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J. Biol. Chem., Vol. 277, Issue 46, 44278-44284, November 15, 2002
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From the Departments of We carried out in vitro
phosphorylation assays to determine whether ROMK1 is a substrate of
protein kinase C (PKC) and used the two-electrode voltage clamp
method to investigate the role of serine residues 4, 183, and 201, the
three putative PKC phosphorylation sites, in the regulation of ROMK1
channel activity. Incubation of the purified His-tagged ROMK1 protein
with PKC and radiolabeled ATP resulted in 32P incorporation
into ROMK1 detected by autoradiography. Moreover, the in
vitro phosphorylation study of three synthesized peptides corresponding to amino acids 1-16, 174-189, and 196-211 of ROMK1 revealed that serine residues 4 and 201 of ROMK1 were the two main PKC
phosphorylation sites. In contrast, 32P incorporation of
peptide 174-189 was absent. In vitro phosphorylation studies with ROMK1 mutants, R1S4/201A, R1S4/183A, and R1S183/201A, demonstrated that the phosphorylation levels of R1S4/201A were significantly lower than those of the other two mutants. Also, the
Ba2+-sensitive K+ current in oocytes injected
with green fluorescent protein (GFP)-R1S4/201A was only 5% of that in
oocytes injected with wild type GFP-ROMK1. In contrast, the
K+ current in oocytes injected with GFP-ROMK1 mutants
containing either serine residue 4 or 201 was similar to those injected
with wild type ROMK1. Confocal microscope imaging shows that the
surface expression of the K+ channels was significantly
diminished in oocytes injected with R1S4/201A and completely absent in
oocytes injected with R1S4/183/201A. Furthermore, the biotin labeling
technique confirmed that the membrane fraction of ROMK channels was
almost absent in HEK293 cells transfected with either R1S4/201A or
R1S4/183/201A. However, when serine residues 4 and 201 were mutated to
aspartate, the K+ currents and the surface expression were
completely restored. Finally, addition of calphostin C in the
incubation medium significantly decreased the K+
current in comparison with that under control conditions. Biotin labeling technique further indicated that inhibition of PKC decreases the surface ROMK1 expression in human embryonic kidney (HEK) cells transfected with ROMK1. We conclude that ROMK1 is a substrate of PKC
and that serine residues 4 and 201 are the two main PKC phosphorylation
sites that are essential for the expression of ROMK1 in the cell surface.
Protein Kinase C (PKC)-induced Phosphorylation of ROMK1 Is
Essential for the Surface Expression of ROMK1 Channels*
,,
Pharmacology and
§ Anatomy and Cell Biology, New York Medical College,
Valhalla, New York 10595 and the ¶ Department of Cellular and
Molecular Physiology, Yale University School of Medicine, New
Haven, Connecticut 06511
*
This work was supported by National Institutes of Health
Grants DK 47402 and DK 54983.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
Pharmacology, New York Medical College, BSB Rm. 537, Valhalla, NY
10595. Tel.: 914-594-4120; Fax: 914-347-4956; E-mail:
wenhui_wang@nymc.edu.
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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