Human Cdc34 and Rad6B ubiquitin-conjugating enzymes target repressors of cyclic AMP-induced transcription for proteolysis

Ubiquitin-mediated proteolysis controls diverse physiological processes in eukaryotes. However, few in vivo targets of the mammalian Cdc34 and Rad6 ub iquitin-conjugating enzymes are known. A yeast-based genetic assay to ident ify proteins that interact with human Cdc34 resulted in three cDNAs encodin g bZIP DNA binding motifs. Two of these interactants are repressors of cycl ic AMP (cAMP)-induced transcription: hICERII gamma, a product of the CREM g ene, and hATF5, a novel ATF homolog. Transfection assays with mammalian cel ls demonstrate both hCdc34- and hRad6B-dependent ubiquitin-mediated proteol ysis of hICERII gamma and hATF5. This degradation requires an active ubiqui tin-conjugating enzyme and results in abrogation of ICERII gamma- and ATP5- mediated repression of cAMP-induced transcription. Consistent with these re sults, the endogenous ICER protein is elevated in cells which are null for murine Rad6B (mHR6B(-/-)) or transfected with dominant negative and antisen se constructs of human CDC34. Based on the requirement for CREM/ICER and Ra d6B proteins in spermatogenesis, we determined expression of Cdc34, Rad6B, CREM/ICER isoforms, and the Skp1-Cullin-F-box ubiquitin protein ligase subu nits Cul-1 and Cul-2, which are associated with Cdc34 activity during murin e testicular development. Cdc34, Rad6B, and the Cullin proteins are express ed in a developmentally regulated manner, with distinctly different pattern s for Cdc34 and the Cullin proteins in germ cells. The Cdc34 and Rad6B prot eins are significantly elevated in meiotic and postmeiotic haploid germ cel ls when chromatin modifications occur. Thus, the stability of specific mamm alian transcription factors is the result of complex targeting by multiple ubiquitin-conjugating enzymes and may have an impact on cAMP-inducible gene regulation during both meiotic and mitotic cell cycles.