Insulin-Induced Phosphorylation and Activation of Cyclic Nucleotide Phosphodiesterase 3B by the Serine-Threonine Kinase Akt

Abstract

Cyclic nucleotide phosphodiesterase (PDE) is an important regulator of the cellular concentrations of the second messengers cyclic AMP (cAMP) and cGMP. Insulin activates the 3B isoform of PDE in adipocytes in a phosphoinositide 3-kinase-dependent manner; however, downstream effectors that mediate signaling to PDE3B remain unknown. Insulin-induced phosphorylation and activation of endogenous or recombinant PDE3B in 3T3-L1 adipocytes have now been shown to be inhibited by a dominant-negative mutant of the serine-threonine kinase Akt, suggesting that Akt is necessary for insulin-induced phosphorylation and activation of PDE3B. Serine-273 of mouse PDE3B is located within a motif (RXRXXS) that is preferentially phosphorylated by Akt. A mutant PDE3B in which serine-273 was replaced by alanine was not phosphorylated either in response to insulin in intact cells or by purified Akt in vitro. In contrast, PDE3B mutants in which alanine was substituted for either serine-296 or serine-421, each of which lies within a sequence (RRXS) preferentially phosphorylated by cAMP-dependent protein kinase, were phosphorylated by Akt in vitro or in response to insulin in intact cells. Moreover, the serine-273 mutant of PDE3B was not activated by insulin when expressed in adipocytes. These results suggest that PDE3B is a physiological substrate of Akt and that Akt-mediated phosphorylation of PDE3B on serine-273 is important for insulin-induced activation of PDE3B.

ACKNOWLEDGMENTS

We thank I. Saito and M. D. Waterfield for pAxCALacZ and a baculovirus that encodes p110α, respectively.

This work was supported by a grant-in-aid for the Research for the Future Program from the Japan Society for the Promotion of Science (to M.K.); by Health Sciences Research Grants (Research on Human Genome and Gene Therapy) from the Ministry of Health and Welfare (to M.K.); by grants from the Ministry of Education, Science, Sports, and Culture of Japan (to M.K. and W.O.); by a grant from the Uehara Memorial Foundation (to M.K.); by a grant for studies on the pathophysiology and complications of diabetes from Tsumura Pharma Ltd. (to M.K.); by a grant from Takeda Science Foundation; and by a grant from ONO Medical Research Foundation (to W.O.). T.K. is a Japan Health Sciences Foundation (JHSF) Fellow.