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KMID : 0617320010100010106
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Journal of Pharmacetical Sceiences Ewha Womans University
2001 Volume.10 No. 1 p.106 ~ p.114
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Interaction of the α subunit of Na,K-ATPase with cofilin
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LEE, Kyunglim
JUNG, Jaehoon/KIM, Miyoung/GUIDOTTI, Guido
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Abstract
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The α1 subunit of rat Na,K-ATPase, composed of 1018 amino acids, is arranged in the membrane so that the middle third of the polypeptide forms a large cytoplasmic loop bordered on both sides by multiple transmenbrane segments. to identify proteins that might interact with the large cytoplasmic loop of Na,K-ATPase and potentially affect the function and/or the disposition of the pump in the cell, the yeast two-hybrid system was used to screen a rat skeletal muscle cDNA library. Several cDNA clones were isolated, some of which coded for coffilin, an actin-binding protein. Cofilin was co-immunoprecipitated with the αsubunit of Na,K-ATPase from extracts of COS-7 cells transiently transfected with haemagglutinin-epitope-tagged cofilin cDNA as well as from yeast extracts. By means of deletion analysis we showed. that the segment f cofilin between residues 45 and 99 is essential for functional association with the large cytoplasmic loop of Na,K-ATPase. Recombinant cofilin was shown to bind to the membrane-bound Na, K-APase; the association between the two proteins was demonstrated by confocal microscopy. The increased level of cofilin in transfected COS-7 cells caused an increase in the rate of ouabain-sensitive Rb^+ uptake, indicating that cofilin elicits, either directly or indirectly, enhanced Na, K-APase activity and that the interaction occurs in vivo.
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