FIG. 2.

Lipid-kinase-dependent phenotypic effects generated by expression of PI3K_68D. (A). Mutation of Asp1457 to Ala within the active site of PI3K_68D generates a catalytically inactive protein. Detergent lysates were prepared from adult flies expressing UAS-PI3K_68D constructs under the control of Gal4-heat shock promoters. The extracts were immunoprecipitated with an antibody to the Myc (9E10) epitope and assayed for PI3K activity with PI as a substrate. Equivalent levels of the two constructs were induced as shown by immunodetection with 9E10, but only the wild-type construct possessed PI3K activity (lane 2). Flies were of the following genotypes: lane 1, Gal4-hs/CyO; lane 2, Gal4-hs, UAS-WT-PI3K_68D/CyO; lane 3 Gal4-hs/TM3; lane 4, Gal4-hs, UAS-KD-PI3K_68D/TM3. Arrows mark the positions of the PI3K_68D protein at ∼210 kDa, of PI3P, and of the origin. For each sample the equivalent of 0.5 or 2 female flies were used for the immunoblot (from extracts) and for the PI3K assay (from immunoprecipitations), respectively. Similar results were obtained in three separate experiments. (B to D) Ectopic expression of PI3K_68D affects the number of scutellar sensory bristles in a PI3K-dependent manner. Images of the thoraxes of adult female flies of genotypes Gal4-ptc/+ (B), Gal4-ptc/UAS-WT-PI3K_68D (C), and Gal4-ptc/+; UAS-KD-PI3K_68D/+ (D) are shown. The mean number of scutellar bristles for n = 40 nota is shown. The scutellum is indicated by a bracket. Normally, the four scutellar bristles point distally. In panel D some of these bristles remain curved upwards as in late stages of normal pupae, presumably because they fail to reorient themselves when the flies emerge. Crosses were performed at 25°C.