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Molecular and Cellular Biology, November 2007, p. 7906-7917, Vol. 27, No. 22
0270-7306/07/$08.00+0     doi:10.1128/MCB.01369-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Regulation of Neuron Survival through an Intersectin-Phosphoinositide 3'-Kinase C2ß-AKT Pathway ^,

Margaret Das,¹ Erica Scappini,^1,2 Negin P. Martin,^1,2 Katy A. Wong,³ Sara Dunn,³ Yun-Ju Chen,³ Stephanie L. H. Miller,^1,4 Jan Domin,⁵ and John P. O'Bryan^1,3*

Laboratory of Signal Transduction,¹ Laboratory of Neurobiology, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, North Carolina 27709,² Department of Pharmacology, University of Illinois College of Medicine, Chicago, Illinois 60612,³ Department of Biomedical Engineering, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599,⁴ Division of Medicine, Imperial College School of Medicine, London W12 0NN, United Kingdom⁵

Received 30 July 2007/ Accepted 4 September 2007

While endocytosis attenuates signals from plasma membrane receptors, recent studies suggest that endocytosis also serves as a platform for the compartmentalized activation of cellular signaling pathways. Intersectin (ITSN) is a multidomain scaffolding protein that regulates endocytosis and has the potential to regulate various biochemical pathways through its multiple, modular domains. To address the biological importance of ITSN in regulating cellular signaling pathways versus in endocytosis, we have stably silenced ITSN expression in neuronal cells by using short hairpin RNAs. Decreasing ITSN expression dramatically increased apoptosis in both neuroblastoma cells and primary cortical neurons. Surprisingly, the loss of ITSN did not lead to major defects in the endocytic pathway. Yeast two-hybrid analysis identified class II phosphoinositide 3'-kinase C2ß (PI3K-C2ß) as an ITSN binding protein, suggesting that ITSN may regulate a PI3K-C2ß-AKT survival pathway. ITSN associated with PI3K-C2ß on a subset of endomembrane vesicles and enhanced both basal and growth factor-stimulated PI3K-C2ß activity, resulting in AKT activation. The use of pharmacological inhibitors, dominant negatives, and rescue experiments revealed that PI3K-C2ß and AKT were epistatic to ITSN. This study represents the first demonstration that ITSN, independent of its role in endocytosis, regulates a critical cellular signaling pathway necessary for cell survival.

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* Corresponding author. Mailing address: Department of Pharmacology, University of Illinois College of Medicine, 835 S. Wolcott, E403 M/C 868, Chicago, IL 60612. Phone: (312) 996-6221. Fax: (312) 996-1225. E-mail: obryanj{at}uic.edu

Published ahead of print on 17 September 2007.

Supplemental material for this article may be found at http://mcb.asm.org/.

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Molecular and Cellular Biology, November 2007, p. 7906-7917, Vol. 27, No. 22
0270-7306/07/$08.00+0     doi:10.1128/MCB.01369-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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