UBIQUITINYLATION OF TRANSCRIPTION FACTORS C-JUN AND C-FOS USING RECONSTITUTED UBIQUITINYLATING ENZYMES

Recombinant c-Jun and c-Fos were ubiquitinylated by the ubiquitin carr ier enzymes E2(14K), E2(20K), or E2(32K) in the presence of the ubiqui tin-activating enzyme, E1. Addition of ubiquitin protein ligase E3 sub stantially enhanced the E2(14K)-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 ubiquitinylat ion of c-Jun was inhibited upon the heterodimerization of c-Jun with c -Fos. Further addition of E2(20K) significantly enhanced ubiquitinylat ion of c-Jun in the heterodimer suggesting a regulatory role of E2(20K ). Polyubiquitinylated c-Jun, wbFos, and wbJun, but not E2(20K)-ubiqui tinylated c-Jun, were readily degraded by the ATP-dependent 26 S multi catalytic proteases. These results suggest that the temporal control o f c-Jun and c-Fos may be regulated through the ubiquitinylation pathwa ys, and the ubiquitinylation of c-Jun and c-Fos may in turn be regulat ed in response to the heterodimerization between them and the cooperat ion between E2(20K) and E3 mediated polyubiquitinylation.