Characterization of a highly effective protein substrate for analysis of JAK2V617F activity
Characterization of a highly effective protein substrate for analysis of JAK2V617F activity
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DOI：
10.1016/j.exphem.2007.07.003
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发表时间：
2007-11-01
影响因子：
2.6
通讯作者：
Zhao, Zhizhuang Joe
中科院分区：
文献类型：
--
作者：
Li, Zhe;Xing, Shu;Zhao, Zhizhuang Joe
Objective. JAK 2（V617 F）在骨髓增生性疾病中的鉴定使JAK 2成为疾病诊断的重要标志物和治疗药物开发的高度有吸引力的靶标。本研究旨在确定一种灵敏和特异的底物，用于JAK 2酶活性的测定。我们表达了谷胱甘肽S-转移酶（GST）融合蛋白命名为GST-JAKS，它携带的肽序列来自人类JAK 2的自磷酸化位点。从大肠杆菌细胞中纯化该蛋白，并用于分析纯化的酶和来自细胞（包括JAK 2（V617 F）阳性的真性红细胞增多症血液的单核细胞）的粗细胞提取物的酪氨酸激酶活性。它也被用来进行JAK 2激酶试验，以筛选JAK 2的抑制剂。GST-JAKS被JAK 2的活化形式强烈磷酸化，包括JAK 2（V617 F）和仅含有其催化结构域的重组蛋白。它对野生型JAK 2的反应最小，并且不被表皮生长受体和胰岛素受体酪氨酸激酶磷酸化。使用GST-JAKS的激酶测定提供了野生型和突变型JAK 2（V617 F）之间的鲜明对比，并且足够灵敏以检测在粗细胞提取物中发现的微量JAK 2（V617 F）。可以将试验放大以斑点印迹形式筛选JAK 2抑制剂。GST-JAKS是JAK 2检测的敏感和特异的蛋白底物。它可能在诊断与JAK 2活性异常相关的疾病方面具有临床应用价值。它也是开发筛选JAK 2抑制剂的大规模测定的优良底物。(C)2007 ISEH -血液学和干细胞学会。爱思唯尔公司出版
Objective. Identification of JAK2 (V617F) in myeloproliferative disorders makes JAK2 an important marker for disease diagnosis and a highly attractive target for therapeutic drug development. This study is intended to identify a sensitive and specific substrate for assays of the JAK2 enzymatic activity.Methods. We expressed a glutathione S-transferase (GST) fusion protein designated GST-JAKS, which carries a peptide sequence derived from the autophosphorylation sites of human JAK2. The protein was purified from Escherichia coli cells and was used to analyze to tyrosine kinase activities of purified enzymes and crude cell extracts from cells, including mononuclear cells of JAK2 (V617F) -positive polycythemia vera blood. It was also used to perform JAK2 kinase assays to screen inhibitors of JAK2.Results. GST-JAKS is strongly phosphorylated by activated forms of JAK2 including JAK2 (V617F) and recombinant protein containing its catalytic domain alone. It showed minimal responses to wild-type JAK2 and was not phosphorylated by the epidermal growth receptor and the insulin receptor tyrosine kinases. Kinase assays with GST-JAKS provide a sharp contrast between wild-type and mutant JAK2,(V617F) and are sensitive enough to detect minute amounts of JAK2 (V617F) found in crude cell extracts. Assays can be scaled up to screen for inhibitors of JAK2 in a dot blot format.Conclusion. GST-JAKS is sensitive and specific protein substrate for JAK2 assays. It may have clinical applications in diagnosis of diseases related to abnormal JAK2 activity. It is also an excellent substrate for development of large scale assays to screen JAK2 inhibitors. (C) 2007 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc.