Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The . gov means it’s official. Federal government websites often end in . gov or . mil. Before sharing sensitive information, make sure you’re on a federal government site. VSports app下载.

Https

The site is secure. The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely V体育官网. .

. 2018 Mar 1;69(5):773-786.e6.
doi: 10.1016/j.molcel.2018.01.038.

V体育官网入口 - Cand1-Mediated Adaptive Exchange Mechanism Enables Variation in F-Box Protein Expression

Xing Liu  1 , Justin M Reitsma  1 , Jennifer L Mamrosh  1 , Yaru Zhang  1 , Ronny Straube  2 , Raymond J Deshaies  3
Affiliations

"VSports手机版" Cand1-Mediated Adaptive Exchange Mechanism Enables Variation in F-Box Protein Expression

Xing Liu et al. Mol Cell. .

Abstract (VSports手机版)

Skp1⋅Cul1⋅F-box (SCF) ubiquitin ligase assembly is regulated by the interplay of substrate binding, reversible Nedd8 conjugation on Cul1, and the F-box protein (FBP) exchange factors Cand1 and Cand2. Detailed investigations into SCF assembly and function in reconstituted systems and Cand1/2 knockout cells informed the development of a mathematical model for how dynamical assembly of SCF complexes is controlled and how this cycle is coupled to degradation of an SCF substrate. Simulations predicted an unanticipated hypersensitivity of Cand1/2-deficient cells to FBP expression levels, which was experimentally validated VSports手机版. Together, these and prior observations lead us to propose the adaptive exchange hypothesis, which posits that regulation of the koff of an FBP from SCF by the actions of substrate, Nedd8, and Cand1 molds the cellular repertoire of SCF complexes and that the plasticity afforded by this exchange mechanism may enable large variations in FBP expression during development and in FBP gene number during evolution. .

Keywords: Cand1; SCF cycle; exchange factor; mathematical modeling; ubiquitin ligase V体育安卓版. .

PubMed Disclaimer

Conflict of interest statement

DECLARATION OF INTERESTS

Raymond J. Deshaies is an employee and shareholder of Amgen VSports最新版本.

VSports在线直播 - Figures

Figure 1
Figure 1. Properties of interactions among Cul1, Cand1 and Skp1•F-box protein revealed by FRET
(A) FRET assay for Cand1•Cul1 complex formation. Fluorescence emission spectra from excitation at 350 nm of 70 nM Cul1AMC•Rbx1, 70 nM FlAsHΔH1Cand1, a mixture of the two (FRET), chase control for FRET, or buffer alone. +Chase indicates 700 nM Cand1. Proteins were added in the indicated order. Addition of chase to Cul1+Cand1 had a negligible effect on FRET due to the long t1/2 of the Cand1•Cul1 complex as shown previously (Pierce et al., 2013). (B) kon for Cand1 binding to Cul1. The observed rates of Cand1•Cul1 assembly at different concentrations of Cand1 are plotted. Linear slope gives kon of 1.7 × 107 M−1s−1. Error bars, ± SEM, n = 5 (see also Fig S1A). (C) kon for Cand1 binding to Cul1•Rbx1 preassembled with FBP. Similar to Fig 1B, except with 100 nM Skp1•Skp2 preincubated with 50 nM Cul1AMC•Rbx1. Linear slope gives kon of 2.0 × 106 M−1s−1. Error bars, ± SEM, n ≥ 4 (see also Fig S1B). (D) Disruption of Cand1•Cul1 by Skp1•Skp2. The change in donor fluorescence versus time was measured following addition of 75 nM Skp1•Skp2 or 75 nM Skp1 to 25 nM FlAsHΔH1Cand1•Cul1AMC•Rbx1. (E) koff of Cand1 from ternary exchange intermediate. The single exponential observed rates of Cand1 dissociation from 10 nM FlAsHΔH1Cand1•Cul1AMC•Rbx1 in the presence of increasing concentrations of Skp1•Skp2 were measured (see Fig S1C) and plotted. Fitting of the curve predicts a rate plateau at 67 sec−1. Error bars, ± SEM, n ≥ 3. (F) Kinetic model of the exchange cycle. The number in parentheses indicates the koff of 2.9 s−1 calculated from detailed balance relations (see Fig S4C). (G) Deletion of β hairpin in Cand1 enables formation of a stable complex comprising Cul1, Skp1•Fbxw7, and Cand1. Cand1 or Cand1Δβ (100 nM) was added to 70 nM CFPCul1•Rbx1•Skp1•Fbxw7TAMRA, and formation of SCFFbxw7 was monitored by FRET. + Chase indicates 700 nM Skp1•Skp2. (H) Deletion of loop regions in Skp1 enables formation of a stable complex comprising Cul1, Skp1•Skp2, and Cand1. Skp1•Skp2 or Skp1ΔΔ•Skp2 (700 nM) was added to 70 nM FlAsHCand1•Cul1AMC•Rbx1 and the persistence of the latter complex was monitored by FRET.
Figure 2
Figure 2. Cand1/2 double knockout (DKO) cells display defects in IκBα degradation and SCFβ-TrCP assembly
(A–C) Cand1/2 DKO cells display defects in IκBα degradation. IκBα levels in indicated cell lines were monitored by western blot (WB) at indicated time points after TNFα treatment. Both phospho-IκBα (pIκBα, upper band) and unmodified IκBα (lower band) were detected by the anti-IκBα antibody. Here and elsewhere in this work, we blotted for GAPDH as a loading control. (B) WB analysis of Cand1 and β-TrCP in cell lysates from (A). A more intense exposure (dark) of the Cand1 blot and relative levels of Cand1 are also shown. (C) Quantification of IκBα t1/2 from panel A. (D) Ubiquitination of pIκBα is significantly reduced in DKO cells. WB analysis (with anti-pIκBα antibody) of the ubiquitination of pIκBα in WT and DKO cells upon TNFα treatment. DKO36 from (A) was used in this experiment and thereafter. (E–F) TNFα promotes formation of SCFβ-TrCP in WT but not DKO cells. Schematic workflow of the experiment is depicted in (E), and WB analysis of endogenous SCFβ-TrCP in WT and DKO cells before and after 10-min TNFα treatment is shown in (F). Relative levels of SCFβ-TrCP were calculated as the β-TrCP:Cul1 ratio in 3xFLAGCul1 immunoprecipitations (IPs), and all ratios were normalized to that obtained for the IP from WT cells not treated with TNFα. Average fold increase of SCFβ-TrCP induced by TNFα treatment is shown in the graph. Error bars, ± SEM, n = 3, P value = 0.001. (G–H) Recruitment of Cul1 to pIκBα•β-TrCP is inefficient in DKO cells. Schematic workflow of the experiment is depicted in (G), and WB analysis of the recruitment of β-TrCP and Cul1 to pIκBα following 10-min TNFα treatment is shown in (H). Expression of 3xFLAGIκBα was induced by 100 ng/ml tetracycline for 24 hours. Relative levels of β-TrCP and Cul1 recruited to pIκBα were calculated as the β-TrCP:pIκBα (see also Fig S2I) and Cul1:β-TrCP ratios in the IPs. Average levels of Cul1 recruited to pIκBα•β-TrCP are shown in the graph. Error bars, ± SEM, n = 3, P value = 0.0001.
Figure 3
Figure 3. Cand1 enhances formation of Cul1•Dcn1 complex and subsequent neddylation of Cul1 stabilizes newly formed SCF
(A) Stable Cul1•Dcn1 complex is dramatically reduced in DKO cells. IP-WB analysis of interactions between endogenous 3xFLAGCul1 and Dcn1 in WT and DKO cells pre-treated with either 0.1% DMSO or 1 μM MLN4924 for 1 hour. (B) Cand1 stabilizes Cul1•Dcn1 complex in vitro. Pulldown-WB analysis of recombinant Dcn1 (0.2 μM) and Ubc12 (0.2 μM) bound to recombinant Cul1•GSTRbx1 (0.4 μM) in the presence and absence of recombinant Cand1, Cand11-603, or Cand1604-1230 (all 0.4 μM). A more intense exposure (dark) of the Dcn1 blot is also shown. (C) The Cul1•GSTDcn1 complex is stabilized by Cand1 in vitro. Pulldown (PD) analysis of recombinant Cul1•Rbx1 (1 μM) bound to recombinant GSTDcn1 (0.6 μM) in the presence of 0–3 μM Cand1. Protein samples were fractionated on a SDS-PAGE gel and stained with Coomassie Blue. Normalized levels of Cul1 recovered were calculated as the ratio of Cul1 to GSTDcn1. (See also Fig S3A) (D) Thermodynamic cycle of Dcn1, Cul1•Rbx1 and Cand1 binding. All numbers are KD values. The KD of 1.8 × 10−6 M for Dcn1 and Cul1•Rbx1 was reported previously (Monda et al., 2013); the KD of 5 × 10−8 M for Dcn1 and Cand1•Cul1•Rbx1 was estimated based on results in Fig 3C and S3A; the KD of 7 × 10−13 M for Cul1•Rbx1 and Cand1 was from Fig 1B; and the KD in parentheses was calculated from detailed balance considerations (see Fig S4C). (E–F) Cand1-bound Cul1 is neddylated faster than free Cul1 in the presence of FBPs. Schematic workflow of a competitive Cul1 neddylation assay is shown in (E). Free Cul1•Rbx1 and Cand1•Cul1•Rbx1 in which the different Cul1 species are labeled with different fluors (FAM or TAMRA) compete for limiting Dcn1, and neddylation enzymes are provided by DKO lysate. 1x represents 50 nM protein in the final sample mixture. Fluorescence scan of the SDS-PAGE gel containing samples prepared as described in (E) is shown in (F). “Fold increase with Cand1” was calculated as the ratio of percent neddylation of Cand1-bound Cul1 to free Cul1 (see Fig S3B–C for negative controls). A representative result of three replicates is shown. (G) Neddylation increases the assembly of FBP with Cand1-bound Cul1. Cand1, Dcn1 and Cul1•GSTRbx1 were pre-incubated and then mixed 1:1 (v:v) with Skp1•β-TrCP and Ubc12 or Ubc12 charged to Nedd8 (Ubc12~Nedd8). After 15 min incubation, the protein mixture was incubated with glutathione beads and immobilized proteins were analyzed by WB. (See also Fig S3D.)
Figure 4
Figure 4. Mathematical model of the SCF cycle
(A) Simplified scheme illustrating the main processes and interactions considered in the mathematical model (see Fig S4 for a detailed reaction scheme). Lines with unidirectional arrows represent irreversible reactions. FB1 stands for Skp1•β-TrCP whereas FB2 represents a pool of auxiliary Skp1•F-box proteins that compete for access to Cul1•Rbx1. Both F-box proteins form SCF ligases with Cul1•Rbx1 that undergo the same cycle of processes including F-box exchange, neddylation, deneddylation, substrate binding and substrate degradation. (B–F) Model simulations and predictions. Simulations labeled in orange color were used to estimate unknown parameters. Remaining simulations represent model predictions. Error bars for predictions were obtained from a profile likelihood analysis (Fig S5A). Experimental results are shown as thin bars. To simulate inhibition of Nedd8 conjugation by MLN4924 we set knedd=0. As a result the fraction of Cul1•Rbx1 bound to Cand1 increased while the fraction of Cul1•Rbx1 bound to Skp1•FBP decreased (Reitsma et al. 2017) (B). If Cand proteins are absent (DKO) the latter fraction is predicted to increase to 100% in agreement with observations. The model confirms (C, upper panel) that re-expression of Cand1 (13% of WT level) in a DKO cell line reduces the half-life (t1/2) for IκBα degradation back to WT levels (Fig 2C). The half-life for substrate degradation is predicted to exhibit a U-shaped dependence on the cellular Cand1 concentration with an extended valley where t1/2 ≈20min remains approximately constant (C, lower panel). Dashed lines indicate the Cand1 concentration in WT (black) and DKO cells with Cand1 re-expressed to 13% of WT level (red). When substrate is added the fraction of β-TrCP bound to Cul1 increases ~1.7-fold (D) from its steady state level (46%) as observed in WT cells (Fig 2F). Cul1 overexpression is predicted to reduce the fraction of neddylated Cul1 (E) in agreement with observations. Also, Cul1 overexpression should have no effect on the half-life for IκBα degradation in WT, but should reduce t1/2 in DKO cells back to WT level (F). In contrast, overexpression of β-TrCP is predicted to have no effect on t1/2 in DKO cells (F).
Figure 5
Figure 5. Experimental concordance with mathematical predictions
(A) 3xFLAGCul1 overexpression rescues the IκBα degradation defect of DKO cells. IκBα levels were monitored by western blot (WB) at indicated time points after TNFα treatment. Overexpression of 3xFLAGCul1 was induced by tetracycline. Average relative t1/2 of IκBα are shown in the graph. Error bars: range of values, n = 2. (B) 3xFLAGCul1 overexpression impedes cullin neddylation. WB analysis of cullins in cell lysates from (A). Fold increase in total Cul1 levels and percent neddylation of overexpressed 3xFLAGCul1 and endogenous Cul4a are indicated. A representative result of two replicates is shown. (C) β-TrCP overexpression does not rescue the IκBα degradation defect of DKO cells. IκBα levels were monitored by western blot (WB) at indicated time points after TNFα treatment. Overexpression of β-TrCP was induced by tetracycline. Average relative t1/2 of IκBα are shown in the graph. Error bars: range of values, n = 2. (See Fig S5E for WB of β-TrCP) (D–E) Overexpression of Cul1 significantly depletes free β-TrCP in the DKO cells. As illustrated in (D), cells with/without tetracycline induced 3xFLAGCul1 were lysed in buffer containing Cul1•GSTRbx1 sponge protein and subjected to GST pulldown, which probes changes in levels of unbound cellular proteins capable of binding to sponge in cell lysate (see Fig S5F for WB images). Average changes in protein levels compared to non-tetracycline induced control are shown in the graph. Overexpression of 3xFLAGCul1 depleted the pool of free β-TrCP in DKO cells by 80%. Error bars: range of values, n = 2. (F–G) Overproduction of β-TrCP modestly reduces the efficiency of its assembly with Cul1. As illustrated in (F), cells containing endogenous 3xFLAGCul1 were lysed in buffer containing Cul1•GSTRbx1 sponge protein. β-TrCP bound to endogenous 3xFLAGCul1 was probed by anti-FLAG beads, and free cellular β-TrCP capable of binding to sponge in cell lysate was probed by GST beads (see Fig S5G for WB images). Percentage of β-TrCP bound to endogenous 3xFLAGCul1 in WT and DKO cells with or without tetracycline-induced overexpression of β-TrCP is graphed. Error bars: range of values, n = 2.
Figure 6
Figure 6. Overexpression of single F-box proteins suppresses cell proliferation in DKO cells by sequestering Cul1
(A) Overexpression of Fbxo6 increases the t1/2 of IκBα only in DKO cells (see Fig S6A–B for WB images). Fbxo6 was overexpressed by transduction with a recombinant lentivirus expressing HAFbxo6. The assay was performed four days after the viral transduction. Average fold increase of IκBα t1/2 by Fbxo6 overexpression in WT and DKO cells are graphed. Error bar: ± SD, n = 3, P value < 0.01. (B) Overexpression of Fbxo6 in DKO cells reduces degradation of SCF substrates. All samples were analyzed on the same gel and blot, but one lane between WT and DKO samples on the blot image was eliminated and indicated as a space. (C) Overexpressed Fbxo6 sequesters Cul1 in DKO cells. Cells were infected with recombinant lentiviruses carrying the HAFbox6 gene five days before HAFbxo6 was IP’d from WT3xFLAG-Cul1 and DKO3xFLAG-Cul1 cells in the presence of recombinant Cul1•GSTRbx1 (+ sponge). Equal percent volumes of Input (In), immunoprecipitation eluent (IP), and flow-through (FT) were analyzed by WB. Long (L) and short (S) exposures of endogenous 3xFLAGCul1 are shown. Quantifications of percent Cul1 in the HAFbxo6 IPs are graphed. Error bars: ± SD, n = 3, P value < 0.01. (D) Fbxo6 overexpression reduces proliferation of DKO cells in a specific, FBP-dependent manner. Cells were treated with recombinant lentiviruses carrying different FBP constructs as indicated. Three days after lentiviral infection, cells were equally seeded and counted every 24 hrs for 4 days. Average cell doubling time is graphed. Error bars: ± SD, n = 3, P value < 0.01. Note that Fbxl16 bound at least as much Skp1 as Fbxo6 but did not bind Cul1 (compare Fig S6G with panel D), and that re-introduction of Cand1 rescued the DKO cells. (E) Overexpression of Cul1 partially rescues toxicity of overproduced Fbxo6 in DKO cells. Cul1 overexpression was induced by tetracycline. Cell doubling was measured as in (D). Error bars: ± SD, n = 3. (F) Overexpression of HASkp2 or HASkp2ΔLRR slows cellular proliferation in DKO cells. Cells were infected by lentiviruses and cell doubling was measured as in (D). Error bars: ± SD, n = 3, P value < 0.01. (G) Overexpression of HASkp2ΔLRR increased the level of apoptosis marker in DKO cells. A representative result of two replicates is shown.
Figure 7
Figure 7. Rapid cycling of Cul1 in human cells
(A) Cycling of Cul1 summarized from biophysical, cellular and computational studies. Association rates are computed based on kon and steady state cellular concentrations of unbound proteins, and the cycle time for Cul1 is computed using effective rates for the reversible binding steps (see also Fig S5D). The reversal of the de-neddylation reaction by Dcn1 (dashed lines) is discouraged in WT cells due to preferential association of Dcn1 with Cand1•Cul1, but is expected to occur more frequently in DKO cells. The substrate of the SCF complex can bind the FBP either in its free or assembled state. Substrate binding stabilizes the SCF complex by preventing CSN from binding. The 55 s cycle time for Cul1 represents the average time it takes a Cul1 molecule to be deneddylated and exchanged into a different SCF if it is not bound by substrate. (B) Deneddylation of Cul1 is fast in human cells. HEK293 cells were treated with 3 μM MLN4924 to inhibit the Nedd8 E1 and were maintained at 37°C fo r the indicated time before being directly lysed on culture plates. Average t1/2 for deneddylation is shown. Error bars, ± SD, n = 3. (C) Neddylation of Cul1 is fast in human cells. Assay condition was similar to (B) but 3 μM CSN5i-3 was used to inhibit CSN (Schlierf et al., 2016). Average t1/2 for neddylation is shown. Error bars, ± SD, n = 3.
See this image and copyright information in PMC

Comment in

References

    1. Bennett EJ, Rush J, Gygi SP, Harper JW. Dynamics of cullin-RING ubiquitin ligase network revealed by systematic quantitative proteomics. Cell. 2010;143:951–965. - PMC - PubMed
    1. Bosu DR, Feng H, Min K, Kim Y, Wallenfang MR, Kipreos ET. C. elegans CAND-1 regulates cullin neddylation, cell proliferation and morphogenesis in specific tissues. Developmental biology. 2010;346:113–126. - "VSports手机版" PMC - PubMed
    1. Chen W, Xiong S, Li J, Li X, Liu Y, Zou C, Mallampalli RK. The ubiquitin E3 ligase SCF-FBXO24 recognizes deacetylated nucleoside diphosphate kinase A to enhance its degradation. Molecular and cellular biology. 2015;35:1001–1013. - PMC - PubMed
    1. Cheng Y, Dai X, Zhao Y. AtCAND1, a HEAT-repeat protein that participates in auxin signaling in Arabidopsis. Plant Physiol. 2004;135:1020–1026. - PMC - PubMed
    1. Chuang HW, Zhang W, Gray WM. Arabidopsis ETA2, an apparent ortholog of the human cullin-interacting protein CAND1, is required for auxin responses mediated by the SCF(TIR1) ubiquitin ligase. The Plant cell. 2004;16:1883–1897. - PMC - PubMed

Publication types

V体育平台登录 - MeSH terms