Impaired activation of PI 3-K/Akt pathway in the v-Abl Δ858-1080 mutant.
(A) Tyrosine phosphorylation of the p85 subunit of PI 3-K is affected downstream of the v-Abl Δ858-1080. Extracts prepared from the indicated cell lines were immunoprecipitated with an anti-p85 antibody and immunoblotted with antiphosphotyrosine antisera. (B) Deletion of the Jak1-binding domain or expression of a kinase-deficient Jak1 reduces PI 3-K activation downstream of v-Abl in vitro. In vitro PI 3-K activation assays were performed by assessing lipid kinase activity in phosphotyrosine immunoprecipitates prepared from the indicated cell lines. L-α phosphatidylinositol phosphate (PIP) was used as substrate. (C) Jak1 kinase activity is required for maximum induction of PIP3 in vivo. In vivo PIP3 levels were measured by extracting lipids from the p160 v-Abl–expressing Ba/F3 cell line prior to (lane 1) or after induction of kinase-inactive Jak1 (lane 2). (D) Activation of Akt downstream of the v-Abl Δ858-1080. Eighty micrograms of whole cell extracts was fractionated on 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and immunoblotted with an antibody specific for activated Akt. For loading control, the blot was stripped and reprobed with an antibody to pan-Akt, which recognizes Akt regardless of its state of activation.
