Structural basis of the phosphorylation-independent recognition of cyclin D1 by the SCFFBXO31

Yunfeng Lia, Kai Jina, Eric Bunkerb, Xiaojuan Zhangb, Xuemei Luoc, Xuedong Liub, and Bing Haoa,1

aDepartment of Molecular Biology and Biophysics, University of Connecticut Health Center, Farmington, CT 06030; bDepartment of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309; and cBiomolecular Resource Facility, University of Texas Medical Branch, Galveston, TX 77555

Edited by Brenda A. Schulman, St. Jude Children’s Research Hospital, Memphis, TN, and approved November 16, 2017 (received for review May 30, 2017) Ubiquitin-dependent proteolysis of cyclin D1 is associated with and other different or unknown domains (FBXO subfamily) (8). normal and tumor cell proliferation and survival. The SCFFBXO31 Four F-box , Skp2 (9), FBXW8 (10), FBXO4 (11), and (Skp1–Cul1–Rbx1–FBXO31) ubiquitin ligase complex mediates geno- FBXO31 (12), are involved in regulation of cyclin D1 ubiquiti- toxic stress-induced cyclin D1 degradation. Previous studies have sug- nation and subsequent degradation. A recent genetic study of gested that cyclin D1 levels are maintained at steady state by these F-Box proteins has led to the view that other unidentified phosphorylation-dependent nuclear export and subsequent proteol- ubiquitin ligases also appear to control cyclin D1 accumulation ysis in the cytoplasm. Here we present the crystal structures of the (13). Although Skp2 and FBXW8 possess the respective LRR – Skp1 FBXO31 complex alone and bound to a phosphorylated cyclin and WD40 repeat domains, FBXO4 and FBXO31 belong to the D1 C-terminal peptide. FBXO31 possesses a unique substrate-binding third class of F-box proteins, whose C-terminal domains exhibit domain consisting of two β-barrel motifs, whereas cyclin D1 binds to no to WD40 or LRR repeats (14). The crystal FBXO31 by tucking its free C-terminal carboxylate tail into an open – cavity of the C-terminal FBXO31 β-barrel. Biophysical and functional structure of the Skp1 FBXO4 complex reveals that FBXO4 adopts studies demonstrate that SCFFBXO31 is capable of recruiting and ubiq- an unexpected small GTPase-like fold (15, 16). Whereas Skp2, uitinating cyclin D1 in a phosphorylation-independent manner. Our FBXO4, and FBXW8 are involved in the normal cell cycle- findings provide a conceptual framework for understanding the sub- dependent oscillation of cyclin D1, FBXO31 is a DNA damage- strate specificity of the F-box FBXO31 and the mechanism of induced checkpoint protein that promotes cyclin D1 degradation FBXO31-regulated cyclin D1 protein turnover. and subsequent G1 cell-cycle arrest in response to genotoxic stress (12). FBXO31 was first identified as a candidate tumor suppressor Skp1–FBXO31–cyclin D1 structure | ubiquitin system | cell cycle in several cancers, and its expression is down-regulated in breast cancer cell lines and primary breast tumors as well as in he eukaryotic ubiquitin–proteasome system has long been hepatocellular carcinoma (17, 18). In addition to cyclin D1, five Trecognized as an integral part of cellular protein turnover other FBXO31 substrates have been identified (19–23). Despite and homeostasis, and, in many cases, plays a pivotal role in the its clear roles in different cellular processes, the mechanism regulation of normal and cancer-related cellular processes (1). In whereby FBXO31 renders substrate specificity to the SCF com- particular, ubiquitin-mediated proteolysis provides a mechanism plex remains largely unknown. of temporal enforcement and coordination of cell cycle transi- tions by working in concert with cyclin-dependent kinases (Cdks) Significance to regulate the levels of various cyclin proteins and Cdk inhibi- tors (2). D-type cyclins encoded by three closely related

The cyclin D1 proto-oncoprotein is a crucial regulator of cell BIOCHEMISTRY (cyclins D1, D2, and D3) are major downstream targets of ex- cycle progression, and excessive cyclin D1 expression and/or tracellular signaling pathways that transduce mitogenic signals to activity is a hallmark of many human cancers. The SCFFBXO31 the core oscillatory cell-cycle engine (3). Through their associa- ubiquitin ligase complex is responsible for targeting cyclin D1 for tion with and activation of Cdk4 and Cdk6, as well as seques- ubiquitination and degradation, thereby regulating its turnover tration of the Cdk inhibitors p21 and p27, D-type cyclins serve as after DNA damage. In this work, we report the structural, bio- key activators of G1 phase reentry and progression in response to physical, and functional characterization of the interaction be- mitogenic stimuli (4). Cyclin D1, the most intensively in- tween cyclin D1 and Skp1–FBXO31. We find that the F-box protein vestigated D-type cyclin, is frequently deregulated in cancer, and FBXO31 targets cyclin D1 for phosphorylation-independent ubiq- its overexpression directly contributes to genomic instability and uitination by recognizing and binding to its extreme C terminus. tumorigenesis (5). Altered ubiquitination and stability of cyclin This knowledge enhances our understanding of the molecular D1 are largely responsible for its elevated levels in tumor cells mechanism of FBXO31-mediated cyclin D1 proteolysis following and were found to be a biomarker of cancer phenotype and genotoxic stress and may facilitate efforts to identify a novel disease progression (6). Thus, a deeper understanding of regu- molecular target for anticancer drug discovery. latory mechanisms of ubiquitin-dependent cell cycle control may potentially assist in development of novel anticancer therapies. Author contributions: Y.L., X. Liu, and B.H. designed research; Y.L., E.B., X.Z., X. Luo, and Ubiquitin–protein ligases (i.e., E3s) recognize and interact X. Liu performed research; Y.L., K.J., E.B., X.Z., X. Luo, X. Liu, and B.H. analyzed data; and with target proteins, thereby controlling substrate specificity for X. Liu and B.H. wrote the paper. ubiquitination reactions. The multimeric SCF (Skp1–Cul1–F-box The authors declare no conflict of interest. protein) complexes are the key ubiquitin–protein ligases that This article is a PNAS Direct Submission. have been linked to cell cycle regulation (7). While interacting Published under the PNAS license. with the adaptor protein Skp1 through the N-terminal F-box Data deposition: The atomic coordinates and structure factors have been deposited in the motif, the F-box protein components of the SCF complexes , www.wwpdb.org (PDB ID codes 5VZT and 5VZU). recognize specific substrates through their variable C-terminal 1To whom correspondence should be addressed. Email: [email protected]. – protein protein interaction domains, including WD40 repeats This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. (FBXW subfamily), leucine-rich repeats (LRRs; FBXL subfamily), 1073/pnas.1708677115/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1708677115 PNAS | January 9, 2018 | vol. 115 | no. 2 | 319–324 Downloaded by guest on September 27, 2021 Ubiquitination of cyclin D1 is believed to be triggered by the Skp1–FBXO31core complex was determined at 2.7-Å resolution phosphorylation of a single threonine residue (Thr286) near the by the single-wavelength anomalous dispersion (SAD) method by C terminus of the protein (24) by glycogen synthase kinase 3β using an Hg derivative (Table S1). The resultant electronic map (GSK3β) (25) and through a MAPK pathway (10, 12). Mutation allowed unambiguous tracing of the two polypeptide chains and the of Thr286 to alanine (i.e., T286A) was shown to reduce cyclin positioning of most side chains, except for a 59-residue disordered D1 polyubiquitination and stabilize the protein in proliferating region in FBXO31 (residues 384–442). The final model of the binary core and quiescent fibroblasts (24). GSK3β-mediated phosphorylation structure contains two Skp1–FBXO31 molecules in the asym- core of cyclin D1 Thr286 was also found to promote cyclin D1 nuclear metric unit. The crystal structure of the ternary Skp1–FBXO31 – export by facilitating its interaction with the nuclear exportin cyclin D1 complex was solved at 2.7-Å resolution by molecular CRM1 (26). The highly stable cyclin D1 T286A mutant remains in replacement by using the binary structure as a search model (Table – core the nucleus throughout the cell cycle (25). As cyclin D1 accumulates S1). It also contains two Skp1 FBXO31 monomers, each bound in the nucleus during G1 phase and exits into the cytoplasm as cells to the phosphorylated cyclin D1 peptide. proceed into S phase (27), it is possible that the phosphorylated cyclin The Skp1–FBXO31core Complex. The Skp1–FBXO31core complex has D1 at Thr286 is targeted for nuclear export for subsequent degra- an elongated structure that is highly analogous to the overall spatial dation in the cytoplasm. In this paper, we report crystal structures of arrangement of other Skp1–F-boxproteincomplexes(29)(Fig. the human Skp1–FBXO31core complex and this complex bound to a 1A). FBXO31core consists of an N-terminal F-box domain (resi- Thr286-phosphorylated C-terminal peptide of cyclin D1, revealing a – – α β – dues 66 101; helices H1 H3), an -helical linker domain (resi- zinc ion-binding, -barrel containing substrate-recognition domain in dues 102–146; helices H4–H6), and a C-terminal domain (residues FBXO31 that binds to the extreme C terminus of cyclin D1 inde- 147–539; helices H7–H12 and strands S1−S13; Fig. 1 A and B). pendent of phosphorylation at Thr286. Our in vitro biochemical FBXO31 The F-box domain folds into the same three-helix cluster con- studies show that the SCF ubiquitin ligase can ubiquitinate formation as in other reported F-box protein structures (29). The both unphosphorylated forms of cyclin D1 and its T286A mutant. molecular interaction between the F-box and Skp1 is also very Taken together, these studies provide fundamental structural in- similar to other Skp1–F-box protein complexes. The linker do- – sights into the FBXO31 cyclin D1 interaction and delineate a main of FBXO31 forms a three-helix platform to position the C- FBXO31 mechanism for SCF -mediated ubiquitination of cyclin terminal domain well away from Skp1 and the F-box domain. D1 in a phosphorylation-independent manner. Our results reinforce the view that F-box proteins are key com- ponents responsible for positioning and orienting the substrate Results and Discussion optimally for ubiquitination by the SCF-bound E2 (29). Crystallization and Structure Determination. We produced the bi- The C-terminal domain of FBXO31 is composed of two dis- core nary Skp1–FBXO31 complexbycoexpressingatruncatedhu- tinct α/β mixed motifs, each adopting a highly curved eight- man Skp1 protein (28) and a core fragment of human FBXO31 stranded antiparallel β-sheet structure (Fig. 1 A and B). The two (residues 66–539) that lacks the N-terminal 65 residues preceding β-sheets are twisted with each strand in a right-handed helical the F-box domain. The Skp1–FBXO31core construct was crystallized manner to form a classic up-and-down continuous β-barrel by a alone and bound to a 17-residue phosphorylated peptide cor- succession of +1 connections, in which the first and last strands responding to the extreme C-terminal region of cyclin D1 associate to close the barrel. The N-terminal β-barrel motif (residues 279–295; phosphorylated at Thr286). The structure of (residues 147−290; helices H7−H10 and strands S1−S8) sits on

Fig. 1. Crystal structures of Skp1–FBXO31core and its complex with the cyclin D1 peptide. (A) Ribbon dia- gram of the Skp1–FBXO31core complex, with the secondary structure elements of FBXO31 labeled. Skp1 and FBXO31 are shown in blue and green, re- + spectively. The bound Zn2 ion is depicted as a red sphere. Dashed lines indicate disordered regions. (B) Topology diagram of FBXO31core. Cylinders and ar- rows represent α-helices and β-strands, respectively. (C) Ribbon diagram of the Skp1–FBXO31core complex bound to the cyclin D1 peptide (orange “licorice sticks”). (D) Sequence alignment of the cyclin D1 pep- tide (residues 279–295) used in crystallization and its corresponding cyclin D2 (residues 273–289) and cyclin D3 (residues 276–292) peptides. Their C-terminal car- boxylate groups are indicated. Cylinder indicates the

310-helix (residues 292–294), dashed lines disordered regions, and red crosses the residues of cyclin D1 that contact FBXO31. Residues conserved among D-type cyclins are highlighted in yellow. The phosphorylated Thr286 in cyclin D1 and its corresponding residue in cyclin D2 (Thr280) and cyclin D3 (Thr283) are high- lighted in red. The predicted PEST-like sequence motif is underlined.

320 | www.pnas.org/cgi/doi/10.1073/pnas.1708677115 Li et al. Downloaded by guest on September 27, 2021 the triple-helix platform formed by the linker domain and cov- present within more mobile extended structures and loop re- ered by a four-helix lid at the opposite end. Interestingly, a zinc gions. In the Zn-β motif, the H7, H8, H9, and H10 helices ion is found at the edge of the N-terminal β-barrel connecting to connecting the S7 and S8 strands, along with the extended loop the four-helix lid and binds to two cysteine and two histidine between the S3 and S4 strands, form a lid that folds back to cap residues (Fig. 1A). Hereafter we refer to the N-terminal β-barrel the open cavity at the end of the barrel, whereas the other end of motif as the Zn-β motif. Unlike the covered Zn-β motif, the the barrel is closed off by the H4, H5, and H6 helices from the C-terminal β-barrel motif (residues 291−539; helices H11−H13 and linker domain. By contrast, the β-barrel of the β-motif has a well- strands S9−S16) forms a largely open-end barrel surrounded by defined cavity at each end, and the H11 and H12 helices wrap three helices (hereafter called the β-motif), in which one end around the upper side of the barrel (Fig. 2B). The C-terminal hubs the substrate-binding cavity. As far as we know, the overall α-helix H13 situated between the Zn-β motif and the β-motif folding topology of the C-terminal domain of FBXO31 has not serves as a molecular strut to support the backsides of the two β been seen before. barrels. Both -barrels in these motifs are largely amphipathic, with polar and charged residues on the outside and nonpolar side The Skp1–FBXO31core–Cyclin D1 Complex. The structure of the chains buried on the inside. A cluster of aromatic side chains Skp1–FBXO31core–cyclin D1 complex reveals that the extreme (Phe305, Tyr309, Phe321, Phe467, Phe488, Phe496, Phe498, and C-terminal nine-residue region (residues 287−295) of cyclin Trp500) form a twisted ladder in the central core of the β-barrel D1 adopts a helical conformation and binds to the β-motif of in the β-motif (Fig. 2C). FBXO31, in which the C-terminal carboxylate group of the Structural similarity search by Dali (30) for the Zn-β and β peptide inserts into the open cavity in the β-barrel (Fig. 1 C and -motifs individually has identified small extracellular proteins in D). (No interpretable electron density was observed for the the lipocalin family that bind and transport largely small hy- N-terminal eight residues including the phosphorylation site.) drophobic molecules (31). Despite the low degree of overall The association between cyclin D1 and FBXO31 buries a total of sequence conservation, lipocalin proteins share a conserved core β ∼570 Å2 of solvent-accessible surface on the two proteins, or fold characterized by a single eight-stranded up-and-down -barrel. ∼ For example, the β-motif of FBOX31 can be superimposed onto 49% of total surface area of the cyclin D1 peptide. Cyclin β D1 peptide binding does not induce significant conformational the -barrel structure of the bilin-binding protein (BBP) bound to the biliverdin IXγ chromophore with an rmsd of 3.5 Å for 72 changes in FBXO31, with FBXO31 in the binary and ternary α ∼ complex structures superimposing with a Cα rmsd of 0.36 Å. C atoms, even though they share only 10% sequence identity (32) (Fig. 2D). Interestingly, the pigment molecule occupies a The C-Terminal Domain of FBXO31. Two β-barrels in the C-terminal central cleft at the upper end of the BBP barrel, in a manner β domain of FBXO31 are arranged ∼65° apart and can be super- similar to the binding of the cyclin D1 peptide to the -motif of imposed onto each other with a 2.1-Å rmsd over 73 Cα atoms FBXO31. Thus, FBXO31 possesses a previously uncharacterized (Fig. 2A). The major differences in the two FBXO31 β-motifs are type of substrate recognition domain in SCF E3s. Molecular Interactions Between Cyclin D1 and FBXO31. The cyclin D1 peptide binds to the upper cavity of the β-barrel in the β-motif of FBXO31 extending from strands 9 and 16 at one side of the surface to the center of the cavity (Fig. 3A and Fig. S1). The middle portion of the peptide (residues 289−292) adopts a turn-like conformation so that the peptide chain stays on top of the barrel, whereas a 310 helix formed by residues 292−294 is tucked into the cavity. Nearly all of the FBXO31 contacts are made by the last four C-terminal cyclin D1 residues (residues 292− 295), and all eight strands of the C-terminal β-barrel of FBXO31 participate in interactions with cyclin D1. The side chain of BIOCHEMISTRY Asp292 and the backbone carbonyl groups of Val293 and Asp294 bind the rim of the barrel with their contacts partially solvent- exposed. Ile295 and the C-terminal carboxylate group insert the furthest into the hub of the barrel, making intermolecular contacts in a mostly buried environment. The cyclin D1-binding cavity in FBXO31 is lined by hydrophobic residues on one side and polar residues on the other side (Fig. 3B and Fig. S1). The C-terminal carboxylate group of cyclin D1 is well positioned to make bi- furcated hydrogen bonds with side chains of Lys330 and Asp334 and also receives a water-mediated hydrogen bond with Thr343 and Leu472. Consequently, the side chain of cyclin D1 Ile295 fits into a hydrophobic pocket flanked by side chains of Tyr309, Ile473, Trp500, and Leu503. These packing interactions rational- ize the preference of a hydrophobic C-terminal residue in the recognition sequence. Fig. 2. The C-terminal domain of FBXO31 features two β-barrel–containing The three residues preceding Ile295 contribute additional motifs. (A) Superimposition of the Zn-β and β-motifs of FBXO31, colored in contacts to FBXO31. The γ-carboxylate group of Asp292 is hy- olive and bright green, respectively. (B) Molecular surface representation of drogen-bonded with the main chain NH of Ser311. The back- β β the inner cavities of the -barrel in the FBXO31 -motif defined by HOLLOW bone carbonyl group of Val293 makes bidentate hydrogen bonds (36). Volumes were calculated with CASTp (37). (C) Licorice-stick represen- with side chains of His312 and Asn336, whereas the main chain tation of side chains of the aromatic and hydrophobic residues lining the inner cavities of the β-barrel in the FBXO31 β-motif. (D) Superimposition of carbonyl of Asp294 forms a hydrogen bond with the main chain NH the FBXO31 β-motif (green) with the BBP protein (32) (magenta; Protein of Ala474. In addition, the hydroxyl group of Thr288 is hydrogen- Data Bank ID code 1BPP). Cyclin D1 (orange) and the biliverdin IXγ chro- bonded with the main chain CO of Leu503. Alignment of 39 mophore (purple) are shown as licorice sticks. FBXO31 orthologs shows that surface residues involved in the

Li et al. PNAS | January 9, 2018 | vol. 115 | no. 2 | 321 Downloaded by guest on September 27, 2021 by ERK2-mediated phosphorylation (Fig. S7A). Moreover, elim- ination of two minor phosphorylation sites (T156A/S219A) on cyclin D1 has no effect on its ubiquitination in the unphosphory- lated and phosphorylated states (Fig. S7B). As cyclin D1 binds and activates Cdk4 and Cdk6, its ability to complex with the kinase may impact its ubiquitination by SCFFBXO31. To address this question, we isolated specific binary complexes of the unphosphorylated and phosphorylated WT and T286A mu- tant cyclin D1 proteins with the purified Cdk6 by using gel- filtration chromatography and comparatively evaluated for their degree of ubiquitination by SCFFBXO31. The levels of the poly- Fig. 3. Molecular interactions between cyclin D1 and FBXO31. (A) Close-up ubiquitinated cyclin D1 bound to Cdk6 are similar to free cyclin view of the FBXO31–cyclin D1 interface shows interacting residues of FBXO31 D1 in the unphosphorylated and phosphorylated states (Fig. S8). (green) and cyclin D1 (orange). Hydrogen bonds are shown as black dashed Together, these data indicate that phosphorylation of Thr286 is lines. (B) Molecular surface representation of the FBXO31 region involved in cyclin D1 binding colored according to the local electrostatic potential. The not essential for the FBXO31-mediated ubiquitination of cyclin cyclin D1 residues are labeled. D1 alone and in complex with Cdk6.

FBXO31 Determinants for Cyclin D1 Recognition. Our structural and interaction with cyclin D1 are conserved across species, whereas biochemical studies suggest that FBXO31 specifically recognizes residues making direct contacts with cyclin D1 are invariant and binds cyclin D1 in a manner independent of phosphoryla- (Figs. S2 and S3). tion. To study the relation between the abilities of FBXO31 to interact with cyclin D1 and mediate its ubiquitination, we mu- Phosphorylation-Independent Cyclin D1 Binding and Ubiquitination. tated three FBXO31 residues individually to alanine and tested The fact that the phosphorylated Thr286 does not participate in the capacity of the resulting variants (S311A, H312A, and K330A) to the cyclin D1–FBXO31 interaction in the ternary complex promote cyclin D1 polyubiquitination in our in vitro ubiquitination structure suggests that phosphorylation is not required for assay (Fig. 4C). Alanine substitutions of His312 and Lys330, whose binding and ubiquitination of cyclin D1 by the SCFFBXO31 side chains are involved in hydrogen-bonding interactions with the ubiquitin ligase. To test this hypothesis, we measured and com- respective Val293 and the terminal carboxylate group of the cyclin pared the binding affinity of Skp1–FBXO31core for the Thr286- D1 peptide, result in significant loss of ubiquitination activity (Fig. phosphorylated cyclin D1 peptide and the corresponding 4C, lanes 6 and 7). In contrast, alanine replacement of Ser311, whose unphosphorylated peptide by using isothermal titration calo- main chain amide hydrogen-bonds to cyclin D1 Asp292, has much rimetry (ITC; Fig. S4). As anticipated, the phosphorylated and less effect on the rate and extent of the FBXO31-mediated ubiq- core unphosphorylated peptides bind to Skp1-FBXO31 with dissoci- uitination reaction (Fig. 4C, lane 5). This result supports our ation constants (Kd)of3.5± 0.6 μMand2.7± 0.4 μM, respectively. This result, in conjunction with the structural data, clearly impli- cates FBXO31 recognition of cyclin D1 in a phosphorylation- independent manner. We next investigated the effects of the Thr286 phosphoryla- tion on the ubiquitination of purified WT and mutant cyclin D1 proteins by using an in vitro assay reconstituted with purified human Uba1 E1, UbcH5a E2, Cul1–Rbx1, and Skp1–FBXO31. Because MAPK/ERK kinase plays an important role in phos- phorylation of cyclin D1 after γ-irradiation (12), we therefore phosphorylated cyclin D1 by using the purified active recombi- nant ERK2 kinase. ERK2 phosphorylates cyclin D1 on Thr286 as well as on Thr156 and Ser219, two minor sites that also match the proline-directed phosphorylation site consensus. We confirmed phosphorylation levels and sites of cyclin D1 by tryptic digestion and LC-MS/MS (Fig. S5) and by immunoblotting with a cyclin D1 phospho-Thr286–specific antibody (Fig. 4A). As expected, addition of Skp1–FBXO31core to reaction mixtures containing the unphos- phorylated cyclin D1 significantly stimulates formation of high mo- lecular weight cyclin D1–ubiquitin conjugates (Fig. 4A,lane5).The SCF ligase activity with FBXO31core is comparable with that of the control reaction in the presence of the full-length FBXO31 (named FBXO31FL;Fig.4A, lane 4). Interestingly, the levels of cyclin D1 ubiquitination are largely similar between the Thr286-phosphorylated and the unphosphorylated proteins (lane 5 vs. lane 10). As GSK3β Fig. 4. In vitro ubiquitination of cyclin D1 by SCFFBXO31 does not require can also phosphorylate cyclin D1 on Thr286 (25), we used a con- phosphorylation of cyclin D1 on Thr286. (A) In vitro ubiquitination of the stitutively active GSK3β mutant (i.e., S9A) to phosphorylate cyclin untreated and ERK2-phosphorylated WT cyclin D1 proteins by Skp1–FBXO31FL D1. The phosphorylated protein displaysasimilarubiquitination and Skp1–FBXO31core.(Top) Cyclin D1 and cyclin D1–ubiquitin conjugates de- level to that of the unphosphorylated cyclin D1 (Fig. S6). tected by Western blots with an anti-cyclin D1 antibody. (Bottom)Phosphor- We next sought to measure the effects of the T286A mutation ylation of Thr286 by Western blots with an anti-cyclin D1 phospho-Thr286 antibody. Asterisks indicate the likely high molecular weight cyclin D1 aggre- in cyclin D1 ubiquitination by using our in vitro assay. We found gates. (B) In vitro ubiquitination of the WT and the T286A mutant cyclin D1 by that the T286A mutant is polyubiquitinated at a level similar to Skp1–FBXO31core.(C) In vitro ubiquitination of cyclin D1 by WT and mutant WT cyclin D1 (Fig. 4B, lane 4 vs. lane 8). Like the WT protein, FBXO31core proteins. In B and C, cyclin D1 and cyclin D1–ubiquitin conjugates the extent of ubiquitination of the T286A mutant is not affected were detected by Western blots with an anti-cyclin D1 antibody.

322 | www.pnas.org/cgi/doi/10.1073/pnas.1708677115 Li et al. Downloaded by guest on September 27, 2021 + protein preparations has a [Zn2 ]/protein ratio of 1.06 ± 0.16, whereas the ratio decreases to 0.40 ± 0.12 by mutation of Cys206 to serine (i.e., C206S; Fig. 6B). As a control, the Cul1– Rbx1 component of the SCF E3 complex that contains a three + zinc-containing RING finger has a [Zn2 ]/protein ratio of 2.48 ± 0.18. Amino acid spacing between the ligands of the zinc ion (Cys-X7-His-X15-Cys-X5-His, where X represents a variable amino acid) is quite different from the consensus sequences of other zinc fingers (33). Cys206 and His214 at the end of the S5–S6 loop form a lasso-like structure, whereas Cys230 and His236 lie at the end of the S7 strand and the C-terminal turn of the short H7 helix, re- C Fig. 5. Subcellular localization of cyclin D1 and FBXO31-mediated cyclin spectively (Fig. 6 ). The side chains of Cys206 and His235 form a D1 degradation in HeLa cells. (A–C) Venus-tagged WT and T286A mutant sandwich-like structure into which the imidazole ring of His214 cyclin D1 were transiently coexpressed with mock or mTurquoise-tagged fits, where the carbonyl oxygen of Cys206 forms a hydrogen bond FBXO31 in HeLa cells. SiR-Hoechst was added to the culture 48 h after trans- with the amine nitrogen of His214. The four zinc ligands are in- fection. More than 800 cells were scored for each measurement, and error bars variant in all FBXO31 orthologs (Fig. S2). < < represent the SD (*P 0.05 and **P 0.01). (A) Subcellular localization of cyclin The role of zinc binding in tethering the β-barrel of the Zn-β + D1 was quantified by segmenting images with the Hoechst signal, and the nu- motifofFBXO31suggeststhatZn2 helps rigidify the antiparallel clear/cytoplasmic fluorescence intensity ratios were determined from triplicate β C wells by using Matlab scripts. (B) Representative fluorescent images showing -sheet surrounded by connecting loops (Fig. 6 ). If so, one would subcellular localization of the Venus-tagged WT cyclin D1 and the T286A mu- anticipate that the folding and activity of FBXO31 would be com- 2+ tant. (Scale bars, 200 μm.) (C) Degradation rate of the Venus-tagged WT cyclin promised when its Zn binding is abolished. To address this issue, D1 and the T286A mutant in the absence or presence of FBOX31. Cycloheximide we generated seven single-point mutants of FBXO31 (C206A, (100 μg/mL) was added to each well 48 h after transfection, and cells were im- H214A, C230A, H236A, C206S, H214F, and H214N), four of which aged every 15 min for at least 3 h. Single-cell degradation analysis was con- fail to express in Escherichia coli or form insoluble aggregates. C206S, ducted by using custom Matlab scripts, and the average rate of decay for each H214F, and H214N mutants were produced, albeit at a much lower experimental condition for defined cell populations was calculated by fitting a linear decay curve (Movies S1–S12). level than the WT protein. Although they tend to aggregate as judged by gel-filtration chromatography, the purified mutant proteins are fully active in vitro with regard to their ubiquitination activity toward structure-based conclusion that the very C terminus of cyclin D1 in cyclin D1 (Fig. 6D,lane5–7 vs. lane 4). This result implicates zinc the absence of phosphorylation is essential for its recognition and binding in facilitating the proper folding of FBXO31. ubiquitination by FBXO31. FBXO31 Binds both Cyclin D2 and Cyclin D3. Various combinations of Cyclin D1 Subcellular Localization and Stability. To address whether D-type cyclins were shown to be expressed in different cell types (3). the SCFFBXO31 complex mediates the phosphorylation-independent Cyclin D1, cyclin D2, and cyclin D3 have high degrees of sequence ubiquitination and degradation of cyclin D1 in mammalian cells, we conservation (52–64% identity) and exhibit extensive homology in transiently coexpressed fluorescent Venus-tagged cyclin D1 with or the extreme C-terminal region, including the signature threonine without mTurquoise-tagged FBXO31 in HeLa cells and measured residue embedded in a PEST-like (proline-, glutamic acid-, serine-, the subcellular localization and degradation rates of WT cyclin and threonine-rich) sequence motif (5). The PEST motif is con- D1 and the T286A mutant by using live-cell imaging techniques. In sidered a characteristic property of short-lived proteins degraded agreement with previous observations (25), WT Venus-cyclin D1 is via the ubiquitin–proteasome pathway (34). Three of the five cyclin localized in nucleus and cytoplasm, whereas the T286A mutant re- D1 residues that directly interact with FBXO31 are variably con- sides a slightly higher ratio in the nucleus (Fig. 5 A and B). As served hydrophobic amino acids in cyclin D2 and cyclin D3 (Fig. FBXO31 itself is distributed in nucleus and cytoplasm (Fig. S9), the 1D). Furthermore, the main chain atoms of the cyclin D1 peptide BIOCHEMISTRY coexpression of cyclin D1 with FBXO31 results in a higher nucleus/cytoplasm expression ratio of WT cyclin D1 but has no effect on the localization of the T286A mutant (Fig. 5A), presumably reflecting the greater activity of FBXO31 in the cytoplasm. To determine the effects of FBXO31 on the stability of the cyclin D1 protein, cells transfected with WT cyclin D1 and the T286A mutant or cotransfected with cyclin D1 and FBXO31 were treated with cycloheximide, and fluorescent signals in each individual cell were measured by time-lapse imaging (Fig. 5C). In the absence of FBXO31, WT cyclin D1 has an approximately twofold higher decay rate than the T286A mutant. Coexpression of FBXO31 with cyclin D1 causes a 10-fold increase in the degradation of WT and mutant cyclin D1 proteins, whereas the decay rate of WT cyclin D1 is approximately twofold of that of the T286A mutant. These results support the notion that FBOX31 promotes phosphorylation- independent degradation of cyclin D1. Fig. 6. The zinc-binding structural motif of FBXO31. (A) The sequence of the zinc-binding motif in FBXO31 with the four zinc ligands highlighted in yellow. The Zinc-Binding Structural Motif of FBXO31. The FBXO31 struc- Cylinders and arrows indicate α-helices and β-strands. (B) ICP-OES analysis shows 2+ core ture reveals a previously unknown, unique CHCH-type zinc- the ratio of Zn to the WT and mutant FBXO31 proteins. The ratios were binding site in its Zn-β motif (Fig. 6A and SI Materials and calculated as the average and SD from four independent measurements. Methods (C) Close-up view of the zinc-binding site of FBXO31 shows the coordinating ). The identity of the metal ion was confirmed by in- residues as licorice sticks. (D) In vitro ubiquitination of cyclin D1 by the WT and ductively coupled plasma-optical emission spectrometry (ICP- mutant FBXO31core proteins. Cyclin D1 and cyclin D1–ubiquitin conjugates OES) analysis of multielement contents of the WT and mutant were detected by Western blots with an anti-cyclin D1 antibody. Asterisks core core FBXO31 proteins. WT Skp1–FBXO31 from four different indicate the likely high molecular weight cyclin D1 aggregates.

Li et al. PNAS | January 9, 2018 | vol. 115 | no. 2 | 323 Downloaded by guest on September 27, 2021 make significant contributions to its interactions with FBXO31 as proteolysis by SCFFBXO31 (12), our structural and biochemical described here earlier. FBXO31 is therefore likely able to recognize data clearly show that Thr286 phosphorylation is dispensable for and bind to cyclin D2 and cyclin D3 in a manner analogous to cyclin cyclin D1 association with FBXO31 and ubiquitination by D1. To this end, we measured binding affinities of Skp1–FBXO31core SCFFBXO31. Moreover, our cell-based experiments demonstrate to the corresponding unphosphorylated C-terminal cyclin D2 (res- that the cyclin D1-T286A mutant is localized more in the nucleus idues 273–289) and cyclin D3 (residues 276–292) peptides by using and FBOX31 can promote phosphorylation-independent degra- ITC. Indeed, we found that the cyclin D2 and cyclin D3 peptides dation of WT cyclin D1 and the T286A mutant. These results core bind Skp1–FBXO31 with Kd values of 3.0 ± 0.5 μMand1.9± reinforce the conclusion that phosphorylation of Thr286 directs 0.5 μM, respectively, compared with a Kd of 2.7 ± 0.4 μMforthe nuclear export of cyclin D1 (25, 26) but is not associated with its cyclin D1 peptide (Fig. S10). subsequent ubiquitination and degradation by SCFFBXO31.FBXW8 (10) and FBXO4 (11) have been reported to recognize phos- Implications for FBXO31-Substrate Recognition and Cyclin D1 Regulation phorylated cyclin D1. It is also worthwhile to consider the possi- FBXO31 by SCF . In many of the well-characterized F-box protein–sub- bility that phosphorylation of cyclin D1 on Thr286 acts to induce strate interactions, F-box proteins recognize the internal sequences its ubiquitination by other ubiquitin ligases. of their targets for rapid degradation. In our crystal structure of the Skp1–FBXO31core–cyclin D1 complex, the free carboxylate tail of Materials and Methods cyclin D1 inserts into the open cavity on the surface of the FBXO31 Proteins were expressed and purified by using standard methods. Crystalli- C-terminal β-barrel motif. By restricting the major interactions to zation was performed by using vapor diffusion in hanging drops. X-ray dif- the extreme C-terminal four residues, FBXO31 appears to recog- fraction data were collected at the National Synchrontron Light Source (NSLS) nizeandbindtoitssubstratesbasedonthesesequencedetermi- beamline X29A and the NSLS-II beamline 17-ID-1. The binary structure was nants. Nonpolar residues (such as Ala, Val, Ile, or Leu) are likely solved by SAD and the ternary structure by molecular replacement. ITC ex- preferred at the C-terminal position such as in the −2 position (i.e., periments were performed with a TA Nano ITC Low Volume calorimeter. In vitro two residues from the C terminus; Fig. 1D). A similar peptide cyclin D1 ubiquitination assays were performed by using purified proteins. Live- recognition mechanism has been discovered in the PDZ domains cell imaging analyses were done by using the ImageXpress MicroXL system. Details of all experimental procedures are provided in SI Materials and Methods. that recognize their target proteins by incorporating the extreme β C-termini of the target into a -sheet in PDZ through antiparallel ACKNOWLEDGMENTS. We thank W. Shi at X29A beamline of NSLS and β-augmentation (35). Nonetheless, it is possible that FBXO31 can J. Jakoncic and A. Soares at 17-ID-1 beamline of NSLS-II for help with data recognize internal sequence motifs present in its other substrates collection; O. Vinogradova, X. Lin, and K. Nguyen for technical assistance with (20, 21). Further studies are needed to fully elucidate the molecular ITC experiments; and N. Ahn for her gift of the ERK2 expression plasmid. This work was supported by National Institutes of Health Grants GM099948 (to basis for substrate selectivity and specificity by FBXO31. B.H.) and GM113141 (to X. Liu), and Connecticut Regenerative Medicine Re- In contrast to the current model in which phosphorylation search Fund Grant 15-RMA-UCHC-04 (to B.H.). The ImageXpress MicroXL was of cyclin D1 on Thr286 is required for its ubiquitin-mediated supported by National Center for Research Resources Grant S10 RR026680.

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