Akt/protein kinase B up-regulates Bcl-2 expression through cAMP-response element-binding protein

In our previous study we showed that insulin-like growth factor-I induces a cAMP-response element (CRE) site-containing Bcl-2 promoter through a novel signaling pathway involving mitogen-activated protein kinase kinase 6/p38 beta mitogen-activated protein kinase/MAP kinase-activated protein kinase-3 /cAMP-response element-binding protein (CREB) (Pugazhenthi, S., Miller, E., Sable, C., Young, P., Heidenreich, K. A., Boxer, L. M., and Reusch, J. E.- B. (1999) J. Biol. Chem. 274, 27529-27535), In the present investigation, w e define a second pathway contributing to CREB-dependent up-regulation of B cl-2 expression as a novel anti-apoptotic function of Akt signaling. To exa mine the role of Akt on Bcl-2 expression, a series of transient transfectio ns using a luciferase reporter gene driven by the promoter region of Bcl-2 containing a CRE were carried out. Pharmacological inhibition of phosphatid ylinositol (PI) 3-kinase, the upstream kinase of Akt, with LY294002 led to a 45% decrease in Bcl-2 promoter activity. The reporter activity was enhanc ed 2.3-fold by overexpression of active p110 subunit of PI 3-kinase and inh ibited 44% by the dominant negative p85 subunit of PI 3-kinase, Cotransfect ion with 3-phosphoinositide-dependent kinase (PDK1), which is required for the full activation of Akt, resulted in enhanced luciferase activity. Insul in-like growth factor-I-mediated induction of Bcl-2 promoter activity was d ecreased significantly (p < 0.01) by the dominant negative forms of p85 sub unit of PI 3-kinase, PDK1, and Akt, These data indicate that regulation of Bcl-2 expression by IGF-I involves a signaling cascade mediated by PI 3-kin ase/PDK1/Akt/CREB. Furthermore, we measured the Bcl-2 mRNA in PC12 cells ov erexpressing Akt by real-time quantitative reverse transcription-polymerase chain reaction using the TaqMan(TM) fluorogenic probe system. We observed a 2.1-fold increase in Bcl-2 mRNA levels in the Akt cell line compared with control PC12 cells, supporting the observation that enhanced CREB activity by Akt signaling leads to increased Bcl-2 promoter activity and cell survi val.