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J. Biol. Chem., Vol. 283, Issue 4, 2407-2417, January 25, 2008

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Inhibition of Raf-1 Alters Multiple Downstream Pathways to Induce Pancreatic β-Cell Apoptosis^*

From the Laboratory of Molecular Signaling in Diabetes, Diabetes Research Group, Departments of Cellular and Physiological Sciences and Surgery, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada

The serine threonine kinase Raf-1 plays a protective role in many cell types, but its function in pancreatic β-cells has not been elucidated. In the present study, we examined whether primary β-cells possess Raf-1 and tested the hypothesis that Raf-1 is critical for β-cell survival. Using reverse transcriptase-PCR, Western blot, and immunofluorescence, we identified Raf-1 in human islets, mouse islets, and in the MIN6 β-cell line. Blocking Raf-1 activity using a specific Raf-1 inhibitor or dominant-negative Raf-1 mutants led to a time- and dose-dependent increase in cell death, assessed by real-time imaging of propidium iodide incorporation, TUNEL, PCR-enhanced DNA laddering, and Caspase-3 cleavage. Although the rapid increase in apoptotic cell death was associated with decreased Erk phosphorylation, studies with two Mek inhibitors suggested that the classical Erk-dependent pathway could explain only part of the cell death observed after inhibition of Raf-1. An alternative Erk-independent pathway downstream of Raf-1 kinase involving the pro-apoptotic protein Bad has recently been characterized in other tissues. Inhibiting Raf-1 in β-cells led to a striking loss of Bad phosphorylation at serine 112 and an increase in the protein levels of both Bad and Bax. Together, our data strongly suggest that Raf-1 signaling plays an important role regulating β-cell survival, via both Erk-dependent and Bad-dependent mechanisms. Conversely, acutely inhibiting phosphatidylinositol 3-kinase Akt had more modest effects on β-cell death. These studies identify Raf-1 as a critical anti-apoptotic kinase in pancreatic β-cells and contribute to our understanding of survival signaling in this cell type.

Received for publication, May 1, 2007 , and in revised form, October 18, 2007.

^* This work was supported in part by operating grants from the Canadian Institutes of Health Research (to J. D. J.) and the Canadian Diabetes Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Cordula and Gunter Paetzold Fellow and supported by National Institutes of Health National Research Service Award F31DK079346.

² Supported by salary awards from the Michael Smith Foundation for Health Research, the Juvenile Diabetes Research Foundation, the Canadian Institutes of Health Research, and the Canadian Diabetes Association. To whom correspondence should be addressed: 5358 Life Sciences Bldg., 2350 Health Sciences Mall, Vancouver, British Columbia V6T 1Z3, Canada. Fax: 604-822-6048; E-mail: jimjohn{at}interchange.ubc.ca.

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