The renal-specific NKCC2 (Na+–K+–2Cl− co-transporter 2) is regulated by changes in phosphorylation state, however, the phosphorylation sites and kinases responsible have not been fully elucidated. In the present study, we demonstrate that the metabolic sensing kinase AMPK (AMP-activated protein kinase) phosphorylates NKCC2 on Ser126in vitro. Co-precipitation experiments indicated that there is a physical association between AMPK and the N-terminal cytoplasmic domain of NKCC2. Activation of AMPK in the MMDD1 (mouse macula densa-derived 1) cell line resulted in an increase in Ser126 phosphorylation in situ, suggesting that AMPK may phosphorylate NKCC2 in vivo. The functional significance of Ser126 phosphorylation was examined by mutating the serine residue to an alanine residue resulting in a marked reduction in co-transporter activity when exogenously expressed in Xenopus laevis oocytes under isotonic conditions. Under hypertonic conditions no significant change of activity was observed. Therefore the present study identifies a novel phosphorylation site that maintains NKCC2-mediated transport under isotonic or basal conditions. Moreover, the metabolic-sensing kinase, AMPK, is able to phosphorylate this site, potentially linking the cellular energy state with changes in co-transporter activity.
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Research Article|
June 13 2007
Regulation of the renal-specific Na+–K+–2Cl− co-transporter NKCC2 by AMP-activated protein kinase (AMPK)
Scott A. Fraser;
Scott A. Fraser
1
*The Burnet Research Institute, Austin Health, Studley Road, Heidelberg, Victoria 3084, Australia
1To whom correspondence should be addressed (email sfraser@burnet.edu.au).
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Ignacio Gimenez;
Ignacio Gimenez
†Department of Pharmacology and Physiology, School of Medicine, University of Zaragoza, 50009 Zaragoza, Spain
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Natasha Cook;
Natasha Cook
*The Burnet Research Institute, Austin Health, Studley Road, Heidelberg, Victoria 3084, Australia
‡Department of Nephrology, Austin Health, Studley Road, Heidelberg, Victoria 3084, Australia
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Ian Jennings;
Ian Jennings
§St. Vincent's Institute of Medical Research, St. Vincent's Hospital, 41 Victoria Parade, Fitzroy, Victoria 3065, Australia
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Marina Katerelos;
Marina Katerelos
*The Burnet Research Institute, Austin Health, Studley Road, Heidelberg, Victoria 3084, Australia
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Frosa Katsis;
Frosa Katsis
§St. Vincent's Institute of Medical Research, St. Vincent's Hospital, 41 Victoria Parade, Fitzroy, Victoria 3065, Australia
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Vicki Levidiotis;
Vicki Levidiotis
*The Burnet Research Institute, Austin Health, Studley Road, Heidelberg, Victoria 3084, Australia
‡Department of Nephrology, Austin Health, Studley Road, Heidelberg, Victoria 3084, Australia
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Bruce E. Kemp;
Bruce E. Kemp
§St. Vincent's Institute of Medical Research, St. Vincent's Hospital, 41 Victoria Parade, Fitzroy, Victoria 3065, Australia
∥CSIRO Molecular and Health Technologies (CMHT), Parkville, Victoria 3052, Australia
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David A. Power
David A. Power
*The Burnet Research Institute, Austin Health, Studley Road, Heidelberg, Victoria 3084, Australia
‡Department of Nephrology, Austin Health, Studley Road, Heidelberg, Victoria 3084, Australia
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Publisher: Portland Press Ltd
Received:
December 12 2006
Accepted:
March 07 2007
Accepted Manuscript online:
March 07 2007
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© 2007 Biochemical Society
2007
Biochem J (2007) 405 (1): 85–93.
Article history
Received:
December 12 2006
Accepted:
March 07 2007
Accepted Manuscript online:
March 07 2007
Citation
Scott A. Fraser, Ignacio Gimenez, Natasha Cook, Ian Jennings, Marina Katerelos, Frosa Katsis, Vicki Levidiotis, Bruce E. Kemp, David A. Power; Regulation of the renal-specific Na+–K+–2Cl− co-transporter NKCC2 by AMP-activated protein kinase (AMPK). Biochem J 1 July 2007; 405 (1): 85–93. doi: https://doi.org/10.1042/BJ20061850
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