VHL-box and SOCS-box domains determine binding specificity for Cul2-Rbx1 and Cul5-Rbx2 modules of ubiquitin ligases.
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-2alpha, 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.
Kamura
Takumi
T
Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, Higashi-ku, Fukuoka, Fukuoka 812-8582, Japan.
Maenaka
Katsumi
K
Kotoshiba
Shuhei
S
Matsumoto
Masaki
M
Kohda
Daisuke
D
Conaway
Ronald C
RC
Conaway
Joan Weliky
JW
Nakayama
Keiichi I
KI
D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D044842:Cullin Proteins / Q000235:genetics / Q000378:metabolism*; D040461:Gene Components; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D013058:Mass Spectrometry; D008956:Models, Chemical; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000378:metabolism; D034622:RNA Interference; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D000071222:Suppressor of Cytokine Signaling 1 Protein; D050826:Suppressor of Cytokine Signaling Proteins; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D051794:Von Hippel-Lindau Tumor Suppressor Protein
C105153:CUL2 protein, human; C496871:CUL5 protein, human; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D044842:Cullin Proteins; C403492:FEM1B protein, human; D047908:Intracellular Signaling Peptides and Proteins; C464446:LRR1 protein, human; D011506:Proteins; C479529:RBX1 protein, human; D012097:Repressor Proteins; C108944:SOCS1 protein, human; D000071222:Suppressor of Cytokine Signaling 1 Protein; D050826:Suppressor of Cytokine Signaling Proteins; D025521:Tumor Suppressor Proteins; D044767:Ubiquitin-Protein Ligases; D051794:Von Hippel-Lindau Tumor Suppressor Protein; C497446:VHL protein, human