Human CUL-1, however, not other cullin family members, selectively interacts with SKP1 to form a complex with SKP2 and cyclin A

Human CUL-1, however, not other cullin family members, selectively interacts with SKP1 to form a complex with SKP2 and cyclin A. 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. Regulation of protein stability by ubiquitin (Ub)-dependent proteolysis plays major roles in the control of multiple aspects of cell function, such as transcriptional control, cell cycle progression, and signal transduction (17). Ubiquitination involves three distinct enzymatic events that ultimately lead to the covalent linkage of Ub polymers to the lysine ?-amino groups of substrate proteins. Ub is initially charged in an ATP-dependent fashion by the Ub-activating enzyme, E1, to form a high-energy thiol-ester bond between the carboxyl group of its C-terminal glycine residue and E1. The thiol-ester-linked Ub is then transferred to an E2, which cooperates with an E3 ligase to catalyze the formation of an isopeptide bond between Ub and the substrate (16). One well-characterized Ub-proteasome pathway is the degradation of IB required for the activation of the transcription factor NF-B (26). IB, which sequesters NF-B in the cell cytoplasm (3), is phosphorylated by the IKK kinase complex that is activated in response to proinflammatory cytokines (4, 9, 28, 36, 48, 53). Such phosphorylation triggers the rapid ubiquitination and subsequent degradation of IB, resulting in the release of NF-B (5, 7, 37). Recent studies have demonstrated that the -TRCP/HOS F-box protein family plays a direct role in targeting IB for ubiquitination and degradation (12, 15, 21, 22, 30, 42, 47, 50) by binding to this inhibitor at its N-terminally located DS(PO3)GXS(PO3) motif (1, 47, 49). More recently, the ubiquitination of IB has been reconstituted in vitro with purified components including SCFHOS/-TRCP-ROC1 as the E3 ligase complex (44). SCFHOS/-TRCP-ROC1 contains Skp1, cullin 1 (CUL1), HOS/-TRCP, and the newly identified ROC1 (also called Rbx1 or Hrt1). ROC1 is a novel RING-H2 finger protein that was initially isolated as a CUL4A-interacting protein by a yeast two-hybrid screen (30). It has also been biochemically purified as a common component of both the human (44) and yeast (38) SCF complexes, as well as the native human von Hippel-Lindau (VHL) tumor suppressor complex (19). In addition, the ROC1 homologue, ROC2 (also called SAG), was isolated as a redox-agent-induced gene product that protects cells from apoptosis (10). ROC1 plays an essential role in the Cdc34/SCF-mediated ubiquitination-degradation pathway. Yeast ROC1 encodes an essential gene whose reduced expression led to the accumulation of Sic1 and Clns (19, 30, 38, 40), both of which are previously identified substrates of the Cdc34-SCF ubiquitination apparatus (11, 35, 39). Yeast ROC1 is a component of the SCF complexes that mediates the in vitro ubiquitination of Sic1 by Cdc4 (19, 38) and Clns by Grr1 (40). The in vitro reconstitution experiments (44) reveal that the human ROC1 protein is recruited by CUL1 to form the SCFHOS/-TRCP-ROC1 complex (with Skp1 and HOS/-TRCP). Like its yeast counterpart, human Skp1 links CUL1 to the F-box protein HOS/-TRCP. In addition, Skp1 enhances the ability of HOS/-TRCP to interact with phosphorylated IB. The purified recombinant SCFHOS/-TRCP-ROC1 complex specifically binds IKK-phosphorylated IB and catalyzes its ubiquitination in the presence of Ub, E1, and Cdc34 as the E2-conjugating enzyme. Each of the four subunits within SCFHOS/-TRCP-ROC1 Rabbit Polyclonal to ARC is required for the substrate ubiquitination reaction. These studies suggest that SCFHOS/-TRCP-ROC1 acts as an E3 holoenzyme that is both necessary and sufficient to initiate and catalyze ubiquitination. A recent study has identified Sgt1p as a novel protein that interacts with Skp1 and which is required for assembling the yeast kinetochore complex (20). However, the biochemical role of Sgt1p in the SCF-ROC1-mediated ubiquitination reaction remains to be determined. In addition to its role in the SCF pathway, ROC1 may mediate other ubiquitin-dependent proteolysis events in the cell. It has been shown by cotransfection experiments that ROC1 Risedronate sodium binds five cullin family members (CUL1, CUL2, CUL3, CUL4A, and CUL4B), whereas ROC2 preferentially interacts with CUL5 (30). Consistent with this, ROC1 is found to be a component of the pVHL complex (19), whose additional subunits include pVHL, Risedronate sodium CUL2, and elongins C and B (24, 33, 43). Furthermore, ROC1 shares extensive homology with APC11 (19, 30, 38), a subunit of the anaphase-promoting complex (35), which interacts with the cullin-related protein APC2 (30). These findings suggest that ROC/APC11, through its combinatorial interaction with cullin/APC2, forms a dimeric Risedronate sodium core component common to a large family of multisubunit Ub.