Title: Ribosomal Protein S9 Is a Novel B23/NPM-binding Protein Required for Normal Cell Proliferation
Abstract: B23 (NPM/nucleophosmin) is a multifunctional nucleolar protein and a member of the nucleoplasmin superfamily of acidic histone chaperones. B23 is essential for normal embryonic development and plays an important role in genomic stability, ribosome biogenesis, and anti-apoptotic signaling. Altered protein expression or genomic mutation of B23 is encountered in many different forms of cancer. Although described as multifunctional, a genuine molecular function of B23 is not fully understood. Here we show that B23 is associated with a protein complex consisting of ribosomal proteins and ribosome-associated RNA helicases. A novel, RNA-independent interaction between ribosomal protein S9 (RPS9) and B23 was further investigated. We found that S9 binding requires an intact B23 oligomerization domain. Depletion of S9 by small interfering RNA resulted in decreased protein synthesis and G1 cell cycle arrest, in association with induction of p53 target genes. We determined that S9 is a short-lived protein in the absence of ribosome biogenesis, and proteasomal inhibition significantly increased S9 protein level. Overexpression of B23 facilitated nucleolar storage of S9, whereas knockdown of B23 led to diminished levels of nucleolar S9. Our results suggest that B23 selectively stores, and protects ribosomal protein S9 in nucleoli and therefore could facilitate ribosome biogenesis. B23 (NPM/nucleophosmin) is a multifunctional nucleolar protein and a member of the nucleoplasmin superfamily of acidic histone chaperones. B23 is essential for normal embryonic development and plays an important role in genomic stability, ribosome biogenesis, and anti-apoptotic signaling. Altered protein expression or genomic mutation of B23 is encountered in many different forms of cancer. Although described as multifunctional, a genuine molecular function of B23 is not fully understood. Here we show that B23 is associated with a protein complex consisting of ribosomal proteins and ribosome-associated RNA helicases. A novel, RNA-independent interaction between ribosomal protein S9 (RPS9) and B23 was further investigated. We found that S9 binding requires an intact B23 oligomerization domain. Depletion of S9 by small interfering RNA resulted in decreased protein synthesis and G1 cell cycle arrest, in association with induction of p53 target genes. We determined that S9 is a short-lived protein in the absence of ribosome biogenesis, and proteasomal inhibition significantly increased S9 protein level. Overexpression of B23 facilitated nucleolar storage of S9, whereas knockdown of B23 led to diminished levels of nucleolar S9. Our results suggest that B23 selectively stores, and protects ribosomal protein S9 in nucleoli and therefore could facilitate ribosome biogenesis. Ribosome biogenesis is a complex process involving many different proteins acting at various stages from early ribosomal RNA (rRNA) synthesis, followed by processing, subunit assembly, and nuclear export of the ribosome (1Boisvert F.M. van Koningsbruggen S. Navascues J. Lamond A.I. Nat. Rev. Mol. Cell Biol. 2007; 8: 574-585Crossref PubMed Scopus (1138) Google Scholar). Synthesis of rRNA and assembly of the ribosomal subunits occurs in the nucleolus. B23 (also known as NPM, nucleophosmin or NO38) is an essential protein that associates with the ribosomal subunits and accumulates predominantly in the nucleolus (2Grisendi S. Mecucci C. Falini B. Pandolfi P.P. Nat. Rev. Cancer. 2006; 6: 493-505Crossref PubMed Scopus (679) Google Scholar). B23 is essential for normal embryonic development, maintenance of genomic stability, and normal ribosome biogenesis as was shown by knocking out the B23 (Npm1) gene in mice (3Grisendi S. Bernardi R. Rossi M. Cheng K. Khandker L. Manova K. Pandolfi P.P. Nature. 2005; 437: 147-153Crossref PubMed Scopus (468) Google Scholar). Indeed, B23 is a multifunctional protein containing an N-terminal oligomerization domain harboring most of the in vitro chaperone activity, a central acidic domain that is required for its ribonuclease activity, and a C-terminal domain mediating binding to nucleic acids (4Herrera J.E. Savkur R. Olson M.O. Nucleic Acids Res. 1995; 23: 3974-3979Crossref PubMed Scopus (133) Google Scholar, 5Hingorani K. Szebeni A. Olson M.O. J. Biol. Chem. 2000; 275: 24451-24457Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 6Szebeni A. Olson M.O. Protein Sci. 1999; 8: 905-912Crossref PubMed Scopus (206) Google Scholar). B23 forms oligomers and the crystal structure of the Xenopus B23 (NO38) core has been solved (7Namboodiri V.M. Akey I.V. Schmidt-Zachmann M.S. Head J.F. Akey C.W. Structure (Camb.). 2004; 12: 2149-2160Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). The oligomers are cyclic pentamers, similar to other members of the nucleoplasmin family (7Namboodiri V.M. Akey I.V. Schmidt-Zachmann M.S. Head J.F. Akey C.W. Structure (Camb.). 2004; 12: 2149-2160Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 8Namboodiri V.M. Dutta S. Akey I.V. Head J.F. Akey C.W. Structure (Camb.). 2003; 11: 175-186Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). The oligomer is the predominant form in cells and is thermostable as well as resistant to disruption by low concentrations of SDS (9Chan P.K. Chan F.Y. Biochim. Biophys. Acta. 1995; 1262: 37-42Crossref PubMed Scopus (60) Google Scholar, 10Herrera J.E. Correia J.J. Jones A.E. Olson M.O. Biochemistry. 1996; 35: 2668-2673Crossref PubMed Scopus (66) Google Scholar, 11Yung B.Y. Chan P.K. Biochim. Biophys. Acta. 1987; 925: 74-82Crossref PubMed Scopus (100) Google Scholar). B23 interacts with other nucleolar proteins, for example, the alternative reading frame (ARF), 3The abbreviations used are: ARF, alternative reading frame; GST, glutathione S-transferase; GFP, green fluorescent protein; WT, wild type; IVT, in vitro translation/transcription; IP, immunoprecipitation; siRNA, small interfering RNA; r-protein, ribosomal protein. tumor suppressor protein (12Bertwistle D. Sugimoto M. Sherr C.J. Mol. Cell. Biol. 2004; 24: 985-996Crossref PubMed Scopus (329) Google Scholar, 13Itahana K. Bhat K.P. Jin A. Itahana Y. Hawke D. Kobayashi R. Zhang Y. Mol. Cell. 2003; 12: 1151-1164Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar), and nucleolin/C23 (14Li Y.P. Busch R.K. Valdez B.C. Busch H. Eur. J. Biochem. 1996; 237: 153-158Crossref PubMed Scopus (144) Google Scholar). B23 can also bind viral proteins, such as Rev and Rex, to promote their nucleolar localization (15Adachi Y. Copeland T.D. Hatanaka M. Oroszlan S. J. Biol. Chem. 1993; 268: 13930-13934Abstract Full Text PDF PubMed Google Scholar, 16Szebeni A. Mehrotra B. Baumann A. Adam S.A. Wingfield P.T. Olson M.O. Biochemistry. 1997; 36: 3941-3949Crossref PubMed Scopus (87) Google Scholar). More recently, B23 has been implicated in pre-mRNA splicing (17Tarapore P. Shinmura K. Suzuki H. Tokuyama Y. Kim S.H. Mayeda A. Fukasawa K. FEBS Lett. 2006; 580: 399-409Crossref PubMed Scopus (48) Google Scholar) and in nuclear export of the L5/5S ribosomal RNA complex (18Yu Y. Maggi Jr., L.B. Brady S.N. Apicelli A.J. Dai M.S. Lu H. Weber J.D. Mol. Cell. Biol. 2006; 26: 3798-3809Crossref PubMed Scopus (166) Google Scholar). B23 plays an important role in tumor development through several distinct mechanisms. First, translocations in which the B23 oligomerization domain is fused to transcription factors or kinases are common in lymphomas and leukemias (19Morris S.W. Kirstein M.N. Valentine M.B. Dittmer K.G. Shapiro D.N. Saltman D.L. Look A.T. Science. 1994; 263: 1281-1284Crossref PubMed Scopus (1980) Google Scholar). Second, C-terminal mutations have been reported to frequently occur in acute myelogenic leukemia with a normal karyotype. These mutations create a novel nuclear export signal, and disrupt a nucleolar localization motif (20Falini B. Mecucci C. Tiacci E. Alcalay M. Rosati R. Pasqualucci L. La Starza R. Diverio D. Colombo E. Santucci A. Bigerna B. Pacini R. Pucciarini A. Liso A. Vignetti M. Fazi P. Meani N. Pettirossi V. Saglio G. Mandelli F. Lo-Coco F. Pelicci P.G. Martelli M.F. N. Engl. J. Med. 2005; 352: 254-266Crossref PubMed Scopus (1461) Google Scholar). Third, increased levels of B23 protein have been noted in transformed cells, but whether this is a contributing factor to, or merely a consequence of increased cancer cell growth remains unclear (2Grisendi S. Mecucci C. Falini B. Pandolfi P.P. Nat. Rev. Cancer. 2006; 6: 493-505Crossref PubMed Scopus (679) Google Scholar). Fourth, loss of chromosome 5q that encodes for B23 occurs in myelodysplastic syndromes (2Grisendi S. Mecucci C. Falini B. Pandolfi P.P. Nat. Rev. Cancer. 2006; 6: 493-505Crossref PubMed Scopus (679) Google Scholar). This loss of B23 causes genomic instability perhaps due to unrestricted centrosome duplication (3Grisendi S. Bernardi R. Rossi M. Cheng K. Khandker L. Manova K. Pandolfi P.P. Nature. 2005; 437: 147-153Crossref PubMed Scopus (468) Google Scholar, 21Okuda M. Horn H.F. Tarapore P. Tokuyama Y. Smulian A.G. Chan P.K. Knudsen E.S. Hofmann I.A. Snyder J.D. Bove K.E. Fukasawa K. Cell. 2000; 103: 127-140Abstract Full Text Full Text PDF PubMed Scopus (565) Google Scholar), and reduces stability of the ARF tumor suppressor (22Colombo E. Marine J.C. Danovi D. Falini B. Pelicci P.G. Nat. Cell Biol. 2002; 4: 529-533Crossref PubMed Scopus (443) Google Scholar, 23Korgaonkar C. Hagen J. Tompkins V. Frazier A.A. Allamargot C. Quelle F.W. Quelle D.E. Mol. Cell. Biol. 2005; 25: 1258-1271Crossref PubMed Scopus (251) Google Scholar). An interesting novel B23 function is its ability to mediate the anti-apoptotic effects of nerve growth factor by acting as a receptor for phosphatidylinositol (3,4,5)-triphosphate in the nucleus (24Ahn J.Y. Liu X. Cheng D. Peng J. Chan P.K. Wade P.A. Ye K. Mol. Cell. 2005; 18: 435-445Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). In line with these findings, it was demonstrated that B23 overexpression increases cell survival after ultraviolet irradiation, possibly in collaboration with proliferating cell nuclear antigen (25Wu M.H. Chang J.H. Yung B.Y. Carcinogenesis. 2002; 23: 93-100Crossref PubMed Scopus (94) Google Scholar, 26Wu M.H. Yung B.Y. J. Biol. Chem. 2002; 277: 48234-48240Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Paradoxically, in some cell types, loss of B23 can result in apoptosis (3Grisendi S. Bernardi R. Rossi M. Cheng K. Khandker L. Manova K. Pandolfi P.P. Nature. 2005; 437: 147-153Crossref PubMed Scopus (468) Google Scholar, 13Itahana K. Bhat K.P. Jin A. Itahana Y. Hawke D. Kobayashi R. Zhang Y. Mol. Cell. 2003; 12: 1151-1164Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar), which is probably related to the important role of B23 in normal cell function. B23 can bind to histones H3, H4, and H2B, and assemble nucleosomes in vitro, like other members of the nucleoplasmin superfamily (7Namboodiri V.M. Akey I.V. Schmidt-Zachmann M.S. Head J.F. Akey C.W. Structure (Camb.). 2004; 12: 2149-2160Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 27Okuwaki M. Matsumoto K. Tsujimoto M. Nagata K. FEBS Lett. 2001; 506: 272-276Crossref PubMed Scopus (221) Google Scholar). These findings have led to the interesting idea that B23 could be a histone chaperone in the nucleolus (7Namboodiri V.M. Akey I.V. Schmidt-Zachmann M.S. Head J.F. Akey C.W. Structure (Camb.). 2004; 12: 2149-2160Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Indeed, B23 can associate with chromatin to stimulate acetylation-dependent transcription (28Swaminathan V. Kishore A.H. Febitha K.K. Kundu T.K. Mol. Cell. Biol. 2005; 25: 7534-7545Crossref PubMed Scopus (149) Google Scholar). To further investigate the physiological function of B23, we set out to identify novel B23-associated proteins using an unbiased approach. We identified ribosomal protein S9 as being one of the most abundant soluble B23-binding proteins, supporting a role for B23 in ribosome biogenesis. Furthermore, we could show an important role of S9 in cell growth and proliferation and that B23 promotes the stability and nucleolar localization of S9. Cell Culture—U2OS osteosarcoma, H1299 lung carcinoma, and normal human fibroblasts WI38 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mm l-glutamine, and penicillin-streptomycin in a humidified incubator. To generate stable S9-FLAG or S9-GFP cell lines, parental U2OS cells were transfected with each plasmid separately and selected in 400 μg/ml of G418 (Sigma). Stable clones were expanded and analyzed for S9 expression. Immunoprecipitation—For mass spectrometry analysis, sub-confluent U2OS cells were cultured in p100 plates and infected with adenovirus expressing Myc-tagged B23. Cells were lysed in 0.1% Nonidet P-40 lysis buffer followed by immunoprecipitation (IP) using a rabbit Myc polyclonal antibody as indicated. In some experiments RNase A (100 μg/ml) (Sigma) was included during the preparation of the lysate and during co-IP. Proteins were resolved on SDS-PAGE and the gels were stained with either silver or Coomassie Blue (Bio-Rad) according to standard protocols. Visible bands were cut out and analyzed using mass spectrometry at the University of North Carolina Chapel Hill core facility. Details for Western blotting and IP have been published (29Enomoto T. Lindstrom M.S. Jin A. Ke H. Zhang Y. J. Biol. Chem. 2006; 281: 18463-18472Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Plasmids and Transfections—Full-length cDNA encoding human ribosomal protein S9 (RPS9) cDNA (clone MGC, 5482; IMAGE, 3452221; accession number, BC000802) was subcloned using standard PCR methods from pCMV-SPORT6 (Invitrogen) and into the pCDNA3 FLAG-3C, pCDNA3, pEGFP-N1, and pGEX-3X plasmids. Primer sequences and PCR conditions are available upon request. Cells were transfected using FuGENE 6 reagent according to the manufacturer's protocol (Roche). Plasmids encoding B23 were recently described (29Enomoto T. Lindstrom M.S. Jin A. Ke H. Zhang Y. J. Biol. Chem. 2006; 281: 18463-18472Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). RNA Interference—Three small interfering RNA (siRNA) oligos targeting human S9 were purchased from Ambion and three others were synthesized using the siRNA construction kit (Ambion). For initial screening of siRNAs stable U2OS S9-GFP cells were transfected and the knockdown was estimated by using Western blotting and cell imaging. From this screen, Ambion RPS9 siRNA number 9201 (sense 5′-GGAUUUCUUAGAGAGACGCTT-3′) was chosen as the main siRNA with the most efficient knockdown. A similar phenotype as described was observed with three other oligos. The siScr and siB23 sequences have been described (13Itahana K. Bhat K.P. Jin A. Itahana Y. Hawke D. Kobayashi R. Zhang Y. Mol. Cell. 2003; 12: 1151-1164Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar). Transfection of siRNA oligos was performed using Oligofectamine according to the manufacturer's instructions (Invitrogen). Antibodies—Two S9-derived peptides synthesized by the UNC Chapel Hill peptide core facility were conjugated to keyhole limpet hemocyanin and used for rabbit immunization to generate novel S9 antisera. The S9 antisera were purified using the Sulfolink column purification kit (Pierce). The specificity and affinity were tested on recombinant GST-S9 using whole cell extracts. Antiserum S9-009 (CRKTYTPRRPFEKSR) was used for straight Western blotting and immunofluorescence. Antiserum S9-162 (RSPYGGGRPGRVKRKNC) was used in Western blotting, co-immunoprecipitation, and immunofluorescence applications. Purified antisera toward L5, L11, and L23 have been described (30Jin A. Itahana K. O'Keefe K. Zhang Y. Mol. Cell. Biol. 2004; 24: 7669-7680Crossref PubMed Scopus (314) Google Scholar, 31Zhang Y. Wolf G.W. Bhat K. Jin A. Allio T. Burkhart W.A. Xiong Y. Mol. Cell. Biol. 2003; 23: 8902-8912Crossref PubMed Scopus (460) Google Scholar). Rabbit anti-Myc and rabbit anti-FLAG antibodies were generously provided by Yue Xiong (UNC Chapel Hill). Monoclonal antibodies toward Myc (clone 9E10), anti-FLAG clone M2 (Sigma), β-actin (Sigma), α,β-tubulin (Santa Cruz), ribosomal protein S6 (Cell Signaling Technology), MDM2 (clone 2A10), p53 (clone DO1, Lab Vision-Neomarkers), p21 (HZ52, LabVision-Neomarkers), B23/NPM (Zymed Laboratories Inc.), and C23/nucleolin (Santa Cruz) were purchased commercially. In Vitro Translation and Glutathione S-Transferase (GST) Pull-down Assay—Promegas TnT-coupled kit was used for in vitro transcription and translation (IVT) according to the manufacturer's instructions. Purification, expression, and elution of GST fusion proteins from beads were carried out according to standard protocols (Amersham Biosciences). For the in vitro binding assay, GST fusion proteins bound to glutathione-Sepharose 4B beads were incubated with 2–4 μl of IVT product in a final volume of 200 μl of binding buffer (29Enomoto T. Lindstrom M.S. Jin A. Ke H. Zhang Y. J. Biol. Chem. 2006; 281: 18463-18472Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar) and rotated at +4 °C overnight followed by extensive washing in binding buffer. Bound material was eluted from the beads in sample buffer and resolved using SDS-PAGE followed by Coomassie Blue staining, gel drying, and autoradiography. Immunofluorescence—Procedures for immunofluoresence have been described (29Enomoto T. Lindstrom M.S. Jin A. Ke H. Zhang Y. J. Biol. Chem. 2006; 281: 18463-18472Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). In brief, cells growing in 6-well plates were washed in phosphate-buffered saline, fixed with 4% paraformaldehyde (Sigma), permeabilized with 0.2% Triton X-100 (Sigma) for 3 min, and kept 30 min in blocking buffer (0.5% bovine serum albumin in 1× phosphate-buffered saline). Following primary and secondary antibody incubations in blocking buffer with extensive washings in between, the stained cells were analyzed using an Olympus IX-81 microscope with SPOT-camera and software as described. Infection—To generate Ad-S9, Ad-S9-EGFP-N1, and Ad-S9-FLAG3C the corresponding DNA was amplified using PCR and inserted into the pCMV-Shuttle vector. Adenoviruses were produced in 293QBT cells according to the manufacturer's instructions (Stratagene). Cells were infected in Dulbecco's modified Eagle's medium with 0.1% fetal bovine serum for 2 h at which time fresh complete medium was added. Ad-MycB23 and Ad-GFP have been described (13Itahana K. Bhat K.P. Jin A. Itahana Y. Hawke D. Kobayashi R. Zhang Y. Mol. Cell. 2003; 12: 1151-1164Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar). RNA and Protein Synthesis Measurements—For estimation of de novo protein synthesis cells were starved in methionine-free Dulbecco's modified Eagle's medium supplemented with dialyzed fetal bovine serum for 30 min followed by a 2-h pulse with [35S]Met (Amersham Biosciences). Protein extracts were prepared in 0.5% Nonidet-P40 lysis buffer and total [35S]Met incorporation was measured using liquid scintillation counting and expressed as counts per minute/μg of protein. Protein concentrations were determined using the Bradford reagent and readings at 595 nm. To determine the half-life of S9, U2OS cells were harvested at the indicated time points, followed by Western blotting and autoradiography. Actinomycin D (Sigma) at a final concentration of 5 nm or dimethyl sulfoxide (DMSO) as vehicle control were added and maintained throughout the assay. For estimation of the ribosomal RNA synthesis rate, pulse-chase experiments using [methyl-3H]methionine were carried out. Cells on 60-mm plates were starved of methionine for 30 min with methionine-free medium and then labeled with l-[methyl-3H]methionine (Amersham Biosciences) for 30 min. After washing with phosphate-buffered saline, total RNA was purified using TRIzol reagent (Invitrogen) and the incorporated radioactivity was measured by a liquid scintillation counter. Cell Proliferation and Flow Cytometry—For measurements of proliferation, cells were seeded in 6-well plates and transfected followed by counting each day using trypan blue in triplicate. For flow cytometric analysis, cells were collected after the indicated treatments by trypsinization, fixed with 70% ethanol, stained with propidium iodide, and analyzed as described previously (32Zhang Y. Xiong Y. Yarbrough W.G. Cell. 1998; 92: 725-734Abstract Full Text Full Text PDF PubMed Scopus (1411) Google Scholar). B23 Is Associated with a Ribosomal Protein-RNA Helicase Complex—To identify novel B23 interacting proteins we performed a co-IP experiment using lysates from U2OS osteosarcoma tumor cells that had been infected with adenovirus expressing Myc-tagged B23 (Ad-Myc-B23). We found that soluble Myc-B23 protein was associated with a protein complex composed of ribosomal proteins (r-proteins) and RNA helicases, most likely representing pre-ribosomal particles at various stages of their maturation (Fig. 1A). Among the most prominent non-ribosomal proteins associated with B23 were RNA helicase A (DHX9), RNA helicase Gu/II (DDX21), and DEAD box RNA helicase 1 (DDX1). In addition, we were able to identify over 15 r-proteins of both the large and small ribosome subunit (Table 1). Not all bands were chosen for analysis as they indeed were assumed r-proteins. Given the suspected RNA-dependent interactions within this complex we included RNase A (100 μg/ml) before and during immunoprecipitation. We could then observe Myc-B23 in a complex with much fewer proteins and the most prominent of those were endogenous B23, which can oligomerize with Myc-B23 (5Hingorani K. Szebeni A. Olson M.O. J. Biol. Chem. 2000; 275: 24451-24457Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 29Enomoto T. Lindstrom M.S. Jin A. Ke H. Zhang Y. J. Biol. Chem. 2006; 281: 18463-18472Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), and ribosomal protein S9 (Fig. 1B). In total, over 42 peptides matching S9 was identified by mass spectrometry (Table 2).TABLE 1B23 associated proteinsProteinAccessionMol. mass (Da)PeptidesaNumber of peptides that match the theoretical digest of the primary protein identified.MS and MS/MS score UbScore of the quality of the peptide-mass fingerprint match and the quality of the MS/MS peptide fragment ion matches (if MS/MS data was generated). Scores of 95 or greater are considered significant.RNA helicase AAAB48855141,979.79172DDX21AAH08071872,90.428461DDX1Q6PJR177,811.524387RPS3ACAE9897429,286.4494RPS6AAH0942728,403.85117RPL8R5RTL828,007.37308RPL7aR5HU7A29,9777188RPL7R5HU729,207.210300RPS9BAC3433022,577.614353RPL18S3835221,621.17123RPL21S5591318,553.14159RPL11AA1897020,111.6370RPL26AAA6027917,277.56123RPL12Q9NQ0217,831.4398RPS11R3HU1118,419584RPS13R3RT1317,211.710233RPL28RL28_HUMAN15,606.6591a Number of peptides that match the theoretical digest of the primary protein identified.b Score of the quality of the peptide-mass fingerprint match and the quality of the MS/MS peptide fragment ion matches (if MS/MS data was generated). Scores of 95 or greater are considered significant. Open table in a new tab TABLE 2Examples of S9 peptides identified by MS/MSPositionSequence110-116LQTQVFK156-162HIDFSLR18-24RPFEKSR163-172SPYGGGRPGR71-79LFEGNALLR102-109IEDFLERR140-150QVVNIPSFIVR59-69ELLTLDEKDPR139-150KQVVNIPSFIVR Open table in a new tab Ribosomal Protein S9 Is a Novel B23 Interacting Protein—To further investigate the interaction between S9 and B23, a reciprocal co-IP using lysates from adenovirus-infected U2OS cells was carried out. We could easily detect an interaction between FLAG-S9 and Myc-B23 using antibodies against either tag in co-IP (Fig. 2A). To estimate the strength of the interaction we incubated bacterially produced and purified S9-GST protein with HeLa cell lysate in increasing concentrations of NaCl in the presence of RNase A and DNase. An interaction between S9-GST and B23 could be formed in 0.6 m NaCl (Fig. 2B), a salt concentration many protein-protein interactions that rely only on weak ionic interactions cannot withstand (33Huang N. Negi S. Szebeni A. Olson M.O. J. Biol. Chem. 2005; 280: 5496-5502Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). To demonstrate the interaction between endogenous S9 and B23, we raised and validated specific antisera against human S9. Antibodies toward S9, but not preimmune serum, could co-IP B23 (Fig. 2C). Similarly, immunoprecipitation of B23 could bring down endogenous S9, whereas no interaction was seen between B23 and ribosomal proteins S6 or L11, when RNase was included (Fig. 2D). In a co-IP using FLAG antibody and lysate from U2OS cells infected with Ad-S9-FLAG followed by silver staining we could estimate that S9 brings down B23 in an approximate 1:1 ratio (Fig. 2E). We next analyzed the consequences of RNase A treatment on the solubility of endogenous S9 in the Nonidet P-40 extraction buffer. We found that whereas p53, B23, and other r-proteins (L5, L11, and L23) remained soluble in the presence of RNase A, S9 protein instead become more insoluble. Hence, more S9 protein was found in the pellet after extraction in lysis buffer supplemented with RNase A (supplemental Fig. S1). This suggests that S9 is prone to aggregation in the absence of RNA. Importantly, we also detected an increased binding between S9 and B23 in serum-deprived U2OS cells, without the use of RNase A, indicating that a pool of S9 protein becomes tethered to B23 during cellular growth arrest (supplemental Fig. S2). An Intact Oligomerization Domain of B23 Is Required for Efficient S9 Binding—To define the protein domain in B23 that is required for binding S9 we carried out co-IP and GST pull-down experiments using a series of B23 deletion mutants. Whereas WT B23, and B23(10–294) bound to S9-FLAG in cells (Fig. 3A) and on S9-GST beads (Fig. 3B), further sequential deletions of the N-terminal region of B23 abolished most, if not all, B23-S9 binding (Fig. 3, A and B). These results may suggest that the binding region for S9 resides within amino acid residues 10–25 in B23. However, since WT and B23(10–294) mutant form oligomers whereas the B23(25–294) mutant and others are severely impaired in oligomerization activity (5Hingorani K. Szebeni A. Olson M.O. J. Biol. Chem. 2000; 275: 24451-24457Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 7Namboodiri V.M. Akey I.V. Schmidt-Zachmann M.S. Head J.F. Akey C.W. Structure (Camb.). 2004; 12: 2149-2160Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 29Enomoto T. Lindstrom M.S. Jin A. Ke H. Zhang Y. J. Biol. Chem. 2006; 281: 18463-18472Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), the B23-S9 interaction can also depend on B23 oligomerization. In fact, the binding of S9 to B23 was completely eliminated when removing the 100–117 highly conserved region in the B23 core region (Fig. 3B, lane 6), or when introducing point mutations in the B23 oligomerization domain (data not shown), similar to that which was observed in the case of the ARF tumor suppressor (29Enomoto T. Lindstrom M.S. Jin A. Ke H. Zhang Y. J. Biol. Chem. 2006; 281: 18463-18472Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Strong binding was seen between S9 and the B23(1–192) mutant suggesting that the B23 oligomerization domain cooperates with the central acidic domains of B23 in binding S9 (Fig. 3A, lane 9). Binding data and status of B23 oligomerization is summarized in Fig. 3, C and D. We conclude that B23 binding to S9 is dependent on an intact B23 oligomerization domain. S9 Is Required for Normal Cell Proliferation—To investigate the physiological role of S9 in cell growth we used siRNA to deplete S9 in primary human fibroblasts (WI38) and the WT p53 containing tumor cell line U2OS. From an initial screening of six siRNA oligonucleotides one S9 siRNA oligo was chosen with 90% knockdown of free (non-ribosome bound) endogenous S9 and without adverse toxicity. 72 h after transfection the cell proliferation rate was reduced by more than 50% compared with control cells (siScr) (Fig. 4B), and S9 knockdown cells exhibited a flat morphology (Fig. 4A). This apparent growth arrest indeed corresponded to an increase in the G1 phase cell population and a decreased number of cells in the S and G2 phase according to flow cytometric analysis (Fig. 4C). Similar results were also obtained using a second siRNA with an approximate 60% S9 silencing (data not shown). Next, we examined whether the knockdown of S9 was associated with p53 activation and induction of p53 target genes p21(CDKN1A) and MDM2. This was investigated because the p53 tumor suppressor pathway often becomes activated after nucleolar or ribosomal stress and mediates a G1 cell cycle arrest (31Zhang Y. Wolf G.W. Bhat K. Jin A. Allio T. Burkhart W.A. Xiong Y. Mol. Cell. Biol. 2003; 23: 8902-8912Crossref PubMed Scopus (460) Google Scholar, 34Lindstrom M.S. Deisenroth C. Zhang Y. Cell Cycle. 2007; 6: 434-437Crossref PubMed Scopus (53) Google Scholar). As is shown in Fig. 4D, G1 cell cycle arrest in U2OS was associated with a pronounced increase in p21 and a minor increase in MDM2 (Fig. 4D, left panel). The basal level of the WT p53 protein is elevated in the U2OS tumor cell line compared with primary WI38 cells and did not further increase upon S9 depletion or siRNA transfection per se. Primary WI38 fibroblasts showed both an increase in p53 protein level and a modest p21 increase but markedly elevated levels of MDM2 protein (Fig. 4D, right panel). To investigate whether ribosome function could be altered as a consequence of S9 loss, we measured protein synthesis in cells. I