Functional analysis of the p300 acetyltransferase domain: the PHD finger of p300 but not of CBP is dispensable for enzymatic activity.
Acetylation of nucleosomal histones is a major regulatory step during activation of eukaryotic gene expression. Among the known acetyltransferase (AT) families, the structure-function relationship of the GNAT superfamily is the most well understood. In contrast, less information is available regarding mechanistic and regulatory aspects of p300/CBP AT function. In this paper, we investigate in closer detail the structure and sequence requirements for p300/CBP enzymatic activity. Unexpectedly, we find that the PHD finger of p300, but not of CBP, is dispensable for AT activity. In order to identify residues involved in substrate or acetyl-coenzyme A (acetyl-CoA) recognition, we have introduced 19 different amino acid substitutions in segments that are highly conserved between animal and plant p300/CBP proteins. By performing acetylation reactions with histones, a p53 peptide or the AT domain itself, we define several residues required for histone and p53 substrate recruitment but not for acetyl-CoA binding. Finally, we show that identical mutations in the p300 and CBP AT domain impair AT activity differently. This latter result combined with the finding of a differential requirement for the PHD finger provides evidence for structural differences between p300 and CBP that may in part underlie a previously reported functional specialization of the two proteins.
Bordoli
L
L
Institute for Molecular Biology, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.
Hüsser
S
S
Lüthi
U
U
Netsch
M
M
Osmani
H
H
Eckner
R
R
D000105:Acetyl Coenzyme A / Q000378:metabolism; D000107:Acetylation; D000123:Acetyltransferases / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics*; D050882:CREB-Binding Protein; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002460:Cell Line; D017124:Conserved Sequence; D051548:Histone Acetyltransferases; D006657:Histones / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010618:Phenanthrolines / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017384:Sequence Deletion / Q000235:genetics; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D015534:Trans-Activators / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D016335:Zinc Fingers; D050880:p300-CBP Transcription Factors; D000099224:p300-CBP-Associated Factor
D000105:Acetyl Coenzyme A; D000123:Acetyltransferases; D050882:CREB-Binding Protein; D018797:Cell Cycle Proteins; D051548:Histone Acetyltransferases; D006657:Histones; D009687:Nuclear Proteins; D010446:Peptide Fragments; D010618:Phenanthrolines; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D016159:Tumor Suppressor Protein p53; D050880:p300-CBP Transcription Factors; D000099224:p300-CBP-Associated Factor; C494363:CREBBP protein, human; C025205:1,10-phenanthroline