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• W2017321408 abstract "F-box proteins are the substrate-recognition components of the Skp1-Cul1-F box protein (SCF) E3 ubiquitin ligases. Here we report a structural relationship between Fbxo7, a component of the SCFFbxo7 E3 ligase, and the proteasome inhibitor PI31. SCFFbxo7 is known to catalyze the ubiquitination of hepatoma-up-regulated protein (HURP) and the inhibitor of apoptosis (IAP) protein but also functions as an activator of cyclin D-Cdk6 complexes. We identify PI31 as an Fbxo7·Skp1 binding partner and show that this interaction requires an N-terminal domain present in both proteins that we term the FP (Fbxo7/PI31) domain. The crystal structure of the PI31 FP domain reveals a novel α/β-fold. Biophysical and mutational analyses are used to map regions of the PI31 FP domain mediating homodimerization and required for heterodimerization with Fbxo7·Skp1. Equivalent mutations in Fbxo7 ablate interaction with PI31 and also block Fbxo7 homodimerization. Knockdown of Fbxo7 does not affect PI31 levels arguing against PI31 being a substrate for SCFFbxo7. We present a model for FP domain-mediated dimerization of SCFFbxo7 and PI31. F-box proteins are the substrate-recognition components of the Skp1-Cul1-F box protein (SCF) E3 ubiquitin ligases. Here we report a structural relationship between Fbxo7, a component of the SCFFbxo7 E3 ligase, and the proteasome inhibitor PI31. SCFFbxo7 is known to catalyze the ubiquitination of hepatoma-up-regulated protein (HURP) and the inhibitor of apoptosis (IAP) protein but also functions as an activator of cyclin D-Cdk6 complexes. We identify PI31 as an Fbxo7·Skp1 binding partner and show that this interaction requires an N-terminal domain present in both proteins that we term the FP (Fbxo7/PI31) domain. The crystal structure of the PI31 FP domain reveals a novel α/β-fold. Biophysical and mutational analyses are used to map regions of the PI31 FP domain mediating homodimerization and required for heterodimerization with Fbxo7·Skp1. Equivalent mutations in Fbxo7 ablate interaction with PI31 and also block Fbxo7 homodimerization. Knockdown of Fbxo7 does not affect PI31 levels arguing against PI31 being a substrate for SCFFbxo7. We present a model for FP domain-mediated dimerization of SCFFbxo7 and PI31. The levels of many regulatory and misfolded proteins are controlled by the ubiquitin-proteasome system (1Hershko A. Curr. Opin. Cell Biol. 1997; 9: 788-799Crossref PubMed Scopus (319) Google Scholar, 2Hershko A. Ciechanover A. Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6959) Google Scholar). A series of enzymes termed E1 (ubiquitin-activating enzyme), E2 (ubiquitin carrier protein), and E3 6The abbreviations used are: E3ubiquitin ligaseHURPhepatoma up-regulated proteinIAPinhibitor of apoptosisITCisothermal calorimetrySCFSkp1-Cul1-F box proteinUblubiquitin-likePRRproline-rich regionFPFbxo7/PI31GSTglutathione S-transferase. (ubiquitin ligase) act in the ubiquitin-proteasome system as part of a concerted cascade to activate and conjugate ubiquitin via an isopeptide linkage to protein substrates (2Hershko A. Ciechanover A. Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6959) Google Scholar). Ubiquitin modification can affect the activity, localization, sorting, and stability of protein substrates (2Hershko A. Ciechanover A. Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6959) Google Scholar, 3Hicke L. Dunn R. Annu. Rev. Cell Dev. Biol. 2003; 19: 141-172Crossref PubMed Scopus (965) Google Scholar, 4d'Azzo A. Bongiovanni A. Nastasi T. Traffic. 2005; 6: 429-441Crossref PubMed Scopus (202) Google Scholar). Recognition and recruitment of protein substrates to the ubiquitin-proteasome system machinery resides within the multisubunit E3 ubiquitin ligases, the largest group of which is the Skp1-Cullin1-F-box protein (SCF) family (1Hershko A. Curr. Opin. Cell Biol. 1997; 9: 788-799Crossref PubMed Scopus (319) Google Scholar, 5Nakayama K.I. Nakayama K. Semin. Cell Dev. Biol. 2005; 16: 323-333Crossref PubMed Scopus (292) Google Scholar, 6Petroski M.D. Deshaies R.J. Nat. Rev. Mol. Cell. Biol. 2005; 6: 9-20Crossref PubMed Scopus (1692) Google Scholar). All known SCF E3 ligases bind an E2 (ubiquitin carrier protein) enzyme through a RING finger-containing subunit (Rbx1) and utilize their F-box subunit to recruit substrates. Although classified by the presence of an F-box (Fbx) motif (7Bai C. Sen P. Hofmann K. Ma L. Goebl M. Harper J.W. Elledge S.J. Cell. 1996; 86: 263-274Abstract Full Text Full Text PDF PubMed Scopus (991) Google Scholar), F-box proteins can be further divided into three classes depending on additional structural elements: Fbxw (WD40 motifs), Fbxl (with leucine-rich repeats), and Fbxo (F-box domain only) (8Cenciarelli C. Chiaur D.S. Guardavaccaro D. Parks W. Vidal M. Pagano M. Curr. Biol. 1999; 9: 1177-1179Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar, 9Winston J.T. Koepp D.M. Zhu C. Elledge S.J. Harper J.W. Curr. Biol. 1999; 9: 1180-1182Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar). Recent structural data have shown how the Fbxw protein CDC4 (Fbw7) uses the center of the WD40 propeller to recognize the phosphorylated epitope of its substrate, cyclin E (10Orlicky S. Tang X. Willems A. Tyers M. Sicheri F. Cell. 2003; 112: 243-256Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar, 11Hao B. Oehlmann S. Sowa M.E. Harper J.W. Pavletich N.P. Mol. Cell. 2007; 26: 131-143Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar). Structural data also exist for the Fbxl protein Skp2 (Fbl1) as part of the SCFSkp2 complex bound to phosphorylated p27 (12Hao B. Zheng N. Schulman B.A. Wu G. Miller J.J. Pagano M. Pavletich N.P. Mol. Cell. 2005; 20: 9-19Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar). In contrast, little is known about how Fbxo proteins recognize their substrates or indeed whether other substrate-targeting domains exist within this subgroup. ubiquitin ligase hepatoma up-regulated protein inhibitor of apoptosis isothermal calorimetry Skp1-Cul1-F box protein ubiquitin-like proline-rich region Fbxo7/PI31 glutathione S-transferase. Fbxo7 has been characterized recently as a member of the Fbxo subgroup and is conserved among higher eukaryotes (8Cenciarelli C. Chiaur D.S. Guardavaccaro D. Parks W. Vidal M. Pagano M. Curr. Biol. 1999; 9: 1177-1179Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar, 13Laman H. Funes J.M. Ye H. Henderson S. Galinanes-Garcia L. Hara E. Knowles P. McDonald N. Boshoff C. EMBO J. 2005; 24: 3104-3116Crossref PubMed Scopus (72) Google Scholar). As well as an F-box motif (residues 329-375), which interacts with Skp1 (8Cenciarelli C. Chiaur D.S. Guardavaccaro D. Parks W. Vidal M. Pagano M. Curr. Biol. 1999; 9: 1177-1179Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar, 9Winston J.T. Koepp D.M. Zhu C. Elledge S.J. Harper J.W. Curr. Biol. 1999; 9: 1180-1182Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar, 13Laman H. Funes J.M. Ye H. Henderson S. Galinanes-Garcia L. Hara E. Knowles P. McDonald N. Boshoff C. EMBO J. 2005; 24: 3104-3116Crossref PubMed Scopus (72) Google Scholar, 14Ilyin G.P. Rialland M. Pigeon C. Guguen-Guillouzo C. Genomics. 2000; 67: 40-47Crossref PubMed Scopus (57) Google Scholar), Fbxo7 has an N-terminal ubiquitin-like (Ubl) domain (residues 1-78) (13Laman H. Funes J.M. Ye H. Henderson S. Galinanes-Garcia L. Hara E. Knowles P. McDonald N. Boshoff C. EMBO J. 2005; 24: 3104-3116Crossref PubMed Scopus (72) Google Scholar, 15Kelley L.A. MacCallum R.M. Sternberg M.J. J. Mol. Biol. 2000; 299: 499-520Crossref PubMed Scopus (1121) Google Scholar) (Fig. 1A), which is thought to mediate interactions with ubiquitin receptor proteins bearing ubiquitin binding domains (16Hicke L. Schubert H.L. Hill C.P. Nat. Rev. Mol. Cell. Biol. 2005; 6: 610-621Crossref PubMed Scopus (639) Google Scholar). The C terminus of Fbxo7 contains a proline-rich region (PRR) but lacks any predicted secondary structure. HURP (hepatoma up-regulated protein) and cIAP1 (an inhibitor of apoptosis protein) have both been reported as substrates for SCFFbxo7-mediated ubiquitination leading to proteasome-mediated degradation (17Hsu J.M. Lee Y.C. Yu C.T. Huang C.Y. J. Biol. Chem. 2004; 279: 32592-325602Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 18Chang Y.F. Cheng C.M. Chang L.K. Jong Y.J. You C.Y. Biochem. Biophys. Res. Commun. 2006; 342: 1022-1026Crossref PubMed Scopus (54) Google Scholar). Each binds to Fbxo7 through its C-terminal proline-rich region (17Hsu J.M. Lee Y.C. Yu C.T. Huang C.Y. J. Biol. Chem. 2004; 279: 32592-325602Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 18Chang Y.F. Cheng C.M. Chang L.K. Jong Y.J. You C.Y. Biochem. Biophys. Res. Commun. 2006; 342: 1022-1026Crossref PubMed Scopus (54) Google Scholar). Fbxo7 also acts as an assembly factor for cyclin D-Cdk6 complexes by virtue of its interaction with D-type cyclins and Cdk6 (13Laman H. Funes J.M. Ye H. Henderson S. Galinanes-Garcia L. Hara E. Knowles P. McDonald N. Boshoff C. EMBO J. 2005; 24: 3104-3116Crossref PubMed Scopus (72) Google Scholar). In this study a combination of structural, biochemical, and genetic approaches were used to identify and characterize regions of Fbxo7 involved in protein interaction as a means to identify putative substrates of SCFFbxo7. Unexpectedly we uncovered a structural link between a domain from Fbxo7 and a related domain from PI31, a regulatory subunit of the immunoproteasome which is an in vitro inhibitor of the 20 S proteasome (19Chu-Ping M. Slaughter C.A. DeMartino G.N. Biochim. Biophys. Acta. 1992; 1119: 303-311Crossref PubMed Scopus (115) Google Scholar, 20Zaiss D.M. Standera S. Holzhutter H. Kloetzel P.M. Sijts A.J. FEBS Lett. 1999; 457: 333-338Crossref PubMed Scopus (77) Google Scholar, 21McCutchen-Maloney S.L. Matsuda K. Shimbara N. Binns D.D. Tanaka K. Slaughter C.A. DeMartino G.N. J. Biol. Chem. 2000; 275: 18557-18565Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 22Zaiss D.M. Standera S. Kloetzel P.M. Sijts A.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14344-14349Crossref PubMed Scopus (83) Google Scholar). The crystal structure of this domain from PI31 revealed a novel α/β-fold and two distinct intermolecular contact surfaces. We used this structure to guide the design of site-specific substitutions to probe surfaces of the FP domain from PI31 responsible for homodimerization and for heterodimeric interaction with Fbxo7. We discuss a possible role for FP domain-mediated dimerization for SCFFbxo7 and PI31 function. Protein Expression and Purification—A bicistronic expression vector was constructed to prepare recombinant GST-tagged Fbxo7·Skp1 complexes for affinity purification. For all experiments the Skp1 cDNA encompassed residues 1-146 and lacked the C-terminal H8 helix as previously described (23Schulman B.A. Carrano A.C. Jeffrey P.D. Bowen Z. Kinnucan E.R. Finnin M.S. Elledge S.J. Harper J.W. Pagano M. Pavletich N.P. Nature. 2000; 408: 381-386Crossref PubMed Scopus (493) Google Scholar). Human Fbxo7 (residues 129-398) (Swiss-Prot Q9Y3I1)-Skp1 (residues 1-146) (Swiss-Prot P63208), and Fbxo7 (residues 169-398)-Skp1 (residues 1-146) constructs were produced as follows. The FBXO7 gene was cloned into pGEX(KG) (24Guan K.L. Dixon J.E. Anal. Biochem. 1991; 192: 262-267Crossref PubMed Scopus (1641) Google Scholar). The SKP1 gene was amplified by PCR from a human cDNA library using a 5′ primer which contained a ribosome binding site and then subcloned into BlueScript (Stratagene). The RBS-SKP1 insert was then subcloned into pGEX(KG)-Fbxo7. For all experiments the Skp1 encompassed residues 1-146 (Skp1ΔH8), which lacks the C-terminal H8 helix aiding expression and solubility (23Schulman B.A. Carrano A.C. Jeffrey P.D. Bowen Z. Kinnucan E.R. Finnin M.S. Elledge S.J. Harper J.W. Pagano M. Pavletich N.P. Nature. 2000; 408: 381-386Crossref PubMed Scopus (493) Google Scholar). Fbxo7(169-398)-Skp1ΔH8 (V253E) mutant was produced using the QuikChange system (Stratagene). Residues 1-151 of human PI31 (Swiss-Prot Q92530) were amplified by PCR and cloned into the pET28a expression plasmid (Novagen). Mutant PI31 constructs (L7M, V6R, and I83E/I90E) and mutant Fbxo7 constructs (V253E and V175E) were produced using the QuikChange system (Stratagene). All constructs were verified by DNA sequencing. The expression plasmids described above were transformed into Escherichia coli strain BL21(DE3), and cultures were grown in Luria broth at 37 °C to an A600 of 0.6. Recombinant protein expression was induced by the addition of isopropyl-β-d-1-thiogalactopyranoside to a final concentration of 250 μm for 3 h at 37 °C. Bacteria were harvested by centrifugation and resuspended in lysis buffer (100 mm NaCl, 50 mm Tris-HCl, pH 8, 10 mm benzimidine, 5 mm β-mercaptoethanol, 1 mm phenylmethylsulfonyl fluoride). Proteins were purified from the soluble fraction using either glutathione-Sepharose (Fbxo7·Skp1) or nickel-nitrilotriacetic acid affinity beads (PI31) by batch purification and cleaved from the beads with thrombin at 4 °C. PI31 and Fbxo7·Skp1 proteins were then purified by size exclusion chromatography using Superdex 75 and Superdex 200 columns, respectively. Columns were equilibrated and run in 20 mm Tris-HCl, 50 mm NaCl, pH 8, and 1 mm dithiothreitol. Selenomethionine was incorporated by transforming the expression plasmid into the E. coli methionine auxotroph strain B834(DE3) grown on minimal medium supplemented by selenium methionine using standard procedures. Selenomethionine-labeled protein and mutants were prepared using a similar procedure to the native protein as described above. Crystallization and Data Collection—The PI31 FP domain was crystallized by vapor diffusion in sitting drops containing 2 μl of protein (8 mg/ml in 20 mm Tris-HCl, pH 8.0) and 2 μl of well solution (20% polyethylene glycol 3350 and 0.1 m ammonium iodide) at room temperature. Crystals grew within 1 week to roughly 0.2 mm. Selenomethionine-substituted wild type FP domain and L7M mutant protein crystallized under the same conditions as the native protein. Data were collected for crystals of human PI31 FP domain, selenomethionine-substituted FP domain, and the selenomethionine-substituted L7M mutant (Table 1). In each case a single crystal was flash-cooled to 100 K in a nitrogen gas stream using 20% (v/v) ethylene glycol as a cryoprotectant. The CCP4 suite of programs (25Collaborative Computational Project, Number 4 Acta Crystallogr. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar) were used for data processing, and REFMAC and COOT were used for refinement and model building (26Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. D. Biol Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13914) Google Scholar, 27Emsley P. Cowtan K. Acta Crystallogr. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (23628) Google Scholar).TABLE 1Values in parenthesis correspond to the highest resolution shellSampleNative PI31 (residues 1-151)L7M PI31 (residues 1-151)L7M PI31 (residues 1-151)Dataset123Diffraction dataNativeSelenomethionineSelenomethionineSpace groupC2C2C2Cell constants (Å; °)a = 109.5, b = 45.2 c = 67.3; β = 111.5°a = 108.4, b = 42.8, c = 66.6; β = 109.1°a = 108.7, b = 42.3, c = 66.3; β = 109.4°Za222Wavelength (Å)1.5410.97855 (peak)0.97855 (peak)Resolution (Å)28.1-2.64 (2.74-2.64)39.75-2.5 (2.64-2.5)27.12-2.50 (2.64-2.5)Completeness (%)99.698.499.9Multiplicity7.1 (6.5)7.3 (7.4)7.2 (7.3)Rmeas (%)7.6 (38.8)7.2 (16.9)4.7 (15.1)Rp.i.m.aRp.i.m. is the precision R-factor. (%)3.2 (15.4)3.3 (9.5)2.4 (6.1)Rano (%)4.5 (8.0)4.7 (8.4)<I>/sd21.1 (4.8)25.5 (12.5)31.9 (11.8)RefinementRfact (%)20.2 (29.5)Rfree (%)25.3 (40.0)Reflections9115 (648)Number of protein atoms3144Number of solvent atoms36Wilson B factor38Average B factor (Å2)42.8Root mean square deviation bonds (Å), angles (°)0.024, 2.262Ramachandran plot (%) (core, allowed, generously allowed, disallowed)85.1/12.4/0.8/1.6a Rp.i.m. is the precision R-factor. Open table in a new tab Structure Determination—Initial attempts to solve the structure using selenomethionine single wavelength anomalous diffraction data were unsuccessful due to the centrosymetric arrangement of the two selenium sites from two methionines in native PI31 FP domain (data not shown). A third methionine site was engineered by an L7M mutation leading to crystals of selenomethionyl-labeled protein, and subsequently datasets were collected at the selenium peak wavelength (Table 1, datasets 2 and 3). The selenium substructure from dataset 2 was found by SHELXD/E and used to calculate SAD phases with a contrast of 0.403 and connectivity of 0.877 (incorrect hand had a contrast of 0.314 and connectivity of 0.845). Phases were improved by 2-fold non-crystallographic averaging within dataset 2 (initial FOM 0.45, final FOM 0.64) using DM (28Cowtan K. Main P. Acta Crystallogr. D Biol. Crystallogr. 1998; 54: 487-493Crossref PubMed Scopus (309) Google Scholar) and cross-crystal averaging with dataset 3 (Table 1, datasets 2 and 3) and density modification using RESOLVE (29Terwilliger T.C. Acta Crystallogr. D Biol. Crystallogr. 2000; 56: 965-972Crossref PubMed Scopus (1636) Google Scholar). To obtain the native PI31 FP domain structure free of introduced mutations, a partial model was built into this map and was subsequently positioned by molecular replacement into the native unit cell (dataset 1). Refinement against this native dataset collected in house produced the final model (Table 1, dataset 1). The final model lacks 9 residues from the C termini of both copies (chain A and B) and residue Ser-75 in chain A. Residues in loop 72-76 and 96-98 are relatively mobile as reflected in high temperature factors and poor electron density. Chain A and B are essentially identical (root mean square deviation of 0.419 Å over 141 Cα residues). Four of the cysteine residues in each chain appear to have undergone partial oxidation and have been modeled as sulfenic acids. Coordinates have been deposited in the Protein Data Bank data base with accession code 2VT8. Isothermal Calorimetry—Isothermal calorimetry experiments used a MicroCal VP-ITC according to manufacturer's instructions. All proteins analyzed by isothermal calorimetry (ITC) were purified by size exclusion chromatography and dialyzed into the same buffer (20 mm Hepes, pH 8, 50 mm NaCl, and 1 mm β-mercaptoethanol) before measurement of their molar concentration. The Fbxo7·Skp1 concentration within the sample cell was 20 μm, and the PI31 titrant concentration was 100 μm. The experiments were carried out at 30 °C with 28 injections each of 10 μl measured with stirring, and all settings were constant for both wild type and mutant experiments. Results were analyzed using the ORIGIN7 software provided by MicroCal and using a one-binding-site model. Analytical Ultracentrifugation—For analytical ultracentrifugation (AUC) experiments, recombinant proteins were stored and analyzed in a buffer containing 20 mm Tris-HCl, pH 8, 50 mm NaCl, and 5 mm β-mercaptoethanol. A Beckman XL-I analytical ultracentrifuge was used to measure both absorbance and interference data according to the manufacturer's instructions. Protein parameters used for data analysis and the calculation of initial experimental parameters were calculated using the program SEDNTERP (30Cole J.L. Lary J.W. Moody P. Laue T.M. Methods Cell Biol. 2008; 84: 143-179Crossref PubMed Scopus (301) Google Scholar). Data were analyzed using the AUC machine software and the programs SEDFIT and SEDPHAT (31Schuck P. Biophys. J. 2000; 78: 1606-1619Abstract Full Text Full Text PDF PubMed Scopus (3089) Google Scholar) using the model monomer-dimer equilibrium in the final analysis. Yeast Two-hybrid Screens—Fbxo7 129-522 and 129-398 were subcloned into pGBDU-C1 and co-transformed with library plasmid (Clontech) into PJ69-4a yeast two-hybrid strain (32James P. Halladay J. Craig E.A. Genetics. 1996; 144: 1425-1436Crossref PubMed Google Scholar). Cell Culture—Human osteosarcoma (U2OS) and Jurkat E6 cell lines were obtained from Cancer Research UK (LRI Cell Production). U2OS cells were grown in Dulbecco's modified Eagle's medium, whereas Jurkat E6 cells were grown in RPMI media. Both media were supplemented with 10% fetal calf serum (Helena Biosciences), 2 mm glutamine, 100 units/ml penicillin, and streptomycin at 37 °C in a humidified 5% CO2 atmosphere. U2OS cells were seeded the day before transfection using FuGENE (catalog #1814443, Roche Applied Science). To visualize the subcellular localization of Fbxo7, cells were transfected with a construct encoding a fusion of dsRED with full-length Fbxo7. 24 h post-transfection, live cells were visualized by confocal microscopy. For immunofluorescence assays on PI31, cells were grown on glass coverslips, fixed in 3% paraformaldehyde, permeabilized with 0.1% Triton X, and blocked with 5% fetal bovine serum before incubation with primary antibody against PI31 (1:200) and secondary rhodamine-conjugated anti-mouse antibody (1:500). The fluorescent dye 3,3′-dihexyloxacarbocyanine iodide (DiOC6(3Hicke L. Dunn R. Annu. Rev. Cell Dev. Biol. 2003; 19: 141-172Crossref PubMed Scopus (965) Google Scholar)) (Invitrogen) was included in the final wash to permit visualization of the endoplasmic reticulum by confocal microscopy. For co-immunofluorescence assays, cells were grown on glass coverslips, fixed in 70% ethanol, permeabilized with 0.1% Triton-X, and blocked with 0.1% gelatin before staining with affinity-purified Fbxo7 polyclonal antibody (1:40) and PI31 monoclonal antibody (1:10). Secondary antibodies sheep anti-rabbit conjugated to AlexaFluor 488 (Molecular Probes) and goat anti-mouse conjugated to Cy5 (DAKO) were both used at 1:300. Coverslips were mounted with Vectashield (Vector) mounting media containing 4′,6-diamidino-2-phenylindole. Cells were visualized by epifluorescence microscopy. Fractionation, Immunoprecipitation, and Western Blotting—U2OS cells were fractionated by resuspending in 100 mm Tris-HCl, pH 7.4, 100 mm NaCl, 25 mm MgCl2, and adding 40 μg/ml digitonin and incubating on ice for 10 min to lyse plasma membrane. Nuclei were separated from cytoplasmic fraction by centrifugation at 3000 rpm for 10 min at 4 °C and lysed in radioimmune precipitation assay buffer (50 mm Tris, pH 7.5, 150 mm NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, protease inhibitor mixture). For immunoprecipitation, cells were lysed in hypotonic lysis buffer (10 mm Tris-HCL, pH 7.5, 10 mm NaCl, 2 mm EDTA, 0.5% Triton X-100, 1 mm phenylmethylsulfonyl fluoride) and protease inhibitor mixture for 10 min on ice before the addition of NaCl to a final concentration of 150 mm. Lysates were centrifuged for 15 min at 13,000 rpm and then immunoprecipitated with anti-FLAG-M2-agarose slurry for 3 h at 4 °C ona rotating wheel. Agarose beads were then washed 4 times with NET2 buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, and 0.05% Triton X-100) before being resuspended in 4× Laemmli buffer and resolved by SDS-PAGE. The following antibodies were used for immunoprecipitation or Western blotting: Cdk6 (SC177) (Santa Cruz Biotechnology Inc.), FLAG (F3165, Sigma), and T7 (catalog #69522-3, Novagen). The antibody against Fbxo7 has been previously described (13Laman H. Funes J.M. Ye H. Henderson S. Galinanes-Garcia L. Hara E. Knowles P. McDonald N. Boshoff C. EMBO J. 2005; 24: 3104-3116Crossref PubMed Scopus (72) Google Scholar). The monoclonal antibody raised against the FP domain of PI31 was made by Monoclonal Antibody Service, Cancer Research UK. Anti-rabbit IgG and anti-mouse IgG antibodies conjugated to horseradish peroxidase were obtained from Santa Cruz Biotechnology, Inc. and Jackson ImmunoResearch Laboratories. Mapping Protein Interaction Sites within Fbxo7 and Identification of PI31 as an Fbxo7 Binding Partner—To map regions within Fbxo7 responsible for mediating its in vivo interactions with known protein partners, we prepared several deletion constructs based on predicted domain boundaries of Fbxo7 and incorporated an N-terminal T7 epitope. These included constructs lacking the N-terminal Ubl domain, the F-box motif, or the C-terminal PRR (Fig. 1A). We first sought to map the binding site for Cdk6, a validated binding partner for Fbxo7 (13Laman H. Funes J.M. Ye H. Henderson S. Galinanes-Garcia L. Hara E. Knowles P. McDonald N. Boshoff C. EMBO J. 2005; 24: 3104-3116Crossref PubMed Scopus (72) Google Scholar). Cdk6 was efficiently co-immunoprecipitated by full-length Fbxo7-(1-522) and by a mutant of Fbxo7 lacking the F-box domain (ΔF-box) indicating that the interaction did not require the F-box (Fig. 1, A and B). We next deleted the first 129 amino acids of Fbxo7 which includes the Ubl domain, designated as Fbxo7-(129-522). However, by SDS-PAGE, this mutant co-migrated with IgG heavy chain (molecular mass, 50 kDa), making it unusable in our co-immunoprecipitation assays. Fbxo7-(129-398) (lacking both the Ubl domain and the PRR region) was also able to co-immunoprecipitate Cdk6 in vivo (Fig. 1, A and B), showing neither region was essential for binding. Because Fbxo7-(129-398) was predicted to have an unstructured sequence of 40 amino acids at the N terminus (129-169), we therefore, truncated the construct further to produce Fbxo7-(169-398). This protein was now unable to bind Cdk6 (Fig. 1B), indicating that within the context of Fbxo7-(129-398), amino acids 129-169 were necessary to bind Cdk6. We then made further deletions from the N terminus including Fbxo7-(239-522), which surprisingly, was able to bind Cdk6 despite the absence of residues 129-169. This indicated another region within 239-522 could mediate Cdk6 interaction. This region contains the F-box domain and the PRR sequence. We deleted the unstructured C terminus of Fbxo7, creating Fbxo7-(239-381) and also attempted to express the C terminus, Fbxo7-(419-522), but this protein could not be produced (Fig. 1, A and B). Fbxo7-(239-381) did not interact with Cdk6, suggesting that the sequences from 381-522, which includes the PRR, previously proposed to mediate substrate recognition for HURP and c-IAP, could also contribute to Cdk6 interaction (17Hsu J.M. Lee Y.C. Yu C.T. Huang C.Y. J. Biol. Chem. 2004; 279: 32592-325602Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 18Chang Y.F. Cheng C.M. Chang L.K. Jong Y.J. You C.Y. Biochem. Biophys. Res. Commun. 2006; 342: 1022-1026Crossref PubMed Scopus (54) Google Scholar). Our data indicate that two regions from Fbxo7 can independently bind to Cdk6, suggesting Fbxo7 has a bipartite Cdk6 binding site. By mapping the binding site of a known Fbxo7-interacting partner to domains within Fbxo7, we validated the use of this panel of Fbxo7-truncated constructs to search for new protein partners of Fbxo7 and, thus, for SCFFbxo7. Closer examination of Fbxo7 sequence conservation and predicted secondary structure identified a putative globular domain (residues 180-324) that precedes the F-box and follows the Ubl and the N-terminal Cdk6 binding sequence. We also identified a highly conserved R(Ar)DP motif (where Ar indicates any aromatic amino acid) within residues 466-496 of the PRR, with an as yet undetermined function. To investigate whether residues 180-324 could function as a protein interaction module and to identify putative partner proteins, we prepared GST-tagged recombinant Fbxo7-(129-398)·Skp1 complexes for ex vivo affinity pulldown experiments from Jurkat cell lysates as described under “Experimental Procedures” (33Schneider U. Schwenk H.U. Bornkamm G. Int. J. Cancer. 1977; 19: 621-626Crossref PubMed Scopus (543) Google Scholar). Skp1 was co-expressed to engage the F-box motif and was used as a positive control to demonstrate proper folding of Fbxo7 (Fig. 1C). After incubation of recombinant GST-Fbxo7-(129-398)·Skp1 immobilized on glutathione-Sepharose beads with the Jurkat cell lysate, the beads were washed and treated with thrombin to release Fbxo7-(129-398)·Skp1. Co-eluting proteins were then identified by mass spectrometry. Using this approach we identified PI31 (proteasome inhibitor 31kDa) as co-eluting with Fbxo7·Skp1 (Fig. 1C) but not from a GST control elution. In addition, the known Skp1-binding protein Cullin1 and endogenous Skp1 also co-eluted with Fbxo7·Skp1. The latter was present as excess recombinant Fbxo7 was present in the protein preparations. In parallel to the affinity pulldown experiments, we undertook yeast two-hybrid screens of a human cDNA library using Fbxo7-(129-398) and Fbxo7-(129-522) as bait (Fig. 1D). In both screens more than 60% of the library plasmids that were isolated had PI31 fused with Gal4 activation domain (GAD), whereas 7% of the clones contained Skp1. Assaying for activation of the HIS3 and ADE2 reporter genes tested the specificity of the pGAD-PI31 interaction with the pGBD-Fbxo7 bait plasmid. Yeast co-transformed with Fbxo7 plasmid and pGAD-PI31 grew on media lacking histidine and adenine. However, yeast transformed with either plasmid alone or a different bait plasmid encoding a viral cyclin fused to GBD together with pGAD-PI31 failed to grow under the same conditions (Fig. 1D). Taken together, the results from these independent screens and affinity pulldown experiments indicate that PI31 can interact with Fbxo7 and is a previously uncharacterized binding partner for Fbxo7. Fbxo7 and PI31 Are Structurally Related—After the identification of PI31 as a putative Fbxo7 binding partner, we analyzed sequences of Fbxo7 and PI31 and noticed that these proteins are related at both a sequence and structural level despite their involvement in quite different multiprotein complexes. Both contain a domain equivalent to residues 180-324 of Fbx" @default.
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• W2017321408 title "Structure of a Conserved Dimerization Domain within the F-box Protein Fbxo7 and the PI31 Proteasome Inhibitor" @default.
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