Profiling The Interactome Of Protein Kinase C ? By Proteomics And Bioinformatics

Background and objective:Protein kinase C ?(PKC?),an isoform of the atypical protein kinase C,is a pivotal regulator in cancer.However,the molecular and cellular mechanisms whereby PKC? 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 PKC?,laying a stepping stone to further understand the diverse biological roles of PKC?.Methods:Protein complexes associated with PKC? 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 PKC? 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 expressions of these two proteins in clinical samples.Results:The PKC? centered PPI network containing 178 nodes and 1225 connections was built.Network analysis showed that the identified proteins were significantly associated with several key signaling pathways regulating cancer related cellular processes.Conclusions:Through combining the proteomics and bioinformatics analyses,a PKC? centered PPI network was constructed,providing a more complete picture regarding the biological roles of PKC? in both breast cancer regulation and other aspects of cellular biology.