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J. Biol. Chem., Vol. 283, Issue 12, 8005-8013, March 21, 2008

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Characterization of Cullin-box Sequences That Direct Recruitment of Cul2-Rbx1 and Cul5-Rbx2 Modules to Elongin BC-based Ubiquitin Ligases^*

Nawel Mahrour, William B. Redwine¹, Laurence Florens, Selene K. Swanson, Skylar Martin-Brown, William D. Bradford, Karen Staehling-Hampton, Michael P. Washburn, Ronald C. Conaway, and Joan W. Conaway²

From the Stowers Institute for Medical Research, Kansas City, Missouri 64110 and Department of Biochemistry and Molecular Biology, Kansas University Medical Center, Kansas City, Kansas 66160

The Elongin BC-box protein family includes the von Hippel-Lindau tumor suppressor and suppressor of cytokine signaling proteins, which are substrate recognition subunits of structurally related classes of E3 ubiquitin ligases composed of Elongin C-Elongin B-Cullin 2-Rbx1 (Cul2 ubiquitin ligases) or of Elongin C-Elongin B-Cullin 5-Rbx2 (Cul5 ubiquitin ligases). The Elongin BC complex acts as an adaptor that links a substrate recognition subunit to heterodimers of either Cullin 2 (Cul2) and RING finger protein Rbx1 or Cullin 5 (Cul5) and Rbx2. It has been shown ( Kamura, T., Maenaka, K., Kotoshiba, S., Matsumoto, M., Kohda, D., Conaway, R. C., Conaway, J. W., and Nakayama, K. I. (2004) Genes Dev. 18, 3055-3065[Abstract/Free Full Text] ) that interaction of BC-box proteins with their cognate Cul-Rbx module is determined by specific regions, called Cul2- or Cul5-boxes, located immediately downstream of their BC-boxes. Here, we investigate further the mechanisms governing assembly of BC-box proteins with their specific Cul-Rbx modules. Through purification and characterization of a larger collection of BC-box proteins that serve as substrate recognition subunits of Cul2 and Cul5 ubiquitin ligases and through structure-function studies, we define Cul2- and Cul5-boxes in greater detail. Although it previously appeared that there was little sequence similarity between Cul5- and Cul2-box motifs, analyses of newly identified BC-box proteins reveal that residues conserved in the Cul2-box represent a subset of those conserved in the Cul5-box. The sequence motif LPP, which is conserved in most Cul5-boxes and has been suggested to specify assembly of Cul5 ligases, is compatible with Cul2 interaction. Finally, the spacing between BC- and Cullin-boxes is much more flexible than has been appreciated and can vary from as few as 3 and as many as 80 amino acids. Taken together, our findings shed new light on the mechanisms by which BC-box proteins direct recruitment of Cullin-Rbx modules during reconstitution of ubiquitin ligases.

Received for publication, August 21, 2007 , and in revised form, January 10, 2008.

^* This work was supported in part by National Institutes of Health Grant GM41628 (to R. C. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables S1-S5 and supplemental references.

¹ Present address: Dept. of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115.

² To whom correspondence should be addressed: Stowers Inst. for Medical Research, 1000 E. 50th St., Kansas City, MO 64110. Tel.: 816-926-4091; Fax: 816-926-2091; E-mail: jlc{at}stowers-institute.org.

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