Akt/Protein Kinase B Up-regulates Bcl-2 Expression through cAMP-response Element-binding Protein

A novel approach to quantitative reverse transcriptase polymerase chain reaction (QC RT-PCR) using real time detection and the 5' nuclease assay has been developed. Cystic fibrosis transmembrane transductance regulator (CFTR) target mRNA is reverse transcribed, amplified, detected, and quantitated in real time. A fluorogenic probe was designed to detect the CFTR amplicon. Relative increase in 6-carboxy-fluorescein reporter fluorescent emission is monitored during PCR amplification using an analytical thermal cycler. An internal control template containing the same primer sequences as the CFTR amplicon, but a different internal sequence, has been designed as a control. An internal control probe with a reporter fluorescent dye tetrachloro-6-carboxy-fluorescein was designed to hybridize to the internal control amplicon. The internal control template is placed in each reaction tube and is used for quantitative analysis of the CFTR mRNA. This method provides a convenient and high-throughput format for QC RT-PCR.

The nuclear factor CREB stimulates the expression of cellular genes following its protein kinase A-mediated phosphorylation at Ser-133. Ser-133 phosphorylation, in turn, activates target gene expression by promoting recruitment of the co-activator CBP. Recent studies showing that CREB and its paralog CREM are required for survival of certain cell types prompted us to examine whether CREB is a nuclear target for activation via the growth factor-dependent Ser/Thr kinase Akt/PKB. When overexpressed in serum-stimulated cells, Akt/PKB potently induced Ser-133 phosphorylation of CREB and promoted recruitment of CBP. Correspondingly, Akt/PKB stimulated target gene expression via CREB in a phospho(Ser-133)-dependent manner. Akt/PKB induced CREB activity only in response to serum stimulation, and this effect was suppressed by the phosphatidylinositol 3-kinase inhibitor LY 294002. Our results support the notion that Akt/PKB promotes cell survival, at least in part, by stimulating the expression of cellular genes via the CREB/CBP nuclear transduction pathway.

Protein kinase B (PKB)/Akt is a growth-factor-regulated serine/threonine kinase which contains a pleckstrin homology domain. Binding of phosphoinositide 3-OH kinase products to the pleckstrin homology domain results in translocation of PKB/Akt to the plasma membrane where it is activated by phosphorylation by upstream kinases including the phosphoinoside-dependent kinase 1 (PDK1). Activated PKB/Akt provides a survival signal that protects cells from apoptosis induced by various stresses, and also mediates a number of metabolic effects of insulin.