Cyclic Nucleotide Phosphodiesterase 3B Is a Downstream Target of Protein Kinase B and May Be Involved in Regulation of Effects of Protein Kinase B on Thymidine Incorporation in FDCP2 Cells

Abstract

Wild-type (F/B), constitutively active (F/B*), and three kinase-inactive (F/Ba−, F/Bb−, F/Bc−) forms of Akt/protein kinase B (PKB) were permanently overexpressed in FDCP2 cells. In the absence of insulin-like growth factor-1 (IGF-1), activities of PKB, cyclic nucleotide phosphodiesterase 3B (PDE3B), and PDE4 were similar in nontransfected FDCP2 cells, mock-transfected (F/V) cells, and F/B and F/B− cells. In F/V cells, IGF-1 increased PKB, PDE3B, and PDE4 activities ∼2-fold. In F/B cells, IGF-1, in a wortmannin-sensitive manner, increased PKB activity ∼10-fold and PDE3B phosphorylation and activity (∼4-fold), but increased PDE4 to the same extent as in F/V cells. In F/B* cells, in the absence of IGF-1, PKB activity was markedly increased (∼10-fold) and PDE3B was phosphorylated and activated (3- to 4-fold); wortmannin inhibited these effects. In F/B* cells, IGF-1 had little further effect on PKB and activation/phosphorylation of PDE3B. In F/B− cells, IGF-1 activated PDE4, not PDE3B, suggesting that kinase-inactive PKB behaved as a dominant negative with respect to PDE3B activation. Thymidine incorporation was greater in F/B* cells than in F/V cells and was inhibited to a greater extent by PDE3 inhibitors than by rolipram, a PDE4 inhibitor. In F/B cells, IGF-1-induced phosphorylation of the apoptotic protein BAD was inhibited by the PDE3 inhibitor cilostamide. Activated PKB phosphorylated and activated rPDE3B in vitro. These results suggest that PDE3B, not PDE4, is a target of PKB and that activated PDE3B may regulate cAMP pools that modulate effects of PKB on thymidine incorporation and BAD phosphorylation in FDCP2 cells.

Novel mechanisms of the regulation of Protein kinase B in adipocytes; implications for Protein kinase A, Epac, Phosphodiesterases 3 and 4

“…Interestingly, PDE3B which seems to regulate a pool of cAMP mediating inhibition of PKB phosphorylation, is at the same time a substrate for PKB [35]. Our results show that stimuli that alter PKB phosphorylation, also affect PDE3B activity.…”

“…In summary, these data suggest that PDE3B mediates the regulation of Ser 473 PKB phosphorylation. However, the opposite situation also appears to occur as PKB has previously been shown to regulate PDE3B phosphorylation and activation [16,35].…”

High Density Lipoprotein-induced Endothelial Nitric-oxide Synthase Activation Is Mediated by Akt and MAP Kinases

“…A mutant of eNOS with Ser-1179 converted to alanine (S1179A eNOS) is unable to be phosphorylated at that site. The triple Akt mutant AktAAA (K179A,T308A,S403A) is enzymatically inactive (29,30), whereas the Akt mutant generated by fusing a myristoylation signal to its amino terminus (AktMyr) is membrane-bound and constitutively active (31,32). The mutant form of PI3 kinase lacking the domain in the p85 subunit that is required for interaction with the catalytic subunit (Sr␣⌬85) works as a dominant negative mutant (33).…”

Akt Activation Protects Hippocampal Neurons from Apoptosis by Inhibiting Transcriptional Activity of p53

“…In Vivo Kinase Assay-To examine the effect of Akt activation on the phosphorylation status of p53, in vivo kinase assays were performed according to the published method (29). Neurons derived from rat fetal brains were seeded in 35-mm dishes at the density of 1-2 ϫ 10 6 cells/dish.…”