The BCR/ABL-activated MAPK^ERK1/2 is responsible for enhanced hnRNP-E2 expression. (A) Effect of different chemical kinase inhibitors on hnRNP-E2 protein levels in Ph1(+) EM-3, K562, 32D-BCR/ABL, and primary CML-BC^CD34+ progenitors. Graph shows mean plus and minus standard deviation (SD) of hnRNP-E2 levels after normalization with HSP90 in untreated and U0126-treated primary CML-BC^CD34+ progenitors (n = 3). Effect of wild-type and dominant-negative K71R ERK1 and K52R ERK2 expression on hnRNP-E2 levels in 32D-BCR/ABL cells. (B) Schematic diagram shows ERK phosphorylation sites (S/T-P) in hnRNP-E2. (C) In vivo MAPK-dependent phosphorylation of hnRNP-E2. Autoradiography (top) shows phosphorylated HA-hnRNP-E2 in ³²P-labeled 32D-BCR/ABL cells ectopically expressing HA-hnRNP-E2 (untreated and U0126-treated) and HA-hnRNP-E2 ^{S173A, S189A, T213A, S272A}. Anti-HA Western blot (bottom) shows expression of HA-hnRNP-E2 and HA-hnRNP-E2 ^{S173A, S189A, T213A, S272A}. Asterisks indicate IgG chains. (D) In vitro MAPK-dependent phosphorylation of hnRNP-E2. (top panel). Bacterially expressed and purified wild-type MBP-hnRNP-E2 was subjected to an in vitro kinase assay and Western blot analysis. Autoradiography (top) shows phosphorylated MBP-hnRNP-E2 by the anti-ERK1/2 immunoprecipitates. Western blot (bottom) shows expression of MBP-hnRNP-E2 and MBP. Effect of serine/threonine to alanine mutations on hnRNP-E2 phosphorylation (bottom panel). Wild-type MBP-hnRNP-E2 and its serine/threonine to alanine mutants were subjected to a kinase assay with recombinant ERK1 (rERK1) and ERK2 (rERK2), respectively. Autoradiography show phosphorylated MBP-hnRNP-E2 by ERK1 and ERK2, respectively. Coomassie blue–stained gels were used as controls for equal loading.