Rational design and validation of an anti-protein kinase C active-state specific antibody based on conformational
Darlene Aparecida Pena1, Victor Piana de Andrade2, Gabriela Ávila Fernandes Silva1
1Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, SP, Brazil.
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.
Insights
Researchers developed a novel antibody to detect active Protein Kinase C (PKC) in cancer tissues. This tool aids in studying PKC activation, revealing higher active PKC levels in aggressive triple-negative breast cancers.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Protein Kinase C (PKC) is crucial in cancer pathways.
- Existing tools cannot reliably detect active classical PKC (cPKC) in tissues.
- Phosphorylation marks cPKC maturation but not activation.
Purpose of the Study:
- To develop a tool for detecting active cPKC.
- To distinguish between active and inactive cPKC states.
- To investigate cPKC activation in cancer.
Main Methods:
- Structure-based rational design of a peptide (C2Cat).
- Generation of an antibody targeting the active cPKC conformation.
- ELISA, immunoprecipitation, and human tissue analysis.
Main Results:
- The anti-C2Cat antibody specifically recognizes active cPKC.
- It detects PKC activation dynamics upon stimulation.
- Higher active cPKC levels correlate with aggressive triple-negative breast cancer.
Conclusions:
- The anti-C2Cat antibody is a valuable tool for studying cPKC activation.
- This antibody enables analysis of active PKC in human tissues.
- Structure-based antibody design can be applied to other signaling molecules.
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