Cell Cycle-dependent Interaction of Mad2 with Conserved Box1/2 Region of Human Granulocyte-Macrophage Colony-stimulating Factor Receptor Common βc
2001; Elsevier BV; Volume: 276; Issue: 45 Linguagem: Inglês
10.1074/jbc.m101488200
ISSN1083-351X
AutoresMitsuo Takeda, Naoshi Dohmae, Koji Takio, Ken‐ichi Arai, Sumiko Watanabe,
Tópico(s)Immune Cell Function and Interaction
ResumoBox1 and 2 (box1/2) are conserved cytoplasmic motifs located in the membrane proximal region of cytokine receptors, including the human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor common βc. Deletion of box1/2 abrogated all the examined activities of GM-CSF, and this phenomenon is explained by the loss of binding by Jak2. To test if a molecule other than Jak2 interacting with the box1/2 region plays a role in GM-CSF receptor signal transduction, we screened for molecules interacting with the box1/2 region by a pull-down assay using recombinant purified protein of GST fused with the βc box1/2 region and a Ba/F3 cell lysate. The mouse homologue of Mad2 protein, which plays an important role in the M phase of the cell cycle, was revealed to associate with the box1/2 region specifically. Peptides corresponding to the box1 sequence also bound to Mad2, and mutation of the box1 decreased the Mad2 interaction. Deletion analysis indicated that interaction with box1/2 occurred through the C-terminal portion of Mad2. Mad2 is known to change affinity for binding partners cell cycle dependently. Binding affinity of Mad2 to box1/2 increased in the late M phase, suggesting the possibility that GM-CSF participates in regulation of the M phase check point through interaction with Mad2. Box1 and 2 (box1/2) are conserved cytoplasmic motifs located in the membrane proximal region of cytokine receptors, including the human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor common βc. Deletion of box1/2 abrogated all the examined activities of GM-CSF, and this phenomenon is explained by the loss of binding by Jak2. To test if a molecule other than Jak2 interacting with the box1/2 region plays a role in GM-CSF receptor signal transduction, we screened for molecules interacting with the box1/2 region by a pull-down assay using recombinant purified protein of GST fused with the βc box1/2 region and a Ba/F3 cell lysate. The mouse homologue of Mad2 protein, which plays an important role in the M phase of the cell cycle, was revealed to associate with the box1/2 region specifically. Peptides corresponding to the box1 sequence also bound to Mad2, and mutation of the box1 decreased the Mad2 interaction. Deletion analysis indicated that interaction with box1/2 occurred through the C-terminal portion of Mad2. Mad2 is known to change affinity for binding partners cell cycle dependently. Binding affinity of Mad2 to box1/2 increased in the late M phase, suggesting the possibility that GM-CSF participates in regulation of the M phase check point through interaction with Mad2. granulocyte-macrophage colony-stimulating factor human granulocyte-macrophage colony-stimulating factor granulocyte-macrophage colony-stimulating factor receptor interleukin anaphase promoting complex glutathione S-transferase polymerase chain reaction polyacrylamide gel electrophoresis tetratrico peptide repeat Hop1p, Rev7p, andMad2 Granulocyte-macrophage colony-stimulating factor (GM-CSF)1 is a cytokine that stimulates the proliferation and differentiation as well as survival of various hematopoietic cells (1Arai K. Lee F. Miyajima A. Miyatake S. Arai N. Yokota T. Annu. Rev. Biochem. 1990; 59: 783-836Crossref PubMed Scopus (1176) Google Scholar). The receptor of human (h) GM-CSF (GM-CSFR) consists of two subunits, α and β, both of which are members of the cytokine receptor superfamily (2Miyajima A. Mui A.L.-F. Ogorochi T. Sakamaki K. Blood. 1993; 82: 1960-1974Crossref PubMed Google Scholar). The α subunit is specific to hGM-CSFR, whereas the β subunit (βc) is shared by IL-3, GM-CSF, and IL-5 receptors (2Miyajima A. Mui A.L.-F. Ogorochi T. Sakamaki K. Blood. 1993; 82: 1960-1974Crossref PubMed Google Scholar). GM-CSF induces tyrosine phosphorylation of βc and various cellular proteins and activates early response genes such as c-fos, c-jun, and c-myc, as well as stimulates cell proliferation in hematopoietic cells and fibroblasts (3Watanabe S. Mui A.L.-F. Muto A. Chen J.X. Hayashida K. Miyajima A. Arai K. Mol. Cell. Biol. 1993; 13: 1440-1448Crossref PubMed Scopus (74) Google Scholar). GM-CSF activates various signaling molecules, including Jak2, STAT5, mitogen-activated protein kinase cascade kinases, and phosphatidylinositol 3-kinase (2Miyajima A. Mui A.L.-F. Ogorochi T. Sakamaki K. Blood. 1993; 82: 1960-1974Crossref PubMed Google Scholar). βc contains box1 and box2 regions (box1/2), which are conserved among the cytokine receptor superfamily, and 8 tyrosine residues located in its cytoplasmic region (4Hayashida K. Kitamura T. Gorman D.M. Arai K. Yokota T. Miyajima A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9655-9659Crossref PubMed Scopus (520) Google Scholar, 5Murakami M. Hibi M. Nakagawa N. Nakagawa T. Yasukawa K. Yamanishi K. Taga T. Kishimoto T. Science. 1993; 260: 1808-1810Crossref PubMed Scopus (645) Google Scholar). To better comprehend signaling events involved in cell proliferation, we and others analyzed biological activities of various mutants of βc (6Sakamaki K. Miyajima I. Kitamura T. Miyajima A. EMBO J. 1992; 11: 3541-3550Crossref PubMed Scopus (295) Google Scholar, 7Watanabe S. Itoh T. Arai K. J. Biol. Chem. 1996; 271: 12681-12686Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 8Okuda K. Griffin J. Blood. 1997; 90: 4759-4760Crossref PubMed Google Scholar) and found that the box1 region was essential for all of the hGM-CSFR signals we examined. Although the box1/2 region is essential and sufficient for cell proliferation and survival, a tyrosine residue(s) is also required for mitogen-activated protein kinase and c-fos promoter activation (8Okuda K. Griffin J. Blood. 1997; 90: 4759-4760Crossref PubMed Google Scholar, 9Itoh T. Liu R. Yokota T. Arai K. Watanabe S. Mol. Cell. Biol. 1998; 18: 742-752Crossref PubMed Google Scholar). Because box1 is assumed to bind Jak2, it is likely that activation of Jak2 is sufficient and essential for hGM-CSF signals through interaction with the box1 region of βc. This notion was supported by observations that dominant negative Jak2 suppressed all the GM-CSFR signaling and activities we investigated (7Watanabe S. Itoh T. Arai K. J. Biol. Chem. 1996; 271: 12681-12686Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). In addition, Jak2 knockout mice showed defects in GM-CSF-dependent colony formation (10Neubauer H. Cumano A. Muller M. Wu H. Huffstadt U. Pfeffer K. Cell. 1998; 93: 397-409Abstract Full Text Full Text PDF PubMed Scopus (680) Google Scholar, 11Parganas E. Wang D. Stravopodis D. Topham D.J. Marine J.-C. Teglund S. Vanin E.F. Bodner S. Colamonici O.R. van Deursen J.M. Grosveld G. Ihle J.N. Cell. 1998; 93: 385-395Abstract Full Text Full Text PDF PubMed Scopus (911) Google Scholar). The box1 region contains the proline-X-proline sequence, which is conserved among members of the cytokine receptor superfamily (12Murakami M. Narazali M. Hibi M. Yawata H. Yasukawa K. Hamaguchi M. Taga T. Kishimoto T. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 11349-11353Crossref PubMed Scopus (491) Google Scholar). Mutation analysis of the motif suggested a crucial role for the motif in a variety of receptor-mediated signalings. The G-CSF receptor mutant in which the conserved proline-X-proline is substituted by Ala-X-Ala can induce neither DNA synthesis nor cell proliferation (13Avalos B.R. Hunter M.G. Parker J.M. Ceselski S.K. Druker B.J. Corey S.J. Mehta V.B. Blood. 1995; 85: 3117-3126Crossref PubMed Google Scholar). The mutant growth hormone receptor failed to induce either cell proliferation or spi-1 induction (14Dinerstein H. Lago F. Goujon L. Ferrag F. Esposito N. Finidori J. Kelly P.A. Postel-Vinay M.C. Mol. Endocrinol. 1995; 9: 1701-1707Crossref PubMed Google Scholar, 15Wang Y.D. Wood W.I. Mol. Endocrinol. 1995; 9: 303-311Crossref PubMed Google Scholar). Similar results were obtained in the case of gp130 and the prolactin receptor (12Murakami M. Narazali M. Hibi M. Yawata H. Yasukawa K. Hamaguchi M. Taga T. Kishimoto T. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 11349-11353Crossref PubMed Scopus (491) Google Scholar, 16O'Neal K.D. Shearer W.T. Mol. Cell. Biol. 1995; 15: 4657-4658Crossref PubMed Scopus (4) Google Scholar, 17O'Neal K.D., Yu- Lee L.Y. Shearer W.T. Ann. N. Y. Acad. Sci. 1995; 766: 282-284Crossref PubMed Scopus (6) Google Scholar). Although the importance of the box1 region in various receptors has been noted, structural information about this region has been awaited. Thus, the role and mechanism of box1 function have remained unsolved. We have now identified Mad2 (mitotic arrest-deficient 2) protein as a box1/2-binding protein. Mad2 was first discovered as a gene responsible for the mitotic checkpoint in yeast (18Li R. Murray A.W. Cell. 1991; 66: 519-531Abstract Full Text PDF PubMed Scopus (935) Google Scholar). The knockout of Mad2 in mice showed that Mad2 is essential in mouse cells after embryonic day 6.5, although it is dispensable for normal cell division in yeast (19Dobles M. Liberal V. Scott M.L. Benezra R. Sorger P.K. Cell. 2000; 101: 635-645Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar). During mitosis, Mad2 localized at an unattached kinetochore monitors for correct spindle-kinetochore attachment, a prerequisite for initiation of anaphase (20Pennisi E. Science. 1998; 279: 477-478Crossref PubMed Scopus (33) Google Scholar). The Mad2-dependent monitoring system, maintained until the completion of spindle attachment, prevents anaphase initiation through Mad2-Cdc20 complex formation (21Fang G., Yu, H. Kirschner M.W. Genes Dev. 1998; 12: 1871-1883Crossref PubMed Scopus (495) Google Scholar, 22Hwang L.H. Lau L.F. Smith D.L. Mistrot C.A. Hardwick K.G. Hwang E.S. Amon A. Murray A.W. Science. 1998; 279: 1041-1044Crossref PubMed Scopus (464) Google Scholar, 23Elledge S.J. Science. 1998; 279: 999-1000Crossref PubMed Scopus (53) Google Scholar). Cdc20 activates the ubiquitin ligase activity of anaphase promoting complex (APC), after which the activated APC promotes the initiation of anaphase. There are a variety of Mad2 binding partners, including molecules not related to the mitotic checkpoint (24Liu Y.C. Pan J. Zhang C. Fan W. Collinge M. Bender J.R. Weissman S.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4313-4318Crossref PubMed Scopus (144) Google Scholar, 25Poelzl G. Kasai Y. Mochizuki N. Shaul P.W. Brown M. Mendelsohn M.E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2836-2839Crossref PubMed Scopus (58) Google Scholar, 26Nelson K.K. Schlondorff J. Blobel C.P. Biochem. J. 1999; 343: 673-680Crossref PubMed Scopus (76) Google Scholar, 27O'Neill T.J. Zhu Y. Gustafson T.A. J. Biol. Chem. 1997; 272: 10035-10040Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Here, we found that hGM-CSFR βc acts as one of such partners of Mad2 and that their interaction is regulated in a cell cycle-dependent manner. Recombinant murine IL-3 (mIL-3) expressed in silkworms, Bombyx mori, was purified as described (28Miyajima A. Schreurs J. Otsu K. Kondo A. Arai K. Maeda S. Gene (Amst.). 1987; 58: 273-281Crossref PubMed Scopus (135) Google Scholar). Recombinant hGM-CSF was a gift from Schering-Plough (Madison, NJ). Antibodies anti-hGM-CSF receptor βc (S-16, C-20) and anti-GST (B-14) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Mad2 was from Transduction Laboratory (Lexington, KY), and anti-phosphotyrosine (4G10) from Upstate Biotechnology Inc (Lake Placid, NY). Peptides were synthesized by using Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry with a Shimadzu PSSM-8 synthesizer (Shimadzu, Kyoto, Japan). All the peptides were designed to have a cysteine at their N terminus for iodoacetamido-based sulfhydryl conjugation. Each peptide was conjugated with SulfoLink coupling gel (Pierce) according to the manufacturer’s instruction. Peptide corresponding to CAGGPPGGPQVNPIPVTDEVV served as a control. The plasmids encoding GST fusion protein were constructed by using pGEX vectors (Amersham Pharmacia Biotech, Uppsala, Sweden). GST-box1/2 was constructed by PCR amplification of the box1/2 region corresponding to amino acids 453–544, and termination was generated after residue 544 (aspartic acid) by PCR mutagenesis. Sequences of primers used for introduction of mutation were 5′-ATGTGATCAGGCTGCGCAGAAAGTG-3′ and 5′-CGGAATTCTAATCTGAGGCAGCTGGAG-3′. Amplified fragments were inserted into BclI and EcoRI sites of pGEX-3X. For GST-α, hGM-CSFR α subunit was digested with CvnI and XhoI. The resulting fragment, which contained almost the entire region of the cytoplasmic region of the α subunit, was inserted into pBS-SK (Stratagene, La Jolla, CA) at SmaI (blunted) and XhoI sites. Then, the fragment was isolated by BamHI and XhoI digestion and inserted into BamHI and XhoI sites of the pGEX-5X-1 vector. His-tagged recombinant proteins were constructed by using the QIAexpressionist type IV pQE vector (Qiagen, Chatsworth, CA) to contain 6 histidines at their N terminus. An XhoI site was created immediately before the initiation codon of Mad2 by PCR mutagenesis, and the entire region of Mad2 was isolated by cleavage at XhoI and HindIII sites. The fragment was inserted into pQE-31 at SalI and HindIII sites. The N terminus deletion mutant was made by insertion of Sau3AI and SacI fragment into pQE-31 BamHI and SacI sites. C terminus deletion was made by deletion of the region after the DpnI site. The DpnI site was blunted and ligated to the blunted SmaI site of the pQE-31 vector. For mammalian expression, the fragment encoding full-length wild-type Mad2 (prepared by XhoI and SpeI sites from pCR2.1 vector; Invitrogen, Carlsbad, CA) was inserted into XhoI and SpeI sites of the pKU2 vector, which contains the SRα promoter (29Takebe Y. Seiki M. Fujisawa J. Hoy P. Yokota K. Arai K. Yoshida M. Arai N. Mol. Cell. Biol. 1988; 8: 466-472Crossref PubMed Google Scholar). Plasmids encoding GST fusion proteins were introduced into BL21 cells (Escherichia coli strain), and GST fusion protein expression was induced by adding 1 mm IPTG. The cells were lysed by sonication, and the overexpressed protein was purified on an affinity column packed with glutathione-Sepharose4B (Amersham Pharmacia Biotech), according to the manufacturer's instruction. The histidine-tagged proteins were expressed in M15 cells (E. coli strain carrying a pREP4 plasmid), and purification of the protein was done by using an Ni2+-nitrilotriacetic acid-agarose (Qiagen, Chatsworth, CA) affinity column, according to the manufacturer's instruction. The eluents were dialyzed against 20 mm Tris-HCl, pH 7.5, and protein concentrations were determined by using a BCA protein assay kit (Pierce). The mouse IL-3-dependent, pro-B cell line Ba/F3 stably expressing both α and β subunits of hGM-CSFR (Ba/F-wild) was maintained in RPMI 1640 containing 5% fetal calf serum, 0.25 ng/ml mIL-3, 50 units/ml penicillin, and 50 μg/ml streptomycin. HeLa cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum, 50 units/ml penicillin, and 50 μg/ml streptomycin. Whole cell lysates used for pull-down assay and immunoprecipitation were prepared by using lysis buffer (40 mm Tris-HCl, pH 7.6, 50 mm KCl, 0.25% Nonidet P-40, 2 mm EDTA, 50 mm NaF, 20 mmβ-glycerophosphate, 1 mm NaVO4, 10 mm 2-mercaptoethanol, 1 mm phenylmethylsulfonyl fluoride, 2 μg/ml aprotinin, 1 μg/ml pepstatin A, 10 μg/ml leupeptin). The undissolved fraction of the lysate was removed by centrifugation (15,000 × g for 30 min). Fractionation of HeLa cell lysates into cytoplasm and nucleus portions was done as follows. Briefly, the cells were incubated in hypotonic buffer (10 mm Hepes, pH 7.9, 10 mm KCl, 2 mmMgCl2, 0.1 mm EDTA, 1 mmdithiothreitol, 0.1 mm phenylmethylsulfonyl fluoride) for 15 min. Nonidet P-40 was added to the cell suspension at a final concentration of 0.6%. The Nonidet P-40-insoluble fraction (enriched in nuclei) was separated by centrifugation (15,000 × g for 30 s). The pellets were washed with hypotonic buffer two times and incubated in extraction buffer (50 mm Hepes, pH 7.9, 50 mm KCl, 300 mm NaCl, 0.1 mm EDTA, 1 mm dithiothreitol, 0.1 mm phenylmethylsulfonyl fluoride, 10% glycerol) for 20 min. Both Nonidet P-40-soluble and -insoluble fractions were diluted with an excess amount of the lysis buffer used in the pull-down assay. The lysate (108 cells/ml) prepared from Ba/F wild cells was incubated overnight at 4 °C with GST fusion proteins and glutathione Sepharose. GST fusion protein and associated protein(s) were eluted with 15 mm glutathione and dialyzed against 20 mm Tris-HCl, pH 7.5. The samples were lyophilized and dissolved in suitable buffer for further analyses. For immunoprecipitation analysis, cell lysates (2 × 107 cells/samples) were incubated with an appropriate antibody and protein G-Sepharose 4 FF (Amersham Pharmacia Biotech) overnight at 4 °C. The bands were visualized by using an ECL Western blotting detection system (Amersham Pharmacia Biotech). For preparation of cell cycle-synchronized cell lysates, HeLa cells were arrested at the G1/S boundary by a double-thymidine block (30Wassmann K. Benezra R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11193-11198Crossref PubMed Scopus (111) Google Scholar). Briefly, HeLa cells were treated for 14 h with Dulbecco’s modified Eagle’s medium containing 2 mmthymidine, released for 10 h in normal medium, and then treated again with thymidine for 14 h. There after, the cells were released in normal medium and harvested after incubation for the times indicated in the figures. For Ba/F3 cells, cells were depleted of mIL-3 for 12 h to become arrested in the G1 phase. hGM-CSF (2 ng/ml) was then added, and the cells were harvested at the indicated time points. The phase of the cell cycle was confirmed by examining DNA contents by propidium iodide staining and flow cytometry (FACScan, Becton Dickinson, San Jose, CA). The lysates from each time point were prepared as described above. Protein amounts of the lysates were quantified with a BCA protein assay kit (Pierce) to confirm equal extraction efficiency among the samples. The lysate from each time point was divided into two tubes, and incubated with anti-βc antibody (2 μg, for Ba/F3 cells), GST-box1/2 purified protein (for HeLa cells), or anti-p55CDC antibody (2 μg). Associated proteins were precipitated either with glutathione-Sepharose beads or protein G beads and separated by SDS-PAGE followed by Western blotting using anti-Mad2 antibody. Two-dimensional electrophoresis, a combination of isoelectric focusing and SDS-PAGE, was carried out according to O'Farrell (31O'Farrell P.H. J. Biol. Chem. 1975; 250: 4007-4021Abstract Full Text PDF PubMed Google Scholar). Protein spots were detected either by silver staining or Western blotting. Silver staining of the PAGE gel was done with a 2D Silver Stain II kit (Daiichi Pure Chemicals Co. Ltd., Tokyo, Japan). For Western blotting, proteins were transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA) by using a semidry type apparatus (model BE-320; Bio-Craft Co. Ltd., Tokyo, Japan) and the blotted membrane was incubated with appropriate antibodies. For amino acid sequencing, two-dimensional separated gels were stained with 0.25% Coomassie Brilliant Blue and bands of 25 and 75 kDa were excised and treated with 0.2 μg of Achromobacter protease I (a gift from Dr. Masaki, Ibaraki University (Ref. 32Masaki T. Tanabe M. Nakamura K. Soejima M. Biochim. Biophys. Acta. 1981; 660: 44-50Crossref PubMed Scopus (157) Google Scholar)) at 37 °C for 12 h in 0.1 m Tris-HCl, pH 9.0, containing 0.1% SDS. The peptides generated were extracted from the gel and separated on columns of DEAE-5PW (2 × 20 mm; Tosoh, Tokyo, Japan) and Mightysil RP-18 (2 × 50 mm; Kanto Chemical, Tokyo, Japan) connected in series with a model 1100 (Hewlett Packard, Palo Alto, CA) liquid chromatography system. Peptides were eluted at a flow rate of 0.1 ml/min, with a linear gradient of 0–60% solvent B, where solvents A and B were 0.09% (v/v) aqueous trifluoroacetic acid and 0.075% (v/v) trifluoroacetic acid in 80% (v/v) acetonitrile, respectively. Selected peptides were subjected to Edman degradation in a model 477A automated protein sequencer (PerkinElmer Life Sciences) connected on-line to a model 120A PTH analyzer (PerkinElmer Life Sciences) and also examined by matrix-assisted laser desorption ionization time of flight mass spectrometry with a Reflex MALDI-TOF (Bruker-Franzen Analytik, Bremen, Germany) in linear mode, with 2-mercaptobenzothiazole used as a matrix. For cloning of full-length mouse Mad2 cDNA, PCR primers were designed according to the GenBank™ mouse Mad2 equivalent sequence of expressed sequence tag. PCR fragments were recovered by used of a TA vector (pCR2.1, Invitrogen, Carlsbad, CA), and the sequence was confirmed by using an ABI PRISM 310 sequencer (PerkinElmer Life Sciences). To determine whether proteins other than Jak2 bind to the box1/2 region, we conducted pull-down assays using the recombinant box1/2 region of βc and Ba/F3 cell lysates. GST protein fused with the βc box1/2 region (GST-box1/2) or the cytoplasmic region of α subunit (GST-α) were constructed, as shown in Fig.1 A. GST-box1/2, GST-α, and GST protein were purified by using a glutathione column and then incubated with the Ba/F3 cell lysate. Proteins were precipitated by glutathione-Sepharose 4B beads and analyzed by two-dimensional electrophoresis. To identify GST protein-binding proteins derived from E. coli, purified GST-box1/2, GST-α fusion proteins, and GST protein were also subjected to two-dimensional electrophoresis, and all the gels were silver-stained. We then compared the six panels precisely and found two proteins that specifically bound to GST-box1/2. Fig. 1 B shows the pattern of GST-box1/2-binding proteins, and arrows indicate specific binding proteins with approximate molecular masses of 25 and 75 kDa. The 25-kDa protein was not detected in samples prepared with GST or GST-α, and only a residual amount of the 75-kDa one co-precipitated with GST or GST-α. Because these proteins were not observed in the gel with GST-box1/2 protein alone, these were probably specific binding proteins that had originated from Ba/F3 cells. We excised these spots and subjected them to microsequencing. The amino acid sequence revealed that two fragments derived from the 75-kDa protein were contained in GRP78 protein (33Munro S. Pelham H.R.B. Cell. 1986; 46: 291-300Abstract Full Text PDF PubMed Scopus (1058) Google Scholar). Because GRP78 exclusively localizes in the endoplasmic reticulum and its structure is closely related to Hsp-70 (33Munro S. Pelham H.R.B. Cell. 1986; 46: 291-300Abstract Full Text PDF PubMed Scopus (1058) Google Scholar), we speculate that the binding of GRP78 to GM-CSFR is an artificial event only observed in an in vitro system. A fragment derived from the 25-kDa protein corresponded to amino acids 193–199 of the human Mad2 protein. As the 25-kDa spot was recognized by an anti-Mad2 antibody in two-dimensional Western blotting analysis (data not shown), this 25-kDa protein is probably the mouse counterpart of the human Mad2. We also analyzed the immunoreactivity of the anti-Mad2 antibody toward GST protein-precipitated proteins by SDS-PAGE followed by Western blotting. The anti-Mad2 antibody recognized a band with a molecular mass of 25 kDa, and this band was observed only with the GST-box1/2 samples incubated with the Ba/F3 cell lysate (Fig.1 C, lane 7). To determine whether the endogenous full-length βc protein binds to Mad2, we carried out co-immunoprecipitation analysis, using anti-βc antibody (specific for human βc) and Ba/F3 cells expressing hGM-CSFR. As shown in Fig. 1 D, Mad2 protein co-immunoprecipitated with βc, thereby suggesting an association of Mads with the native receptor. We next asked whether this association is direct, and for this we used a binding assay of recombinant Mad2 and box1/2 proteins. Full-length mouse Mad2 coding region cDNA was isolated by PCR using synthesizing primers according to GenBank™ mouse Mad2 equivalent sequences. The recently published sequence of the mouse Mad2 (19Dobles M. Liberal V. Scott M.L. Benezra R. Sorger P.K. Cell. 2000; 101: 635-645Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar) completely matched the sequence of our PCR product. To obtain purified Mad2 for the binding assay, we constructed histidine-tagged Mad2 protein (His-Mad2) and purified it. Then, binding activity of His-Mad2 protein and GST-box1/2, GST-α, or GST proteins was examined. As shown in Fig. 2 (lanes 1–3), when the protein complex was precipitated with glutathione-Sepharose 4B beads, only the GST-box1/2 co-precipitated with His-Mad2. Likewise, when proteins were precipitated by Ni2+ beads, again only the GST-box1/2 co-precipitated with the His-Mad2 protein (lanes 10–12). In contrast, neither GST-α nor GST was precipitated together with His-Mad2. Lanes 4–6 and 7–9indicate that nearly the same amount of proteins precipitated in each lane. These results indicate that Mad2 association with box1/2 is specific and direct. We next examined the requirement of the box1/2 sub-region for binding to Mad2 protein, using a βc-derived peptide conjugated with SulfoLink beads. Various peptides corresponding to a portion of the box1/2 region or its derivatives were synthesized and conjugated with SulfoLink beads at their N-terminal cysteine residue (Fig.3 A). The association with Mad2 was examined by incubating these peptides with Ba/F3 cell lysate, and the proteins that bound to the peptide beads were precipitated and separated by SDS-PAGE. Fig. 3 B shows the Western blotting pattern obtained by using anti-Mad2 antibody for reaction with the precipitated proteins. Mad2 co-precipitated with the peptide-beads that covered the entire box1 region (lane 2). We next examined the binding potential of various box1/2-derived peptides. Neither box2 peptide nor control peptide precipitated Mad2 protein (lanes 4 and 7). Amino acids “PNP” conserved within box1 region of many cytokine receptors are thought to be critical for signal transduction as determined by mutation analysis of several receptors (12Murakami M. Narazali M. Hibi M. Yawata H. Yasukawa K. Hamaguchi M. Taga T. Kishimoto T. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 11349-11353Crossref PubMed Scopus (491) Google Scholar). When a peptide carrying a mutation within this conserved PNP motif (box1/ANA) was used for precipitation, the amount of co-precipitated Mad2 was significantly reduced (lane 3). It was also reported that 8 amino acid residues are critical for receptor activation (7Watanabe S. Itoh T. Arai K. J. Biol. Chem. 1996; 271: 12681-12686Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 34Quelle F.W. Sato N. Witthuhn B.A. Inhorn R.C. Eder M. Miyajima A. Griffin J. Ihle J.N. Mol. Cell. Biol. 1994; 14: 4335-4341Crossref PubMed Google Scholar). The box1 peptide, which lacks these critical residues (ΔBox1), abrogated Mad2 precipitation (lane 6). The peptide corresponds to a joint region of box1 and box2 also could not precipitate Mad2 (lane 5). These results suggest that the conserved region of box1 plays an important role in binding to Mad2 protein. We also checked direct binding between recombinant purified His-Mad2 and these peptides, and essentially the same results (data not shown) as obtained with the Ba/F3 cell lysate emerged. To analyze the region of Mad2 required for binding to βc, we next constructed deletion mutants of Mad2, as schematically shown in Fig.4 A. Mutants were fused at their N terminus with a histidine tag for purification. Purified histidine-tagged Mad2 mutant proteins were incubated with purified GST box1/2, and the proteins were precipitated using glutathione-Sepharose 4B beads and analyzed by Coomassie Brilliant Blue staining (Fig.4 B). The GST-box1/2 could bind wild type (lane 2) as well as mutant Mad2 lacking the N-terminal portion (ΔN, lane 4), but the amount of precipitated ΔN-Mad2 was clearly less than that of the wild-type Mad2, thus indicating that the N-terminal region of Mad2 was not essential for but influenced the binding affinity between Mad2 and box1/2. In contrast, mutant Mad2 lacking the C terminus (ΔC) did not bind to GST-box1/2 (lane 3). In view of these data, we speculate that the C-terminal portion (54 residues, amino acid positions 152–205) is sufficient and is required to interact with GST-box1/2. The anti-Mad2 antibody, which recognizes the ΔN mutant but not the ΔC mutant interfered with the association between box1/2 protein and Mad2 of Ba/F3 lysates, in a dose-dependent manner (Fig. 4C). In contrast, control antibodies, anti-γ-catenin, and anti-phosphotyrosine (4G10), did not affect Mad2 binding, thereby supporting the conclusion that the C-terminal region of Mad2 is important and sufficient for binding between box1/2 and Mad2. Mad2 associates with various cell cycle-related proteins in a cell cycle phase-dependent manner (21Fang G., Yu, H. Kirschner M.W. Genes Dev. 1998; 12: 1871-1883Crossref PubMed Scopus (495) Google Scholar, 22Hwang L.H. Lau L.F. Smith D.L. Mistrot C.A. Hardwick K.G. Hwang E.S. Amon A. Murray A.W. Science. 1998; 279: 1041-1044Crossref PubMed Scopus (464) Google Scholar, 30Wassmann K. Benezra R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11193-11198Crossref PubMed Scopus (111) Google Scholar, 35Li Y. Gorbea C. Mahaffey D. Rechsteiner M. Benezra R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12431-12436Crossref PubMed Scopus (184) Google Scholar, 36Kallio M. Weinstein J. Daum J.R. Burke D.J. Gorbsky G.J. J. Cell Biol. 1998; 141: 1393-1406Crossref PubMed Scopus (221) Google Scholar). To determine whether the association between Mad2 and βc is also affected during the cell cycle, we prepared HeLa cell lysates at various phases of the cell cycle. HeLa cells were arrested at G1 by a thymidine double block and released from arrest by
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