Cyclic AMP inhibits Akt activity by blocking the membrane localization of PDK1

Akt is a protein serine/threonine kinase that plays an important role in th e mitogenic responses of cells to variable stimuli. Akt contains a pleckstr in homology (PH) domain and is activated by phosphorylation at threonine 30 8 and serine 473, Binding of 3 ' -OH phosphorylated phosphoinositides to th e PH domain results in the translocation of Akt to the plasma membrane wher e it is activated by upstream kinases such as (phosphoinositide-dependent k inase-l (PDK1), Over-expression of constitutively active forms of Akt promo tes cell proliferation and survival, and also stimulates p70 S6 kinase (p70 S6K), In many cells, an increase in levels of intracellular cyclic AMP (cAM P) diminishes cell growth and promotes differentiation, and in certain cond itions 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 threon ine 308 and serine 473 of Akt, which is required for the protein kinase act ivities of Akt, cAMP also negatively regulates PDK1 by inhibiting its trans location to the plasma membrane, despite not affecting its protein kinase a ctivities. Furthermore, when we co-expressed myristoylated Akt and PDK1 mut ants which constitutively co-localize in the plasma membrane, Akt activity was no longer sensitive to raised intracellular cAMP concentrations. Finall y, 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, whi ch 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.