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The selective and inducible activation of endogenous PI 3-kinase in PC12 cells results in efficient NGF-mediated survival but defective neurite outgrowth

Oncogene volume 18, pages 4586–4597 (1999)Cite this article

Abstract

The Trk/Nerve Growth Factor receptor mediates the rapid activation of a number of intracellular signaling proteins, including phosphatidylinositol 3-kinase (PI 3-kinase). Here, we describe a novel, NGF-inducible system that we used to specifically address the signaling potential of endogenous PI 3-kinase in NGF-mediated neuronal survival and differentiation processes. This system utilizes a Trk receptor mutant (Trkdef) lacking sequences Y490, Y785 and KFG important for the activation of the major Trk targets; SHC, PLC-γl, Ras, PI 3-kinase and SNT. Trkdef was kinase active but defective for NGF-induced responses when stably expressed in PC12nnr5 cells (which lack detectable levels of TrkA and are non-responsive to NGF). The PI 3-kinase consensus binding site, YxxM (YVPM), was introduced into the insert region within the kinase domain of Trkdef. NGF-stimulated tyrosine phosphorylation of the Trkdef+PI 3-kinase addback receptor, resulted in the direct association and selective activation of PI 3-kinase in vitro and the production of PI(3,4)P2 and PI(3,4,5)P3 in vivo (comparable to wild-type). PC12nnr5 cells stably expressing Trkdef+PI 3-kinase, initiated neurite outgrowth but failed to stably extend and maintain these neurites in response to NGF as compared to PC12 parental cells, or PC12nnr5 cells overexpressing wild-type Trk. However, Trkdef+PI 3-kinase was fully competent in mediating NGF-induced survival processes. We propose that while endogenous PI 3-kinase can contribute in part to neurite initiation processes, its selective activation and subsequent signaling to downstream effectors such as Akt, functions mainly to promote cell survival in the PC12 system.

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Acknowledgements

We especially thank KH Vousden at the ABL-Basic Research Program for helpful discussion and support. We thank G Vande Woude at NCI for interest in this study. We thank LA Greene for interesting discussion, TF Franke for advice on performing Akt assays, and DK Morrison for 4G10. This research was sponsored in part by the National Cancer Institute, DHHS under contract with ABL (M Ashcroft, RM Stephens, DR Kaplan), and by the National Cancer Institute of Canada (DR Kaplan). DR Kaplan is the recipient of the Harold E Johns Award from the National Cancer Institute of Canada.

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  1. Bengt Hallberg

    Present address: Umeå University, 87 Umeå, Sweden

  2. David R Kaplan

    Present address: Montreal Neurological Institute, McGill University, Montreal, PQ H3A 2B4, Canada

Authors and Affiliations

  1. ABL-Basic Research Program, NCI-FCRDC, West 7th Street, Frederick, MD 21702, Maryland, USA

    Margaret Ashcroft, Robert M Stephens & David R Kaplan

  2. Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London, WC2A 3PX, UK

    Bengt Hallberg & Julian Downward

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Ashcroft, M., Stephens, R., Hallberg, B. et al. The selective and inducible activation of endogenous PI 3-kinase in PC12 cells results in efficient NGF-mediated survival but defective neurite outgrowth. Oncogene 18, 4586–4597 (1999). https://doi.org/10.1038/sj.onc.1202814

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