Ubiquitinylation of transcription factors c-Jun and c-Fos using reconstituted ubiquitinylating enzymes.
Recombinant c-Jun and c-Fos were ubiquitinylated by the ubiquitin carrier enzymes E214K, E220K, or E232K in the presence of the ubiquitin-activating enzyme, E1. Addition of ubiquitin protein ligase E3 substantially enhanced the E214K-mediated ubiquitinylation of c-Jun and c-Fos. Truncated c-Jun and c-Fos mutant proteins including wbJun and wbFos were also ubiquitinylated under the same conditions, suggesting the sites of ubiquitinylation are located within the dimerization and DNA binding domains of c-Jun and c-Fos. The E3-dependent ubiquitinylation of c-Jun was inhibited upon the heterodimerization of c-Jun with c-Fos. Further addition of E220K significantly enhanced ubiquitinylation of c-Jun in the heterodimer suggesting a regulatory role of E220K. Polyubiquitinylated c-Jun, wbFos, and wbJun, but not E220K-ubiquitinylated c-Jun, were readily degraded by the ATP-dependent 26 S multicatalytic proteases. These results suggest that the temporal control of c-Jun and c-Fos may be regulated through the ubiquitinylation pathways, and the ubiquitinylation of c-Jun and c-Fos may in turn be regulated in response to the heterodimerization between them and the cooperation between E220K and E3 mediated polyubiquitinylation.
Hermida-Matsumoto
M L
ML
Department of Chemistry, Georgetown University, Washington, D. C. 20057, USA.
Chock
P B
PB
Curran
T
T
Yang
D C
DC
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D003546:Cysteine Endopeptidases / Q000378:metabolism; D011756:Diphosphates / Q000378:metabolism; D006801:Humans; D008025:Ligases / Q000302:isolation & purification / Q000378:metabolism*; D046911:Macromolecular Substances; D009097:Multienzyme Complexes / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D016760:Proto-Oncogene Proteins c-fos / Q000302:isolation & purification / Q000378:metabolism*; D016755:Proto-Oncogene Proteins c-jun / Q000302:isolation & purification / Q000378:metabolism*; D011817:Rabbits; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism*; D012156:Reticulocytes / Q000378:metabolism; D013379:Substrate Specificity; D014157:Transcription Factors / Q000378:metabolism; D044764:Ubiquitin-Activating Enzymes; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000378:metabolism*
D011756:Diphosphates; D046911:Macromolecular Substances; D009097:Multienzyme Complexes; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D011994:Recombinant Proteins; D014157:Transcription Factors; D014452:Ubiquitins; D000255:Adenosine Triphosphate; D044767:Ubiquitin-Protein Ligases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D008025:Ligases; D044764:Ubiquitin-Activating Enzymes