Phosphatidylinositol 3-kinase mediates activation of ATM by high NaCl and by ionizing radiation: Role in osmoprotective transcriptional regulation

Abstract

High NaCl causes DNA double-strand breaks and activates the transcription factor, TonEBP/OREBP, resulting in increased transcription of several protective genes, including those involved in accumulation of compatible organic osmolytes. Several kinases are known to contribute to signaling activation of TonEBP/OREBP, including ATM, which is a member of the phosphatidylinositol 3-kinase (PI3K)-like kinase family and is activated by DNA double-strand breaks. The purpose of the present studies was to investigate a possible role of PI3K Class IA (PI3K-IA). We found that high NaCl increases PI3K-IA lipid kinase activity. Inhibiting PI3K-IA either by expressing a dominant negative of its regulatory subunit, p85, or by small interfering RNA-mediated knockdown of its catalytic subunit, p110alpha, reduces high NaCl-induced increases in TonEBP/OREBP transcriptional activity and transactivation, but not nuclear translocation of TonEBP/OREBP, or increases in its abundance. Further, suppression of PI3K-IA inhibits the activation of ATM that is caused by either ionizing radiation or high NaCl. High NaCl-induced increase in TonEBP/OREBP activity is reduced equally by inhibition of ATM or PI3K-IA, and the effects are not additive. The conclusions are as follows: (i) PI3K-IA activity is necessary for both high NaCl- and ionizing radiation-induced activation of ATM and (ii) high NaCl activates PI3K-IA, which, in turn, contributes to full activation of TonEBP/OREBP via ATM.

Fig. 1.

PI3K-IA is activated by high NaCl. (A) PI3K-IA activity was measured in p85 immunoprecipitates 1 h after increasing osmolality to 500 mosmol/kg by adding NaCl to Jurkat cells expressing active PI3K-IA; i.e., tetracycline was present, which prevented expression of the c-Myc Δp85, PI3K-IA DN construct. (A Lower) Western blot showing that there were equal amounts of p85 protein in reactions from the cells at 300 and 500 mosmol/kg. (B and C) Osmolality bathing HEK293 was increased to 500 mosmol/kg for 1 h by adding NaCl. (B) p85 protein abundance and phosphorylation on Y508. (C) Time course of high NaCl-induced phosphorylation of p85 (Y508-P) in HEK293 cells. The cells were grown at 300 mosmol/kg. For the “control” at 300 mosmol/kg, that medium was not changed. For the points at 0 min, we changed the medium to a new one at 300 or 500 mosmol/kg, then immediately washed with ice-cold PBS, followed immediately by the protein extraction buffer. (D) GSK-3β phosphorylation on S9. All values are normalized to the activity in cells kept at 300 mosmol/kg. Mean ± SEM (∗, P < 0.05; n = 3).

Fig. 2.

Inhibition of PI3K-IA decreases TonEBP/OREBP transcriptional activity but does not affect its subcellular localization. To inhibit PI3K-IA, the c-Myc Δp85 (PI3K-IA DN) construct was expressed in the Jurkat Tet-off cells by removing tetracycline for 48 h, or in the HEK293 Tet-on cells, by adding 2.0 μg/ml doxycycline for 24 h. (A) TonEBP/OREBP transcriptional activity in Tet-off Jurkat cells. The cells were transfected with ORE-X reporter plasmids. Twenty-four hours later, osmolality either was kept at 300 mosmol/kg or was increased to 500 mosmol/kg for 16 h by adding NaCl before measuring luciferase reporter activity. (B) TonEBP/OREBP transcriptional activity in HEK293 Tet-on cells. The cells were transiently cotransfected with ORE-X and c-Myc Δp85 constructs. Twenty-four hours later, osmolality either was kept at 300 mosmol/kg or was increased to 500 mosmol/kg for 16 h by adding NaCl before measuring luciferase activity. (C) Effect of siRNA targeting p110α on TonEBP/OREBP transcriptional activity. HEK293 cells stably transfected with ORE-X (HEK293 ORE-X cells) were transfected with 20 nM siRNA, either control (random, nontargeting) or targeting p110α for 48 h. After transfection, osmolality either was kept at 300 mosmol/kg or was increased to 500 mosmol/kg for 24 h by adding NaCl before measuring luciferase reporter activity. In the blots, abundance of TonEBP/OREBP and p110α was determined by Western analysis. (D) TonEBP/OREBP transactivation in Tet-off Jurkat cells. The cells were cotransfected with the GAL4 UAS reporter and GAL4dbd-548–1531. Twenty-four hours later, osmolality either was kept at 300 mosmol/kg or was increased to 500 mosmol/kg for 16 h by adding NaCl before measuring luciferase activity. In A–D, values are normalized to cells kept at 300 mosmol/kg. (E) TonEBP/OREBP intracellular location in Jurkat cells. Cells were exposed to 200, 300, or 500 mosmol/kg by varying NaCl concentration (“2,” “3,” or “5”) for 30 min before measuring TonEBP/OREBP protein by Western analysis in cytoplasmic and nuclear extracts and calculating TonEBP/OREBP nuclear/cytoplasmic ratio. A representative Western blot is shown. All values are mean ± SEM (∗, P < 0.05; n = 3).

Fig. 3.

Effects of PI3K-IA DN (Δp85) or p110α siRNA on ATM activation (phosphorylation of S1981). The cells were exposed to 5 Gy of IR, then analyzed 1 h later or exposed to high NaCl for 2 h. (A) Time course of high NaCl-induced phosphorylation of ATM (S1981-P) in HEK293 cells. The cells were grown at 300 mosmol/kg. For the control at 300 mosmol/kg, that medium was not changed. For the points at 0 min, we changed the medium to a new one at 300 or 500 mosmol/kg, then immediately washed with ice-cold PBS, followed also immediately by the protein extraction buffer. (B) Jurkat Tet-off cells. The c-Myc Δp85 (PI3K-IA DN) construct was expressed by removing tetracycline for 48 h. (B Lower) Western blots analysis of Δp85 expression and of ATM and p38 phosphorylation. (C) HEK293 Tet-on cells. The c-Myc Δp85 construct was expressed by adding 2.0 μg/ml doxycycline for 24 h. (C Lower) Western blot analysis of Δp85 expression and ATM phosphorylation. (D) Effect of siRNA targeting p110α on ATM activation. HEK293 cells stably transfected with ORE-X (HEK293 ORE-X cells) were transfected with 20 nM siRNA for 48 h, either control (random, nontargeting) or targeting p110α. (D Lower) Western blot analysis of p110α and p110γ expression and the phosphorylation of ATM and GSK-3β. (E) Effects of expression of PI3K-IA and/or ATM on TonEBP/OREBP transcriptional activity. PI3K-IA activity in the tetracycline-regulated PI3K mutant Jurkat cells was modified by the presence or absence of tetracycline for 48 h, then the cells were cotransfected by electroporation with ORE-X reporter and with wild type (wt) or kinase dead (KD) ATM, which is a dominant negative. Twenty-four hours later, osmolality either was kept at 300 mosmol/kg or was increased to 500 mosmol/kg (NaCl added) for 2 h before measuring reporter activity. All values are relative to control kept at 300 mosmol/kg. Mean ± SEM (∗, P < 0.05; n = 3).