High-throughput identification of FLT3 wild-type and mutant kinase substrate preferences and application to design of sensitive in vitro kinase assay substrates

Abstract

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-offunction 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 (wildtype, 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.

Lipid Droplet-Derived Monounsaturated Fatty Acids Traffic via PLIN5 to Allosterically Activate SIRT1

“…Peptides were synthesized using a Protein Technologies SymphonyX synthesizer and using 4-methylbenzydrylamine hydrochloride resin (Iris Biotech GMBH) (Perez et al, 2019). Standard Fmoc-protected amino acid (AA) coupling occurred in the presence of 95 mM HCTU (Iris Biotech GMBH) and 200 mM N-methylmorpholine (Gyros Protein Technologies; S-1L-NMM) over two 20-min coupling cycles.…”

Assays for tyrosine phosphorylation in human cells

“…Combinatorial screening has identified a number of useful sequences for efficient assays for many kinases, however many of these are not sufficiently specific for cell-based use, since screening strategies rarely counter-screen for off-targets. We have employed a combination prediction/ design workflow using known substrate information that may be useful to others in identifying a sequence to use, but for which specificity would still need to be determined on a peptide-by-peptide basis (Lipchik et al, 2015;Perez, Blankenhorn, Murray, & Parker, 2019). Accordingly, there is no one-size-fits-all solution, and researchers will need to put time and effort into identifying the best substrate for their kinase target and cell-based application.…”

Novel Bruton’s Tyrosine Kinase (BTK) substrates for time-resolved luminescence assays

“…Therefore, we employed a modified phosphoproteomics workflow designed to determine the substrate profile of a kinase (similar to our previous reports for developing FLT3 substrates). [24][25][26] In brief, we treated trypsin-digested cell lysate with phosphatase to remove endogenous modifications, followed by reaction with recombinant BTK for two hours. Phosphopeptides resulting from this reaction were enriched and identified using an Orbitrap Fusion mass spectrometer (Figure 1).…”