The ATF3 Transcription Factor Is a Short-Lived Substrate of the Arg/N-Degron Pathway.

The Arg/N-degron pathway targets proteins for degradation by recognizing their specific N-terminal residues or, alternatively, their non-N-terminal degrons. In mammals, this pathway is mediated by the UBR1, UBR2, UBR4, and UBR5 E3 ubiquitin ligases, and by the p62 regulator of autophagy. UBR1 and UBR2 are sequelogous, functionally overlapping, and dominate the targeting of Arg/N-degron substrates in examined cell lines. We constructed, here, mouse strains in which the double mutant [UBR1(-/-)UBR2(-/-)] genotype can be induced conditionally, in adult mice. We also constructed human [UBR1(-/-)UBR2(-/-)] HEK293T cell lines that unconditionally lack UBR1/UBR2. ATF3 is a basic leucine zipper transcription factor that regulates hundreds of genes and can act as either a repressor or an activator of transcription. Using the above double-mutant mice and human cells, we found that the levels of endogenous, untagged ATF3 were significantly higher in both of these [UBR1(-/-)UBR2(-/-)] settings than in wild-type cells. We also show, through chase-degradation assays with [UBR1(-/-)UBR2(-/-)] and wild-type human cells, that the Arg/N-degron pathway mediates a large fraction of ATF3 degradation. Furthermore, we used split-ubiquitin and another protein interaction assay to detect the binding of ATF3 to both UBR1 and UBR2, in agreement with the UBR1/UBR2-mediated degradation of endogenous ATF3. Full-length 24 kDa ATF3 binds to similar to 100 kDa fragments of 200 kDa UBR1 and UBR2 but does not bind (in the setting of interaction assays) to full-length UBR1/UBR2. These and other binding patterns, whose mechanics remain to be understood, may signify a conditional (regulated) degradation of ATF3 by the Arg/N-degron pathway.