Endothelial cell retraction is induced by PAK2 monophosphorylation of myosin II.
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.
Zeng
Q
Q
Department of Pathology, St Louis University School of Medicine St Louis, Missouri 63104-1028, USA.
Lagunoff
D
D
Masaracchia
R
R
Goeckeler
Z
Z
Côté
G
G
Wysolmerski
R
R
D000199:Actins / Q000378:metabolism; D000470:Alkaloids / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D002227:Carbazoles; D002417:Cattle; D002460:Cell Line; D048429:Cell Size / Q000187:drug effects; D003599:Cytoskeleton / Q000187:drug effects* / Q000648:ultrastructure; D003931:Diacetyl / Q000031:analogs & derivatives / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007211:Indoles; D008845:Microinjections; D008969:Molecular Sequence Data; D018994:Myosin Light Chains / Q000378:metabolism*; D009219:Myosin-Light-Chain Kinase / Q000037:antagonists & inhibitors; D009218:Myosins / Q000378:metabolism*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011817:Rabbits; D019311:Staurosporine / Q000494:pharmacology; D054462:p21-Activated Kinases
D000199:Actins; D000470:Alkaloids; D002227:Carbazoles; D004791:Enzyme Inhibitors; D007211:Indoles; D018994:Myosin Light Chains; C063769:KT 5926; C004717:diacetylmonoxime; D017346:Protein Serine-Threonine Kinases; D054462:p21-Activated Kinases; D009219:Myosin-Light-Chain Kinase; D009218:Myosins; D019311:Staurosporine; D003931:Diacetyl