Regulation of neddylation and deneddylation of cullin1 in SCFSkp2 ubiquitin ligase by F-box protein and substrate Gil Bornstein, Dvora Ganoth, and Avram Hershko* Unit of Biochemistry, Rappaport Faculty of Medicine, Technion–Israel Institute of Technology, Haifa 31096, Israel Contributed by Avram Hershko, May 17, 2006 The activity of cullin-containing ubiquitin protein ligase complexes lated cullins bind tightly to a protein called CAND1 (cullin- is stimulated by linkage to cullin of the ubiquitin-like protein associated and neddylation-dissociated 1). Binding of CAND1 to Nedd8 (‘‘neddylation’’). Neddylation is inhibited by the tight bind- Cul1 prevents its neddylation and also the binding of Skp1 to ing of cullins to CAND1 (cullin-associated and neddylation-disso- Cul1 and thus the assembly of the SCF complex (6–9). The ciated 1) protein, and Nedd8 is removed from cullins by specific crystal structure of the CAND1–Cul1 complex showed tight isopeptidase activity of the COP9͞signalosome (CSN) complex. The binding of CAND1 to both N-terminal and C-terminal domains mechanisms that regulate neddylation and deneddylation of cul- of Cul1, thus accounting for both actions of CAND1 (10). lins were unknown. We examined this problem for the case of Although it was initially reported that neddylation of Cul1 in SCFSkp2, a cullin1 (Cul1)-containing ubiquitin ligase complex that complex with CAND1 caused the dissociation of the complex contains the S phase-associated protein Skp2 as the substrate- (6), subsequent experiments with more purified preparations did binding F-box protein subunit. SCFSkp2 targets for degradation the not detect neddylation or dissociation of Cul1 in complex with cyclin-dependent kinase (cdk) inhibitor p27 in the G1-to-S phase CAND1 (10). Because the initial experiments were carried out transition, a process that requires its phosphorylation and binding with Cul1–Cand1 complex immunoprecipitated from mamma- to cdk2-cyclin E. Because levels of Skp2, cyclin E, and the accessory lian cells (6), it was suggested that some cellular factors may be protein Cks1 (cyclin kinase subunit 1) all rise at the end of G1 phase, required for the dissociation of the complex (10). Thus, an it seemed possible that the neddylation of Cul1 in SCFSkp2 is important problem is how the dissociation of Cul1 from CAND1 regulated by the availability of the F-box protein and͞or the and its neddylation are initiated. The extent of the neddylation substrate. We found that the supplementation of Skp2–Skp1 and of cullins is also affected by the COP9͞signalosome (CSN) substrate (along with further components necessary for substrate complex, a large multiprotein complex that has a specific isopep- presentation to the ubiquitin ligase) to extracts of HeLa cells tidase activity that deneddylates cullins (reviewed in ref. 11). synergistically increased levels of neddylated Cul1. Skp2–Skp1 Although neddylation of cullins is important for the assembly abrogates the inhibitory influence of CAND1 on the neddylation of and activity of cullin-based ubiquitin ligases, it is not clear how Cul1 by promoting the dissociation of the cullin–CAND1 complex, neddylation is regulated. It was previously suggested that the whereas substrate, together with substrate-presenting compo- availability of a Skp1–F-box protein–substrate complex may nents, prevents the action of CSN to deneddylate cullin. We trigger the neddylation of Cul1 (6, 11), but experimental evi- propose a sequence of events in which the increased availability of dence for this notion was very scanty. It was observed that the Skp2 and substrate in the transition of cells to S phase promotes von Hippel–Lindau tumor suppressor gene product (pVHL), the neddylation and assembly of the SCFSkp2 ubiquitin ligase which is the substrate-binding subunit of a Cul2-containing complex. ubiquitin ligase complex, stimulates the neddylation of Cul2 (12, 13). It was reported also that SCF␤TrCP that contains the cell cycle ͉ Nedd8 neddylated form of Cul1 preferentially binds to phosphorylated substrates of this ubiquitin ligase complex (see figure 1 in ref. 14). The molecular mechanisms responsible for the regulation of ullin-containing multiprotein complexes comprise the larg- cullin neddylation were unknown and are the subject of this est family of ubiquitin protein ligases. The best studied of C investigation. these complexes is the SCF (Skp1-cullin1-F-box protein) class of ubiquitin ligases. They consist of cullin1 (Cul1) scaffold protein, Results the N-terminal domain of which binds the S phase-associated Levels of Neddylated Cullin1 in Extracts of HeLa Cells Are Increased by Skp1 adaptor protein while the C-terminal domain binds the Skp2–Skp1 and p27 Substrate of SCFSkp2. Initially, we examined the ROC1 RING finger protein. Skp1 binds variable substrate- notion that increased availability of Skp2 and of phosphorylated binding F-box proteins that may also interact directly with Cul1 p27 in transition to S phase stimulated the neddylation, and thus (reviewed in refs. 1 and 2). We have been studying an SCF the activity, of the newly assembled SCFSkp2 ubiquitin ligase ubiquitin ligase that contains the F-box protein Skp2 (SCFSkp2) complex. As shown in Fig. 1A, the combined addition to HeLa and that targets the cyclin-dependent kinase (cdk) inhibitor p27 cell extracts of Skp2–Skp1, its substrate p27, and further com- for degradation in the transition of cells from G0͞G1 to S phase Skp2 ponents necessary for the binding of phosphorylated p27 to the of the cell cycle (3, 4). SCF is assembled at the end of G1 by SCFSkp2 ubiquitin ligase (3, 4) markedly increased levels of BIOCHEMISTRY the rise in levels of its specific F-box protein, Skp2, and of its neddylated Cul1 (lane 2). Because free Skp2 is not soluble, Skp2 accessory protein, Cks1 (cyclin kinase subunit 1) (2, 4). As is the case with other cullin-based ubiquitin ligases, the assembly and Skp2 activity of SCF is regulated by the ligation of the small, Conflict of interest statement: No conflicts declared. ubiquitin-like protein Nedd8 to a specific lysine residue at the Abbreviations: Cul1, cullin1; SCF, Skp1-Cul1-F-box protein; CAND1, cullin-associated and C-terminal domain of Cul1 (1). This process, also called ‘‘ned- neddylation-dissociated; CSN, COP9͞signalosome; cdk, cyclin-dependent kinase; Cks1, dylation,’’ increases the ubiquitin ligase activity of SCF, in part cyclin kinase subunit 1. by increasing its affinity for E2 (5). Neddylation also controls the *To whom correspondence should be addressed. E-mail: [email protected]. assembly of cullin-based ubiquitin ligases, because nonneddy- © 2006 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0603921103 PNAS ͉ August 1, 2006 ͉ vol. 103 ͉ no. 31 ͉ 11515–11520 Downloaded by guest on October 1, 2021 ‘‘substrate’’ and to Skp2–Skp1 as ‘‘Skp2.’’ Although the stimu- latory effects of Skp2 and substrate could be readily observed in crude extracts of HeLa cells, they had no significant influence on a purified neddylation system consisting of recombinant His-6- Cul1, Nedd8, the E1-like enzyme APP-BP1 (amyloid-␤ precur- sor protein binding protein 1)͞Uba3, and the Nedd8-specific E2 Ubc12 (data not shown), which suggested the involvement of additional cellular factors in the control of cullin neddylation. To identify the cellular factors that may be involved in the regulation of the neddylation of Cul1, we fractionated extracts of HeLa cells. In this fractionation, we tested the possible involve- ment of two factors that are known to have a negative influence on cullin neddylation: the CSN complex, which has deneddyla- tion activity (11), and the protein CAND1, which binds to cullins and prevents their neddylation (6–9). Extracts of HeLa cells were subjected to chromatography on a heparin-Sepharose column, a procedure previously used for the purification of CSN (17). The position of known proteins in column fractions was followed by immunoblotting. As shown in Fig. 1B, the Jab1 subunit of the CSN complex eluted mainly in fractions 3–8. The Jab1-containing peak fractions coincided with enzyme activity that deneddylates Cul1 (data not shown). Cul1 eluted in a broad region of this column, spanning from fraction 5 to the end of the salt gradient (Fig. 1B), possibly because of its binding to different proteins in different complexes. APP-BP1, a subunit of the E1-like enzyme of Nedd8, eluted from the heparin column at low salt concentrations in fractions 1–3, whereas CAND1 eluted in a broader region in fractions 2–6 (Fig. 1B). Fig. 1. Skp2 and substrate synergistically increase levels of neddylated Cul1 Because the strong deneddylating activity concentrated in in extracts and fractions from HeLa cells. (A) Increase in levels of neddylated CSN peak fractions interfered with the Cul1 neddylation assay, Cul1 in extracts of HeLa cells by combined addition of Skp2–Skp1, p27 sub- we tried to reconstitute the effects of F-box protein and substrate strate, and other components required for the interaction of substrate with with fractions from both sides of the CSN peak. As the source SCFSkp2. The neddylation of endogenous Cul1 in extracts of HeLa cells (3 ␮gof of endogenous Cul1 for neddylation, we used fraction 10 from protein) was assayed as described in Materials and Methods, except that the the heparin column. The neddylation of endogenous Cul1 was concentration of APP-BP1-Uba3 was 0.01 nM. Skp2–Skp1 was added as indi- assayed in the presence of recombinant Nedd8 and Ubc12. When cated in the presence of either complete substrate mixture (lane 2) or sub- strate mixtures from which the indicated components were omitted (lanes a small amount of fraction 2 of the heparin column (containing 3–5). (B) Fractionation of HeLa cell extracts on a heparin-Sepharose column. endogenous E1-like enzyme; Fig. 1B) was added to this system, Extracts of HeLa cells were fractionated as described in Materials and Meth- significant neddylation of Cul1 could be seen (Fig.