Cullin 3-Based Ubiquitin Ligases as Master Regulators of Mammalian Cell Differentiation.

Specificity of the ubiquitin proteasome system is controlled by ubiquitin E3 ligases, including their major representatives, the multisubunit cullin-RING ubiquitin (Ub) ligases (CRLs). More than 200 different CRLs are divided into seven families according to their cullin scaffolding proteins (CUL1-7) around which they are assembled. Research over two decades has revealed that different CRL families are specialized to fulfill specific cellular functions. Whereas many CUL1-based CRLs (CRL1s) ubiquitylate cell cycle regulators, CRL4 complexes often associate with chromatin to control DNA metabolism. Based on studies about differentiation programs of mesenchymal stem cells (MSCs), including myogenesis, neurogenesis, chondrogenesis, osteogenesis and adipogenesis, we propose here that CRL3 complexes evolved to fulfill a pivotal role in mammalian cell differentiation.

Cul3 neddylation is crucial for gradual lipid droplet formation during adipogenesis.

Cullin 3 (Cul3) belongs to the family of cullins (Cul1-7) providing the scaffold for cullin-RING ubiquitin (Ub) ligases (CRLs), which are activated by neddylation and represent essential E3 ligases of the Ub proteasome system. During adipogenic differentiation neddylated Cul3 accumulates in LiSa-2 preadipocytes. Downregulation of Cul3 and inhibition of neddylation by MLN4924 blocks the formation of lipid droplets (LDs), the lipid storage organelles and markers of adipogenesis. Neddylation of Cul3 coincides with an increase of Rab18, a GTPase associated with LDs. Immunoprecipitation and confocal fluorescence microscopy revealed physical association of Cul3 and Rab18 at the membrane of LDs. RhoA, a suppressor of adipogenesis decreased during differentiation. Our results in LiSa-2 cells, but also mouse embryonic fibroblasts revealed a connection between Cul3, Rab18 and RhoA. Downregulation of Cul3 led to a marked increase in RhoA protein expression after 6days of LiSa-2 cell differentiation, suggesting that Cul3 is involved in the regulation of RhoA stability.

CAND1-dependent control of cullin 1-RING Ub ligases is essential for adipogenesis.

Cullin-RING ubiquitin (Ub) ligases (CRLs) are responsible for ubiquitinylation of approximately 20% of all proteins degraded by the Ub proteasome system (UPS). CRLs are regulated by the COP9 signalosome (CSN) and by Cullin-associated Nedd8-dissociated protein 1 (CAND1). The CSN is responsible for removal of Nedd8 from cullins inactivating CRLs. CAND1 modulates the assembly of F-box proteins into cullin 1-RING Ub ligases (CRL1s). We show that CAND1 preferentially blocks the integration of Skp2 into CRL1s. Suppression of CAND1 expression in HeLa cells leads to an increase of the Skp2 assembly into CRL1s and to significant reduction of the cyclin-dependent kinase (CDK) inhibitor p27. In contrary, CAND1 overexpression causes elevation of p27. The observed CAND1-dependent effects and CAND1 expression are independent of the CSN as demonstrated in CSN1 knockdown cells. Increase of p27 is a hallmark of preadipocyte differentiation to adipocytes (adipogenesis). We demonstrate that the accumulation of p27 is associated with an increase of CAND1 and a decrease of Skp2 during adipogenesis of human LiSa-2 preadipocytes. CAND1 knockdown reduces p27 and blocks adipogenesis. Due to the impact of CAND1 on Skp2 control, CAND1 could represent an important effector molecule in adipogenesis, but also in cancer development.

Latent CSN-CRL complexes are crucial for curcumin-induced apoptosis and recruited during adipogenesis to lipid droplets via small GTPase RAB18.

The COP9 signalosome (CSN) and cullin-RING ubiquitin ligases (CRLs) form latent CSN-CRL complexes detectable in cells. We demonstrate that the CSN variants CSNCSN7A and CSNCSN7B preferentially bind to CRL3 or CRL4A and CRL4B, respectively. Interestingly, the interacting protein ubiquitin-specific protease 15 exclusively binds to latent CSNCSN7A-CRL3, while p27KIP attaches to latent CSNCSN7B-CRL4A complex. Inhibition of deneddylation by CSN5i-3 or neddylation by MLN4924 do not impede the formation of latent complexes. Latent CSNCSN7A-CRL3 and latent CSNCSN7B-CRL4A/B particles are essential for specific cellular functions. We found that curcumin-induced cell death requires latent CSNCSN7B-CRL4A. Knockout of CSN7B in HeLa cells leads to resistance against curcumin. Remarkably, the small GTPase RAB18 recruits latent CSNCSN7A-CRL3 complex to lipid droplets (LDs), where CRL3 is activated by neddylation, an essential event for LD formation during adipogenesis. Knockdown of CSN7A or RAB18 or destabilization of latent complexes by cutting off CSN7A C-terminal 201-275 amino acids blocks adipogenesis.

CSN7B defines a variant COP9 signalosome complex with distinct function in DNA damage response.

Mammalian COP9 signalosome (CSN) exists as two variant complexes containing either CSN7A or CSN7B paralogs of unknown functional specialization. Constructing knockout cells, we found that CSN7A and CSN7B have overlapping functions in the deneddylation of cullin-RING ubiquitin ligases. Nevertheless, CSNCSN7B has a unique function in DNA double-strand break (DSB) sensing, being selectively required for ataxia telangiectasia mutated (ATM)-dependent formation of NBS1S343p and γH2AX as well as DNA-damage-induced apoptosis triggered by mitomycin C and ionizing radiation. Live-cell microscopy revealed rapid recruitment of CSN7B but not CSN7A to DSBs. Resistance of CSN7B knockout cells to DNA damage is explained by the failure to deneddylate an upstream DSB signaling component, causing a switch in DNA repair pathway choice with increased utilization of non-homologous end joining over homologous recombination. In mice, CSN7B knockout tumors are resistant to DNA-damage-inducing chemotherapy, thus providing an explanation for the poor prognosis of tumors with low CSN7B expression.

The COP9 Signalosome: A Multi-DUB Complex.

The COP9 signalosome (CSN) is a signaling platform controlling the cellular ubiquitylation status. It determines the activity and remodeling of ~700 cullin-RING ubiquitin ligases (CRLs), which control more than 20% of all ubiquitylation events in cells and thereby influence virtually any cellular pathway. In addition, it is associated with deubiquitylating enzymes (DUBs) protecting CRLs from autoubiquitylation and rescuing ubiquitylated proteins from degradation. The coordination of ubiquitylation and deubiquitylation by the CSN is presumably important for fine-tuning the precise formation of defined ubiquitin chains. Considering its intrinsic DUB activity specific for deneddylation of CRLs and belonging to the JAMM family as well as its associated DUBs, the CSN represents a multi-DUB complex. Two CSN-associated DUBs, the ubiquitin-specific protease 15 (USP15) and USP48 are regulators in the NF-κB signaling pathway. USP15 protects CRL1ß-TrCP responsible for IκBα ubiquitylation, whereas USP48 stabilizes the nuclear pool of the NF-κB transcription factor RelA upon TNF stimulation by counteracting CRL2SOCS1. Moreover, the CSN controls the neddylation status of cells by its intrinsic DUB activity and by destabilizing the associated deneddylation enzyme 1 (DEN1). Thus, the CSN is a master regulator at the intersection between ubiquitylation and neddylation.

Diversity of COP9 signalosome structures and functional consequences.

The COP9 signalosome (CSN) is a regulator of the ubiquitin (Ub) proteasome system (UPS). It interacts with hundreds of cullin-RING ubiquitin E3 ligases (CRLs) and regulates their activity by removing the Ub-like protein Nedd8 from cullins. In mammalian cells 7 different cullins exist which form CRLs with adaptor proteins and with a large number of substrate recognition subunits such as F-box and BTB proteins. This large variety of CRL-complexes is deneddylated by the CSN. The capacity of the CSN to interact with numerous types of CRL complexes can be explained by its structural diversity, which allows different CSN variants to interact with different binding partners and substrates and enables different subunit expression profiles. Diversity of CSN complexes presumably occurs by: (1) flexibility of CSN holo complex structure; (2) formation of CSN mini complexes and free CSN subunits and (3) generation of CSN variants via integration of CSN subunit isoforms. In this review we will discuss the structural diversity of the CSN complex and possible functional consequences.

CAND1 exchange factor promotes Keap1 integration into cullin 3-RING ubiquitin ligase during adipogenesis.

Adipogenesis is governed by a plethora of regulatory proteins which are most commonly controlled by the ubiquitin proteasome system. Here, we show that the differentiation of LiSa-2 preadipocytes is associated with an increase of cullin-associated and neddylation-dissociated 1 (CAND1), COP9 signalosome (CSN), neddylated cullin 3 (Cul3) and the BTB protein Keap1. Silencing of CAND1 leads to a decrease and reduced integration of Keap1 into Cul3-RING ubiquitin ligases (CRL3) and to a retardation of adipogenesis. Transient transfection of LiSa-2 cells with CAND1 targeting miRNA148a also reduces Keap1 and slowed down adipogenesis of LiSa-2 cells. These results demonstrate for the first time that CAND1 acts as a BTB-protein exchange factor for CRL3 complexes. The specific increase of neddylated Cul3 might be explained by the recruitment of Cul3 or CRL3 in a membrane-bound location during adipogenesis. Together, the results show that during adipogenesis in LiSa-2 cells a CAND1-dependent remodeling and activation/neddylation of CRL3 complexes take place.