Activation of Protein Kinase B Induced by H2O2 and Heat Shock through Distinct Mechanisms Dependent and Independent of Phosphatidylinositol 3-Kinase

Abstract

Protein kinase B (PKB) is a downstream target of phosphatidylinositol (PI) 3-kinase in the signaling pathway of growth factors, and is activated by cellular stress such as H 2 O 2 and heat shock. To study the mechanism of the stress-induced activation of PKB, PI 3-kinase products were measured in stress-stimulated cells. Both PI 3,4-bisphosphate and PI 3,4,5-trisphosphate increased in H 2 O 2 -treated cells, and the elevation of these phospholipids and activation of PKB were concurrently blocked by wortmannin, a potent inhibitor of PI 3-kinase. In heat-shocked cells, the level of PI 3,4-bisphosphate did not change while that of PI 3,4,5-trisphosphate increased slightly, and an association between PKB molecules was observed. Two active PKB fractions, presumably monomeric and oligomeric forms, were resolved from heat-shocked cells by gel filtration column chromatography. Activation of the former was suppressed by pretreatment with wortmannin, whereas the generation and activation of the latter were not blocked by the PI 3-kinase inhibitor. Only the monomeric form, but not the oligomeric form, was recovered from H 2 O 2 -treated cells, and its activation was prevented by wortmannin. These results indicate that PKB is activated by two distinct mechanisms that are dependent and independent of PI 3-kinase in stress-stimulated cells.Key words: heat shock, hydrogen peroxide (H 2 O 2 ), phosphatidylinositol 3-kinase, pleckstrin homology domain, protein kinase B.Protein kinase B (PKB, also named Akt and RAC-protein phosphatidylinositol (PI) 3-kinase in the growth factor kinase) is a serine/threonine protein kinase with a plecksignaling pathway {3-6). Namely, PKB was first reported strin homology (PH) domain in its arnino-tenninal region to be activated by direct interaction of PI 3,4-bisphosphate and a catalytic domain in its carboxyl-terminal region (1, (PI 3,4-P 2 ) with its PH domain (7-9), and then the phos-2). This protein kinase was identified as an enzyme with a phorylation of PKB was shown to be indispensable for its catalytic domain closely related to both . In PKBa, Thr 308 in the activation loop protein kinase and protein kinase C, and also as a cellular of its catalytic domain and Ser 473 in the carboxyl-terminal counterpart of a rodent viral oncogene v-akt. Thus far, end region were identified as the phosphorylation sites three mammalian PKB genes, a, fi, and y, have been (12). Later, PDK1 (3-phosphoinositide-dependent protein isolated. Studies on physiological roles of PKB have revealkinase) and related enzymes were isolated that catalyze the ed that this protein kinase is a downstream target of phosphorylation of Thr 308 of PKB a-in the presence of PI 3, 4,5-trisphosphate (PI 3,4,5-P 3 ) and PI 3,4-P 2 (13-16). 'This study was supported in part by research grants from the Thus, the direct association of the PI 3 -kinase products with Scientific Research Funds of the Ministry of Education, Science, the PH domain and phosphorylation by upstream protein Sports and Culture of Japan, the Suntory Institute for Bi...

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“…However, the opposite results were observed by other groups (19,20). Konishi et al also provide evidence of AKT1 activation by H 2 O 2 and heat shock through both PI3K-dependent and -independent pathways (18). We previously demonstrated that activation of AKT2 by growth factors required PI3K activity, whereas both PI3K-dependent and -independent pathways contributed to AKT2 activation by Ras (26).…”

“…Two previous reports showed that PI3K inhibitors did not block heat shock-or H 2 O 2 -induced activation of AKT1 and, thus, suggested that stress (unlike growth factors) activates AKT1 in a PI3K-indpendent manner (17,18). However, the opposite results were observed by other groups (19,20).…”

“…2A). As described above, stress has been shown to activate AKT1 by both PI3K-dependent and -independent pathways (17,18). To assess the role of PI3K in the stress-induced activation of AKT2, A2780 cells transfected with HA-AKT2 were exposed to 25 M LY294002, a specific PI3K inhibitor, for 30 min before stress or TNF␣ treatments.…”

“…It is controversial whether stress-induced AKT1 activation is mediated by the PI3K pathway (17)(18)(19). Two previous reports showed that PI3K inhibitors did not block heat shock-or H 2 O 2 -induced activation of AKT1 and, thus, suggested that stress (unlike growth factors) activates AKT1 in a PI3K-indpendent manner (17,18).…”

“…Two separate studies demonstrated that AKT1 is activated when NIH 3T3 fibroblasts are stressed in a variety of ways (17,18). Based on data showing that PI3K inhibitors do not prevent AKT1 activation by stress, these studies concluded that stressinduced AKT1 activation was PI3K-independent.…”