Fig 2: Protein stability of ERa is enhanced in cells with AKTIP loss through CAND1(A) MCF7 cells stably expressing AKTIP shRNA or vector were treated with 10 µg/mL cycloheximide (CHX) for the indicated hours before being subjected to western blotting. ERK2 was loading control. The graph shows band intensities quantified by densitometry in ImageJ and normalized to that at 0 h (n = 3). *p < 0.05 using two-way ANOVA for comparison between groups with Sidak’s multiple comparison test.(B) Lysates of AKTIP-depleted or vector control cells were subjected to immunoprecipitation (IP) with anti-ERa antibody before western blotting (immunoblotting [IB]). ERa protein levels were normalized prior to IP by using proportionally different amounts of lysates. IP with normal immunoglobulin G (IgG) was control.(C) AKTIP-bound proteins pulled down in MCF7 cells were identified by mass spectrometry. Protein interaction network of the protein partners was generated by STRING. ERa, which was not pulled down in the IP, was included in the STRING analysis to reveal proteins that potentially interact with ERa. Only proteins in the ubiquitin/proteasome degradation pathway are shown in the network map. Red dots: hits known to inhibit protein ubiquitination. Line thickness reflects the degree of confidence of the interaction.(D–G) MCF7 cells stably expressing AKTIP shRNA or vector were transfected with (D) a pool of 4 siRNA of each gene indicated, (E and F) individual siRNA of CAND1, or (G) OX plasmid of CUL2 or NEDD8 for 72 h. Protein lysates were harvested for western blotting or IP.(H) Lysates of AKTIP-depleted or vector control cells were subjected to IP with anti-CUL2 antibody (top) or anti-CAND1 antibody (bottom) using equal amounts of lysates.(I) Lysates of AKTIP-depleted or vector control cells were subjected to IP with anti-ERa antibody using proportionally different amounts of lysates for equal ERa levels (top) or anti-CUL2 antibody using equal amounts of lysates (bottom). IP with IgG was control. Input was lysate without the IP procedure. Relative densitometric values (after normalization with the immunoprecipitated proteins) are provided below the blots. *p < 0.05; ***p < 0.001; ns, no significant difference compared with mock using t test (n = 3). The western blots shown are representatives of three independent experiments.

Fig 3: CAND2 accelerates the disassembly of SCFSKP2 with a koff similar to that of CAND1 but a KM greater than that of CAND1.A FRET assay reporting the interaction between CUL1 and SKP1·SKP2. Fluorescence emission spectra from excitation at 430 nm of 100 nM CFPCUL1, 100 nM FlAsHSKP1·SKP2, a mixture of the two (FRET), the chase control for FRET, and the buffer alone. Signals were normalized to the peak donor emission. Chase: 1 μM SKP1·SKP2. Proteins were added in the indicated order and analyzed within 1 min after mixing. B Binding (kon) of SKP1·SKP2 to CUL1 or CUL1 preassembled with CAND1/2. kobs of CFPCUL1·FlAsHSKP1·SKP2 association when 5 nM CFPCUL1 alone or 5 nM CFPCUL1 preincubated with 20 nM CAND1/2 was mixed with increasing concentrations of FlAsHSKP1·SKP2 (Supplementary Fig. 9). The linear slope gave the kon. Data are presented as mean ± SEM; n = 5 independent experiments. C, D Accelerated disassembly of SCFSKP2 by CAND1 vs. CAND2. C Real-time detection of CFP fluorescence in a stopped-flow apparatus upon mixing 40 nM CAND1 or CAND2 with 5 nM preassembled CFPCUL1·FlAsHSKP1·SKP2. D kobs of CFPCUL1·FlAsHSKP1·SKP2 dissociation when increasing concentrations of CAND1 or CAND2 were mixed with 5 nM CFPCUL1·FlAsHSKP1·SKP2 (Supplementary Fig. 10). Fitting with hyperbola gave the koff and KM. Data are presented as mean ± SEM; n = 5 independent experiments for CAND1 and 4 for CAND2. E Kinetic model of CUL1 substrate receptor (SR) exchange mediated by CAND1 vs. CAND2. Source data are provided as a Source Data file.

Fig 4: FBP changes the koff but not the kon of CUL1·CAND2.A Binding (kon) of CAND1 or CAND2 to CUL1 preassembled with SKP1·SKP2. kobs of CFPCAND1·CUL1FlAsH or CFPCAND2·CUL1FlAsH association when 5 nM CFPCAND1 or CFPCAND2 was mixed with increasing concentrations of SKP1·SKP2-bound CUL1FlAsH (Supplementary Fig. 7). The linear slope gave the kon. Data are presented as mean ± SEM; n = 3 independent experiments. B–D SKP1·SKP2 increases the koff of CUL1·CAND1 and CUL1·CAND2 to similar degrees. Real-time detection of CFP fluorescence in a stopped-flow apparatus upon the addition of 80 nM SKP1·SKP2 to 5 nM CFPCAND1·CUL1FlAsH (B) or CFPCAND2·CUL1FlAsH (C). D kobs of CFPCAND1·CUL1FlAsH or CFPCAND2·CUL1FlAsH dissociation when increasing concentrations of SKP1·SKP2 were mixed with 5 nM CFPCAND1·CUL1FlAsH or CFPCAND2·CUL1FlAsH (Supplementary Fig. 8). Fitting with hyperbola gave the koff and KM. Data are presented as mean ± SEM; n = 5 independent experiments. Source data are provided as a Source Data file.

Fig 5: CAND2 promotes SCF-mediated protein degradation.A CAND2 binds to unneddylated CUL1 in human cell lysates. Whole-cell lysates (WCL) from HEK293 cells of the indicated genotypes were immunoprecipitated (IP) with an anti-HA antibody. Cells were treated with 1 μM MLN4924 for 1 h before harvesting. Samples were immunoblotted with anti-CUL1, anti-HA, and anti-GAPDH antibodies. DKO: CAND1 and CAND2 double knockout cells; DKOCAND1-HA: DKO cells expressing transgenic CAND1HA; DKOCAND2-HA: DKO cells expressing transgenic CAND2HA. Representative results from three independent experiments are shown. B, C CAND2 rescues the TNFα-induced degradation of p-IκBα in DKO cells. B Immunoblotting of HA (CAND1 or CAND2), IκBα, and GAPDH at the indicated time points after the addition of TNFα to the indicated cells in the presence of cycloheximide (CHX). C Quantification of the relative p-IκBα levels in (B). Data are presented as mean ± SEM; n = 3 independent experiments. D, E Both CAND1 and CAND2 are required for proper degradation of IRP2 in HEK293 cells. D Immunoblotting of IRP2, FBXL5, and GAPDH at the indicated time points after the addition of ferric ammonium citrate (FAC) to induce the accumulation of FBXL5 and the degradation of IRP2 in the indicated cells. KO: knockout. E Quantitative analyses of IRP2 degradation in (D). Data are presented as mean ± SEM; n = 3 independent experiments except for DKO (n = 4). F–H CAND2-KO impaired the enrichment of the IRP2-bound active SCF complex. F Schematic diagram of the experimental procedure. G Samples from cells of the indicated genotypes were prepared as described in (F) and immunoblotted with anti-HA (FBXL5), anti-FLAG (IRP2), and anti-CUL1 antibodies. The normalized signal intensity ratios of co-immunoprecipitated neddylated CUL1 (CUL1N8) and FLAGIRP2 are shown below the blots. H Quantitation of results in (G). Data are presented as means of two independent experiments distinguished by dots with different shapes and colors. Source data are provided as a Source Data file.