Activation of the Ras/Mitogen-Activated Protein Kinase Pathway by Kinase-Defective Epidermal Growth Factor Receptors Results in Cell Survival but Not Proliferation

Signalling by the epidermal growth factor (EGF) receptor (EGFR) has been studied intensively, but for most cell types the analysis is complicated by the fact that EGFR not only homodimerizes but can also form heterodimers with other EGFR family members. Heterodimerization is a particular problem in the study of EGFR mutants, where the true phenotype of the mutants is confounded by the contribution of the heterodimer partner to signal transduction. We have made use of the murine hemopoietic cell line BaF/3, which does not express EGFR family members, to express wild-type (WT) EGFR, three kinase-defective EGFR mutants (V741G, Y740F, and K721R), or a C-terminally truncated EGFR (CT957) and have measured their responses to EGF. We found that under the appropriate conditions EGF can stimulate cell proliferation of BaF/3 cells expressing WT or CT957 EGFRs but not that of cells expressing the kinase-defective mutants. However, EGF promotes the survival of BaF/3 cells expressing either of the kinase-defective receptors (V741G and Y740F), indicating that these receptors can still transmit a survival signal. Analysis of the early signalling events by the WT, V741G, and Y740F mutant EGF receptors indicated that EGF stimulates comparable levels of Shc phosphorylation, Shc–GRB-2 association, and activation of Ras, B-Raf, and Erk-1. Blocking the mitogen-activated protein kinase (MAPK) signalling pathway with the specific inhibitor PD98059 abrogates completely the EGF-dependent survival of cells expressing the kinase-defective EGFR mutants but has no effect on the EGF-dependent proliferation mediated by WT and CT957 EGFRs. Similarly, the Src family kinase inhibitor PP1 abrogates EGF-dependent survival without affecting proliferation. However blocking phosphatidylinositol-3-kinase or JAK-2 kinase with specific inhibitors does arrest growth factor-dependent cell proliferation. Thus, EGFR-mediated mitogenic signalling in BaF/3 cells requires an intact EGFR tyrosine kinase activity and appears to depend on the activation of both the JAK-2 and PI-3 kinase pathways. Activation of the Src family of kinases or of the Ras/MAPK pathway can, however, be initiated by a kinase-impaired EGFR and is linked to survival.

表皮生长因子（epidermal growth factor, EGF）受体（EGFR）的信号转导已得到广泛研究，但在大多数细胞类型中，相关分析因EGFR不仅可发生同源二聚化，还能与其他EGFR家族成员形成异源二聚体而变得复杂。异源二聚化在EGFR突变体研究中尤为棘手，因为异源二聚体伙伴对信号转导的贡献会干扰突变体真实表型的解析。本研究使用不表达EGFR家族成员的小鼠造血细胞系BaF/3，分别表达野生型（wild-type, WT）EGFR、三种激酶缺陷型EGFR突变体（V741G、Y740F与K721R）以及C端截短型EGFR（CT957），并检测这些细胞对EGF的应答反应。研究发现，在适宜条件下，EGF可促进表达WT或CT957 EGFR的BaF/3细胞增殖，但无法促进表达激酶缺陷型突变体的细胞增殖。然而，EGF可提升表达两种激酶缺陷型受体（V741G与Y740F）的BaF/3细胞的存活率，表明此类受体仍可传递存活信号。对WT、V741G及Y740F突变型EGF受体介导的早期信号事件分析显示，EGF可诱导相似水平的Shc磷酸化、Shc–GRB-2结合以及Ras、B-Raf与Erk-1的激活。使用特异性抑制剂PD98059阻断丝裂原活化蛋白激酶（mitogen-activated protein kinase, MAPK）信号通路，可完全取消表达激酶缺陷型EGFR突变体细胞的EGF依赖性存活，但对WT与CT957 EGFR介导的EGF依赖性增殖无影响。同样，Src家族激酶抑制剂PP1可取消EGF依赖性存活，却不影响细胞增殖。但使用特异性抑制剂阻断磷脂酰肌醇3-激酶（phosphatidylinositol-3-kinase, PI3K）或JAK-2激酶，则会抑制生长因子依赖性的细胞增殖。综上，BaF/3细胞中EGFR介导的有丝分裂原信号转导需要完整的EGFR酪氨酸激酶活性，且似乎依赖JAK-2与PI3K通路的激活。而Src家族激酶或Ras/MAPK通路的激活可由激酶缺陷型EGFR启动，并与细胞存活相关。