NPM-ALK activates JNK and cJun and Induces AP-1 transcriptional activity. (A) HEK 293T cells were stably transfected with expression plasmids including empty vector (pDest40), active NPM-ALK, or mutant NPM-ALK (K210R) with a kinase-dead domain. Expression of NPM-ALK, in cells transfected with functional or kinase-dead mutant NPM-ALK, was confirmed by Western blot analysis using the ALK1 antibody. ALK activity in cells transfected with the functional NPM-ALK construct was confirmed using an antibody specific for phosphorylated ALK. Stable expression of functional NPM-ALK in HEK 293T cells resulted in JNK phosphorylation/activation, which was associated with phosphorylation of cJun at serine 73 and an increase in total c-Jun levels that is attributable to positive autoregulation of cJun transcription. Total JNK1 expression served as a protein loading control in this experiment. (B) Jurkat cells were transiently transfected with 50 μg of empty vector (pDest40), active NPM-ALK, or mutant, kinase-dead NPM-ALK (210K>R), and whole-cell lysates were prepared at 48 hours after transfection. Immunoblots showed that transient expression of the functional NPM-ALK in Jurkat cells resulted in a substantial increase of p-JNK, which was associated with increased phosphorylation of cJun. (C) To study the NPM-ALK–induced AP-1 transcriptional activity, Jurkat cells were transiently transfected with a luciferase reporter gene under the control of a promoter that contains 3 successive AP-1 specific binding sites (3×AP-Luc) together with an empty expression vector or expression plasmids encoding for the functional or kinase-dead mutant NPM-ALK. Two sets of experiments were performed with or without cotransfection of a full-length cJun expression plasmid (pHA-cJun). After 48 hours the cells were collected to determine luciferase activity. The results showed an increase in relative luciferase units in cells expressing the functional NPM-ALK, indicating an increase in AP-1 transcriptional activity. The transcriptional activity was enhanced in Jurkat cells by coexpression of full-length cJun (right panel). The fold activation compared with the basal activity of the AP-1 promoter sites in cells transfected with the empty vector, which was set to 1. All measurements were performed in triplicate; bars indicate standard error. (D) Coimmunoprecipitaion studies were performed in SU-DHL1 and Karpas 299 cells. Whole-cell lysates were first immunoprecipitated with JNK1, JNK2, or control IgG1 antibodies, and then immunoblotted using specific ALK antibody. Conversely, whole-cell lysates were also immunoprecipitated with ALK or control IgG1 antibodies and then immunoblotted using a JNK1/2 antibody that detects both JNK1 and JNK2. The same membrane was also probed with ALK antibody. The top 2 arrows show a specific band at 80 kDa (NPM-ALK), indicating that NPM-ALK physically interacts with JNK1 and JNK2 in ALK⁺ ALCL cells. The bottom arrow indicates detection of JNK1/2 by Western blot analysis after inverse coimmunoprecipitation that further confirmed the physical interaction between NPM-ALK and JNKs. Immunoglobulin heavy chain (HC) served as a loading control. (E) SU-DHL1 and Karpas 299 cells were treated with the inhibitor WHI-P154 at concentrations (0, 0.5, 2.5, 5, or 10 μM), previously shown to inhibit JAK3 and ALK enzymatic activity. Whole-cell lysates were prepared at 24 hours after treatment. Immunoblots demonstrate that ALK phosphorylation is decreased at a concentration of 2.5 μm, and correlates with decreased phosphorylation (activation) of JNK. (F) Inhibition of ALK enzymatic activity in NPM-ALK⁺ ALCL cells resulted in decreased AP-1 DNA binding activity in a concentration-dependent manner as shown by EMSA and autoradiography (black arrow). SU-DHL1 and Karpas 299 cells were treated with the inhibitor WHI-P154 at concentrations of 0, 5, or 10 μM. After incubation for 24 hours, nuclear extracts were prepared and assessed by EMSA using double-stranded consensus AP-1 oligonucleotide. Free DNA (free probe, FP) is shown in the bottom of the gel.