The Ser/Thr-specific phosphatase PHLPP (pleckstrin homology domain leucine-rich repeat protein phosphatase) regulates the amplitude and duration of agonist-evoked Akt signaling by dephosphorylating the hydrophobic motif (Ser473) of Akt, therefore inactivating Akt. We recently reported that gene transfer of adenylyl cyclase type 6 (AC6) into neonatal rat cardiac myocytes was associated with increased Akt phosphorylation and activity. To determine the underlying mechanisms for AC6-associated increase in Akt activation, we determined how AC6 gene transfer regulated the activity of PHLPP2 (one of the three PHLPP family phosphatases) in neonatal rat cardiac myocytes. We found that increased Akt activity was associated with inhibition of PHLPP2 activity by AC6. AC6 was physically associated with PHLPP2, which prevents PHLPP2-mediated Akt dephosphorylation. However, isoproterenol or forskolin stimulation immediately activated PHLPP2, which resulted in markedly dephosphorylation of Akt at Ser473. Activation of PHLPP2 by isoproterenol and forskolin was cAMP-independent, but required an intact cytoplasmic domain of AC6. Mutation in the cytoplasmic domain of AC6 abolished agonist-induced PHLPP2 activation. This novel bidirectional regulation of Akt activity may contribute to the unexpected favorable effects of AC6 on the failing heart. (C) 2009 Elsevier Inc. All rights reserved.