Inactivation and dephosphorylation of protein kinase Balpha (PKBalpha) promoted by hyperosmotic stress.

To study the role of protein kinase B (PKB) in response to cellular stress, we examined PKBalpha activity following different stress treatments. Hyperosmotic but not chemical stress resulted in inactivation of PKBalpha and prevented activation by pervanadate and mitogens. Hyperosmotic shock did not affect the MAP kinase pathway, suggesting that this inhibitory effect was specific for PKB. Our data further indicate that downregulation occurs via dephosphorylation of Thr308 and Ser473, the major regulatory phosphorylation sites of PKBalpha. Indeed, calyculin A, which inhibits protein phosphatases 1 and 2A, effectively blocked hyperosmotic stress-mediated inactivation (dephosphorylation) of PKBalpha. High osmolarity did not affect phosphatidylinositol 3-kinase activity but led to a marked increase in PI(3,4,5)P3 and a decrease in PI(3,4)P2 formation after pervanadate stimulation, suggesting that hyperosmotic stress has an inhibitory effect on a phosphatidylinositol 5-phosphatase which converts PI(3,4,5)P3 into PI(3,4)P2. Immunofluorescence studies revealed that membrane translocation, a prerequisite for PKB activation, was not affected by hyperosmotic stress. Our results indicate that hyperosmotic stress can act at two levels: (i) inhibition of phosphorylation of Thr308 and Ser473 by upstream kinases and (ii) by promoting rapid dephosphorylation of these regulatory sites.