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. 1997 Oct 14;94(21):11345-50.
doi: 10.1073/pnas.94.21.11345.

Interleukin 3-dependent survival by the Akt protein kinase

Z Songyang  1 , D Baltimore, L C Cantley, D R Kaplan, T F Franke
Affiliations

Interleukin 3-dependent survival by the Akt protein kinase

Z Songyang et al. Proc Natl Acad Sci U S A. .

Abstract

Interleukin 3 (IL-3)-dependent survival of hematopoietic cells is known to rely on the activity of multiple signaling pathways, including a pathway leading to activation of phosphoinositide 3-kinase (PI 3-kinase), and protein kinase Akt is a direct target of PI 3-kinase. We find that Akt kinase activity is rapidly induced by the cytokine IL-3, suggesting a role for Akt in PI 3-kinase-dependent signaling in hematopoetic cells. Dominant-negative mutants of Akt specifically block Akt activation by IL-3 and interfere with IL-3-dependent proliferation. Overexpression of Akt or oncogenic v-akt protects 32D cells from apoptosis induced by IL-3 withdrawal. Apoptosis after IL-3 withdrawal is accelerated by expression of dominant-negative mutants of Akt, indicating that a functional Akt signaling pathway is necessary for cell survival mediated by the cytokine IL-3. Thus Akt appears to be an important mediator of anti-apoptotic signaling in this system.

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Figures

Figure 1
Figure 1
Akt is activated by IL-3 depending upon the activity of PI-3 kinase. (A) 32D cells were starved of IL-3 as described above and stimulated with 1 ng/ml IL-3 (Materials and Methods). Subsequently, the kinase activity of Akt was determined by histone H2B phosphorylation in immunoprecipitates using anti-Akt-CT. Akt kinase activity also was determined in immunoprecipitates from cells that were pretreated with 100 nM wortmannin before IL-3 stimulation. Subsequently, the immunoprecipitates were probed with anti-Akt-CT to ensure equal protein loading. (B) 32D cells expressing HA-Akt or HA-Akt(K179M) were starved of IL-3 and stimulated for various times with 2 ng/ml IL-3. HA-Akt and HA-Akt(K179M) proteins were immunoprecipitated with monoclonal anti-HA before and after stimulation, and their kinase activity was monitored by in vitro kinase. The lower panel shows an anti-Akt-CT immunoblot of the immunoprecipitates.
Figure 2
Figure 2
Activation of endogenous Akt by IL-3 is inhibited by expression of the Akt N terminus. The kinase activity of endogenous Akt was determined by histone H2B phosphorylation in anti-Akt-CT immunoprecipitates from 32D cells stimulated with IL-3 (1 ng/ml). Akt activity was measured in immunoprecipitates from cells transfected with pBabe vector alone as well as from cells transfected with HA-Akt(PH) in pBabe. Whole-cell lysates were blotted with anti-Akt-CT and anti-Akt-NT to determine expression of Akt and the Akt N terminus, respectively. In parallel, MAPK mobility shifts were determined by anti-Erk immunoblotting of whole-cell lysates after electrophoresis in 7.5% SDS/PAGE.
Figure 3
Figure 3
Proliferation of cells expressing Akt constructs. The proliferation of 32D cells expressing various Akt constructs was measured at various levels of IL-3 using the [3H]thymidine uptake assay (see Materials and Methods). The error bars indicate the standard error of four experiments. (Left) [3H]thymidine uptake was determined in cells transfected with vector alone or v-akt in pLXSN. (Right) [3H]thymidine uptake also was measured in cells transfected with pBabe alone and HA-Akt or HA-Akt(K179M) in pBabe.
Figure 4
Figure 4
Overexpressed Akt and oncogenic v-akt prevent apoptosis that is induced by IL-3 withdrawal. The viability of cells was measured by the trypan blue exclusion assays. The percentages of surviving cells at different times after IL-3 withdrawal are presented. (A) Control cells transfected with vector alone and cells expressing Bcl-2 or v-akt were starved of IL-3 for 24 hr and then assayed for trypan blue exclusion. The error bars indicate the standard error of four experiments. (B) Cells transfected with pBabe alone and cells expressing HA-Akt, HA-Akt(PH), HA-Akt (K179M), or Bcl-2 were starved of IL-3 for 24 hr, and subsequently assayed by trypan blue dye exclusion assays. The survival of cells that expressed Bcl-2 and wild-type HA-Akt or kinase-deficient HA-Akt(K179M) also was estimated 24 hr after IL-3 depletion. Error bars indicate the standard error of three experiments.
Figure 5
Figure 5
v-akt protects cells from apoptosis induced by the DNA damaging reagent etoposide. Percentages of surviving cells were estimated at 16 hr post-etoposide addition to IL-3-containing medium. Error bars indicate the difference between two experiments.
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