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Mechanism of D-type cyclins recognition by the AMBRA1 E3 ligase receptor

ABSTRACT: AMBRA1 is a tumour suppressor protein that functions as a substrate receptor in the ubiquitin conjugation system and regulates the stability of D-type cyclins and cell proliferation. Here, we present the cryo-EM structure of cyclin D1 bound AMBRA1-DDB1 complex at 3.55 Å resolution. The structure reveals a substrate interaction surface on the AMBRA1 WD40 domain that specifically binds to the C-terminal region of D-type cyclins. This interaction is dependent on the phosphorylation of Thr286 residue in the C-terminal phosphodegron site of D-type cyclins. The phosphodegron motif folds into a turn-like conformation followed by a 310 helix that promotes its assembly with AMBRA1. Additionally, we show that AMBRA1 mutants, which are defective in cyclin D1 binding, lead to cyclin D1 accumulation and DNA damage. Understanding the AMBRA1-D-type cyclins structure enhances the knowledge of the molecular mechanisms that govern the cell cycle control and may lead to new therapeutic approaches for cancers linked to abnormal cyclin D activity.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Goran Stjepanovic

LAB HEAD: Goran Stjepanovic

PROVIDER: PXD062122 | Pride | 2025-04-17

REPOSITORIES: Pride

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			Action	DRS
	AMBRA1_WD40_cyclinD1.pep.xml	Pepxml
	AMBRA1_WD40_cyclinD1.raw	Raw
	AMBRA1_WD40_cyclinD1_2.raw	Raw
	AMBRA1_WD40_cyclinD1_plus_STREP_crapome.fasta	Fasta
	checksum.txt	Txt

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Structure of the DDB1-AMBRA1 E3 ligase receptor complex linked to cell cycle regulation

Project description:AMBRA1 is a tumor suppressor protein that functions as a substrate receptor of the ubiquitin conjugation system with roles in autophagy and the cell cycle regulatory network. The intrinsic disorder of AMBRA1 has thus far precluded its structural determination. To solve this problem, we analyzed the dynamics of AMBRA1 using hydrogen deuterium exchange mass spectrometry (HDX-MS). The HDX results indicated that AMBRA1 is a highly flexible protein and can be stabilized upon interaction with DDB1, the adaptor of the Cullin4A/B E3 ligase. Here, we present the cryo-EM structure of AMBRA1 in complex with DDB1 at 3.08 Å resolution. The structure shows that parts of the N- and C-terminal structural regions in AMBRA1 fold together into the highly dynamic WD40 domain and reveals how DDB1 engages with AMBRA1 to create a binding scaffold for substrate recruitment. The N-terminal helix-loop-helix motif and WD40 domain of AMBRA1 associate with the double-propeller fold of DDB1. We also demonstrate that DDB1 binding-defective AMBRA1 mutants prevent ubiquitination of the substrate Cyclin D1 in vitro and increase cell cycle progression. Together, these results provide structural insights into the AMBRA1-ubiquitin ligase complex and suggest a mechanism by which AMBRA1 acts as a hub involved in various physiological processes.

2023-11-16 | PXD046056 | Pride

The AMBRA1 E3 ligase adaptor regulates the stability of cyclin D

Project description:The initiation of cell division integrates a large number of intra- and extracellular inputs. D-type cyclins (hereafter, cyclin D) couple these inputs to the initiation of DNA replication1. Increased levels of cyclin D promote cell division by activating cyclin-dependent kinases 4 and 6 (hereafter, CDK4/6), which in turn phosphorylate and inactivate the retinoblastoma tumour suppressor. Accordingly, increased levels and activity of cyclin D-CDK4/6 complexes are strongly linked to unchecked cell proliferation and cancer2,3. However, the mechanisms that regulate levels of cyclin D are incompletely understood4,5. Here we show that autophagy and beclin 1 regulator 1 (AMBRA1) is the main regulator of the degradation of cyclin D. We identified AMBRA1 in a genome-wide screen to investigate the genetic basis of the response to CDK4/6 inhibition. Loss of AMBRA1 results in high levels of cyclin D in cells and in mice, which promotes proliferation and decreases sensitivity to CDK4/6 inhibition. Mechanistically, AMBRA1 mediates ubiquitylation and proteasomal degradation of cyclin D as a substrate receptor for the cullin 4 E3 ligase complex. Loss of AMBRA1 enhances the growth of lung adenocarcinoma in a mouse model, and low levels of AMBRA1 correlate with worse survival in patients with lung adenocarcinoma. Thus, AMBRA1 regulates cellular levels of cyclin D, and contributes to cancer development and the response of cancer cells to CDK4/6 inhibitors.

2021-02-04 | GSE146303 | GEO

The AMBRA1 E3 ligase adaptor regulates Cyclin D protein stability [U2OS cells]

Project description:The initiation of cell division is a fundamental process that integrates a large number of intra- and extra-cellular inputs. In mammalian cells, D-type cyclins (Cyclin D) couple these inputs to the decision to initiate DNA replication. Increased levels of Cyclin D promote cell cycle progression by activating cyclin-dependent kinases 4 and 6 (CDK4/6). Accordingly, increased levels and activity of Cyclin D and their associated kinases are strongly linked to unchecked cell proliferation and tumor development. Despite this central role of Cyclin D in cell cycle progression, the mechanisms regulating their levels remain incompletely understood. Here we describe AMBRA1 as the main regulator of Cyclin D protein degradation. We first identify AMBRA1 as the top candidate in a genome-wide CRISPR/Cas9 loss-of-function screen investigating the genetic basis of resistance to chemical CDK4/6 inhibition. AMBRA1 loss results in high protein levels of Cyclin D in cells and in mice. AMBRA1 loss further promotes lung cancer development in a mouse model, and low levels of AMBRA1 correlate with worse survival in lung cancer patients. Mechanistically, AMBRA1 acts as a substrate receptor for the Cullin 4 E3 ligase complex to promote ubiquitylation and proteasomal degradation of the three Cyclin D family members. Thus, AMBRA1 regulates Cyclin D protein levels and contributes to the development of cancer as well as the response of cancer cells to CDK4/6 inhibitors.

2021-02-05 | GSE159920 | GEO

The AMBRA1 E3 ligase adaptor regulates Cyclin D protein stability [U937 cells]

2021-02-05 | GSE146588 | GEO

Differential expression between FHL2-/- and WT MEFs.

Project description:The LIM-only protein FHL2 acts as a transcriptional modulator that positively or negatively regulates multiple signaling pathways. We recently reported that FHL2 cooperates with CBP/p300 in the activation of ß-catenin/TCF target gene cyclin D1. In this paper, we demonstrate that FHL2 is associated with the cyclin D1 promoter at the TCF/CRE site, providing evidence that cyclin D1 is a direct target of FHL2. We show that deficiency of FHL2 greatly reduces the proliferative capacity of spontaneously immortalized mouse fibroblasts which is associated with decreased expression of cyclin D1 and p16INK4a, and hypophosphorylation of Rb. Reexpression of FHL2 in FHL2-null fibroblasts efficiently restores cyclin D1 levels and cell proliferative capacity, indicating that FHL2 is critical for cyclin D1 activation and cell growth. Moreover, ectopic cyclin D1 expression is sufficient to override growth inhibition of immortalized FHL2-null fibroblasts. Gene expression profiling revealed that FHL2 deficiency triggers a broad change of the cell cycle program that is associated with downregulation of several G1/S and G2/M cyclins, E2F transcription factors and DNA replication machinery, thus correlating with reduced cell proliferation. This change also involves downregulation of the negative cell cycle regulators, particularly INK4 inhibitors, which could counteract the decreased expression of cyclins, allowing cells to grow. Our study illustrates that FHL2 can act on different aspects of the cell cycle program to finely regulate cell proliferation. Keywords: mouse embryonic fibroblasts derived from FHL2-/- and WT mouse embryos (14.5 days)

2008-06-06 | GSE10902 | GEO

Transcription profiling of mouse embryonic fibroblasts from FHL2-/- and wild type animals

Project description:The LIM-only protein FHL2 acts as a transcriptional modulator that positively or negatively regulates multiple signaling pathways. We recently reported that FHL2 cooperates with CBP/p300 in the activation of Ã-catenin/TCF target gene cyclin D1. In this paper, we demonstrate that FHL2 is associated with the cyclin D1 promoter at the TCF/CRE site, providing evidence that cyclin D1 is a direct target of FHL2. We show that deficiency of FHL2 greatly reduces the proliferative capacity of spontaneously immortalized mouse fibroblasts which is associated with decreased expression of cyclin D1 and p16INK4a, and hypophosphorylation of Rb. Reexpression of FHL2 in FHL2-null fibroblasts efficiently restores cyclin D1 levels and cell proliferative capacity, indicating that FHL2 is critical for cyclin D1 activation and cell growth. Moreover, ectopic cyclin D1 expression is sufficient to override growth inhibition of immortalized FHL2-null fibroblasts. Gene expression profiling revealed that FHL2 deficiency triggers a broad change of the cell cycle program that is associated with downregulation of several G1/S and G2/M cyclins, E2F transcription factors and DNA replication machinery, thus correlating with reduced cell proliferation. This change also involves downregulation of the negative cell cycle regulators, particularly INK4 inhibitors, which could counteract the decreased expression of cyclins, allowing cells to grow. Our study illustrates that FHL2 can act on different aspects of the cell cycle program to finely regulate cell proliferation. Experiment Overall Design: Two biological genotypes, FHL2-/- and WT MEF cells. Three spontaneously immortalized clones of each genotype were analyzed using Affymetrix arrays.

2008-06-18 | E-GEOD-10902 | biostudies-arrayexpress

Gene expression profile of mouse breast cancer V720 cells treated with vehicle or PD 0332991

Project description:D-cyclins represent components of cell cycle machinery. To test the efficacy of targeting D-cyclins in cancer treatment, we engineered mouse strains which allow acute and global ablation of individual D-cyclins in a living animal. Ubiquitous shutdown of cyclin D1 or inhibition of cyclin D associated kinase activity in mice bearing ErbB2-driven mammary carcinomas halted cancer progression and triggered tumor-specific senescence, without compromising the animals' health. Ablation of cyclin D3 in mice bearing T-cell acute lymphoblastic leukemias (T-ALL) triggered tumorspecific apoptosis. Such selective killing of leukemic cells can be also achieved by inhibiting cyclin D associated kinase activity in mouse and human T-ALL models. Hence, contrary to what one might expect from ablation of a cell cycle protein, acute shutdown of a D-cyclin leads not only to cell cycle arrest, but it also triggers tumor cell senescence or apoptosis, and it affects different tumor types through distinct cellular mechanisms. Inhibiting cyclin D-activity represents a highly-selective anticancer strategy which specifically targets cancer cells without significantly affecting normal tissues.

2012-10-16 | GSE40513 | GEO

Gene expression profile of human T-ALL cell line KOPTK1 treated with vehicle or PD 0332991

2012-10-16 | GSE40512 | GEO

Gene expression profile of mouse breast cancer V720 cells treated with vehicle or PD 0332991

2012-10-16 | E-GEOD-40513 | biostudies-arrayexpress

Analyses of cyclin D1 function using a "genetic-proteomic" approach

Project description:We examined the transcriptional function of cyclin D1 in mouse development using two approaches. First, we queried association of cyclin D1 with the genome of E14.5 mouse embryos using ChIP-on-chip approach. We observed binding of cyclin D1 to several promoter regions. Second, we compared gene expression profiles between wild-type and cyclin D1-null retinas. We observed several transcripts with altered levels in cyclin D1-null organs. This SuperSeries is composed of the SubSeries listed below.

2010-01-29 | GSE13636 | GEO

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