Related Experiment Videos
Akt/protein kinase B up-regulates Bcl-2 expression through cAMP-response element-binding protein
S Pugazhenthi1, A Nesterova, C Sable
1Department of Endocrinology, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
The Journal of Biological Chemistry
|February 7, 2001
Summary
Insulin-like growth factor-I (IGF-I) activates a second pathway, involving Akt signaling, to increase Bcl-2 expression and promote cell survival. This Akt/CREB pathway complements the previously identified MAPK pathway, highlighting a dual mechanism for IGF-I in regulating apoptosis.
Area of Science:
- Molecular Biology
- Cell Signaling
- Biochemistry
Background:
- Insulin-like growth factor-I (IGF-I) is known to influence cell survival.
- A previously identified pathway involving MAPK kinase 6/p38beta MAPK/MAP kinase-activated protein kinase-3/cAMP-response element-binding protein (CREB) mediates IGF-I-induced Bcl-2 promoter activity.
- The precise signaling cascades regulating Bcl-2 expression, a key anti-apoptotic protein, require further elucidation.
Purpose of the Study:
- To identify and characterize a second signaling pathway through which IGF-I up-regulates Bcl-2 expression.
- To investigate the role of Akt signaling in the regulation of Bcl-2 promoter activity and expression.
- To determine the involvement of phosphatidylinositol 3-kinase (PI 3-kinase)/Akt/CREB cascade in IGF-I-mediated Bcl-2 induction.
Main Methods:
- Transient transfections using a luciferase reporter gene driven by the Bcl-2 promoter containing a cAMP-response element (CRE).
- Pharmacological inhibition of PI 3-kinase using LY294002.
- Overexpression of active and dominant-negative subunits of PI 3-kinase, PDK1, and Akt.
- Real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) to measure Bcl-2 mRNA levels.
Main Results:
- Inhibition of PI 3-kinase decreased Bcl-2 promoter activity by 45%.
- Overexpression of active PI 3-kinase enhanced reporter activity, while dominant-negative PI 3-kinase inhibited it.
- Dominant-negative forms of PI 3-kinase, PDK1, and Akt significantly reduced IGF-I-mediated Bcl-2 promoter activity.
- Overexpression of Akt led to a 2.1-fold increase in Bcl-2 mRNA levels.
Conclusions:
- IGF-I regulates Bcl-2 expression through a PI 3-kinase/PDK1/Akt/CREB signaling cascade.
- Akt signaling contributes to CREB-dependent up-regulation of Bcl-2, representing a novel anti-apoptotic function.
- This Akt-mediated pathway, alongside the previously identified MAPK pathway, provides a comprehensive understanding of IGF-I's role in cell survival regulation.