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J. Biol. Chem., Vol. 282, Issue 32, 23205-23218, August 10, 2007

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Interaction of Integrin-linked Kinase with the Kidney Chloride/Bicarbonate Exchanger, kAE1^*

Thitima Keskanokwong^¶1, Haley J. Shandro², Danielle E. Johnson², Saranya Kittanakom^||, Gonzalo L. Vilas³, Paul Thorner^**, Reinhart A. F. Reithmeier^||, Varaporn Akkarapatumwong⁴, Pa-thai Yenchitsomanus⁴, and Joseph R. Casey⁵

From the Membrane Protein Research Group, Department of Physiology and Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada, the Institute of Molecular Biology and Genetics, Mahidol University, Salaya Campus, Nakorn Pathom 73170, Thailand, the ^¶Department of Research and Development, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand, the ^||Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada, the ^**Division of Pathology, Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada, and the Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario M5G 1L5, Canada

Kidney anion exchanger 1 (kAE1) mediates chloride/bicarbonate exchange at the basolateral membrane of kidney -intercalated cells, thereby facilitating bicarbonate reabsorption into the blood. Human kAE1 lacks the N-terminal 65 residues of the erythroid form (AE1, band 3), which are essential for binding of cytoskeletal and cytosolic proteins. Yeast two-hybrid screening identified integrin-linked kinase (ILK), a serine/threonine kinase, and an actin-binding protein as an interacting partner with the N-terminal domain of kAE1. Interaction between kAE1 and ILK was confirmed in co-expression experiments in HEK 293 cells and is mediated by a previously unidentified calponin homology domain in the kAE1 N-terminal region. The calponin homology domain of kAE1 binds the C-terminal catalytic domain of ILK to enhance association of kAE1 with the actin cytoskeleton. Overexpression of ILK increased kAE1 levels at the cell surface as shown by flow cytometry, cell surface biotinylation, and anion transport activity assays. Pulse-chase experiments revealed that ILK associates with kAE1 early in biosynthesis, likely in the endoplasmic reticulum. ILK co-localized with kAE1 at the basolateral membrane of polarized Madin-Darby canine kidney cells and in -intercalated cells of human kidneys. Taken together these results suggest that ILK and kAE1 traffic together from the endoplasmic reticulum to the basolateral membrane. ILK may provide a linkage between kAE1 and the underlying actin cytoskeleton to stabilize kAE1 at the basolateral membrane, resulting in higher levels of cell surface expression.

Received for publication, March 12, 2007 , and in revised form, May 31, 2007.

^* This work was supported in part by Canadian Institutes of Health Research operating grants (to J. R. C. and R. A. F. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1–3.

¹ Supported by a scholarship award from the Royal Golden Jubilee-Ph.D. Program of the Thailand Research Fund.

² Supported by a Strategic Training Grant from the Canadian Institutes of Health Research.

³ Supported by a postdoctoral fellowship from Alberta Heritage Foundation for Medical Research.

⁴ Supported by Thailand Research Fund Grant BRG4880007.

⁵ Scientist of the Alberta Heritage Foundation for Medical Research. To whom correspondence should be addressed. Tel.: 780-492-7203; Fax: 780-492-8915; E-mail: joe.casey{at}ualberta.ca.

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