title: Opposing effects of PKCalpha and PKCepsilon on basolateral membrane dynamics in intestinal epithelia.
abstract: PKC is a critical effector of plasma membrane dynamics, yet the mechanism and isoform-specific role of PKC are poorly understood. We recently showed that the phorbol ester PMA (100 nM) induces prompt activation of the novel isoform PKCepsilon followed by late activation of the conventional isoform PKCalpha in T84 intestinal epithelia. PMA also elicited biphasic effects on endocytosis, characterized by an initial stimulatory phase followed by an inhibitory phase. Activation of PKCepsilon was shown to be responsible for stimulation of basolateral endocytosis, but the role of PKCalpha was not defined. Here, we used detailed time-course analysis as well as selective activators and inhibitors of PKC isoforms to infer the action of PKCalpha on basolateral endocytosis. Inhibition of PKC by the selective conventional PKC inhibitor Gö-6976 (5 microM) completely blocked the late inhibitory phase and markedly prolonged the stimulatory phase of endocytosis measured by FITC-dextran uptake. The PKCepsilon-selective agonist carbachol (100 microM) induced prolonged stimulation of endocytosis devoid of an inhibitory phase. Actin disassembly caused by PMA was completely blocked by Gö-6850 but not by Gö-6976, implicating PKCepsilon as the key isoform responsible for actin disruption. The Ca2+ agonist thapsigargin (5 microM) induced early activation of PKC when added simultaneously with PMA. This early activation of PKCalpha blocked the ability of PMA to remodel basolateral F-actin and abolished the stimulatory phase of basolateral endocytosis. Activation of PKCalpha stabilizes F-actin and thereby opposes the effect of PKCepsilon on membrane remodeling in T84 cells.
authors.affiliation: Department of Surgery, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267, USA.
authors.forename: Jaekyung Cecilia
authors.initials: JC
authors.lastname: Song
authors.forename: Patangi K
authors.initials: PK
authors.lastname: Rangachari
authors.forename: Jeffrey B
authors.initials: JB
authors.lastname: Matthews
mesh_terms: D000199:Actins / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002227:Carbazoles / Q000494:pharmacology; D002273:Carcinogens / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology; D016764:Cell Polarity / Q000502:physiology; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000187:drug effects / Q000201:enzymology; D004347:Drug Interactions; D004705:Endocytosis / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000201:enzymology*; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D007413:Intestinal Mucosa / Q000166:cytology* / Q000201:enzymology; D007527:Isoenzymes / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D051744:Protein Kinase C-epsilon; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D019284:Thapsigargin / Q000494:pharmacology
chemical_list: D000199:Actins; D002227:Carbazoles; D002273:Carcinogens; D004791:Enzyme Inhibitors; D007211:Indoles; D007527:Isoenzymes; C081021:Go 6976; D019284:Thapsigargin; C496628:PRKCA protein, human; C497255:PRKCE protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D051744:Protein Kinase C-epsilon; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium