title: Regulation of the renal-specific Na+-K+-2Cl- co-transporter NKCC2 by AMP-activated protein kinase (AMPK).
abstract: 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 Ser126 in 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.
authors.affiliation: The Burnet Research Institute, Austin Health, Studley Road, Heidelberg, Victoria 3084, Australia. sfraser@burnet.edu.au
authors.forename: Scott A
authors.initials: SA
authors.lastname: Fraser
authors.forename: Ignacio
authors.initials: I
authors.lastname: Gimenez
authors.forename: Natasha
authors.initials: N
authors.lastname: Cook
authors.forename: Ian
authors.initials: I
authors.lastname: Jennings
authors.forename: Marina
authors.initials: M
authors.lastname: Katerelos
authors.forename: Frosa
authors.initials: F
authors.lastname: Katsis
authors.forename: Vicki
authors.initials: V
authors.lastname: Levidiotis
authors.forename: Bruce E
authors.initials: BE
authors.lastname: Kemp
authors.forename: David A
authors.initials: DA
authors.lastname: Power
mesh_terms: D055372:AMP-Activated Protein Kinases; D000595:Amino Acid Sequence; D000818:Animals; D045163:Antibodies, Phospho-Specific / Q000378:metabolism; D002460:Cell Line; D004789:Enzyme Activation; D006801:Humans; D007668:Kidney / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism*; D009865:Oocytes / Q000166:cytology / Q000502:physiology; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D012413:Rubidium / Q000378:metabolism; D016415:Sequence Alignment; D012694:Serine / Q000378:metabolism; D028021:Sodium-Potassium-Chloride Symporters / Q000235:genetics / Q000378:metabolism*; D064466:Solute Carrier Family 12, Member 1; D014982:Xenopus laevis
chemical_list: D045163:Antibodies, Phospho-Specific; D009097:Multienzyme Complexes; D011993:Recombinant Fusion Proteins; C578714:SLC12A1 protein, human; C578715:Slc12a1 protein, mouse; D028021:Sodium-Potassium-Chloride Symporters; D064466:Solute Carrier Family 12, Member 1; D012694:Serine; D017346:Protein Serine-Threonine Kinases; D055372:AMP-Activated Protein Kinases; D012413:Rubidium