Fig. 4.

VEGF increased ETS1 acetylation and interaction with BRD4. a ClustalW alignment of a region within the ETS1 NT domain. K8 and K18 were major sites of acetylation, as determined by mass spectrometry. b ETS1 NT acetylation enhances BRD4 binding. Peptides corresponding to the N-terminus of ETS1 were synthesized with either lysine or acetyl-lysine (@) at positions 8 and 18. Immobilized peptides were incubated with V5-BRD4-containing cell lysates. K8 or K18 acetylation enhanced BRD4 binding. c ETS1–BRD4 interaction is modulated by K8 and K18 acetylation. The ETS1 NT domain was expressed with mutations that abrogate (R) or mimic (Q) lysine acetylation. Interaction with co-transfected V5-BRD4 was assessed by co-immunoprecipitation. d CBP acetylates ETS1 at K8 and K18. ETS1 acetylation was assessed by immunoprecipitation followed by immunoblotting with acetyl-lysine specific antibody. ETS1 acetylation required K8 and K18, and ERK phosphorylation sites T38 and S41. e CBP stimulates BRD4 binding to ETS1 NT domain. Expression constructs were co-transfected into 293T cells. BRD4 co-precipitated by ETS1 was detected by immunoblotting. CBP stimulated ETS1–BRD4 interaction. f VEGF stimulates ETS1 phosphorylation. HUVEC cells were treated with 50 ng ml⁻¹ VEGF for the indicated time. ETS1 T38 phosphorylation (p-ETS1) was detected using specific antibody. g ERK is required to phosphorylate ETS1 downstream of VEGF. ERK pathway inhibitor PD598059 blocked ETS1 phosphorylation in HUVECs stimulated by VEGF, but inhibitors of other ETS1 kinases (KN93 or Dasatinib) did not. h VEGF induced ETS1 NT acetylation at K8 and K18. In HUVEC cells, VEGF treatment induced robust acetylation of the ETS1 NT domain, but this was abolished by K8;18R mutation. i VEGF-induced ETS1–BRD4 interaction. ETS1 was immunoprecipitated from HUVEC cells treated with VEGF for the indicated time. Co-precipitated BRD4 was measured by immunoblotting. j VEGF-induced ETS1–BRD4 interaction requires ERK activity. Co-immunoprecipitation assay as in i was performed with or without ERK inhibitor PD598059