Protein phosphorylation is a key mechanism in several cell functions such as cytoskeletal protein regulation and cell contraction. A critical event of agonist-induced cytoskeletal reorganization of endothelial cells (EC) is the phosphorylation of myosin light chains (MLC), which catalyzes by MLC kinase and triggers EC contraction and permeability Biochemical counterpoint to MLC phosphorylation, ie, MLC dephosphorylation, catalyzed by MLC phosphatase (MLCP), has received much less attention in studies of EC barrier regulation. To evaluate the role of MLCP in EC barrier regulation we cloned MLCP targeting subunit 1 (MYPT1) from pulmonary endothelium.
Purpose The purpose of our study was to ectopically express MYPT1 construct in pulmonary EC with and without thrombin stimulation in order to examine the role of MLCP in EC cytoskeletal regulation. Methods: cDNA encoding MYPT1 was generated from bovine pulmonary artery EC (BPAEC), then it was subcloned into pcDNA3.1 vector using KpnI. and Eco RI. restriction sites. BPAEC grown on coverslips were transfected with resulting plasmid and stained for actin, diphospho MLC and c-myc-tag., and slides were analyzed by Nikon video-imaging system. Level of MYPT1 expression was analyzed by Western immnoblotting with c-myc-tag-specific antibody.
Summary of Results As expected, thrombin (100 nM for 5 min) induces significant increase in ppMLC staining and F-actin stress fiber formation indicating EC contraction. Surprisingly, however, MYPT1 overexpression has no effect on the level of either basal or thrombin-induced MLC phosphorylation or stress fiber formation. Overexpressed MYPT1 was diffusively distributed in the cytoplasm and did not co-localize with stress fibers. These data indicate that expression of MYPT1 alone is not sufficient to affect thrombin-induced cytoskeletal rearrangement in EC and further, more complex studies are needed to evaluate the role of MLCP in EC barrier regulation.