Preparation of Synchronized Human Cell Extracts to Study Ubiquitination and Degradation

Williamson, Adam; Jin, Lingyan; Rapé, Michael

doi:10.1007/978-1-60327-993-2_19

Cited by 16 publications

(20 citation statements)

References 14 publications

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“…G1 HeLa extract was prepared as described (Williamson et al, 2009b). APC/C was captured on Protein A/G beads with an anti-Cdc27 antibody (Santa Cruz Biotech), by rotating the bead slurry with G1 extract on a rotary mixer for ~4h.…”

Section: Methodsmentioning

confidence: 99%

APC/C and SCF cyclin F Constitute a Reciprocal Feedback Circuit Controlling S-Phase Entry

et al. 2016

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SUMMARY The Anaphase Promoting Complex/Cyclosome (APC/C) is an ubiquitin ligase and core component of the cell cycle oscillator. During G1-phase APC/C binds to its substrate receptor Cdh1 and APC/CCdh1 plays an important role in restricting S-phase entry and maintaining genome integrity. We describe a reciprocal feedback circuit between APC/C and a second ubiquitin ligase, the SCF (Skp1-Cul1-F box). We show that Cyclin F, a cell cycle regulated substrate receptor (F-box protein) for the SCF, is targeted for degradation by APC/C. Furthermore, we establish that Cdh1 is itself a substrate of SCFCyclin F. Cyclin F loss impairs Cdh1 degradation and delays S-phase entry, and this delay is reversed by simultaneous removal of Cdh1. These data indicate that the coordinated, temporal ordering of Cyclin F and Cdh1 degradation, organized in a double-negative feedback loop, represents a fundamental aspect of cell cycle control. This mutual antagonism could be a feature of other oscillating systems.

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Section: Methodsmentioning

confidence: 99%

APC/C and SCF cyclin F Constitute a Reciprocal Feedback Circuit Controlling S-Phase Entry

et al. 2016

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“…BMH-21 treatment increased polyubiquitination of RPA194 as detected by a polyubiquitin antibody (Figure 8A). We then used an in vitro degradation assay to examine the ubiquitin-dependence of RPA194 degradation (Williamson et al, 2009). Ubiquitin, recombinant E1 and E2 enzymes and BMH-21-treated cellular lysate as source of E3-ligase were mixed to assess the degradation of in vitro-translated RPA194.…”

Section: Resultsmentioning

confidence: 99%

“…In vitro degradation of RPA194 was as in Williamson et al, 2009 with slight modifications. Cells were treated with BMH-21 for 3 hr and extracts were prepared in SB-buffer (20 mM HEPES pH 7.5, 1.5 mM MgCl 2 , 5 mM KCl, 1 mM DTT, protease inhibitors (Roche), 15 mM creatine phosphate, 2 mM ATP).…”

Section: Methodsmentioning

confidence: 99%

A Targeting Modality for Destruction of RNA Polymerase I that Possesses Anticancer Activity

et al. 2014

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SUMMARY We define here the activity and mechanisms of action of a small molecule lead compound for cancer targeting. We show that the compound, BMH-21, has wide and potent antitumorigenic activity across NCI60 cancer cell lines and represses tumor growth in vivo. BMH-21 binds GC-rich sequences, which is present at high frequency in ribosomal DNA genes, and potently and rapidly represses RNA polymerase I (Pol I) transcription. Strikingly, we find that BMH-21 causes proteasome-dependent destruction of RPA194, the large catalytic subunit protein of Pol I holocomplex, and this correlates with cancer cell killing. Our results show that Pol I activity is under proteasome-mediated control, which reveals an unexpected therapeutic opportunity.

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“…Below we discuss a few important parameters and some problems that may arise when performing this assay. Further troubleshooting tips for both extract preparation and the protein microarrays may be found in 12 and 15 , respectively. See Table 2 for description of a few common troubleshooting tips.…”

Section: Commentarymentioning

confidence: 99%

Post‐Translational Modification Profiling—a High‐Content Assay for Identifying Protein Modifications in Mammalian Cellular Systems

Merbl

Kirschner

2014

CP Protein Science

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Protein microarrays are extremely useful for detecting substrates of protein kinases, substrates of ubiquitin ligases and other post translational modifications. The ability to screen binding interactions as well as post-translational modifications of thousands of proteins at once has improved our ability to identify their targets. Utilizing such systems in combination with functional mammalian cell extracts that preserve enzymatic activity offers advantages in identifying semi-quantitative changes of these interactions in the context of specific cellular conditions. This article provides a detailed procedure for setting up an extract-based activity assay for high content detection of protein post-translation modifications. It also provides basic guidelines for data analysis.

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Preparation of Synchronized Human Cell Extracts to Study Ubiquitination and Degradation

Cited by 16 publications

References 14 publications

APC/C and SCF cyclin F Constitute a Reciprocal Feedback Circuit Controlling S-Phase Entry

APC/C and SCF cyclin F Constitute a Reciprocal Feedback Circuit Controlling S-Phase Entry

A Targeting Modality for Destruction of RNA Polymerase I that Possesses Anticancer Activity

Post‐Translational Modification Profiling—a High‐Content Assay for Identifying Protein Modifications in Mammalian Cellular Systems

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