Multiple roles for acetylation in the interaction of p300 HAT with ATF-2.
The transcriptional coactivator paralogues p300 and CBP contain acetyltransferase domains (HAT) and catalyze the lysine acetylation of histones and other proteins as an important aspect of their functions. Prior studies revealed that the basic leucine zipper domain (b-ZIP) of transcription factor ATF-2 (also called CRE-BP1) can interact with the CBP HAT domain. In this study, we have examined the ATF-2 b-ZIP interaction with the p300 HAT domain and shown that p300 HAT autoacetylation can enhance the binding affinity. Pull-down assays revealed that hyperacetylated p300 HAT is more efficiently retained by immobilized ATF-2 b-ZIP than hypoacetylated p300 HAT. Loop deleted p300 HAT lacking autoacetylation was retained about as well as hyperacetylated p300 HAT, suggesting that the loop and ATF-2 compete for p300 HAT binding. While ATF-2 b-ZIP is a weak inhibitor of hypoacetylated p300 HAT acetylation of a histone H4 peptide, hyperacetylated p300 HAT is much more potently inhibited by ATF-2 b-ZIP. Moreover, we showed that ATF-2 b-ZIP could serve as an acetyltransferase substrate for p300 HAT. Using mass spectrometry, two p300 HAT lysine acetylation sites were mapped in ATF-2 b-ZIP. Immunoprecipitation-Western blot analysis with anti-acetyl-lysine antibody revealed that ATF-2 can undergo reversible acetylation in vivo. Mutational analysis of the two ATF-2 b-ZIP acetylation sites revealed their potential contributions to ATF-2-mediated transcriptional activation. Taken together, these studies suggest multiple roles for protein acetylation in the regulation of transcription by p300/CBP and ATF-2.
Karanam
Balasubramanyam
B
Department of Pharmacology and Molecular Sciences, Johns Hopkins School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205, USA.
Wang
Ling
L
Wang
Dongxia
D
Liu
Xin
X
Marmorstein
Ronen
R
Cotter
Robert
R
Cole
Philip A
PA
D000107:Acetylation; D051698:Activating Transcription Factor 2 / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000378:metabolism*; D002522:Chlorocebus aethiops; D051548:Histone Acetyltransferases / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D016350:Leucine Zippers; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D014158:Transcription, Genetic; D050880:p300-CBP Transcription Factors; D000099224:p300-CBP-Associated Factor
D051698:Activating Transcription Factor 2; D018797:Cell Cycle Proteins; D051548:Histone Acetyltransferases; D014157:Transcription Factors; D050880:p300-CBP Transcription Factors; D000099224:p300-CBP-Associated Factor; C497105:ATF2 protein, human