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.
Mesh Terms:
Amino Acid Sequence, Carrier Proteins, Cell Cycle Proteins, Cells, Cultured, Cullin Proteins, Gene Components, Humans, Immunoblotting, Immunoprecipitation, Intracellular Signaling Peptides and Proteins, Mass Spectrometry, Models, Chemical, Molecular Sequence Data, Protein Binding, Protein Structure, Tertiary, Proteins, RNA Interference, Repressor Proteins, Sequence Alignment, Suppressor of Cytokine Signaling Proteins, Tumor Suppressor Proteins, Ubiquitin-Protein Ligases, Von Hippel-Lindau Tumor Suppressor Protein
Amino Acid Sequence, Carrier Proteins, Cell Cycle Proteins, Cells, Cultured, Cullin Proteins, Gene Components, Humans, Immunoblotting, Immunoprecipitation, Intracellular Signaling Peptides and Proteins, Mass Spectrometry, Models, Chemical, Molecular Sequence Data, Protein Binding, Protein Structure, Tertiary, Proteins, RNA Interference, Repressor Proteins, Sequence Alignment, Suppressor of Cytokine Signaling Proteins, Tumor Suppressor Proteins, Ubiquitin-Protein Ligases, Von Hippel-Lindau Tumor Suppressor Protein
Genes Dev.
Date: Dec. 15, 2004
PubMed ID: 15601820
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