Identification of Elongin C and Skp1 sequences that determine Cullin selection.
The multiprotein von Hippel-Lindau (VHL) tumor suppressor and Skp1-Cul1-F-box protein (SCF) complexes belong to families of structurally related E3 ubiquitin ligases. In the VHL ubiquitin ligase, the VHL protein serves as the substrate recognition subunit, which is linked by the adaptor protein Elongin C to a heterodimeric Cul2/Rbx1 module that activates ubiquitylation of target proteins by the E2 ubiquitin-conjugating enzyme Ubc5. In SCF ubiquitin ligases, F-box proteins serve as substrate recognition subunits, which are linked by the Elongin C-like adaptor protein Skp1 to a Cul1/Rbx1 module that activates ubiquitylation of target proteins, in most cases by the E2 Cdc34. In this report, we investigate the functions of the Elongin C and Skp1 proteins in reconstitution of VHL and SCF ubiquitin ligases. We identify Elongin C and Skp1 structural elements responsible for selective interaction with their cognate Cullin/Rbx1 modules. In addition, using altered specificity Elongin C and F-box protein mutants, we investigate models for the mechanism underlying E2 selection by VHL and SCF ubiquitin ligases. Our findings provide evidence that E2 selection by VHL and SCF ubiquitin ligases is determined not solely by the Cullin/Rbx1 module, the target protein, or the integrity of the substrate recognition subunit but by yet to be elucidated features of these macromolecular complexes.
Yan
Qin
Q
Stowers Institute for Medical Research, Kansas City, Missouri 64110, USA.
Kamura
Takumi
T
Cai
Yong
Y
Jin
Jingji
J
Ivan
Mircea
M
Mushegian
Arcady
A
Conaway
Ronald C
RC
Conaway
Joan Weliky
JW
D000595:Amino Acid Sequence; D064173:Anaphase-Promoting Complex-Cyclosome; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D002460:Cell Line; D044842:Cullin Proteins / Q000737:chemistry* / Q000378:metabolism; D000075065:Elongin; D004952:Esters / Q000378:metabolism; D044783:F-Box Proteins / Q000737:chemistry; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D047468:Immunoprecipitation; D007313:Insecta; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D044786:S-Phase Kinase-Associated Proteins / Q000737:chemistry*; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000737:chemistry; D025801:Ubiquitin / Q000737:chemistry; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes / Q000737:chemistry; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry; D051794:Von Hippel-Lindau Tumor Suppressor Protein
D044842:Cullin Proteins; C000616395:ELOC protein, human; D000075065:Elongin; D004952:Esters; D044783:F-Box Proteins; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D011994:Recombinant Proteins; D044786:S-Phase Kinase-Associated Proteins; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; D025801:Ubiquitin; C577447:CDC34 protein, human; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome; D044767:Ubiquitin-Protein Ligases; D051794:Von Hippel-Lindau Tumor Suppressor Protein; C497446:VHL protein, human