PT - JOURNAL ARTICLE AU - Minervo Perez AU - John Blankenhorn AU - Kevin J. Murray AU - Laurie L. Parker TI - High-throughput identification of FLT3 wild-type and mutant kinase substrate preferences and application to design of sensitive <em>in vitro</em> kinase assay substrates AID - 10.1101/457689 DP - 2018 Jan 01 TA - bioRxiv PG - 457689 4099 - http://biorxiv.org/content/early/2018/10/31/457689.short 4100 - http://biorxiv.org/content/early/2018/10/31/457689.full AB - 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 haematopoiesis, 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.List of abbreviationsAMLacute myeloid leukemiaRTKreceptor tyrosine kinaseITDinternal tandem duplicationTKDtyrosine kinase domainWTwild typeTKItyrosine kinase inhibitorKALIPKinase Assay Linked PhosphoproteomicsFBSfetal bovine serumPBSphosphate buffered salineACNacetonitrileDTTdithiothreitolTFAtrifluoroacetic acidHLBhydrophilic-lipophilic balancedTris-HCLtris(hydroxymethyl)aminomethane hydrochlorideEDTAethylenediaminetetraacetic acidFAformic acidFDRfalse discovery rateMCCMatthews Correlation CoefficientAAamino acidDMFdimethylformamideDMSOdimethyl sulfoxidePDGFRβplatelet derived growth factor betaALKanaplastic leukemia kinaseBTKburton’s tyrosine kinaseMOPS4-morpholinepropanesulfonic acidBSAbovine serum albuminHEPES4-(2-hydroxyethyl)-1-piperzoneethanesulfonic acidTBStris buffered salineARamplex red2Htwo hours16H16 hoursSDVstandard deviation valuesPPMpositional probability matrixSSMsite selectivity matrixDRdose response