Generation and characterization of a highly effective protein substrate for analysis of FLT3 activity

详细信息    查看全文

• 作者：Yun Chen (1)
  Yao Guo (3)
  Jiayu Han (1)
  Wanting Tina Ho (1)
  Shibo Li (2)
  Xueqi Fu (3)
  Zhizhuang Joe Zhao (1) (3)
  
• 关键词：Tyrosine kinase ; FLT3 ; Activity assay ; Inhibitor screening ; Acute myeloid leukemia
• 刊名：Journal of Hematology & Oncology
• 出版年：2012
• 出版时间：December 2012
• 年：2012
• 卷：5
• 期：1
• 全文大小：450KB
• 参考文献：1. Gilliland DG, Griffin JD: The roles of FLT3 in hematopoiesis and leukemia. / Blood 2002, 100:1532鈥?542. CrossRef
  2. Masson K, R枚nnstrand L: Oncogenic signaling from the hematopoietic growth factor receptors c-Kit and Flt3. / Cell Signal 2009, 21:1717鈥?726. CrossRef
  3. Stirewalt DL, Radich JP: The role of FLT3 in hematopoietic malignancies. / Nat Rev Cancer 2003, 3:650鈥?65. CrossRef
  4. Takahashi S: Current findings for recurring mutations in acute myeloid leukemia. / J Hematol Oncol 2011, 4:36. CrossRef
  5. Takahashi S: Downstream molecular pathways of FLT3 in the pathogenesis of acute myeloid leukemia: biology and therapeutic implications. / J Hematol Oncol 2011, 4:13. CrossRef
  6. Schlenk RF, D枚hner K, Krauter J, Fr枚hling S, Corbacioglu A, Bullinger L, Habdank M, Sp盲th D, Morgan M, Benner A, Schlegelberger B, Heil G, Ganser A, D枚hner H: Mutations and treatment outcome in cytogenetically normal acute myeloid leukemia. / N Engl J Med 2008, 358:1909鈥?918. CrossRef
  7. Lowenberg B: Diagnosis and prognosis in acute myeloid leukemia鈥搕he art of distinction. / N Engl J Med 2008, 358:1960鈥?962. CrossRef
  8. Gregory TK, Wald D, Chen Y, Vermaat JM, Xiong Y, Tse W: Molecular prognostic markers for adult acute myeloid leukemia with normal cytogenetics. / J Hematol Oncol 2009, 2:23. CrossRef
  9. Zhu X, Ma Y, Liu D: Novel agents and regimens for acute myeloid leukemia: 2009 ASH annual meeting highlights. / J Hematol Oncol 2010, 3:17. CrossRef
  10. Fathi A, Levis M: FLT3 inhibitors: a story of the old and the new. / Curr Opin Hematol 2011, 18:71鈥?6. CrossRef
  11. Pratz KW, Levis MJ: Bench to Bedside Targeting of FLT3 in Acute Leukemia. / Curr Drug Targets 2010, 11:781鈥?89. CrossRef
  12. Smith BD, Levis M, Beran M, Giles F, Kantarjian H, Berg K, Murphy KM, Dauses T, Allebach J, Small D: Single-agent CEP-701, a novel FLT3 inhibitor, shows biologic and clinical activity in patients with relapsed or refractory acute myeloid leukemia. / Blood 2004, 103:3669鈥?676. CrossRef
  13. Fathi AT, Chabner BA: FLT3 inhibition as therapy in acute myeloid leukemia: a record of trials and tribulations. / Oncologist 2011, 16:1162鈥?174. CrossRef
  14. Li Z, Xu M, Xing S, Ho WT, Ishii T, Li Q, Fu X, Zhao ZJ: Erlotinib effectively inhibits JAK2V617F activity and polycythemia vera cell growth. / J Biol Chem 2007, 282:3428鈥?432. CrossRef
  15. Li Z, Xing S, Wang S, Ho WT, Zhao ZJ: Characterization of a highly effective protein substrate for analysis of JAK2(V617F) Activity. / Exp Hematol 2007, 35:1624鈥?632. CrossRef
  
• 作者单位：Yun Chen (1)
  Yao Guo (3)
  Jiayu Han (1)
  Wanting Tina Ho (1)
  Shibo Li (2)
  Xueqi Fu (3)
  Zhizhuang Joe Zhao (1) (3)
  
  1. Department of Pathology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, 73104, USA
  3. Edmond H. Fischer Signal Transduction Laboratory, College of Life Sciences, Jilin University, Changchun, China
  2. Department of Pediatrics, University of Oklahoma Health Sciences Center, Oklahoma City, OK, 73104, USA
  
• ISSN：1756-8722

文摘

Background Gain-of-function mutations of tyrosine kinase FLT3 are frequently found in acute myeloid leukemia (AML). This has made FLT3 an important marker for disease diagnosis and a highly attractive target for therapeutic drug development. This study is intended to generate a sensitive substrate for assays of the FLT3 enzymatic activity. Methods We expressed in Escherichia coli cells a glutathione S-transferase (GST) fusion protein designated GST-FLT3S, which contains a peptide sequence derived from an autophosphorylation site of FLT3. The protein was used to analyze tyrosine kinase activity of baculovirus-expressed FLT3 and crude cell extracts of bone marrow cells from AML patients. It was also employed to perform FLT3 kinase assays for FLT3 inhibitor screening. Results GST-FLT3S in solution or on beads was strongly phosphorylated by recombinant proteins carrying the catalytic domain of wild type FLT3 and FLT3D835 mutants, with the latter exhibiting much higher activity and efficiency. GST-FLT3S was also able to detect elevated tyrosine kinase activity in bone marrow cell extracts from AML patients. A small-scale inhibitor screening led to identification of several potent inhibitors of wild type and mutant forms of FLT3. Conclusions GST-FLT3S is a sensitive protein substrate for FLT3 assays. It may find applications in diagnosis of diseases related to abnormal FLT3 activity and in inhibitor screening for drug development.