Endothelial cell retraction is induced by PAK2 monophosphorylation of myosin II

2000 ◽  
Vol 113 (3) ◽  
pp. 471-482 ◽  
Author(s):  
Q. Zeng ◽  
D. Lagunoff ◽  
R. Masaracchia ◽  
Z. Goeckeler ◽  
G. Cote ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Actin Cytoskeleton ◽  
Light Chain ◽  
Myosin Ii ◽  
Specific Inhibitor ◽  
Regulatory Light Chain ◽  
Atpase Inhibitor ◽  
Cytoskeletal Rearrangements ◽  
Cell Retraction

The p21-activated kinase (PAK) family includes several enzyme isoforms regulated by the GTPases Rac1 and Cdc42. PAK1, found in brain, muscle and spleen, has been implicated in triggering cytoskeletal rearrangements such as the dissolution of stress fibers and reorganization of focal complexes. The role of the more widely distributed PAK2 in controlling the cytoskeleton has been less well studied. Previous work has demonstrated that PAK2 can monophosphorylate the myosin II regulatory light chain and induce retraction of permeabilized endothelial cells. In this report we characterize PAK2's morphological and biochemical effect on intact endothelial cells utilizing microinjection of constitutively active PAK2. Under these conditions we observed a modification of the actin cytoskeleton with retraction of endothelial cell margins accompanied by an increase in monophosphorylation of myosin II. Selective inhibitors were used to analyze the mechanism of action of PAK2. Staurosporine, a direct inhibitor of PAK2, largely prevented the action of microinjected PAK2 in endothelial cells. Butanedione monoxime, a non-specific myosin ATPase inhibitor, also inhibited the effects of PAK2 implicating myosin in the changes in cytoskeletal reorganization. In contrast, KT5926, a specific inhibitor of myosin light chain kinase was ineffective in preventing the changes in morphology and the actin cytoskeleton. The additional finding that endogenous PAK2 associates with myosin II is consistent with the proposal that cell retraction and cytoskeletal rearrangements induced by microinjected PAK2 depend on the direct activation of myosin II by PAK2 monophosphorylation of the regulatory light chain.

Adenosine induces dephosphorylation of myosin II regulatory light chain in cultured bovine corneal endothelial cells

2004 ◽  
Vol 79 (4) ◽  
pp. 543-551 ◽  
Author(s):  
S.P. Srinivas ◽  
M. Satpathy ◽  
P. Gallagher ◽  
E. Larivière ◽  
W. Van Driessche
Keyword(s):  
Endothelial Cells ◽  
Light Chain ◽  
Myosin Ii ◽  
Regulatory Light Chain ◽  
Corneal Endothelial Cells

scholarly journals Nonmuscle myosin II is responsible for maintaining endothelial cell basal tone and stress fiber integrity

2008 ◽  
Vol 295 (4) ◽  
pp. C994-C1006 ◽  
Author(s):  
Zoe M. Goeckeler ◽  
Paul C. Bridgman ◽  
Robert B. Wysolmerski
Keyword(s):  
Endothelial Cell ◽  
Light Chain ◽  
Actin Polymerization ◽  
Myosin Ii ◽  
Rho Kinase ◽  
Specific Inhibitor ◽  
Isometric Tension ◽  
Stress Fiber ◽  
Tension Development ◽  
Basal Tone

Cultured confluent endothelial cells exhibit stable basal isometric tone associated with constitutive myosin II regulatory light chain (RLC) phosphorylation. Thrombin treatment causes a rapid increase in isometric tension concomitant with myosin II RLC phosphorylation, actin polymerization, and stress fiber reorganization while inhibitors of myosin light chain kinase (MLCK) and Rho-kinase prevent these responses. These findings suggest a central role for myosin II in the regulation of endothelial cell tension. The present studies examine the effects of blebbistatin, a specific inhibitor of myosin II activity, on basal tone and thrombin-induced tension development. Although blebbistatin treatment abolished basal tension, this was accompanied by an increase in myosin II RLC phosphorylation. The increase in RLC phosphorylation was Ca2+ dependent and mediated by MLCK. Similarly, blebbistatin inhibited thrombin-induced tension without interfering with the increase in RLC phosphorylation or in F-actin polymerization. Blebbistatin did prevent myosin II filament incorporation and association with polymerizing or reorganized actin filaments leading to the disappearance of stress fibers. Thus the inhibitory effects of blebbistatin on basal tone and induced tension are consistent with a requirement for myosin II activity to maintain stress fiber integrity.

scholarly journals Schizosaccharomyces pombe Pak-related protein, Pak1p/Orb2p, phosphorylates myosin regulatory light chain to inhibit cytokinesis

2008 ◽  
Vol 183 (5) ◽  
pp. 785-793 ◽  
Author(s):  
Tsui-Han Loo ◽  
Mohan Balasubramanian
Keyword(s):  
Actin Cytoskeleton ◽  
Schizosaccharomyces Pombe ◽  
Light Chain ◽  
Myosin Ii ◽  
Regulatory Light Chain ◽  
Eukaryotic Cells ◽  
Myosin Regulatory Light Chain ◽  
Related Protein ◽  
Filamentous Actin

p21-activated kinases (Paks) have been identified in a variety of eukaryotic cells as key effectors of the Cdc42 family of guanosine triphosphatases. Pak kinases play important roles in regulating the filamentous actin cytoskeleton. In this study, we describe a function for the Schizosaccharomyces pombe Pak-related protein Pak1p/Orb2p in cytokinesis. Pak1p localizes to the actomyosin ring during mitosis and cytokinesis. Loss of Pak1p function leads to accelerated cytokinesis. Pak1p mediates phosphorylation of myosin II regulatory light chain Rlc1p at serine residues 35 and 36 in vivo. Interestingly, loss of Pak1p function or substitution of serine 35 and serine 36 of Rlc1p with alanines, thereby mimicking a dephosphorylated state of Rlc1p, leads to defective coordination of mitosis and cytokinesis. This study reveals a new mechanism involving Pak1p kinase that helps ensure the fidelity of cytokinesis.

Regulation of permeabilized endothelial cell retraction by myosin phosphorylation

1991 ◽  
Vol 261 (1) ◽  
pp. C32-C40 ◽  
Author(s):  
R. B. Wysolmerski ◽  
D. Lagunoff
Keyword(s):  
Endothelial Cell ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Specific Inhibitor ◽  
Light Chains ◽  
Myosin Light Chains ◽  
Inhibitory Peptide ◽  
Endothelial Cell Retraction ◽  
Cell Retraction ◽  
Gamma S

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.

scholarly journals Citron Kinase, a Rho-dependent Kinase, Induces Di-phosphorylation of Regulatory Light Chain of Myosin II

2003 ◽  
Vol 14 (5) ◽  
pp. 1745-1756 ◽  
Author(s):  
Shigeko Yamashiro ◽  
Go Totsukawa ◽  
Yoshihiko Yamakita ◽  
Yasuharu Sasaki ◽  
Pascal Madaule ◽  
...  
Keyword(s):  
Light Chain ◽  
Myosin Ii ◽  
Specific Inhibitor ◽  
Kinase Domain ◽  
Regulatory Light Chain ◽  
Myosin Phosphatase ◽  
Fiber Assembly ◽  
Citron Kinase

Citron kinase is a Rho-effector protein kinase that is related to Rho-associated kinases of ROCK/ROK/Rho-kinase family. Both ROCK and citron kinase are suggested to play a role in cytokinesis. However, no substrates are known for citron kinase. We found that citron kinase phosphorylated regulatory light chain (MLC) of myosin II at both Ser-19 and Thr-18 in vitro. Unlike ROCK, however, citron kinase did not phosphorylate the myosin binding subunit of myosin phosphatase, indicating that it does not inhibit myosin phosphatase. We found that the expression of the kinase domain of citron kinase resulted in an increase in MLC di-phosphorylation. Furthermore, the kinase domain was able to increase di-phosphorylation and restore stress fiber assembly even when ROCK was inhibited with a specific inhibitor, Y-27632. The expression of full-length citron kinase also increased di-phosphorylation during cytokinesis. These observations suggest that citron kinase phosphorylates MLC to generate di-phosphorylated MLC in vivo. Although both mono- and di-phosphorylated MLC were found in cleavage furrows, di-phosphorylated MLC showed more constrained localization than did mono-phosphorylated MLC. Because citron kinase is localized in cleavage furrows, citron kinase may be involved in regulating di-phosphorylation of MLC during cytokinesis.

Characterization of myosin II regulatory light chain isoforms in HeLa cells

Cytoskeleton ◽  
2015 ◽  
Vol 72 (12) ◽  
pp. 609-620 ◽  
Author(s):  
Tomo Kondo ◽  
Morihiro Okada ◽  
Kayo Kunihiro ◽  
Masayuki Takahashi ◽  
Yoshio Yaoita ◽  
...  
Keyword(s):  
Light Chain ◽  
Hela Cells ◽  
Myosin Ii ◽  
Regulatory Light Chain
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