VCAM-1 signals activate endothelial cell protein kinase Calpha via oxidation.
Lymphocyte binding to VCAM-1 activates endothelial cell NADPH oxidase, resulting in the generation of 1 muM H(2)O(2). This is required for VCAM-1-dependent lymphocyte migration. In this study, we identified a role for protein kinase Calpha (PKCalpha) in VCAM-1 signal transduction in human and mouse endothelial cells. VCAM-1-dependent spleen cell migration under 2 dynes/cm(2) laminar flow was blocked by pretreatment of endothelial cells with dominant-negative PKCalpha or the PKCalpha inhibitors, Rö-32-0432 or Gö-6976. Phosphorylation of PKCalpha(Thr638), an autophosphorylation site indicating enzyme activity, was increased by Ab cross-linking of VCAM-1 on endothelial cells or by the exogenous addition of 1 muM H(2)O(2). The anti-VCAM-1-stimulated phosphorylation of PKCalpha(Thr638) was blocked by scavenging of H(2)O(2) and by inhibition of NADPH oxidase. Furthermore, anti-VCAM-1 signaling induced the oxidation of endothelial cell PKCalpha. Oxidized PKCalpha is a transiently active form of PKCalpha that is diacylglycerol independent. This oxidation was blocked by inhibition of NADPH oxidase. In summary, VCAM-1 activation of endothelial cell NADPH oxidase induces transient PKCalpha activation that is necessary for VCAM-1-dependent transendothelial cell migration.
Abdala-Valencia
Hiam
H
Allergy-Immunology Division, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA.
Cook-Mills
Joan M
JM
D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D002227:Carbazoles / Q000494:pharmacology; D002465:Cell Movement; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology* / Q000502:physiology; D004789:Enzyme Activation; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D051379:Mice; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000378:metabolism; D010084:Oxidation-Reduction; D010766:Phosphorylation; D051571:Protein Kinase C-alpha / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000494:pharmacology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011758:Pyrroles / Q000494:pharmacology; D015398:Signal Transduction; D013154:Spleen / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology; D013912:Threonine / Q000378:metabolism; D019010:Vascular Cell Adhesion Molecule-1 / Q000187:drug effects / Q000502:physiology*
D000906:Antibodies; D002227:Carbazoles; D007211:Indoles; D047428:Protein Kinase Inhibitors; D011758:Pyrroles; D019010:Vascular Cell Adhesion Molecule-1; C081021:Go 6976; C085989:Ro 32-0432; D013912:Threonine; D006861:Hydrogen Peroxide; D019255:NADPH Oxidases; D051571:Protein Kinase C-alpha