Profiling the interactome of protein kinase C ζ by proteomics and bioinformatics

Proteome Science
Chunyu HouRuibing Chen

Abstract

Protein kinase C ζ (PKCζ), an isoform of the atypical protein kinase C, is a pivotal regulator in cancer. However, the molecular and cellular mechanisms whereby PKCζ regulates tumorigenesis and metastasis are still not fully understood. In this study, proteomics and bioinformatics analyses were performed to establish a protein-protein interaction (PPI) network associated with PKCζ, laying a stepping stone to further understand the diverse biological roles of PKCζ. Protein complexes associated with PKCζ were purified by co-immunoprecipitation from breast cancer cell MDA-MB-231 and identified by LC-MS/MS. Two biological replicates and two technical replicates were analyzed. The observed proteins were filtered using the CRAPome database to eliminate the potential false positives. The proteomics identification results were combined with PPI database search to construct the interactome network. Gene ontology (GO) and pathway analysis were performed by PANTHER database and DAVID. Next, the interaction between PKCζ and protein phosphatase 2 catalytic subunit alpha (PPP2CA) was validated by co-immunoprecipitation, Western blotting and immunofluorescence. Furthermore, the TCGA database and the COSMIC database were used to analyze the ex...Continue Reading

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Methods Mentioned

BETA
co-immunoprecipitation
PCR
Transfection
reverse transcription PCR
Co-IP
two hybrid

Software Mentioned

DAVID
CoIP
InACT
Systems Biology and Evolution MATLAB Toolbox ( SBEToolbox )
Excel
Protein Discoverer
MS
SPSS
PANTHER
PPI

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