Characterization and differential expression of human vascular smooth muscle myosin light chain 2 isoform in nonmuscle cells

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Abstract
The 20-kDa regulatory myosin light chain (MLC), also known as MLC-2, plays an important role in the regulation of both smooth muscle and nonmuscle cell contractile activity. Phosphorylation of MLC-2 by the enzyme MLC kinase increases the actin-activated myosin ATPase activity and thereby regulates the contractile activity. We have isolated and characterized an MLC-2 cDNA corresponding to the human vascular smooth muscle MLC-2 isoform from a cDNA library derived from umbilical artery RNA. The translation of the in vitro synthesized mRNA, corresponding to the cDNA insert, in a rabbit reticulocyte lysate results in the synthesis of a 20,000-dalton protein that is immunoreactive with antibodies raised against purified chicken gizzard MLC-2. The derived amino acid sequence of the putative human smooth muscle MLC-2 shows only three amino acid differences when compared to chicken gizzard MLC-2. However, comparison with the human cardiac isoform reveals only 48% homology. Blot hybridizations and S1 nuclease analysis indicate that the human smooth muscle MLC-2 isoform is expressed restrictively in smooth muscle tissues such as colon and uterus and in some, but not all, nonmuscle cell lines. Previously reported MLC-2 cDNA from rat aortic smooth muscle cells in culture is ubiquitously expressed in all muscle and nonmuscle cells, and it was suggested that both smooth muscle and nonmuscle MLC-2 proteins are identical and are probably encoded by the same gene. In contrast, the human smooth muscle MLC-2 cDNA that we have characterized from an intact smooth muscle tissue is not expressed in skeletal and cardiac muscles and also in a number of nonmuscle cells. Nevertheless, MLC-2 protein species is readily detectable in all the nonmuscle cell lines using antibodies to smooth muscle MLC-2 protein. Two-dimensional gel analysis of the 35S-labeled proteins from a nonmuscle cell line indicates three protein species that are immunoprecipitated with MLC-2 antibodies. Comparison of the two-dimensional gel pattern indicates the absence of one MLC-2 protein species in the cell lines that do no express the smooth muscle MLC-2 mRNA. Together, these results suggest that the smooth muscle and nonmuscle MLC-2 isoforms are separate and are possibly encoded by separate genes. Hence, the MLC-2 cDNA sequence reported in this paper corresponds to a novel and distinct smooth muscle isoform.