Mammalian mediator subunit mMED8 is an Elongin BC-interacting protein that can assemble with Cul2 and Rbx1 to reconstitute a ubiquitin ligase.
The heterodimeric Elongin BC complex has been shown to interact in vitro and in cells with a conserved BC-box motif found in an increasing number of proteins including RNA polymerase II elongation factor Elongin A, suppressor of cytokine signaling (SOCS)-box proteins, and the von Hippel-Lindau tumor suppressor protein. Recently, the Elongin BC complex was found to function as an adaptor that links these BC-box proteins to a module composed of Cullin family members Cul2 or Cul5 and RING-H2 finger protein Rbx1 to reconstitute a family of E3 ubiquitin ligases that activate ubiquitylation by the E2 ubiquitin-conjugating enzyme Ubc5. As part of our effort to understand the functions of Elongin BC-based ubiquitin ligases, we exploited a modified yeast two-hybrid screen to identify a mammalian BC-box protein similar in sequence to Saccharomyces cerevisiae Mediator subunit Med8p. In this report we demonstrate (i) that mammalian MED8 is a subunit of the mammalian Mediator complex and (ii) that MED8 can assemble with Elongins B and C, Cul2, and Rbx1 to reconstitute a ubiquitin ligase. Taken together, our findings are consistent with the model that MED8 could function to recruit ubiquitin ligase activity directly to the RNA polymerase II transcriptional machinery.
Brower
Christopher S
CS
Stowers Institute for Medical Research, Kansas City, MO 64110, USA.
Sato
Shigeo
S
Tomomori-Sato
Chieri
C
Kamura
Takumi
T
Pause
Arnim
A
Stearman
Robert
R
Klausner
Richard D
RD
Malik
Sohail
S
Lane
William S
WS
Sorokina
Irina
I
Roeder
Robert G
RG
Conaway
Joan Weliky
JW
Conaway
Ronald C
RC
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002460:Cell Line; D002461:Cell Line, Transformed; D044842:Cullin Proteins; D018076:DNA, Complementary; D019281:Dimerization; D000075065:Elongin; D005656:Fungal Proteins / Q000378:metabolism; D006801:Humans; D008025:Ligases / Q000235:genetics / Q000378:metabolism*; D008099:Liver / Q000378:metabolism; D008322:Mammals; D056892:Mediator Complex; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D051381:Rats; D029701:Saccharomyces cerevisiae Proteins; D018411:Spodoptera; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D051794:Von Hippel-Lindau Tumor Suppressor Protein
C105153:CUL2 protein, human; D018797:Cell Cycle Proteins; D044842:Cullin Proteins; D018076:DNA, Complementary; C000616397:ELOA protein, human; C000616403:Eloa protein, mouse; C000616404:Eloa protein, rat; D000075065:Elongin; D005656:Fungal Proteins; C465434:MED8 protein, rat; D056892:Mediator Complex; C113770:NUT2 protein, S cerevisiae; D009687:Nuclear Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; C489270:UBC5 protein, S cerevisiae; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D051794:Von Hippel-Lindau Tumor Suppressor Protein; D008025:Ligases; C497446:VHL protein, human