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

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 wa...

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Bibliographic Details
Published in:Scientific reports 2016-02, Vol.6 (1), p.22114-22114, Article 22114
Main Authors: Pena, Darlene Aparecida, Andrade, Victor Piana de, Silva, Gabriela Ávila Fernandes, Neves, José Ivanildo, Oliveira, Paulo Sergio Lopes de, Alves, Maria Julia Manso, Devi, Lakshmi A., Schechtman, Deborah
Format: Article
Language:English
Subjects:
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Antibodies
Antibodies - metabolism
Binding Sites - immunology
Breast cancer
Breast Neoplasms - immunology
Breast Neoplasms - metabolism
Breast Neoplasms - pathology
Carcinogenesis
Cell Line, Tumor
Crystal structure
Enzyme Activation
Enzyme-linked immunosorbent assay
Female
Humanities and Social Sciences
Humans
Immunoprecipitation
Isoenzymes - immunology
Kinases
multidisciplinary
Neoplasm Staging
Neuroblastoma - immunology
Neuroblastoma - metabolism
Neuroblastoma - pathology
Peptide Fragments - immunology
Phosphorylation
Protein Conformation
Protein Domains - genetics
Protein kinase C
Protein Kinase C beta - genetics
Protein Kinase C beta - immunology
Protein Kinase C beta - metabolism
Receptors, Estrogen - metabolism
Science
Signal Transduction
Structure-Activity Relationship
Transduction
Tumors
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Summary: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.
ISSN:2045-2322
2045-2322
DOI:10.1038/srep22114