Identification of a Binding Motif for Ankyrin on the -Subunit of Na,K-ATPase

doi:10.1074/jbc.270.50.29971

Identification of a Binding Motif for Ankyrin on the -Subunit of Na,K-ATPase (*)

From the Institute of Anatomy, University of Würzburg, D-97070 Würzburg, Germany

^§ To whom correspondence should be addressed:
Institute of Anatomy, University of Würzburg, Koellikerstr. 6, D-97070 Würzburg, Germany.
Tel.: 49-931-31-27-03; Fax: 49-931-15-988.

Abstract

Cytoskeleton membrane associations are important for a variety of cellular functions. The anion exchanger of erythrocytes (AE1) and Na,K-ATPase of polarized epithelial cells provide well studied examples of how integral membrane proteins are anchored via the linker molecule ankyrin to the spectrin-based membrane cytoskeleton. In the present study we have generated several recombinant fragments of the large (third) cytoplasmic domain (CD3) of Na,K-ATPase to define binding sites of ankyrin on CD3 at a molecular level. We provide evidence that a cluster of four amino acids, ALLK, is essential for binding of ankyrin to both recombinant CD3 and to native Na,K-ATPase. Once bound, conformational changes might uncover further binding sites for ankyrin on Na,K-ATPase. A motif related to the ALLK cluster is also present in the cytoplasmic domain of AE1 where this sequence (ALLLK) turned out to be also important for ankyrin binding. These motifs are highly conserved during evolution of both Na,K-ATPase and AE1, further underlining their potential role in cytoskeleton to membrane linkage.

Footnotes

↵* Supported by grants of the Deutsche Forschungsgemeinschaft (to R. K. and D. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

AE1

anion exchanger 1

CD3

third cytoplasmic domain of Na,K-ATPase α-subunit

CD2

second cytoplasmic domain of Na,K-ATPase α-subunit

MDCK

Mardin Darby canine kidney

PAGE

polyacrylamide gel electrophoresis

RT

room temperature

PBS

phosphate-buffered saline

HRP

horseradish peroxidase

BSA

bovine serum albumin

CD-AE1

cytoplasmic domain of AE1.
- Received August 3, 1995.
- Revision received September 26, 1995.