Sakai Kazuko, Yokote Hideyuki, Murakami-Murofushi Kimiko, Tamura Tomohide, Saijo Nagahiro, Nishio Kazuto
Shien-Lab, National Cancer Center Research Institute, Tsukiji 5-1-1, Chuo-ku, Tokyo 104-0045, Japan.
Biochem J. 2006 Aug 1;397(3):537-43. doi: 10.1042/BJ20051962.
The existence of an in-frame deletion mutant correlates with the sensitivity of lung cancers to EGFR (epidermal growth factor receptor)-targeted tyrosine kinase inhibitors. We reported previously that the in-frame 15-bp deletional mutation (delE746-A750 type deletion) was constitutively active in cells. Kinetic parameters are important for characterizing an enzyme; however, it remains unclear whether the kinetic parameters of deletion mutant EGFR are similar to those of wild-type EGFR. We analysed autophosphorylation in response to ATP and inhibition of gefitinib for deletion mutant EGFR and wild-type EGFR. Kinetic studies, examining autophosphorylation, were carried out using EGFR fractions extracted from 293-pDelta15 and 293-pEGFR cells transfected with deletion mutant EGFR and wild-type EGFR respectively. We demonstrated the difference in activities between unstimulated wild-type (K(m) for ATP=4.0+/-0.3 microM) and mutant EGFR (K(m) for ATP=2.5+/-0.2 microM). There was no difference in K(m) values between EGF-stimulated wild-type EGFR (K(m) for ATP=1.9+/-0.1 microM) and deletion mutant EGFR (K(m) for ATP=2.2+/-0.2 microM). These results suggest that mutant EGFR is active without ligand stimulation. The K(i) value for gefitinib of the deletion mutant EGFR was much lower than that of wild-type EGFR. These results suggest that the deletion mutant EGFR has a higher affinity for gefitinib than wild-type EGFR.
框内缺失突变体的存在与肺癌对表皮生长因子受体（EGFR）靶向酪氨酸激酶抑制剂的敏感性相关。我们之前报道过，框内15个碱基对的缺失突变（delE746 - A750型缺失）在细胞中具有组成型活性。动力学参数对于表征一种酶很重要；然而，目前尚不清楚缺失突变体EGFR的动力学参数是否与野生型EGFR相似。我们分析了缺失突变体EGFR和野生型EGFR对ATP的自磷酸化反应以及吉非替尼的抑制作用。分别使用从转染了缺失突变体EGFR和野生型EGFR的293 - pDelta15和293 - pEGFR细胞中提取的EGFR组分进行了检测自磷酸化的动力学研究。我们证明了未刺激的野生型（ATP的K(m)=4.0±0.3 microM）和突变体EGFR（ATP的K(m)=2.5±0.2 microM）之间活性的差异。在表皮生长因子（EGF）刺激的野生型EGFR（ATP的K(m)=1.9±0.1 microM）和缺失突变体EGFR（ATP的K(m)=2.2±0.2 microM）之间，K(m)值没有差异。这些结果表明，突变体EGFR在没有配体刺激的情况下具有活性。缺失突变体EGFR对吉非替尼的K(i)值远低于野生型EGFR。这些结果表明，缺失突变体EGFR对吉非替尼的亲和力高于野生型EGFR。
