Protein kinase C-alpha signals rho-guanine nucleotide dissociation inhibitor phosphorylation and rho activation and regulates the endothelial cell barrier function.
The Rho-GDP guanine nucleotide dissociation inhibitor (GDI) complexes with the GDP-bound form of Rho and inhibits its activation. We investigated the role of protein kinase C (PKC) isozymes in the mechanism of Rho activation and in signaling the loss of endothelial barrier function. Thrombin and phorbol 12-myristate 13-acetate induced rapid phosphorylation of GDI and the activation of Rho-A in human umbilical venular endothelial cells. Inhibition of PKC by chelerythrine chloride abrogated the thrombin-induced GDI phosphorylation and Rho activation. Depletion of PKC prevented the thrombin-induced GDI phosphorylation and Rho activation, thereby indicating that these events occurred downstream of phorbol ester-sensitive PKC isozyme activation. The depletion of PKC or inhibition of Rho by C3 toxin also prevented the thrombin-induced decrease in transendothelial electrical resistance (a measure of increased transendothelial permeability), thus indicating that PKC-induced barrier dysfunction was mediated through Rho-dependent pathway. Using inhibitors and dominant-negative mutants, we found that Rho activation was regulated by PKC-alpha. Moreover, the stimulation of human umbilical venular endothelial cells with thrombin induced rapid association of PKC-alpha with Rho. Activated PKC-alpha but not PKC-epsilon induced marked phosphorylation of GDI in vitro. Taken together, these results indicate that PKC-alpha is critical in regulating GDI phosphorylation, Rho activation, and in signaling Rho-dependent endothelial barrier dysfunction.
Mehta
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Department of Pharmacology, The University of Illinois College of Medicine, Chicago, Illinois 60612, USA. dmehta@uic.edu
Rahman
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Malik
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D002199:Capillary Permeability; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D017930:Genes, Reporter; D020730:Guanine Nucleotide Dissociation Inhibitors / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism*; D008564:Membrane Potentials / Q000187:drug effects; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology
D004791:Enzyme Inhibitors; D020730:Guanine Nucleotide Dissociation Inhibitors; D007527:Isoenzymes; C496628:PRKCA protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D013917:Thrombin; D019204:GTP-Binding Proteins; D013755:Tetradecanoylphorbol Acetate