Abstract
Previous studies have demonstrated that AKT1 and AKT3 are activated by heat shock and oxidative stress via both phosphatidylinositol 3-kinase-dependent and -independent pathways. However, the activation and role of AKT2 in the stress response have not been fully elucidated. In this study, we show that AKT2 in epithelial cells is activated by UV-C irradiation, heat shock, and hyperosmolarity as well as by tumor necrosis factor alpha (TNFalpha) through a phosphatidylinositol 3-kinase-dependent pathway. The activation of AKT2 inhibits UV- and TNF alpha-induced c-Jun N-terminal kinase (JNK) and p38 activities that have been shown to be required for stress- and TNF alpha-induced programmed cell death. Moreover, AKT2 interacts with and phosphorylates I kappa B kinase alpha. The phosphorylation of I kappa B kinase alpha and activation of NF kappa B mediates AKT2 inhibition of JNK but not p38. Furthermore, phosphatidylinositol 3-kinase inhibitor or dominant negative AKT2 significantly enhances UV- and TNF alpha-induced apoptosis, whereas expression of constitutively active AKT2 inhibits programmed cell death in response to UV and TNFalpha -induced apoptosis by inhibition of stress kinases and provide the first evidence that AKT inhibits stress kinase JNK through activation of the NF kappa B pathway.
Highlights
AKT3/protein kinase B␥ (AKT3) are activated by heat shock and oxidative stress derived growth factor, tumor necrosis factor ␣ (TNF␣), epidervia both phosphatidylinositol 3-kinase-dependent and mal growth factor, and insulin-like growth factor 1 (IGF1)
We show that AKT2 in epithe- nase (PI3K) [1], and of the downstream targets of PI3K, AKT is lial cells is activated by UV-C irradiation, heat shock, thought to play an essential role in the cellular response to and hyperosmolarity as well as by tumor necrosis factor stress
AKT2 Is Activated by UV Irradiation, Heat Shock, Hyperosmolarity, and TNF␣—Previous studies showed that stress activates AKT1 and AKT3 but not AKT2 in fibroblasts [19]
Summary
Cell Lines, Transfection, and Stimulation—The human epithelial cancer cell lines, A2780, OVCAR3, and human embryonic kidney (HEK) 293 cells were cultured at 37 °C and 5% CO2 in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum. Protein expression was determined by probing Western blots of immunoprecipitates or total cell lysates with the antibodies described above or with the appropriate antibodies as noted in figure legends. The presence of PI3K activity in the immunoprecipitates was determined by incubating the beads in reaction buffer (10 mM HEPES (pH 7.4), 10 mM MgCl2, 50 M ATP) containing 20 Ci [␥-32P]ATP and 10 g L-␣-phosphatidylinositol 4,5-bisphosphate (Biomol) for 20 min at 25 °C. Terminal Deoxynucleotidyltransferase-mediated dUTP Nick End Labeling (TUNEL) Assay—AKT2 stably transfected A2780 cells were seeded into 60-mm dishes and grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum for 24 h and pretreated with or without LY294002 for 2 h before exposure to UV, heat shock, NaCl, or TNF␣.
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