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Distinct phases of cellular signaling revealed by time-resolved protein synthesis

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Abstract

The post-translational regulation of protein function is involved in most cellular processes. As such, synthetic biology tools that operate at this level provide opportunities for manipulating cellular states. Here we deploy proximity-triggered protein trans-splicing technology to enable the time-resolved synthesis of target proteins from premade parts. The modularity of the strategy allows for the addition or removal of various control elements as a function of the splicing reaction, in the process permitting the cellular location and/or activity state of starting materials and products to be differentiated. The approach is applied to a diverse set of proteins, including the kinase oncofusions breakpoint cluster region–Abelson (BCR–ABL) and DNAJ–PKAc where dynamic cellular phosphorylation events are dissected, revealing distinct phases of signaling and identifying molecular players connecting the oncofusion to cancer transformation as new therapeutic targets of cancer cells. We envision that the tools and control strategies developed herein will allow the activity of both naturally occurring and designer proteins to be harnessed for basic and applied research.

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Fig. 1: CAGE modules regulate the subcellular location of SPs via control elements.
Fig. 2: Conditional generation of oncofusion transcription factors.
Fig. 3: Post-translational generation of BCR–ABL allows dissection of dynamic cellular phosphorylation events.
Fig. 4: Dynamic cellular phosphorylation events induced by post-translational generation of DNAJ–PKAc.

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Acknowledgements

We thank members of the Muir Lab for their many helpful comments and insights. We thank S. Kyin and H. H. Shwe at the Princeton University Proteomics and Mass Spectrometry Core Facility. We thank C. J. DeCoste and K. Rittenbach at the Princeton University Flow Cytometry Resource Facility for cell sorting services funded by Rutgers Cancer Institute of New Jersey (P30CA072720-5921). We thank G. S. Laevsky and S. Wang at the Princeton University Confocal Imaging Facility. We also thank J. M. Miller at the Princeton University Genomics Core Facility. The research reported in this publication was supported by the National Institutes of Health (NIH; grant R01GM086868 to T.W.M.).

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  1. Department of Chemistry, Princeton University, Princeton, NJ, USA

    Gihoon Lee & Tom W. Muir

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  1. Gihoon Lee
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G.L. and T.W.M. conceived the work, designed and performed the experiments, and wrote and reviewed the manuscript.

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Correspondence to Tom W. Muir.

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Extended data

Extended Data Fig. 1 CAGE modules generate the splice products in live cells by non-toxic rapamycin analog treatment.

a, Mechanism of the protein trans-splicing reaction by split inteins. b, Schematic of the CPS reaction using CAGE modules. Rapalog-induced FKBP-FRB dimerization is followed by a domain-swap** event that yields an active split intein complex. c, Immunoblots of phosphorylation levels at mTOR S2448 and AKT S473 sites in HEK293T cells (left) and AML12 cells (right) treated with DMSO, non-toxic rapamycin analog AP21967 (rapalog) or rapamycin at the indicated dose for 18 hours. Data are representative of n = 3 independent experiments. d, Left—immunoblots (top) of splice product (P300 catalytic subunit, FLAG-tag) generation via CAGE modules incorporating FRB T2098L mutant (FRB*) in HEK293T cells. Cells were treated with DMSO, rapamycin (100 nM) or rapalog (100 nM) for 18 hours. Starting constructs originated from the previous work19. The bar graph below shows quantified splice product levels following the indicated treatments normalized to the product band intensity of the condition ‘rapamycin’ (bottom) (n = 4 independent experiments, mean and s.e.m.). Right—as in the left-hand but employing wild-type FRB in the CAGE constructs. Since rapalog was found to be specific to FRB* and does not affect mTOR signaling, FRB* was incorporated into the CAGE system and used in all subsequent studies with rapalog. CAGEN refers to ‘NpuNCage-FKBP’, and CAGEC refers to ‘FRB-NpuCCage’.

Source data

Extended Data Fig. 2 Engineering the CAGE toolbox via charge-swap mutations of the NrdJ-1 cages.

a, Schematic showing engineering strategy used to tune the responsiveness of the CAGE via charge-swap mutations. The interaction between the appended caging unit and the split intein can be strengthened or weakened by manipulating key salt bridges. b, A predicted structure of the NrdJ-1 intein generated via AlphaFold2 modeling, with the amino acids for charge-swap mutations in the cages marked in green circles. c, The amino acid sequences of both NrdJ-1NCage and NrdJ-1CCage constructs (intein fragment + appended cage) with the sites altered through charge-swap mutations indicated as in b marked in green boxes and underlined in red. d, Schematic of MBP–GFP splice product generated by proximity-triggered CPS between complementary MBP-CAGEN and CAGEC-GFP with rapalog treatment in cells. Flanking extein sequences at the splice site were either native NrdJ-1 (NPC/SEI) or AAA/SAA. e, Immunoblots of the MBP–GFP splice product (SP) in HEK293T cells co-expressing MBP-CAGEN and CAGEC-GFP, treated with either DMSO or rapalog (100 nM) for 4 hours. Relative splice product levels were screened with every single mutant in the cages of the CAGEN or CAGEC indicated in b and c and the combination of #2E/#5A in the cage of CAGEN and #1K in the cage of CAGEC. f,g, Immunoblots of the MBP–GFP splice product (SP) in HEK293T cells co-expressing MBP-CAGEN and CAGEC-GFP, treated with either DMSO or rapalog (100 nM) for indicated time points (f) or treated with DMSO or increasing dose of rapalog for 4 hours (g). Left—CAGE without charge-swap mutations, NPC/SEI extein sequence. Middle—engineered CAGE (#2E/#5A in the cage of CAGEN and #1K in the cage of CAGEC) NPC/SEI extein sequence. Right—engineered CAGE (#2E/#5A in the cage of CAGEN and #1K in the cage of CAGEC), AAA/SAA extein sequence. Data in e–g are representative of n = 3 independent experiments. CAGEN refers to ‘NrdJ-1NCage-FKBP’, and CAGEC refers to ‘FRB-NrdJ-1CCage’ in e–g. Importantly, the engineered NrdJ-1 CAGE (#2E/#5A in the cage of CAGEN and #1K in the cage of CAGEC) was found to be optimal and used in all subsequent studies.

Source data

Extended Data Fig. 3 AML1-ETO splice product generated from co-expressed CAGE modules in cells.

a, Protein splicing junctions employed in the generation of AML1-ETO using the caged Npu and NrdJ-1 split inteins. The caged Npu system required the insertion of a ‘CF’ di-peptide sequence to facilitate efficient protein trans-splicing. This ‘scar’ is added to the N-terminus of ETO segment. By contrast, the caged NrdJ-1 split intein supports traceless splicing using the native serine at position 36 of ETO. b, Immunoblots of HA-tagged AML1-ETO splice product (SP) generated in HEK293T cells co-expressing AML1-CAGEN (Myc-tag) and CAGEC-ETO (HA-tag). Cells were treated with either DMSO or rapalog (100 nM) for 4 hours before analysis. HEK293T cells expressing a HA-tagged AML1-ETO fusion (WT) are used as a positive control (rightmost lane). c, Immunoblots of HA-tagged AML1-ETO splice product (SP) generated HEK293T cells co-expressing AML1-CAGEN (wild-type or inactive C1A mutant) and CAGEC-ETO. Cells were treated with either DMSO or rapalog (100 nM) for 4 hours before analysis. d,e, Characterization of the AML1-ETO splice product by mass spectrometry. AML1-ETO splice product generated in HEK293T cells was subjected to trypsinolysis, and targeted proteomics was used to identify the tryptic peptide spanning the splice junction (calculated m/z = 519.517, z = 4). Overlaid PRM chromatograms for the six parent-to-daughter transitions in targeted proteomics runs (d) and annotated MS/MS spectrum from the peptide (e). f, Immunoblots of AML1-ETO generation in HEK293T cells co-expressing AML1-CAGEN and CAGEC-ETO. Cells were treated with DMSO or increasing doses of rapalog for 18 hours before analysis. g,h, Immunoblots of AML1-ETO generation in HEK293T cells co-expressing AML1-CAGEN and CAGEC-ETO. Cells were treated with DMSO or rapalog (100 nM) for indicated time points before analysis. i, Subcellular location of the AML1-CAGEN (C1A mutant) and CAGEC-ETO in HEK293T cells treated with DMSO or rapalog (100 nM) for 18 hours before immunoblotting. The C1A mutation of the CAGEN module blocks the splice product generation. CAGEN refers to ‘NrdJ-1NCage-FKBP-NES’, and CAGEC refers to ‘NES-FRB-NrdJ-1CCage’. Data in b,c and f–i are representative of n = 3 independent experiments. In b–h, CAGEN refers to ‘NpuNCage-FKBP-NES’ and CAGEC refers to ‘NES-FRB-NpuCCage.

Source data

Extended Data Fig. 4 MLL-AF9 and NUP98-HOXA9 splice products generated from co-expressed CAGE modules in cells.

a,g, Protein splicing junctions employed in the generation of MLL-AF9 (a) and NUP98-HOXA9 (g) using the caged Npu and NrdJ-1 split inteins. The caged Npu system required the insertion of a ‘CFN’ tri-peptide sequence to facilitate efficient protein trans-splicing. This ‘scar’ is added to the N-terminus of AF9 segment (a) and HOXA9 segment (g). The caged NrdJ-1 split intein supports traceless splicing using the native serine at positions 490 of AF9 (a) and 176 of HOXA9 (g). b,h, Immunoblots of MLL-AF9 (HA-tag) splice product (SP) generation in HEK293T cells co-expressing MLL-CAGEN (Myc-tag) and CAGEC-AF9 (HA-tag) (b) and NUP98-HOXA9 (HA-tag) splice product (SP) generation in HEK293T cells co-expressing NUP98-CAGEN (Myc-tag) and CAGEC-HOXA9 (HA-tag) (h). Cells were treated with DMSO or rapalog (100 nM) for indicated time points before analysis. CAGEN refers to ‘NpuNCage-FKBP-NES’, and CAGEC refers to ‘NES-FRB-NpuCCage’. c,d,i,j, Immunoblots of MLL-AF9 generation in HEK293T cells co-expressing MLL-CAGEN and CAGEC-AF9 (c,d) and NUP98-HOXA9 generation in HEK293T cells co-expressing NUP98-CAGEN and CAGEC-HOXA9 (i,j). Cells were treated with DMSO or increasing doses of rapalog for 18 hours before analysis. CAGE constructs incorporated either NpuCage (c,i) or NrdJ-1Cage (d,j). e,f,k,l, Immunoblots of the HA-tagged MLL-AF9 splice product generated in HEK293T cells co-expressing MLL-CAGEN and CAGEC-AF9 (e,f) and the HA-tagged NUP98-HOXA9 splice product generated in HEK293T cells co-expressing NUP98-CAGEN and CAGEC-HOXA9 (k,l) treated with rapalog (100 nM) for 18 hours followed by a subcellular fraction to separate cytosolic and nuclear proteins. Tubulin and histone H4 serve as cytosolic and nuclear markers, respectively. CAGE constructs incorporated either NpuCage (e,k) or NrdJ-1Cage (f,l). Data in b–f and h–l are representative of n = 3 independent experiments. ‘Anti-HA (dark)’ indicates longer exposure to the immunoblots against anti-HA bands.

Source data

Extended Data Fig. 5 Multi-omics validation of the scarless AML1-ETO splice product.

a, Screenshots of ChIP–seq results from three cited references displayed from the UCSC genome browser. Shown are six different regions with associated transcripts. The x-axis represents the genomic positions in base pairs: negative control_chr21: 30,032,740–30,032,921; GREB1 (negative control)_chr2: 11,498,624–11,498,837; GAPDH-TSS_chr12: 6,534,775–6,534,872; PTEN-promoter_chr10: 87,863,242–87,863,355; LNPEP-promoter_chr5: 96,935,104–96,935,201; EMILIN1-enhancer_chr2: 27,081,454–27,081,577. The first two regions scored negative for AML1-ETO occupancy, and the last four scored positive for AML1-ETO occupancy in patient-derived Kasumi-1 cells. b, Schematic of the quantitative AP-MS workflow using SILAC-labeled HEK293T cells stably expressing AML1-CAGEN and CAGEC-ETO constructs. c, Immunoblots of the abundance of AML1-ETO splice product (HA-tag) and HDAC1 in the SILAC inputs (top) and outputs (bottom) (n = 2, biological replicate samples). d, Gene Ontology (GO) biological process categories enriched among the proteins in quantitative AP-MS workflow, satisfying average SILAC ratio over two-fold and P values less than 0.05. The statistical significance of quantitative proteomics data was determined by two-way Student’s t tests, and the statistical significance of GO analysis was determined by Fisher’s exact tests via Metascape. CAGEN refers to ‘NrdJ-1NCage-FKBP-NES’, and CAGEC refers to ‘NES-FRB-NrdJ-1CCage’ in c.

Source data

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 (autoregulatory domain, N) and the linker connecting the autoregulatory 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 HA-tagged 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 were treated with DMSO or rapalog (100 nM) for 18 hours before analysis. The optimal CAGEC-ABL1 construct contained a ‘L79 + N24’ combination. f, Schematic of the N-myristoylation metabolic labeling workflow. 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). Cells 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. ‘Anti-HA (dark)’ indicates longer exposure to the immunoblots against anti-HA bands and ‘anti-FLAG (dark)’ against anti-FLAG bands.

Source data

Extended Data Fig. 7 Dynamic tyrosine phosphorylation events following BCR-ABL activation.

a, Cluster map of the proteomics dataset representing the time-resolved phosphorylation at protein tyrosine sites, combined from three biological replicates. Data processing employing the stringency filters described in Supplementary Fig. 2b results in 39 representative dynamics within 6-hour timeslots. b, Phosphorylation fold-change plots of representative protein tyrosine sites as a function of time. Phosphorylation levels at each time point are normalized to the values at 0 hours (n = 3, mean with s.e.m.). c, Immunoblots of the tyrosine phosphorylation levels in HEK293T cells co-expressing BCR-CAGEN and CAGEC-ABL1. Cells were treated with rapalog (100 nM) for indicated time points before analysis with the indicated antibodies. d, Immunoblots of tyrosine phosphorylation levels in HEK293T cells containing a Dox-inducible BCR-ABL gene expression system. BCR-ABL expressed via doxycycline (200 ng/mL) treatment for indicated time points before analysis with the indicated antibodies. Data in a,b are generated from n = 3 independent biological replicates. Data in c,d are representative of n = 3 independent experiments. CAGEN refers to ‘NpuNCage-FKBP’, and CAGEC refers to ‘ABLauto-FRB-NpuCCage’.

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Extended Data Fig. 8 DNAJB1-CAGEN and CAGEC-PRKACA for inducible kinase activation.

a, DNAJ-PKAc (PDB: 4WB7). The sites of native gene fusion and protein trans-splicing are indicated. b, Left—mouse PRKACA (PDB: 4DFX) highlighting the myristate tail allosterically bound to the catalytic domain. A linker colored in red is for inserting the CAGEC. Right—PKA tetrameric complex (PDB: 3TNP). The location of the CAGEC insertion site is indicated in red. c, Schematic depicting the CAGEC-PRKACA. The N-terminal alpha-helix of PRKACA containing the N-myristate (1–37 aa) is connected to the N-terminus of the CAGEC via a linker. d, Schematic of the split site within DNAJ-PKAc. A38 site in the PRKACA is mutated to serine in the N-terminus of split PRKACA for protein trans-splicing by NrdJ-1Cage. e,f, Immunoblots of the HA-tagged CAGEC-PRKACA expressed in HEK293T cells following YnMyr metabolic labeling and biotin conjugation using the workflow described in Extended Data Fig. 6f. Wild-type CAGEC-PRKACA was enriched (e), and CAGEC-PRKACA with G2A mutation failed to be enriched (f). g, Proximity-induced CPS in HEK293T cells co-expressing DNAJB1-CAGEN (Myc-tag) and CAGEC-PRKACA (HA-tag). Cells were treated with DMSO or rapalog (100 nM) for indicated time points before immunoblotting. X refers to non-transfected cells, whereas DNAJ-PKAc refers to cells expressing DNAJ-PKAc (HA-tagged). h, Similar to g but showing the dose response to rapalog at 24 hours. i, Proximity-induced CPS in AML12 cells co-expressing DNAJB1-CAGEN (Myc-tag) and CAGEC-PRKACA (HA-tag). Cells were treated with DMSO or rapalog (100 nM) for indicated time points before immunoblotting. j, Similar to i but showing the dose response to rapalog at 6 hours. k, Immunoblots of the phosphorylation levels in AML12 cells, treated with rapalog (100 nM) for indicated time points before analysis with the indicated antibodies. l, Immunoblots of HSP70 co-immunoprecipitated with HA-tagged splice product. A pulldown assay was performed using the lysates from stable AML12 cells treated with DMSO or rapalog (100 nM) for 24 hours. Data in e–l are representative of n = 3 independent experiments. CAGEN refers to ‘NrdJ-1NCage-FKBP’, and CAGEC refers to ‘PKAcauto-FRB-NrdJ-1CCage’ in e–j and l. ‘Anti-HA (dark)’ indicates longer exposure to the immunoblots against anti-HA bands.

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Extended Data Fig. 9 MS2 and MS3 analysis of phosphorylated Serine 401 SGK1 peptide.

a, Immunoblots of the abundance of SGK1 and phospho-PKA substrates in the inputs. WT refers to non-transformed AML12 cells, whereas DNAJ-PKAc refers to the AML12 cells stably expressing DNAJ-PKAc fusion. b–d, Quantification of SGK1 phosphorylated serine 401 levels in non-transformed and DNAJ-PKAc-expressing AML12 cells by mass spectrometry. Targeted proteomics was performed to identify and quantify the tryptic peptide spanning the SGK1 phosphorylated serine 401 after being subjected to trypsinolysis, phosphopeptide enrichment and isobaric labeling with TMTpro reagents (calculated m/z = 635.691, z = 3). Annotated MS2 and MS3 spectrum from the peptide (b), overlaid chromatograms for the six parent-to-daughter transitions in targeted proteomics runs (c) and the bar charts showing quantified SGK1 pS401 levels in non-transformed and DNAJ-PKAc-expressing AML12 cells normalized by the SGK1 intensity in (a) (n = 2 independent experiments, means). d, Statistical significance was determined by two-way Student’s t tests: 0.005 < *P < 0.05.

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Extended Data Fig. 10 Pharmacologically inhibiting or overexpressing SGK1 in DNAJ-PKAc-expressing AML12 cells.

a,b, Immunoblots of phospho-ERK1/2 in non-transformed (a) and DNAJ-PKAc-expressing (b) AML12 cells. Cells were treated with DMSO or increasing doses of GS-9007 for 24 hours followed by immunoblotting using the indicated antibodies. c, Bar charts showing quantified pERK levels of indicated treatments in non-transformed and DNAJ-PKAc-expressing AML12 cells normalized by the ERK intensity (n = 4 independent experiments, means and s.e.m.). d,e, Immunoblots of phospho-ERK1/2 in non-DNAJ-PKAc-expressing (d) and DNAJ-PKAc-expressing (e) AML12 cells. Cellular SGK1 levels were further increased by overexpression (OE) or decreased by knock-down (KD) before analysis. f,g, Left—representative wells for non-DNAJ-PKAc-expressing (f) and DNAJ-PKAc-expressing (g) AML12 cells, either nontransformed or overexpressing SGK1, growth for 10 days and stained with crystal violet. Right—clonogenic cell growth of non-DNAJ-PKAc-expressing (f) and DNAJ-PKAc-expressing (g) AML12 cells quantified by the area covered by colonies and normalized by the average colony area of non-SGK1-transformed cells of each plate (n = 24 independent experiments, means and s.d.). h, Schematic of a proposed molecular link between DNAJ-PKAc activation and ERK phosphorylation through SGK1. Data in d–e are representative of n = 3 independent experiments. In c, f and g, statistical significance was determined by two-way Student’s t tests: 0.005 < *P < 0.05; ***P < 0.001.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–10 and Supplementary Tables 1–5.

Reporting Summary

Supplementary Data 1

ChIP–qPCR data processing.

Supplementary Data 2

AML1–ETO SP qAP-MS profile.

Supplementary Data 3

Time-resolved tyrosine phosphorylation profile (BCR–ABL, HEK293T).

Supplementary Data 4

Time-resolved phosphorylation profile (DNAJ–PKAc, HEK293T).

Supplementary Data 5

Time-resolved phosphorylation profile (DNAJ–PKAc, AML12).

Supplementary Data 6

Phosphosite local sequences (DNAJ–PKAc, HEK293T).

Supplementary Data 7

Phosphosite local sequences (DNAJ–PKAc, AML12).

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Lee, G., Muir, T.W. Distinct phases of cellular signaling revealed by time-resolved protein synthesis. Nat Chem Biol (2024). https://doi.org/10.1038/s41589-024-01677-3

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