Overexpression of nPKC theta is inhibitory for agrin-induced nicotinic acetylcholine receptor clustering in C2C12 myotubes.
Protein kinase C (PKC) activity has been implicated in nicotinic acetylcholine receptor (nAChR) cluster disruption but the specific PKC isoforms involved have not been identified. We first tested whether phorbol esters, which activate PKCs, regulate agrin-induced nAChR clustering in C(2)C(12) cells. We found that extended phorbol ester treatment (6 hr) increased nAChR clustering by two-fold. This increase correlated in time with downregulation of PKCs, as indicated by the disappearance of cPKC alpha, suggesting that the presence of PKCs is inhibitory for maximal nAChR clustering. To address the question whether nPKC theta, a specific PKC isoform restricted in expression to skeletal muscle and localized to neuromuscular junctions, regulates agrin-induced nAChR cluster formation we overexpressed an nPKC theta -green fluorescent protein (GFP) fusion protein in C(2)C(12) myotubes. The number of nAChR clusters was significantly reduced in nPKC theta-GFP compared to GFP overexpressing myotubes at less-than-maximal clustering concentrations of agrin. These data indicate that nPKC theta activity inhibits nAChR cluster formation. To examine whether nPKC theta activation by phorbol esters regulates agrin-induced nAChR clustering, we treated overexpressing myotubes overnight with maximal agrin concentrations followed by phorbol esters for 1 hr. Phorbol ester treatment reduced preexisting nAChR cluster numbers in nPKC theta-GFP compared to GFP-overexpressing myotubes, suggesting that stimulating nPKC theta activity disrupts nAChR clusters in the presence of maximal clustering concentrations of agrin. Together these findings, that nPKC theta activity inhibits agrin-induced nAChR cluster formation and disrupts preexisting agrin-induced nAChR clusters, suggest that nPKC theta activity is inhibitory for agrin function.
Miles
Kathryn
K
Department of Anatomy and Cell Biology, State University of New York Health Science Center at Brooklyn, Brooklyn, New York 11203, USA. kathryn.miles@downstate.edu
Wagner
Michael
M
D018171:Agrin / Q000494:pharmacology; D000818:Animals; D019556:COS Cells / Q000378:metabolism; D002273:Carcinogens / Q000494:pharmacology; D018929:Cell Culture Techniques; D017077:Culture Media, Conditioned / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005455:Fluorescent Antibody Technique; D015971:Gene Expression Regulation, Enzymologic; D049452:Green Fluorescent Proteins; D007527:Isoenzymes / Q000187:drug effects / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D018485:Muscle Fibers, Skeletal / Q000187:drug effects / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000494:pharmacology; D010703:Phorbol Esters / Q000494:pharmacology; D020033:Protein Isoforms / Q000494:pharmacology; D011493:Protein Kinase C / Q000187:drug effects / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D000074121:Protein Kinase C-theta; D011940:Receptor Aggregation / Q000187:drug effects; D011978:Receptors, Nicotinic / Q000187:drug effects / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection
D018171:Agrin; D002273:Carcinogens; D017077:Culture Media, Conditioned; D007527:Isoenzymes; D008164:Luminescent Proteins; D009419:Nerve Tissue Proteins; D010703:Phorbol Esters; D020033:Protein Isoforms; D011978:Receptors, Nicotinic; D049452:Green Fluorescent Proteins; C496630:Prkca protein, mouse; C489593:Prkcq protein, mouse; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D000074121:Protein Kinase C-theta