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Showing content from https://pubmed.ncbi.nlm.nih.gov/23535663/ below:

CAND1 controls in vivo dynamics of the cullin 1-RING ubiquitin ligase repertoire

Affiliations

Affiliation

¹ Signal Transduction Program, Sanford-Burnham Medical Research Institute, La Jolla, California 92037, USA.

PMID: 23535663
PMCID: PMC3637025
DOI: 10.1038/ncomms2636

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CAND1 controls in vivo dynamics of the cullin 1-RING ubiquitin ligase repertoire

Shuangding Wu et al. Nat Commun. 2013.

Affiliation

¹ Signal Transduction Program, Sanford-Burnham Medical Research Institute, La Jolla, California 92037, USA.

PMID: 23535663
PMCID: PMC3637025
DOI: 10.1038/ncomms2636

Item in Clipboard

Abstract

The combinatorial architecture of cullin 1-RING ubiquitin ligases, in which multiple F-box containing substrate receptors compete for access to CUL1, poses special challenges to assembling cullin 1-RING ubiquitin ligase complexes through high affinity protein interactions while maintaining the flexibility to dynamically sample the entire F-box containing substrate receptor repertoire. Here, using highly quantitative mass spectrometry, we demonstrate that this problem is addressed by CAND1, a factor that controls the dynamics of the global cullin 1-RING ubiquitin ligase network by promoting the assembly of newly synthesized F-box containing substrate receptors with CUL1-RBX1 core complexes. Our studies of in vivo cullin 1-RING ubiquitin ligase dynamics and in vitro biochemical findings showing that CAND1 can displace F-box containing substrate receptors from Cul1p suggest that CAND1 functions in a cycle that serves to exchange F-box containing substrate receptors on CUL1 cores. We propose that this cycle assures comprehensive sampling of the entire F-box containing substrate receptor repertoire in order to maintain the cullin 1-RING ubiquitin ligase landscape, a function that we show to be critical for substrate degradation and normal physiology.

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Figures
Figure 1. Effect of Knd1p on the…

Figure 1. Effect of Knd1p on the CRL1 repertoire

(a) Wildtype (WT) S. pombe cells…

Figure 1. Effect of Knd1p on the CRL1 repertoire

(a) Wildtype (WT) S. pombe cells and cells deleted for knd1 (Δknd1), which also carry a Myc-tagged allele of endogenous Cul1p, were grown in medium containing either ammonium-¹⁴N chloride or ammonium-¹⁵N chloride as the nitrogen sources. Equal numbers of cells were mixed at a ratio of 1:1, followed by the isolation of CRL1 complexes through immunopurification (IP) of Cul1p-Myc. The purified material was analyzed by LC-MS/MS to identify Cul1p-associated proteins and to quantify their relative abundance in WT and Δknd1 samples based on ¹⁴N/¹⁵N peptide/protein ratios. (b) Relative abundance of Cul1p and Cul1p interacting proteins in WT versus Δknd1 cells. Triplicate datasets (Supplementary Data File 1) were averaged and standard deviations are indicated. Statistically significant changes (t-test, p ≤ 0.05) are indicated by asterisks. (c) Absolute amounts of Cul1p, Pof1p, and Pof10p retrieved from a 1:1 mixture of ¹⁵N-labeled WT and ¹⁴N-labeled Δknd1 cells by Cul1p IP and mass spectrometry-based quantification by selected ion monitoring. The right hand panel shows the same measurements for Cul1p and Pof15p from an independent Cul1p IP sample. (d) Lysate of WT and Δknd1 cells expressing Pof1p or Pof10p tagged with the c-Myc epitope were used in immunoprecipitation with anti-Myc antibodies, followed by detection of co-precipitated proteins with either anti-Myc or anti-Cul1p antibodies. The neddylated and unneddylated forms of Cul1p are indicated. Total cell lysate is shown in the bottom two panels. The asterisk denotes a cross-reactivity of the anti-Cul1p antibody.

Figure 2. Effect of Knd1p on CRL1…

Figure 2. Effect of Knd1p on CRL1 dynamics

(a) Steady-state levels of FBPs in WT…

Figure 2. Effect of Knd1p on CRL1 dynamics

(a) Steady-state levels of FBPs in WT and Δknd1 cells. The expression of Pof1p, Pof9p, and Pof10p modified with c-Myc epitopes at the endogenous genomic locus was determined by immunoblotting with Myc antibodies. Blots were reprobed with anti-Skp1p and anti-Cdc2p (PSTAIR) antibodies as a reference. (b) The first 3 bars show a quantification of the FBP ratios in Δknd1 versus WT cells apparent from the immunoblots in (A) as determined by film densitometry. Ratios are from values normalized to Cdc2p. The fourth bar shows the average Skp1p ratio obtained from 4 independent immunoblot measurements including standard deviation. The fifth bar shows the Δknd1/WT ratio of Skp1p quantified by LC-MS/MS. Skp1p was immunopurified independently four times from 1:1 mixtures of ¹⁵N (WT) and ¹⁴N (Δknd1) labeled cells. The average ratio and the standard deviation were plotted. The sixth bar shows the average ratio of all proteins identified in the Skp1p purification. The ratio of these background proteins was close to 1.0 with a narrow standard deviation, supporting the significance of the ~1.5-fold increase in Skp1p levels in Δknd1 cells. (c) Schematic outline of the ¹⁵N pulse labeling strategy. Cells maintained in routine ¹⁴N media were shifted to ¹⁵N media for 120 or 240 minutes prior to preparation of cell lysate, Cul1p-IP, and analysis by LC-MS/MS. (d) Relative fractions of newly synthesized FBPs and CSN subunits associated with Cul1p in wildtype and Δknd1 cells. Data represent averages and standard deviations of three independent experiments (see Supplementary Data 4).

Figure 3. CRL1 complexes are stabilized in…

Figure 3. CRL1 complexes are stabilized in the absence of Knd1p

(a) Myc epitope-tagged Pof15p…

Figure 3. CRL1 complexes are stabilized in the absence of Knd1p

(a) Myc epitope-tagged Pof15p was expressed from a pREP3 plasmid containing an inducible promoter in strains that harbor endogenously tagged Pof10p-Myc. The strains were constructed in two backgrounds, wildtype (WT) and Δknd1. Myc-Pof15p expression was switched on by the removal of thiamine for 14 – 24 h as indicated. CRL1 complexes were immunoprecipitated with anti-Cul1p antibodies and monitored for the levels of co-precipitated Pof10p-Myc and Myc-Pof15p. As a specificity control, the 24 h lysate was used for immunoprecipitation with rabbit IgG. The level of Pof10p bound to Cul1p was quantified and plotted in a bar chart. The bottom panel shows total lysates. (b) Same experiment as in (a) but with Myc-Pof15p expressed at low, non-competing levels. The level of Pof15p bound to Cul1p was quantified and plotted in a bar chart.

Fig. 4. CAND1 displaces F-box proteins from…

Fig. 4. CAND1 displaces F-box proteins from Cul1p

(a) Cul1p- and Pof10p-associated protein complexes were…

Fig. 4. CAND1 displaces F-box proteins from Cul1p

(a) Cul1p- and Pof10p-associated protein complexes were immunopurified from Δknd1 cells and incubated with 1 µg of recombinant His-tagged human CAND1 for 30 minutes. 1 µg of bovine serum albumin was used as control instead of CAND1. The complexes were analyzed for the levels of Cul1p and Pof10p-Myc by immunoblotting with Cul1p and Myc antibodies, and signals were quantified. The asterisk denotes a band present in the CAND1 preparation that weakly cross-reacts with the α-Cul1p antiserum (possibly insect cell CUL1). (b) Pof10p-Myc complexes were isolated from Δknd1 and Δknd1 csn5 cells as in (a) and incubated for the indicated times with 1 ug BSA or CAND1 as indicated. The complexes were analyzed for the levels of Cul1p and Pof10p-Myc by immunoblotting with Cul1p and Myc antibodies, and signals were quantified.

Fig. 5. Knd1p is required for efficient…

Fig. 5. Knd1p is required for efficient CRL1-mediated substrate degradation

(a) Wildtype and Δ knd1…

Fig. 5. Knd1p is required for efficient CRL1-mediated substrate degradation

(a) Wildtype and Δknd1 cells were incubated with 100 ug/ml cycloheximide for the indicated times and Ams2p levels were determined by immunoblotting relative to the loading control Cdc2p. The graph on the right shows a quantification of the results. Exponential trend lines were fitted through the data points in Excel, and the resulting equations were used to calculate half-lives. (b) Hydroxyurea sensitivity of Δknd1 cells. 5-fold serial dilutions of wildtype and Δknd1 cells were spotted onto media with (bottom) or without (top) 10 mM hydroxyurea (HU).