HER2/Neu-mediated activation of the ETS transcription factor ER81 and its target gene MMP-1

Abstract

In this study, we show that the ETS transcription factor ER81 directly binds to and activates the promoter of the matrix metalloproteinase gene, MMP-1. Further, the oncoprotein HER2/Neu synergizes with ER81 to stimulate MMP-1 transcription. The activation of ER81 by HER2/Neu is mediated by MAP kinases, which phosphorylate ER81 in its N-terminal activation domain. Four respective phosphorylation sites have been identified. Blocking phosphorylation at these sites decreases ER81 transcriptional activity, which can be further diminished by abolishment of phosphorylation at two non-MAP kinase sites. Altogether, our results reveal mechanisms of how phosphorylation of ER81 regulates the expression of target genes such as MMP-1, which may be important for many physiological processes from embryogenesis to adulthood as well as for tumor metastasis.

HER2/Neu- and TAK1-mediated Up-regulation of the Transforming Growth Factor β Inhibitor Smad7 via the ETS Protein ER81

“…This suggests that DNA binding of ER81 is intramolecularly inhibited by N-terminal amino acids that contain its phosphorylation sites, similar to what has been observed for the related ETS protein PEA3 (65,66). HER2/Neu induces the phosphorylation of ER81 within its N terminus (42) and may thereby alleviate this intramolecular repression.…”

“…However, ER81 readily associated with the ETS2/3 probe containing the closely juxtaposed ETS2 and ETS3 sites, and an anti-ER81 antibody elicited a respective supershift formation. Interestingly, much more DNA binding was observable in the presence of the anti-ER81 antibody that probably stabilizes DNA binding by relieving an intramolecular repression (42,53). However, when the ETS2 site was mutated, ER81 binding was no longer observable (see ETSm2/3 in Fig.…”

“…4, C and D). Moreover, the 6xA mutant of ER81, in which all six ERK MAPK-dependent phosphorylation sites in ER81 have been mutated to alanine residues (42,59), was unable to stimulate the Smad7 promoter, nor did it support HER2/Neu to do so (Fig. 4E).…”

“…After incubation for 1 h on ice, the debris was removed by centrifugation (20,800 ϫ g, 10 min, 4°C), and the resulting supernatant was frozen in liquid nitrogen and stored at Ϫ80°C. Bacterially expressed ER81 (249 -477) was generated and purified using the "IMPACT-CN System" (New England Biolabs) as described before (42). Radiolabeled probes plus 0.25 g of poly(dI-dC)⅐poly(dI-dC) (sonicated to an average length of ϳ500 bp) were suspended in 10 l of 20 mM Hepes, pH 7.4, 25 mM NaCl, 2 mM dithiothreitol, 0.5 mM EDTA, 12% glycerol, 0.01% Tween 20, 0.1 g/ l bovine serum albumin.…”

“…Clinically, HER2/Neu overexpression in tumors is associated with high grade, advanced stage, and poor prognosis. One downstream target of HER2/Neu is the ETS transcription factor ER81 (42,43), which has a highly conserved ETS DNA binding domain (44). In particular, ER81 is overexpressed in breast cancer cells (45) and may collaborate with HER2/Neu in breast cancer formation.…”