Abstract
Protein kinase C (PKC) plays a regulatory role in key pathways in cancer. However, since phosphorylation is a step for classical PKC (cPKC) maturation and does not correlate with activation, there is a lack of tools to detect active PKC in tissue samples. Here, a structure-based rational approach was used to select a peptide to generate an antibody that distinguishes active from inactive cPKC. A peptide conserved in all cPKCs, C2Cat, was chosen since modeling studies based on a crystal structure of PKCβ showed that it is localized at the interface between the C2 and catalytic domains of cPKCs in an inactive kinase. Anti-C2Cat recognizes active cPKCs at least two-fold better than inactive kinase in ELISA and immunoprecipitation assays, and detects the temporal dynamics of cPKC activation upon receptor or phorbol stimulation. Furthermore, the antibody is able to detect active PKC in human tissue. Higher levels of active cPKC were observed in the more aggressive triple negative breast cancer tumors as compared to the less aggressive estrogen receptor positive tumors. Thus, this antibody represents a reliable, hitherto unavailable and a valuable tool to study PKC activation in cells and tissues. Similar structure-based rational design strategies can be broadly applied to obtain active-state specific antibodies for other signal transduction molecules.
Highlights
Most antibodies that are thought to detect active kinases are directed against phosphorylation sites considered essential for kinase activation such as the activation loop[5]
(2015) the reinterpretation of a previously reported crystal structure of PKCβ II combined with docking of the different domains in the kinase, proposed an alternative structure for inactive Protein kinase C (PKC) with the C2 domain interacting with the carboxy-terminus (V5) and the catalytic domain of the kinase further contributing to the maintenance of the kinase in an inactive conformation[10]
Based on the crystal structure coordinates[13], the follow up study reinterpreting the crystal structure and validating that the C2 domain interacts with the catalytic domain in PKCβ II10, and other biochemical studies, about the conformational changes suffered by PKC upon activation[14,15], we propose a model for PKCβ ΙΙ similar to the one proposed by Antal and collaborators[10] in which the kinase domain interacts with the C2 domain
Summary
Most antibodies that are thought to detect active kinases are directed against phosphorylation sites considered essential for kinase activation such as the activation loop[5]. A peptide derived from an interaction region between the C2 and catalytic domain of cPKC in an inactive kinase was chosen and used to generate active-state specific polyclonal antibodies. These antibodies preferentially recognize active cPKCs confirming that this region is exposed in the active PKC. Higher levels of active PKC was found in neuroblastoma cells upon phorbol or receptors stimulation, in triple negative breast cancer cells and in patient tissues as compared to estrogen receptor positive cells These antibodies can be used to monitor PKC activation status and help elucidate the temporal dynamics of PKC activation and mechanisms that lead to tumorigenesis
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