DNA bound RUNX1/ PEBP2-β complex promotes RUNX1 and p300 phosphorylation. (A) Coupling of RUNX1 and p300 phosphorylation. HEK293 cells were transfected with RUNX1 or phosphorylation-defective point mutant constructs (100 ng) together with FLAG-tagged p300 (300 ng) in the absence or presence of PEBP2-β (300 ng). Cells were lysed 24 hours after transfection, and lysates were analyzed by Western blotting with antibodies against FLAG (top panel) and RUNX1 (middle panel) and PEPB2β (bottom panel), respectively. (B) HEK293 cells were cotransfected with wild-type RUNX1 or heterologous DNA-binding domain (DBD) mutants (100 ng), FLAG-tagged p300 (300 ng), and PEBP2-β (300 ng) as indicated. Cells were lysed 36 hours after transfection and analyzed by Western blotting using antibodies against RUNX1 (top panel) or FLAG (bottom panel). (C) Schematic representation of domain structure and phosphorylation sites of RUNX1 and mutants used in panels B and E. (D) Mapping of functional domains necessary for the phosphorylation of RUNX1 and p300. Wild-type or mutant RUNX1 were coexpressed with FLAG-p300 and PEBP2-β and analyzed by Western blotting 24 hours after transfection as described in panel A. (E) Mutation of putative phosphorylation target sites on RUNX1. Proline (P)-directed serine (S) and threonine (T) residues targeted in mutations m1 (S249A), m2 (S266A), and m3 (T273A/276A) are denoted by asterisks and mutated to alanine (A). (F) Transient transfection and Western blot analyses of RUNX1 point mutants in HEK293 cells. Wild-type, leukemogenic, and engineered point mutants of RUNX1 were coexpressed with p300 and PEBP2-β and analyzed by Western blotting as described in panel A.