Generation and characterization of a highly effective protein substrate for analysis of FLT3 activity
- Yun Chen†1,
- Yao Guo†3,
- Jiayu Han1,
- Wanting Tina Ho1,
- Shibo Li2,
- Xueqi Fu3 and
- Zhizhuang Joe Zhao1, 3Email author
© Chen et al. licensee BioMed Central Ltd. 2012
Received: 7 June 2012
Accepted: 30 June 2012
Published: 16 July 2012
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.
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.
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.
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.
KeywordsTyrosine kinase FLT3 Activity assay Inhibitor screening Acute myeloid leukemia
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]. Mutations of FLT3 have been detected in approximately 30% of patients with acute myelogenous leukemia (AML) and in a small number of patients with acute lymphocytic leukemia or myelodysplastic syndrome [3, 4]. The most common mutations of FLT3 found in hematopoietic malignancies involve internal tandem duplications (FLT3-ITD) within the juxtamembrane domain, while point mutations within the tyrosine kinase domain (FLT3-TKD) is found in about 7% of AML patients. Both types of mutations cause constitutive activation of FLT3 kinase activity, thereby turning on downstream signaling proteins and resulting in uncontrolled cell proliferation [3–5].
FLT3 mutations are important markers for AML diagnosis, and FLT3-ITD is strongly associated with the poor prognosis of AML patients . In fact, detection of FLT3-ITD is currently a routine diagnostic practice, and the presence of FLT3-ITD guides therapeutic decisions in AML patients with a normal karyotype [7, 8]. As gain-of-function mutants, FLT3-ITD and FLT3-TKD are obvious targets for therapeutic kinase inhibitors. Indeed, inhibiting FLT3 tyrosine kinase activity has been the focus of both preclinical and clinical research in AML. Many potent FLT3 inhibitors have been identified, and some have been clinically tested as single agents and in combination with chemotherapy, but thus far clinical responses have been limited [9, 10]. Currently, sorafenib, an inhibitor of tyrosine protein kinases (VEGFR and PDGFR) and Raf kinases, is the only approved FLT3 inhibitor for clinical use. Sorafenib is available for off-label use although it does not usually lead to a complete response . Further effort in FLT3 inhibitor screening is clearly needed. For this purpose, more effective methods for FLT3 kinase activity assays are highly desirable.
In this study, we have generated a glutathione S-transferase (GST) fusion protein carrying a peptide sequence derived from an autophosphorylation site of human FLT3. This protein designated GST-FLT3S, can be effectively phosphorylated by recombinant FLT3 enzymes. It can be used to detect elevated FLT3 activity in bone marrow cells from AML patients and to test inhibitory effects of various protein kinase inhibitors. We believe GST-FLT3S should find broad applications in detecting increased FLT3 activity from clinical samples for diagnostic purposes and for identifying effective FLT3 inhibitors through small-scale testing and large-scale screening.
Results and discussion
GST-FLT3S is a highly effective substrate for assays of FLT3 activity
GST-FLT3S can be used to detect increased tyrosine kinase activity in AML samples
FLT3 mutations have been found in many patients with AML and ALL. Currently, various gene-based analyses have been developed to detect such mutations, and this has provided valuable information for diagnosis of the diseases and has helped in designing proper treatments [5–7]. However, the gene-based assays reveal only the presence, not the activity, of the mutant FLT3. Because GST-FLT3S directly detects enzymatic activity, it should identify abnormal elevations of the FLT3 activity due to mutations of the enzyme at unknown sites or to malfunction of other signaling components that regulate FLT3.
GST-FLT3S can be used to screen FLT3 inhibitors
Known targets of protein kinase inhibitors tested
VEGFRs, PDGFRs, KIT, and FLT3
BCR-ABL, KIT, DDRs, PDGFRs, and CSF-1R
EGFR and JAK2
EGFR, Her2, and Her3
BCR-ABL, SRCs, and KIT
BCR-ABL, KIT, and PDGFRs
FLT3, JAK2, TrkA, TrkB, and TrkC
B-Raf, C-Raf, PDGFRs, VEGFRs, KIT, and FLT3
Identifying potent inhibitors of oncogenic protein kinases is a new trend in anticancer drug development [9–11]. Our current study provides a unique protein substrate for FLT3 kinase assays to test inhibitory effects of existing protein kinase inhibitors and to screen chemical libraries for new inhibitors. It is suitable for assays with the substrate in solution or immobilized on a solid surface, in small scale or in high throughput. It allows direct measurement of phosphorylation of added proteins substrate with high sensitivity, serving as an alternative to inhibitor screening assays by determining phosphorylation of synthetic peptide substrates, autophopshorylation of kinases, and competition with ATP or analogs for binding to kinases.
We have developed a robust protein substrate designated GST-FLT3S for assays of FLT3 kinase activity. GST-FLT3S can be produced economically in large quantities and used for assays of FLT3 in solution and when immobilized on beads. It reveals activation of FLT3 by mutations in the catalytic domain and detects increased tyrosine kinase activities in the cell extracts from AML patients. It can be used to test the effectiveness of existing protein kinase inhibitors of FLT3 and for large-scale inhibitor screening. We believe GST-FLT3S should serve as an important tool for future studies related to FLT3 and for disease diagnosis and therapeutic drug development. Finally, our study also demonstrates that GST is an excellent carrier for expression of short peptide sequences to serve as substrates of protein kinases.
Materials and clinical samples
Monoclonal anti-phosphotyrosine antibody PY20 was purchased from BD Biosciences. Protein kinase inhibitors were from ChemieTek. De-identified normal and AML bone marrow samples were collected from local clinical laboratories. The samples were residues from routine cytogenetic tests. Institutional review board approval was obtained before these samples were collected and analyzed. The bone marrow samples were treated with red cell lysis buffer, and proteins were extracted from white cells with a whole-cell extraction buffer containing 25 mM β-glycerophosphate (pH 7.3), 5 mM EDTA, 2 mM EGTA, 5 mM β-mercaptoethanol, 1% Triton X-100, 0.1 M NaCl, 1 mM sodium vanadate, and a protease inhibitor cocktail (Roche Applied Science). Cell lysates were cleared by centrifugation in a microfuge at 13,000g, and clear extracts were used directly for FLT3 activity assays. The remaining pellets were used for DNA isolation by using phenol/chloroform extraction after digestion of proteins with proteinase K.
Expression and purification of GST-FLT3S and recombinant FLT3
To make the GST-FLT3S construct, DNA oligos encoding a peptide with the sequence SDNEYFYVD were synthesized and ligated into the pGex-2T vector, following a similar strategy previously described for a JAK2 substrate . The peptide sequence was derived from human FLT3 and contains the tyrosine 589 autophosphorylation site. The recombinant fusion protein was expressed in E. coli cells and then purified by using a glutathione-Sepharose column. We employed the baculovirus expression system to express various wild type and mutant forms of FLT3 as described for other tyrosine kinases . In brief, a DNA fragment encoding the entire intracellular portion (amino acid residues 573–993) of human FLT3 was cloned into the pBluebacHis2 vector (Invitrogen). D835H and D835Y and FLT3 kinase domain mutations were introduced by using site-specific mutagenesis. The resultant plasmid DNAs, together with Bac-N-blue DNA, were used to transfect Sf9 insect cells to generate recombinant viruses according to manufacturer’s protocol (Invitrogen). Recombinant proteins were purified from cell extracts of baculovirus-infected Sf9 cells by using Ni-NTA columns (Qiagen).
Tyrosine kinase activity assays
Phosphorylation of GST-FLT3S by isolated tyrosine kinases or cell extracts was carried out in a buffer system containing 25 mM Tris–HCl (pH 7.5), 10 mM MgCl2, 0.2 mM adenosine 5’-triphosphate, and 2 mM dithiothreitol. Reactions were usually run for 20 minutes at room temperature and were stopped by SDS gel sample buffer. Tyrosine phosphorylation of proteins was determined by immunoblotting analyses with anti-phosphotyrosine antibody PY20, followed by horseradish peroxidase-conjugated secondary antibodies. Detection by the electrochemiluminescence method, capture of immunoblot images, and quantification of band signals were carried out by using FluorChem SP imaging system from Alpha Innotech [14, 15]. When the activity assays were performed with GST-FLT3S bound to glutathione-Sepharose beads, reactions were stopped by 15 mM EDTA, and tyrosine phosphorylation was detected by using anti-phosphotyrosine antibody PY20 followed by a fluorescein-conjugated secondary antibody. All microscopic analysis was performed with an Olympus BX51 microscope equipped with a DP71 camera.
Acute myeloid leukemia
This work was supported by grants HL079441 and HL094591 from the National Institutes of Health, and a grant from Oklahoma Center for the Advancement of Science & Technology (to ZJ Zhao).
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