Kinetic and Mass Spectrometric Analysis of p300 Histone Acetyltransferase Domain Autoacetylation

Balasubramanyam Karanam,Lihua Jiang,Ling Wang,Neil L Kelleher,Philip A Cole

doi:10.1074/jbc.m608813200

Abstract

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.

Highlights

Tones and other proteins [1]
A recombinant N-terminal thioester fragment is produced by intein fusion, and it is ligated via a chemoselective reaction to a C-terminal peptide fragment to produce the semisynthetic p300 histone acetyltransferase (HAT) protein
Intramolecular, or both? If intermolecular, can the p300-associated HAT PCAF catalyze p300 acetylation? Are there preferences for the order of sites of autoacetylation, or is it purely stochastic? Here we have investigated the biochemistry of p300 HAT domain autoacetylation to attempt to address these issues

Summary

Introduction

Tones and other proteins [1]. Among the HAT families, p300 and its paralog, CREB-binding protein (CBP), have been identified as major enzymes in the acetylation of a myriad of protein targets with a range of biological functions [2, 3]. By Lys-CoA [28], varying concentrations of Lys-CoA (0 –2 ␮M) were used with fixed p300 HAT domain (40 nM) and acetyl-CoA (20 ␮M) for 30 s. For the electrospray ionization quantitative-Fourier transform mass spectrometry (FTMS) mapping experiments, wt p300 HAT (50 –58 ␮M) along with acetyl-CoA (1.6 or 6 mM) were incubated in reaction buffer (20 mM sodium HEPES, pH 7.9, 200 mM NaCl, 10 mM dithiothreitol) for the time indicated and quenched with 1% aqueous trifluoroacetic acid.

Results

Conclusion