Differential Regulation of Alternatively Spliced Endothelial Cell Myosin Light Chain Kinase Isoforms by p60Src

Abstract

The Ca 2؉ /calmodulin-dependent endothelial cell myosin light chain kinase (MLCK) triggers actomyosin contraction essential for vascular barrier regulation and leukocyte diapedesis. Two high molecular weight MLCK splice variants, EC MLCK-1 and EC MLCK-2 (210 -214 kDa), in human endothelium are identical except for a deleted single exon in MLCK-2 encoding a 69-amino acid stretch ( 20 by Ca 2ϩ /calmodulin-dependent enzyme MLCK is essential for the initiation of nonmuscle and smooth muscle contraction (1, 3, 9 -11). In specific nonmuscle tissues, such as the vascular endothelium, this kinase is known to be involved in endothelial cell migration, cell retraction (12), endothelial cell barrier regulation (13), transendothelial migration of neutrophils (14, 15), and possibly apoptosis (16). The MLCK isoform abundantly expressed in smooth muscle (SM MLCK) generally exists as a 130-to 150-kDa protein that has been well characterized (for review see Refs. 3 and 11). However, Western blot screening of a variety of embryonic and adult smooth muscle and nonmuscle tissues revealed expression of a high molecular weight MLCK variant with electrophoretic mobility in the range of [208][209][210][211][212][213][214]. Garcia and colleagues (20) subsequently sequenced the high molecular weight MLCK isoform cloned from a human endothelial cell cDNA library, revealing an open reading frame, which encodes a protein of 1914 amino acids. Both the low molecular mass (130 -150 kDa) and high molecular mass (208 -214 kDa) MLCK isoforms share essentially identical actin binding, MLC binding, catalytic, and Ca 2ϩ /CaM-regulatory domains. The extreme C-terminal kinase-related protein (KRP) domain, which binds myosin, is contained within both EC MLCK and SM MLCK but can be also expressed as an independent protein capable of stabilizing myofilaments in vitro (3,(21)(22)(23). The C-terminal half of the endothelial MLCK isoform (residues 923-1914) exhibits 99.8% homology to the human low molecular weight MLCK from hippocampus and substantial homology to published SM MLCK sequences from rabbit (94% homology), bovine (95% homology), and chicken (85% homology) (5, 6, 24, 25). However, the exact biological function of the 922-amino acid N-terminal portion, which is unique to the high

A Differentiation-dependent Splice Variant of Myosin Light Chain Kinase, MLCK1, Regulates Epithelial Tight Junction Permeability

“…Long MLCK is expressed as multiple splice variants; among these MLCK2 predominates, whereas the fulllength transcript, MLCK1, is a minor form (15). The transcripts for these isoforms differ by only 207 base pairs, but insertion of this 69-amino acid domain can increase MLCK1 activity after phosphorylation by Src family kinases (16). This 69-amino acid region also contains a potential SH2-binding domain, raising the possibility that there are protein interac- FIG.…”

“…Immunoblotting-Human endothelial cell MLCK1 and MLCK2 were prepared using the Bac-to-Bac baculovirus expression system (Invitrogen) as described previously (16). For large scale expression and purification, Sf9 cells were infected with baculovirus (multiplicity of infection ϭ 1), and the cells producing human endothelial MLCK1 and MLCK2 were harvested.…”

“…BLAST searches confirmed that this sequence was not expressed in other known or predicted sequences in the human genome. To avoid generating phosphospecific antiserum, described phosphorylation sites at Tyr 464 and Tyr 471 (16) were excluded from the peptide. The peptide was conjugated to keyhole limpet hemocyanin (Calbiochem) using glutaraldehyde.…”

“…The corresponding 69-amino acid region contains Src kinase phosphorylation sites and a potential SH2-binding domain (15). In vitro enzymatic assays have shown that phosphorylation by Src kinase increases the V max of MLCK1, whereas the activity of unphosphorylated MLCK1 is similar to MLCK2 (16).…”

Molecular Basis of Epithelial Barrier Regulation

“…58,59 Alternative splicing of long MLCK transcripts leads to translation of two different MLCK isoforms in intestinal epithelia, 58 of which one can be regulated by Src kinase as well as Ca ϩ2 /calmodulin. 60 Thus, whereas the mechanisms by which TNF activates MLCK activity in intestinal epithelia are not yet described, both Src kinase and Ca ϩ2 / calmodulin are potential signaling intermediates.…”