Gandhi S, Lorimer D D, de Lanerolle P
Department of Physiology and Biophysics, University of Illinois at Chicago, 60612, USA.
Am J Physiol. 1997 Feb;272(2 Pt 2):F214-21. doi: 10.1152/ajprenal.1997.272.2.F214.
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.
构建了一种鼠白血病逆转录病毒载体，使其包含编码野生型大鼠主动脉20 kDa肌球蛋白轻链（MLC20）或MLC20突变形式的DNA，其中Thr18和Ser19突变为丙氨酸。这些突变导致MLC20不能被肌球蛋白轻链激酶磷酸化。在两个MLC20序列中都添加了来自c-myc的11个氨基酸的表位，以方便鉴定这些蛋白质。稳定转导了Madin-Darby犬肾细胞，并使用myc特异性抗体通过蛋白质印迹分析证明了MLC20的表达。通过从转导细胞中纯化肌球蛋白并重复蛋白质印迹分析来证明MLC20交换。对纯化的肌球蛋白进行肌动蛋白激活的腺苷三磷酸酶测定表明，含有突变型MLC20的肌球蛋白的ATP水解速率降低了约50%。在表达突变型MLC20的细胞单层上，跨上皮电阻降低，甘露醇通量增加。这些数据表明，MLC20磷酸化参与调节细胞旁通透性和上皮屏障功能。
