The protein kinase activity of PI3K promotes Ser585 phosphorylation and cell survival.

<p>(A) Factor-deprived TF-1 cells were stimulated for 20 min with GM-CSF, the cells lysed and p85 or βc immunoprecipitates were subjected to immunoblot analysis. (B) TF-1 cells were stimulated as in (A) and immunoblotted with an anti-phospho-Ser473 Akt pAb, anti-phospho-Ser21/9-GSK3α/β pAb or total Akt or GSK pAb. (C) TF-1 cells were stimulated as in (A) and subjected to 4G10 immunoprecipitation and lipid kinase assays using [γ-³²P]ATP and PIP as substrates. Error bars represent standard error of the mean from three independent experiments with non-significant (ns, <i>p</i>&gt;0.05) and significant differences (*<i>p</i>&lt;0.05) indicated. (D) Primary mouse Lin⁻ hemopoietic progenitor BM cells from wt and AKT1−/− mice were plated in murine GM-CSF and cell survival was determined after 72 h (left panel). The survival of Lin− cells isolated from wt mice in the presence or absence of murine GM-CSF was also assessed in the presence (black bars) and absence (grey) of 10 µM Akt inhibitor (AKTI-1) (right panel). (E) FDM cells expressing either GFP or a tamoxifen-inducible constitutively active form of myristolated Akt1 (myr-Akt1) (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001515#pbio.1001515.s003" target="_blank">Figure S3A</a>) were plated in the absence of murine IL-3 and ±4-hydroxy tamoxifen (4HT) and cell survival was assessed by propidium iodine (PI) exclusion and flow cytometry. (F) TF-1 cells were co-transfected with GFP alone or myr-Akt1 and GFP and then plated in 1 pM GM-CSF and either 5 µM YM024 or vehicle (DMSO, -) and cell survival was assessed by PI staining after 48 h. (G) TF-1 cells were transfected with plasmids encoding either GFP or Akt1-PH-GFP and cell survival was assessed after 48 h. (H, I) TF-1 cells were transduced with constructs for the doxycyclin-inducible expression of either p110α-CAAX or p110α-4KA-CAAX. After 2 d, cells were plated in doxycycline and/or 5 µM YM024 and cytokine as indicated. (H) Cell lysates were subjected to immunoblot analysis after 12 h. (I) Cell survival was analysed after 48 h. Error bars represent standard deviations (* <i>p</i>&lt;0.05).