Fig. 2

PKC and PP2A-dependent PI3K inactivation by autophosphorylation of Ser608-p85. a, b Gq-dependent inactivation of PI3K. Serum starved αT3-1 (a) and SVOG-40 (b) cells were stimulated with 0.1 µM GnRH-a or 10 μM PGF2α for 30 min. The cells were then harvested, and PI3K was IPed with anti-PI3K (p85) Ab, followed by PI3K lipid phosphorylation assay. The amounts of PI(3)P (p-PI) produced were detected by autography (upper panels), and the amount of p85 IPed was detected by anti-p85 Ab (lower panels). c, d The inactivation of PI3K is dependent on PKC and PP2A. Serum starved αT3-1 (c) and SVOG-40 (d) cells were pretreated with either the PKC inhibitor GFx (GF, 3 µM, 20 min) or the PP2A inhibitor okadaic acid (OA, 0.5 µM, 20 min), treated with 0.1 µM GnRH-a or 10 μM PGF2α for 30 min, and then subjected to lipid phosphorylation assay as in a, b. e, f Anti-phospho Ab reveals a PKC dependent phosphorylation of Ser608-p85. Serum starved αT3-1 (e) or PC3 (f) cells were stimulated with TPA (250 nM) for the indicated time, cells were harvested and analyzed by Western Blotting with the newly developed anti-pPI3K (pSer608-p85) and anti-p85 Abs. The graphs in the bottom panels represent means ± standard errors of three experiments. * p < 0.01