Model for ER81 as a target of HER2/Neu-induced acetylation and phosphorylation events. MAPKAPK, MAPK-activated protein kinase.

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-ER81_2-477 (1 μg) and 0.5 μg of HER2/Neu-V664E. (B) One microgram of wild-type or mutated CMV-ER81_2-477 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.

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-ER81_1-210 constructs (0.2 μg) with the Gal4₂-tk80-luc luciferase reporter (A) or as CMV-ER81_2-477 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).

Acetylation of K116 potentiates DNA binding of ER81. Lysates were prepared from 293T cells transiently transfected with either wild-type or mutant CMV-ER81_2-477 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.

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 ER81_2-477. (D) 293T cells were cotransfected with wild-type Myc-tagged ER81_2-477 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.

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.

ER81 interacts with P/CAF in vivo and in vitro. (A) Coimmunoprecipitations (IP) were done with lysates of 293T cells transfected with Myc-tagged ER81_2-477 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-ER81_1-210, or GST-ER81_2-477. (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.

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.

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-ER81_2-477 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-ER81_2-477 or pEV3S and treated with the indicated HDAC inhibitors 12 h prior to lysis. (C) CV-1 cells were cotransfected with TORU-luc, CMV-ER81_2-477, 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.

P/CAF stimulates transcriptional activity of ER81 and acetylates K116. (A) CV-1 cells were cotransfected with TORU-luc, CMV-ER81_2-477 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-ER81_2-477 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-ER81_63-210 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.

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-ER81_2-477, GST-ER81_1-249, 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 ³²P_i, 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-ER81_2-477 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 ER81_2-477 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-ER81_2-477 (top) or histones (bottom).