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

Abstract

Ubiquitin-dependent proteolysis of cyclin D1 is associated with normal and tumor cell proliferation and survival. The SCF^FBXO31 (Skp1-Cul1-Rbx1-FBXO31) ubiquitin ligase complex mediates genotoxic stress-induced cyclin D1 degradation. Previous studies have suggested that cyclin D1 levels are maintained at steady state by phosphorylation-dependent nuclear export and subsequent proteolysis in the cytoplasm. Here we present the crystal structures of the Skp1-FBXO31 complex alone and bound to a phosphorylated cyclin D1 C-terminal peptide. FBXO31 possesses a unique substrate-binding domain consisting of two β-barrel motifs, whereas cyclin D1 binds to FBXO31 by tucking its free C-terminal carboxylate tail into an open cavity of the C-terminal FBXO31 β-barrel. Biophysical and functional studies demonstrate that SCF^FBXO31 is capable of recruiting and ubiquitinating cyclin D1 in a phosphorylation-independent manner. Our findings provide a conceptual framework for understanding the substrate specificity of the F-box protein FBXO31 and the mechanism of FBXO31-regulated cyclin D1 protein turnover.

Keywords: Skp1–FBXO31–cyclin D1 structure; cell cycle; ubiquitin system.

Fig. 1.

Crystal structures of Skp1–FBXO31^core and its complex with the cyclin D1 peptide. (A) Ribbon diagram of the Skp1–FBXO31^core complex, with the secondary structure elements of FBXO31 labeled. Skp1 and FBXO31 are shown in blue and green, respectively. The bound Zn²⁺ ion is depicted as a red sphere. Dashed lines indicate disordered regions. (B) Topology diagram of FBXO31^core. Cylinders and arrows represent α-helices and β-strands, respectively. (C) Ribbon diagram of the Skp1–FBXO31^core complex bound to the cyclin D1 peptide (orange “licorice sticks”). (D) Sequence alignment of the cyclin D1 peptide (residues 279–295) used in crystallization and its corresponding cyclin D2 (residues 273–289) and cyclin D3 (residues 276–292) peptides. Their C-terminal carboxylate 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 highlighted in red. The predicted PEST-like sequence motif is underlined.

Fig. 2.

The C-terminal domain of FBXO31 features two β-barrel–containing motifs. (A) Superimposition of the Zn-β and β-motifs of FBXO31, colored in olive and bright green, respectively. (B) Molecular surface representation of the inner cavities of the β-barrel in the FBXO31 β-motif defined by HOLLOW (36). Volumes were calculated with CASTp (37). (C) Licorice-stick representation 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 the FBXO31 β-motif (green) with the BBP protein (32) (magenta; Protein Data Bank ID code 1BPP). Cyclin D1 (orange) and the biliverdin IXγ chromophore (purple) are shown as licorice sticks.

Fig. 3.

Molecular interactions between cyclin D1 and FBXO31. (A) Close-up view of the FBXO31–cyclin D1 interface shows interacting residues of FBXO31 (green) and cyclin D1 (orange). Hydrogen bonds are shown as black dashed lines. (B) Molecular surface representation of the FBXO31 region involved in cyclin D1 binding colored according to the local electrostatic potential. The cyclin D1 residues are labeled.

Fig. 4.

In vitro ubiquitination of cyclin D1 by SCF^FBXO31 does not require phosphorylation of cyclin D1 on Thr286. (A) In vitro ubiquitination of the untreated and ERK2-phosphorylated WT cyclin D1 proteins by Skp1–FBXO31^FL and Skp1–FBXO31^core. (Top) Cyclin D1 and cyclin D1–ubiquitin conjugates detected by Western blots with an anti-cyclin D1 antibody. (Bottom) Phosphorylation of Thr286 by Western blots with an anti-cyclin D1 phospho-Thr286 antibody. Asterisks indicate the likely high molecular weight cyclin D1 aggregates. (B) In vitro ubiquitination of the WT and the T286A mutant cyclin D1 by Skp1–FBXO31^core. (C) In vitro ubiquitination of cyclin D1 by WT and mutant FBXO31^core proteins. In B and C, cyclin D1 and cyclin D1–ubiquitin conjugates were detected by Western blots with an anti-cyclin D1 antibody.

Fig. 5.

Subcellular localization of cyclin D1 and FBXO31-mediated cyclin D1 degradation in HeLa cells. (A–C) Venus-tagged WT and T286A mutant cyclin D1 were transiently coexpressed with mock or mTurquoise-tagged FBXO31 in HeLa cells. SiR-Hoechst was added to the culture 48 h after transfection. More than 800 cells were scored for each measurement, and error bars represent the SD (*P < 0.05 and **P < 0.01). (A) Subcellular localization of cyclin D1 was quantified by segmenting images with the Hoechst signal, and the nuclear/cytoplasmic fluorescence intensity ratios were determined from triplicate wells by using Matlab scripts. (B) Representative fluorescent images showing subcellular localization of the Venus-tagged WT cyclin D1 and the T286A mutant. (Scale bars, 200 μm.) (C) Degradation rate of the Venus-tagged WT cyclin D1 and the T286A mutant in the absence or presence of FBOX31. Cycloheximide (100 µg/mL) was added to each well 48 h after transfection, and cells were imaged every 15 min for at least 3 h. Single-cell degradation analysis was conducted by using custom Matlab scripts, and the average rate of decay for each experimental condition for defined cell populations was calculated by fitting a linear decay curve (Movies S1–S12).

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. Cylinders and arrows indicate α-helices and β-strands. (B) ICP-OES analysis shows the ratio of Zn²⁺ to the WT and mutant FBXO31^core proteins. The ratios were calculated as the average and SD from four independent measurements. (C) Close-up view of the zinc-binding site of FBXO31 shows the coordinating residues as licorice sticks. (D) In vitro ubiquitination of cyclin D1 by the WT and mutant FBXO31^core proteins. Cyclin D1 and cyclin D1–ubiquitin conjugates were detected by Western blots with an anti-cyclin D1 antibody. Asterisks indicate the likely high molecular weight cyclin D1 aggregates.