areas of membrane ruffling and that both proteins relocate in a Ca2+-dependent manner within newly formed vesicle-like structures. Our data identify IQGAP1 as a potential target protein of S100B during processes of dynamic rearrangement of cell membrane morphology. They also reveal an additional cellular function for IQGAP1 associated with Zn2+/Ca2+-dependent relocation of S100B. S100B is a member of the S100 family of proteins containing two EF-hand-type calcium-binding domains (1Schäfer B.W. Heizmann C. Trends Biochem. Sci. 1996; 21: 134-140Abstract Full Text PDF PubMed Scopus (885) Google Scholar). This protein interacts not only with Ca2+ but also with Zn2+ ions, binding Zn2+ ions with an affinity in the nanomolar range (2Baudier J. Glasser N. Gérard D. J. Biol. Chem. 1986; 261: 8192-8203Abstract Full Text PDF PubMed Google Scholar). The capacity of S100B to bind and release Zn2+ suggests that Zn2+ may not only play a structural role but might also be involved, together with Ca2+, in concerted regulation of S100B function. The S100B protein is naturally highly expressed in the vertebrate nervous system, where it is present in astrocytes and Schwann cells (3Haglid K.G. Hamberger A. Hansson H.A. Hyden H. Persson L. Ronnback L. Nature. 1975; 258: 748-749Crossref PubMed Scopus (12) Google Scholar). In the adult central nervous system, the S100B protein is present in the nuclei and cytoplasm of astrocytes and accumulates in the astrocytic dendrites in the perivascular processes (4Rickmann M. Wolff J.R. Histochemistry. 1995; 103: 135-145Crossref PubMed Scopus (27) Google Scholar). Studies in different laboratories suggest a variety of intracellular regulations by S100B, including negative cell growth regulation (5Scotto C. Delphin C. Deloulme J.C. Baudier J. Mol. Cell. Biol. 1999; 19: 7168-7180Crossref PubMed Scopus (57) Google Scholar), cell structure (6Selinfreund R.H. Barger S.W. Welsh M.J. Van Eldik L.J. J. Cell Biol. 1990; 111: 2021-2028Crossref PubMed Scopus (111) Google Scholar), and calcium homeostasis (7Xiong Z. O'Hanlon D. Becker L.E. Roder J. MacDonald J.F. Marks A. Exp. Cell Res. 2000; 257: 281-289Crossref PubMed Scopus (100) Google Scholar). The S100B protein is also secreted from astrocytes and has extracellular functions (8Donato R. Int. J. Biochem. Cell Biol. 2001; 33: 637-668Crossref PubMed Scopus (1328) Google Scholar). Extracellular S100B acts as a modulator of neuronal synaptic plasticity (9Nishiyama H. Knopfel T. Endo S. Itohara S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4037-4042Crossref PubMed Scopus (226) Google Scholar). Although nanomolar quantities have beneficial neurotrophic effects on nerve cells, high levels of this protein have been implicated in glia activation and could contribute to the development of brain pathology as observed in Down's syndrome and Alzheimer's disease (10Griffin W.S. Stanley L.C. Ling C. White L. MacLeod V. Perrot L.J. White C.L. Araoz C. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7611-7615Crossref PubMed Scopus (1652) Google Scholar). The recent observation that S100B triggers activation of the pro-inflammatory cell surface receptor receptor for advanced glycation end products has shed more light on its extracellular function (11Hofmann M.A. Drury S. Fu C. Qu W. Taguchi A. Lu Y. Avila C. Kambham N. Bierhaus A. Nawroth P. Neurath M.F. Slattery T. Beach D. McClary J. Nagashima M. Morser J. Stern D. Schmidt A.M. Cell. 1999; 97: 889-901Abstract Full Text Full Text PDF PubMed Scopus (1609) Google Scholar). In cultured human astrocytoma U87 cells, S100B secretion is dependent on relocation of S100B toward vesicle-like structures at the periphery of the cells and is regulated by Ca2+ and Zn2+ (12Davey G.E. Murmann P. Heizmann C.W. J. Biol. Chem. 2001; 276: 30819-30826Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). S100B can also be secreted into the bloodstream and cerebrospinal fluid and is a biochemical marker of brain damage or dysfunction in acute and chronic diseases (13Peskind E.R. Griffin W.S. Akama K.T. Raskind M.A. Van Eldik L.J. Neurochem. Int. 2001; 39: 409-413Crossref PubMed Scopus (165) Google Scholar, 14Portela L.V. Brenol J.C. Walz R. Bianchin M. Tort A.B. Canabarro U.P. Beheregaray S. Marasca J.A. Xavier R.M. Neto E.C. Goncalves C.A. Souza D.O. Clin. Diagn. Lab. Immunol. 2002; 9: 164-166PubMed Google Scholar). A relationship between S100B accumulation in the astrocytic end-feet and morphological changes of astrocytes in the perivascular regions has been reported previously (15Abdul-Khaliq H. Schubert S. Stoltenburg-Didinger G. Troitzsch D. Bottcher W. Hubler M. Meissler M. Grosse-Siestrop C. Alexi-Meskishvili V. Hetzer R. Lange P.E. Clin. Chem. Lab. Med. 2000; 38: 1169-1172PubMed Google Scholar). These changes may be related to the release of S100B into the blood stream (15Abdul-Khaliq H. Schubert S. Stoltenburg-Didinger G. Troitzsch D. Bottcher W. Hubler M. Meissler M. Grosse-Siestrop C. Alexi-Meskishvili V. Hetzer R. Lange P.E. Clin. Chem. Lab. Med. 2000; 38: 1169-1172PubMed Google Scholar). Consistent with dynamic regulation of astrocyte cell shape by S100B, antisense inhibition of S100B production in cultured rat glial C6 cells is correlated with alterations in cellular morphology (6Selinfreund R.H. Barger S.W. Welsh M.J. Van Eldik L.J. J. Cell Biol. 1990; 111: 2021-2028Crossref PubMed Scopus (111) Google Scholar). The mechanisms of regulation of astrocyte cell morphology by S100B and its secretion pathway remain unclear. By analogy with other EF-hand Ca2+-binding proteins, such as calmodulin, one might suppose that the biological activity of S100B is related to Ca2+/Zn2+-dependent interaction with target proteins. In this study, we identify IQGAP1 protein as the first S100B target protein identified to date whose interaction with S100B is regulated by Zn2+ and Ca2+. IQGAP1 is also the major specific cytoplasmic S100B target protein present in both rat glial C6 and human U373 or U87 astrocytoma cell lines. We also provide evidence that cytoplasmic S100B specifically binds to a sub-population of IQGAP1 molecules that localize at the polarized leading edge and areas of membrane ruffling and that both S100B and IQGAP1 proteins are relocated in a Ca2+-dependent manner within vesicle-like structures. The interaction of S100B with IQGAP1 may have important implications for understanding the roles played by S100B in processes of dynamic rearrangement of cell membranes and in the mechanisms of Zn2+/Ca2+-dependent relocation and secretion of S100B. Human astrocytoma U-373MG, U-87MG cells, rat glioma C6 cells, mammary carcinoma MCF7 cells, and NIH 3T3 fibroblasts were cultured in Dulbecco's modified Eagle's medium with Glutamax (Invitrogen) supplemented with 10% fetal calf serum (Invitrogen). Cells were labeled in methionine-free minimal essential medium, 5% fetal calf serum supplemented with 35SMet/Cys mix (50 μCi/ml) for 6 h. U-373MG, U-87MG, NIH 3T3, and MCF7 cells were transfected with the pcDNA-Neo containing the wild-type or C-terminal deleted S100B cDNA (17Deloulme J.C. Assard N. Ouengue Mbele G. Mangin C. Kuwano R. Baudier J. J. Biol. Chem. 2000; 275: 35302-35310Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar) using FuGENETM 6 reagent transfection according to manufacturer's protocol. For stably transfected S100B-MCF7 cell lines, cells were incubated, 48 h after transfection, in complete medium supplemented with 500 μg ml 1The abbreviations used are: CaM, calmodulin; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; MS/MS, tandem mass spectrometry; GTPγS, guanosine 5′-3-O-(thio)triphosphate; GFP, green fluorescent protein neomycin (G418), and neomycin-resistant S100B-MCF7 clones were selected. S100B and CaM1 were purified from bovine brain to homogeneity (16Baudier J. Mochly-Rosen D. Newton A. Lee S.H. Koshland Jr., D.E. Cole R.D. Biochemistry. 1987; 26: 2886-2893Crossref PubMed Scopus (123) Google Scholar). S100B- and CaM-Sepharose were prepared by reaction of bovine brain S100B and CaM with CnBr-Sepharose in 20 mm HEPES, pH 7.8, 0.5 mmCaCl2. Both S100B- and CaM-Sepharose contained 2 mg of protein per milliliter of beads. The purification protocols for human S100A1, S100A6, S100B, and S100A11 recombinant proteins have been described previously (17Deloulme J.C. Assard N. Ouengue Mbele G. Mangin C. Kuwano R. Baudier J. J. Biol. Chem. 2000; 275: 35302-35310Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Recombinant human S100B, S100A1, S100A6, and S100A11 were coupled to CnBr-Sepharose (1 mg of protein per milliliter of beads) as described above. Monoclonal anti-β-tubulin antibody was a gift from Drs L. Paturle and D. Job (Laboratoire du cytosquelette, CEN-Grenoble). Polyclonal rabbit anti-S100B antibodies (Z0311 and A5110) were from Dako. Purified S100B monoclonal antibody S16 was previously described (18Takahashi M. Chamczuk A. Hong Y. Jackowski G. Clin. Chem. 1999; 45: 1307-1311Crossref PubMed Scopus (17) Google Scholar). Monoclonal anti-calmodulin (C-7055) and monoclonal anti-S100A6 antibody (S5046) were from Sigma. The monoclonal mouse anti-calmodulin (05-173) and the monoclonal mouse anti-IQGAP1 AF4 (05-504) antibodies were from Upstate Biotechnology. IQGAP1 AF4 antibody was used for immunoprecipitation experiments. The mouse monoclonal anti-IQGAP1 (mAb IgG1, I53820) antibody was from Transduction Laboratories and used at 1:2000 in Western blot analysis. Polyclonal rabbit anti-IQGAP1 antibody was a gift from Dr. J. Ericksson (Cornell University, Ithaca, NY) and was used at 1:2000 for immunofluorescence. Mouse monoclonal anti β-catenin antibody was from Transduction Laboratories (C19220). Polyclonal rabbit anti-Cdc42 antibody was from Santa Cruz Biotechnology (SC-87). MyoD monoclonal antibody 5.8A was from H. Weintraub (Seattle, WA). S100B protein was resolved with SDS-Tris-Tricine-11%-PAGE (17Deloulme J.C. Assard N. Ouengue Mbele G. Mangin C. Kuwano R. Baudier J. J. Biol. Chem. 2000; 275: 35302-35310Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). β-tubulin, β-catenin, and IQGAP1 were analyzed using Laemmli SDS-PAGE. The proteins were transferred to nitrocellulose membrane and incubated with the primary antibodies. Proteins were visualized using an ECL kit (PerkinElmer Life Sciences). The following constructs were used for in vitro translation using the TnT T7 Quick system (Promega): (i) pCAN-myc-IQGAP1 containing a cDNA encoding IQGAP1 was a gift from J. Erickson (Cornell University, Ithaca, NY); (ii) the pcDNA vector containing a cDNA coding for the IQGAP1-N terminus (amino acids 1–863) was generated by digesting pCAN-myc-IQGAP1 with BamHI and subcloning theBamHI-BamHI fragment into a BamHI site in pcDNA3.1(+) (Invitrogen); (iii) the pcDNA containing a cDNA coding for the IQGAP1-IQ domains (amino acids 740–869) was generated by digesting the pcDNA/N-ter with StuI andBamHI and subcloning the StuI/BamHI fragment into the EcoRV/BamHI sites in pcDNA3.1(−) (Invitrogen); (iv) the pcDNA/CHD-IQ was obtained by digesting pcDNA/N-ter with StuI and by religating the vector; (v) the pcDNA/IR-IQ (amino acids 232–740) was obtained by digesting pcDNA/N-ter with StuI and BamHI. Then the StuI/StuI andStuI/BamHI fragments were subcloned intoEcoRV/BamHI sites in pcDNA3.1(−). The 170-kDa protein that binds to S100B-Sepharose in the absence of calcium was excised from Coomassie Blue-stained gels and washed with 50% acetonitrile. Gel pieces were dried in a vacuum centrifuge and re-hydrated in 20 μl of 25 mmNH4HCO3 containing 0.5 μg of trypsin (Promega, sequencing grade). After 4-h incubation at 37 °C, a 0.5-μl aliquot was removed for MALDI-TOF analysis and spotted onto the MALDI sample probe on top of a dried 0.5-μl mixture of 4:3 saturated α-cyano-4-hydroxy-trans-cinnamic acid in acetone/10 mg/ml nitrocellulose in acetone/isopropanol 1:1. Samples were rinsed by placing a 5-μl volume of 0.1% trifluoroacetic acid on the matrix surface after the analyte solution had dried completely. After 2 min, the liquid was blown off by pressurized air. MALDI mass spectra of peptide mixtures were obtained using a Bruker Biflex mass spectrometer (Bruker-Franzen Analityk, Bremen, Germany). Internal calibration was applied to each spectrum using trypsin autodigestion peptides (MH+ 842.50, MH+ 1045.55, MH+ 2211.11). Protein identification was confirmed by tandem mass spectrometry experiments. After in-gel tryptic digestion, the gel pieces were extracted with 5% formic acid solution and then with acetonitrile. The extracts were combined with the original digest, and the sample was evaporated to dryness in a vacuum centrifuge. The residues were dissolved in 0.1% formic acid and desalted using a Zip Tip (Millipore). Elution of the peptides was performed with 5–10 μl of 50% acetonitrile/0.1% formic acid solution. The peptide solution was introduced into a glass capillary (Protana) for nanoelectrospray ionization. Tandem mass spectrometry experiments were carried out on a quadruple time-of-flight hybrid mass spectrometer (Micromass, Altrincham, UK) to obtain sequence information. Collision-induced dissociation of selected precursor ions was performed using argon as the collision gas and collision energies of 40–60 eV. Protein identification was achieved using both MALDI peptide mass fingerprints and MS/MS sequence information. Mass spectrometric data were compared with known sequences using the programs MS-Fit and MS-Edman located at the University of California San Francisco (available at prospector.ucsf.edu/). Tandem mass spectrometry sequencing of three different peptides (LGLAPQIQDLYGK, LEGVLAEVAQHYQDTLIR, and FPDAGEDELLK) confirmed the strict identity of the 170-kDa protein with IQGAP1. These peptides are not found in other human protein sequences, including IQGAP2 and the putative IQGAP3 sequence. For binding assays and co-immunoprecipitation experiments, cells were lysed at 4 °C in TTBS buffer (40 mm Tris-HCl, pH 7.5, 150 mm NaCl, 0.3% Triton X-100) plus protease inhibitors (leupeptin, aprotinin, pepstatin, and 4-(2-aminoethyl)benzenesulfonylfluoride hydrochloride, 10 μg/ml each) and centrifuged for 10 min. Cell lysates were precleared by incubation for 10 min with 50 μl of protein A-Sepharose. 500-μl aliquots of precleared supernatant were supplemented with either 5 mmEDTA/5 mm EGTA, or with 20 μmZnSO4, or with 0.3 mm CaCl2/10 μm ZnSO4 and mixed with 20 μl of affinity beads equilibrated in the same buffers. After mixing at 4 °C for 15 min, the beads were spun down and the supernatant was removed. The beads were washed three times with 1 ml of binding buffers. At the last wash, beads were transferred to new Eppendorf tubes and boiled in SDS-sample buffer. For binding assays using recombinant IQGAP1 or IQGAP1 domains, 20 μl of reaction lysates were diluted in 500 μl of TTBS and processed as described for cell extracts. 500-μl aliquots of pre-cleared supernatant were mixed with 40 μm GTPγS (Sigma) plus 5 mm MgCl2, if needed, and with either 5 mm EDTA/5 mm EGTA or 0.3 mm CaCl2/10 μm ZnSO4and mixed with the appropriate antibodies (5 μg) plus 20 μl of protein A- or protein G-Sepharose equilibrated in the same buffers. After mixing at 4 °C for 50 min, the beads were centrifuged briefly (5 s, 13,000 rpm), and the supernatant was removed. The beads were washed three times with 1 ml of appropriate incubation buffers. At the last wash, beads were transferred to new microcentrifuge tubes and boiled in SDS-sample buffer containing 5 mm EGTA and 5 mm EDTA. cells grown on permanox slides from Nunc, Inc. were fixed for 30 min with 4% paraformaldehyde in HBS (10 mm HEPES, pH 7.4, 130 mm NaCl, 15 mm KCl, 5 mm MgCl2) and permeabilized for 5 min with TBS (30 mm Tris-HCl, pH 7.4, 150 mm NaCl) containing 0.2% Triton X-100 plus 1 mm CaCl2. After washing with TBS/1 mm CaCl2, cells were incubated for 30 min in TBS/1 mm CaCl2 containing 10% normal goat serum and then incubated with primary antibodies for 90 min in the same buffer containing 5% normal goat serum. The cells were then washed with TBS/1 mm CaCl2 and incubated for 1 h with the appropriate secondary antibodies conjugated with cyanin3 (Jackson ImmunoResearch Laboratories) or with Alexa488 (Molecular Probes, Inc., Eugene, OR) in the same buffer as described for the primary antibodies. After washing with TBS/1 mmCaCl2, cells were incubated in a solution of Hoechst 33258 (2 μg/ml) for 5 min and placed under coverslips. Preparations were analyzed with a Zeiss fluorescence microscope (Axiovert 200M) or a Leica confocal microscope (TCS-SP2). In the transformed human astrocytoma U373 cell line, the expression of S100B is up-regulated in post-confluent cells (Fig.1). Up-regulation of S100B is specific, because it is not observed with β-tubulin, calmodulin (CaM), and IQGAP1. Indirect immunofluorescence analysis of post-confluent U373 cells, double-stained with polyclonal rabbit anti-human S100B (red) and mouse monoclonal anti-S100A6 (green), reveals heterogeneity in S100B staining among cells (Fig.2 A). Although all cells are uniformly immunostained with S100A6 antibodies (Fig. 2 B), considerable variation in S100B immunostaining characterized post-confluent U373 cells. In post-confluent culture, the strongest S100B-positive cells had grown on top of the cell layer. These cells are characterized by intense cytoplasmic S100B immunoreactivity and have adopted a less flattened morphology with long processes. Confocal microscopy analysis of S100B immunostaining in confluent and post-confluent U373 cells confirmed a relationship between S100B overexpression and change in cell shape (Fig. 2, C andD). In these experiments, cells were double-labeled with S100B polyclonal antibodies (red) and IQGAP1 monoclonal antibody (green). U373 cells that enter confluence have a flattened morphology. In these cells, the weak S100B immunoreactivity is mostly nuclear and IQGAP1 accumulates at the cell periphery (Fig.2 C). In post-confluent culture, cells characterized by intense cytoplasmic S100B immunoreactivity have adopted a less flattened morphology with long processes (Fig. 2 D). Overlapping of the S100B and IQGAP stainings (white pixels) reveals that some of the S100B colocalizes with IQGAP1 at the cytoplasmic membrane and within processes. The correlation between cytoplasmic S100B overexpression with changes in cell shape is consistent with previous studies that showed that selective inhibition of S100B production by antisense strategies in rat glioma C6 cells resulted in a more flattened cellular morphology (6Selinfreund R.H. Barger S.W. Welsh M.J. Van Eldik L.J. J. Cell Biol. 1990; 111: 2021-2028Crossref PubMed Scopus (111) Google Scholar). In rat C6 glioma cells, S100B is also up-regulated in post-confluent cells, and its up-regulation correlates with drastic cell morphological changes (data not shown). In an attempt to identify specific S100B target proteins that could mediate the effect of S100B on cell morphology, we compared proteins in astrocytoma U373 and glial C6 cell extracts that bind to S100B-Sepharose beads. A major S100B-binding protein that migrated with an apparent molecular mass of 170 kDa was identified in both cell lines (Fig.3 A). The 170-kDa protein binds to S100B-Sepharose beads in EGTA/EDTA- and Ca2+/Zn2+-containing buffer. The 170-kDa human protein from U373 MG cells was further characterized by mass spectrometry. Protein identification was achieved using both MALDI peptide mass fingerprints and MS/MS sequence information (see “Materials and Methods”). Results revealed that it corresponds to human IQGAP1. IQGAP1 is a widely expressed protein that acts as a scaffold in recruiting and maintaining the organization of cytoskeletal proteins at the plasma membrane (19Bashour A.M. Fullerton A.T. Hart M.J. Bloom G.S. J. Cell Biol. 1997; 137: 1555-1566Crossref PubMed Scopus (212) Google Scholar, 20Erickson J.W. Cerione R.A. Hart M.J. J. Biol. Chem. 1997; 272: 24443-24447Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 21Fukata M. Kuroda S. Fujii K. Nakamura T. Shoji I. Matsuura Y. Okawa K. Iwamatsu A. Kikuchi A. Kaibuchi K. J. Biol. Chem. 1997; 272: 29579-29583Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 22Fukata M. Kuroda S. Nakagawa M. Kawajiri A. Itoh N. Shoji I. Matsuura Y. Yonehara S. Fujisawa H. Kikuchi A. Kaibuchi K. J. Biol. Chem. 1999; 274: 26044-26050Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 23Fukata M. Nakagawa M. Itoh N. Kawajiri A. Yamaga M. Kuroda S. Kaibuchi K. Mol. Cell. Biol. 2001; 21: 2165-2183Crossref PubMed Scopus (78) Google Scholar, 24Kuroda S. Fukata M. Nakagawa M. Fujii K. Nakamura T. Ookubo T. Izawa I. Nagase T. Nomura N. Tani H. Shoji I. Matsuura Y. Yonehara S. Kaibuchi K. Science. 1998; 281: 832-835Crossref PubMed Scopus (428) Google Scholar, 25Li Z. Kim S.H. Higgins J.M. Brenner M.B. Sacks D.B. J. Biol. Chem. 1999; 274: 37885-37892Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 26Mateer S.C. McDaniel A.E. Nicolas V. Habermacher G.M. Lin M.J. Cromer D.A. King M.E. Bloom G.S. J. Biol. Chem. 2002; 277: 12324-12333Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 27Fukata M. Watanabe T. Noritake J. Nakagawa M. Yamaga M. Kuroda S. Matsuura Y. Iwamatsu A. Perez F. Kaibuchi K. Cell. 2002; 109: 873-885Abstract Full Text Full Text PDF PubMed Scopus (491) Google Scholar). The other high molecular weight Ca2+/Zn2+-dependent S100B-binding protein present in glial C6 cells, but not in U373 cell extract, has been previously identified as AHNAK (28Gentil B. Delphin C. Ouengue Mbele G. Deloulme J.C. Ferro M. Garin J. Baudier J. J. Biol. Chem. 2001; 276: 23253-23261Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). The binding of IQGAP1 to S100B is specific, because it is not observed with S100A6 (Fig. 3 B, lanes 5 and 6), and S100A11 (Fig. 3 C, lanes 3 and 7), two other S100 species expressed in U373 cells (17Deloulme J.C. Assard N. Ouengue Mbele G. Mangin C. Kuwano R. Baudier J. J. Biol. Chem. 2000; 275: 35302-35310Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). S100A1, the closest S100B homologue that is not expressed in U373 cells (17Deloulme J.C. Assard N. Ouengue Mbele G. Mangin C. Kuwano R. Baudier J. J. Biol. Chem. 2000; 275: 35302-35310Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar), also binds IQGAP1 (Fig. 3 B, lanes 3 and 4). IQGAP1 in U373 cell extract also binds to calmodulin-Sepharose beads (Fig.3 C, lanes 4 and 8). A physical interaction between S100B with IQGAP1 was confirmed by co-immunoprecipitation of a S100B/IQGAP1 complex from confluent U373 cell extract (Fig. 4). In a first set of experiments, S100B was immunoprecipitated with S16 monoclonal S100B antibody that recognizes an epitope located within the N terminus of S100B (18Takahashi M. Chamczuk A. Hong Y. Jackowski G. Clin. Chem. 1999; 45: 1307-1311Crossref PubMed Scopus (17) Google Scholar). The presence of IQGAP1 in the S100B immunoprecipitate was revealed with anti-IQGAP1 polyclonal antibodies. A small but detectable amount of IQGAP1 is found in the S100B immunoprecipitates in EDTA/EGTA buffer (Fig. 4 A, lane 4). The amount of IQGAP1 immunoprecipitated with S100B monoclonal antibody increased substantially in buffer containing Ca2+/Zn2+(Fig. 4 A, lane 5). The co-immunoprecipitation of IQGAP1 with S100B is specific, because it is not observed with control anti-MyoD antibodies (Fig.4 A, lanes 2 and 3). The Ca2+/Zn2+ requirement for the interaction between soluble S100B and IQGAP1 contrasts with the apparent divalent ion-independent interaction observed with S100B cross-linked to Sepharose beads (see below). In a second set of experiments, IQGAP1 was immunoprecipitated with anti-IQGAP1 AF4 monoclonal antibody (Fig. 4 B). S100B is found associated with IQGAP1 immunoprecipitates when using Ca2+/Zn2+-containing buffer. Cdc42 and β-catenin, two other IQGAP1 target proteins (22Fukata M. Kuroda S. Nakagawa M. Kawajiri A. Itoh N. Shoji I. Matsuura Y. Yonehara S. Fujisawa H. Kikuchi A. Kaibuchi K. J. Biol. Chem. 1999; 274: 26044-26050Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 23Fukata M. Nakagawa M. Itoh N. Kawajiri A. Yamaga M. Kuroda S. Kaibuchi K. Mol. Cell. Biol. 2001; 21: 2165-2183Crossref PubMed Scopus (78) Google Scholar, 24Kuroda S. Fukata M. Nakagawa M. Fujii K. Nakamura T. Ookubo T. Izawa I. Nagase T. Nomura N. Tani H. Shoji I. Matsuura Y. Yonehara S. Kaibuchi K. Science. 1998; 281: 832-835Crossref PubMed Scopus (428) Google Scholar) are also found associated with IQGAP1 immunoprecipitates in both EGTA/EDTA and Ca2+/Zn2+ buffer. Several laboratories have also shown intracellular interactions between CaM and IQGAP1 (19Bashour A.M. Fullerton A.T. Hart M.J. Bloom G.S. J. Cell Biol. 1997; 137: 1555-1566Crossref PubMed Scopus (212) Google Scholar, 20Erickson J.W. Cerione R.A. Hart M.J. J. Biol. Chem. 1997; 272: 24443-24447Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar,26Mateer S.C. McDaniel A.E. Nicolas V. Habermacher G.M. Lin M.J. Cromer D.A. King M.E. Bloom G.S. J. Biol. Chem. 2002; 277: 12324-12333Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 29Ho Y.D. Joyal J.L. Li Z. Sacks D.B. J. Biol. Chem. 1999; 274: 464-470Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). In Fig. 4 C, we compared the association of calmodulin (CaM) and S100B with immunoprecipitate IQGAP1 out of exponentially growing and post-confluent U373 cells in EGTA/EDTA- and Ca2+/Zn2+-containing buffer. CaM and S100B that co-immunoprecipitated with IQGAP1 were sequentially revealed using the same nitrocellulose transfer membrane. CaM co-immunoprecipitates with IQGAP1 in all conditions tested, whereas S100B only co-immunoprecipitates with IQGAP1 from post-confluent cell extracts in buffer containing Ca2+/Zn2+(compare lanes 2 and 3). It is noteworthy that, although other laboratories reported that Ca2+ enhances the interaction between CaM and IQGAP1 (26Mateer S.C. McDaniel A.E. Nicolas V. Habermacher G.M. Lin M.J. Cromer D.A. King M.E. Bloom G.S. J. Biol. Chem. 2002; 277: 12324-12333Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 29Ho Y.D. Joyal J.L. Li Z. Sacks D.B. J. Biol. Chem. 1999; 274: 464-470Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 30Joyal J.L. Annan R.S. Ho Y.D. Huddleston M.E. Carr S.A. Hart M.J. Sacks D.B. J. Biol. Chem. 1997; 272: 15419-15425Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar), we found more CaM immunoreactivity associated with IQGAP1 in U373 cell extracts containing EGTA and EDTA (compare lanes 2 and 3 or lanes 5 and6). This unexpected observation cannot solely be explained by a competition with S100B, because it is also observed with sub-confluent culture characterized by low S100B expression. In a pull-down assay using S100B cross-linked onto Sepharose beads, the S100B/IQGAP1 interaction can be detected independently of the presence of EGTA/EDTA or Ca2+/Zn2+ in binding buffer (Fig. 3). In contrast, co-immunoprecipitation experiments with endogenous cellular proteins revealed that IQGAP1/S100B interaction is markedly strengthened when Ca2+ and Zn2+ are included in binding buffer (Fig. 4). One factor that might explain this apparent discrepancy is the high S100B protein concentration used in the pull-down assay (1.5–3 μm) compared with the soluble S100B in cell extracts. To evaluate the effect of S100B concentration on complex formation with IQGAP1, we performed co-immunoprecipitation analysis using MCF7 cells extracts (which do not express the S100B protein) supplemented with increasing concentrations of recombinant human S100B (Fig. 5 A). Results confirm that the S100B/IQGAP1 interaction is regulated by divalent ions and that high concentrations of S100B are not sufficient to promote ion-independent interactions. Previous studies from our laboratory have shown that chemical modifications within the S100B molecule may have profound effects on the protein quaternary and tertiary structures (2Baudier J. Glasser N. Gérard D. J. Biol. Chem." @default.
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