Regulation of permeabilized endothelial cell retraction by myosin phosphorylation.

Permeabilized endothelial cell monolayers retracted on exposure to ATP and Ca2+. ADP, inosine triphosphate (ITP), GTP, adenosine 5'-(gamma-thio)triphosphate (ATP-gamma S), and 5'-adenylylimidodiphosphate failed to support retraction. However, ATP gamma S, a substrate for myosin light-chain kinase (MLCK) but not myosin adenosinetriphosphatase (ATPase), combined with ITP, a substrate for myosin ATPase but not MLCK, supported retraction. Two MLCK pseudosubstrate peptides, M5 and SM-1, inhibited endothelial cell retraction equally and more effectively than myosin kinase-inhibitory peptide with a sequence based on the phosphorylated site of myosin light chain. M5 was shown to inhibit thiophosphorylation of endothelial cell myosin light chains. Endothelial cells incubated with exogenous unregulated kinase in the presence of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetra-acetic acid retracted on addition of ATP. This retraction was accompanied by thiophosphorylation of the 19 kDa myosin light chains in the presence of ATP gamma 35S. The N-ethylmaleimide-modified subfragment 1 of myosin heads, a specific inhibitor of actin-myosin interaction, prevented retraction. These data add support to the proposal of a central role for MLCK activation of myosin in endothelial retraction.

[1]  M. Ikebe.  Mode of inhibition of smooth muscle myosin light chain kinase by synthetic peptide analogs of the regulatory site. , 1990, Biochemical and biophysical research communications.

[2]  T. Itoh,et al.  Effects of modulators of myosin light-chain kinase activity in single smooth muscle cells , 1989, Nature.

[3]  H Charbonneau,et al.  Synthetic peptides based on the calmodulin-binding domain of myosin light chain kinase inhibit activation of other calmodulin-dependent enzymes. , 1988, Biochemical and biophysical research communications.

[4]  A. Means,et al.  Autoregulation of enzymes by pseudosubstrate prototopes: myosin light chain kinase. , 1988, Science.

[5]  R. Wysolmerski,et al.  Inhibition of endothelial cell retraction by ATP depletion. , 1988, The American journal of pathology.

[6]  F. Fay,et al.  Physiological and structural properties of saponin-skinned single smooth muscle cells , 1987, The Journal of general physiology.

[7]  A. Means,et al.  The calmodulin binding domain of chicken smooth muscle myosin light chain kinase contains a pseudosubstrate sequence. , 1987, The Journal of biological chemistry.

[8]  D. Rotrosen,et al.  Histamine type I receptor occupancy increases endothelial cytosolic calcium, reduces F-actin, and promotes albumin diffusion across cultured endothelial monolayers , 1986, The Journal of cell biology.

[9]  R. Wysolmerski,et al.  The effect of ethchlorvynol on cultured endothelial cells. A model for the study of the mechanism of increased vascular permeability. , 1985, The American journal of pathology.

[10]  B. Burnside,et al.  Regulation of reactivated contraction in teleost retinal cone models by calcium and cyclic adenosine monophosphate , 1984, The Journal of cell biology.

[11]  K. Weber,et al.  Calcium control of actin-myosin based contraction in triton models of mouse 3T3 fibroblasts is mediated by the myosin light chain kinase (MLCK)-calmodulin complex. , 1983, Experimental cell research.

[12]  W. Kerrick,et al.  Superprecipitation of gizzard actomyosin, and tension in gizzard muscle skinned fibers in the presence of nucleotides other than ATP. , 1982, Biochimica et biophysica acta.

[13]  James R. Smith,et al.  Expression of angiotensin‐converting enzyme activity in cultured pulmonary artery endothelial cells , 1981, Journal of cellular physiology.

[14]  S. Perry,et al.  Purification and properties of myosin light-chain kinase from fast skeletal muscle. , 1977, The Biochemical journal.

[15]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[16]  R. Wysolmerski,et al.  Involvement of myosin light-chain kinase in endothelial cell retraction. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[17]  D. Blumenthal,et al.  [10] Preparation and properties of the calmodulin-binding domain of skeletal muscle myosin light chain kinase , 1987 .

[18]  R. Ezzell,et al.  Evidence for regulation of lamellipodial and tail contraction of glycerinated chicken embryonic fibroblasts by myosin light chain kinase. , 1986, Cell motility and the cytoskeleton.