Identification of the cofilin-binding sites in the large cytoplasmic domain of Na,K-ATPase.
Na,K-ATPase, an alpha, beta heterodimer, is found in the plasma membrane of all animal cells. The alpha chain is believed to have 10 transmembrane regions and a large cytoplasmic domain between the 4th and 5th transmembrane regions (H4-H5). In our previous report, the large (3rd) cytoplasmic domains of the alpha1 and alpha2 isoform were found to interact with cofilin, an actin-modulating protein, by the yeast two-hybrid system. Here we show that cofilin interacts only with the 3rd cytoplasmic domain of the alpha2 subunit but not with the 2nd, 4th, and 5th cytoplasmic domains or the cytoplasmic region of the beta subunit of Na,K-ATPase. We also demonstrate that cofilin interacts with the large cytoplasmic domains of the alpha1, alpha2 and alpha3 isoforms of Na,K-ATPase, but not with those of glucose transporter 1, glucose transporter 4, cystic fibrosis transmembrane conductance regulator and plasma membrane Ca-ATPase. We introduced 10 mutations into the 3rd cytoplasmic domain of Na,K-ATPase to identify the binding sites with cofilin. Eight of these mutants were single amino acid substitutions (R417Q, K470Q, K654G, D672A, K691A, R700G, R700A and D710G) and two were double mutant (K654GR700G and K719AK720A). Analysis of the activity of the reporter gene of these mutants shows that residues D672 and R700 of the 3rd cytoplasmic domain of Na,K-ATPase are involved in the interaction with cofilin.
Kim
Miyoung
M
College of Pharmacy, Center for Cell Signaling Research and Division of Molecular Life Sciences, Ewha Womans University, Seoul 120-750, South Korea.
Jung
Jaehoon
J
Park
Chul Seung
CS
Lee
Kyunglim
K
D051339:Actin Depolymerizing Factors; D001483:Base Sequence; D001665:Binding Sites; D003593:Cytoplasm / Q000378:metabolism; D004926:Escherichia coli; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D008840:Microfilament Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D017354:Point Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D051339:Actin Depolymerizing Factors; D007527:Isoenzymes; D008840:Microfilament Proteins; D021122:Protein Subunits; D000254:Sodium-Potassium-Exchanging ATPase