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Revisado por pares
Ki-1/57 Interacts with RACK1 and Is a Substrate for the Phosphorylation by Phorbol 12-Myristate 13-Acetate-activated Protein Kinase C
2004; Elsevier BV; Volume: 279; Issue: 12 Linguagem: Inglês
10.1074/jbc.m306672200
ISSN1083-351X
AutoresFlávia C. Nery, Dario Oliveira Passos, Vera S. Garcia, Jörg Kobarg,
Tópico(s)Cancer-related Molecular Pathways
ResumoKi-1/57, the 57-kDa human protein antigen recognized by the CD30 antibody Ki-1, is a cytoplasmic and nuclear protein that is phosphorylated on serine and threonine residues. When isolated from the Hodgkin's lymphoma analogous cell line L540 Ki-1/57 co-immunoprecipitated with a Thr/Ser protein kinase activity. It has been also found to interact with hyaluronic acid and has therefore been termed intracellular IHABP4 (hyaluronan-binding protein 4). Recent studies demonstrated, however, that Ki-1/57 engages in specific interaction with the chromo-helicase-DNA-binding domain protein 3, a nuclear protein involved in chromatin remodeling and transcription regulation. We used the yeast two-hybrid system to find proteins interacting with Ki-1/57 and identified the adaptor protein RACK1 (receptor of activated kinase 1). Next, we confirmed this interaction in vitro and in vivo, performed detailed mapping studies of the interaction sites of Ki-1/57 and RACK-1, and demonstrated that Ki-1/57 also co-precipitates with protein kinase C (PKC) when isolated from phorbol 12-myristate 13-acetate (PMA)-activated L540 tumor cells and is a substrate for PKC phosphorylation in vitro and in vivo. Interestingly, the interaction of Ki-1/57 with RACK1 is abolished upon activation of L540 cells with PMA, which results in the phosphorylation of Ki-1/57 and its exit from the nucleus. Taken together, our data suggest that Ki-1/57 forms a stable complex with RACK-1 in unstimulated cells and upon PMA stimulation gets phosphorylated on threonine residues located at its extreme C terminus. These events associate Ki-1/57 with the RACK1/PKC pathway and may be important for the regulation of its cellular functions. Ki-1/57, the 57-kDa human protein antigen recognized by the CD30 antibody Ki-1, is a cytoplasmic and nuclear protein that is phosphorylated on serine and threonine residues. When isolated from the Hodgkin's lymphoma analogous cell line L540 Ki-1/57 co-immunoprecipitated with a Thr/Ser protein kinase activity. It has been also found to interact with hyaluronic acid and has therefore been termed intracellular IHABP4 (hyaluronan-binding protein 4). Recent studies demonstrated, however, that Ki-1/57 engages in specific interaction with the chromo-helicase-DNA-binding domain protein 3, a nuclear protein involved in chromatin remodeling and transcription regulation. We used the yeast two-hybrid system to find proteins interacting with Ki-1/57 and identified the adaptor protein RACK1 (receptor of activated kinase 1). Next, we confirmed this interaction in vitro and in vivo, performed detailed mapping studies of the interaction sites of Ki-1/57 and RACK-1, and demonstrated that Ki-1/57 also co-precipitates with protein kinase C (PKC) when isolated from phorbol 12-myristate 13-acetate (PMA)-activated L540 tumor cells and is a substrate for PKC phosphorylation in vitro and in vivo. Interestingly, the interaction of Ki-1/57 with RACK1 is abolished upon activation of L540 cells with PMA, which results in the phosphorylation of Ki-1/57 and its exit from the nucleus. Taken together, our data suggest that Ki-1/57 forms a stable complex with RACK-1 in unstimulated cells and upon PMA stimulation gets phosphorylated on threonine residues located at its extreme C terminus. These events associate Ki-1/57 with the RACK1/PKC pathway and may be important for the regulation of its cellular functions. The first monoclonal antibody that specifically detected the malignant Hodgkin's and Sternberg-Reed cells in Hodgkin's lymphoma was called Ki-1 and binds to the 120-kDa lymphocyte co-stimulatory molecule CD30 (Ki-1/120) on the surface of the Hodgkin's cells (1Schwab U. Stein H. Gerdes J. Lemke H. Kirchner H. Schaadt M. Diehl V. Nature. 1982; 299: 65-67Crossref PubMed Scopus (741) Google Scholar, 2Dürkop H. Latza U. Hummel M. Eitelbach F. Seed B. Stein H. Cell. 1992; 68: 421-427Abstract Full Text PDF PubMed Scopus (602) Google Scholar). It has however been noticed early on that this antibody also cross-reacts with an intracellular phosphoprotein antigen of 57 kDa termed Ki-1/57 (3Hansen H. Lemke H. Bredfeldt G. Könnecke I. Havsteen B. Biol. Chem. Hoppe Seyler. 1989; 370: 409-416Crossref PubMed Scopus (23) Google Scholar, 4Froese P. Lemke H. Gerdes J. Havsteen B. Schwarting R. Hansen H. Stein H. J. Immunol. 1987; 139: 2081-2087PubMed Google Scholar). In vitro phosphorylation experiments performed with the Ki-1/57 antigen isolated from tumor cells demonstrated that it is associated with a serine/threonine protein kinase activity (5Hansen H. Bredfeldt G. Havsteen B. Lemke H. Res. Immunol. 1990; 141: 13-31Crossref PubMed Scopus (21) Google Scholar). Electron microscopic analysis showed that the Ki-1/57 antigen is located in the cytoplasm, at the nuclear pores, and in the nucleus, where it is frequently found in association with the nucleolus and other nuclear bodies (6Rohde D. Hansen H. Hafner M. Lange H. Mielke V. Hansmann M.L. Lemke H. Am. J. Pathol. 1992; 140: 473-482PubMed Google Scholar). Tryptic digestion of the Ki-1/57 antigen resulted in the cloning of a partial cDNA encoding Ki-1/57 (7Kobarg J. Schnittger S. Fonatsch C. Lemke H. Bowen M.A. Buck F. Hansen H.P. Exp. Clin. Immunogenet. 1997; 14: 273-280PubMed Google Scholar). The isolated contig 1The abbreviations used are: contig, group of overlapping clones; AR, autoradiography; CHD3, chromo-helicase-DNA-binding domain protein 3; PMA, phorbol 12-myristate 13-acetate; PKC, protein kinase C; PDE, phosphodiesterase; IHABP4, intracellular hyaluronan-binding protein 4; PAI, plasminogen activator inhibitor; Ni-NTA, nickel-nitrilotriacetic acid; RACK1, receptor of activated kinase 1; Ki-1/57, the 57 kDa protein antigen detected by antibody Ki-1; WD repeat, denotes conserved tryptophan and aspartic acid residues within this domain. of 1380 bp length encodes the C-terminal 60% of the Ki-1/57 protein. Later, another group cloned the full-length Ki-1/57 cDNA (8Huang L. Grammatikakis N. Yoneda M. Banerjee S.D. Toole B.P. J. Biol. Chem. 2000; 275: 29829-29839Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Huang et al. (8Huang L. Grammatikakis N. Yoneda M. Banerjee S.D. Toole B.P. J. Biol. Chem. 2000; 275: 29829-29839Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar) found that Ki-1/57 has a hyaluronan binding activity and gave it the second name, intracellular hyaluronan-binding protein 4 (IHABP4). They also found that IHABP4/Ki-1/57 binds to other negatively charged glycosaminoglycans like chondroitin sulfate, heparane sulfate, and RNA, although with lower affinity. The functional meaning of Ki-1/57 interaction with these macromolecules remains open. When we were searching the sequence data bank for Ki-1/57 related molecules, we found the human protein CGI-55, which amino acid sequence has 40.7% identity and 67.4% similarity with that of Ki-1/57 (9Lemos T. Passos D.O. Nery F.C. Kobarg J. FEBS Lett. 2003; 533: 14-20Crossref PubMed Scopus (49) Google Scholar). This high degree of similarity suggests that both proteins might be paralogues and may have related functions. CGI-55 has also been described to bind to the 3′-region of the mRNA encoding the plasminogen activator inhibitor (PAI) type 1 (10Heaton J.H. Dlakic W.M. Dlakic M. Gelehrter T.D. J. Biol. Chem. 2001; 276: 3341-3347Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Heaton et al. (10Heaton J.H. Dlakic W.M. Dlakic M. Gelehrter T.D. J. Biol. Chem. 2001; 276: 3341-3347Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar) have therefore termed CGI-55 as PAI RNA-binding protein 1 and suggested that it could be involved in the regulation of the stability of the PAI mRNA, although they do not provide experimental data to support this hypothesis. We explored the yeast two-hybrid system to identify possible interacting proteins for both Ki-1/57 and CGI-55 and in this way obtain clues for the functional context of these proteins. Our analysis resulted in the identification of the human protein chromo-helicase-DNA-binding domain protein 3 (CHD3) as a partner for both proteins (9Lemos T. Passos D.O. Nery F.C. Kobarg J. FEBS Lett. 2003; 533: 14-20Crossref PubMed Scopus (49) Google Scholar). The CHD proteins are members of the chromo domain family, a class of proteins that are involved in transcriptional regulation and chromatin remodeling (11Koonin E.V. Zhou S. Lucchesi J.C. Nucleic Acids Res. 1995; 23: 4229-4233Crossref PubMed Scopus (161) Google Scholar, 12Cavalli G. Paro R. Curr. Opin. Cell Biol. 1998; 10: 354-360Crossref PubMed Scopus (154) Google Scholar, 13Aubry F. Mattei M.G. Galibert F. Eur. J. Biochem. 1998; 254: 558-564Crossref PubMed Scopus (19) Google Scholar, 14Woodage T. Basrai M.A. Baxevanis A.D. Hieter P. Collins F.S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11472-11477Crossref PubMed Scopus (307) Google Scholar, 15Tong J.K. Hassig C.A. Schnitzler G.R. Kingston R.E. Schreiber S.L. Nature. 1998; 395: 917-921Crossref PubMed Scopus (547) Google Scholar, 16Ogas J. Kaufmann S. Henderson J. Somerville C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13839-13844Crossref PubMed Scopus (409) Google Scholar, 17Zhang Y. LeRoy G. Seelig H.P. Lane W.S. Reinberg D. Cell. 1998; 95: 279-289Abstract Full Text Full Text PDF PubMed Scopus (686) Google Scholar). The binding of the proteins Ki-1/57 and CGI-55 to CHD3 might define them as a family of CHD3-binding proteins and suggested the possibility that they could be involved in nuclear functions associated with the remodeling of chromatin and the regulation of transcription. Whereas in the case of the CGI-55, 42% of the found interacting clones represented CHD3, only 4% of the clones interacting with Ki-1/57 represented CHD3 (9Lemos T. Passos D.O. Nery F.C. Kobarg J. FEBS Lett. 2003; 533: 14-20Crossref PubMed Scopus (49) Google Scholar). Here we report that the vast majority of clones (54%) found to interact with Ki-1/57 represent the scaffold and regulatory protein RACK-1 (receptor of activated kinase 1), a protein that we did not identify in the interaction screen of the putative Ki-1/57 paralogue CGI-55. RACK1 has a molecular mass of 36 kDa and is composed of seven WD repeats (18McCahill A. Warwicker J. Bolger G.B. Houslay M.D. Yarwood S.J. Mol. Pharmacol. 2002; 62: 1261-1273Crossref PubMed Scopus (333) Google Scholar, 19Schechtmann D. Mochly-Rosen D. Oncogene. 2001; 20: 6339-6347Crossref PubMed Scopus (284) Google Scholar). Its overall structure resembles that of the β-subunit of G proteins (20Garcia-Higuera I. Fenoglio J. Li Y. Lewis C. Panchenko M.P. Reiner O. Smith T.F. Neer E.J. Biochemistry. 1996; 35: 13985-13994Crossref PubMed Scopus (164) Google Scholar, 21Neer E.J. Smith T.F. Cell. 1996; 84: 175-178Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). RACK1 has been reported to interact with PKCβ (22Ron D. Luo J. Mochly-Rosen D. J. Biol. Chem. 1995; 270: 24180-24187Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, 23Csukai M. Mochly-Rosen D. Pharmacol. Res. 1999; 39: 253-259Crossref PubMed Scopus (116) Google Scholar, 24Stebbins E.G. Mochly-Rosen D. J. Biol. Chem. 2001; 276: 29644-29650Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar); Src (25Chang B.Y. Conroy K.B. Machleder E.M. Cartwright C.A. Mol. Cell. Biol. 1998; 18: 3245-3256Crossref PubMed Google Scholar); β-integrins (26Liliental J. Chang D.D. J. Biol. Chem. 1998; 273: 2379-2383Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar); PDE4D5 (27Yarwood S.J. Steele M.R. Scotland G. Houslay M.D. Bolger G.B. J. Biol. Chem. 1999; 274: 14909-14917Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar); the β-subunit of the granulocyte-macrophage colony-stimulating factor, interleukin 3, and interleukin 5 receptors (28Geijsen N. Spaargaren M. Raaijmakers J.A. Lammers J.W. Koenderman L. Coffer P.J. Oncogene. 1999; 18: 5126-5130Crossref PubMed Scopus (79) Google Scholar); type 1 interferon receptor (29Croze E. Usachewa A. Asarnow D. Minshall R.D. Perez H.D. Colamonici O. J. Immunol. 2000; 165: 5127-5132Crossref PubMed Scopus (47) Google Scholar); STAT1 (30Usachewa A. Smith R. Minshall R. Baida G. Seng S. Croze E. Colamonici O. J. Biol. Chem. 2001; 27: 22948-22953Abstract Full Text Full Text PDF Scopus (68) Google Scholar); and a number of viral proteins (31Gallina A. Rossi F. Milanesi G. Virology. 2001; 238: 7-18Crossref Scopus (37) Google Scholar, 32Sang N Severino A. Russo P. Baldi A. Giordano A. Mileo A.M. Paggi M.G. De Luca A. J. Biol. Chem. 2001; 276: 27026-27033Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 33Baumann M. Gires O. Kolch W. Mischak H. Zeidler R. Pich D. Hammerschmidt W. Eur. J. Biochem. 2000; 267: 3891-3901Crossref PubMed Scopus (49) Google Scholar). RACK1 is up-regulated in human carcinomas and during tissue regeneration after ischemic renal injury (34Padanilam B.J. Hammerman M.R. Am. J. Physiol. 1997; 272: F160-F166PubMed Google Scholar, 35Berns H. Humar R. Hengerer B. Kiefer F.N. Battegay E.J. FASEB J. 2000; 14: 2549-2558Crossref PubMed Scopus (107) Google Scholar). Furthermore, RACK1 has been functionally implicated in the development of cardiac hypertrophy (36Pass J.M. Gao J. Jones W.K. Wead W.B. Wu X. Zhang J. Baines C.P. Bolli R. Zheng Y.T. Joshua I.G. Ping P. Am. J. Physiol. 2001; 281: H2500-H2510Crossref PubMed Google Scholar), the regulation of cell adhesion (37Tscherkasowa A.E. Adams-Klages S. Kruse A.-L. Wiegmann K. Mathieu S. Kolanus W. Krönke M Adam D. J. Immunol. 2002; 169: 5161-5170Crossref PubMed Scopus (39) Google Scholar), the increase of focal adhesion (38Cox E.A. Benin D. Doan A.T. O'Toole T. Huttenlocher A. Mol. Biol. Cell. 2003; 14: 658-669Crossref PubMed Scopus (123) Google Scholar), and the protection from viral, E1A protein-induced apoptosis (32Sang N Severino A. Russo P. Baldi A. Giordano A. Mileo A.M. Paggi M.G. De Luca A. J. Biol. Chem. 2001; 276: 27026-27033Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). On a molecular level the interaction of RACK1 with Src has been described to result in an inhibition of the kinase activity of Src (25Chang B.Y. Conroy K.B. Machleder E.M. Cartwright C.A. Mol. Cell. Biol. 1998; 18: 3245-3256Crossref PubMed Google Scholar). The activity of PDE4E5 on the other hand was unaffected by the binding of RACK1 (27Yarwood S.J. Steele M.R. Scotland G. Houslay M.D. Bolger G.B. J. Biol. Chem. 1999; 274: 14909-14917Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). Although RACK1 has been reported to have a stimulatory effect on the substrate phosphorylation by PKC (22Ron D. Luo J. Mochly-Rosen D. J. Biol. Chem. 1995; 270: 24180-24187Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, 39Csukai M. Chen C.H. DeMatteis M.A. Mochly-Rosen D. J. Biol. Chem. 1997; 272: 29200-29206Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar), others found that RACK1 does not influence the kinase activity of serine/threonine kinases such as PKC, cAMP-dependent protein kinase, and casein kinase II (25Chang B.Y. Conroy K.B. Machleder E.M. Cartwright C.A. Mol. Cell. Biol. 1998; 18: 3245-3256Crossref PubMed Google Scholar), indicating that the RACK1 activity on PKC may be influenced by the kind of substrate involved. Here we show that RACK1 interacts with Ki-1/57, confirm this interaction in vitro and in vivo, and map the interaction sites of Ki-1/57 and RACK-1 in detail. Furthermore, we found that Ki-1/57 is a substrate for PKC and that its interaction with RACK1 is abolished in the course of the PMA activation of the cells. Our data suggest that Ki-1/57 is involved in specific protein-protein interactions and provide a plausible explanation for the long known fact that Ki-1/57, which does not contain a kinase domain, in fact co-precipitates with kinase activity. The co-precipitated kinase activity appears to be PKC. This could be confirmed by the co-immunoprecipitation of Ki-1/57 with PKC, which associates with Ki-1/57 after PMA stimulation of the cells. Our results further suggest that the cellular functions of Ki-1/57 may be subject to regulation via a PKC/RACK1 pathway. Plasmid Constructions—Several sets of oligonucleotides were designed to allow subcloning of the complete Ki-1/57 coding region in different expression vectors. Insertion into pGEX-2TK (Amersham Biosciences) allowed expression of Ki-1/57(1–413) as a C-terminal fusion to GST (GST-Ki-1/57). The cDNAs encoding full-length Ki-1/57, Ki-1/57(122–413), and the eight other indicated deletion constructs (numbered in the same way) were inserted into the yeast two-hybrid expression vector pBTM-116 (40Bartel P.L. Fields S. Methods Enzymol. 1995; 254: 241-263Crossref PubMed Scopus (303) Google Scholar, 41Vojtek A.B. Hollenberg S.M. Methods Enzymol. 1995; 255: 331-342Crossref PubMed Scopus (238) Google Scholar, 42Durfee T. Becherer K. Chen P.L. Yeh S.H. Yang Y. Kilburn A.E. Lee W.H. Elledge S.J. Genes Dev. 1993; 7: 555-569Crossref PubMed Scopus (1299) Google Scholar, 43Hollenberg S.M. Sternglanz R. Cheng P.F. Weintraub H. Mol. Cell. Biol. 1995; 15: 3813-3822Crossref PubMed Scopus (585) Google Scholar). Other deletions were also subcloned into bacterial expression vectors peT28a, pProEx, or pGEX to allow their expression as His6-tagged or GST-tagged fusion proteins. In a similar fashion the cDNAs encoding the indicated deletions of the protein RACK-1 were amplified and inserted into the yeast two-hybrid vector pGAD424 (Clontech), and full-length RACK1 was inserted into the bacterial expression vector pET-28a (Novagen) to allow expression of the His6-RACK1 fusion protein. Yeast Two-hybrid Screening and Interaction Analysis—The pBTM116-Ki-1/57(122–413) (40Bartel P.L. Fields S. Methods Enzymol. 1995; 254: 241-263Crossref PubMed Scopus (303) Google Scholar) vector was used to express a fragment spanning 60% of the C terminus of the protein Ki-1/57 linked to the C terminus of LexA DNA-binding domain peptide in Saccharomyces cerevisiae strain L40. A human fetal brain cDNA library (Clontech) expressing GAL4 activation domain fusion proteins was co-transformed with the recombinant pBTM116-Ki-1/57 vector. Selection of transformants, the β-galactosidase activity test, plasmid DNA extraction, and sequencing were performed as described previously (9Lemos T. Passos D.O. Nery F.C. Kobarg J. FEBS Lett. 2003; 533: 14-20Crossref PubMed Scopus (49) Google Scholar). Bacterial Expression and Protein Purification—GST, GST-Ki-1/57, and His6-RACK-1 proteins were expressed in Escherichia coli BL21-CodonPlus-RIL (Stratagene) and purified using glutathione-Sepharose 4B (Amersham Biosciences) or Ni-NTA-Sepharose as described before (44Moraes K.C.M. Quaresma A.J.C. Maehnss K Kobarg J. Biol. Chem. 2002; 384: 25-37Google Scholar). In Vitro Binding Assay, Western Blot Analysis, Antibodies, and Cell Culture—GST or GST-Ki-1/57 fusion proteins were coupled to glutathione-Sepharose beads. After washing, the beads were incubated with His6-RACK-1 for 2 h at 4 °C and then washed with buffer (20 mm Tris-HCl, pH 7.5, 150 mm NaCl). The proteins bound to the beads were separated by SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and visualized by immunochemiluminescence using a mouse anti-GST antibody (for control of equal loading of beads) or anti-His5 monoclonal antibody (Qiagen) and secondary anti-mouse IgG-horseradish peroxidase conjugate. The anti-RACK-1 monoclonal antibody was from Transduction Laboratories. The specific anti-Ki-1/57 monoclonal antibodies A26, E203 (7Kobarg J. Schnittger S. Fonatsch C. Lemke H. Bowen M.A. Buck F. Hansen H.P. Exp. Clin. Immunogenet. 1997; 14: 273-280PubMed Google Scholar), and Ki-1 (1Schwab U. Stein H. Gerdes J. Lemke H. Kirchner H. Schaadt M. Diehl V. Nature. 1982; 299: 65-67Crossref PubMed Scopus (741) Google Scholar) have been described previously. Anti-Ki-1/67 control antibody had been provided by Prof. Dr. Hilmar Lemke (45Diehl V. Kirchner H.H. Schaadt M. Fonatsch C. Stein H. Gerdes J. Boie C. J. Cancer Res. Clin. Oncol. 1981; 101: 111-124Crossref PubMed Scopus (124) Google Scholar). An anti-phospho-PKC antibody sampler kit was purchased from Cell Signaling Technology. L540 Hodgkin's analogous cells (46Gerdes J. Schwab U. Lemke H. Stein H. Int. J. Cancer. 1983; 31: 13-20Crossref PubMed Scopus (2318) Google Scholar) were cultivated in RPMI 1640 medium, supplemented with 20% fetal calf serum, 2 mml-glutamine, penicillin (100 units/ml), and streptomycin (100 μg/ml) at 37 °C and 5% CO2 (L540 standard medium). HeLa cells were cultivated in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 2 mml-glutamine, penicillin (100 units/ml), and streptomycin (100 μg/ml) under equal conditions. In Vivo Binding Assay—5.0 × 107 L540 cells were or were not stimulated with PMA (100 ng/ml) for 4 h (33Baumann M. Gires O. Kolch W. Mischak H. Zeidler R. Pich D. Hammerschmidt W. Eur. J. Biochem. 2000; 267: 3891-3901Crossref PubMed Scopus (49) Google Scholar). The cells were lysed in 1 ml of buffer NaCl/Tris (25 mm Tris, pH 7.5, 137 mm NaCl, 2.7 mm KCl, 1% Triton X-100, protease inhibitors). The lysates were treated with DNase (Promega) and cleared at 14.000 × g for 30 min. Next 20 μl of protein A-Sepharose beads (Amersham Biosciences) were loaded with the indicated antibodies overnight at 4 °C, washed in buffer NaCl/Tris, and incubated with the L540 lysate overnight at 4 °C. After further three washes with the buffer Tris/EDTA (10 mm Tris, pH 7.5, 1 mm EDTA, 0.5 m NaCl), the beads were resuspended in SDS-PAGE loading buffer, boiled, and analyzed by SDS-PAGE and Western blot using the indicated antibodies. Western blots were developed by chemiluminescence as described previously (44Moraes K.C.M. Quaresma A.J.C. Maehnss K Kobarg J. Biol. Chem. 2002; 384: 25-37Google Scholar). Loading controls consisted either of protein detection by SDS-PAGE or of Western blot development with control antibodies as indicated in the figures. In Vitro Phosphorylation Assay and Phosphoamino Acid Analysis— 5 × 107 L540 cells were treated or not with PMA, collected, and lysed as described above. Endogenous PKC was immunoprecipitated from these lysates with anti-phospho-PKC-Pan antibody (Cell Signaling) coupled to protein A-Sepharose beads. Next these beads were incubated with purified GST-Ki-1/57, His6-RACK-1, both, or GST in kinase buffer (25 mm Tris, pH 7.5, 1.32 mm CaCl2, 5 mm MgCl2, 1 mm EDTA, 1.25 EGTA, 1 mm dithiothreitol) containing 10 nm PMA, 5 μm ATP, and 0.5 μCi of [γ-32P]ATP, in a total volume of 25 μl for 30 min at 30 °C. Phosphorylated proteins were run out by SDS-PAGE. The gel was stained, dried, and exposed to x-ray film. In other experiments purified GST, GST-Ki-1/57, His6-RACK1, and deletion constructs of Ki-1/57 were phosphorylated in complete kinase buffer in a final volume of 50 μl at 30 °C with purified PKC-Pan, PKCζ, or PKCθ for 15 min. The PKCζ or PKCθ are human recombinant His-tagged and affinity-purified proteins (Promega). PKC-Pan was purified from rat brain and consists predominantly of the PKC isoforms α, β, and γ (Promega). Radioactively labeled proteins were visualized as described above. Phosphoamino acid analysis was basically performed as described in Machado et al. (47Machado E.L. Silva A.C. Silva M. Leite A. Ottoboni L.M.M. Phytochemistry. 2002; 61: 835-842Crossref PubMed Scopus (5) Google Scholar). Briefly, the 32P-radiolabeled phosphorylated proteins were hydrolyzed with 6 n HCl for 60 min at 90 °C. The hydrolysate was lyophilized, dissolved in water, and spotted onto Sigma cell type 100 cellulose thin layer chromatography plates (Sigma). The solvent system was isobutyric acid, 0.5 m ammonium hydroxide (5:3). Phosphoserine, phosphothreonine, and phosphotyrosine standards (2 μg) (Sigma) were mixed with the radiolabeled protein hydrolysate and spotted together on the TLC plates. Amino acids were visualized with 0.2% ninhydrin in ethanol, and radiolabeled residues were detected by autoradiography (AR). Theoretical phosphorylation site prediction was performed by the software NetPhos 2.0 Prediction server available at the web site of the Center for Biological Sequence Analysis (www.cbs.dtu.dk/services/NetPhos). Metabolic Labeling, in Vivo Phosphorylation Assay, and Kinase Inhibitors—5 × 106 L540 cells were preincubated or not for 1 h with protein kinase inhibitors: Ro-32-0432 (28 nm), and staurosporine (0.7 nm) (Calbiochem). This inhibitor incubation was performed with phosphate-free L540 standard medium (the fetal calf serum in this medium had been dialyzed against a 150 mm NaCl solution). Next the cells were activated or not by the addition of 100 ng/ml of PMA for a second hour. In parallel to the PMA treatment, the cells were metabolically labeled by the addition of 0.4 mCi of radioactive 32P-labeled inorganic phosphate (Amersham Biosciences). After lysis Ki-1/57 was immunoprecipitated from the lysates of the metabolically labeled L540 cells with anti-Ki-1 antibody A26 coupled to protein A-Sepharose beads and analyzed by autoradiography and SDS-PAGE. Preparation of Cytoplasmic and Nuclear Cell Fractions—L540 cells were harvested and incubated with 300 μl of hypotonic buffer A (10 mm Tris, pH 7.5, 10 mm KCl, 0.1 mm EDTA, 1.5 mm MgCl2, 0.5 mm dithiothreitol, and a mixture of protease inhibitors) for 30 min on ice (33Baumann M. Gires O. Kolch W. Mischak H. Zeidler R. Pich D. Hammerschmidt W. Eur. J. Biochem. 2000; 267: 3891-3901Crossref PubMed Scopus (49) Google Scholar). The nuclei were recovered by centrifugation at 14,000 rpm for 10 min. The supernatant represents the cytoplasmic fraction. To obtain the nuclear fraction, the crude nuclear pellet was resuspended in 200 μl of hypertonic buffer B (20 mm Tris, pH 8.0, 0.4 m NaCl, 0.1 mm EDTA, 1.5 mm MgCl2, 0.5 mm dithiothreitol, 25% v/v glycerol) followed by incubation on ice for 30 min. After centrifugation, the fractions were incubated with the antibodies at 4 °C overnight. On the next day 20 μl of protein A-Sepharose were added for 2 h. Immunofluorescence Analysis—HeLa cells grown on glass coverslips were stimulated or not with PMA for 4 h at 37 °C. The cells were fixed with 100% methanol and immunostained with primary antibody monoclonal mouse Ki-1, mouse anti-RACK1, or rabbit anti-Phospho-PKC, and secondary antibody fluorescein anti-mouse or rhodamine anti-rabbit antibody. The cells were examined with a Nikon microscope. DAPI staining was used to show the positions of the nuclei. The cells were examined with Nikon fluorescence microscope. Immunolabeled proteins were presented with the respective color. Superimposing the two colors (merge) results in a yellow/orange signal. Yeast Two-hybrid Screen—To identify Ki-1/57 interacting proteins, the yeast two-hybrid system (40Bartel P.L. Fields S. Methods Enzymol. 1995; 254: 241-263Crossref PubMed Scopus (303) Google Scholar, 41Vojtek A.B. Hollenberg S.M. Methods Enzymol. 1995; 255: 331-342Crossref PubMed Scopus (238) Google Scholar, 42Durfee T. Becherer K. Chen P.L. Yeh S.H. Yang Y. Kilburn A.E. Lee W.H. Elledge S.J. Genes Dev. 1993; 7: 555-569Crossref PubMed Scopus (1299) Google Scholar, 43Hollenberg S.M. Sternglanz R. Cheng P.F. Weintraub H. Mol. Cell. Biol. 1995; 15: 3813-3822Crossref PubMed Scopus (585) Google Scholar) was employed, utilizing a human fetal brain cDNA library (Clontech). In a first screen we used a fragment of the Ki-1/57 cDNA that encodes its C-terminal 60% as a bait. 2.0 × 106 screened co-transformants yielded 250 clones positive for both His3 and LacZ reporter constructs. Library plasmids DNA of 80 clones were sequenced. 54% of the sequenced clones all encoded the full-length protein RACK1 (48Ron 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). Another protein identified was CHD3, which had already been described previously elsewhere (9Lemos T. Passos D.O. Nery F.C. Kobarg J. FEBS Lett. 2003; 533: 14-20Crossref PubMed Scopus (49) Google Scholar) and represented 4% of the interacting clones. Other nuclear proteins involved in the regulation of transcription have also been identified but will be described elsewhere. Mapping the Interaction Sites of Ki-1/57 and RACK1—Next, we mapped the Ki-1/57 region required for the interaction with RACK1 using the yeast two-hybrid method (Fig. 1). N- and C-terminal deletion constructs of the Ki-1/57 protein were fused to the LexA DNA-binding domain (Fig. 1A) and tested for their ability to bind full-length RACK1 (Fig. 1B). The two constructs that encompass the N-terminal and central regions: Ki-1/57(1–150) and Ki-1/57(151–263) failed to bind to RACK1. Full-length Ki-1/57, the C-terminal construct used in the two hybrid screen Ki-1/57(122–413) as well as the C-terminal deletion Ki-1/57(264–413) all interacted with RACK1. This suggests that the RACK1-binding site is located at the Ki-1/57 C terminus. The co-transformation of pBTM116-Ki-1/57 with several unrelated "bait" constructions, including pACT2-AUF1 (44Moraes K.C.M. Quaresma A.J.C. Maehnss K Kobarg J. Biol. Chem. 2002; 384: 25-37Google Scholar) (not shown) and with empty pBTM116 vector (Fig. 1B), showed no interaction. Furthermore, we mapped the RACK1 regions that are required for the interaction with Ki-1/57. N- and C-terminal deletion constructs of the RACK1 protein were fused to the Gal4 activation domain (vector pACT2; Fig. 1C) and tested for their ability to bind to full-length Ki-1/57 (Fig. 1D). None of the four different deletion constructs of RACK1 interacted with Ki-1/57. This shows that full-length RACK1 is required for an interaction with Ki-1/57. In Vitro Confirmation of the Ki-1/57-RACK1 Interaction with Purified Fusion Proteins—To confirm the interaction between Ki-1/57 and RACK1 in vitro, we next performed in vitro pull-down assays with purified recombinant proteins that had been
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