Expression of a mutant myosin light chain that cannot be phosphorylated increases paracellular permeability

Abstract

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.

Pulmonary Endothelial Cell Barrier Enhancement by Sphingosine 1-Phosphate

“…The blot shown (IB) is representative of two independent experiments. expression of mutant MLC that cannot be phosphorylated by MLCK leads to decreased resistance across epithelial monolayers (38), providing further support for the importance of MLCK activity in the maintenance of barrier function.…”

Requirement for Rho-mediated Myosin Light Chain Phosphorylation in Thrombin-stimulated Cell Rounding and Its Dissociation from Mitogenesis

“…Phosphorylation of myosin light chains by MLCK stimulates the actinactivated ATPase in smooth muscle and non-muscle myosin (31)(32)(33). Myosin phosphorylation has been shown to regulate smooth muscle contractility (31)(32)(33) and non-muscle cell responses such as cell motility (63,64), epithelial barrier function (65,66), and endothelial contractility (67). In addition to MLCK, myosin light chain can be phosphorylated by PKC, PKA, and PAK (p21-activated kinase 1) (68 -70), although the physiological significance of these phosphorylation reactions is unclear.…”

Myosin light chain phosphorylation regulates barrier function by remodeling tight junction structure

“…These studies used epithelial cell lines stably transfected to express either a non-phosphorylatable MLC or a constitutively active truncated MLCK (tMLCK). This resulted in markedly reduced or increased MLC phosphorylation-dependent actomyosin contraction, respectively (Gandhi et al, 1997;Hecht et al, 1996). In both cases, epithelial cells lines with altered MLC phosphorylation were unable to develop significant barrier function, suggesting that TJ assembly was disrupted (Gandhi et al, 1997;Hecht et al, 1996).…”

“…Two previous studies have examined the role of myosin in epithelial TJ biogenesis and assembly (Gandhi et al, 1997;Hecht et al, 1996). These studies used epithelial cell lines stably transfected to express either a non-phosphorylatable MLC or a constitutively active truncated MLCK (tMLCK).…”

“…This resulted in markedly reduced or increased MLC phosphorylation-dependent actomyosin contraction, respectively (Gandhi et al, 1997;Hecht et al, 1996). In both cases, epithelial cells lines with altered MLC phosphorylation were unable to develop significant barrier function, suggesting that TJ assembly was disrupted (Gandhi et al, 1997;Hecht et al, 1996). Unfortunately, structure of these incompletely developed TJs was not studied, probably reflecting the relatively limited understanding of TJ protein composition at the time.…”