In vitro activation of Stat3 by epidermal growth factor receptor kinase

Stat proteins are SH2 domain-containing transcription factors that are activated in cells by various cytokines and growth factors. In the case of cytokines whose receptors lack protein kinase activity, phosphorylation–activation is mediated by members of the JAK family of tyrosine protein kinases. In the case of growth factors whose receptors have intrinsic tyrosine protein kinase activity, it is thought that Stat proteins can be activated either directly by the receptor or indirectly through JAK proteins. To test the possibility of direct activation, we have used purified Stat3α, Stat3β, and epidermal growth factor receptor kinase produced in recombinant baculovirus-infected Sf9 insect cells. The Stat proteins formed a stable complex with the receptor kinase, and they were phosphorylated on tyrosine by the receptor kinase and activated for binding to DNA, properties shared with Stat proteins purified from Sf9 cells coexpressing JAK1 or JAK2. Both JAK-phosphorylated Stat3β and Stat3β phosphorylated in vitro by the receptor kinase were 20–50 times more active on a molar basis for DNA binding than phosphorylated Stat3α. We conclude that Stat3 isoforms can be directly phosphorylated and thereby activated in vitro by the epidermal growth factor receptor kinase.

信号转导与转录激活因子（Stat proteins）是一类含SH2结构域（SH2 domain）的转录因子，可在细胞内被多种细胞因子与生长因子激活。针对受体缺乏蛋白激酶活性的细胞因子，其介导的Stat蛋白磷酸化激活过程由酪氨酸蛋白激酶JAK家族成员完成；而对于受体具有内在酪氨酸蛋白激酶活性的生长因子，现有研究认为Stat蛋白可直接被受体激酶激活，或通过JAK蛋白间接激活。为验证直接激活的可能性，我们采用了在重组杆状病毒感染的Sf9昆虫细胞中重组表达并纯化的Stat3α、Stat3β与表皮生长因子受体激酶。实验结果显示，Stat蛋白可与该受体激酶形成稳定复合物，且能被该受体激酶在酪氨酸残基上磷酸化，同时获得DNA结合能力；该特性与共表达JAK1或JAK2的Sf9细胞中纯化得到的Stat蛋白一致。活性检测表明，经JAK磷酸化的Stat3β，以及经受体激酶体外磷酸化的Stat3β，其DNA结合活性的摩尔活性均为磷酸化Stat3α的20~50倍。综上，我们得出结论：Stat3同工型可在体外被表皮生长因子受体激酶直接磷酸化并激活。