Fig. 1. Protein kinase B phosphorylates recombinant PIKfyve on serine318 and stimulates its 5-kinase activity. (A) Recombinant GST-PIKfyve fusion protein was incubated in the absence or presence of recombinant active PKB as indicated and with [-³²P]ATP. The upper panel shows an autoradiogram of the resulting gel. The lower panel shows a western blot of the same samples probed with anti-pS318 antibody. Data are representative of three separate experiments. (B) Recombinant GST-PIKfyve was incubated in the absence or presence of recombinant active [-³²P]ATP and PKB, as indicated. The activity of PIKfyve was determined using PtdIns(3)P as substrate according to Materials and Methods. The phosphorylated lipids were then separated by thin layer chromatography. The upper panel shows an autoradiogram of an experiment performed in triplicate. The lower panel shows data from three independent experiments. ^*P<0.05 compared to activity in the control.