Angus Chen

Recruitment of a ROC1–CUL1 Ubiquitin Ligase by Skp1 and HOS to Catalyze the Ubiquitination of IκBα

Abstract Activation of the transcription factor NF-κB in response to proinflammatory stimuli requires the phosphorylation-triggered and ubiquitin-dependent degradation of the NF-κB inhibitor, IκBα. Here, we show the in vitro reconstitution of the phosphorylation-dependent ubiquitination of IκBα with purified components. ROC1, a novel SCF-associated protein, is recruited by cullin 1 to form a quaternary SCF HOS –ROC1 holoenzyme (with Skp1 and the β-TRCP homolog HOS). SCF HOS –ROC1 binds IKKβ-phosphorylated IκBα and catalyzes its ubiquitination in the presence of ubiquitin, E1, and Cdc34. ROC1 plays a unique role in the ubiquitination reaction by heterodimerizing with cullin 1 to catalyze ubiquitin polymerization.

Inhibition of p21-mediated ROS accumulation can rescue p21-induced senescence

The cyclin‐dependent kinase (CDK) inhibitor p21Waf1/Cip1/Sdi1 was identified initially as a gene induced in senescent cells and itself has been shown to cause permanent growth arrest/senescence. Reactive oxygen species (ROS), a byproduct of oxidative processes, can also induce an irreversible growth arrest similar to senescence. Here we show that p21 increased intracellular levels of ROS both in normal fibroblasts and in p53‐negative cancer cells. N‐acetyl‐L‐cysteine, an ROS inhibitor, rescued p21‐induced senescence, showing that ROS elevation is necessary for induction of the permanent growth arrest phenotype. p16Ink4a, a CDK4‐ and CDK6‐specific inhibitor, failed to increase ROS levels, and cell cycle arrest induced by p16 was reversible following its down‐regulation, demonstrating the specificity of this p21 effect. A p21 mutant that lacked the ability to bind proliferating cell nuclear antigen (PCNA) retained the ability to induce both ROS and permanent growth arrest. All of these findings establish that p21 mediates senescence by a mechanism involving ROS accumulation which does not require either its PCNA binding or the CDK inhibitory functions shared with p16.

Conjugation of Nedd8 to CUL1 Enhances the Ability of the ROC1-CUL1 Complex to Promote Ubiquitin Polymerization

The SCF-ROC1 ubiquitin-protein isopeptide ligase (E3) ubiquitin ligase complex targets the ubiquitination and subsequent degradation of protein substrates required for the regulation of cell cycle progression and signal transduction pathways. We have previously shown that ROC1-CUL1 is a core subassembly within the SCF-ROC1 complex, capable of supporting the polymerization of ubiquitin. This report describes that the CUL1 subunit of the bacterially expressed, unmodified ROC1-CUL1 complex is conjugated with Nedd8 at Lys-720 by HeLa cell extracts or by a purified Nedd8 conjugation system (consisting of APP-BP1/Uba3, Ubc12, and Nedd8). This covalent linkage of Nedd8 to CUL1 is both necessary and sufficient to markedly enhance the ability of the ROC1-CUL1 complex to promote ubiquitin polymerization. A mutation of Lys-720 to arginine in CUL1 eliminates the Nedd8 modification, abolishes the activation of the ROC1-CUL1 ubiquitin ligase complex, and significantly reduces the ability of SCFHOS/β -TRCP-ROC1 to support the ubiquitination of phosphorylated IκBα. Thus, although regulation of the SCF-ROC1 action has been previously shown to preside at the level of recognition of a phosphorylated substrate, we demonstrate that Nedd8 is a novel regulator of the efficiency of polyubiquitin chain synthesis and, hence, promotes rapid turnover of protein substrates.

HOS, a human homolog of Slimb, forms an SCF complex with Skp1 and Cullin1 and targets the phosphorylation-dependent degradation of IκB and β-catenin

SCF E3 ubiquitin ligases mediate ubiquitination and proteasome-dependent degradation of phosphorylated substrates. We identified a human F-box/WD40 repeats protein (HOS), which is homologous to Slimb/hβTrCP. Being a part of SCF complex with Skp1 and Cullin1, HOS specifically interacted with the phosphorylated IκB and β-catenin, targeting these proteins for proteasome-dependent degradation in vivo. This targeting required Cullin1 as expression of a mutant Cullin1 abrogated the degradation of IκB and of β-catenin. Mutant HOS which lacks the F-box blocked TNFα-induced degradation of IκB as well as GSK3β-mediated degradation of β-catenin. This mutant also inhibited NF-κB transactivation and increased the β-catenin-dependent transcription activity of Tcf. These results demonstrate that SCFHOS E3 ubiquitin ligase regulate both NF-κB and β-catenin signaling pathways.

The SCFHOS/β-TRCP-ROC1 E3 Ubiquitin Ligase Utilizes Two Distinct Domains within CUL1 for Substrate Targeting and Ubiquitin Ligation

ABSTRACT We describe a purified ubiquitination system capable of rapidly catalyzing the covalent linkage of polyubiquitin chains onto a model substrate, phosphorylated IκBα. The initial ubiquitin transfer and subsequent polymerization steps of this reaction require the coordinated action of Cdc34 and the SCFHOS/β-TRCP-ROC1 E3 ligase complex, comprised of four subunits (Skp1, cullin 1 [CUL1], HOS/β-TRCP, and ROC1). Deletion analysis reveals that the N terminus of CUL1 is both necessary and sufficient for binding Skp1 but is devoid of ROC1-binding activity and, hence, is inactive in catalyzing ubiquitin ligation. Consistent with this, introduction of the N-terminal CUL1 polypeptide into cells blocks the tumor necrosis factor alpha-induced and SCF-mediated degradation of IκB by forming catalytically inactive complexes lacking ROC1. In contrast, the C terminus of CUL1 alone interacts with ROC1 through a region containing the cullin consensus domain, to form a complex fully active in supporting ubiquitin polymerization. These results suggest the mode of action of SCF-ROC1, where CUL1 serves as a dual-function molecule that recruits an F-box protein for substrate targeting through Skp1 at its N terminus, while the C terminus of CUL1 binds ROC1 to assemble a core ubiquitin ligase.