Protein Phosphorylation Profiling Using an in Situ Proximity Ligation Assay: Phosphorylation of AURKA-elicited EGFR-Thr654 and EGFR-Ser1046 in Lung Cancer Cells

PLoS One. 2013;8(3):e55657. doi: 10.1371/journal.pone.0055657. Epub 2013 Mar 8.

Abstract

The epidermal growth factor receptor (EGFR), which is up-regulated in lung cancer, involves the activation of mitogenic signals and triggers multiple signaling cascades. To dissect these EGFR cascades, we used 14 different phospho-EGFR antibodies to quantify protein phosphorylation using an in situ proximity ligation assay (in situ PLA). Phosphorylation at EGFR-Thr654 and -Ser1046 was EGF-dependent in the wild-type (WT) receptor but EGF-independent in a cell line carrying the EGFR-L858R mutation. Using a ProtoAarray™ containing ∼5000 recombinant proteins on the protein chip, we found that AURKA interacted with the EGFR-L861Q mutant. Moreover, overexpression of EGFR could form a complex with AURKA, and the inhibitors of AURKA and EGFR decreased EGFR-Thr654 and -Ser1046 phosphorylation. Immunohistochemical staining of stage I lung adenocarcinoma tissues demonstrated a positive correlation between AURKA expression and phosphorylation of EGFR at Thr654 and Ser1046 in EGFR-mutant specimens, but not in EGFR-WT specimens. The interplay between EGFR and AURKA provides an explanation for the difference in EGF dependency between EGFR-WT and EGFR-mutant cells and may provide a new therapeutic strategy for lung cancer patients carrying EGFR mutations.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenocarcinoma / enzymology*
  • Adenocarcinoma / genetics
  • Adenocarcinoma / pathology
  • Antibodies / chemistry
  • Aurora Kinase A
  • Aurora Kinases
  • Cell Line, Tumor
  • ErbB Receptors / genetics
  • ErbB Receptors / metabolism*
  • Female
  • HEK293 Cells
  • Humans
  • Immunohistochemistry / methods
  • Lung Neoplasms / enzymology*
  • Lung Neoplasms / genetics
  • Lung Neoplasms / pathology
  • Male
  • Phosphorylation / genetics
  • Protein Array Analysis / methods
  • Protein-Serine-Threonine Kinases / genetics
  • Protein-Serine-Threonine Kinases / metabolism*

Substances

  • Antibodies
  • EGFR protein, human
  • ErbB Receptors
  • AURKA protein, human
  • Aurora Kinase A
  • Aurora Kinases
  • Protein-Serine-Threonine Kinases

Grant support

This research was supported by grants from the National Science Council (NSC101-2325-B-010-011 and NSC101-2627-B-010-001), the Center of Excellence for Cancer Research at Taipei Veterans General Hospital (DOH101-TD-C-111-007), and the Ministry of Education, Aim for the Top University Plan (National Yang Ming University) to CYH. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.