BCR/ABL- and MAPK-dependent phosphorylation of HNRPK regulates Myc mRNA translation in BCR/ABL-transformed myeloid cells. (A) Western blots show Myc levels in parental 32Dcl3 cells (lane 1), untreated 32D-BCR/ABL cells (lane 6); or cells treated with imatinib (lane 7) or with the MEK1 inhibitor PD098059 (lane 8); or cells transduced with the empty retrovirus (lane 2) or with wild-type and mutant HNRPK (lanes 3-5) or with MAPK ERK1 (lanes 9-10) and ERK2 (lanes 11-12) cDNAs. All cell lines were cultured in the presence of IL-3. (B) Northern blots show Myc mRNA levels in the indicated cell lines either maintained in IL-3 or IL-3 deprived for 12 hours in the presence or absence of imatinib. (C) Autoradiography and anti-MYC Western blot show levels of ³⁵S-labeled newly synthesized and total immunoprecipitated Myc protein in parental 32Dcl3, untreated, and imatinib-treated 32D-BCR/ABL and S284/353A expressing 32D-BCR/ABL cells. (D) Levels of Myc protein, expressed as arbitrary densitometric units after normalization with GRB2 levels, in cycloheximide-treated parental 32Dcl3, 32D-BCR/ABL, and its derivative cell lines expressing wild-type or mutant HNRPK proteins. Half-life was calculated using the algorithm t½ = 0.693t_n/ln(C₀/C_n), where n represents the last time point. (E) Histograms show levels of MYC mRNA (red bars) and protein (blue bars) after normalization with GAPDH mRNA and GRB2 protein levels, respectively. Values in samples 2 to 7 represent arbitrary densitometric units expressed as percentage of those of sample 1. Western blot and RT-PCR were performed with lysate and total RNA, respectively, of CD34⁺marrow cells from healthy individuals (lanes 1-2) and from CML-CP (lanes 3-4), CML-AP (lane 5), and CML-BC (lanes 6-7) patients.