High-throughput Identification of FLT3 Wild-type and Mutant Kinase Substrate Preferences and Application to Design of Sensitive In Vitro Kinase Assay Substrates

Mol Cell Proteomics. 2019 Mar;18(3):477-489. doi: 10.1074/mcp.RA118.001111. Epub 2018 Dec 12.


Acute myeloid leukemia (AML) is an aggressive disease that is characterized by abnormal increase of immature myeloblasts in blood and bone marrow. The FLT3 receptor tyrosine kinase plays an integral role in hematopoiesis, and one third of AML diagnoses exhibit gain-of-function mutations in FLT3, with the juxtamembrane domain internal tandem duplication (ITD) and the kinase domain D835Y variants observed most frequently. Few FLT3 substrates or phosphorylation sites are known, which limits insight into FLT3's substrate preferences and makes assay design particularly challenging. We applied in vitro phosphorylation of a cell lysate digest (adaptation of the Kinase Assay Linked with Phosphoproteomics (KALIP) technique and similar methods) for high-throughput identification of substrates for three FLT3 variants (wild-type, ITD mutant, and D835Y mutant). Incorporation of identified substrate sequences as input into the KINATEST-ID substrate preference analysis and assay development pipeline facilitated the design of several peptide substrates that are phosphorylated efficiently by all three FLT3 kinase variants. These substrates could be used in assays to identify new FLT3 inhibitors that overcome resistant mutations to improve FLT3-positive AML treatment.

Keywords: Acute Myeloid Leukemia; Assay development; FLT3; FLT3-ITD; KINATEST-ID; Kinase Assay-Linked with Phosphoproteomics; Mass Spectrometry; Phosphorylation; Substrate identification; Tyrosine Kinases*.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Cell Line, Tumor
  • High-Throughput Screening Assays
  • Humans
  • Leukemia, Myeloid, Acute / genetics
  • Leukemia, Myeloid, Acute / metabolism*
  • Mutation*
  • Phosphorylation
  • Protein Domains
  • Protein Interaction Maps
  • Proteomics / methods*
  • Tandem Repeat Sequences
  • fms-Like Tyrosine Kinase 3 / genetics
  • fms-Like Tyrosine Kinase 3 / metabolism*


  • FLT3 protein, human
  • fms-Like Tyrosine Kinase 3