Mutations of the potential p85-binding site (Y418) and the binding site for IRS-1 (Y152 and Y156) have no effect on the binding of NPM-ALK with the p85-CSH2 domain.
Mutants of NPM-ALK were generated by site-directed mutagenesis as described in “Materials and methods.” The wild-type or mutated NPM-ALK constructs were transfected into Ba/F3 cells and stably selected by G418. The expression levels of the different NPM-ALK constructs were demonstrated by anti-ALK immunoblotting of the total cell lysates (A and B, left panels). For each binding experiment, 5 × 106 Ba/F3 cell lysates were incubated with p85-CSH2 or p85-NSH2 GST-fusion protein for 1 hour at 4°C. The bound protein complexes were collected with glutathione-beads, washed thoroughly, and resolved by 7.5% SDS-PAGE. Immunoblotting was performed with anti-ALK antibody (A and B, right panels).
