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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We have screened for a factor that induces neurite outgrowth by PC12 cells only after NGF pretreatment in the supernatants of 38 transformed cell lines, finding three positive clones. The factor showing the strongest activity was purified and identified as IL6. In this NGF-IL6 system, the process of neurite outgrowth by PC12 cells can be separated into NGF-dependent and IL6-dependent steps. The IL6-dependent step requires RNA synthesis, suggesting that IL6 induces new gene expression depending on NGF-priming. These findings suggest that the gene expression during the differentiation process is regulated by at least two signals.