Using in Vivo Biotinylated Ubiquitin to Describe a Mitotic Exit Ubiquitome from Human Cells

Min, Mingwei; Mayor, Ugo; Dittmar, Gunnar; Lindon, Catherine

doi:10.1074/mcp.m113.033498

Cited by 36 publications

(57 citation statements)

References 71 publications

(77 reference statements)

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“…To compare the ubiquitination capacity of these two proteins in vivo , we expressed them in a human U2OS cell line [44] also expressing biotin-tagged ubiquitin [45] and synchronized the cells in mitotic exit. Taking advantage of the affinity purification reagent GFP-Trap, that we have found to bind GFP derivatives even under denaturing conditions, we purified Aurora A-Venus from these synchronized cells.…”

Section: Resultsmentioning

confidence: 99%

Ubiquitination site preferences in anaphase promoting complex/cyclosome (APC/C) substrates

2013

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Ordered progression of mitosis requires precise control in abundance of mitotic regulators. The anaphase promoting complex/cyclosome (APC/C) ubiquitin ligase plays a key role by directing ubiquitin-mediated destruction of targets in a temporally and spatially defined manner. Specificity in APC/C targeting is conferred through recognition of substrate D-box and KEN degrons, while the specificity of ubiquitination sites, as another possible regulated dimension, has not yet been explored. Here, we present the first analysis of ubiquitination sites in the APC/C substrate ubiquitome. We show that KEN is a preferred ubiquitin acceptor in APC/C substrates and that acceptor sites are enriched in predicted disordered regions and flanked by serine residues. Our experimental data confirm a role for the KEN lysine as an ubiquitin acceptor contributing to substrate destruction during mitotic progression. Using Aurora A and Nek2 kinases as examples, we show that phosphorylation on the flanking serine residue could directly regulate ubiquitination and subsequent degradation of substrates. We propose a novel layer of regulation in substrate ubiquitination, via phosphorylation adjacent to the KEN motif, in APC/C-mediated targeting.

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Section: Resultsmentioning

confidence: 99%

Ubiquitination site preferences in anaphase promoting complex/cyclosome (APC/C) substrates

2013

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“…Protein degradation removes proteins and hence their phosphorylation status, ensuring that mitotic exit continues in one direction and does not reverse (11,12). However, currently only ϳ170 proteins have been found to be targeted for degradation during mitotic exit (13), which although likely to be a significant underestimation, is only well short of the 5000ϩ proteins phosphorylated during mitosis (14). Therefore, during exit a substantial number of proteins must be dephosphorylated by phosphatases in preparation for the next G1 phase.…”

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confidence: 99%

Global Phosphoproteomic Mapping of Early Mitotic Exit in Human Cells Identifies Novel Substrate Dephosphorylation Motifs

McCloy

Parker

Rogers

et al. 2015

Molecular & Cellular Proteomics

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Entry into mitosis is driven by the coordinated phosphorylation of thousands of proteins. For the cell to complete mitosis and divide into two identical daughter cells it must regulate dephosphorylation of these proteins in a highly ordered, temporal manner. There is currently a lack of a complete understanding of the phosphorylation changes that occur during the initial stages of mitotic exit in human cells. Therefore, we performed a large unbiased, global analysis to map the very first dephosphorylation events that occur as cells exit mitosis. We identified and quantified the modification of >16,000 phosphosites on >3300 unique proteins during early mitotic exit, providing up to eightfold greater resolution than previous studies. The data have been deposited to the ProteomeXchange with identifier PXD001559. Only a small fraction (ϳ10%) of phosphorylation sites were dephosphorylated during early mitotic exit and these occurred on proteins involved in critical early exit events, including organization of the mitotic spindle, the spindle assembly checkpoint, and reformation of the nuclear envelope. Surprisingly this enrichment was observed across all kinase consensus motifs, indicating that it is independent of the upstream phosphorylating kinase. Therefore, dephosphorylation of these sites is likely determined by the specificity of phosphatase/s rather than the activity of kinase/s. Dephosphorylation was significantly affected by the amino acids at and surrounding the phosphorylation site, with several unique evolutionarily conserved amino acids correlating strongly with phosphorylation status. These data provide a potential mechanism for the specificity of phosphatases, and how they co-ordinate the ordered events of mitotic exit. In summary, our results provide a global overview of the phosphorylation changes that occur during the very first stages of mitotic exit, providing novel mechanistic insight into how phosphatase/s specifically regulate this critical transition. Molecular & Cellular

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“…KIFC1 can bind to survivin and prevents survivin from poly-ubiquitination [23], and, therefore, protects the cells from apoptosis. As mentioned before, KIFC1 itself also possess the potential to be bound and degraded by APC/C [54]. It is, therefore, possible that the protection relationship between KIFC1 and survivin is mutual.…”

Section: Evidence For Applicability Of Kifc1-related Systems In Anti-mentioning

confidence: 98%

“…In the human cancer cell line U2OS, KIFC1 has been found to possess an ubiquitination site which is then degraded by the 26s proteasome. Here, KIFC1 was found to be able to bind to E3 ligase APC/C at its destruction box (D box), which is also thought necessary for the degradation process [54]. In addition, CDK-1 (MPF) functions as a KIFC1 stabilizer during ubiquitination and degradation by phosphorylating KIFC1 at the Ser6 site [55] (Figure 2D).…”

Section: Evidence For Applicability Of Kifc1-related Systems In Anti-mentioning

confidence: 99%

KIFC1: a promising chemotherapy target for cancer treatment?

Xiao

Yang

2016

Oncotarget

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The kinesin motor KIFC1 has been suggested as a potential chemotherapy target due to its critical role in clustering of the multiple centrosomes found in cancer cells. In this regard, KIFC1 seems to be non-essential in normal somatic cells which usually possess only two centrosomes. Moreover, KIFC1 is also found to initiatively drive tumor malignancy and metastasis by stabilizing a certain degree of genetic instability, delaying cell cycle and protecting cancer cell surviving signals. However, that KIFC1 also plays roles in other specific cell types complicates the question of whether it is a promising chemotherapy target for cancer treatment. For example, KIFC1 is found functionally significant in vesicular and organelle trafficking, spermiogenesis, oocyte development, embryo gestation and double-strand DNA transportation. In this review we summarize a recent collection of information so as to provide a generalized picture of ideas and mechanisms against and in favor of KIFC1 as a chemotherapy target. And we also drew the conclusion that KIFC1 is a promising chemotherapy target for some types of cancers, because the side-effects of inhibiting KIFC1 mentioned in this review are theoretically easy to avoid, while KIFC1 is functionally indispensable during mitosis and malignancy of multi-centrosome cancer cells. Further investigations of how KIFC1 is regulated throughout the mitosis in cancer cells are needed for the understanding of the pathways where KIFC1 is involved and for further exploitation of indirect KIFC1 inhibitors.

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Using in Vivo Biotinylated Ubiquitin to Describe a Mitotic Exit Ubiquitome from Human Cells

Cited by 36 publications

References 71 publications

Ubiquitination site preferences in anaphase promoting complex/cyclosome (APC/C) substrates

Ubiquitination site preferences in anaphase promoting complex/cyclosome (APC/C) substrates

Global Phosphoproteomic Mapping of Early Mitotic Exit in Human Cells Identifies Novel Substrate Dephosphorylation Motifs

KIFC1: a promising chemotherapy target for cancer treatment?

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