PKCdelta influences p190 phosphorylation and activity: events independent of PKCdelta-mediated regulation of endothelial cell stress fiber and focal adhesion formation and barrier function.
BACKGROUND
We have shown that protein kinase Cdelta (PKCdelta) inhibition results in increased endothelial cell (EC) permeability and decreased RhoA activity; which correlated with diminished stress fibers (SF) and focal adhesions (FA). We have also shown co-precipitation of p190RhoGAP (p190) with PKCdelta. Here, we investigated if PKCdelta regulates p190 and whether PKCdelta-mediated changes in SF and FA or permeability were dependent upon p190.


METHODS
Protein-protein interaction and activity analyses were performed using co-precipitation assays. Analysis of p190 phosphorylation was performed using in vitro kinase assays. SF and FA were analyzed by immunofluorescence analyses. EC monolayer permeability was measured using electrical cell impedance sensor (ECIS) technique.


RESULTS
Inhibition of PKCdelta increased p190 activity, while PKCdelta overexpression diminished p190 activity. PKCdelta bound to and phosphorylated both p190FF and p190GTPase domains. p190 protein overexpression diminished SF and FA formation and RhoA activity. Disruption of SF and FA or increased permeability induced upon PKCdelta inhibition, were not attenuated in EC in which the p190 isoforms were suppressed individually or concurrently.


CONCLUSIONS
Our findings suggest that while PKCdelta can regulate p190 activity, possibly at the FF and/or GTPase domains, the effect of PKCdelta inhibition on SF and FA and barrier dysfunction occurs through a pathway independent of p190.
Fordjour
Akua K
AK
Vascular Research Laboratory, Providence Veterans Affairs Medical Center, Department of Medicine, Alpert Medical School of Brown University, Providence, RI 02908, USA.
Harrington
Elizabeth O
EO
D000098:Acetophenones / Q000494:pharmacology; D000818:Animals; D001578:Benzopyrans / Q000494:pharmacology; D001665:Binding Sites; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D022001:Focal Adhesions / Q000187:drug effects / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D015151:Immunoblotting; D008856:Microscopy, Fluorescence; D010766:Phosphorylation; D011485:Protein Binding; D051745:Protein Kinase C-delta / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011651:Pulmonary Artery / Q000166:cytology; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D022502:Stress Fibers / Q000378:metabolism*; D014162:Transfection
D000098:Acetophenones; C513040:Arhgap35 protein, rat; D001578:Benzopyrans; D004791:Enzyme Inhibitors; D034741:RNA, Small Interfering; D012097:Repressor Proteins; D049452:Green Fluorescent Proteins; C085746:rottlerin; D051745:Protein Kinase C-delta