Ahmad F, Cong L N, Stenson Holst L, Wang L M, Rahn Landstrom T, Pierce J H, Quon M J, Degerman E, Manganiello V C
Pulmonary/Critical Care Medicine Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
J Immunol. 2000 May 1;164(9):4678-88. doi: 10.4049/jimmunol.164.9.4678.
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 approximately 2-fold. In F/B cells, IGF-1, in a wortmannin-sensitive manner, increased PKB activity approximately 10-fold and PDE3B phosphorylation and activity ( approximately 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 ( approximately 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.
野生型（F/B）、组成型激活型（F/B*）以及三种激酶失活型（F/Ba-、F/Bb-、F/Bc-）的Akt/蛋白激酶B（PKB）在FDCP2细胞中持续过表达。在缺乏胰岛素样生长因子-1（IGF-1）的情况下，非转染的FDCP2细胞、mock转染（F/V）细胞以及F/B和F/B-细胞中，PKB、环核苷酸磷酸二酯酶3B（PDE3B）和PDE4的活性相似。在F/V细胞中，IGF-1使PKB、PDE3B和PDE4的活性增加约2倍。在F/B细胞中，IGF-1以渥曼青霉素敏感的方式使PKB活性增加约10倍，PDE3B的磷酸化和活性增加（约4倍），但PDE4的增加程度与F/V细胞相同。在F/B细胞中，在缺乏IGF-1的情况下，PKB活性显著增加（约10倍），PDE3B被磷酸化并激活（3至4倍）；渥曼青霉素抑制了这些作用。在F/B细胞中，IGF-1对PKB以及PDE3B的激活/磷酸化几乎没有进一步影响。在F/B-细胞中，IGF-1激活PDE4，而非PDE3B，这表明激酶失活的PKB在PDE3B激活方面表现为显性负性。F/B*细胞中的胸苷掺入比F/V细胞中的更多，并且与PDE4抑制剂咯利普兰相比，PDE3抑制剂对其抑制作用更大。在F/B细胞中，PDE3抑制剂西洛酰胺抑制了IGF-1诱导的凋亡蛋白BAD的磷酸化。激活的PKB在体外使rPDE3B磷酸化并激活。这些结果表明，PKB的作用靶点是PDE3B而非PDE4，并且激活的PDE3B可能调节cAMP池，从而调节PKB对FDCP2细胞中胸苷掺入和BAD磷酸化的影响。
