Concerted Activation of ETS Protein ER81 by p160 Coactivators, the Acetyltransferase p300 and the Receptor Tyrosine Kinase HER2/Neu

Apollina Goel,Ralf Janknecht

doi:10.1074/jbc.m400036200

Abstract

Activator of thyroid and retinoic acid receptor (ACTR) is overexpressed in approximately 60% of primary human breast tumors and belongs to the p160 steroid receptor coactivator family. In this study, we identified a novel interaction partner of ACTR, the ETS transcription factor ER81 that is also heavily implicated in mammary tumor formation. ACTR and related p160 family members (steroid receptor coactivator-1 and glucocorticoid receptor-interacting protein-1 (GRIP-1)) augment ER81-mediated transcription. Although ACTR and GRIP-1 can acetylate ER81, this posttranslational modification of ER81 is not required for its stimulation by ACTR or GRIP-1. In addition, ACTR collaborates with the p300 coactivator, a joint interaction partner of ACTR and ER81, to stimulate ER81 function and the ability of p300 to acetylate ER81 is indispensable for this collaboration. Furthermore, the receptor tyrosine kinase HER2/Neu, an oncoprotein particularly found overexpressed in breast tumors, cooperates with both ACTR and p300 to stimulate ER81-mediated transcription. Thus, oncogenic HER2/Neu and ACTR may synergize to orchestrate mammary tumorigenesis through the dysregulation of the transcription factor ER81 and its target genes.

Highlights

The p160 steroid receptor coactivator (SRC)1 family gained much attention as pivotal coactivators for nuclear hormone receptors
SRCs Are Coactivators of the PEA3 Subfamily of ETS Proteins—To analyze whether the p160 family of SRCs can affect the function of the ETS protein ER81, we studied ER81-mediated gene transcription with a luciferase reporter construct driven by the promoter of the matrix metalloproteinase-1 (MMP-1) gene, an established target gene of ER81 [30, 47]
To further prove that p160 proteins are coactivators of ER81, we examined their role in the activation of the endogenous MMP-1 gene (Fig. 1B)

Summary

EXPERIMENTAL PROCEDURES

Plasmids—The mammalian expression vectors for ER81 and ER81K33R/K116R [37], SRC-1 [39], ACTR [3], p300-HA, and p300⌬HAT-HA (lacking amino acids 1430 –1504) [40], p300⌬SRC (lacking amino acids 2042–2157) [41], FLAG-P/CAF [42], HER2/Neu-V664E [43], GST-p300HAT [37], GST-GRIP-11158–1432 [12], HA-CARM1 [44], and HA-PRMT1 [44] were as reported. 50 ␮l of lysates from 293T cells transiently transfected with Myc6-ACTR (2.5 ␮g), HA-CARM1 (2.5 ␮g), or HA-PRMT1 (2.5 ␮g) expression plasmid were incubated for 2 h at 4 °C with approximately equimolar quantities of GST fusion proteins pre-bound to glutathione-agarose beads (Sigma) in 2.5 mM Tris-HCl, 7.5 mM Na4P2O7, 12.5 mM NaCl, 12.5 mM NaF, 1 mM DTT, and 0.25% Triton X-100, pH 7.1, containing protease inhibitor mixture. Coimmunoprecipitations—293T cells were transiently transfected with vectors for FLAG3-ER81 (1.5 ␮g) and 2.5 ␮g of full-length or various truncations of Myc6-ACTR and lysed in 2.5 mM Tris-HCl, 7.5 mM Na4P2O7, 12.5 mM NaCl, 12.5 mM NaF, and 0.25% Triton X-100, pH 7.1, supplemented with 10 ␮g/ml leupeptin, 2 ␮g/ml aprotinin, 1 ␮g/ml pepstatin A, 1 mM phenylmethylsulfonyl fluoride, 0.5 mM Na3VO4, 10 mM sodium butyrate, and 0.2 mM DTT. Whole cell lysates were prepared and assayed for luciferase activity essentially as described previously [46]

RESULTS

DISCUSSION

Apollina Goel and Ralf Janknecht