Akt is a protein serine/threonine kinase that plays an important role in the mitogenic responses of cells to variable stimuli. Akt contains a pleckstrin homology (PH) domain and is activated by phosphorylation at threonine 308 and serine 473. Binding of 3-OH phosphorylated phosphoinositides to the PH domain results in the translocation of Akt to the plasma membrane where it is activated by upstream kinases such as (phosphoinositide-dependent kinase-1 (PDK1). Over-expression of constitutively active forms of Akt promotes cell proliferation and survival, and also stimulates p70 S6 kinase (p70S6K). In many cells, an increase in levels of intracellular cyclic AMP (cAMP) diminishes cell growth and promotes differentiation, and in certain conditions cAMP is even antagonistic to the effect of growth factors. Here, we show that cAMP has inhibitory effects on the phosphatidylinositol 3-kinase/PDK/Akt signaling pathway. cAMP potently inhibits phosphorylation at threonine 308 and serine 473 of Akt, which is required for the protein kinase activities of Akt. cAMP also negatively regulates PDK1 by inhibiting its translocation to the plasma membrane, despite not affecting its protein kinase activities. Furthermore, when we co-expressed myristoylated Akt and PDK1 mutants which constitutively co-localize in the plasma membrane, Akt activity was no longer sensitive to raised intracellular cAMP concentrations. Finally, cAMP was also found to inhibit the lipid kinase activity of PI3K and to decrease the levels of phosphatidylinositol 3,4,5-triphosphate in vivo, which are required for the membrane localization of PDK1. Collectively, these data strongly support the theory that the cAMP-dependent signaling pathway inhibits Akt activity by blocking the coupling between Akt and its upstream regulators, PDK, in the plasma membrane.The phosphatidylinositol 3-kinase (PI3K) 1 -dependent cell signaling pathway has emerged as a key regulatory pathway involved in a number of cellular events (1). Upon activation of growth factor tyrosine kinase receptors, the p85 regulatory subunit of PI3K recruits the p110 catalytic subunit to the plasma membrane (2). The p110 catalytic subunit increases the level of PtdIns-3,4,5-P 3 and phosphatidylinositol 3,4-bisphosphate (PtdIns-3,4-P 2 ), which induce the membrane translocation of PDK1 and Akt (also called PKB or RAC-PK) by binding to the pleckstrin homology domain (3). In the membrane, PDK1 phosphorylates and activates Akt in a PtdIns-3,4,5-P 3 -or PtdIns-3,4-P 2 -dependent manner (4, 5). By a mechanism that involves phospholipase C (6), activated Akt is released from the membrane and phosphorylates various targets.This complex and unique signaling pathway has been implicated in a variety of cellular events such as cell proliferation and survival (1, 7). Previously, it has been shown that various survival factors, such as nerve growth factor, require the activation of PI3K to prevent various cell types from undergoing apoptosis (8, 9). The mechanism by which the PI3K pathway protects cells from programm...

Akt is stimulated by several growth factors, and mediates their cell survival signals. Recent studies have shown that Akt may play an intermediate role between phosphatidylinositol 3-kinase (PI3K) and p70 S6 kinase (p70S6K). Here we show that a novel nuclear p70S6K-related kinase (SRK) exists and that its in vivo function is also augmented by over-expression of Akt. Conceptual translation of the SRK cDNA revealed that the catalytic domain of SRK was highly homologous to that of p70S6K, and that the treatment of wortmannin or rapamycin strongly inhibited the phosphorylation and the activation of SRK, as in p70S6K. However, the Nand C-terminal domains of SRK were quite di erent from those of p70S6K. In immunolocalization analyses, we demonstrated a constitutive nuclear localization of SRK and the presence of a nuclear localization signal in its C-terminus. In vitro S6 phosphotransferase activities of SRK were stimulated with a slower kinetics by a variety of agonists to p70S6K. Interestingly, overexpression of the proto-oncogene Akt resulted in EGFindependent activation of SRK, while over-expression of kinase-dead Akt actually had an inhibitory e ect. This relationship between Akt and SRK suggests that SRK may be a novel target of Akt and perhaps an important downstream component in the nuclear function of Akt.