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.
Highlights
The serine threonine kinase Akt/protein kinase B is an important mediator of metabolic as well as survival responses to insulin and growth factors [1]
In our previous studies in PC12 cells, we identified that three post-receptor pathways activated by IGF-I through extracellular-regulated kinase, p38 MAPK, and PI 3-kinase are capable of mediating Ser-133 phosphorylation of CREB [13, 16]
The objective of the present study was to examine whether IGF-I mediated signaling through PI 3-kinase and Akt leads to a CREBdependent increase in Bcl-2 promoter activity
Summary
The serine threonine kinase Akt/protein kinase B is an important mediator of metabolic as well as survival responses to insulin and growth factors [1]. In addition to its metabolic actions, Akt/protein kinase B has been shown to promote cell survival by growth factors against several apoptotic stimuli [3, 4]. In our previous studies in PC12 cells, we identified that three post-receptor pathways activated by IGF-I through extracellular-regulated kinase, p38 MAPK, and PI 3-kinase are capable of mediating Ser-133 phosphorylation of CREB [13, 16]. Du and Montminy [15] demonstrated recently that Akt stimulates the phosphorylation and the transcriptional activity of the CREB in HEK 293 cells These reports clearly raise the possibility that Akt could mediate part of the IGF-I-induced increase in the expression of Bcl-2 at the transcriptional level. The objective of the present study was to examine whether IGF-I mediated signaling through PI 3-kinase and Akt leads to a CREBdependent increase in Bcl-2 promoter activity
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