S163 is critical for FXYD5 modulation of wound healing in airway epithelial cells.
The FXYD family, which contains seven members, are tissue specific regulators of the Na,K-ATPase. Increased expression of FXYD5, a cancer-cell-associated membrane glycoprotein, has been associated with increased cell motility and metastatic potential. To better understand how FXYD5 may modulate cell motility, we analyzed S163, a conserved residue in all FXYD family members located in the C-terminus. Ectopic expression of human FXYD5 S163 mutants in HEK 293 cells showed that negative charge at S163 (S163D) decreased membrane localization, assessed by immunofluorescence. Coimmunoprecipitation studies revealed decreased FXYD5/Na,K-ATPase interaction for S163D compared with wild-type or S163A mutants. Interestingly, FXYD5 overexpression induced expression of vimentin, a marker of epithelial-mesenchymal transition, in murine airway epithelial cells. Because Na,K-ATPase expression is decreased in some forms of cancer and is critical for establishing cell polarity and suppressing cell motility, we analyzed S163 mutants in an epithelial cell scratch-wound model as a measure of cell migration. Wild-type FXYD5 overexpression increased reepithelialization (p<0.0001), which was further increased in S163D mutants (p<0.005). However, S163A mutants inhibited epithelial cell migration compared with wild-type FXYD5 overexpression (p<0.0001). We conclude that negative charge at S163 regulates FXYD5/Na,K-ATPase interaction and that this interaction modulates cell migration across a wound in airway epithelial cells.
Miller
Timothy J
TJ
Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106, USA. txm20@case.edu
Davis
Pamela B
PB
D000818:Animals; D002465:Cell Movement; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000502:physiology*; D007473:Ion Channels; D008562:Membrane Glycoproteins / Q000235:genetics / Q000502:physiology*; D051379:Mice; D008840:Microfilament Proteins; D009154:Mutation; D009363:Neoplasm Proteins / Q000235:genetics / Q000502:physiology*; D020545:Respiratory Mucosa / Q000166:cytology; D000254:Sodium-Potassium-Exchanging ATPase / Q000502:physiology; D014945:Wound Healing / Q000502:physiology*
C451410:FXYD5 protein, human; D007473:Ion Channels; D008562:Membrane Glycoproteins; D008840:Microfilament Proteins; D009363:Neoplasm Proteins; D000254:Sodium-Potassium-Exchanging ATPase