Fig 1: FES mediates E. coli phagocytosis by HL-60 neutrophils via SYK activation.a, b Complete FESS700C inhibition at 100 nM WEL028 in HL-60 neutrophils. Cells were differentiated into neutrophils (1 µM ATRA, 1.25% DMSO, 72–96 h), treated with WEL028 (100 nM, 1 h) and lysates were post-labeled (1 µM WEL033). Band intensities normalized to vehicle-treated control (n = 3). c Identification of WEL028 (100 nM) kinase targets in FESS700C HL-60 neutrophils using chemical proteomics. Kinases with >2-fold enrichment compared to vehicle control (q < 0.05) were designated as targets (means of fold enrichment, n = 4). d–e WEL028 reduces phagocytic uptake in FESS700C but not WT neutrophils. Treatment as in (a)–(b), followed by addition of GFP-expressing E. coli B834 (MOI = 30, 1 h, 37 °C) and flow cytometry analysis (n = 5). Phagocytic index: fraction GFP-positive cells (number of phagocytic cells) multiplied by GFP MFI (number of phagocytized bacteria). f SYK Y352 as proposed FES phosphorylation site (bold red), based on phosphorylation of homologous ZAP70 peptide (PamChip® microarray, n = 3). g FES phosphorylates SYK Y352 in situ. U2OS cells co-expressing FESS700C-FLAG and SYK-V5 were incubated with vehicle or WEL028 (200 nM, 1 h). Lysates were labeled (250 nM WEL033) and analyzed by in-gel fluorescence and immunoblot (n = 3). h SYK interacts with FES. Co-transfected U2OS cells were incubated as in (g), followed by anti-FLAG immunoprecipitation and immunoblot analysis (n = 3). i Phagocytosis of E. coli by HL-60 neutrophils depends on FES, SYK and PLCγ2 activity and actin polymerization. Incubations as in (d)–(e) (WEL028: 100 nM, R406: 1 µM, U-73122: 5 µM, cytochalasin D: 10 µM; n = 5). j Phosphorylation of SYK Y352 and downstream substrates is inhibited by WEL028 in infected FESS700C but not WT neutrophils, and absent in FESKO neutrophils. Inhibitor incubations as in (d)-(e), followed by addition of GFP-expressing E. coli B834 (MOI = 30, 2 min, 37 °C) and immunoblot analysis (n = 3). Data represent means ± SEM. Statistical analysis: ANOVA with Holm-Sidak’s multiple comparisons correction: ***P < 0.001; *P < 0.05; NS if P > 0.05. Source data are provided as Source Data file.
Fig 2: Visualization of endogenous FESS700C in CRISPR/Cas9-edited HL-60 cells.a HL-60 cells can differentiate into macrophages upon treatment with phorbol 12-myristate 13-acetate (PMA) for 48 h or into neutrophils with all-trans-retinoic acid (ATRA, 1 µM) and DMSO (1.25%) for 72–96 h. b CRISPR/Cas9 gene editing strategy. Selected sgRNA (bold blue) directs Cas9 to cleave at predicted site (red triangle). A ssODN homology-directed repair (HDR) donor template (red) flanks introduced mutations with 80 bp homology arms. The S700C mutation generates a BglII restriction site along with three silent mutations (orange) to remove PAM sites. Of note, two of the three mutated PAM sites correspond to sgRNAs not used in this study. PAM: protospacer-adjacent motif. c Restriction-fragment length polymorphism (RFLP) assay for identification of HL-60 FESS700C clone. Genomic region was amplified by PCR and amplicons were digested with BglII. Expected fragment size after digestion: 365 + 133 bp. d Sanger sequencing traces of WT HL-60 cells and homozygous FESS700C HL-60 clone. No deletions, insertions or undesired mutations were detected. e CD11b surface expression of HL-60 cells prior to and after differentiation, analyzed by flow cytometry. Threshold for CD11b-positive cells was determined using isotype control antibody (n = 3). f Reduction in proliferation upon differentiation is similar for WT and FESS700C cells. Proliferation ratio: cell number after differentiation divided by cell number before differentiation (n = 3). g Transcriptome profile of WT and FESS700C HL-60 macrophages analyzed by TempO-Seq. Differentially expressed genes (shown in black) were identified using following cut-offs: log2 of fold change FESS700C over WT HL-60 cells < −1 or > 1 with q-value < 0.05 (n = 3). h Endogenous FES is visualized by WEL033 in differentiated HL-60 FESS700C cells. WT or FESS700C HL-60 cells promyeloblasts, macrophages or neutrophils were lysed, followed by labeling with WEL033 (1 μM). FES expression increases upon differentiation (anti-FES immunoblot). Data represent means ± SEM. Statistical analysis: two-tailed t-test with Holm-Sidak’s multiple comparisons correction: NS if P > 0.05. Source data are provided as a Source Data file.
Fig 3: Design and characterization of FES cysteine point mutants.a Location of mutated active-site residues (green) in FES crystal structure with bound reversible inhibitor TAE684 (PDB code: 4e93). b Activity of recombinantly expressed FES mutants compared to WT, determined as relative amount of phosphorylated peptide substrate after 60 min incubation using TR-FRET assay. c Reaction progress kinetics for FESWT and FESS700C. d Determination of ATP KM for FESWT and FESS700C. Enzyme reactions in TR-FRET assay were performed with ULight-TK peptide (50 nM) and ATP (b, c: 100 µM, d: variable) and quenched (b, d: after 60 min, c: variable). e Peptide phosphorylation substrate profile for FESWT and FESS700C as determined in PamChip® microarray. Peptides were filtered for those with ATP-dependent signal and log2 of signal intensity >3. The peptide substrates were identical for FESWT and FESS700C (Venn diagram, inset). f Preferred substrate consensus sequence based on FESWT substrate profile. Illustration was rendered using Enologos (http://www.benoslab.pitt.edu). g SH2 domain binding profile for FESWT and FESS700C as determined in PamChip® microarray. Peptides with non-specific antibody binding were excluded. The peptide SH2 binding partners were identical for FESWT and FESS700C (Venn diagram, inset). Data represent means ± SEM (n = 3). Statistical analysis: ANOVA with Holm–Sidak’s multiple comparisons correction: *** P < 0.001; NS if P > 0.05. Source data are provided as a Source Data file.
Fig 4: FESS700C can be visualized by fluorescent probe WEL033.a Design of fluorescent probe WEL033 (Cy5-conjugate). b Dose-dependent labeling of full-length FESS700C by WEL033 in HEK293T cell lysate. c Labeling by WEL033 is specific, exclusive for FESS700C and dependent on catalytic lysine 590. Lysates were preincubated with vehicle or 4 and labeled by WEL033 (250 nM). d, e Visualization of FESS700C target engagement by WEL028 and TAE684. Lysate was preincubated with WEL028 or TAE684 and labeled by WEL033 (250 nM). Band intensities were normalized to vehicle-treated control (n = 3). f, g WEL028 engages recombinantly expressed FESS700C in live cells. Transfected HEK293T cells were treated with WEL028 and labeled proteins were visualized using click chemistry. Band intensities were normalized to highest concentration (n = 3). h, i WEL028 blocks FESS700C but not FESWT autophosphorylation (visualized by immunoblot using anti-phospho-FES Y713). Band intensities were normalized to vehicle-treated control (n = 3). Data represent means ± SEM. Statistical analysis: two-tailed t-test: *** P < 0.001. Source data are provided as a Source Data file.
Fig 5: FESS700C is selectively and covalently inhibited by WEL028.a Proposed covalent binding mode of compound 4 to Cys700 in crystal structure of FES (PDB code: 4e93). b In vitro inhibition profile of FESWT and FESS700C by WEL028 (TR-FRET assay, n = 3). c Inhibition profile of WEL028 (100 nM) on FESWT and FESS700C on peptide substrates (PamChip® microarray, normalized to vehicle-treated control, n = 3). Scale shows residual activity from minimum (0%, red) to maximum (100%, gray). d MS/MS-based identification of WEL028 covalently bound to Cys700. Precursor ion (m/z [M + 2H]2+ = 689.2671) was fragmented and signature ions are shown. Precursor ion was not observed in vehicle-treated control. e In vitro selectivity profile of WEL028 (1 µM, 1 h preincubation) on 380 recombinant kinases, visualized as waterfall plot (each data point is an individual kinase; n = 2). Data represent means ± SEM. Source data are provided as a Source Data file.
from Cell Signaling Technology for FES (D5B4Y) Rabbit mAb