Acetylation-mediated transcriptional activation of the ETS protein ER81 by p300, P/CAF, and HER2/Neu - PubMed (original) (raw)
Acetylation-mediated transcriptional activation of the ETS protein ER81 by p300, P/CAF, and HER2/Neu
Apollina Goel et al. Mol Cell Biol. 2003 Sep.
Abstract
The regulated expression of the ETS transcription factor ER81 is a prerequisite for normal development, and its dysregulation contributes to neoplasia. Here, we demonstrate that ER81 is acetylated by two coactivators/acetyltransferases, p300 and p300- and CBP-associated factor (P/CAF) in vitro and in vivo. Whereas p300 acetylates two lysine residues (K33 and K116) within the ER81 N-terminal transactivation domain, P/CAF targets only K116. Acetylation of ER81 not only enhances its ability to transactivate but also increases its DNA binding activity and in vivo half-life. Furthermore, oncogenic HER2/Neu, which induces phosphorylation and thereby activation of ER81, was less able to activate acetylation-deficient ER81 mutants, indicating that both acetyltransferase and protein kinase-specific regulatory mechanisms control ER81 activity. Importantly, HER2/Neu overexpression stimulates the ability of p300 to acetylate ER81, likely by inducing phosphorylation of p300 through the Ras-->Raf-->mitogen-activated protein kinase pathway. This represents a novel mechanism by which oncogenic HER2/Neu, Ras, or Raf may promote tumor formation by enhancing acetylation not only of ER81 but also of other downstream effector transcription factors as well as histones.
Figures
FIG. 1.
Augmentation of ER81-mediated transcription by HDAC inhibitors and p300. (A) Mv1Lu cells were cotransfected with the TORU-luc reporter construct and either 0.5 μg of CMV-ER812-477 or empty expression vector pEV3S. Cells were treated with sodium butyrate (NaB) or TSA for 12 h prior to lysis. (B) Detection of MMP-1 and GAPDH gene expression in 293T cells by RT-PCR. Cells were transiently transfected with either 0.5 μg of CMV-ER812-477 or pEV3S and treated with the indicated HDAC inhibitors 12 h prior to lysis. (C) CV-1 cells were cotransfected with TORU-luc, CMV-ER812-477, or pEV3S vector (0.2 μg) and p300 or p300Δ1430-1504 (0.4 μg) as indicated. (D) 293T cell extracts were immunoprecipitated with no antibody or protein A-coupled anti-GAL4 or antiacetyllysine antibodies (06-933; Upstate Biotechnology). Subsequently, immunoblotting was performed with anti-ER81 antibodies (anti-ETV-1 C20; Santa Cruz Biotechnology). Input levels of ER81 were detected by immunoblotting ER81 immunoprecipitated with anti-ETV-1 antibodies. Where indicated, cells were treated with sodium butyrate or cotransfected with 1 μg of the HDAC3 expression plasmid.
FIG. 2.
ER81 is acetylated by p300. (A) 293T cells were cotransfected with Myc-tagged ER81 without or with the p300-HA expression plasmid. Immunoprecipitations were performed with either a control antibody or antiacetyllysine antibodies (AcK 1, PAN-AC1 and Abcam; AcK 2, 06-933; Upstate Biotechnolgy), followed by anti-Myc Western blotting (top). ER81 protein levels are shown by anti-Myc Western blotting (bottom). (B) Antiacetyllysine immunoprecipitations similar to those in panel A in the presence of Flag-HDAC3. (C) In vitro acetylation assays. Various GST-ER81 chimeras were incubated with p300-HAT and [14C]acetyl coenzyme A and then separated by SDS-PAGE. Acetylated ER81 was detected by autoradiography. Asterisk, autoacetylated p300 HAT. (Bottom) Coomassie blue-stained protein gel with purified recombinant proteins. AD, activation domain; ID, inhibitory domain; ETS, DNA binding domain of ER81.
FIG. 3.
Lysines 33 and 116 of ER81 are acetylated by p300. (A) All lysine residues in the region between amino acids 1 to 210 of ER81 (asterisks). (B) In vitro acetylation assays of the indicated GST-ER81 mutant proteins. (C) In vitro acetylation of ER812-477. (D) 293T cells were cotransfected with wild-type Myc-tagged ER812-477 or the indicated mutant versions (0.5 μg) and the p300-HA expression plasmid (3 μg). Immunoprecipitations were performed with the antiacetyllysine antibody (06-933; Upstate Biotechnology) as detailed in the legend for Fig. 2A.
FIG. 4.
ER81 interacts with P/CAF in vivo and in vitro. (A) Coimmunoprecipitations (IP) were done with lysates of 293T cells transfected with Myc-tagged ER812-477 and either p300-HA or Flag-P/CAF. ER81 was detected by anti-Myc Western blotting. (B) GST pull-down assays were carried out by incubating whole-cell extract of 293T cells expressing Flag-P/CAF with glutathione-agarose beads prebound to control GST protein, GST-ER811-210, or GST-ER812-477. (C) Coimmunoprecipitation of endogenous P/CAF and ER81. 293T cell extracts were immunoprecipitated with the indicated antibodies and P/CAF was revealed with anti-P/CAF antibodies (kindly provided by Yoshihiro Nakatani). (D) Mapping the ER81 domain responsible for interaction with P/CAF. Immunoprecipitations were performed with anti-Flag antibodies by using lysates of 293T cells transfected with Flag-P/CAF and various Myc-tagged ER81 truncations (middle). The expression levels of ER81 proteins were assessed by anti-Myc Western blotting (bottom). IgH, immunoglobulin H; AD, ID, and ETS are as defined for Fig. 2.
FIG. 5.
P/CAF stimulates transcriptional activity of ER81 and acetylates K116. (A) CV-1 cells were cotransfected with TORU-luc, CMV-ER812-477 or pEV3S vector (0.2 μg), p300-HA (0.4 μg), and Flag-P/CAF or Flag-P/CAFΔ497-526 (0.5 μg) as indicated. The resulting luciferase activities were measured. (B) Anti-Flag Western blot of immunoprecipitated Flag-P/CAF and Flag-P/CAFΔ497-526 expressed in CV-1 cells. (C) Expression of MMP-1 and GAPDH assessed by RT-PCR in 293T cells transiently transfected with 0.2 μg of CMV-ER812-477 or empty vector pEV3S and 0.5 μg of the indicated wild-type or mutated cofactors. (D) In vitro acetylation assays were performed with various GST-ER81 fusion proteins and the HAT domain of P/CAF. (E) In vitro acetylation assay of wild-type or mutated GST-ER8163-210 similar to those of panel D. (F) 293T cells were cotransfected with wild-type Myc-tagged ER81 or indicated mutant versions and the P/CAF-Flag expression plasmid. Immunoprecipitations were performed with either the control or antiacetyllysine antibody (06-933; Upstate Biotechnology), followed by anti-Myc Western blotting (top). The input levels of ER81 are shown at the bottom.
FIG. 6.
Acetylation of K116 potentiates DNA binding of ER81. Lysates were prepared from 293T cells transiently transfected with either wild-type or mutant CMV-ER812-477 and with or without p300-HA. The 32P-labeled E74 oligonucleotide was used to perform electrophoretic mobility shift assays. The lysates were normalized for equal expression of ER81 protein by anti-ER81 (anti-ETV-1 C20; Santa Cruz Biotechnology) Western blotting (bottom). (Right) Proof of the identity of ER81 by supershift experiments utilizing anti-ER81 or control anti-GAL4 antibodies.
FIG. 7.
Acetylation-mediated stabilization of ER81. Pulse-chase experiments were performed with 293T cells transfected with Myc-tagged ER81 plus either p300-HA or Flag-P/CAF (A) or Myc-tagged ER81 mutant proteins K33R, K116R, and K33/116R without (B) or with (C) p300-HA. Cells were pulsed with [35S]methionine and chased for up to 8 h, followed by immunoprecipitations with anti-Myc antibodies. The relative amount of radioactive ER81 (logarithmic scale) is plotted as a function of time with the signal at time zero set to 100%. The results of three independent experiments are shown.
FIG. 8.
Role of K33 and K116 acetylation in p300- and P/CAF-mediated activation of ER81. Wild-type ER81 or the indicated mutant proteins were cotransfected into CV-1 cells either as Gal4-ER811-210 constructs (0.2 μg) with the Gal42-tk80-luc luciferase reporter (A) or as CMV-ER812-477 expression vectors (0.2 μg) with TORU-luc (B). p300 (0.4 μg) and P/CAF (0.5 μg) were coexpressed as indicated, and the resulting luciferase activities were measured. (C) RT-PCR analysis of MMP-1 gene transcription in 293T cells. Where indicated, wild-type or acetylation-deficient ER81 mutant proteins (0.2 μg) were coexpressed in the presence of p300 or P/CAF (0.5 μg).
FIG. 9.
ER81 mutant proteins K33 and K116 show impaired HER2/Neu-stimulated transactivation. (A) ER81 activity measured in CV-1 cells with the TORU-luc reporter. Cells were transiently transfected with wild-type or mutant CMV-ER812-477 (1 μg) and 0.5 μg of HER2/Neu-V664E. (B) One microgram of wild-type or mutated CMV-ER812-477 expression vectors was cotransfected with the MMP-1 luc reporter and HER2/Neu-V664E (0.5 μg) as indicated, and resulting luciferase activities in RK13 cells were measured.
FIG. 10.
HER2/Neu potentiates p300 HAT activity. (A) 293T cells were transfected with p300-HA (1 μg) with or without HER2/Neu-V664E (1 μg). Immunoprecipitations were performed with anti-HA antibodies, and products were used either directly or after phosphatase treatment for in vitro acetylation of GST, GST-ER812-477, GST-ER811-249, or histones (upper left). The expression levels of p300 were compared by anti-HA Western blotting (lower left). Changes in HAT activity were determined by measuring the incorporation of radioactivity with a PhosphorImager normalized to densitometric determination of p300 protein amount and are graphically depicted on the right. (B) In vivo phosphorylation of p300. Mv1Lu cells were transfected with 4 μg of p300-HA and 0.5 μg of HER2/Neu-V664E as indicated. After in vivo labeling with 32Pi, p300-HA was immunoprecipitated and incorporation of radioactivity was assessed by autoradiography. (Right) Corresponding anti-HA immunoblot. (C) MMP-1 gene expression evaluated by RT-PCR. 293T cells were transfected with 0.5 μg of CMV-ER812-477 or empty vector pEV3S, 0.4 μg of p300 or p300Δ1430-1504, and 1 μg of HER2/Neu-V664E as indicated. (D) 293T cells were transfected with p300-HA (1 μg), HER2/Neu-V664E (1 μg), and either wild-type Myc-tagged ER812-477 or the indicated mutant versions (0.5 μg). Immunoprecipitations were performed with the antiacetyllysine antibody (06-933; Upstate Biotechnology), followed by Western blotting with anti-Myc antibodies (top). Input levels of Myc-tagged ER81 proteins are shown at the bottom. (E) 293T cells were transfected with or without p300-HA (1 μg) and with or without HER2/Neu-V664E (1 μg), H-Ras-G12V (0.05 μg), or BXB (0.5 μg). Equal amounts of p300 immunoprecipitated with anti-HA antibodies were used for in vitro acetylation of GST-ER812-477 (top) or histones (bottom).
FIG. 11.
Model for ER81 as a target of HER2/Neu-induced acetylation and phosphorylation events. MAPKAPK, MAPK-activated protein kinase.
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