Sumoylation promotes optimal APC/C activation and timely anaphase

Lee, Christine C.; Li, Bing; Yu, Hongtao; Matunis, Michael J.

doi:10.7554/elife.29539

Cited by 30 publications

(43 citation statements)

References 78 publications

(118 reference statements)

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“…With in vitro analysis of a substrate, mutants can also be particularly valuable in verifying specific modification sites. Here we describe a protocol for in vitro SUMO modification routinely used in our laboratory ( Zhu et al ., 2008 ; Reiter et al ., 2016 ), as recently illustrated by our analysis of the APC4 subunit of APC/C ( Lee et al ., 2018 ).…”

Section: [Background]supporting

confidence: 72%

“…To directly observe SUMO modification of specific substrates, immunoblots may also be performed with substrate-specific antibodies if modification levels are sufficiently high. For example, APC4 is a subunit of the Anaphase Promoting Complex/Cyclosome (APC/C) that is SUMO-modified robustly during the mitotic stage of the cell cycle as observed by immunoblot analysis of whole cell lysates from synchronized cells ( Lee et al ., 2018 ). Although SUMO modification by a single protein subunit can be detected as a ~12 kDa shift in molecular weight ( Figure 1 ), this shift cannot be distinguished from modification by other ubiquitin-like proteins without further validation.…”

Section: [Background]mentioning

confidence: 99%

“…Although SUMO modification by a single protein subunit can be detected as a ~12 kDa shift in molecular weight ( Figure 1 ), this shift cannot be distinguished from modification by other ubiquitin-like proteins without further validation. An alternative method for demonstrating SUMO modification utilizes a cell line with His-SUMO, followed by Ni-NTA purification under denaturing conditions and immunoblotting for co-purifying proteins of interest ( Lee et al ., 2018 ). Complimentary approaches for detecting and validating SUMO-modified proteins utilize overexpression of SUMO E1 and E2 enzymes, or the depletion of SENPs, followed by immunoblotting for the protein of interest.…”

Section: [Background]mentioning

confidence: 99%

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A Method for SUMO Modification of Proteins in vitro

Lee

et al. 2018

BIO-PROTOCOL

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The Small Ubiquitin-related Modifier (SUMO) is a protein that is post-translationally added to and reversibly removed from other proteins in eukaryotic cells. SUMO and enzymes of the SUMO pathway are well conserved from yeast to humans and SUMO modification regulates a variety of essential cellular processes including transcription, chromatin remodeling, DNA damage repair, and cell cycle progression. One of the challenges in studying SUMO modification in vivo is the relatively low steady-state level of a SUMO-modified protein due in part to the activity of SUMO deconjugating enzymes known as SUMO Isopeptidases or SENPs. Fortunately, the use of recombinant SUMO enzymes makes it possible to study SUMO modification in vitro. Here, we describe a sensitive method for detecting SUMO modification of target human proteins using an in vitro transcription and translation system derived from rabbit reticulocyte and radiolabeled amino acids.

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Section: [Background]supporting

confidence: 72%

Section: [Background]mentioning

confidence: 99%

Section: [Background]mentioning

confidence: 99%

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A Method for SUMO Modification of Proteins in vitro

Lee

et al. 2018

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“…Yeast SUMOylation mutants showed a large budded phenotype and fail to properly degrade APC/C substrates Pds1 and Clb3, indicating an essential role for SUMOylation during the mitotic transition 36 . Recently, APC4, one subunit of APC/ C complex, has been identified to be SUMOylated during mitosis and is required for timely anaphase onset 37,38 . Proteomic studies have also identified SUMOylation sites on other subunits of the APC/C 39 .…”

Section: Discussionmentioning

confidence: 99%

P53 suppresses SENP3 phosphorylation to mediate G2 checkpoint

et al. 2020

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In response to DNA damage, p53-mediated signaling is regulated by protein phosphorylation and ubiquitination to precisely control G2 checkpoint. Here we demonstrated that protein SUMOylation also engaged in regulation of p53mediated G2 checkpoint. We found that G2 DNA damage suppressed SENP3 phosphorylation at G2/M phases in p53dependent manner. We further found that the suppression of SENP3 phosphorylation was crucial for efficient DNA damage/p53-induced G2 checkpoint and G2 arrest. Mechanistically, we identified Cdh1, a subunit of APC/C complex, was a SUMOylated protein at G2/M phase. SENP3 could de-SUMOylate Cdh1. DNA damage/p53-induced suppression of SENP3 phosphorylation activated SENP3 de-SUMOylation of Cdh. De-SUMOylation promoted Cdh1 dephosphorylation by phosphatase Cdc14B, and then activated APC/C Cdh1 E3 ligase activity to ubiquitate and degrade Polo-like kinase 1 (Plk1) in process of G2 checkpoint. These data reveal that p53-mediated inhibition of SENP3 phosphorylation regulates the activation of Cdc14b-APC/C Cdh1 -Plk1 axis to control DNA damage-induced G2 checkpoint.

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“…Sumoylation also regulates ubiquitination enzymes in other cellular contexts. For example, sumoylation of a subunit of the APC ubiquitin E3 in mitosis was recently suggested to help activate the latter for timely anaphase onset (Eifler et al, 2018; Lee et al, 2018). Of note, SUMO and ubiquitin can also antagonize each other’s effects, sometimes via modification of the same site(s), as has been shown for DNA replication factor PCNA (Zilio et al, 2017).…”

Section: Crosstalk Between Sumo-based and Other Signaling Processesmentioning

confidence: 99%

SUMO-Mediated Regulation of Nuclear Functions and Signaling Processes

2018

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Summary Since the discovery of SUMO twenty years ago, SUMO conjugation has become a widely-recognized post-translational modification that targets a myriad of proteins in many processes. Great progress has been made in understanding the SUMO pathway enzymes, substrate sumoylation, and the interplay between sumoylation and other regulatory mechanisms in a variety of contexts. As these research directions continue to generate insights into SUMO-based regulation, several mechanisms by which sumoylation and desumoylation can orchestrate large biological effects are emerging. These include the ability to target multiple proteins within the same cellular structure or process, respond dynamically to external and internal stimuli, and modulate signaling pathways involving other post-translational modifications. Focusing on nuclear function and intracellular signaling, this review highlights a broad spectrum of historical data and recent advances with the aim of providing an overview of mechanisms underlying SUMO-mediated global effects to stimulate further inquiry into intriguing roles of SUMO.

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Sumoylation promotes optimal APC/C activation and timely anaphase

Cited by 30 publications

References 78 publications

A Method for SUMO Modification of Proteins in vitro

A Method for SUMO Modification of Proteins in vitro

P53 suppresses SENP3 phosphorylation to mediate G2 checkpoint

SUMO-Mediated Regulation of Nuclear Functions and Signaling Processes

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