Dissociation of cytokine-induced phosphorylation of Bad and activation of PKB/akt: involvement of MEK upstream of Bad phosphorylation - PubMed (original) (raw)

Dissociation of cytokine-induced phosphorylation of Bad and activation of PKB/akt: involvement of MEK upstream of Bad phosphorylation

M P Scheid et al. Proc Natl Acad Sci U S A. 1998.

Abstract

The phosphatidylinositol 3-kinase (PI3K)-signaling pathway has emerged as an important component of cytokine-mediated survival of hemopoietic cells. Recently, the protein kinase PKB/akt (referred to here as PKB) has been identified as a downstream target of PI3K necessary for survival. PKB has also been implicated in the phosphorylation of Bad, potentially linking the survival effects of cytokines with the Bcl-2 family. We have shown that granulocyte/macrophage colony-stimulating factor (GM-CSF) maintains survival in the absence of PI3K activity, and we now show that when PKB activation is also completely blocked, GM-CSF is still able to stimulate phosphorylation of Bad. Interleukin 3 (IL-3), on the other hand, requires PI3K for survival, and blocking PI3K partially inhibited Bad phosphorylation. IL-4, unique among the cytokines in that it lacks the ability to activate the p21ras-mitogen-activated protein kinase (MAPK) cascade, was found to activate PKB and promote cell survival, but it did not stimulate Bad phosphorylation. Finally, although our data suggest that the MAPK pathway is not required for inhibition of apoptosis, we provide evidence that phosphorylation of Bad may be occurring via a MAPK/ERK kinase (MEK)-dependent pathway. Together, these results demonstrate that although PI3K may contribute to phosphorylation of Bad in some instances, there is at least one other PI3K-independent pathway involved, possibly via activation of MEK. Our data also suggest that although phosphorylation of Bad may be one means by which cytokines can inhibit apoptosis, it may be neither sufficient nor necessary for the survival effect.

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Figures

Figure 1

Cell death resulting from PI3K inhibition. MC/9 cells were washed three times and resuspended in complete medium containing the indicated cytokine, or medium without cytokine (starved). LY-294002 (25 μM; closed circles) or vehicle (open circles) were added at time 0, and at the indicated hours duplicate aliquots of cells were removed, washed, stained with annexin-V-FITC and propidium iodide, and analyzed by using flow cytometry. We found that cells undergoing apoptosis quickly showed an increase in annexin-V binding but excluded propidium iodide (early apoptosis). At later time points the percentage of propidium iodide staining cells gradually increased (late apoptosis). We are reporting here the total amount of annexin-V-FITC and propidium iodide staining, which is representative of populations containing cells at both stages of apoptosis. Results are representative of at least four independent experiments.

Figure 2

Cytokine activation of PKB and requirement for PI3K. (A) MC/9 cells starved of cytokine for 3–5 hr were stimulated with the indicated cytokines at concentrations that have been shown previously to induce maximal tyrosine phosphorylation and PI3K activity, for the indicated times. Also, some cells were pretreated with LY-294002 (25 μM) for 10 min and stimulated for 5 min with cytokine. Cells were pelleted and lysed in ice-cold solubilization buffer (see Materials and Methods). PKB was immunoprecipitated and its activity was measured in an in vitro kinase assay by using Crosstide as substrate. Individual experiments have been normalized to the percentage of stimulation induced by a 5-min treatment with IL-3, which was always performed concurrently. Typical maximum stimulations for IL-3, IL-4, and GM-CSF ranged between 8- and 12-fold above unstimulated samples, which generally were in the 2,000–3,000 cpm range. (B) Cells were prepared as above and pretreated with LY-294002 (25 μM) or vehicle alone for 10 min followed by stimulation with GM-CSF for the indicated times. PKB immunoprecipitation and kinase assay was performed as above. The results presented are from three experiments using duplicate samples, with error bars representing SEM.

Figure 3

Bad mobility shift is induced by treatment with IL-3, GM-CSF, or SCF but not IL-4. (A) Cells were starved of cytokine for 3–5 hr and stimulated with IL-3 for the indicated times, or were left unstimulated. Cells were isolated and solubilized as described in Materials and Methods. Bad was immunoprecipitated (5 μg of B36420, Transduction Laboratories) and separated by SDS/PAGE followed by transfer to nitrocellulose and immunoblotting for Bad. (B) Immunoprecipitated Bad from cells stimulated with IL-3, IL-4, or GM-CSF for 10 min and immunoblotted with B36420. (C) Whole-cell lysates from a similar experiment immunoblotted with anti-Bad antibody SC-943 (Santa Cruz Biotechnology). Results shown are representative of at least four independent experiments.

Figure 4

IL-4 does not stimulate Bad phosphorylation. (A) Cells were starved of cytokine for 3–5 hr and then metabolically labeled with 32P-labeled orthophosphate for 2 hr, followed by stimulation with the indicated cytokine for 10 min. Bad was immunoprecipitated as described in Fig. 3 and fractionated by SDS/PAGE. 32P-labeled Bad was detected by autoradiography and quantitated by either a PhosphorImager or by excising the bands and counting by liquid scintillation. This experiment was performed in duplicate, with one set of samples shown. (B) Identical experiment as A, but SCF was tested instead of GM-CSF. The numbers beneath each lane correspond to the average stimulation above untreated for each set of duplicates.

Figure 5

PI3K inhibition partially blocks IL-3- but not GM-CSF-induced Bad phosphorylation. (A) Cells were preincubated with LY-294002 (25 μM) or vehicle alone for 10 min, followed by stimulation with IL-3 or GM-CSF for 10 min. Bad was immunoprecipitated as described in Fig. 3 and mobility shift was examined by immunoblotting with a polyclonal anti-Bad antibody (SC-943). (B) Cells were pretreated with the concentrations of LY-294002 indicated above the lanes for 10 min and stimulated with IL-3 or GM-CSF for 10 min. Bad was immunoprecipitated and immunoblotted with B36420 (Transduction Laboratories) to determine mobility shift. These results are representative of three independent experiments.

Figure 6

MEK inhibition blocks Bad phosphorylation without effect on survival. (A) Cells were preincubated with PD98059 (PD) at the indicated concentrations for 30 min followed by stimulation with IL-3 or GM-CSF for 5 min. Detergent solubilized cell lysates were fractionated by SDS/PAGE and immunoblotted with anti-phospho-MAPK. (B) Bad was immunoprecipitated from the same cells lysates and immunoblotted to determine mobility shift. These results are representative of three independent experiments. (C) MC/9 cells were washed and resuspended in IL-3, GM-CSF, or media alone (Unstim) and incubated with 50 μM PD98059 (solid bars) or vehicle (shaded bars) for 11 hr. Apoptosis was determined by annexin V binding by using flow cytometry. Results shown are averages of duplicate samples and are representative of two independent experiments.

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Dissociation of cytokine-induced phosphorylation of Bad and activation of PKB/akt: involvement of MEK upstream of Bad phosphorylation - PubMed (original) (raw)