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J. Biol. Chem., Vol. 275, Issue 37, 28599-28606, September 15, 2000
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From the The activity of the
Na+/H+ exchanger NHE3 isoform, which is
found primarily in epithelial cells, is sensitive to the state of actin
polymerization. Actin assembly, in turn, is controlled by members of
the small GTPase Rho family, namely Rac1, Cdc42, and RhoA. We therefore
investigated the possible role of these GTPases in modulating NHE3
activity. Cells stably expressing NHE3 were transiently transfected
with inhibitory forms of Rac1, Cdc42, or RhoA and transport activity
was assessed using microfluorimetry. NHE3 activity was not adversely
affected by either dominant-negative Rac1 or Cdc42. By contrast, the
inhibitory form of RhoA greatly depressed NHE3 activity, without
noticeably altering its subcellular distribution. NHE3 activity was
equally reduced by inhibiting p160 Rho-associated kinase I (ROK), a
downstream effector of RhoA, with the selective antagonist Y-27632 and
a dominant-negative form of ROK. Furthermore, inhibition of ROK reduced
the phosphorylation of myosin light chain. A comparable net
dephosphorylation was achieved by the myosin light chain kinase
inhibitor ML9, which similarly inhibited NHE3. These data suggest that
optimal NHE3 activity requires a functional RhoA-ROK signaling pathway
which acts, at least partly, by controlling the phosphorylation of
myosin light chain and, ultimately, the organization of the actin cytoskeleton.
RhoA and Rho Kinase Regulate the Epithelial
Na+/H+ Exchanger NHE3
ROLE OF MYOSIN LIGHT CHAIN PHOSPHORYLATION*
§¶,§,
,,, and Cell Biology Programme, The Hospital for
Sick Children, Toronto, Ontario M5G 1X8, Canada, the Department
of Surgery, The Toronto Hospital and University of Toronto, Toronto,
Ontario M5G 1L7, Canada, the ** Division of Signal Transduction, Nara
Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara
630-0101, Japan, and the §§ Department of
Physiology, McGill University, Montreal, Quebec H3G 1Y6, Canada
*
This work was supported in part by the Medical Research
Council of Canada and the Kidney Foundation of Canada.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.
International Scholar of the Howard Hughes Medical Institute
and the current holder of the Pitblado Chair in Cell Biology at The
Hospital for Sick Children. Cross-appointed to the Department of
Biochemistry, University of Toronto.
¶¶
Medical Research Council of Canada Scientist. To whom
correspondence should be addressed: Dept. of Physiology, McGill
University, McIntyre Medical Science Bldg., 3655 Promenade
Sir-William-Osler, Montreal, Quebec H3G 1Y6, Canada. Tel.:
514-398-8335; Fax: 514-398-7452; E-mail: orlowski@med.mcgill.ca.
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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