Mutation of the Protein Kinase C Phosphorylation Site on Rat α1 Na+,K+-ATPase Alters Regulation of Intracellular Na+ and pH and Influences Cell Shape and Adhesiveness

doi:10.1074/jbc.272.32.20179

Mutation of the Protein Kinase C Phosphorylation Site on Rat α1 Na⁺,K⁺-ATPase Alters Regulation of Intracellular Na⁺ and pH and Influences Cell Shape and Adhesiveness*

From the ^‡Department of Woman and Child Health, Karolinska Institute, S-112 81 Stockholm, Sweden, the ^¶Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, New York 10021, and the ^‖La Jolla Cancer Research Center, The Burnham Institute, La Jolla, California 92037

Abstract

The enzyme Na⁺,K⁺-ATPase creates the transmembrane Na⁺ gradient that is of vital importance for functioning of all eukaryotic cells. Na⁺,K⁺-ATPase can be phosphorylated by protein kinase A (PKA) and protein kinase C (PKC), and these sites of phosphorylation have been identified. In the present study, we have examined the physiological significance of PKC phosphorylation of rat Na⁺,K⁺-ATPase. In COS cells transfected with wild type rat Na⁺,K⁺-ATPase α1, intracellular Na⁺ was higher and pH was lower than in cells transfected with rat Na⁺,K⁺-ATPase α1 in which the PKC phosphorylation site, Ser-23, had been mutated into alanine. Phorbol dibutyrate inhibited Na⁺,K⁺-ATPase-dependent ATP hydrolysis and Rb⁺ uptake in cells expressing wild type Na⁺,K⁺-ATPase but not in cells expressing S23A Na⁺,K⁺-ATPase. Cells expressing the S23A mutant had a more rounded appearance and attached less well to fibronectin than did untransfected cells or cells transfected with wild type rat Na⁺,K⁺-ATPase α1. These results indicate a functional role for PKC-mediated phosphorylation of rat Na⁺,K⁺-ATPase α1 and suggest a connection between this enzyme and cell adhesion.

Footnotes

↵* This work was supported by grants from the Swedish Medical Research Council (Project number 03644) and the Swedish Heart Lung Foundation (to A. A.) and by United States Public Health Service Grant MH-40899 (to P. G.).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.
↵§ The first two authors contributed equally to this work.
↵** Participation to this work was initiated during stay at the Karolinska Institute as a Nobel Fellow.
↵‡ To whom correspondence should be addressed. Tel.: 46-8-672-2222; Fax: 46-8-672-1941.
↵1 The abbreviations used are: PKA, cAMP-dependent protein kinase; PKC, protein kinase C; AM, acetoxymethyl; BCECF, 2′,7′-bis(carboxyethyl)-5(6)-carboxyfluorescein; DMEM, Dulbecco’s modified Eagle’s medium; PDBu, phorbol 12,13-dibutyrate; PBS, phosphate-buffered saline; SBFI, Na⁺-binding benzofuran isophthalate; Na⁺ _i, intracellular Na⁺ concentration; NHE, Na⁺/H⁺ exchanger.
- Received January 28, 1997.
- Revision received March 31, 1997.