The Nedd8-conjugated ROC1-CUL1 Core Ubiquitin Ligase Utilizes Nedd8 Charged Surface Residues for Efficient Polyubiquitin Chain Assembly Catalyzed by Cdc34

Abstract

Lysine 48-linked polyubiquitin chains are the principle signal for targeting proteins for degradation by the 26 S proteasome. Here we report that the conjugation of Nedd8 to ROC1-CUL1, a subcomplex of the SCF-ROC1 E3 ubiquitin ligase, selectively stimulates Cdc34-catalyzed lysine 48-linked multiubiquitin chain assembly. We have further demonstrated that separate regions within the human Cdc34 C-terminal tail are responsible for multiubiquitin chain assembly and for physical interactions with the Nedd8-conjugated ROC1-CUL1 to assemble extensive ubiquitin polymers. Structural comparisons between Nedd8 and ubiquitin reveal that six charged residues (Lys 4 , Glu 12 , Glu 14 , Arg 25 , Glu 28 , and Glu 31 ) are uniquely present on the surface of Nedd8. Replacement of each of the six residues with the corresponding amino acid in ubiquitin decreases the ability of Nedd8 to activate the ubiquitin ligase activity of ROC1-CUL1. Moreover, maintenance of the proper charges at amino acid positions 14 and 25 are necessary for retaining wild type levels of activity, whereas introduction of the opposite charges at these positions abolishes the Nedd8 activation function. These results suggest that Nedd8 charged surface residues mediate the activation of ROC1-CUL1 to specifically support Cdc34-catalyzed ubiquitin polymerization.Nedd8 (or its orthologue Rub1) is a small ubiquitin (Ub)-like molecule that modifies all members of the Cullin/Cdc53 protein family (1, 2), resulting in the formation of an isopeptide bond linkage between the ⑀-amino group of a conserved Cullin lysine residue and the C-terminal carboxyl group of Nedd8 glycine 76. The conjugation is an ATP-dependent reaction that requires a Nedd8-specific E1 activating enzyme, composed of the APP-BP1 and Uba3 heterodimer, and Ubc12 as the E2 conjugating enzyme (3). In addition, the Cullin-interacting RING finger protein, ROC1/Rbx1/Hrt1, is required for the reaction as disruption of the RING domain abolishes the modification (4).Two well characterized members of the Cullin family, CUL1 and CUL2, serve as subunits of the two multisubunit E3 Ub ligase complexes, SCF-ROC1 (5-8) and pVHL-elongin C/B-CUL2-ROC1 (9), respectively. It has now been well established that the ROC1-CUL1 subassembly acts as a core ubiquitin ligase, capable of supporting Ub polymerization (5,6,8,10,11). In addition, results from transient transfection experiments have shown that ROC1 and its homologue ROC2 interact with all members of the Cullin protein family and that the resulting ROC-Cullin complex is active in supporting Ub polymerization (6). Thus, while the biological roles of Cullin members including CUL3, CUL4A, CUL4B, and CUL5 remain elusive, it is highly likely that all of the ROC-Cullin based complexes are involved in cellular Ub-dependent proteolysis pathways. It is, therefore, conceivable that Nedd8, through its conjugation to Cullins, functions to regulate the stability of cellular proteins.Accumulating genetic evidence has demonstrated a critical role for Nedd8 in the regulation of cell pro...

The Acidic Tail domain of Human Cdc34 is Required for p27Kip1 Ubiquitination and Complementation of a cdc34 Temperature Sensitive Yeast Strain

“…This is contrary to published studies of Cdc34 binding to Cul1 and Roc1. 15 Paradoxically, the binding of the Cdc34 5 PT MUT A to Cul1 and Roc1 was reduced compared to WT Cdc34 and the Cdc34 1-200 mutant, while the binding of the Cdc34 5 PT MUT E to Cul1 and Roc1 was similar to WT Cdc34 (Fig. 3A, B).…”

“…In vitro binding studies of Cdc34 with Cul1 and Roc1 and Cdc34 auto-ubiquitination assays suggest that Cdc34 is recruited to the SCF through an interaction with a Cul1-Roc1 heterodimer. 15,36 More specifically, in vitro binding studies indicate that residues 195-236 of Cdc34 are required for maximal binding to GST-Roc1-Cul1 . 15 This suggested to us that the Cdc34 1-200 defect in complementation and p27 Kip1 ubiquitination might be attributable to an inability of the truncated Cdc34 to bind efficiently to the SCF through its association with Cul1-Roc1.…”

“…10 In addition, past studies have indicated that residues 195-208 of human Cdc34 were required for a stable interaction with Cul-Roc1, while residues 209-236 enhanced this interaction significantly and were required for maximal binding to Cul1-Roc1. 15 These results were based on in vitro binding studies of human Cdc34 with Cul1-Roc1 and on in vitro substrate-independent polyubiquitin chain assembly (or Cdc34 auto-ubiquitination) studies. 15 Recent studies in budding yeast have also demonstrated that the critical step in the ubiquitination of the yeast CDK inhibitor, p40 Sic1 , is actually the efficient dissociation of yeast Cdc34p from SCF Cdc4 .…”

“…15 These results were based on in vitro binding studies of human Cdc34 with Cul1-Roc1 and on in vitro substrate-independent polyubiquitin chain assembly (or Cdc34 auto-ubiquitination) studies. 15 Recent studies in budding yeast have also demonstrated that the critical step in the ubiquitination of the yeast CDK inhibitor, p40 Sic1 , is actually the efficient dissociation of yeast Cdc34p from SCF Cdc4 . 16 These studies indicated that both charged and uncharged Cdc34p efficiently associated with the SCF, but only charged Cdc34p efficiently dissociated from the SCF and supported the ubiquitination of p40 Sic1 .…”

Drosophila homolog of APP-BP1 (dAPP-BP1) interacts antagonistically with APPL during Drosophila development

“…Recent studies have shown that NEDD8 modification facilitates the recruitment of the E2 ubiquitinconjugating enzyme to the SCF complex and mediates the dissociation of p120 CAND1 an inhibitor of SCF ligase activity. 11,12 In Drosophila NEDD8 mutants, there is an accumulation of protein levels for the cell cycle regulator, Cyclin E (CycE), and for the signal transduction effectors, Cubitus interruptus (Ci), and Armadillo (Arm). 13 Recently, NEDD8 has been shown to be incorporated into a variety of ubiquitinated inclusions, including the neurofibrillary tangles present in AD.…”