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DeKinomics pulse-chases kinase functions in living cells

Nature Chemical Biology (2024)Cite this article

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Abstract

Cellular context is crucial for understanding the complex and dynamic kinase functions in health and disease. Systematic dissection of kinase-mediated cellular processes requires rapid and precise stimulation (‘pulse’) of a kinase of interest, as well as global and in-depth characterization (‘chase’) of the perturbed proteome under living conditions. Here we developed an optogenetic ‘pulse-chase’ strategy, termed decaging kinase coupled proteomics (DeKinomics), for proteome-wide profiling of kinase-driven phosphorylation at second-timescale in living cells. We took advantage of the ‘gain-of-function’ feature of DeKinomics to identify direct kinase substrates and further portrayed the global phosphorylation of understudied receptor tyrosine kinases under native cellular settings. DeKinomics offered a general activation-based strategy to study kinase functions with high specificity and temporal resolution under living conditions.

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Fig. 1: Establishment and characterization of DeKinomics’ temporal resolution in vitro and in living cells.
Fig. 2: Characterization of DeKinomics’ temporal resolution in phosphoproteome.
Fig. 3: DeKinomics-coupled ultra-deep pTyr proteomics revealed direct SRC substrates.
Fig. 4: SRC regulates UBA1 activity through Y55 phosphorylation.
Fig. 5: DeKinomics profiled dynamic phosphorylation of the understudied RTK TYRO3.

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Data availability

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the iProX partner repository72,73 with the dataset identifier PXD039316. Source data are provided with this paper.

Code availability

The custom computer code has been deposited to Zenodo at https://doi.org/10.5281/zenodo.8365329.

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Acknowledgements

This work was supported by grant nos. 2021YFA1302603 (P.R.C.), 2021YFA1301601 (R.T.), 2021YFA1301602 (R.T.), 2021YFA1302603 (R.T.), 2022YFC3401104 (R.T.) and 2020YFE0202200 (R.T.) from the National Key Research and Development Program of China; grant nos. 21937001 (P.R.C.), 22137001 (P.R.C.), 91753000 (P.R.C.), 92253304 (R.T.) and 22125403 (R.T.) from the National Natural Science Foundation of China; grant no. 2019B151502050 (R.T.) from the Guangdong Provincial Fund for Distinguished Young Scholars; grant nos. JCYJ20200109141212325 (R.T.), JSGGZD20220822095200001 (R.T.) and JCYJ20210324120210029 (R.T.) from the Shenzhen Innovation of Science and Technology Commission; grant no. Z200010 from the Beijing Natural Science Foundation (P.R.C.); New Cornerstone Science Foundation through the New Cornerstone Investigator Program (P.R.C.) and the XPLORER PRIZE (P.R.C.). We thank J. Guo (University of Nebraska-Lincoln) for sharing the CMF-RS plasmid. We thank M. Pan and Q. Zheng (Protein Design and Synthesis Center, Institute of Translational Medicine, Shanghai Jiao Tong University) for providing recombinant E2 and Ub-FITC proteins. We thank Y. Sun and P. Bai (Southern University of Science and Technology) for providing primary PDAC cells.

Author information

Author notes

  1. These authors contributed equally: Yicheng Weng, Wendong Chen, Qian Kong.

Authors and Affiliations

  1. New Cornerstone Science Laboratory, Synthetic and Functional Biomolecules Center, Beijing National Laboratory for Molecular Sciences, New Cornerstone Science Laboratory, College of Chemistry and Molecular Engineering, Peking University, Beijing, China

    Yicheng Weng, Ruixiang Wang, Ruxin Zeng, Yanjun Liu & Peng R. Chen

  2. Department of Chemistry and Research Center for Chemical Biology and Omics Analysis, School of Science, Southern University of Science and Technology, Shenzhen, China

    Yicheng Weng, Wendong Chen, Qian Kong, An He, Yiheng Mao, Yunqiu Qin, Mi Ke, Jie Wang & Ruijun Tian

  3. Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China

    Yicheng Weng, Ruixiang Wang & Peng R. Chen

  4. Guangdong Provincial Key Laboratory of Cell Microenvironment and Disease Research, Southern University of Science and Technology, Shenzhen, China

    Yicheng Weng, Wendong Chen, Qian Kong, An He, Yiheng Mao, Yunqiu Qin, Mi Ke & Ruijun Tian

  5. Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China

    Yicheng Weng, Ruixiang Wang & Peng R. Chen

  6. South China Institute of Biomedicine, Academy of Phronesis Medicine, Guangzhou, China

    Wendong Chen

  7. Shenzhen Bay Laboratory, Shenzhen, China

    William Shu Ching Ngai, Heng Zhang & Peng R. Chen

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  1. Yicheng Weng
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Contributions

P.R.C. and R.T. conceived and supervised the study. Y.W. conceived all experiments and performed cell culture and treatment, kinase-ONPK expression and decaging, proteomics data analysis, immunofluorescence experiments, stable cell line constructions, ubiquitin thioester formation assays and other unspecified experiments. Y.W., W.C. and Q.K. performed pTyr proteomics and Flag IP-MS. Y.W., W.C., Y.M. and Y.Q. performed phosphoproteomics experiments. R.Z. and Y.L. synthesized ONPK. A.H. and M.K. performed LC–MS analysis of ONPK decaging rate. Y.W., R.W., W.S.C.N., H.Z. and J.W. performed CMF site-specific incorporation, protein purification and kinase phosphorylation assays. Y.W., R.T. and P.R.C. wrote the paper with inputs from all authors.

Corresponding authors

Correspondence to Ruijun Tian or Peng R. Chen.

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Nature Chemical Biology thanks Kazuya Machida, Yonghao Yu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Characterization of ONPK decaging in vitro and in living cells.

a, Liquid chromatography-mass spectrometry (LC-MS) analysis of remaining ONPK with UV irradiation. Error bars represent mean ± SD (n = 3 independent experiments). b, The MS/MS spectrum of the ONP caged peptide (residue 295–318 of SRC) with neutral loss of ONP group. ONP, ONP modification; OX, oxidation modification.

Source data

Extended Data Fig. 2 Validation of DeKinomics’ temporal resolution in phosphoproteome.

a, The numbers of identified phosphoproteins, phosphopeptides and phosphosites in DeKinomics study of BRAF with phosphorylated residue numbers from different classes according to localization probability and distribution of phosphorylated amino acids. b, Quantitative phosphoproteomics revealed the changes of phosphosites in MAPK signaling pathway upon BRAF activation. c, The KEGG pathway enrichment analysis of phosphosites with the Euclidean distance among the smallest 10%. According to the gene ratio, the top 10 pathways were depicted. Two-sided hypergeometric test followed by Benjamini-Hochberg correlation. KEGG, Kyoto Encyclopedia of Genes and Genomes. d, Dynamics of MAPK signaling pathway phosphosites that increased upon BRAF activation were showed in detail. e, Validation of phosphorylation dynamics in MAPK signaling pathway in repetitious experiments. Error bars represent mean ± SD (n = 3 independent experiments).

Source data

Extended Data Fig. 3 SRC-driven pTyr proteome dynamics were deeply profiled by DeKinomics.

a, Western blotting validation of rapid activation of tyrosine kinase SRC (n = 2). b, The numbers of identified pTyr proteins, peptides and phosphosites in the SRC study, with the pTyr residue number from different classes according to localization probability. c, Schematic illustration of the time-resolved pTyr proteomics upon UV irradiation as a control experiment. d, Bar graph comparing the unchanged and the significantly changed pTyr sites in UV irradiation experiment. e, Violin plot indicating the increased global pTyr intensity upon SRC activation. The counts of quantified pTyr sites (n) in each time point are indicated in brackets. Box plots show median (center line), the upper and lower quantiles (box), and the minima and maxima of the data (whiskers). f, The Pearson correlation of pTyr intensity between the biological replicates. g, GO enrichment analysis of pTyr sites from different clusters.

Source data

Extended Data Fig. 4 Validating SRC substrates with monoclonal stable cell line expressing SRC-ONPK.

a, Volcano plots indicating the increased pTyr sites within 6 s upon SRC activation. n = 3 independent experiments; two-sided Student’s t-test. b, Venn diagram indicating that the SRC substrate candidates were determined with two criteria. c, pPiggyBac plasmid used to generate the HEK293T stable cell line that expressing ONPK-incorporated SRC protein. IRES, internal ribosome entry sites; NeoR, Neomycine Resistance; PuroR, Puromycine Resistance; copGFP, green fluorescent protein from copepod Pontellina plumate. d, Western blotting analysis indicating that the expression level of SRC-ONPK could be tuned by ONPK concentration (n = 2). e, Confocal immunofluorescence imaging confirming same SRC localization between exogenous and endogenous SRC. Scale bars: 5 μm (n = 3). f, Schematic illustration of PRM validation for physiological relevance between SRC and downstream pTyr sites (n = 3). g, Histogram indicating pTyr sites with i) the max intensity of their corresponding precursor, ii) Evidence count > 15 would be conduct to PRM validation. The filters including precursor charge < 4, random selection to shorten the list, manual checking for duplication transition were also applied for determining the final list for PRM assay. h, Veen diagram indicating the number of pTyr sites that were studied, quantified and validated in PRM assay respectively. i, Schematic illustration of biological functions of the validated SRC substrates by PRM.

Source data

Extended Data Fig. 5 Control experiments for in vitro kinase phosphorylation assay.

a, The extracted ion chromatograph (XIC) of the fragment ions of ERK1 pY204 (precursor: VpYENVGLMQQQK++) in compare with PTPN11 pY584 (precursor: IADPEHDHTGFLTEpYVATR++). b, Western blotting estimating the SRC concentration in HEK293T cells (n = 2). Loaded protein amount of #1, #2 and #3 HEK293T lysate were 5.2 μg, 6.0 μg and 6.8 μg respectively. Western blotting bands were quantified by image studio software (LI-COR, v5.2). We took 6.5 μL mg−1 as an estimation for the cell volume of the HEK293T cells74.

Source data

Extended Data Fig. 6 SRC phosphorylated UBA1 and suppressed its enzymatic activity.

a, Dot plot indicating the number of reference reporting post-translational modification (PTM) sites identification on UBA1 in PhosphoSitePlus database (v6.6.0.4). b, Schematic illustration of modified thioester formation assay for measuring the UBA1’s activity. c, Sequence alignment of region where human UBA1 S140 and Y141 (highlighted) located. Asterisks indicate the conserved residues. d, e, Quantitative fluorescence measurements of E2-Ub-FITC thioester formation assay. Error bars represent mean ± SD (n = 3 independent experiments).

Source data

Extended Data Fig. 7 DeKinomics applications on oncogenic RTK variants.

a, Sequence alignment for locating conserved catalytic lysine residue in HER2 and NTRK1. Asterisks indicate the conserved residues. b-c, Schematic illustration of DeKinomics study workflow of HER2-L755S and TPM3-NTRK1 (n = 3). d, Volcano plots indicating the successful profiling of downstream phosphorylation of HER2-L755S. n = 3 independent experiments; two-sided Student’s t-test. e, Fusion protein TPM3-NTRK1 could be rapidly activated and induce downstream Tyr phosphorylation (n = 3 independent experiments). f, Heatmap of pTyr relative abundance indicating autophosphorylation and Tyr phosphorylation on signal transduction protein induced by TPM3-NTRK1 activation.

Source data

Extended Data Fig. 8 DeKinomics study of TYRO3.

a, Sequence alignment for locating conserved catalytic lysine residue in TYRO3. Asterisks indicate the conserved residues. b, Confocal immunofluorescence imaging of exogenous TYRO3-K550ONPK (n = 3). Scale bars: 5 μm. c, Heatmap indicating TYRO3 activation induced TYRO3 autophosphorylation. d, The fuzzy c-means clustering of significantly changed pTyr sites upon TYRO3 activation. e, Venn diagram indicating that TYRO3 candidate substrates were determined with 2 criteria: pTyr sites significantly changed in 0–60 sec (ANOVA) and were in cluster 1 with membership > 0.7 in fuzzy c-means clustering.

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Extended Data Fig. 9 DeKinomics-depicted TYRO3 function.

a, Volcano plot comparisons between TYRO3-activated cells and control cells. n = 3 independent experiments; two-sided Student’s t-test. b, The fuzzy c-means clustering of significantly changed phosphosites in phosphoproteome. c, An overview of early phosphorylation cascades of TYRO3. The annotations of protein function were adopted from Uniprot database.

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Extended Data Fig. 10 Targeting TYRO3-PRKCD axis.

a, Cell proliferation assay to evaluate the effect of inhibitor combination. Primary PDAC cells were isolated from the KPf/fC (KrasLSL-G12D/+;Trp53flox/flox;Pdx1-Cre) PDAC mouse model55. Error bars represent mean ± SD (n = 4 independent experiments); two-sided Student’s t-test. b, Schematic illustration of dual-targeting of the TYRO3-PRKCD signaling axis by inhibitors.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–6.

Reporting Summary

Supplementary Table 1

DeKinomics study of BRAF.

Supplementary Table 2

DeKinomics study of SRC.

Supplementary Table 3

SRC substrate candidates.

Supplementary Table 4

Results of the SRC substrates validation experiment.

Supplementary Table 5

DeKinomics study of HER2 and TPM3-NTRK1.

Supplementary Table 6

TYRO3 study in living cells with DeKinomics.

Supplementary Table 7

KSEA of TYRO3-regulated pSer/pThr sites.

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Weng, Y., Chen, W., Kong, Q. et al. DeKinomics pulse-chases kinase functions in living cells. Nat Chem Biol (2024). https://doi.org/10.1038/s41589-023-01497-x

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