1. p38 MAPK activation is required for phosphorylation of Akt at Ser473.
p38 MAPK activation is required for phosphorylation of Akt at Ser473. A. Effects of inhibition of p38 MAPK on PI3K- and PDK1-mediated phosphorylation of Akt in response to radiation. HeLa cells were exposed to 10 Gy γ-radiation in the presence or absence of 30 μmol/L SB After 48 h, cell lysates were immunoprecipitated with anti-PI3K (anti-p85), PDK1, or Akt antibody. PI3K, PDK1, and Akt kinase assays were done on immune complexes. A representative autoradiogram of PI3K assay is shown, and the position of phosphatidylinositol phosphate is indicated. GST-Akt and GST-GSK3-β fusion proteins were used as substrates for PDK1 and Akt, respectively. Cell lysates were also subjected to Western blot analysis with anti-Thr308-Akt, anti-Ser473-Akt, anti-Akt, and anti-PTEN antibodies. B. Effect of overexpression of dominant-negative p38 MAPK on radiation-induced Akt phosphorylation. HeLa cells were exposed to 10 Gy γ-radiation in the presence or absence of dominant-negative form of p38 MAPK. After 48 h, cell lysates were immunoprecipitated with anti-p38 MAPK or Ser473-Akt antibody, and p38 MAPK and Akt kinase assays were done on immune complexes. ATF2 and GST-GSK3-β fusion proteins were used as substrates for p38 MAPK and Akt, respectively. Cell lysates were also subjected to Western blot analysis with anti-Flag, anti-Thr308-Akt, anti-Ser473-Akt, anti-Akt, and anti-β-actin antibodies. β-Actin was used as a loading control. C. Effect of overexpression of p38 MAPK on radiation-induced Akt phosphorylation. HeLa cells were exposed to 10 Gy γ-radiation in the presence or absence of wild-type p38 MAPK. After 48 h, cell lysates were subjected to Western blot analysis with anti-Thr308-Akt, anti-Ser473-Akt, anti-Akt, anti-Flag, and anti-β-actin antibodies. β-Actin was used as a loading control. Cell lysates were also immunoprecipitated with anti-Ser473-Akt antibody, and Akt kinase assay was done on immune complexes. GST-GSK3-β fusion protein was used as substrates for Akt.
Min-Jung Kim et al. Mol Cancer Res 2008;6:
©2008 by American Association for Cancer Research