Kinetic and mass spectrometric analysis of p300 histone acetyltransferase domain autoacetylation.
Acetylation of proteins by p300 histone acetyltransferase plays a critical role in the regulation of gene expression. The prior discovery of an autoacetylated regulatory loop in the p300 histone acetyltransferase (HAT) domain prompted us to further explore the mechanisms of p300 autoacetylation. Here we have described a kinetic and mass spectrometric analysis of p300 HAT autoacetylation. The rate of p300 HAT autoacetylation was approximately fourth order with respect to p300 HAT domain concentration and thus appeared to be a highly cooperative process. By showing that a catalytically defective p300 HAT domain could be efficiently acetylated by active p300 HAT, we deduced that autoacetylation occurs primarily by an intermolecular mechanism. This was further confirmed using a semisynthetic biotinylated p300 HAT domain that could be physically separated from the catalytically defective p300 HAT by avidin affinity chromatography. Autoacetylation catalyzed by p300 HAT was approximately 1000-fold more efficient than PCAF (p300/CREB-binding protein-associated factor)-mediated acetylation of catalytically defective p300 HAT. Using a novel tandem mass spectrometric approach, it was found to be possible to observe up to 17 autoacetylation events within the intact p300 regulatory loop. Kinetic analysis of the site specificity of p300 autoacetylation reveals a class of rapid events followed by a slower set of modifications. Several of these rapid autoacetylation sites correlate with an acetyltransferase-activating function based on prior mutagenesis analysis.
Karanam
Balasubramanyam
B
Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Jiang
Lihua
L
Wang
Ling
L
Kelleher
Neil L
NL
Cole
Philip A
PA
D000107:Acetylation; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D019921:Biotinylation; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D005583:Fourier Analysis; D051548:Histone Acetyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D017384:Sequence Deletion; D021241:Spectrometry, Mass, Electrospray Ionization; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D050880:p300-CBP Transcription Factors; D000099224:p300-CBP-Associated Factor
D001426:Bacterial Proteins; D018797:Cell Cycle Proteins; D051548:Histone Acetyltransferases; D010455:Peptides; D014157:Transcription Factors; D050880:p300-CBP Transcription Factors; D000099224:p300-CBP-Associated Factor