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Fbx7 Functions in the SCF Complex Regulating Cdk1-Cyclin B-phosphorylated Hepatoma Up-regulated Protein (HURP) Proteolysis by a Proline-rich Region

2004; Elsevier BV; Volume: 279; Issue: 31 Linguagem: Inglês

10.1074/jbc.m404950200

1083-351X

Jung-Mao Hsu, Yuan-Chii G. Lee, Chang‐Tze Ricky Yu, Chi‐Ying F. Huang,

Autophagy in Disease and Therapy

F-box proteins, components of SCF ubiquitin-ligase complexes, are believed to be responsible for substrate recognition and recruitment in SCF-mediated proteolysis. F-box proteins that have been identified to function in the SCF complexes to date mostly have substrate-binding motifs, such as WD repeats or leucine-rich repeats in their C termini. However, many F-box proteins lack recognizable substrate-binding modules; whether they can function in the SCF complexes remains unclear. We show here that Fbx7, an F-box protein without WD repeats and leucine-rich repeats, is required for the proteasome-mediated proteolysis of the hepatoma up-regulated protein (HURP). Depletion of Fbx7 by small interfering RNA leads to depression of HURP ubiquitination and accumulation of HURP abundance. In the SCFFbx7 complex, Fbx7 recruits HURP through its C-terminal proline-rich region in a Cdk1-cyclin B-phosphorylation dependent manner. Mutation of the multiple Cdk1-cyclin B phosphorylation sites on HURP or the proline-rich region of Fbx7 abolishes the association between Fbx7 and HURP. Thus, Fbx7 is a functional adaptor of the SCF complex with a proline-rich region as the substrate-binding module. In addition to Fbx7, data base analyses reveal two putative mammalian proline-rich region-containing F-box proteins, KIAA1783 and RIKEN cDNA 2410015K21. Taken together, these findings further expound the diverse substrate-recognition abilities of the SCF complexes. F-box proteins, components of SCF ubiquitin-ligase complexes, are believed to be responsible for substrate recognition and recruitment in SCF-mediated proteolysis. F-box proteins that have been identified to function in the SCF complexes to date mostly have substrate-binding motifs, such as WD repeats or leucine-rich repeats in their C termini. However, many F-box proteins lack recognizable substrate-binding modules; whether they can function in the SCF complexes remains unclear. We show here that Fbx7, an F-box protein without WD repeats and leucine-rich repeats, is required for the proteasome-mediated proteolysis of the hepatoma up-regulated protein (HURP). Depletion of Fbx7 by small interfering RNA leads to depression of HURP ubiquitination and accumulation of HURP abundance. In the SCFFbx7 complex, Fbx7 recruits HURP through its C-terminal proline-rich region in a Cdk1-cyclin B-phosphorylation dependent manner. Mutation of the multiple Cdk1-cyclin B phosphorylation sites on HURP or the proline-rich region of Fbx7 abolishes the association between Fbx7 and HURP. Thus, Fbx7 is a functional adaptor of the SCF complex with a proline-rich region as the substrate-binding module. In addition to Fbx7, data base analyses reveal two putative mammalian proline-rich region-containing F-box proteins, KIAA1783 and RIKEN cDNA 2410015K21. Taken together, these findings further expound the diverse substrate-recognition abilities of the SCF complexes. The ubiquitin-proteasome pathway plays a central role in regulation of many biological processes, including cell cycle progression, transcription, and signal transduction. The formation of ubiquitin-protein conjugates requires three components: a ubiquitin-activating enzyme (E1), 1The abbreviations used are: E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase; HURP, hepatoma up-regulated protein; PRR, proline-rich region; HA, hemagglutinin; siRNA, small interfering RNA; PIPES, 1,4-pipera-zinediethanesulfonic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. 1The abbreviations used are: E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase; HURP, hepatoma up-regulated protein; PRR, proline-rich region; HA, hemagglutinin; siRNA, small interfering RNA; PIPES, 1,4-pipera-zinediethanesulfonic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. a ubiquitin-conjugating enzyme (E2), and a ubiquitin ligase (E3). Ubiquitinated proteins are rapidly degraded by the 26 S proteasome. In the ubiquitin-mediated proteolysis, E3s play a major role in providing specificity of substrate recognition and timing for ubiquitin-dependent proteolysis (1Laney J.D. Hochstrasser M. Cell. 1999; 97: 427-430Google Scholar, 2Hershko A. Ciechanover A. Annu. Rev. Biochem. 1998; 67: 425-479Google Scholar). The SCF (Skp1, Cdc53/Cullin1, F-box protein) complex, one of the E3s, is a highly diverse multicomponent complex consisting of common components, such as Skp1, Cullin1, and Rbx1, as well as variable components known as F-box proteins (3Tyers M. Jorgensen P. Curr. Opin. Genet. Dev. 2000; 10: 54-64Google Scholar, 4Deshaies R.J. Annu. Rev. Cell Dev. Biol. 1999; 15: 435-467Google Scholar, 5Craig K.L. Tyers M. Prog. Biophys. Mol. Biol. 1999; 72: 299-328Google Scholar). By biochemical and topological studies, the SCF complex is known to utilize F-box protein to recruit phosphorylated substrate to its core (2Hershko A. Ciechanover A. Annu. Rev. Biochem. 1998; 67: 425-479Google Scholar, 4Deshaies R.J. Annu. Rev. Cell Dev. Biol. 1999; 15: 435-467Google Scholar, 6Zheng N. Schulman B.A. Song L. Miller J.J. Jeffrey P.D. Wang P. Chu C. Koepp D.M. Elledge S.J. Pagano M. Conaway R.C. Conaway J.W. Harper J.W. Pavletich N.P. Nature. 2002; 416: 703-709Google Scholar, 7Kipreos E.T. Pagano M. Genome Biol. 2000; 1: 3002.1-3002.7Google Scholar). Thus, the substrate specificity of the SCF complex is thought to depend on the F-box protein component. Each F-box protein contains an N-terminal motif of ∼40 amino acids, known as the F-box, which links the F-box protein to other components of the SCF complex by binding Skp1 (5Craig K.L. Tyers M. Prog. Biophys. Mol. Biol. 1999; 72: 299-328Google Scholar, 8Bai C. Sen P. Hofmann K. Ma L. Goebl M. Harper J.W. Elledge S.J. Cell. 1996; 86: 263-274Google Scholar). In general, F-box proteins have additional C-terminal protein-protein interaction motifs responsible for substrate binding. The two most common motifs are WD repeats (9Neer E.J. Schmidt C.J. Nambudripad R. Smith T.F. Nature. 1994; 371: 297-300Google Scholar) and leucine-rich repeats (10Kobe B. Deisenhofer J. Trends Biochem. Sci. 1994; 19: 415-421Google Scholar), both of which have been found to bind phosphorylated substrates and recruit them to the SCF complexes (11Skowyra D. Craig K.L. Tyers M. Elledge S.J. Harper J.W. Cell. 1997; 91: 209-219Google Scholar). Substrate recognition by the SCF complex is thought to require the same core components (i.e. Cullin1, Skp1, and Rbx1) but to utilize various F-box proteins. The ability of the same core to bind multiple F-box proteins increases the substrate repertoire. Modern advances in genome sequencing and molecular cloning have led to the identification of numerous F-box proteins, which are classified into three subfamilies according to the C-terminal substrate-binding motifs that they contain (7Kipreos E.T. Pagano M. Genome Biol. 2000; 1: 3002.1-3002.7Google Scholar, 12Cenciarelli C. Chiaur D.S. Guardavaccaro D. Parks W. Vidal M. Pagano M. Curr. Biol. 1999; 9: 1177-1179Google Scholar, 13Winston J.T. Koepp D.M. Zhu C. Elledge S.J. Harper J.W. Curr. Biol. 1999; 9: 1180-1182Google Scholar). The Fbw and Fbl families represent the F-box proteins containing WD repeats and leucine-rich repeats, respectively, and have been shown to function in the SCF complexes and recruit substrates for ubiquitination. The Fbx family represents the F-box proteins without known substrate-binding motifs and includes the majority of F-box proteins. However, only limited numbers of Fbx family members, such as Fbx2, Fbx4, and Fbx6b (14Yoshida Y. Chiba T. Tokunaga F. Kawasaki H. Iwai K. Suzuki T. Ito Y. Matsuoka K. Yoshida M. Tanaka K. Tai T. Nature. 2002; 418: 438-442Google Scholar, 15den Engelsman J. Keijsers V. de Jong W.W. Boelens W.C. J. Biol. Chem. 2003; 278: 4699-4704Google Scholar, 16Yoshida Y. Tokunaga F. Chiba T. Iwai K. Tanaka K. Tai T. J. Biol. Chem. 2003; 278: 43877-43884Google Scholar), have been recently demonstrated to function in the SCF complexes. The structural and functional characterization of the rest of the Fbx family members still needs to be defined. Whether these novel Fbx family members function in the SCF complexes, as do those of the Fbw and Fbl families, is one of the central issues in current studies of ubiquitin-mediated proteolysis, especially because F-box proteins have also been found to be associated with various biological pathways other than SCF-mediated proteolysis (7Kipreos E.T. Pagano M. Genome Biol. 2000; 1: 3002.1-3002.7Google Scholar, 17Kim J. Kim J.H. Lee S.H. Kim D.H. Kang H.Y. Bae S.H. Pan Z.Q. Seo Y.S. J. Biol. Chem. 2002; 277: 24530-24537Google Scholar, 18Gomes M.D. Lecker S.H. Jagoe R.T. Navon A. Goldberg A.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14440-14445Google Scholar). The gap causing slow progress in this field is due mainly to the lack of substrates. Research to identify the downstream targets of these novel Fbx family members is needed to fill this gap. Hepatoma up-regulated protein (HURP) was initially identified through bioinformatics analysis that was performed to search for potentially important regulatory genes involved in growth control of human hepatocellular carcinoma (19Tsou A.P. Yang C.W. Huang C.Y. Yu R.C. Lee Y.C. Chang C.W. Chen B.R. Chung Y.F. Fann M.J. Chi C.W. Chiu J.H. Chou C.K. Oncogene. 2003; 22: 298-307Google Scholar). Empirical characterization in HeLa cells indicates that HURP is a cell cycle-regulated gene with an elevated expression in the G2/M phase, followed by a sharp decline in early to middle G1 phase. Immunofluorescence studies reveal that HURP localizes to the spindle poles during mitosis. Overexpression of HURP in 293T cells results in an enhanced cell growth at low serum levels and at polyhema-based, anchorage-independent growth conditions (19Tsou A.P. Yang C.W. Huang C.Y. Yu R.C. Lee Y.C. Chang C.W. Chen B.R. Chung Y.F. Fann M.J. Chi C.W. Chiu J.H. Chou C.K. Oncogene. 2003; 22: 298-307Google Scholar). Furthermore, elevated gene expression of HURP and its related truncated form KIAA0008 is highly associated with human hepatocellular carcinoma, colon cancer, breast cancer and transitional cell carcinoma (19Tsou A.P. Yang C.W. Huang C.Y. Yu R.C. Lee Y.C. Chang C.W. Chen B.R. Chung Y.F. Fann M.J. Chi C.W. Chiu J.H. Chou C.K. Oncogene. 2003; 22: 298-307Google Scholar, 20Bassal S. Nomura N. Venter D. Brand K. McKay M.J. van der Spek P.J. Genomics. 2001; 77: 5-7Google Scholar, 21Chiu A.W. Huang Y.L. Huan S.K. Wang Y.C. Ju J.P. Chen M.F. Chou C.K. Urology. 2002; 60: 181-185Google Scholar, 22Huang Y.L. Chiu A.W. Huan S.K. Wang Y.C. Ju J.P. Lu C.L. Anticancer Res. 2003; 23: 2729-2733Google Scholar), suggesting that HURP may have a role in carcinogenesis. Here, we demonstrate that HURP undergoes proteasome-mediated proteolysis and the SCFFbx7 complex is the corresponding upstream E3 ligase. Fbx7, a member of the Fbx family (12Cenciarelli C. Chiaur D.S. Guardavaccaro D. Parks W. Vidal M. Pagano M. Curr. Biol. 1999; 9: 1177-1179Google Scholar, 13Winston J.T. Koepp D.M. Zhu C. Elledge S.J. Harper J.W. Curr. Biol. 1999; 9: 1180-1182Google Scholar), functions as an adaptor recruiting Cdk1-cyclin B-phosphorylated HURP to the core SCF complex. Fbx7 interacts with HURP through its C-terminal proline-rich region (PRR), suggesting that PRR is a new substrate-binding module of F-box proteins other than WD repeats and leucine-rich repeats. By data base search, we further identify two putative PRR-containing F-box proteins, KIAA1783 and RIKEN cDNA 2410015K21. Thus, these PRR-containing molecules might constitute a new subfamily of mammalian F-box proteins. Chemicals and Antibodies—Protease inhibitors: leupeptin, aprotinin, phenylmethylsulfonyl fluoride, and pepstatin; proteasome inhibitors: MG132 (N-CBZ-Leu-Leu-leucinal), proteasome inhibitor I, and lactacystin; calpain inhibitors: ALLN (calpain inhibitor I, MG 101) and ALLM (calpain inhibitor II); and Cdk1-specific inhibitors: roscovitine and olomoucine, were purchased from Sigma and Calbiochem. Cdk1-cyclin B and λ phosphatase were purchased from New England Biolabs. Anti-HURP polyclonal antibody was raised against whole molecular recombinant HURP protein (19Tsou A.P. Yang C.W. Huang C.Y. Yu R.C. Lee Y.C. Chang C.W. Chen B.R. Chung Y.F. Fann M.J. Chi C.W. Chiu J.H. Chou C.K. Oncogene. 2003; 22: 298-307Google Scholar). Anti-Cdk1 monoclonal antibody was purchased from CHEMICON. Anti-Skp1 and anti-Cullin1 monoclonal antibodies were purchased from BD Transduction. Anti-HA monoclonal antibody (3F10) was purchased from Roche Diagnostics. Anti-FLAG monoclonal antibody (M5) was purchased from Sigma. Anti-Fbx7 antibody was purchased from Zymed. Anti-ubiquitin antibody was purchased from Santa Cruz Biotechnology and MPM2 antibody was purchased from Upstate. Alkaline phosphatase-conjugated or horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibody were purchased from PerkinElmer Life Sciences and used in 1:2000-1:5000 dilutions. Cell Culture, Cell-cycle Synchronization, Transfection, and Immunofluorescence Analysis—293T cells were cultured in Dulbecco's modified Eagle's medium with 10% heat-inactivated fetal bovine serum and 2 mm glutamine (Invitrogen) and maintained in a humidified incubator at 37 °C in the presence of 5% CO2. To synchronize 293T cells in the G2/M phase, exponentially growing cells were incubated with 100 ng/ml nocodazole (Sigma) for 16 h. Cells were released from the G2/M block by removing the nocodazole. Continuous time point samples were collected for various assays. Transient transfection of cells was performed with LipofectAMINE (Invitrogen) according to the manufacturer's instructions. Analysis of the subcellular localization of various proteins by indirect immunofluoresence was carried out as previously described (19Tsou A.P. Yang C.W. Huang C.Y. Yu R.C. Lee Y.C. Chang C.W. Chen B.R. Chung Y.F. Fann M.J. Chi C.W. Chiu J.H. Chou C.K. Oncogene. 2003; 22: 298-307Google Scholar). Construction of Expression Vectors and Site-directed Mutagenesis—Human full-length HURP cDNA (designated as HURP-WT) and Cdk1 cDNA were subcloned into FLAG-CMV2 (Kodak) or a modified version of pcDNA3.0 (Invitrogen), with an HA epitope tag for mammalian expression, or T7 promoter-driven vectors for the in vitro coupled transcription-translation reaction. The HURP phosphorylation site mutant (HURP-PM) and various Fbx7-truncated mutants (Fbx7-ΔN, Fbx7-ΔF, Fbx7-ΔC, and Fbx7-ΔP) were generated by PCR-based mutagenesis (QuikChange™ Site-directed mutagenesis kit, Stratagene). FLAG-tagged F-box protein constructs used in this study were kindly provided by Dr. Michele Pagano (New York University School of Medicine). Preparation of Cell Lysates, Immunoprecipitation, and Western Blotting Analysis—To prepare cell-free lysates, cells were harvested, washed with phosphate-buffered saline, and lysed in extraction buffer, which was composed of 50% lysate buffer (20 mm PIPES, pH 7.2, 100 mm NaCl, 1 mm EDTA, 0.1% CHAPS, 10% sucrose, 1 mm phenylmethylsulfonyl fluoride, 1 mm dithiothreitol, 1 mm Na3VO4, and 10 μg/ml each of leupeptin, aprotinin, chymostatin, and pepstatin) and 50% IP washing buffer (10 mm Hepes, pH 7.6, 2 mm MgCl2, 50 mm NaCl, 5 mm EGTA, 0.1% Triton X-100, and 40 mm β-glycerolphosphate). After incubation at 4 °C for 30 min, cellular debris was removed by centrifugation at 13,000 × g for 30 min. Protein concentrations were determined using BCA Protein Assay reagents (Pierce). In general, 1 mg of total cell lysate was incubated with antibodies against target epitopes and Protein A/G-agarose beads (Oncogene Research Product) to immunoprecipitate the target proteins. The immunoprecipitation products were resolved by 8-15% SDS-PAGE. Proteins were transferred to polyvinylidene difluoride membrane (Millipore) and detected with various antibodies. The complexed IgGs were detected by incubation with secondary antibodies conjugated to alkaline phosphatase or horseradish peroxidase, and developed using the nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate or the Western Lighting system (PerkinElmer Life Sciences). Preparation of Recombinant Protein, in Vitro Kinase Reaction, and Phosphorylation Site Determination—Human HURP recombinant proteins were prepared using a bacterial expression system. HURP cDNA was subcloned into expression vector pET32a (Novagen) and expressed as His-tagged fusion protein. The proteins were expressed and trapped in inclusion bodies and solubilized with 2 m urea. The solubilized proteins were partially purified by nickel-agarose (Qiagen) under denaturing conditions. The purified proteins were then dialyzed to remove the denaturant as described in the manufacturer's manual. The purified recombinant HURP proteins were incubated with bauclovirus-expressed human Cdk1-cyclin B complex (New England Biolabs) in kinase buffer (50 mm Tris-HCl, 10 mm MgCl2, 1 mm EGTA, 2 mm dithiothreitol, and 0.01% Brij 35, pH 7.5) containing [γ-33P]ATP. After incubation at 30 °C for 30 min, samples were subjected to SDS-PAGE and phosphorylated bands were visualized by autoradiography. For phosphorylation site determination, Escherichia coli expressed recombinant HURP proteins were incubated with Cdk1-cyclin B in kinase buffer containing ATP. After reaction for 30 min, samples were subjected to SDS-PAGE and stained with Coomassie Blue. HURP proteins were sliced from the gel and phosphorylation sites were determined by LC MS/MS analysis as previously described (23Tsay Y.G. Wang Y.H. Chiu C.M. Shen B.J. Lee S.C. Anal. Biochem. 2000; 287: 55-64Google Scholar). [35S]Methionine-labeled proteins were prepared by coupled transcription-translation reactions in rabbit reticulocyte lysates (Promega). Ubiquitination Assay—293T cells were transfected with expression vectors encoding HA-tagged HURP or Myc-tagged ubiquitin separately or together. 48 h after transfection, cells were harvested and lysed. Equal amounts of total lysates were subjected to immunoprecipitation with anti-HA antibody. The immunoprecipitation products were analyzed by Western blotting with anti-HA or anti-ubiquitin antibodies. Cycloheximide Inhibition Assay—The turnover rates of proteins were determined using cycloheximide inhibition of protein synthesis (24Zhao J. Tenev T. Martins L.M. Downward J. Lemoine N.R. J. Cell Sci. 2000; 113: 4363-4371Google Scholar, 25Patrick G.N. Zhou P. Kwon Y.T. Howley P.M. Tsai L.H. J. Biol. Chem. 1998; 273: 24057-24064Google Scholar). Cycloheximide blocks protein synthesis through interaction with the translocase enzyme in eukaryotic cells. Cells were treated with 50 μg/ml cycloheximide (Sigma) and harvested at different stages. Equal amounts of cell lysates were subjected to SDS-PAGE and analyzed by Western blotting. Data Bases Used in This Study—The data bases used in this study includes BLAST and PROSITE. Small Interference RNA (SiRNA)—The following target sequence was used for Fbx7 siRNA: CCCACACCAUUCCAUUCUA. To generate functional siRNA, we designed the gene-specific insert such that it specifies a 19-nucleotide sequence corresponding to nucleotides 1,136-1,154 (CCCACACCATTCCATTCTA) of Fbx7, separated by a 9-nucleotide non-complementary spacer (TTCAAGAGA) from the reverse complement of the same 19-nucleotide sequence. This insert was then subcloned in a vector system, named pSUPER as described (26Brummelkamp T.R. Bernards R. Agami R. Science. 2002; 296: 550-553Google Scholar) to direct the synthesis of siRNA in mammalian cells. HURP Is a Phosphoprotein Regulated by the Ubiquitin-Proteasome Pathway—Previous studies have shown that in HeLa cells the HURP transcript is low in G1 and S phases, but is predominantly expressed in the G2/M phase (19Tsou A.P. Yang C.W. Huang C.Y. Yu R.C. Lee Y.C. Chang C.W. Chen B.R. Chung Y.F. Fann M.J. Chi C.W. Chiu J.H. Chou C.K. Oncogene. 2003; 22: 298-307Google Scholar). We showed here that HURP protein expression levels also underwent periodic changes along with cell cycle progression. 293T cells were synchronized in the G2/M phase by nocodazole block and subsequently released into cell cycle progression by removing nocodazole. Endogenous HURP protein levels in G2/M-arrested cells were analyzed in comparison to those in exponentially growing cells. Western blot showed that HURP protein expression levels were low in exponentially growing cells but accumulated in G2/M-arrested cells, which was coincident with the expression pattern of cyclin B (Fig. 1, A, lanes 1 and 2; B, lanes 1 and 3). Notably, HURP exhibited various degrees of electrophoretic mobility shifts on SDS-PAGE as cells were synchronized in the G2/M phase (Fig. 1, A, lane 2; B, lane 3). These mobility up-shifts were abolished when λ phosphatase was added to lysates, indicating they were because of protein phosphorylation (Fig. 1B, lane 4). To further support this finding, [35S]methionine-labeled HURP was incubated with nocodazole-arrested 293T lysates, which resulted in a reminiscent mobility up-shift pattern (Fig. 1C). The mobility up-shifts of [35S]methionine-labeled HURP were also sensitive to λ phosphatase treatment (Fig. 1C), characteristic of protein phosphorylation. Moreover, simultaneous decreases in the protein quantity and in the degree of phosphorylation of HURP were observed as G2/M-arrested cells were released into the cell cycle progression by removing nocodazole (Fig. 1A). Taken together, these results demonstrate that HURP is a mitotic phosphoprotein that exhibits periodic variations both in the phosphorylation state and in protein quantity as cells progress through the cell cycle. To determine the molecular mechanism involved in the degradation of HURP, 293T cells were treated with three kinds of conventional proteasome inhibitors (lactacystin, proteasome inhibitor I, and MG132) to block HURP from degradation taking place as G2/M-arrested 293T cells were released into cell cycle progression. Because MG132 exhibits additional inhibitory activities against calpain, two calpain inhibitors (ALLM and ALLN) were used as controls. Treatment with proteasome inhibitors, but not calpain inhibitors, could block HURP degradation, implying the involvement of proteasomes in HURP degradation (Fig. 1D). Because ubiquitination is the signal for effective recognition and degradation of proteins by the proteasome, we then investigated whether HURP is a substrate of ubiquitination. 293T cells were transfected with expression vectors encoding HA-tagged HURP or Myc-tagged ubiquitin separately or together. Equal amounts of lysates were subjected to immunoprecipitation with anti-HA antibody followed by Western blotting with anti-HA and anti-ubiquitin antibodies. High molecular weight products accumulated in cells cotransfected with HURP and ubiquitin but not in cells transfected with ubiquitin alone (Fig. 1E), indicating that HURP is a substrate of ubiquitination. Taken together, these results reveal that HURP is a substrate of the ubiquitin-proteasome pathway. Fbx7 Serves as an Adaptor Linking the SCF Complex with HURP—To identify the upstream E3 ligase responsible for HURP ubiquitination, we collected a set of F-box protein expression constructs and assayed for their association with HURP by immunoprecipitation. 293T cells were transfected with expression vectors encoding HA-tagged HURP or FLAG-tagged F-box proteins (Fbw1a, Fbw2, Fbl3a, Fbx4, and Fbx7) separately or together followed by immunoprecipitation and Western blotting analysis. The results showed that Fbx7, but not any of the other F-box proteins tested, physically associated with HURP (Fig. 2A). To determine whether Fbx7 serves as an adaptor and recruits HURP to a SCF complex, we expressed HA-tagged HURP (HA-HURP) and FLAG-tagged Fbx7 (FLAG-Fbx7) separately or together in 293T cells and immunoprecipitated the cell lysates with anti-FLAG antibody. Then, we examined the existence of other SCF components in the immunoprecipitates by Western blotting analysis. Endogenous Skp1 and Cullin1 were detected in the FLAG-Fbx7 immunoprecipitate (Fig. 2B, lane 2). This data, in agreement with previous reports, represented the reconstitution of the SCFFbx7 complex (12Cenciarelli C. Chiaur D.S. Guardavaccaro D. Parks W. Vidal M. Pagano M. Curr. Biol. 1999; 9: 1177-1179Google Scholar). Moreover, co-expression of HA-HURP and FLAG-Fbx7 followed by immunoprecipitation with anti-FLAG antibody resulted in the complex formation of HURP, Fbx7, Skp1, and Cullin1 (Fig. 2B, lane 3). To further illustrate the role of Fbx7 in this complex and test the significance of Fbx7 in the stability of its putative substrate, HURP, we used the siRNA to reduce the expression of Fbx7 and then assessed its effect on HURP ubiquitination. Results showed that HURP ubiquitination was depressed in cells contransfected with plasmid-based double-stranded RNA against Fbx7 (Fig. 2C). Furthermore, we assessed its effect on HURP abundance. HURP accumulated in siRNA-inhibited 293T cells when compared with cells transfected with a control empty vector (Fig. 2D). Collectively, these data indicate that Fbx7 is involved in the regulation of HURP stability. Fbx7 Associates with Cdk1-Cyclin B-phosphorylated HURP—There is compelling evidence that protein phosphorylation is a regulatory factor in the interactions between the substrate of the SCF complex and the F-box protein (11Skowyra D. Craig K.L. Tyers M. Elledge S.J. Harper J.W. Cell. 1997; 91: 209-219Google Scholar, 27Koepp D.M. Schaefer L.K. Ye X. Keyomarsi K. Chu C. Harper J.W. Elledge S.J. Science. 2001; 294: 173-177Google Scholar, 28Spencer E. Jiang J. Chen Z.J. Genes Dev. 1999; 13: 284-294Google Scholar). As shown in Fig. 1A, HURP is a mitotic phosphoprotein, suggesting that protein phosphorylation may provide a mode of regulating the interaction between Fbx7 and HURP. In fact, MPM2 antibody, an antibody specifically recognizing a subset of mitosis-specific phosphoproteins (29Davis F.M. Tsao T.Y. Fowler S.K. Rao P.N. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 2926-2930Google Scholar, 30Engle D.B. Doonan J.H. Morris N.R. Cell Motil. Cytoskeleton. 1988; 10: 434-437Google Scholar), could recognize the immunoprecipitated HURP from nocodazole-arrested cells (Fig. 3A), suggesting that HURP might be the downstream substrate of Cdk1-cyclin B, which has been shown to be able to generate MPM2 reactivity (31Ye X.S. Xu G. Pu R.T. Fincher R.R. McGuire S.L. Osmani A.H. Osmani S.A. EMBO J. 1995; 14: 986-994Google Scholar, 32Westendorf J.M. Rao P.N. Gerace L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 714-718Google Scholar). Therefore, we next addressed whether HURP is a downstream substrate of Cdk1-cyclin B. First, active Cdk1-cyclin B could phosphorylate HURP in vitro. When recombinant HURP protein was added to the kinase reaction in the presence of recombinant Cdk1-cyclin B and [γ-33P]ATP, an [γ-33P]ATP-incorporated band was observed at the 10-min point in the Cdk1-cyclin B-mediated kinase reaction, but not in the control lanes (Fig. 3B). Second, Cdk1-specific inhibitors, roscovitine (33Deng M.Q. Shen S.S. Biol. Reprod. 2000; 62: 873-878Google Scholar) and olomoucine (34Malecz N. Foisner R. Stadler C. Wiche G. J. Biol. Chem. 1996; 271: 8203-8208Google Scholar), partially abolished the mobility up-shifts of HURP as [35S]methionine-labeled HURP was incubated with nocodazole-arrested 293T lysates (Fig. 3C), suggesting that HURP is a potential substrate of Cdk1-cyclin B in the G2/M phase. Third, overexpression of FLAG-tagged HURP and HA-tagged Cdk1 in 293T cells followed by immunoprecipitation with anti-FLAG showed that HURP was coimmunoprecipitated with Cdk1, indicating that HURP can associate with Cdk1 in culture cells (Fig. 3D). Fourth, immunofluoresence analysis also demonstrated that endogenous Cdk1 and HURP colocalized to the mitotic spindles in the metaphase (Fig. 3E). Taken together, these results indicate that HURP is a potential substrate of Cdk1-cyclin B. Cdk1-cyclin B is known to phosphorylate numerous proteins and regulate their stability (35Thom G. Minshall N. Git A. Argasinska J. Standart N. Biochem. J. 2003; 370: 91-100Google Scholar, 36Ke P.Y. Yang C.C. Tsai I.C. Chang Z.F. Biochem. J. 2003; 370: 265-273Google Scholar, 37Castro A. Peter M. Magnaghi-Jaulin L. Vigneron S. Galas S. Lorca T. Labbe J.C. Mol. Biol. Cell. 2001; 12: 2660-2671Google Scholar). To examine whether Cdk1-cyclin B-mediated HURP phosphorylation is the key step in the interaction between HURP and Fbx7, we determined the in vitro Cdk1-cyclin B phosphorylation sites on HURP by LC MS/MS analysis. Nine sites (Ser67, Thr329, Thr401, Thr402, Ser618, Thr639, Ser642, Thr759, and Ser839) were identified with 82% amino acid sequence coverage, indicating that Cdk1-cyclin B can phosphorylate HURP at multiple sites in vitro (Fig. 4A). Initially, we replaced each phosphorylation site individually with alanine and some combination of two phosphorylation site mutations on HURP. These mutants were assayed for their association with Fbx7; however, none of these mutants tested had a significant difference in terms of association with Fbx7 (data not shown), raising the possibility that multisite phosphorylation of HURP may be required for the association with Fbx7. Next, we constructed a HURP mutant in which all nine phosphorylation sites were replaced with alanines (designated as HURP-PM). HURP-PM exhibited a more depressed phosphorylation state than HURP-WT, both as incubating with nocodazole-arrested cell lysates (Fig. 4B) and as expressing in mitotic cells (Fig. 4C), suggesting that Cdk1-cyclin B-mediated HURP phosphorylation could recapitulate in the in vivo situation. Subsequently, we examined whether a complex of Fbx7 and HURP-PM could be detected in 293T cells. The results showed that mutation of all nine Cdk1-cyclin B phosphorylation sites on HURP abolished the association between Fbx7 and HURP (Fig. 4D). In addition, comparisons of