Identification and characterization of signal transducer and activator of transcription 3 recruitment sites within the epidermal growth factor receptor.
The oncogene signal transducer and activator of transcription 3 (Stat3) is constitutively activated in a wide variety of human cancers, including squamous cell carcinoma of the head and neck. In squamous cell carcinoma of the head and neck, Stat3 activation is mediated by up-regulation of the autocrine ligand-receptor pair, tumor growth factor alpha and epidermal growth factor receptor (EGFR), resulting in cell growth and resistance to apoptosis. The initiating molecular event in Stat3 activation is recruitment to specific phosphotyrosine motifs within signaling complexes. Stat3 activation by the EGFR has been mapped to the COOH-terminal region of the EGFR between amino acid residues 1061 and 1123, which contains Y1068 and Y1086. However, it is not known if Stat3 binds directly to the EGFR or if either of these tyrosines is involved in this interaction. In this study, we demonstrated in stably transfected NIH-3T3 cells that activation of Stat3 by EGFR was eliminated by mutation of all five EGFR tyrosines to phenylalanine and that activation was restored with return of two of the mutated tyrosine sites, Y1068 and Y1086, to wild-type. Stat3 was detected in the activated EGFR complex, and its presence within the complex was dependent on Y1068 and/or Y1086. Phosphododecapeptides spanning Y1068 and Y1086 were able to pull down Stat3 with Y1068 being more effective than Y1086 in this regard. Real-time mirror resonance affinity analysis revealed Stat3 bound to phosphododecapeptide Y1068 with a K(D) of 135 +/- 20 nM and to phosphododecapeptide Y1086 with a K(D) of 243 +/- 36 nM (P = 0.044), consistent with the results of the pull-down assays. The lower K(D) of Y1068 was completely attributable to slower dissociation of Stat3 bound to Y1068 versus Y1086. Each phosphododecapeptide was capable of destabilizing Stat3 homodimers in vitro. When delivered into squamous carcinoma cells, phosphopeptides spanning Y1068 and Y1086 were able to inhibit EGFR-stimulated Stat3 DNA binding activity and cell proliferation.
Shao
Huang
H
Section of Infectious Diseases, Department of Medicine, Baylor College of Medicine, Houston, Texas 77030, USA.
Cheng
Haiyun Y
HY
Cook
Richard G
RG
Tweardy
David J
DJ
D016475:3T3 Cells; D000818:Animals; D015374:Biosensing Techniques; D002294:Carcinoma, Squamous Cell / Q000235:genetics / Q000378:metabolism; D002455:Cell Division / Q000502:physiology; D004273:DNA, Neoplasm / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D051379:Mice; D009154:Mutation; D010449:Peptide Mapping; D010649:Phenylalanine / Q000235:genetics; D010748:Phosphopeptides / Q000378:metabolism; D011485:Protein Binding; D050796:STAT3 Transcription Factor; D015534:Trans-Activators / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000235:genetics
D004273:DNA, Neoplasm; D004268:DNA-Binding Proteins; D010748:Phosphopeptides; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; C494087:Stat3 protein, mouse; D015534:Trans-Activators; D014443:Tyrosine; D010649:Phenylalanine; D066246:ErbB Receptors