State-specific Monoclonal Antibodies Identify an Intermediate State in Epsilon Protein Kinase C Activation

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 ⑀PKC, was raised using the first variable (V1) domain of ⑀PKC as the immunogen. 14E6 binding is specific for ⑀PKC 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 ⑀PKC 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, ⑀RACK, the receptor for activated ⑀PKC, inhibits 14E6 binding to ⑀PKC, suggesting that the 14E6 epitope is lost or hidden when active ⑀PKC binds to its RACK. Therefore, the 14E6 antibody appears to identify a transient state of activated but non-anchored ⑀PKC. Moreover, binding of 14E6 to ⑀PKC only after activation suggests that lipid-dependent conformational changes associated with ⑀PKC activation precede binding of the activated isozyme to its specific RACK, ⑀RACK. Further, monoclonal antibody 14E6 should be a powerful tool to study the pathways that control rapid translocation of ⑀PKC from cytosolic to membrane localization on activation.

Rational design of a selective antagonist of ε protein kinase C derived from the selective allosteric agonist, pseudo-RACK peptide

“…In the absence of treatment (time 0, Fig. 4B), only 5% of the cells showed staining for the transition state as previously published [19]. The inactive peptide, AψεRACK, induced a staining pattern indistinguishable from the control-PMA treated cells (Fig.…”

“…We propose that if ψεRACK peptide binds to this transition state, it should stabilize it and therefore more enzyme in the transition state should be found in the cell. To test this hypothesis, we used our state-specific monoclonal antibody, 14E6, which selectively recognizes active but not anchored εPKC [19]. Since 14E6 recognizes εPKC in a transition state, when the enzyme is activated, but before it anchors to its RACK, we expect to see a fast increase in staining of cells with 14E6 followed by a decrease, when the active enzyme is anchored to its RACK [19].…”

“…To test this hypothesis, we used our state-specific monoclonal antibody, 14E6, which selectively recognizes active but not anchored εPKC [19]. Since 14E6 recognizes εPKC in a transition state, when the enzyme is activated, but before it anchors to its RACK, we expect to see a fast increase in staining of cells with 14E6 followed by a decrease, when the active enzyme is anchored to its RACK [19]. Indeed, 1 min after activation with wild type ψεRACK (DψεRACK) peptide together with 1nM PMA, 72% of the cells showed staining for the transition state, as oppose to only 33% for cells treated with only 1nM PMA (Fig.…”

“…Five days after isolation, cells were treated with the indicated peptide for 15 minutes followed by treatment with 1 nM PMA for the indicated periods of time. Cells were then fixed with cold methanol:acetone (1:1), blocked with 1% normal goat serum and incubated overnight at 4°C with εPKC state-selective antibody, 14E6 [19]. After 2 hours incubation with FITC secondary goat anti-mouse IgM (Jackson ImmunoResearch, West Grove, PA), cells were washed three times with PBS/ 0.1% Triton X-100 and mounted with Vectashield.…”

“…Immunofluorescence staining of cardiac myocytes was performed as previously described [19]. Neonatal cardiac myocytes were cultured onto 8 well chamber slides.…”