Abstract
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.
Highlights
Several isozymes of protein kinase C (PKC),1 lipid-dependent protein kinases, are present within a single cell, each mediat
The V1 domain of this isozyme was chosen as the immunogen because it contains a RACK binding site [7, 10] that is exposed only after ⑀PKC activation
Identification of Antibodies to the V1 Domain of ⑀PKC That Are Isozyme-specific—An IgM antibody, 14E6, was shown by ELISA to be specific for ⑀PKC. 14E6 bound to MBP-⑀PKC-V1 and to full-length recombinant ⑀PKC expressed in Sf9 cells (Sf9-⑀PKC), but not to Sf9-␦ PKC, Sf9-␥ PKC, MBP-␦ PKC-V1, MBP- IIPKC-V5, or to the ⑀RACK fragment that binds the ⑀PKC-V1 domain (MBP-⑀RACK-C) (Fig. 1)
Summary
Several isozymes of protein kinase C (PKC),1 lipid-dependent protein kinases, are present within a single cell, each mediat-. Translocation of ⑀PKC, as determined by the commercial antibodies, persisted at 30 min (Fig. 5A), when activated enzyme was no longer detected by 14E6 (Fig. 5B), suggesting that exposure of the epitope for 14E6 is more transient than is localization to sites usually associated with activated enzyme.
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