Abstract
BackgroundGain-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.MethodsWe 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.ResultsGST-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.ConclusionsGST-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.
Highlights
Gain-of-function mutations of tyrosine kinase FLT3 are frequently found in acute myeloid leukemia (AML)
glutathione S-transferase (GST)-FLT3S is a highly effective substrate for assays of FLT3 activity We constructed a pGex-GST-FLT3S plasmid, which encodes a GST fusion protein carrying the peptide sequence derived from the autophosphorylation site 589 of FLT3
GST-FLT3S was strongly phosphorylated by recombinant proteins containing the catalytic domain of wild type and D835H and D835Y mutant forms of FLT3, while plain GST was not phosphorylated at all it has 14 tyrosyl residues, demonstrating the specificity of the FLT3 kinase and phosphorylation of the FLT3 peptide fused to GST (Figure 1A)
Summary
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. FLT3 is a member of the class III receptor tyrosine kinase (RTK) family. It is expressed in immature hematopoietic cells and plays an important role in the normal development of stem cells and the immune system [1,2]. Further effort in FLT3 inhibitor screening is clearly needed For this purpose, more effective methods for FLT3 kinase activity assays are highly desirable
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