P300 Modulates ATF4 Stability and Transcriptional Activity Independently of Its Acetyltransferase Domain

Irina Lassot,Emilie Estrabaud,Stephane Emiliani,Monsef Benkirane,Richard Benarous,Florence Margottin-Goguet

doi:10.1074/jbc.m505294200

Abstract

ATF4 plays a crucial role in the cellular response to stress and multiple stress responses pathways converge to the translational up-regulation of ATF4. ATF4 is a substrate of the SCF(betaTrCP) ubiquitin ligase that binds to betaTrCP through phosphorylation on a DSGXXXS motif. We show here that ATF4 stability is also modulated by the histone acetyltransferase p300, which induces ATF4 stabilization by inhibiting its ubiquitination. Despite p300 acetylates ATF4, we found that p300-mediated ATF4 stabilization is independent of p300 catalytic activity, using either the inactive form of p300 or the acetylation mutant ATF4-K311R. ATF4 deleted of its p300 binding domain is no more stabilized by p300 nor recruited into nuclear speckles. In consequence of ATF4 stabilization, both p300 and the catalytically inactive enzyme increase ATF4 transcriptional activity.

Highlights

Tional selectivity of ATF4 is modulated by the formation of heterodimers with multiple C/EBP bZIP or AP-1 family members [12,13,14]
We have shown that ATF4 degradation is mediated by the E3 ubiquitin ligase SCF␤TrCP [21] that we first identified as the E3 ubiquitin ligase responsible for the degradation of CD4 induced by the human immunodeficiency virus type 1 protein Vpu [22]
In this work we show that the expression of ATF4 can be regulated at the level of its stability by the acetylase p300

Summary

MATERIALS AND METHODS

Plasmid Construction and Mutagenesis—Vectors for FLAG-P/CAF, FLAG-p300, and FLAG-p300⌬HAT were kindly provided by Y. Cells were washed in phosphatebuffered saline, resuspended in lysis buffer (120 mM NaCl, 50 mM Tris, pH 7.5, 0.5% Triton, pH 8, 1 mM dithiothreitol, 1 mM EDTA, 400 nM trichostatin A, protease inhibitors), and immunoprecipitated using anti-HA antibody (12CA5, Roche Applied Science). HeLa cells were fixed, permeabilized, and incubated with anti-FLAG (M2, Sigma), anti-RNAPollII (8WG16, BabCO), or anti-PML (PG-M3, Santa Cruz Biotechnology) antibodies, washed in phosphate-buffered saline, and incubated with anti-mouse Cy3 antibodies (Jackson ImmunoResearch), or directly incubated with anti-HA-fluorescein isothiocyanate antibody (3F10, Roche Applied Science). Cells were co-transfected with 1 ␮g of 1XAARE-LUC, 30 ng of pRL-TK-Renilla (PRL-TK from Promega), and various amounts of plasmids expressing HA-ATF4 or HA-ATF4 mutants, FLAG-p300 and FLAG-p300⌬HAT, or siRNA against p300 as indicated.

RESULTS

DISCUSSION