Using an in situ proximity ligation assay to systematically profile endogenous protein-protein interactions in a pathway network

J Proteome Res. 2014 Dec 5;13(12):5339-46. doi: 10.1021/pr5002737. Epub 2014 Oct 9.


Signal transduction pathways in the cell require protein-protein interactions (PPIs) to respond to environmental cues. Diverse experimental techniques for detecting PPIs have been developed. However, the huge amount of PPI data accumulated from various sources poses a challenge with respect to data reliability. Herein, we collected ∼ 700 primary antibodies and employed a highly sensitive and specific technique, an in situ proximity ligation assay, to investigate 1204 endogenous PPIs in HeLa cells, and 557 PPIs of them tested positive. To overview the tested PPIs, we mapped them into 13 PPI public databases, which showed 72% of them were annotated in the Human Protein Reference Database (HPRD) and 8 PPIs were new PPIs not in the PubMed database. Moreover, TP53, CTNNB1, AKT1, CDKN1A, and CASP3 were the top 5 proteins prioritized by topology analyses of the 557 PPI network. Integration of the PPI-pathway interaction revealed that 90 PPIs were cross-talk PPIs linking 17 signaling pathways based on Reactome annotations. The top 2 connected cross-talk PPIs are MAPK3-DAPK1 and FAS-PRKCA interactions, which link 9 and 8 pathways, respectively. In summary, we established an open resource for biological modules and signaling pathway profiles, providing a foundation for comprehensive analysis of the human interactome.

Keywords: Reactome; cross-talk protein−protein interaction; in situ proximity ligation assay; protein−protein interaction; signaling pathway.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biological Assay / methods*
  • Caspase 3 / metabolism
  • Cyclin-Dependent Kinase Inhibitor p21 / metabolism
  • Databases, Protein
  • HeLa Cells
  • Humans
  • Models, Biological
  • Oligonucleotide Probes / genetics
  • Oligonucleotide Probes / metabolism
  • Protein Binding
  • Protein Interaction Mapping / methods
  • Protein Interaction Maps*
  • Proteome / metabolism*
  • Proteomics / methods*
  • Proto-Oncogene Proteins c-akt / metabolism
  • Reproducibility of Results
  • Signal Transduction
  • Tumor Suppressor Protein p53 / metabolism
  • beta Catenin / metabolism


  • CDKN1A protein, human
  • CTNNB1 protein, human
  • Cyclin-Dependent Kinase Inhibitor p21
  • Oligonucleotide Probes
  • Proteome
  • Tumor Suppressor Protein p53
  • beta Catenin
  • Proto-Oncogene Proteins c-akt
  • CASP3 protein, human
  • Caspase 3