ERK activation is involved in AML1 transcriptional activation of IEX-1. (A) TPO induces an ERK-dependent increased phosphorylation of AML1. UT7-Mpl cells were stimulated for 1 hour with 10 nM TPO (lanes 2 and 3) in the absence (lane 2) or the presence of 10 μM U0126 (lane 3). Total cell lysates were analyzed by immunoblotting with anti-AML1–, antiphospho-ERK–, and anti-ERK–specific antibodies. (B) TPO induces specific AML1 phosphorylation. UT7-Mpl cells were in vivo labeled with [³²P]Pi and stimulated (lane 2) or not (lane 1) with 10 nM TPO. Endogenous AML1 was oligo-precipitated and detected by Western blot analysis. Radioactive proteins were analyzed with a Typhoon 9400. (C) The ERK-dependent phosphorylation of Ser249 and Ser266 is sufficient to induce a shift in AML1 mobility. HEK293T cells were transfected with mock vector (lane 1), pME18SAMLwt (lane 2), or pME18SAML1S249/266A (lane 3) and oligo-precipitation was performed. Results were analyzed by immunoblot with anti-AML1–specific antibody. (D) IEX-1 promoter induction by AML1 phospho sites mutants. UT7-Mpl cells were cotransfected with 400 ng pLucIEXp and 400 ng either wild-type or mutated AML1 as indicated and stimulated with 10 nM TPO. The cells were harvested 20 hours after transfection for luciferase assays. Data represent means ± SD of at least 3 independent experiments. *P < .05.