Split Chloramphenicol Acetyl-Transferase Assay Reveals Self-Ubiquitylation-Dependent Regulation of UBE3B

Levin-Kravets, Olga; Kordonsky, Alina; Shusterman, Anna; Biswas, Sagnik; Persaud, Avinash K.; Elias, Sivan; Langut, Yael; Florentin, Amir; Simpson-Lavy, Kobi J.; Yariv, Elon; Avishid, Reut; Sror, Mor; Almog, Ofir; Marshanski, Tal; Kadosh, S; David, Nicole Ben; Manori, Bar; Fischer, Zohar; Lilly, Jeremiah; Борисова, Е. В.; Ambrozkiewicz, Mateusz C.; Tarabykin, Victor; Kupiec, Martin; Thaker, Maulik; Rotin, Daniela; Prag, Gali

doi:10.1016/j.jmb.2021.167276

Cited by 5 publications

(4 citation statements)

References 62 publications

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“…Targeted protein degradation represents an emerging therapeutic modality with the potential to tackle disease-causing proteins that have in the past been highly challenging to target with conventional small molecules. Our results pave the way for the development of CRL4 DCAF1 -dependent PROTACs or molecular glue degraders (Bekes et al, 2022; Levin-Kravets et al, 2021) to target PLK4 degradation as a novel modality for cancer therapy.…”

Section: Discussionmentioning

confidence: 82%

The CUL4-DDB1-DCAF1 E3 ubiquitin ligase complex regulates PLK4 protein levels to prevent premature centriole duplication

Grossmann,

Kratz,

Kordonsky

et al. 2023

Preprint

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Centrioles play important roles in the assembly of centrosomes and cilia. Centriole duplication occurs once per cell cycle and is dependent on polo-like kinase 4 (PLK4). To prevent centriole amplification, which is a hallmark of cancer, PLK4 protein levels need to be tightly regulated. Here, we show that the Cullin(CUL)4A/B-DDB1-DCAF1, CRL4DCAF1, E3 ligase targets PLK4 for degradation in human cells. DCAF1 binds and ubiquitylates PLK4 in G2 phase to prevent premature centriole duplication in mitosis. In contrast to the regulation of PLK4 by SCFβ-TrCP, the interaction between PLK4 and DCAF1 is independent of PLK4 kinase activity and mediated by polo-boxes 1 and 2 of PLK4, suggesting that DCAF1 promotes PLK4 ubiquitylation independently of β-TrCP. Thus, the SCFSlimb/β-TrCPpathway, targeting PLK4 for ubiquitylation based on its phosphorylation state and CRL4DCAF1, which ubiquitylates PLK4 by binding to the conserved PB1-PB2 domain, appear to be complementary ways to control PLK4 abundance to prevent centriole overduplication.

show abstract

Section: Discussionmentioning

confidence: 82%

The CUL4-DDB1-DCAF1 E3 ubiquitin ligase complex regulates PLK4 protein levels to prevent premature centriole duplication

Grossmann,

Kratz,

Kordonsky

et al. 2023

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…Targeted protein degradation represents an emerging therapeutic modality with the potential to tackle disease-causing proteins that have in the past been highly challenging to target with conventional small molecules. Our results pave the way for the development of CRL4 DCAF1 -dependent PROTACs or molecular glue degraders (Levin-Kravets et al, 2021;Bekes et al, 2022) to target PLK4 degradation as a novel modality for cancer therapy.…”

Section: Discussionmentioning

confidence: 82%

CRL4^DCAF1ubiquitin ligase regulates PLK4 protein levels to prevent premature centriole duplication

Grossmann,

Kratz,

Kordonsky

et al. 2024

Life Sci. Alliance

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Centrioles play important roles in the assembly of centrosomes and cilia. Centriole duplication occurs once per cell cycle and is dependent on polo-like kinase 4 (PLK4). To prevent centriole amplification, which is a hallmark of cancer, PLK4 protein levels need to be tightly regulated. Here, we show that the Cullin4A/B-DDB1-DCAF1, CRL4DCAF1, E3 ligase targets PLK4 for degradation in human cells. DCAF1 binds and ubiquitylates PLK4 in the G2 phase to prevent premature centriole duplication in mitosis. In contrast to the regulation of PLK4 by SCFβ-TrCP, the interaction between PLK4 and DCAF1 is independent of PLK4 kinase activity and mediated by polo-boxes 1 and 2 of PLK4, suggesting that DCAF1 promotes PLK4 ubiquitylation independently of β-TrCP. Thus, the SCFSlimb/β-TrCPpathway, targeting PLK4 for ubiquitylation based on its phosphorylation state and CRL4DCAF1, which ubiquitylates PLK4 by binding to the conserved PB1-PB2 domain, appear to be complementary ways to control PLK4 abundance to prevent centriole overduplication.

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“…To validate the prediction models, we assessed the ability of the GAP domain of RASA1 to bind ubiquitin. Using a bacterial Split-Chloramphenicol Acetyl Transferase (Split-CAT) assay (Levin-Kravets et al, 2021 ), we detected the binding of wt-RASA1 GAP to ubiquitin, whereas E1015A/ R1016A mutations impaired ubiquitin binding (Fig. 3B ).…”

Section: Resultsmentioning

confidence: 95%

“…The assays were performed as previously described in (Levin-Kravets et al, 2021 ). E. coli Mach1 was co-transformed with pC-CAT-Ubiquitin and pN-CAT-RASA1 GAP or pN-CAT-NF1 GRD vectors and selected on agar plates supplemented with 30 μg/ml kanamycin and 25 μg/ml streptomycin respectively.…”

Section: Methodsmentioning

confidence: 99%

K128 ubiquitination constrains RAS activity by expanding its binding interface with GAP proteins

Magits,

Steklov,

Jang

et al. 2024

EMBO J

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The RAS pathway is among the most frequently activated signaling nodes in cancer. However, the mechanisms that alter RAS activity in human pathologies are not entirely understood. The most prevalent post-translational modification within the GTPase core domain of NRAS and KRAS is ubiquitination at lysine 128 (K128), which is significantly decreased in cancer samples compared to normal tissue. Here, we found that K128 ubiquitination creates an additional binding interface for RAS GTPase-activating proteins (GAPs), NF1 and RASA1, thus increasing RAS binding to GAP proteins and promoting GAP-mediated GTP hydrolysis. Stimulation of cultured cancer cells with growth factors or cytokines transiently induces K128 ubiquitination and restricts the extent of wild-type RAS activation in a GAP-dependent manner. In KRAS mutant cells, K128 ubiquitination limits tumor growth by restricting RAL/ TBK1 signaling and negatively regulating the autocrine circuit induced by mutant KRAS. Reduction of K128 ubiquitination activates both wild-type and mutant RAS signaling and elicits a senescence-associated secretory phenotype, promoting RAS-driven pancreatic tumorigenesis.

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Split Chloramphenicol Acetyl-Transferase Assay Reveals Self-Ubiquitylation-Dependent Regulation of UBE3B

Cited by 5 publications

References 62 publications

The CUL4-DDB1-DCAF1 E3 ubiquitin ligase complex regulates PLK4 protein levels to prevent premature centriole duplication

The CUL4-DDB1-DCAF1 E3 ubiquitin ligase complex regulates PLK4 protein levels to prevent premature centriole duplication

CRL4^DCAF1ubiquitin ligase regulates PLK4 protein levels to prevent premature centriole duplication

K128 ubiquitination constrains RAS activity by expanding its binding interface with GAP proteins

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Split Chloramphenicol Acetyl-Transferase Assay Reveals Self-Ubiquitylation-Dependent Regulation of UBE3B

Cited by 5 publications

References 62 publications

The CUL4-DDB1-DCAF1 E3 ubiquitin ligase complex regulates PLK4 protein levels to prevent premature centriole duplication

The CUL4-DDB1-DCAF1 E3 ubiquitin ligase complex regulates PLK4 protein levels to prevent premature centriole duplication

CRL4DCAF1ubiquitin ligase regulates PLK4 protein levels to prevent premature centriole duplication

K128 ubiquitination constrains RAS activity by expanding its binding interface with GAP proteins

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CRL4^DCAF1ubiquitin ligase regulates PLK4 protein levels to prevent premature centriole duplication