Biochemical and Biophysical Research Communications 270, 1093–1096 (2000)
doi:10.1006/bbrc.2000.2576, available online at http://www.idealibrary.com on

Modification of Cullin-1 by Ubiquitin-like Protein Nedd8
Enhances the Activity of SCF skp2 toward p27 kip1
Mitsuru Morimoto, Tamotsu Nishida, Reiko Honda, 1 and Hideyo Yasuda 2
School of Life Science, Tokyo University of Pharmacy and Life Science,
1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan

Received March 17, 2000

The periodic expression of cell cycle proteins isimportant for the regulation of cell cycle progression. The amount of CDK inhibitor, p27 kip1 , one suchprotein, seems to be regulated by the ubiquitinproteasome system. The ubiquitin ligase (E3) toward
p27 kip1 is thought to be SCF skp2 . The activity of SCF skp2
was increased by the addition of Roc1 protein to thecomplex. Furthermore, the ubiquitination of p27 kip1
seemed to be dependent on the phosphorylation ofT187 of p27 kip1 because the mutant T187A was not
ubiquitinated at all in an in vitro ubiquitination
system. Cullin-1, a component of SCF, is modified byubiquitin-like protein Nedd8. The modification siteof cullin-1 was shown to be K696 because the K696R
mutant was not modified. When the effect of the
Nedd8 modification on the SCF skp2 activity toward
p27 kip1 was investigated, the activity was markedly
decreased by using the Nedd8-unmodified mutant
cullin-1 (K696R), indicating that the modification
may play an important role on the SCF skp2 activity
toward p27 kip1 . © 2000 Academic Press
Key Words: p27 kip1; SCF skp2; Nedd8; cullin-1; ubiquitin;ubiquitination; CDK inhibitor; RUB1; CDC53.

In mammals, cell-cycle progression is thought to bemainly regulated by the activity of CDK. When thecells enter G 0 phase, the amount of CDK inhibitor
p27 kip1 increases, which inhibits the activity of CDK4 orCDK6 (1). On the other hand, when the cells enter G 1
phase the amount of p27 kip1 decreases to activate CDK4or CDK6. Since the p27 kip1 is degraded by the ubiquitinproteasome system, the regulatory mechanism of its
ubiquitination is important to clarify the regulation of1
Present address: Max-Planck Institute for Biochemistry, Martinsried, Germany.
2To whom correspondence should be addressed. Fax: (81) (426)76-7249. E-mail: yasuda@ls.toyaku.ac.jp..

cell-cycle progression (2– 4). The ubiquitination of theprotein is regulated by the phosphorylation of it. The
phosphorylation of T187 of p27 is thought to be aprerequisite for the ubiquitination and is attributed toCyclinE/CDK2 complex (5–7). The SCF skp2, consistingof cullin-1, skp1, and skp2, seems to function as aubiquitin ligase (E3) toward p27 (3, 4). The p27 binding
protein Jab1/Mov34 may function to accelerate thedegradation of p27 by carrying p27 into cytoplasm (8).
Nedd8, a homologue of ubiquitin, is a modifier ofcullin family proteins, and this modification is catalyzed by APPBP1/Uba3 as E1 and Ubc12 as E2 (9, 10).
Recently, the cullin-1 in SCF skp2 was found to be modified by Nedd8 (11, 12). Furthermore, cullin-2 in VBC(11, 13), another E3 complex, is modified by it. Morerecently, cullin-1 to cullin-5 have also been found to bemodified by it (10).
The modified cullin-1 is found in centrosomes, andthe ratio of the modified cullin-1 to total cullin-1 increases when the cullin-1 is expressed together withRoc1 in Sf-9 cells in the baculovirus protein expression
system (14). However, the role of this modification has
not yet been clarified.
Here, we show that Nedd8 modification of cullin-1
enhances the ubiquitination of p27.
MATERIALS AND METHODS
Expression and mutation of the proteins. Cullin-1, skp1, skp2,Roc1, p27 kip1 , cyclinE, CDK2, APPBP1, and hUba3 were expressedin a baculovirus expression system using Sf-9 cells according tothe manufacturer’s protocol (Clontech). In some experiments, theSf-9 cells were co-infected with baculoviruses with inserts of various cDNAs. UbcH5, Ubc12, and Nedd8 were expressed in E. coli
(BL21, LysS) by using the pET vector. Point mutation of p27
cDNA or Nedd8 cDNA was done by use of the PCR method as
described previously (15).
In vitro ubiquitination assay. The Sf-9 cells expressing cullin-1,skp1, and skp2 were lysed in 50 mM Tris–HCl (pH 7.4), 3 mM MgCl 2,1 mM phenylmethyl sulfonyl fluoride (PMSF), 0.1 M NaCl, and 0.1%
NP-40, and the SCF complex (composed of these expressed proteins)

1093

0006-291X/00 $35.00
Copyright © 2000 by Academic Press
All rights of reproduction in any form reserved.

Vol. 270, No. 3, 2000

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

FIG. 1. Roc 1 enhances ubiquitination of p27 kip1 in vitro. SCF skp2,consisting of cullin-1, skp1, and skp2, was mixed with Roc1, and theubiquitination reaction toward p27 was carried out as describedunder Materials and Methods except that the reaction mixture also
contained HeLa cell lysate (4 ⫻ 10 5 cells/ml). Lane 1, the reaction
mixture without Roc1 and with SCF skp2; lane 2, with Roc1 and without SCF skp2; lane 3, with Roc1 and SCF skp2.

was bound to a TALON metal affinity resin (Clontech), which binds
6xHis-tagged cullin-1, and eluted from the resin by use of 2 mM
imidazole. The Sf-9 cells expressing GST-p27 and CyclinE/CDK2
were lysed in 50 mM Tris–HCl (pH 7.4), 3 mM MgCl 2, 1 mM PMSF,
0.1 M NaCl, and 0.1% NP-40, and GST-p27 was purified byglutathione-Sepharose 4B affinity resin. When 6xHis-tagged Roc1
was used, the protein was purified by use of the TALON resin asdescribed above. The in vitro ubiquitination was carried out essentially as described previously, using biotin-labeled ubiquitin (15, 16).
Briefly, the reaction mixture containing 40 mM Tris–HCl, 2 mM
ATP, 1 mM dithiothreitol, mouse E1 (1 ␮g), E2 (UbcH5, 3 ␮g),
SCF skp2 (2 ␮g), GST-tagged p27 kip1 (150 ng), and biotinylated ubiquitin was incubated for 30 min at 25°C. After incubation, the GSTtagged p27 kip1 was pelleted after having been bound by glutathioneSepharose 4B, and the resin was applied to SDS-polyacrylamide gelelectrophoresis (SDS–PAGE). The ubiquitinated p27 kip1 was detectedby Western blotting using avidin-peroxidase and the ECL method(Amersham Pharmacia).

carried out the ubiquitination assay in the presence orabsence of Roc1 protein. As shown in Fig. 1, theubiqutination of p27 increased markedly in the presence of Roc1. Cullin-1, a component of SCF skp2, hasbeen shown to be modified by Nedd8, a ubiquitin-likeprotein. So to determine whether the Nedd8 modification is important for the activity of SCF, we carried out
the ubiquitination of p27 with or without the Nedd8
modification system, i.e., APPBP1/hUba3, hUba12,
and Nedd8. The ubiquitination of p27 was markedly
increased in the presence of this modification system.
In contrast, the p27 (T187A) was not ubiquitinated at
all with or without the Nedd8 modification system (Fig.
2). However, even without the Nedd8 modification system, some ubiquitination of p27 was apparent, perhaps
due to modification of cullin-1 by Nedd8 in Sf-9 cells
during its expression. To confirm the effect of Nedd8
modification of cullin-1 on ubiquitination of p27, we
examined the effect of Nedd8 un-modified form ofcullin-1 on the ubiquitination.
Effect of Nedd8 unmodified mutant cullin-1 on
SCF skp2 activity. Recently, the mammalian cullin-2
was found to be modified at K689 by Nedd8 (13). Whenhuman cullin-1 was aligned with cullin-2, the Nedd8
modification site of cullin-2 corresponded to K696 incullin-1, as shown in Fig. 3. Therefore, the K696 residue was modified to R696, and the Nedd8 modificationof this mutant cullin-1 was tested. After the wildtype or mutant cullin-1 had been subjected to theNedd8 modification system, the modification of it was

Nedd8 modification system. The Sf-9 cells expressing APPBP1and hUba3 were lysed; and the mixture containing APPBP1 (500 ng),hUba3 (50 ng), hUbc12 (3 ␮g), SCF skp2 (2 ␮g), and Nedd8 or GSTNedd8 (1 ␮g) was incubated at 25°C for 30 min. After incubation, theSCF skp2 containing 6xHis-tagged cullin-1 were purified by use ofTALON beads. When the Nedd8-modified SCF skp2 was used for the invitro ubiquitination assay, HeLa cell lysate (1 ⫻ 10 6 cells) was added
to the Nedd8 modification system. The in vitro ubiquitination assay
was performed as described above. The Western blotting of Nedd8 or
6xHis-tagged protein was done by use of anti-Nedd8 antibody(Alexis) or anti-6xHis antibody (Qiagen), respectively, andperoxidase- conjugated anti-mouse rabbit antibody (Dako).

RESULTS AND DISCUSSION
Ubiquitination of p27 by SCF skp2 in the presence ofRoc1 and Nedd8 modification. The Roc1 protein wasfound to be a binding protein of cullin family proteins
and to activate the ubiquitination of I␬B␣ by SCF ␤-TrCP(17, 18). To know whether the Roc1 protein is also
important in the activity of SCF skp2 toward p27, weFIG. 2. Nedd8 modification of cullin-1 and the phosphorylationof T187 of p27 enhance the ubiquitination of p27. The Sf-9 cells wereco-infected with GST-tagged p27 or GST-tagged p27 (T187A) andCyclin E/CDK2. Nedd8 modification and the in vitro ubiquitination
assay were carried out by use of GST-tagged p27 or GST-tagged p27(T187A) as described under Materials and Methods. Lane 1, ubiquitination of GST-tagged p27 without Nedd8 modification; lane 2,ubiquitination of GST-tagged p27 with Nedd8 modification; lane 3,ubiquitination of GST-tagged p27 (187A) without Nedd8 modification; lane 4, ubiquitination of GST-tagged p27 (187A) with Nedd8modification.

1094

Vol. 270, No. 3, 2000BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

FIG. 3. Alignment of Nedd8 modification site of cullin-2 withcullin-1 sequence. The K696 of cullin-1 seems to be the putative
Nedd8 modification site.

checked. By Western blotting using anti-Nedd8 antibody, the modification of wild-type cullin-1 was detected, but that of the mutant cullin-1 was not (Fig.
4A). Also, when the GST-tagged Nedd8 was used in themodification system, the Nedd8 modified wild-typecullin-1 was detected, but the modified mutant cullinwas not (Fig. 4B). The Nedd8-modified form of cullin-1
had a much larger molecular weight than the unmodified cullin-1 (lanes 2, 4, and 6 in Fig. 4B). After theNedd8 modification reaction had been carried out inthe presence of wild-type or mutant cullin-1, the ubiquitination of p27 under the two conditions was com-pared. Although the SCF skp2 containing Nedd8-modified cullin-1 catalyzed the multi-ubiquitinated chain
formation of p27 (lane 2 in Fig. 5), the SCF skp2 containing mutant cullin-1 did not catalyze the multiubiquitinated chain formation of p27 (Fig. 5).
Here we showed that Nedd8 modification of cullin-1
enhanced the activity of SCF skp2 toward p27 kip1. The
in vitro ubiquitination assay in mammalian systemshowed that the modification is essential for theSCF skp2 activity, although the disruption of RUB1, theyeast Saccharomyces cerevisiae homologue of Nedd8gene, showed that the gene was not essential (19). In
the yeast system, the over-expression of Cdc53 or Hrt1,
the yeast homologue of Roc1, may compensate for thedefect of Rub1 modification.
Further, we showed that Roc1 also enhanced theactivity, indicating that SCF skp2 may also use the Roc1/cullin-1 heterodimer as the catalytic site, as shown for
SCF ␤-TrCP. The modification may be necessary for theefficient assembly of SCF components, and/or be necessary for the catalytic activity of the Roc1/cullin-1

FIG. 4. K696 of cullin-1 is the Nedd8 modification site. (A) After the reactions indicated at the top of the figure had been carried out,
the GST-tagged cullin-1 was pelleted by glutathione-Sepharose 4B beads, and the Nedd8-modified cullin-1 was detected withanti-Nedd8 antibody (Alexis). The cullin-1 proteins were detected by staining with coomassie brilliant blue R-250 (CBB). Nedd8modified cullin-1 was only detected in lane 2. (B) After reactions indicated, GST-tagged Nedd8 was pelleted by glutathione-Sepharose
4B beads, and GST-Nedd8-modified 6xHis-tagged cullin-1 was detected with anti-6xHis antibody (Qiagen). The wild-type cullin-1 andmutant-type cullin-1 used in the reaction are shown in lanes 5 and 6 as a control of Western blotting. Nedd8- modified His-cullin-1 wasonly detected in lane 2.

1095

Vol. 270, No. 3, 2000

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONSREFERENCES

FIG. 5. SCF skp2 containing the Nedd8 un-modified form ofcullin-1 did not have ubiquitin ligase activity toward p27. SCF skp2
containing wild-type cullin-1 (lanes 1 and 2), or K696R mutantcullin-1 (lanes 3 and 4) was used in a ubiquitination assay toward
p27 kip1, with (lanes 2 and 4) or without (lanes 1 and 3) the Nedd8
modification reaction. The cullin-1 proteins were detected with anti6xHis antibody (Qiagen). Multi-ubiquitinated forms of p27 wereapparent in lane 2, and some in lane 1, but none were found for themutant cullin-1.

heterodimer. Thus, the Nedd8 modification of SCF skp2
is probably essential for its activity.
ACKNOWLEDGMENTS
We thank Dr. Hirofumi Tanaka for his help to express the proteinsin Sf-9 cells. This work was supported in part by a Grant-in-Aid from
the Ministry of Education, Science, Culture, and Sports in Japan.

1. Sherr, C. J., and Roberts, J. M. (1999) Genes Dev. 13, 1501–1512.
2. Pagano, M., Tam, S. W., Theodoras, A. M., Beer-Romero, P., Del
Sal, G., Chau, V., Yew, P. R., Draetta, G. F., and Rolfe, M. (1995)
Science 269, 682– 685.
3. Carrano, A. C., Eytan, E., Hershko, A., and Pagano, M. (1999)
Nat. Cell Biol. 4, 193–199.
4. Suter, H., Chatelain, E., Marti, A., Wirbelauer, C., Senften, M.,
Muller, U., and Krek, W. (1999) Nat. Cell Biol. 1, 207–214.
5. Vlach, J., Hennecke, S., and Amati, B. (1997) EMBO J. 16,5334 –5344.
6. Nguyen, H., Gitig, D. M., and Koff, A. (1999) Mol. Cell. Biol. 19,1190 –1201.
7. Montagnoli, A., Fiore, F., Eytan, E., Carrano, A. C., Draetta, G. F.,
Hershko, A., and Pagano, M. (1999) Genes Dev. 13, 1181–1189.
8. Tomoda, K., Kubota, Y., and Kato, J. (1999) Nature 398, 160 –165.
9. Osaka, F., Kawasaki, H., Aida, N., Saeki, M., Chiba, T., Kawashima, S., Tanaka, K., and Kato, S. (1998) Genes Dev. 12,2263–2268.
10. Hori, T., Osaka, F., Chiba, T., Miyamoto, C., Okabayashi, K.,
Shimbara, N., Kato, S., and Tanaka, K. (1999) Oncogene 18,6829 – 6834.
11. Liakopoulos, D., Busgen, T., Brychzy, A., Jentsch, S., and Pause,
A. (1999) Proc. Natl. Acad. Sci. USA 96, 5510 –5515.
12. Freed, E., Lacey, K. R., Huie, P., Lyapina, S. A., Deshaies, R. J.,
Stearns, T., and Jackson, P. K. (1999) Genes Dev. 13, 2242–2257.
13. Wada, H., Yeh, E. T., and Kamitani, T. (1999) Biochem. Biophys.
Res. Commun. 257, 100 –105.
14. Kamura, T., Conrad, M. N., Yan, Q., Conaway, R. C., and Conaway, J. W. (1999) Genes Dev. 13, 2928 –2933.
15. Honda, R., Tanaka, H., and Yasuda, H. (1997) FEBS Lett. 420,25–27.
16. Honda, R., and Yasuda, H. (1999) EMBO J. 18, 22–27.
17. Tan, P., Fuchs, S. Y., Chen, A., Wu, K., Gomez, C., Ronai, Z., andPan, Z. Q. (1999) Mol. Cell 3, 527–533.
18. Ohta, T., Michel, J. J., Schottelius, A. J., and Xiong, Y. (1999)
Mol. Cell 3, 535–541.
19. Lammer, D., Mathias, N., Laplaza, J. M., Jiang, W., Liu, Y., Callis,J., Goebl, M., and Estelle, M. (1998) Genes Dev. 7, 914 –926.

1096