Abnormalities of structure, function, expression, and signal transduction of ERK in relation to carcinogenesis

我们在人口腔鳞状细胞癌细胞系（HSC6） ERK2的CD域内发现了一个从谷氨酸(E)到赖氨酸(K)的密码子322点突变。在本研究中，我们旨在阐明(1)ERK2突变体与正常突变体的生化、生物物理和生物学变化，以及(2)此类突变在口腔癌中的发生率。通过这些研究，我们期望得到E322K突变如何在癌症发生中发挥作用的答案。结果表明：(1)E322K蛋白失去了与MAPK磷酸酶结合的活性，导致突变蛋白的组成性激活；(2)外源表达E322K的细胞在软琼脂中获得了生长优势；(3)E322K在PC12细胞中表现出略低的神经突样形成活性；携带E322K的果蝇在复眼发育中表现出微弱的异常；(5)在88份口腔癌样本中，除3例CD结构域多态性碱基改变外，未发现明显的突变。这些结果表明，E322K突变体对细胞转化和异常发育具有较弱的活性，但即使存在突变，其突变发生率也较低。此外，我们获得的结果表明(1)SNT-2与优先磷酸化的ERK和EGF受体结合，通过反馈回路机制下调EGF信号，(2)在口腔癌细胞系中观察到另一种ERK2结合蛋白Naf1的异常表达，该蛋白可能保护G2/ m阻滞细胞免于凋亡

We found a point mutation at the codon 322 from glutamic acid (E) to lysine (K) within the CD domain of ERK2 of a cell line (HSC6) established from human oral squamous cell carcinoma. In this study we aimed to elucidate (1)biochemical, biophysical, and biological changes of ERK2 mutant by comparing with the normal couterpart and (2) incidence of such mutations in oral cancer. By these studies, we anticipated to get an answer how E322K mutation plays a role in the genesis of cancer. The results obtained are (1) E322K protein lost activity to bind MAPK phosphatase, resulting in constitutive activation of mutant protein, (2) cells exogenously expressing E322K gained growth advantage in soft agar, (3) E322K showed slightly less neurite-like forming activity in PC12 cells, (4) transgenic D, rosophila carrying E322K showed weak abnormality in development of compound eye and (5) no significant mutation was found except for 3 cases of polymorphic base changes in the CD domain out of 88 oral cancer samples. These results suggested that E322K mutant has weak activity toward cellular transformation and abnormal development but the incidence of mutation was low if there is.Besides, we obtained results suggesting (1) that SNT-2 binds to preferentially phosphorylated ERK and to EGF receptor, which results in down-regulation of EGF signaling by a feed back loop mechanism, (2) that aberrant expression of Naf1, another ERK2 binding protein, was observed in oral cancer cell lines and this protein may protect cells from apoptosis in G2/M-arrested cells