Co-regulation proteomics reveals substrates and mechanisms of APC/C-dependent degradation - PubMed (original) (raw)

Co-regulation proteomics reveals substrates and mechanisms of APC/C-dependent degradation

Sasha A Singh et al. EMBO J. 2014.

Abstract

Using multiplexed quantitative proteomics, we analyzed cell cycle-dependent changes of the human proteome. We identified >4,400 proteins, each with a six-point abundance profile across the cell cycle. Hypothesizing that proteins with similar abundance profiles are co-regulated, we clustered the proteins with abundance profiles most similar to known Anaphase-Promoting Complex/Cyclosome (APC/C) substrates to identify additional putative APC/C substrates. This protein profile similarity screening (PPSS) analysis resulted in a shortlist enriched in kinases and kinesins. Biochemical studies on the kinesins confirmed KIFC1, KIF18A, KIF2C, and KIF4A as APC/C substrates. Furthermore, we showed that the APC/C(CDH1)-dependent degradation of KIFC1 regulates the bipolar spindle formation and proper cell division. A targeted quantitative proteomics experiment showed that KIFC1 degradation is modulated by a stabilizing CDK1-dependent phosphorylation site within the degradation motif of KIFC1. The regulation of KIFC1 (de-)phosphorylation and degradation provides insights into the fidelity and proper ordering of substrate degradation by the APC/C during mitosis.

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Figures

Figure 1

Figure 1

Overview of cell cycle proteomics experiments of synchronized HeLa cells

  1. A Upper panel: A schematic of APC/C activity with respect to prototypical substrate level profiles. APC/C can complex with either CDC20 or CDH1 as a co-activator. Lower panel: Workflow of the cell synchronization experiment and the subsequent TMT-based quantitative proteomics experiment.
  2. B–D Representative MS/MS spectra and TMT reporter ion traces from the known APC/C substrates NUSAP1 (B), TPX2 (C) and from a non-APC/C substrate, GAPDH (D). Lower case amino acids indicate sites of TMT labels.
  3. E–G The complete TMT-based peptide abundance profiles (after sum normalization) from all MS/MS spectra (peptide-spectrum matches) associated with NUSAP1 (E), TPX2 (F) and GAPDH (G), respectively, are shown in various shades of grey. The red traces indicate the mean.

Figure 2

Figure 2

Protein clustering approach and results

  1. The protein profile similarity screening workflow leading to the identification of novel APC/C substrates.
  2. The six APC/C reference clusters used in the co-regulation analysis as indicated in Steps 4 and 5 of (A). The proteins within each cluster are indicated. The arrowhead indicates a distinct abundance apex, and the asterisk indicates a differential steepness in the ascent and/or descent of the abundance profile.

Figure 3

Figure 3

Co-regulation analysis reveals kinesins as putative novel APC/C targets

  1. Heat map reflecting the percentile (%) in which the kinesins identified in our analysis are ranked in the six reference clusters (C1–C6). Boxed kinesins were flagged as putative APC/C targets based on their median percentile. Bolded values indicate ranks within the 1st percentile of each cluster.
  2. Individual protein abundance profiles for kinesins flagged as candidate (black traces) and non-candidate (green traces) APC/C substrates. KIF22, a known APC/C substrate, is indicated in red. The y-axes indicate relative abundance ranging from 0 to 0.3.

Figure 4

Figure 4

In vitro APC/C-dependent degradation assays on selected kinesins

  1. Results of the degradation assays of 35S-labeled kinesins KIFC1, KIF18A and KIF2C as a function of incubation with G1, i.e. APC/CCDH1-active, HeLa S3 cell extracts in the presence or absence of the APC/C inhibitors EMI1 and SECURIN. The known APC/C substrate SECURIN and kinesin light chain 1 (KLC1) serve as a positive and negative control, respectively. The densitometry-based quantification results of three independent degradation assays are shown.
  2. Results of the degradation assays of 35S-labeled WT or D-box mutants (DM) of KIFC1, KIF18A and SECURIN (positive control) in G1 cell extracts in the presence or absence of the APC/C inhibitors EMI1 and SECURIN. The densitometry-based quantification results of three independent degradation assays are shown. Of note: all lanes for each individual construct come from the same gel, but empty/irrelevant lanes were removed for clarity. The former location of these irrelevant lanes are indicated by dotted black lines; please see accompanying Source Data for scans of the complete gel images.
  3. APC/C-dependent in vitro ubiquitination of either 35S-labeled WT or D-box mutated KIFC1. The high-molecular-weight smearing in the gel indicates ubiquitinated 35S-KIFC1 (the red box indicates the quantified area). The poly-ubiquitinated forms of WT and mutant KIFC1 were quantified and normalized to the 0 h time point.

Source data are available online for this figure.

Figure 5

Figure 5

Identification and quantification of a cell cycle-dependent phosphorylation site in KIFC1

  1. Observed sequence coverage for KIFC1. Sequence stretches in grey were not observed. Sequences indicated in red pertain to the corresponding traces in (B). The FLEXIQuant KIFC1 clone contained an amino acid variant (T368P, italicized tryptic peptide) (Gerhard et al, 2004); this peptide was not used for further analysis.
  2. FLEXIQuant analysis of normalized KIFC1 peptide abundances across the cell cycle. Two different antibodies (A: SIGMA or B: Bethyl Laboratories) were used for immunoprecipitating KIFC1. The red, solid trace highlights the peptide that is post-translationally modified during mitosis to a significant extent (corresponding to phosphopeptide containing pS6). The red, dashed trace highlights the peptide with the known phosphorylation sites S26 or S31 that is not modified in a cell cycle-dependent manner.
  3. Three FLEXIQuant-based KIFC1-derived peptide peak pairs as observed in the sample collected 9 hours post thymidine release: Two peptides (TTLEGHLAK and APSQLPLSGSR) are not significantly modified and thus do not show a deviation from the mean (L:H = 12.4). In contrast, the peptide SPLLEVK is modified to a considerable extent showing a significant deviation from the mean (L:H = 1.6 versus 12.4). The red dashed and solid frames correspond to the peptide trace profiles in (B).

Figure 6

Figure 6

Functional analysis of the Ser6 phosphorylations

  1. Degradation assays of 35S-labeled WT-KIFC1, DM-KIFC1, and various serine-to-alanine and serine-to-aspartic acid mutants in G1 cell extracts in the absence/presence of recombinant EMI1 and SECURIN. Red box indicates mutants (DM and S6D) that are resistant to APC/C-dependent degradation.
  2. Non-degradable KIFC1 induces the monopolar spindle phenotype in mitotic cells. HeLa cells expressing WT or mutant KIFC1-tagged with eGFP were fixed 36 h post transfection, and labeled with DAPI (DNA) and anti-β-TUBULIN antibodies. Representative images from the normal bipolar (WT, S6A) and abnormal monopolar (DM, S6D) phenotypes are shown.
  3. The fraction of bipolar and monopolar spindle cells, 36 h post transfection with the depicted expression constructs.

Source data are available online for this figure.

Figure 7

Figure 7

Model for phosphorylation-dependent inhibition of APC/C-mediated degradation of KIFC1 Phosphorylation of KIFC1 at Ser6 by CDK1 renders KIFC1 incapable of binding to the APC/CCDH1. The phosphate may form a salt bridge with the neighboring arginine which is a key residue in the APC/C recognition motif, the D-box. Removal of the phosphorylation by a phosphatase permits APC/C access to the D-box for subsequent ubiquitination and degradation.

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