A molecular basis for stabilization of the von Hippel-Lindau (VHL) tumor suppressor protein by components of the VHL ubiquitin ligase.

The multiprotein von Hippel-Lindau (VHL) tumor suppressor (CBC(VHL), Cul2-Elongin BC-VHL) and SCF (Skp1-Cul1/Cdc53-F-box protein) complexes are members of structurally related families of E3 ubiquitin ligases that use a heterodimeric module composed of a member of the Cullin protein family and the RING finger protein Rbx1 (ROC1/Hrt1) to activate ubiquitylation of target proteins by the E2 ubiquitin-conjugating enzymes Ubc5 and Cdc34. VHL and F-box proteins function as the substrate recruitment subunits of CBC(VHL) and SCF complexes, respectively. In cells, many F-box proteins are short lived and are proposed to be ubiquitylated by an autocatalytic mechanism and destroyed by the proteasome following assembly into SCF complexes. In contrast, the VHL protein is stabilized by interaction with the Elongin B and C subunits of CBC(VHL) in cells. In this report, we have presented direct biochemical evidence that unlike the F-box protein Cdc4, which is ubiquitylated in vitro by Cdc34 in the context of the SCF, the VHL protein is protected from Ubc5-catalyzed ubiquitylation following assembly into the CBC(VHL) complex. CBC(VHL) is continuously required for negative regulation of hypoxia-inducible transcription factors in normoxic cells and of SCF complexes, many of which function only transiently during the cell cycle or in response to cellular signals. Our findings provide a molecular basis for the different modes of cellular regulation of VHL and F-box proteins and are consistent with the known roles of CBC(VHL).