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J. Biol. Chem., Vol. 281, Issue 23, 15790-15799, June 9, 2006

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Functional Interactions of Phospholemman (PLM) (FXYD1) with Na⁺,K⁺-ATPase

PURIFICATION OF 1/1/PLM COMPLEXES EXPRESSED IN PICHIA PASTORIS^*

From the Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel

Human FXYD1 (phospholemman, PLM) has been expressed in Pichia pastoris with porcine ₁/His₁₀-₁ subunits of Na⁺,K⁺-ATPase or alone. Dodecyl--maltoside-soluble complexes of ₁/₁/PLM have been purified by metal chelate chromatography, either from membranes co-expressing ₁,His₁₀-₁, and PLM or by in vitro reconstitution of PLM with ₁/His₁₀-₁ subunits. Comparison of functional properties of purified ₁/His₁₀-₁ and ₁/His₁₀-₁/PLM complexes show that PLM lowered K_0.5 for Na⁺ ions moderately (30%) but did not affect the turnover rate or K_m of ATP for activating Na⁺,K⁺-ATPase activity. PLM also stabilized the ₁/His₁₀-₁ complex. In addition, PLM markedly (>3-fold) reduced the K_0.5 of Na⁺ ions for activating Na⁺-ATPase activity. In membranes co-expressing ₁/His₁₀-₁ with PLM the K_0.5 of Na⁺ ions was also reduced, compared with the control, excluding the possibility that detergent or lipid in purified complexes compromise functional interactions. When expressed in HeLa cells with rat ₁, rat PLM significantly raised the K_0.5 of Na⁺ ions, whereas for a chimeric molecule consisting of transmembranes segments of PLM and extramembrane segments of FXYD4, the K_0.5 of Na⁺ ions was significantly reduced, compared with the control. The opposite functional effects in P. pastoris and HeLa cells are correlated with endogenous phosphorylation of PLM at Ser⁶⁸ or unphosphorylated PLM, respectively, as detected with antibodies, which recognize PLM phosphorylated at Ser⁶⁸ (protein kinase A site) or unphosphorylated PLM. We hypothesize that PLM interacts with ₁/His₁₀-₁ subunits at multiple locations, the different functional effects depending on the degree of phosphorylation at Ser⁶⁸. We discuss the role of PLM in regulation of Na⁺,K⁺-ATPase in cardiac or skeletal muscle cells.

Received for publication, March 2, 2006 , and in revised form, April 3, 2006.

^* This work was supported by the Weizmann Institute Renal Research Fund, the Josef Cohen Minerva center for Biomembranes, and the Israel Science Foundation. 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.

¹ Incumbent of the Hella and Derrick Kleeman Chair of Biochemistry.

² Incumbent of the William Smithburg Chair of Biochemistry. To whom correspondence should be addressed: Dept. of Biological Chemistry, Weizmann Institute of Science, Rehovot, 76100, Israel. Tel.: 972-8-934-2278; Fax: 972-8-934-4118; E-mail: Steven.Karlish{at}weizmann.ac.il.

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