Extended Data Fig. 6 |. Design of CAGEC-ABL1 with appended native autoregulatory domain for inducible tyrosine phosphorylation in cells upon BCR/ABL activation.
a, Schematic showing the design of the splicing site used for assembly of BCR/ABL using the CAGE system. ‘CFN’ amino acids are added to the N-terminus of the ABL segment to facilitate protein trans-splicing reaction using NpuCage. b, Schematic showing the optimization of the autoinhibited CAGEC-ABL1 construct. Various lengths of the unstructured cap region containing the N-myristate site (Auto-regulatory domain, N) and the flexible linker connecting the auto-regulatory domain with the CAGEC module (Adjacent linker region, L) were screened to find the optimal arrangement needed to suppress ABL kinase activity. c, d, Immunoblots of the basal tyrosine phosphorylation levels in HEK293T cells expressing indicated ABL constructs and CAGEC-ABL1 variants featuring different autoinhibitory domain (N) and adjacent linker (L) lengths. e, Immunoblots of BCR/ABL (HA-tag) and global tyrosine phosphorylation levels in HEK293T cells co-expressing BCR-CAGEN and CAGEC-ABL1 variants. Cells treated with DMSO or rapalog (100 nM) for 18 hours before analysis. From these studies, it was determined that the optimal CAGEC-ABL1 construct contained a L79 + N24 combination. f, Schematic of the N-myristoylation metabolic labeling workflow. HEK293T cells expressing optimal CAGEC-ABL1 (N79/L24) were cultured in growth media with YnMyr (20 μM) for 24 hours and lysed. Labeled proteins were captured by click chemistry reaction with an azide-biotin tag, enriched with streptavidin beads, and analyzed by anti-HA immunoblots. g, Immunoblots of the HA-tagged CAGEC-ABL1 construct expressed in HEK293T cells cultured with YnMyr and enriched by the workflow described in (f). CAGEC-ABL1 construct was enriched following YnMyr metabolic labeling and biotin conjugation (left). By contrast, the G2A mutation of CAGEC-ABL1 failed to be enriched in the workflow (right). h, Proximity-induced CPS in HEK293T cells co-expressing BCR-CAGEN (Myc-tag) and CAGEC-ABL1 (HA-tag). Cell were treated with DMSO or increasing doses of rapalog (100 nM) for 18 hours before immunoblotting using the indicated antibodies. i, Kinetics of BCR/ABL splice product generation in HEK293T cells co-expressing BCR-CAGEN and CAGEC-ABL1. Cells were treated with DMSO or rapalog (100 nM) for indicated time-points, followed by immunoblotting using indicated antibodies. Data in (c-e) and (g-i) are representative of n=3 independent experiments. CAGEN refers to ‘NpuNCage-FKBP,’ and CAGEC refers to ‘ABLauto-FRB-NpuCCage’ in (c-e) and (g-i).