Rational design and validation of an anti-protein kinase C active-state specific antibody based on conformational changes

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.

Generating Conformation‐Specific Polyclonal and Monoclonal Anti‐Protein Kinase C Antibodies and Anti–Active State Specific PKC Antibodies

“…A deletion mutant of the pseudo‐substrate generates a constitutively active kinase, confirming that this region maintains the kinase inactive (Pena et al., ). This deletion mutant can be expressed in cells and used as an antigen to immunize mice with active PKC to obtain anti‐active PKCβΙ monoclonal antibodies.…”

“…In the field of signal transduction, an antibody that recognizes active states of specific kinases is highly desirable, since it could be used for spatial and temporal dynamics studies of a specific pathway as well as to understand the role of a specific kinase in a signaling process. Since many kinases undergo phosphorylation upon activation, phospho‐specific antibodies that recognize the phosphorylated/active kinase have been developed (Pena et al., ). However, since phosphorylation is a labile modification and since some enzymes do not undergo phosphorylation upon activation, other strategies to develop antibodies that recognize specifically an active kinase are desired.…”

“…The first strategy relies on identifying regions that are involved in intramolecular interactions in an inactive state and thus are only exposed in an active kinase. Peptides sufficiently long to maintain at least in part the structure of the interaction surfaces are designed from these regions, and used to immunize animals to obtain antibodies that should preferentially recognize the active kinase (Pena et al., ). The second strategy is based on the fact that it is very difficult to obtain sufficient quantities of pure protein in an active state.…”

“…The second strategy is based on the fact that it is very difficult to obtain sufficient quantities of pure protein in an active state. We designed a strategy of immunizing mice with lysates of cells expressing a mutated form of PKCβΙ that is constitutively active (Pena et al., ). A similar strategy has been used previously to obtain specific antibodies for G‐protein‐coupled receptor heteromers (Gomes, Gupta, Bushlin, & Devi, ).…”

“…When designing peptides for immunization strategies, long peptides have a greater possibility of being presented to the MHC complex in a structure similar to the one in the native protein. Thus, a peptide that was sufficiently long to have a similar structure as the region in the whole protein, involved in the intramolecular interaction between the catalytic and the C2 domains of PKC, was chosen as the immunizing antigen (Pena et al., ).…”