Mutations in the helix αC of the catalytic domain from the EGFR affect its activity in cervical cancer cell lines.

Abstract

The EGFR is a protein that belongs to the ErbB family of tyrosine kinase receptors. The EGFR is often overexpressed in human carcinomas. Amplification of the EGFR gene and mutations in the EGFR tyrosine kinase domain occur in patients with cancer. In cervical cancer, the expression level of the EGFR protein appears to directly associate with human papillomavirus infection. Our previous research demonstrated that in the cervical cancer cell lines, CALO and INBL, the EGFR is non-phosphorylated. The aim of the current study was to analyze the catalytic activity of the isolated EGFR and the presence of mutations in the control region αC. Catalytic activity was assessed by a universal in vitro kinase assay using polyGluTyr as a substrate, and the proteins were visualized by western blotting. For mutation analysis, DNA from CALO and INBL cell lines was isolated, and PCR was used to amplify the exons corresponding to the helix αC in the EGFR. The PCR products were visualized by agarose gel electrophoresis. The bands were isolated using a Zymoclean Gel DNA Recovery kit and directly sequenced. The EGFR, which was isolated and analyzed using the in vitro kinase assay, had catalytic activity. The receptor contained some mutations in the helix αC of the catalytic domain in both cell lines. The observed changes in the amino acid sequence may induce a different spatial arrangement and, therefore, a different conformation, which may confer different activities to this receptor. Thus, it was concluded that non-phosphorylated EGFR has catalytic activity, and it bears some amino acid changes in the helix αC of the catalytic domain in the CALO and INBL cells. These results suggest that the EGFR may function as an activator of other ErbB family receptors in these cervical cancer cells.