Interaction of the alpha subunit of Na,K-ATPase with cofilin.
The alpha1 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 transmembrane 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 cofilin, an actin-binding protein. Cofilin was co-immunoprecipitated with the alpha subunit 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 of 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-ATPase; 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 (86)Rb(+) uptake, indicating that cofilin elicits, either directly or indirectly, enhanced Na,K-ATPase activity and that the interaction occurs in vivo.
Lee
K
K
College of Pharmacy, Center for Cell Signaling Research and Division of Molecular Life Sciences, Ewha Woman's University, Seoul 120-750, Korea. klyoon@mm.ewha.ac.kr
Jung
J
J
Kim
M
M
Guidotti
G
G
D051339:Actin Depolymerizing Factors; D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D019556:COS Cells; D002352:Carrier Proteins; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D018076:DNA, Complementary / Q000302:isolation & purification; D004789:Enzyme Activation; D015870:Gene Expression; D015723:Gene Library; D008840:Microfilament Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012697:Serine Endopeptidases / Q000235:genetics; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D000099160:LexA Repressor Protein
D051339:Actin Depolymerizing Factors; D001426:Bacterial Proteins; D002352:Carrier Proteins; D018076:DNA, Complementary; D008840:Microfilament Proteins; D012697:Serine Endopeptidases; D000254:Sodium-Potassium-Exchanging ATPase; D000099160:LexA Repressor Protein