Cyclic AMP Inhibits Akt Activity by Blocking the Membrane Localization of PDK1

Abstract

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...

Differential Signaling of Cyclic AMP

“…Results from these PKA inhibitor studies further confirm that the inhibitory effect of cAMP on PKB is mediated by PKA. Our evidence that cAMP, acting through PKA, inhibits the endogenous PKB activity in HEK 293 cells is consistent with the recent report by Kim et al (47) showing that cAMP/PKA impairs EGF-induced activation of exogenous PKB activity in a PI3K-dependent manner in a wide variety of cell lines, including HEK293 cells. This inhibitory effect of cAMP on PKB activation is mediated by blocking the coupling between PKB and its upstream regulator PDK in the plasma membrane (47).…”

“…The PI3K/PKB signaling pathway is a key component in control of cell survival and proliferation (43,44). cAMP has been implicated in modulating PKB activity; however, elevation of intracellular cAMP concentration can lead to either inhibitory (45)(46)(47) or stimulatory (35,36,48) effects on PKB activity. At present, the role that Epac plays in this process is unclear.…”

Activation of tumor suppressor protein PTEN and induction of apoptosis are involved in cAMP-mediated inhibition of cell number in B92 glial cells

“…In response to cAMP stimulation, we observed an increase in the activity of PTEN and a decrease in Akt activity in B92 cells that resulted in the induction of apoptosis. An inhibitory effect of cAMP on the activation of Akt has been described in various cell types [2,5,6,12]. However, the mechanism of cAMP-induced inhibition of Akt is not yet fully understood.…”

“…However, the mechanism of cAMP-induced inhibition of Akt is not yet fully understood. Specifically, the role of PI3K in cAMP-mediated Akt inhibition is still controversial; previous studies have indicated either no effect [5] or negative effects [6] of cAMP on PI3K activity. Although PTEN is not a classical target of cAMP action, cAMP-dependent PTEN activation has been reported in alveolar macrophages and human glioma cells [2,12].…”

Deletion of PI3K-p85α gene impairs lineage commitment, terminal maturation, cytokine generation and cytotoxicity of NK cells

“…Akt, PDK1 and Btk are the major downstream signaling molecules of PI3K signalosomes. [24][25][26] Most of the mice deficient of p85a, p55a and p50a die at birth. 20 In this study, we investigated the role of p85a/p55a/ p50a subunits in NK cells.…”