Physical association and reciprocal functional interactions between IGF-IR and NPM-ALK. (A left panel) Immunoprecipitation by anti-IGF-IR antibody followed by WB by anti-ALK antibody shows IGF-IR and NPM-ALK to be physically associated in 5 ALK⁺ ALCL cell lines. P6 and R⁻ cells were included as controls. Vertical lines have been inserted to indicate repositioned gel lanes. (A right panel) To rule out nonspecific binding of the protein of interest, a control was used in all experiments where beads were used without an antibody, and the results from the DEL cell line are shown as a representative example. (B top panel) Transfection studies in 293T cells show that the deletion of the C terminus of NPM-ALK abrogates its ability to bind to IGF-IR, whereas deletion of the NPM portion does not affect this ability, as it appears to be similar to WT NPM-ALK. (B bottom panel) Adequate protein expression of different NPM-ALK constructs after transfection into 293T cell line is shown using coimmunoprecipitation. (C top panel) Transfection experiments in 293T cells show that IGF-IR possesses much less ability to bind to NPM-ALK^Y644F or NPM-ALK^Y664F mutant compared with its ability to bind to WT NPM-ALK, NPM-ALK^Y567F, or NPM-ALK^Y646F. (C bottom panel) WB shows adequate expression of the different NPM-ALK constructs. (D) Suggesting the functional interaction between IGF-IR and NPM-ALK, specific targeting of NPM-ALK using ALK siRNA induces marked down-regulation of pIGF-IR in Karpas 299 and SR786 cells. Changes are not seen in IGF-IR. The findings are consistent when anti-IGF-IR antibody was used for immunoprecipitation followed by anti-pTyr antibody for WB. The experiment was performed in other ALK⁺ ALCL cell lines with consistent results. (E) Although treatment of the Karpas 299 cell line with ALK siRNA induces down-regulation of pAkt and pSTAT3, this effect is diminished when the cells are additionally stimulated with IGF-IR ligand, IGF-I. (F) In support of the reciprocal functional interactions between IGF-IR and NPM-ALK, specific targeting of IGF-IR by siRNA in Karpas 299 cells induces a significant decrease in pNPM-ALK levels at Tyr646 and Tyr664. (G) Treating the ALK⁺ ALCL cell lines with IGF-I induces significant increase in NPM-ALK kinase activity up to 92 and 42% of its baseline levels in SR786 and DEL cell lines, respectively. This effect is largely reversed when these cells are treated with IGF-I and one of the anti–IGF-I–neutralizing antibodies or the anti-IGF-IR blocking antibody. P less than .01 for cells treated with IGF-I compared with controls and other treatments groups. (H) To further demonstrate the functional interaction between the 2 kinases, transfection of P6 cells with WT NPM-ALK induces significant up-regulation of pIGF-IR. In contrast, changes are not noted in pIGF-IR levels when P6 cells were transfected with NPM-ALK^K210R. Transfection of P6 cells with different NPM-ALK constructs did not affect the baseline levels of IGF-IR. R⁻ cells were used as a negative control for IGF-IR expression. Using the anti-ALK antibody confirmed the expression upon transfection of either the WT NPM-ALK or NPM-ALK^K210R. (I) Transfection of Jurkat cell line, which expresses IGF-IR, with WT NPM-ALK induces marked increase in pIGF-IR. In contrast, NPM-ALK^K210A fails to induce a similar effect, and pIGF-IR remains comparable with empty vector. (J left panel) WT NPM-ALK, but not NPM-ALK^K210A, induces up-regulation of pIGF-IR levels in the ALK⁺ TCL cell line DEL. (J right panel) pNPM-ALK levels significantly increase after transfection of DEL cells with WT IGF-IR. Of note is that transfection of this cell line with IGF-IR^{Y1131,1135,1136F}, which possesses significantly less kinase activity compared with WT IGF-IR, does not induce a similar effect on pNPM-ALK levels. (K) Cotransfection of R⁻ cells with WT IGF-IR and WT NPM-ALK is associated with marked increase in NPM-ALK kinase activity compared with cotransfection of these cells with IGF-IR^{Y1131,1135,1136F} and WT NPM-ALK. The results represent one of 2 consistent experiments. (L) Coimmunoprecipitation studies in 293T cell line show that NPM-ALK^Y338F possesses significantly low levels of tyrosine phosphorylation. Similar to WT NPM-ALK, NPM-ALK^Y338F can still efficiently bind to IGF-IR. Nonetheless, in comparison to WT NPM-ALK, NPM-ALK^Y338F lacks the ability to induce the phosphorylation/activation of IGF-IR. (M) NPM-ALK^Y338F does not increase the basal level of IGF-IR kinase activity in 293T cells. The results are representative of 2 consistent experiments.