title: Akt/protein kinase B up-regulates Bcl-2 expression through cAMP-response element-binding protein.
abstract: 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/p38beta 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, we define a second pathway contributing to CREB-dependent up-regulation of Bcl-2 expression as a novel anti-apoptotic function of Akt signaling. To examine the role of Akt on Bcl-2 expression, a series of transient transfections using a luciferase reporter gene driven by the promoter region of Bcl-2 containing a CRE were carried out. Pharmacological inhibition of phosphatidylinositol (PI) 3-kinase, the upstream kinase of Akt, with LY294002 led to a 45% decrease in Bcl-2 promoter activity. The reporter activity was enhanced 2.3-fold by overexpression of active p110 subunit of PI 3-kinase and inhibited 44% by the dominant negative p85 subunit of PI 3-kinase. Cotransfection with 3-phosphoinositide-dependent kinase (PDK1), which is required for the full activation of Akt, resulted in enhanced luciferase activity. Insulin-like growth factor-I-mediated induction of Bcl-2 promoter activity was decreased significantly (p < 0.01) by the dominant negative forms of p85 subunit 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-kinase/PDK1/Akt/CREB. Furthermore, we measured the Bcl-2 mRNA in PC12 cells overexpressing 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 survival.
authors.affiliation: Department of Endocrinology, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
authors.forename: S
authors.initials: S
authors.lastname: Pugazhenthi
authors.forename: A
authors.initials: A
authors.lastname: Nesterova
authors.forename: C
authors.initials: C
authors.lastname: Sable
authors.forename: K A
authors.initials: KA
authors.lastname: Heidenreich
authors.forename: L M
authors.initials: LM
authors.lastname: Boxer
authors.forename: L E
authors.initials: LE
authors.lastname: Heasley
authors.forename: J E
authors.initials: JE
authors.lastname: Reusch
mesh_terms: D000818:Animals; D017362:Cyclic AMP Response Element-Binding Protein / Q000502:physiology*; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D016716:PC12 Cells; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000502:physiology*; D051057:Proto-Oncogene Proteins c-akt; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis* / Q000235:genetics; D051381:Rats; D015854:Up-Regulation
chemical_list: D017362:Cyclic AMP Response Element-Binding Protein; D007334:Insulin-Like Growth Factor I; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D019253:Proto-Oncogene Proteins c-bcl-2; C494920:Akt1 protein, rat