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J. Biol. Chem., Vol. 277, Issue 40, 37551-37558, October 4, 2002
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From the Department of Cellular and Molecular Physiology, Yale
University School of Medicine, New Haven, Connecticut 06520 and the
Mount Desert Island Biological Laboratory,
Salsbury Cove, Maine 04672
The Na-K-Cl cotransporter NKCC1 is activated by
phosphorylation of a regulatory domain in its N terminus. In the
accompanying paper (Darman, R. B., and Forbush, B. (2002)
J. Biol. Chem. 277, 37542-37550), we identify three
phosphothreonines important in this process. Using a phospho-specific
antibody (anti-phospho-NKCC1 antibody R5) raised against a
diphosphopeptide containing Thr212 and Thr217
of human NKCC, we were readily able to monitor the cotransporter activation state. In 32P phosphorylation experiments with
rectal gland tubules, we show that the R5 antibody signal is
proportional to the amount of 32P incorporated into NKCC1;
and in experiments with NKCC1-transfected HEK-293 cells, we demonstrate
that R5-detected phosphorylation directly mirrors functional
activation. Immunofluorescence analysis of shark rectal gland shows
activation-dependent R5 antibody staining along the
basolateral membrane. In perfused rat parotid glands, isoproterenol
induced staining of both acinar and ductal cells along the basolateral
membrane. Isoproterenol also induced basolateral staining of the
epithelial cells in rat trachea, whereas basal cells in the
subepithelial tissue displayed heavy, non-polarized staining of the
cell membrane. In rat colon, agonist stimulation induced staining along
the basolateral membrane of crypt cells. These data provide direct
evidence of NKCC1 regulation in these tissues, and they further link
phosphorylation of NKCC1 with its activation in transfected cells and
native tissue. The high conservation of the regulatory threonine
residues among NKCC1, NKCC2, and NCC family members, together with the
fact that tissues from divergent vertebrate species exhibit similar
R5-binding profiles, lends further support to the role of this
regulatory locus in vivo.
Activation of the Na-K-Cl Cotransporter NKCC1
Detected with a Phospho-specific Antibody*
,
*
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.
Present address: University Children's Hospital Munich,
Lindwurmstrasse 4, 80337 Munich, Germany.
§
Present address: Dept. of Molecular Medicine, Beth Israel
Deaconess Medical Center, Harvard Medical School, Boston, MA 02215.
¶
To whom correspondence and reprint requests should be
addressed: Dept. of Cellular and Molecular Physiology, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06520-0826. Tel.: 203-785-4068; Fax: 203-785-6834; E-mail:
biff.forbush@yale.edu.
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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