Characterization of the Y250F-YMPM and Y250F-YAAA mutants. (A)
In vitro lipid kinase assay. Anti-MT immunoprecipitates (IP) were
analyzed for their ability to phosphorylate
phosphatidylinositol-4,5-bisphosphate. PI3-kinase product
phosphatidylinositol-3,4,5-trisphosphate (PI3,4,5P3) is indicated.
Y250F-YMPM showed more MT-associated PI3-kinase activity than WT MT.
Y250F-YAAA restored a significant amount of PI3-kinase activity compared with
Y250F. (B) Y250F-YMPM and Y250F-YAAA alleles activate Akt. Equal
amounts of cell lysates were analyzed by Western blotting with an
anti-phospho-Akt Ser-473 antibody (Upper) and an anti-Akt antibody
(Lower).