Involvement of protein phosphatase-1 in cytoskeletal organization of cultured endothelial cells.
The phosphorylation and dephosphorylation of cytoskeletal proteins regulate the shape of eukaryotic cells. To elucidate the role of serine/threonine protein phosphatases (PP) in this process, we studied the effects of calyculin A (CLA), a potent and specific inhibitor of protein phosphatases 1 (PP-1) and 2A (PP-2A) on the cytoskeletal structure of cultured human umbilical vein endothelial cells (HUVECs). The addition of CLA (5 min) caused marked alterations in cell morphology, such as cell constriction and bleb formation. Microtubules and F-actin were reorganized, becoming markedly condensed around the nucleus. Although the fluorescence intensity of phosphoamino acids was not significantly different according to immunocytochemistry between cells with and without CLA, polypeptides of 135, 140, 158, and 175 kDa were specifically phosphorylated on serine and/or threonine residues. There was no significant effect on tyrosine residues. The effects of CLA on cytoskeletal changes and protein phosphorylation were almost completely inhibited by the non-selective kinase inhibitor, K-252a. The effect of CLA on cell morphology was at least 100 times more potent than that of okadaic acid, consistent with the inhibitory potency against PP-1. The catalytic subunit of PP-1 was also identified in HUVECs by Western blotting with its monoclonal antibody antibody. These results suggest that PP-1 is closely involved in sustaining the normal structure of the cytoskeleton.
Shinoki
N
N
Department of Surgery II, Osaka University Medical School, Japan.
Sakon
M
M
Kambayashi
J
J
Ikeda
M
M
Oiki
E
E
Okuyama
M
M
Fujitani
K
K
Yano
Y
Y
Kawasaki
T
T
Monden
M
M
D000199:Actins / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D002227:Carbazoles / Q000494:pharmacology; D002467:Cell Nucleus / Q000378:metabolism / Q000648:ultrastructure; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000187:drug effects / Q000648:ultrastructure*; D004730:Endothelium, Vascular / Q000187:drug effects / Q000201:enzymology* / Q000648:ultrastructure*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D026121:Indole Alkaloids; D008387:Marine Toxins; D051379:Mice; D008870:Microtubules / Q000648:ultrastructure; D010080:Oxazoles / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D010769:Phosphothreonine / Q000378:metabolism; D047428:Protein Kinase Inhibitors; D054645:Protein Phosphatase 1; D014471:Umbilical Veins
D000199:Actins; D002227:Carbazoles; D004791:Enzyme Inhibitors; D026121:Indole Alkaloids; D008387:Marine Toxins; D010080:Oxazoles; D047428:Protein Kinase Inhibitors; D010769:Phosphothreonine; D010768:Phosphoserine; C059041:calyculin A; C049985:staurosporine aglycone; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1