Conventional protein kinase C mediates phorbol-dibutyrate-induced cytoskeletal remodeling in a7r5 smooth muscle cells.
Phorbol dibutyrate (PDBu) induced the formation of podosome-like structures together with partial disassembly of actin stress fibers in A7r5 smooth muscle cells. These podosomes contained alpha-actinin, F-actin, and vinculin and exhibit a tubular, column-like structure arising perpendicularly from the bottom of PDBu-treated cells. The conventional protein kinase C (PKC) antagonist, GO6976, inhibited PDBu-induced cytoskeletal remodeling at 0.1 microM, whereas the novel PKC antagonist, rottlerin, was ineffective at 10 microM. PDBu induced the translocation of the conventional PKC-alpha but not the novel PKC-delta to the sites of podosome formation in A7r5 cells. Although partial disassembly of actin stress fibers was observed in both Y-27632- and PDBu-treated cells, focal adhesions were much reduced in number and size only in Y-27632-treated cells. Furthermore, PDBu restored focal adhesions in Y-27632-treated cells. Live video fluorescence microscopy of alpha-actinin GFP revealed a lag phase of about 20 min prior to the rapid formation and dynamic reorganization of podosomes during PDBu treatment. These findings suggest that conventional PKCs mediate PDBu-induced formation of dynamic podosome-like structures in A7r5 cells, and Rho-kinase is unlikely to be the underlying mechanism. The podosome columns could represent molecular scaffolds where PKC-alpha phosphorylates regulatory proteins necessary for Ca(2+) sensitization in smooth muscle cells.
Hai
Chi-Ming
CM
Department of Molecular Pharmacology, Physiology, & Biotechnology, Austrian Academy of Sciences, Salzburg, Austria. chi-ming_hai@brown.edu
Hahne
Penelope
P
Harrington
Elizabeth O
EO
Gimona
Mario
M
D000098:Acetophenones / Q000494:pharmacology; D000185:Actinin / Q000378:metabolism; D000199:Actins / Q000378:metabolism; D000577:Amides / Q000494:pharmacology; D000818:Animals; D001578:Benzopyrans / Q000494:pharmacology; D002227:Carbazoles / Q000494:pharmacology; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000187:drug effects / Q000502:physiology / Q000648:ultrastructure*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D049452:Green Fluorescent Proteins; D007211:Indoles / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000502:physiology / Q000648:ultrastructure*; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*; D051571:Protein Kinase C-alpha; D051745:Protein Kinase C-delta; D021381:Protein Transport / Q000187:drug effects; D011725:Pyridines / Q000494:pharmacology; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection; D016596:Vinculin / Q000378:metabolism
D000098:Acetophenones; D000199:Actins; D000577:Amides; D001578:Benzopyrans; D002227:Carbazoles; D004791:Enzyme Inhibitors; D007211:Indoles; D007527:Isoenzymes; D008164:Luminescent Proteins; D011725:Pyridines; D011993:Recombinant Fusion Proteins; D000185:Actinin; D016596:Vinculin; C081021:Go 6976; C108830:Y 27632; D049452:Green Fluorescent Proteins; D015240:Phorbol 12,13-Dibutyrate; C085746:rottlerin; C497260:Prkcd protein, rat; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D051745:Protein Kinase C-delta