Modulation of FXYD interaction with Na,K-ATPase by anionic phospholipids and protein kinase phosphorylation.
FXYD10 is a 74 amino acid small protein which regulates the activity of shark Na,K-ATPase. The lipid dependence of this regulatory interaction of FXYD10 with shark Na,K-ATPase was investigated using reconstitution into DOPC/cholesterol liposomes with or without the replacement of 20 mol % DOPC with anionic phospholipids. Specifically, the effects of the cytoplasmic domain of FXYD10, which contains the phosphorylation sites for protein kinases, on the kinetics of the Na,K-ATPase reaction were investigated by a comparison of the reconstituted native enzyme and the enzyme where 23 C-terminal amino acids of FXYD10 had been cleaved by mild, controlled trypsin treatment. Several kinetic properties of the Na,K-ATPase reaction cycle as well as the FXYD-regulation of Na,K-ATPase activity were found to be affected by acidic phospholipids like PI, PS, and PG. This takes into consideration the Na+ and K+ activation, the K+-deocclusion reaction, and the poise of the E1/E2 conformational equilibrium, whereas the ATP activation was unchanged. Anionic phospholipids increased the intermolecular cross-linking between the FXYD10 C-terminus (Cys74) and the Cys254 in the Na,K-ATPase A-domain. However, neither in the presence nor in the absence of anionic phospholipids did protein kinase phosphorylation of native FXYD10, which relieves the inhibition, affect such cross-linking. Together, this seems to indicate that phosphorylation involves only modest structural rearrangements between the cytoplasmic domain of FXYD10 and the Na,K-ATPase A-domain.
Cornelius
Flemming
F
Department of Biophysics, Institute of Physiology and Biophysics, University of Aarhus, Aarhus 8000, Denmark. fc@biophys.au.dk
Mahmmoud
Yasser A
YA
D000255:Adenosine Triphosphate / Q000378:metabolism; D000838:Anions; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D010455:Peptides / Q000737:chemistry*; D010743:Phospholipids / Q000737:chemistry*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000737:chemistry / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry*
D000838:Anions; D010455:Peptides; D010743:Phospholipids; D000255:Adenosine Triphosphate; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D000254:Sodium-Potassium-Exchanging ATPase