Transcriptional coregulator SNURF (RNF4) possesses ubiquitin E3 ligase activity.
SNURF/RNF4 has been implicated in transcriptional regulation and growth inhibition in a RING finger-dependent fashion. In this work, we show that SNURF mediates its own ubiquitination in vitro in a ubiquitin-conjugating enzyme (E2)-selective manner: SNURF acts as an E3 ligase with UbcH5A and B, HHR6B (RAD6B), E2-25K, MmUbc7 and UbcH13, but not with UbcH3, UbcM4, MmUbc6 or E2-20K. In contrast, the well-characterized RING E3, AO7, functions only with members of the UbcH5 family. Furthermore, depending on the E2 used, the ubiquitin modification manifests as mono- or multi-ubiquitination. Mutation of conserved cysteine residues within the RING finger motif of SNURF abolishes the ubiquitination in vitro and in intact cells. Size fractionation of murine embryonal carcinoma F9 cell proteins shows that the majority of endogenous SNURF resides in salt-resistant > or =500-kDa complexes, suggesting that SNURF functions as a RING component in a multiprotein complex. Taken together, SNURF/RNF4 functions as an E3 ligase and this activity is closely linked to its transcription regulatory functions.
Häkli
M
M
Biomedicum Helsinki, Institute of Biomedicine, University of Helsinki, FIN-00014 Helsinki, Finland.
Lorick
K L
KL
Weissman
A M
AM
Jänne
O A
OA
Palvimo
J J
JJ
D020816:Amino Acid Motifs; D000818:Animals; D019556:COS Cells; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D002850:Chromatography, Gel; D003545:Cysteine / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D008025:Ligases / Q000235:genetics* / Q000378:metabolism*; D051379:Mice; D017354:Point Mutation; D011233:Precipitin Tests; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D018243:Teratocarcinoma; D014158:Transcription, Genetic; D014162:Transfection; D025801:Ubiquitin / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases
D011993:Recombinant Fusion Proteins; D025801:Ubiquitin; D044767:Ubiquitin-Protein Ligases; D008025:Ligases; D005978:Glutathione; D003545:Cysteine