Covalent modification of cullins by the ubiquitin-like protein NEDD8 (neddylation) regulates protein ubiquitination by promoting the assembly of cullin-RING ligase E3 complexes. Like ubiquitination, neddylation results from an enzymatic cascade involving the sequential activity of a dedicated E1 (APPBP1/ Uba3), E2 (Ubc12), and an ill-defined E3. We show that SCCRO (also known as DCUN1D1) binds to the components of the neddylation pathway (Cullin-ROC1, Ubc12, and CAND1) and augments but is not required for cullin neddylation in reactions using purified recombinant proteins. We also show that SCCRO recruits Ubc12ϳNEDD8 to the CAND1-Cul1-ROC1 complex but that this is not sufficient to dissociate or overcome the inhibitory effects of CAND1 on cullin neddylation in purified protein assays. In contrast to findings in cellular systems where no binding is seen, we show that SCCRO and CAND1 can bind to the neddylated Cul1-ROC1 complex in assays using purified recombinant proteins. Although neddylated (not unneddylated) Cul1-ROC1 is released from CAND1 upon incubation with testis lysate from SCCRO ؉/؉ mice, the addition of recombinant SCCRO is required to achieve the same results in lysate from SCCRO ؊/؊ mice. Combined, these results suggest that SCCRO is an important component of the neddylation E3 complex that functions to recruit charged E2 and is involved in the release of inhibitory effects of CAND1 on cullin-RING ligase E3 complex assembly and activity.Post-translational modification of proteins by ubiquitin (Ub) 4 regulates diverse cellular functions including protein turnover, differentiation, apoptosis, cell cycle, and transcription (1-5). Given its essential role, ubiquitination is a highly regulated process that involves the sequential action of three enzymes termed as E1, E2, and E3. In this enzymatic cascade, E1 initiates the process by forming a high energy thioester bond with Ub in an ATP-coupled reaction. The Ub is then transferred to E2 as a thioester intermediate. Finally, E3s serve as the targeting arm in the ubiquitination process, mediating the transfer of Ub from E2 to the target protein to create an isopeptide bond between the C-terminal glycine in Ub and a lysine residue on the substrate protein. Once attached, the Ub itself can be modified to generate polyubiquitin chains on the target protein (6). The functional effects of ubiquitination are influenced by the chain length and the residue on the Ub to which the chain is attached. Polyubiquitination promotes translocation to the 26 S proteasome for degradation. Other functional effects of mono-and polyubiquitination include protein translocation, interaction, and activation.Although there is only one known E1 (except in plants) and relatively few E2s, E3s exist in multiple forms to allow for specific protein targeting (6). In general, E3s are modular multiprotein complexes that can be divided into two broad categories based on the presence of either a HECT (homologous to E6-AP C terminus) or RING (Really Interesting New Gene)-finger domain-containi...

Previous investigators described a kindred with an X-linked dominant form of phosphate wasting in which affected children did not have radiographic evidence of rickets, whereas older individuals were progressively disabled by severe bowing. They proposed that this kindred suffered from a distinct disorder that they referred to as adult-onset vitamin D-resistant hypophosphatemic osteomalacia (AVDRR). We recently identified a gene, PHEX, that is responsible for the disorder X-linked hypophosphatemic rickets. To determine whether AVDRR is a distinct form of phosphate wasting, we searched for PHEX mutations in affected members of the original AVDRR kindred. We found that affected individuals have a missense mutation in PHEX exon 16 that results in an amino acid change from leucine to proline in residue 555. Clinical evaluation of individuals from this family indicates that some of these individuals display classic features of X-linked hypophosphatemic rickets, and we were unable to verify progressive bowing in adults. In light of the variability in the clinical spectrum of X-linked hypophosphatemic rickets and the presence of a PHEX mutation in affected members of this kindred, we conclude that there is only one form of X-linked dominant phosphate wasting.