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

Abstract

Using multiplexed quantitative proteomics, we analyzed cell cycledependent 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.

Integrated Microfluidics for Protein Modification Discovery

“…1A). Reticulocyte lysate is then applied to the chip for in vitro translation (IVT), an established method in PTM research and beyond (4,11,(17)(18)(19). Protein expression is nearly unaffected by protein length and compatible with transmembrane-and other insoluble proteins (Glick and Gerber; manuscript in preparation).…”

“…The PCR product was cloned in-frame upstream to eGFP in pCS-FA vector. The plasmids: pCS2-FA-Securin, pCS2-FA, pCS2-FA-Kifc1-eGFP, and Emi1 C terminus (amino acids 299 -477) in pGEX, were described elsewhere (4,10,11). pCS2-GFP was kindly provided by Lior Appelbaum (Bar-Ilan University, Ramat Gan, Israel).…”

“…The sequential binding of Ub to itself via one of its 7 K residues or its N-terminus, can generate distinct Ub-chains that differentially regulate proteins' fate, most notably: degradation by the proteasome (25). As proof of concept, we focused on the anaphase-promoting complex/cyclosome (APC/C), a Ub ligase that targets Securin, mitotic cyclins, and other cell cycle proteins for degradation by the proteasome (4,10,26). We based our approach on rhodamine (Rd)-labeled-Ub (Fig.…”

“…We based our approach on rhodamine (Rd)-labeled-Ub (Fig. 4A) and a cell-free system generated from HeLa S3 cells, pre-synchronized to the G1-phase of the cell cycle (G1 extracts) (4,10,11).…”

Arabidopsis Cell Division Cycle 20.1 Is Required for Normal Meiotic Spindle Assembly and Chromosome Segregation

“…The SAC proteins regulate chromosome segregation by sequestering CDC20 (Cell Division Cycle 20), preventing it from serving as a cofactor for the E3 ubiquitin ligase anaphasepromoting complex/cyclosome (APC/C) (Izawa and Pines, 2015). Correction of mistakes in kinetochore attachment to the spindle releases CDC20, which then activates APC/C for the removal of cohesin, thus promoting entry into anaphase (Salah and Nasmyth, 2000;Singh et al, 2014). In mitosis, the SAC proteins, including the protein kinase Aurora B, are recruited to unattached kinetochores to delay chromosome segregation until all chromosomes are correctly attached to the bipolar spindle (Kang and Yu, 2009;Zich and Hardwick, 2010).…”