Expression of a mutant myosin light chain that cannot be phosphorylated increases paracellular permeability.
A murine leukemia retroviral vector was engineered to contain the DNA encoding either the wild-type, rat aorta 20-kDa myosin light chain (MLC20) or a mutant form of MLC20 in which Thr18 and Ser19 were mutated into alanines. These mutations result in a MLC20 that cannot be phosphorylated by myosin light chain kinase. An 11-amino acid epitope from c-myc was added to both MLC20 sequences to facilitate identification of these proteins. Madin-Darby canine kidney cells were stably transduced, and MLC20 expression was demonstrated by Western blot analysis using a myc-specific antibody. MLC20 exchange was demonstrated by purifying myosin from the transduced cells and repeating the Western blot analysis. Actin-activated adenosinetriphosphatase assays on the purified myosins demonstrated approximately 50% decrease in the rate of ATP hydrolysis by the myosin containing the mutant MLC20. Transepithelial electrical resistance was decreased and mannitol flux was increased across monolayers of cells expressing mutant MLC20. These data demonstrate that MLC20 phosphorylation is involved in regulating paracellular permeability and epithelial barrier function.
Gandhi
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S
Department of Physiology and Biophysics, University of Illinois at Chicago, 60612, USA.
Lorimer
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DD
de Lanerolle
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P
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D004285:Dogs; D017097:Electric Impedance; D007668:Kidney / Q000166:cytology / Q000378:metabolism* / Q000502:physiology; D008353:Mannitol / Q000493:pharmacokinetics; D008856:Microscopy, Fluorescence; D009154:Mutation; D018994:Myosin Light Chains / Q000235:genetics* / Q000378:metabolism*; D009218:Myosins / Q000378:metabolism; D010539:Permeability; D010766:Phosphorylation; D051381:Rats; D011994:Recombinant Proteins
D018994:Myosin Light Chains; D011994:Recombinant Proteins; D008353:Mannitol; D000251:Adenosine Triphosphatases; D009218:Myosins