The spatial and temporal regulation of the second messenger PtdIns(4,5)P2 has been shown to be crucial for regulating numerous processes in the cytoplasm and in the nucleus. Three isoforms of PIP5K1 (phosphatidylinositol 4-phosphate 5-kinase), A, B and C, are responsible for the regulation of the major pools of cellular PtdIns(4,5)P2. PIP5K1B is negatively regulated in response to oxidative stress although it remains unclear which pathways regulate its activity. In the present study, we have investigated the regulation of PIP5K1B by protein phosphorylation. Using MS analysis, we identified 12 phosphorylation sites on PIP5K1B. We developed a phospho-specific antibody against Ser413 and showed that its phosphorylation was increased in response to treatment of cells with phorbol ester, H2O2 or energy restriction. Using inhibitors, we define a stress-dependent pathway that requires the activity of the cellular energy sensor AMPK (AMP-activated protein kinase) and PKC (protein kinase C) to regulate Ser413 phosphorylation. Furthermore, we demonstrate that PKC can directly phosphorylate Ser413in vitro. Mutation of Ser413 to aspartate to mimic serine phosphorylation decreased both PIP5K1B activity in vitro and PtdIns(4,5)P2 synthesis in vivo. Our studies show that collaboration between AMPK and PKC dictates the extent of Ser413 phosphorylation on PIP5K1B and regulates PtdIns(4,5)P2 synthesis.
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Research Article|
October 10 2013
Collaboration of AMPK and PKC to induce phosphorylation of Ser413 on PIP5K1B resulting in decreased kinase activity and reduced PtdIns(4,5)P2 synthesis in response to oxidative stress and energy restriction
Iman van den Bout;
Iman van den Bout
*Inositide laboratory, The Paterson Institute for Cancer Research, Wilmslow Road, Manchester M20 4BX, U.K.
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David R. Jones;
David R. Jones
*Inositide laboratory, The Paterson Institute for Cancer Research, Wilmslow Road, Manchester M20 4BX, U.K.
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Zahid H. Shah;
Zahid H. Shah
*Inositide laboratory, The Paterson Institute for Cancer Research, Wilmslow Road, Manchester M20 4BX, U.K.
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Jonathan R. Halstead;
Jonathan R. Halstead
†Syngenta Cereals, Syngenta, 4006 Hawthorne Circle, Longmont, CO, U.S.A.
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Willem-Jan Keune;
Willem-Jan Keune
*Inositide laboratory, The Paterson Institute for Cancer Research, Wilmslow Road, Manchester M20 4BX, U.K.
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Shabaz Mohammed;
Shabaz Mohammed
‡Biomolecular Mass Spectrometry and Proteomics Group, Padualaan 8, Utrecht, 3584 CH, The Netherlands
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Clive S. D’Santos;
Clive S. D’Santos
§Cancer Research UK Cambridge Research Institute, Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE, U.K.
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Nullin Divecha
Nullin Divecha
1
*Inositide laboratory, The Paterson Institute for Cancer Research, Wilmslow Road, Manchester M20 4BX, U.K.
1To whom correspondence should be addressed (email ndivecha@picr.man.ac.uk).
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Publisher: Portland Press Ltd
Received:
February 19 2013
Revision Received:
July 25 2013
Accepted:
August 05 2013
Accepted Manuscript online:
August 05 2013
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2013 Biochemical Society
2013
Biochem J (2013) 455 (3): 347–358.
Article history
Received:
February 19 2013
Revision Received:
July 25 2013
Accepted:
August 05 2013
Accepted Manuscript online:
August 05 2013
Citation
Iman van den Bout, David R. Jones, Zahid H. Shah, Jonathan R. Halstead, Willem-Jan Keune, Shabaz Mohammed, Clive S. D’Santos, Nullin Divecha; Collaboration of AMPK and PKC to induce phosphorylation of Ser413 on PIP5K1B resulting in decreased kinase activity and reduced PtdIns(4,5)P2 synthesis in response to oxidative stress and energy restriction. Biochem J 1 November 2013; 455 (3): 347–358. doi: https://doi.org/10.1042/BJ20130259
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