title: Protein phosphorylation profiling using an in situ proximity ligation assay: phosphorylation of AURKA-elicited EGFR-Thr654 and EGFR-Ser1046 in lung cancer cells.
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.
authors.affiliation: Institute of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan.
authors.forename: Tzu-Chi
authors.initials: TC
authors.lastname: Chen
authors.forename: Yu-Wen
authors.initials: YW
authors.lastname: Liu
authors.forename: Yei-Hsuan
authors.initials: YH
authors.lastname: Huang
authors.forename: Yi-Chen
authors.initials: YC
authors.lastname: Yeh
authors.forename: Teh-Ying
authors.initials: TY
authors.lastname: Chou
authors.forename: Yu-Chung
authors.initials: YC
authors.lastname: Wu
authors.forename: Chun-Chi
authors.initials: CC
authors.lastname: Wu
authors.forename: Yi-Rong
authors.initials: YR
authors.lastname: Chen
authors.forename: Hui-Chuan
authors.initials: HC
authors.lastname: Cheng
authors.forename: Pei-Jung
authors.initials: PJ
authors.lastname: Lu
authors.forename: Jin-Mei
authors.initials: JM
authors.lastname: Lai
authors.forename: Chi-Ying F
authors.initials: CY
authors.lastname: Huang
mesh_terms: D000230:Adenocarcinoma / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D000906:Antibodies / Q000737:chemistry; D064096:Aurora Kinase A; D064127:Aurora Kinases; D045744:Cell Line, Tumor; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism*; D005260:Female; D057809:HEK293 Cells; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D008175:Lung Neoplasms / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D008297:Male; D010766:Phosphorylation / Q000235:genetics; D040081:Protein Array Analysis / Q000379:methods; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*
chemical_list: D000906:Antibodies; C512478:EGFR protein, human; D066246:ErbB Receptors; C576917:AURKA protein, human; D064096:Aurora Kinase A; D064127:Aurora Kinases; D017346:Protein Serine-Threonine Kinases