Matsuo R, Ochiai W, Nakashima K, Taga T
Department of Molecular Cell Biology, Medical Research Institute, Tokyo Medical and Dental University, 2-3-10, Kanda-surugadai, Chiyoda-ku, Tokyo 101-0062, Japan.
J Immunol Methods. 2001 Jan 1;247(1-2):141-51. doi: 10.1016/s0022-1759(00)00313-6.
Signal transduction from cell surface receptors to the nucleus is regulated in most part by protein phosphorylation. For the purpose of identification of kinases which play an important role at a particular phosphorylation step in a series of signal transduction pathways, we have developed a new expression-screening method using a phosphorylation site specific antibody and a vector encoding substrate polypeptide. We have applied this method for screening kinases which phosphorylate STAT3 at serine(727). In this screening, antibody (PS727 antibody) specifically recognizing STAT3 in which serine(727) is phosphorylated was first prepared. Escherichia coli, bacteria expressing a serine(727)-containing fragment of STAT3 which was fused to glutathione-S-transferase (GST) (GST-STAT3-WT) were infected by lambda phage cDNA expression libraries. Phosphorylation of GST-STAT3-WT was effectively performed in E. coli as expected, and clones positive for PS727 antibody immunoreactivity were selected. Isolated 53 clones encode four serine/threonine kinases; extracellular signal regulated kinase 1 (ERK1/p44-MAPK), dual specificity Yak1 related kinase (DYRK), dual specificity Yak1 related kinase 2 (DYRK2) and homeodomain interacting protein kinase 2 (HIPK2). These kinases have a potential to phosphorylate serine(727) in STAT3 protein also in mammalian cells. The present method is considered to be applicable in general to isolate kinases.
从细胞表面受体到细胞核的信号转导在很大程度上受蛋白质磷酸化调控。为了鉴定在一系列信号转导途径中特定磷酸化步骤起重要作用的激酶，我们开发了一种新的表达筛选方法，该方法使用磷酸化位点特异性抗体和编码底物多肽的载体。我们已将此方法应用于筛选能使信号转导和转录激活因子3（STAT3）的丝氨酸（727）位点发生磷酸化的激酶。在该筛选过程中，首先制备了特异性识别丝氨酸（727）磷酸化的STAT3的抗体（PS727抗体）。用λ噬菌体cDNA表达文库感染表达与谷胱甘肽-S-转移酶（GST）融合的含丝氨酸（727）的STAT3片段的大肠杆菌（GST-STAT3-WT）。如预期的那样，GST-STAT3-WT在大肠杆菌中有效地发生了磷酸化，并筛选出对PS727抗体免疫反应呈阳性的克隆。分离出的53个克隆编码四种丝氨酸/苏氨酸激酶：细胞外信号调节激酶1（ERK1/p44-丝裂原活化蛋白激酶）、双特异性Yak1相关激酶（DYRK）、双特异性Yak1相关激酶2（DYRK2）和同源结构域相互作用蛋白激酶2（HIPK2）。这些激酶在哺乳动物细胞中也有可能使STAT3蛋白中的丝氨酸（727）发生磷酸化。本方法被认为一般适用于分离激酶。
