VHL-box and SOCS-box domains determine binding specificity for Cul2-Rbx1 and Cul5-Rbx2 modules of ubiquitin ligases

Genes Dev. 2004 Dec 15;18(24):3055-65. doi: 10.1101/gad.1252404.

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-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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Carrier Proteins / metabolism
  • Cell Cycle Proteins / metabolism*
  • Cells, Cultured
  • Cullin Proteins / genetics
  • Cullin Proteins / metabolism*
  • Gene Components
  • Humans
  • Immunoblotting
  • Immunoprecipitation
  • Intracellular Signaling Peptides and Proteins / genetics
  • Intracellular Signaling Peptides and Proteins / metabolism*
  • Mass Spectrometry
  • Models, Chemical
  • Molecular Sequence Data
  • Protein Binding
  • Protein Structure, Tertiary
  • Proteins / metabolism
  • RNA Interference
  • Repressor Proteins / genetics
  • Repressor Proteins / metabolism*
  • Sequence Alignment
  • Suppressor of Cytokine Signaling 1 Protein
  • Suppressor of Cytokine Signaling Proteins
  • Tumor Suppressor Proteins / genetics
  • Tumor Suppressor Proteins / metabolism*
  • Ubiquitin-Protein Ligases / genetics
  • Ubiquitin-Protein Ligases / metabolism*
  • Von Hippel-Lindau Tumor Suppressor Protein

Substances

  • CUL2 protein, human
  • CUL5 protein, human
  • Carrier Proteins
  • Cell Cycle Proteins
  • Cullin Proteins
  • FEM1B protein, human
  • Intracellular Signaling Peptides and Proteins
  • LRR1 protein, human
  • Proteins
  • RBX1 protein, human
  • Repressor Proteins
  • SOCS1 protein, human
  • Suppressor of Cytokine Signaling 1 Protein
  • Suppressor of Cytokine Signaling Proteins
  • Tumor Suppressor Proteins
  • Ubiquitin-Protein Ligases
  • Von Hippel-Lindau Tumor Suppressor Protein
  • VHL protein, human