A regulatory locus of phosphorylation in the N terminus of the Na-K-Cl cotransporter, NKCC1.
The secretory Na-K-Cl cotransporter NKCC1 is activated by secretagogues through a phosphorylation-dependent mechanism. We found a phosphorylation stoichiometry of 3.0 +/- 0.4 phosphorylated residues/NKCC1 protein harvested from shark rectal gland tubules maximally stimulated with forskolin and calyculin A, showing that at least three sites on the cotransporter are phosphorylated upon stimulation. Three phosphoacceptor sites were identified in the N-terminal domain of the protein (at Thr(184), Thr(189), and Thr(202)) using high pressure liquid chromatography and matrix-assisted laser desorption ionization time-of-flight mass spectrometry to analyze tryptic fragments of the radiolabeled cotransporter. None of these residues occurs in the context of strong consensus sites for known Ser/Thr kinases. The threonines and the surrounding amino acids are highly conserved between NKCC1 and NKCC2, and similarities are also present in the Na-Cl cotransporter NCC (or TSC). This strongly suggests that the phosphoregulatory mechanism is conserved among isoforms. Through expression of shark NKCC1 mutants in HEK-293 cells, Thr(189) was found to be necessary for activation of the protein, whereas phosphorylation at Thr(184) and Thr(202) was modulatory, but not required. In conjunction with the recent finding (Darmen, R. B., Flemmer, A., and Forbush, B. (2001) J. Biol. Chem. 276, 34359-34362) that protein phosphatase-1 binds to residues 107-112 in the shark NKCC1 sequence, these results demonstrate that the N terminus of NKCC1 constitutes a phosphoregulatory domain of the transporter.
Darman
Rachel B
RB
Department of Cellular and Molecular Physiology, Yale University, New Haven, Connecticut 06510, USA. rdarman@aregroup.harvard.edu
Forbush
Biff
B
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D004284:Dogfish; D006801:Humans; D017136:Ion Transport; D007700:Kinetics; D008958:Models, Molecular; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010748:Phosphopeptides / Q000737:chemistry; D010766:Phosphorylation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012491:Salt Gland / Q000378:metabolism; D028021:Sodium-Potassium-Chloride Symporters / Q000737:chemistry / Q000378:metabolism*; D064506:Solute Carrier Family 12, Member 2; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014162:Transfection; D014357:Trypsin
D010446:Peptide Fragments; D010748:Phosphopeptides; D011994:Recombinant Proteins; C578877:SLC12A2 protein, human; D028021:Sodium-Potassium-Chloride Symporters; D064506:Solute Carrier Family 12, Member 2; D014357:Trypsin