Phosphodiesterase (PDE)-3B, a major PDE isoform in adipocytes, plays a pivotal role in the antilipolytic action of insulin. Insulin-induced phosphorylation and activation of PDE3B is phosphatidylinositol 3-kinase (PI3-K) and Akt dependent, but the precise mechanism of PDE3B activation is not fully understood. We have identified 14-3-3 β, a critical scaffolding molecule in signal transduction, as a protein that interacts with PDE3B using the yeast two-hybrid system. The interaction between PDE3B and 14-3-3 β was then confirmed in vitro. The glutathione S-transferase (GST)-tagged 14-3-3 β interacts with endogenous PDE3B of rat adipocytes, and this interaction is enhanced when adipocytes are treated with insulin. Coimmunoprecipitation experiments reveal that endogenous PDE3B also associates with endogenous 14-3-3 β in rat adipocytes, and this interaction is enhanced by insulin. Two different PI3-K inhibitors, wortmannin and Ly294002, block this induction, suggesting that PI3-K is required. Synthetic 15 amino acid peptides of rat PDE3B containing phosphorylated Ser-279 or -302 inhibit this interaction, indicating that the insulin-regulated phosphorylation of these serine residues is involved. Because insulin receptor substrate-1 also associates with 14-3-3, the dimeric 14-3-3 β could function as a scaffolding protein in the activation of PDE3B by insulin.
Address correspondence and reprint requests to Dr. H. Osawa and Dr. H. Makino, Department of Laboratory Medicine, Ehime University School of Medicine, Shigenobu, Ehime 791-0295, Japan. E-mail:, .
Received for publication 7 April 2002 and accepted in revised form 23 August 2002.
β-gal, β-galactosidase; GST, glutathione S-transferase; IRS, insulin receptor substrate, PDE, phosphodiesterase; PI3-K, phosphatidylinositol 3-kinase; PMSF, phenylmethylsulfonyl fluoride.