Human Cdc34 and Rad6B Ubiquitin-Conjugating Enzymes Target Repressors of Cyclic AMP-Induced Transcription for Proteolysis

Abstract

Ubiquitin-mediated proteolysis controls diverse physiological processes in eukaryotes. However, few in vivo targets of the mammalian Cdc34 and Rad6 ubiquitin-conjugating enzymes are known. A yeast-based genetic assay to identify proteins that interact with human Cdc34 resulted in three cDNAs encoding bZIP DNA binding motifs. Two of these interactants are repressors of cyclic AMP (cAMP)-induced transcription: hICERIIγ, a product of the CREM gene, and hATF5, a novel ATF homolog. Transfection assays with mammalian cells demonstrate both hCdc34- and hRad6B-dependent ubiquitin-mediated proteolysis of hICERIIγ and hATF5. This degradation requires an active ubiquitin-conjugating enzyme and results in abrogation of ICERIIγ- and ATF5-mediated repression of cAMP-induced transcription. Consistent with these results, the endogenous ICER protein is elevated in cells which are null for murine Rad6B (mHR6B^−/−) or transfected with dominant negative and antisense constructs of human CDC34. Based on the requirement for CREM/ICER and Rad6B proteins in spermatogenesis, we determined expression of Cdc34, Rad6B, CREM/ICER isoforms, and the Skp1–Cullin–F-box ubiquitin protein ligase subunits Cul-1 and Cul-2, which are associated with Cdc34 activity during murine testicular development. Cdc34, Rad6B, and the Cullin proteins are expressed in a developmentally regulated manner, with distinctly different patterns for Cdc34 and the Cullin proteins in germ cells. The Cdc34 and Rad6B proteins are significantly elevated in meiotic and postmeiotic haploid germ cells when chromatin modifications occur. Thus, the stability of specific mammalian 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.

ACKNOWLEDGMENTS

We are grateful to a large number of investigators who provided reagents for this study. We thank M. Vidal for yeast two-hybrid reagents; L. Prakash, P. Sassone-Corsi, G. McKnight, B. Kelly, D. Bohmann, M. Goebl, and B. Clurman for strains and plasmids; J. La Baer for the cDNA library; C. Molina, H. Roest, J. Hoeijmaker, P. Sassone-Corsi, and W. Krek for antisera; S. Sharan and A. Bradley for mouse tissues; J. Hoeijmaker and H. Roest for mHR6Brecombinant cells; G. Wilson for irradiated testes; G. Shetty for testes from jsd mice; Y. Zhang for elutriated germ cells; and S. Luo for assistance with the Northern analysis. We thank S. Elledge and V. Lundblad for comments on the manuscript and an anonymous reviewer for suggesting the experiments with the results shown in Fig.10.

This study was supported by the grants from the U.S. Army Medical Research and Material Command (DAMD-17-96-1-6087 to D.P.), the American Cancer Society (JFRA-559 to S.E.P.), and the National Institutes of Health (HD16843 to M.L.M.) and by Baylor Child Health Research Center grant P30-HD27832.