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J. Biol. Chem., Vol. 277, Issue 40, 37542-37550, October 4, 2002
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From the Department of Cellular and Molecular Physiology, Yale
University, New Haven, Connecticut 06510 and the Mount Desert
Island Biological Laboratory, Salsbury Cove, Maine 04672
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 Thr184,
Thr189, and Thr202) 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,
Thr189 was found to be necessary for activation of the
protein, whereas phosphorylation at Thr184 and
Thr202 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.
A Regulatory Locus of Phosphorylation in the N
Terminus of the Na-K-Cl Cotransporter, NKCC1*
and
*
This work was supported by National Institutes of Health
Grant DK47661.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Molecular
Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School,
330 Brookline Ave., Boston, MA 02215. Tel.: 617-667-2923; Fax:
617-667-8040; E-mail: rdarman@caregroup.harvard.edu.
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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