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Molecular and Cellular Biology, November 2000, p. 8185-8197, Vol. 20, No. 21
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

The CUL1 C-Terminal Sequence and ROC1 Are Required for Efficient Nuclear Accumulation, NEDD8 Modification, and Ubiquitin Ligase Activity of CUL1

Manabu Furukawa,1 Yanping Zhang,1 Joseph McCarville,2 Tomohiko Ohta,1,dagger and Yue Xiong1,2,3,*

Lineberger Comprehensive Cancer Center,1 Department of Biochemistry and Biophysics,2 and Program in Molecular Biology and Biotechnology,3 University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7295

Received 28 April 2000/Returned for modification 14 June 2000/Accepted 10 August 2000

Members of the cullin and RING finger ROC protein families form heterodimeric complexes to constitute a potentially large number of distinct E3 ubiquitin ligases. We report here that the highly conserved C-terminal sequence in CUL1 is dually required, both for nuclear localization and for modification by NEDD8. Disruption of ROC1 binding impaired nuclear accumulation of CUL1 and decreased NEDD8 modification in vivo but had no effect on NEDD8 modification of CUL1 in vitro, suggesting that ROC1 promotes CUL1 nuclear accumulation to facilitate its NEDD8 modification. Disruption of NEDD8 binding had no effect on ROC1 binding, nor did it affect nuclear localization of CUL1, suggesting that nuclear localization and NEDD8 modification of CUL1 are two separable steps, with nuclear import preceding and required for NEDD8 modification. Disrupting NEDD8 modification diminishes the Ikappa Balpha ubiquitin ligase activity of CUL1. These results identify a pathway for regulation of CUL1 activity---ROC1 and the CUL1 C-terminal sequence collaboratively mediate nuclear accumulation and NEDD8 modification, facilitating assembly of active CUL1 ubiquitin ligase. This pathway may be commonly utilized for the assembly of other cullin ligases.

* Corresponding author. Mailing address: 22-012 Lineberger Comprehensive Cancer Center, Campus Box 7295, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7295. Phone: (919) 962-2142. Fax: (919) 966-8799. E-mail: yxiong{at}emailunc.edu.

dagger Permanent address: Department of Surgery, St. Marianna University School of Medicine, Kawasaki 216, Japan.

Molecular and Cellular Biology, November 2000, p. 8185-8197, Vol. 20, No. 21
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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