VHL-box and SOCS-box domains determine binding specificity for Cul2-Rbx1 and Cul5-Rbx2 modules of ubiquitin ligases (original) (raw)
- Takumi Kamura1,2,
- Katsumi Maenaka3,
- Shuhei Kotoshiba1,2,
- Masaki Matsumoto1,2,
- Daisuke Kohda3,
- Ronald C. Conaway4,5,
- Joan Weliky Conaway4,5, and
- Keiichi I. Nakayama1,2,6
- 1Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, Higashi-ku, Fukuoka, Fukuoka 812-8582, Japan; 2CREST, Japan Science and Technology Corporation, Kawaguchi, Saitama 332-0012, Japan; 3Division of Structural Biology, Research Center for Prevention of Infectious Diseases, Medical Institute of Bioregulation, Kyushu University, Higashi-ku, Fukuoka, Fukuoka 812-8582, Japan; 4Stowers Institute for Medical Research, Kansas City, Missouri 64110, USA; 5Department of Biochemistry and Molecular Biology, Kansas University Medical Center, Kansas City, Kansas 66160, USA
Abstract
The ECS (Elongin B/C-Cul2/Cul5-SOCS-box protein) complex is a member of a family of ubiquitin ligases that share a Cullin-Rbx module. SOCS-box proteins recruit substrates to the ECS complex and are linked to Cullin-Rbx via Elongin B/C. VHL has been implicated as a SOCS-box protein, but lacks a C-terminal sequence (downstream of the BC box) of the SOCS box. We now show that VHL specifically interacts with endogenous Cul2-Rbx1 in mammalian cells, whereas SOCS-box proteins associate with Cul5-Rbx2. We also identify LRR-1 and FEM1B as proteins that share a region of homology with VHL (the VHL box, including the BC box and downstream residues) and associate with Cul2-Rbx1. ECS complexes can thus be classified into two distinct protein assemblies, that is, those that contain a subunit with a VHL box (composed of the BC box and a downstream Cul2 box) that interacts with Cul2-Rbx1, and those that contain a subunit with a SOCS box (BC box and downstream Cul5 box) that interacts with Cul5-Rbx2. Domain-swapping analyses showed that the specificity of interaction of VHL-box and SOCS-box proteins with Cullin-Rbx modules is determined by the Cul2 and Cul5 boxes, respectively. Finally, RNAi-mediated knockdown of the Cul2-Rbx1 inhibited the VHL-mediated degradation of HIF-2α, whereas knockdown of Cul5-Rbx2 did not affect it. These data suggest that the functions of the Cul2-Rbx1 and Cul5-Rbx2 modules are distinct.
Footnotes
Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/gad.1252404.
↵6 Corresponding author. E-MAIL nakayak1{at}bioreg.kyushu-u.ac.jp; FAX 81-92-642-6819.
- Accepted October 14, 2004.
- Received August 20, 2004.
Cold Spring Harbor Laboratory Press