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.