State-specific monoclonal antibodies identify an intermediate state in epsilon protein kinase C activation.
Evaluation of the activation state of protein kinase C (PKC) isozymes relies on analysis of subcellular translocation. A monoclonal antibody, 14E6, specific for the activated conformation of epsilonPKC, was raised using the first variable (V1) domain of epsilonPKC as the immunogen. 14E6 binding is specific for epsilonPKC and is greatly increased in the presence of PKC activators. Immunofluorescence staining by 14E6 of neonatal rat primary cardiac myocytes and the NG108-15 neuroblastoma glioma cell line, NG108-15/D2, increases rapidly following cell activation and is localized to new subcellular sites. However, staining of translocated epsilonPKC with 14E6 is transient, and the epitope disappears 30 min after activation of NG-108/15 cells by a D2 receptor agonist. In contrast, subcellular localization associated with activation, as determined by commercially available polyclonal antibodies, persists for at least 30 min. In vitro, epsilonRACK, the receptor for activated epsilonPKC, inhibits 14E6 binding to epsilonPKC, suggesting that the 14E6 epitope is lost or hidden when active epsilonPKC binds to its RACK. Therefore, the 14E6 antibody appears to identify a transient state of activated but non-anchored epsilonPKC. Moreover, binding of 14E6 to epsilonPKC only after activation suggests that lipid-dependent conformational changes associated with epsilonPKC activation precede binding of the activated isozyme to its specific RACK, epsilonRACK. Further, monoclonal antibody 14E6 should be a powerful tool to study the pathways that control rapid translocation of epsilonPKC from cytosolic to membrane localization on activation.
Souroujon
Miriam C
MC
Department of Molecular Pharmacology, Stanford University School of Medicine, Stanford, California 94305, USA.
Yao
Lina
L
Chen
Haibin
H
Endemann
Gerda
G
Khaner
Hanita
H
Geeraert
Virginie
V
Schechtman
Deborah
D
Gordon
Adrienne S
AS
Diamond
Ivan
I
Mochly-Rosen
Daria
D
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D002460:Cell Line; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D004789:Enzyme Activation; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000737:chemistry; D007313:Insecta; D008055:Lipids / Q000737:chemistry; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D032383:Myocytes, Cardiac / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D011233:Precipitin Tests; D011485:Protein Binding; D011487:Protein Conformation; D020033:Protein Isoforms; D011493:Protein Kinase C / Q000737:chemistry / Q000378:metabolism*; D051744:Protein Kinase C-epsilon; D017434:Protein Structure, Tertiary; D051381:Rats; D000075370:Receptors for Activated C Kinase; D011994:Recombinant Proteins / Q000737:chemistry
D000911:Antibodies, Monoclonal; D000939:Epitopes; D008055:Lipids; D010455:Peptides; D020033:Protein Isoforms; D000075370:Receptors for Activated C Kinase; D011994:Recombinant Proteins; C070301:peptide I; C497257:Prkce protein, rat; D011493:Protein Kinase C; D051744:Protein Kinase C-epsilon