Cell cycle oscillators underlying orderly proteolysis of E2F8

Abstract

We uncovered interlocking mechanisms regulating the temporal proteolysis of the transcriptional repressor E2F8 in cycling cells including SCFCyclin F in G2, dephosphorylation of Cdk1 sites, and activation of APC/CCdh1, but not APC/CCdc20 during mitotic exit and G1. Differential stabilization under limited APC/C activity allows E2F8 to reaccumulate during late G1 and coregulate S-phase entry.

Protein degradation analysis by affinity microfluidics

“…NDB cells are based on the HEK293 cell line. A detailed description of this cell system can be found in Ref [18]. NDB and HeLa S3 (ATCC; #CCL-2.2) cells were maintained in tissue culture dishes containing Dulbecco's Modified Eagles Medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and 1% Penicillin-Streptomycin solution (Biological Industries; #01-055-1A, #04-001-1A, #03-020-1B, #03-031-1B).…”

“…As a proof of concept, we utilized mitotic extracts from HEK293 cells that are blocked in an anaphase-like state due to high levels of non-degradable Cyclin-B1. This mitotic cell-free system, hereafter referred to as NDB, recapitulates APC/C Cdc20 -mediated proteolysis of the cell cycle proteins Securin and Geminin [18,31]. Next, we tested whether pDOC can be utilized to obtain reliable kinetic information on protein degradation.…”

“…Note that reaction cocktails contained 1 μl IVT, 20 μl extracts and 2 μl ubiquitin/E. mix solution, following our standard protocol [18,27,34] For each time point, 5 μl reaction mixes were flowed for a period of 3 min through protein chambers with open MITOMI button valves, allowing immobilization of the target protein, while the neck valves were closed. This loading protocol enabled clear visualization of the target protein without the concern of signal saturation (Figure S2).…”

“…Protein degradation assays in cell-free extracts, also known as 'cell-free systems', have been instrumental in cell biology research, enabling direct and quantitative analyses of ubiquitin-mediated proteolysis in physiologically relevant environments. In fact, much of the cell cycle principles were discovered by monitoring the degradation of cell cycle proteins in extracts from frog eggs or cycling human cells (see for example [14][15][16][17][18][19][20]). The extensive use of these 'degradation assays' in today's modern era, is a testament to their efficacy (see for example [21][22][23]).…”

In silico APC/C substrate discovery reveals cell cycle-dependent degradation of UHRF1 and other chromatin regulators

“…Accordingly, it remains difficult to assess the relative contributions of mRNA expression and PLOS BIOLOGY protein degradation to overall protein expression during cell cycle. It is also unknown if other ubiquitin ligases might control UHRF1 during cell cycle, as is the case for some APC/C substrates [76,77]. The overall contributions of mRNA expression or additional E3s to the dynamics of protein expression during cell cycle are unknown for most APC/C substrates.…”