用酵母双杂交系统研究Smad3和Smad4的相互作用

Homomeric and Heteromeric Interactions of Smad3 and Smad4 Identified with Yeast Two hybrid System

Ｓｍ ａｄ３ 和 Ｓｍ ａｄ４ 是将 Ｔ Ｇ Ｆβ的信号从细胞外传递到细胞核内的重要的信号传导蛋白． Ｔ Ｇ Ｆβ与其受体结合后，激活受体的磷酸激酶，使 Ｓｍ ａｄ３ 发生磷酸化，活化的 Ｓｍ ａｄ３ 与 Ｓｍ ａｄ４ 结合，形成异源复合物，进入到核中．然后 Ｓｍ ａｄ４ 以 Ｄ Ｎ Ａ 结合蛋白的形式与特定的 Ｄ Ｎ Ａ 结合，将 Ｔ Ｇ Ｆβ的信号传到核内．激活转录，诱导背中胚层的形成，抑制细胞的分化等．经研究利用酵母双杂交试验，鉴定了 Ｓｍ ａｄ３ 和 Ｓｍ ａｄ４ 相互作用的功能区域．构建 Ｓｍ ａｄ３ 和 Ｓｍ ａｄ４ 的 Ｃ 端、 Ｎ 端和中间连接区的突变体，将这些突变体克隆到 ｐ Ｇ Ａ Ｄ４２４ 和 ｐ Ｇ Ｂ Ｔ９ 载体中，并转化到 Ｈ Ｆ７ Ｃ 酵母中．通过 Ｌｅｕ－ ／ Ｔｒｐ－ ／ Ｈｉｓ－ Ｓ Ｄ 平板上菌落的形成，和 Ｘ－ ｇａｌ显色反应鉴定转化到酵母中的两个克隆质粒的相互作用．结果显示 Ｓｍ ａｄ４ 与 Ｓｍ ａｄ３ 异源相五作用时，主要是通过 Ｓｍ ａｄ４ 的中间连接区．在同源作用时， Ｓｍ ａｄ３ 是通过 Ｃ 端，而 Ｓｍ ａｄ４ 是通过中间连接区进行的．

Smad3 and Smad4 are newly identified essential intracellular signalling components of TGF β.Smad3 and Smad4 were shown to associate in homomeric and heteromeric interactions as assessed by yeast two hybrid analyses.With Eco R Ⅰ and Sma Ⅰ sites Smad4 and its mutants,such as N termial domain,C terminal domain and linker region were inserted into plasmids pGAD424 and pGBT9.With Sma Ⅰ and Bam H Ⅰ sites Smad3 and its mutants,such as N terminal domain plus linker region,and C terminal domain were inserted into pGAD424 and pGBT9 also.The constructed plasmids were transfected into yeast HF7C as scheduled.The interactions were identified with colonies on Leu -/Trp -/His - SD plates and blue color reaction in X gal containing plates as previously described.The results showed that heteromeric interactions were mediated through the linker region of Smad4 and any domain of Smad3.In Smad3,the homomeric interaction was mediated by the C terminal domain,while the homomeric association of Smad4 was mediated by the linker region.The results suggested that the linker region of Smad4 played the key role in the association of Smad3 and Smad4.