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).</p

Model for the regulation of cell survival by the protein kinase or lipid kinase activities of PI3K.

<p>Left panel: Low physiological concentrations of cytokine in the picomolar range activate the protein kinase activity of PI3K leading to Ser608 phosphorylation of p85 and Ser585 phosphorylation of the GM-CSF/IL-3 βc receptor (red circles) to promote cell survival in the absence of phosphotyrosine pathways and proliferation (survival-only response). Middle panel: Nanomolar concentrations of cytokine result in activation of the JAK2 tyrosine kinase, Tyr577 phosphorylation of βc, the recruitment of a Shc:grb2:GAB2:PI3K signaling complex to Tyr577 (red circle) <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001515#pbio.1001515-Guthridge2" target="_blank">[6]</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001515#pbio.1001515-Gu1" target="_blank">[33]</a>, and the activation of canonical PI3K lipid signaling via Akt to promote cell proliferation and survival. Right panel: Blockade of the p110α catalytic subunit of PI3K inhibits both protein kinase targets (Ser585) and lipid kinase targets (Akt) and induces apoptosis in primary human AML cells.</p

Cell survival is autonomous in human AML and CML cells and is refractory to tyrosine kinase inhibition.

<p>(A) Primary human AML MNCs from patient AML1 (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001515#pbio.1001515.s007" target="_blank">Table S2</a>) or (B) K562 CML cells were cultured in DMSO, 10 µM JAKI or 2 µM imatinib and GM-CSF. Cell survival (annexin V-negative) (grey) or proliferation (BrdU) (black) were measured by flow cytometry. (C) Where indicated, primary AML blasts were preincubated in JAKI (10 µM), src kinase inhibitor, PP1 (10 µM), or vehicle (DMSO) for 20 min following which the cells were stimulated with GM-CSF for 5 min. βc was then immunoprecipitated and subjected to immunoblot analysis with anti-phospho-βc Ser585 pAb, anti-phospho-βcTyr577 pAb, or anti-βc (1C1) mAb. (D) K562 CML cells were preincubated in JAKI (10 µM), src kinase inhibitor, PP1 (10 µM), 2 µM imatinib, 0.1 µM dasatinib, or vehicle (DMSO) for 20 min following which the cells were stimulated with GM-CSF for 5 min and immunoblotted as in (C). Ckl blots were performed to confirm Bcr-Abl inhibition (loss of p-Crkl) by imatinib and dasatinib.</p

PI3K protein kinase substrate phosphorylation sites.

<p>PI3K protein kinase substrate phosphorylation sites.</p

Purification and pharmacological profiling of a Ser585 kinase from AML.

<p>(A) AML MNCs (from patients AML4 and AML8; both samples gave same profile) were subjected to hypotonic lysis and then chromatography on a Superdex 200PC column. Aliquots of eluted fractions were immunoblotted with anti-p85 pAbs. (B) Fraction 8 (the peak of kinase activity) was analysed for kinase activity in vitro using a βcSer585 peptide substrate and 10 µM of the indicated inhibitors. (C) Kinase reactions were performed using fraction 8 in the presence of increasing concentrations of LY294002 (1, 10, 50, and 100 µM), PI3-Kγγ1 (0.01, 0.1, 1, and 10 µM), Wortmannin (0.01, 0.1, 1, and 10 µM), and quercetin (1, 10, 100, and 1,000 nM). Error bars indicate ± standard deviation.</p

Inhibition of the p110α catalytic subunit of PI3K down-regulates Ser585 phosphorylation and induces apoptosis in primary AML MNCs.

<p>(A) HEK 293T cells were transfected with a construct for the expression of the βc subunit of the GM-CSF and IL-3 receptors together with 100 nM of the indicated siRNAs. After 72 h, cells were lysed and blotted with the indicated antibodies. (B) AML MNCs were transfected with 100 nM of siRNA-p110α-1 or control siRNA and after 48 h, cells were lysed and immunoblots performed using the indicated antibodies. Quantified signals are indicated under the immunoblots. (C) The phospho-Ser585 signals from six independent AML samples (AML10–15, from <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001515#pbio-1001515-g006" target="_blank">Figures 6B</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001515#pbio.1001515.s005" target="_blank">S5A</a>) were quantified by laser densitometry and normalized to control siRNA (Ctl) with horizontal lines representing the means (<i>p</i> = 0.001, Mann Whitney U). (D) AML MNCs (AML14 light shade. AML15 dark shade) were plated in 1 µM each of YM024, TGX-221, IC87114, or AS252424 and cell survival examined at 48 h. (E) AML MNCs (AML14 light shade. AML15 dark shade) were transfected with 100 nM control siRNA (Ctl) or siRNA-p110α-1 and cell survival was examined after 48 h.</p

The protein kinase activity of PI 3-kinase can directly phosphorylate Ser585.

<p>(A) PI3K was immunopurified from TF-1 cells by immunoprecipitation with anti-p85 pAb (no anti-p85 was used for the mock) following which in vitro kinase assays were performed using a phospho-βcSer585 peptide (non-phosphorylatable control peptide, Ctl) or a βcSer585 peptide and ±10 µM LY294002 (LY). (B) Kinase activity was examined in either mock or p85 immunoprecipitates using either a phospho-βcSer585 non-phosphorylatable control peptide (Ctl) or a βcSer585 peptide in kinase buffer containing either 10 mM MnCl₂ (Mn⁺⁺), 10 mM MgCl₂ (Mg⁺⁺), or 10 mM MnCl₂ and 0.25 mM DTT (Mn⁺⁺/DTT). (C) Immunoprecipitation of specific isoforms of p110 from TF-1 cells was performed and lipid kinase assays were performed. (D) The same immunoprecipitations were also subjected to in vitro kinase reactions using either control peptide (Ctl) or the βcSer585 peptide ±10 µM LY294002. (E) HEK 293T cells were transfected with wt-p110α (p110α), a lipid-kinase defective form of p110α (p110a-4KA), and/or myc-tagged p85. myc-p85 was immunoprecipitated and subjected to in vitro kinase assays using purified recombinant intracytoplasmic domain of βc (βic) following which reactions were subjected to SDS-PAGE and autoradiography or immunoblot analysis for p110α and myc-p85. (F) Purified recombinant p110α was examined for its ability to phosphorylate Ser585 in purified recombinant βic in vitro and reactions were subjected to immunoblot analysis using anti-phospho-βcSer585 pAb. (G) Purified recombinant p110α was incubated with 50 µM βcSer585 peptide in the presence/absence of 50 nM of the PIK-75 p110α-selective inhibitor. Reactions were spotted onto nitrocellulose and blotted with the anti-phospho-βcSer585 pAb. Phospho-βcSer585 peptide (50 µM) was included as a positive control for the anti-phospho-βcSer585 pAb. The histogram shows laser densitometry quantification of signals. Error bars represent standard deviations.</p

Inhibition of the p110α catalytic subunit of PI3K reduces Ser585 phosphorylation and blocks the survival-only response.

<p>(A) TF-1 cells were plated in GM-CSF for up to 14 d (media changed every 2 d) and cell viability was determined by trypan blue exclusion. On day 10, an aliquot of cells in 1 pM GM-CSF was washed and 1,000 pM GM-CSF added (arrow). (B) TF-1 cells were plated in GM-CSF and proliferation was measured by BrdU-incorporation. (C–F) Factor-deprived TF-1 cells were pre-incubated for 45 min with (C) LY294002, (D) PIK-75, (E) PI-103, (F) YM024 at the concentrations shown, and then stimulated for 5 min with GM-CSF, lysed, and immunoblotted with anti-phospho-βcSer585 pAb and anti-βc mAb. (G) Laser densitometry quantification of the ability of p110 isoform-selective inhibitors to block Ser585 phosphorylation in which the ratio of phospho-Ser585 relative to total βc in the presence of drug is expressed as a percentage of the maximum Ser585 phosphorylation (100%, dotted line). (H) TF-1 cells were cultured in 1 pM GM-CSF in the presence of 100 nM PIK-75, 5 µM YM024, 1 µM TGX-221, 5 µM IC87114, or 100 nM AS252424 and cell viability was determined at 72 h by trypan blue exclusion. (I) Primary mouse lineage-negative BM progenitor cells were cultured in 1 pM murine GM-CSF in the presence of 100 nM PIK-75, 5 µM YM024, 1 µM TGX-221, 5 µM IC87114, or 100 nM AS252424 and cell viability was determined at 48 h. Error bars indicate standard deviations (* <i>p</i>&lt;0.05).</p