Association of activating transcription factor 2 (ATF2) with the ubiquitin-conjugating enzyme hUBC9. Implication of the ubiquitin/proteasome pathway in regulation of ATF2 in T cells.
Activating transcription factor 2 (ATF2) is regulated by phosphorylation via the Jun N-terminal kinase, and its binding activity is markedly induced at late stages of T and B lymphocyte activation (Feuerstein, N., Firestein, R., Aiyer, N., Xiao, H., Murasko, D., and Cristofalo, V. (1996) J. Immunol. 156, 4582-4593). To identify proteins that interact specifically with ATF2 in lymphocytes, the yeast two-hybrid interaction system was employed using ATF2 cDNA as a "bait." In two separate screenings, a clone was identified that revealed a novel sequence with homology to several members of the ubiquitin-conjugating enzyme family. An identical sequence was recently reported as the human homolog of the yeast UBC9, hUBC9. Northern blot analysis revealed a 1.3-kilobase RNA transcript, which showed differential levels of expression in various human tissues and a moderate induction after a 48-h stimulation of peripheral blood T lymphocytes. An antibody that was generated against the bacterially expressed glutathione S-transferase-hUBC9 detected a approximately 19-kDa protein, which localizes predominantly in the nuclei of T cells. Further quantitative assays using the yeast two-hybrid system confirmed a high and specific level of interaction of hUBC9 with ATF2 and lack of interaction with lamin or control vectors. Two other cyclic AMP-responsive element-binding transcription factors, CREB and ATF1, also showed significant levels of interaction with hUBC9. However, this interaction was severalfold lower as compared with ATF2. Far Western blot analysis confirmed the specific binding of ATF2 and hUBC9 also in vitro. Evidence is presented that indicates a physiological significance for the interaction of hUBC9 with ATF2. (a) We show that ATF2 is ubiquitinated in vivo and in vitro, and (b) ATF2 ubiquitination in vitro is facilitated by addition of purified hUBC9. (c) ATF2 is shown to undergo a proteolytic process, which is rapidly regulated upon T cell activation concomitant with induction of ATF2 phosphorylation. (d) A proteasome inhibitor delays the down-regulation of ATF2 phophorylation after T cell activation. Taken collectively, these results implicate a role for hUBC9 and the ubiquitin/proteasome pathway in regulation of ATF2 in T cells.
Firestein
R
R
Center for Gerontology, Allegheny University of the Health Sciences and the Division of Rheumatology, Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
Feuerstein
N
N
D051697:Activating Transcription Factor 1; D051698:Activating Transcription Factor 2; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000502:physiology; D004268:DNA-Binding Proteins; D006801:Humans; D034882:Lamins; D008025:Ligases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000502:physiology; D009687:Nuclear Proteins / Q000032:analysis / Q000378:metabolism; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex; D011485:Protein Binding / Q000502:physiology; D012333:RNA, Messenger / Q000032:analysis; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013601:T-Lymphocytes / Q000502:physiology; D014157:Transcription Factors / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes; D014452:Ubiquitins / Q000502:physiology; D000098843:Ubiquitin-Conjugating Enzyme UBC9
C497105:ATF2 protein, human; D051697:Activating Transcription Factor 1; D051698:Activating Transcription Factor 2; D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D034882:Lamins; D009097:Multienzyme Complexes; D009687:Nuclear Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D014157:Transcription Factors; D014452:Ubiquitins; D044763:Ubiquitin-Conjugating Enzymes; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D008025:Ligases; D000098843:Ubiquitin-Conjugating Enzyme UBC9