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. 2024 May;629(8014):1174-1181.
doi: 10.1038/s41586-024-07407-y. Epub 2024 May 8.

The intrinsic substrate specificity of the human tyrosine kinome

Tomer M Yaron-Barir  1   2   3 , Brian A Joughin  4   5   6   7 , Emily M Huntsman  1   2 , Alexander Kerelsky  1   2 , Daniel M Cizin  1   2 , Benjamin M Cohen  1   2 , Amit Regev  1 , Junho Song  1 , Neil Vasan  8 , Ting-Yu Lin  1   9 , Jose M Orozco  10   11 , Christina Schoenherr  12 , Cari Sagum  13 , Mark T Bedford  13 , R Max Wynn  14   15 , Shih-Chia Tso  14 , David T Chuang  14   15 , Lei Li  16 , Shawn S-C Li  17 , Pau Creixell  4   5   6   7   18 , Konstantin Krismer  4   5   6   19 , Mina Takegami  4   5   6   7 , Harin Lee  20 , Bin Zhang  20 , Jingyi Lu  20 , Ian Cossentino  20 , Sean D Landry  20 , Mohamed Uduman  20 , John Blenis  1   21   22 , Olivier Elemento  2 , Margaret C Frame  12 , Peter V Hornbeck  20 , Lewis C Cantley  23   24   25 , Benjamin E Turk  26 , Michael B Yaffe  27   28   29   30   31 , Jared L Johnson  32   33   34
Affiliations

The intrinsic substrate specificity of the human tyrosine kinome

Tomer M Yaron-Barir et al. Nature. 2024 May.

Abstract

Phosphorylation of proteins on tyrosine (Tyr) residues evolved in metazoan organisms as a mechanism of coordinating tissue growth1. Multicellular eukaryotes typically have more than 50 distinct protein Tyr kinases that catalyse the phosphorylation of thousands of Tyr residues throughout the proteome1-3. How a given Tyr kinase can phosphorylate a specific subset of proteins at unique Tyr sites is only partially understood4-7. Here we used combinatorial peptide arrays to profile the substrate sequence specificity of all human Tyr kinases. Globally, the Tyr kinases demonstrate considerable diversity in optimal patterns of residues surrounding the site of phosphorylation, revealing the functional organization of the human Tyr kinome by substrate motif preference. Using this information, Tyr kinases that are most compatible with phosphorylating any Tyr site can be identified. Analysis of mass spectrometry phosphoproteomic datasets using this compendium of kinase specificities accurately identifies specific Tyr kinases that are dysregulated in cells after stimulation with growth factors, treatment with anti-cancer drugs or expression of oncogenic variants. Furthermore, the topology of known Tyr signalling networks naturally emerged from a comparison of the sequence specificities of the Tyr kinases and the SH2 phosphotyrosine (pTyr)-binding domains. Finally we show that the intrinsic substrate specificity of Tyr kinases has remained fundamentally unchanged from worms to humans, suggesting that the fidelity between Tyr kinases and their protein substrate sequences has been maintained across hundreds of millions of years of evolution.

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Conflict of interest statement

L.C.C. is a founder and member of the board of directors of Agios Pharmaceuticals and is a founder and receives research support from Petra Pharmaceuticals; is listed as an inventor on a patent (WO2019232403A1, Weill Cornell Medicine) for combination therapy for PI3K-associated disease or disorder, and the identification of therapeutic interventions to improve response to PI3K inhibitors for cancer treatment; is a co-founder and shareholder in Faeth Therapeutics; has equity in and consults for Cell Signaling Technologies, Volastra, Larkspur and 1 Base Pharmaceuticals; and consults for Loxo-Lilly. J.L.J. has received consulting fees from Scorpion Therapeutics and Volastra Therapeutics. T.M.Y.-B. is a co-founder of DeStroke. N.V. reports consulting activities for Novartis and is on the scientific advisory board of Heligenics. O.E. is a founder and equity holder of Volastra Therapeutics and OneThree Biotech; is a member of the scientific advisory board of Owkin, Freenome, Genetic Intelligence, Acuamark and Champions Oncology; and receives research support from Eli Lilly, Janssen and Sanofi. M.T.B. is the co-founder of EpiCypher. The other authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Profiling optimal phosphorylation motifs reveals sequence specificity of the human Tyr kinome.
a, Experimental workflow for the PSPA analysis and representative results. Z denotes fixed positions containing one of the 20 natural amino acids, phosphorylated Thr (pT) or phosphorylated Tyr (pY). X denotes unfixed positions containing randomized mixtures of all natural amino acids except for Tyr and Cys. Autoradiograms (right) indicate kinase preferences for specific amino acids at each position; darker spots indicate preferred residues. b, Hierarchical clustering of 93 Tyr kinases on the basis of their amino acid motif selectivity determined from the quantified PSPA data. Kinase names are colour coded according to catalytic domain sequence phylogeny (inset). The diagram in a was created using BioRender.
Fig. 2
Fig. 2. The phosphorylation motifs for the human Tyr kinome enable comparison of all kinases for Tyr phosphorylation sites.
a, Schematic of the substrate-scoring process. b–d, Scoring results and the substrate motif logos for Tyr675 on IRS2 and the insulin receptor kinase (b), Tyr705 on STAT3 and JAK1 (c) and Tyr530 on SRC and C-terminal SRC kinase (CSK) (d). Red text in b–d indicates known upstream kinases.
Fig. 3
Fig. 3. Kinome-wide motif analysis of phosphoproteomic data identifies condition-dependent patterns of kinase regulation and dysregulation.
a, Schematic of the motif enrichment analysis of Tyr phosphoproteomics data. FF, frequency factor. b–g, Results from published datasets in cells after ligand stimulation (b,c), oncogenic mutation (d,e) or targeted inhibition (f,g) of Tyr kinases. b, NIH3T3 fibroblasts after 15 min treatment with 100 ng ml−1 PDGF-ββ. c, Cultured myotubes after treatment for 2 h with 10 nM agrin. d, Ba/F3 cells after expression of BCR–ABL fusion protein. e, HEK293 cells after expression of KIF5B–ALK fusion protein. f, PC-9 cells after treatment for 3 h with 1 μM erlotinib. g, H1781 cells after treatment for 3 h with 1 μM afatinib. Kinases indicated in bold in b–g are discussed in the main text. The enrichments in b–g were determined using one-sided exact Fisher’s tests. Fully annotated versions of these plots are shown in Supplementary Fig. 3. The diagrams in a and d were created using BioRender. Source data
Fig. 4
Fig. 4. Phosphorylation motifs for the human Tyr kinome enable broad categorization of phosphosites and reveal functional correspondence with the SH2-ome.
a, Comprehensive scoring of the Tyr phosphoproteome by all Tyr kinase motifs. b, Annotation of the human Tyr phosphoproteome by percentile scores with all RTK and nRTK motifs. 7,315 known human phosphorylation sites, were sorted along the x axis according to the number of kinases that score the site in the 90th or higher percentile of substrates for that kinase. Independently in each column of the heat map, kinases were ranked by score for that substrate. Examples of experimentally studied kinase–substrate relationships are highlighted (yellow squares). ITAM, immunoreceptor tyrosine-based activation motif. c, The overlap between phosphorylation motifs of kinases and pTyr recognition motifs of SH2 domains. d, Schematic of the calculation of enrichment of kinase phosphorylation and SH2-domain-binding motifs. e–g, Signalling schematics (top) and motif enrichment plots (bottom) of SH2-binding PSSMs for Tyr phosphorylation sites scored according to the kinase PSSMs of ABL (e), PDGFRβ (f), LCK (g) and ZAP70 (g). In the schematics, the arrows represent recruitment of the indicated protein’s SH2 domain by the indicated kinases. The enrichments in e–g were determined using one-sided exact Fisher’s tests and corrected for multiple hypotheses using the Benjamini–Hochberg method. Fully annotated versions of these plots are presented in Supplementary Fig. 4. The diagrams in c and d were created using BioRender.
Fig. 5
Fig. 5. The diversity of intrinsic Tyr kinase substrate specificity is evolutionarily conserved.
a, Comparison of sequence selectivity between the human and C. elegans orthologues of SRC kinase. b, Hierarchical clustering of the substrate motifs (PSSMs) of the human and nematode Tyr kinases. Worm kinase names are denoted with asterisks and colour coded according to their phylogenetic relationships with human Tyr kinase families (inset). Clusters containing distinct orthologous groups are highlighted. The diagram in a was created using BioRender.
Extended Data Fig. 1
Extended Data Fig. 1. Correlation between Tyr kinase PSSMs derived from PSPA assays and bacterial peptide display.
Pearson correlation coefficients for position specific scoring matrices (PSSMs) obtained previously for five kinases screened by bacterial display were calculated against the PSSMs of the 78 conventional RTKs and nRTKs obtained in this study. Correlation coefficients are sorted from lowest to highest with each of the 5 kinases screened by bacterial display with the 5 best-matching kinase selectivities in our study explicitly labelled.
Extended Data Fig. 2
Extended Data Fig. 2. Structural models of kinase-substrate complexes.
a, EGFR (PDB: 2GS6) in complex with synthetic peptide. Dotted green circle shows positive surface potential in the vicinity of the −1 residue. b, Synthetic peptide from its complex with EGFR (PDB: 2GS6) modelled onto ACK (PDB: 1U46). Dotted green circle shows negative surface potential in the vicinity of the −1 residue. c, INSR (PDB: 1IR3) in complex with synthetic peptide. Dotted green circle shows positive surface potential in the vicinity of the substrate N-terminal residues. d, Synthetic peptide from its complex with INSR (PDB: 1IR3) modelled onto DDR2 (PDB: AF-Q16832-K1A). Dotted green circle shows negative surface potential in the vicinity of the substrate N-terminal residues. Surface electrostatics are represented with Coulombic potential values were computed in ChimeraX and represented by scale bars (kcal/mol·e). In all panels, “Tyr” represents the site of phosphorylation and “−1” indicates the residue directly to the N-terminal side of the site of phosphorylation.
Extended Data Fig. 3
Extended Data Fig. 3. Human Tyr kinases display strong selectivities and diverse preferences for the amino acids near their Tyr phosphorylation sites.
a-c, Log-selectivity of the 78 conventional RTKs and nRTKs on PSPA substrate peptides containing Tyr sites flanked by isoleucine (a), serine (b), or glutamate (c) residues relative to the 18 natural amino acids excluding cysteine and tyrosine. d, Kinome-wide variability in log-selectivity for specific amino acid residues at each position surrounding the substrate Tyr phosphorylation site. Horizontal line indicates a value of 0.5 logs, identifying positions −1 to +3 as the most variably selective positions. e, Experimental kinase selectivity for each Tyr kinase on all amino acids across the highly selective substrate positions −1 to +3.
Extended Data Fig. 4
Extended Data Fig. 4. Phosphopriming favorability is a general feature of the human Tyr kinome.
a, Schematic of Tyr substrate phosphopriming. b, PSPA data and simplified sequence logos highlighting various phosphopriming preferences. The Tyr phosphoacceptors in the logos are represented as Y. c, Phosphopriming favorability of the Tyr kinome. The colour scheme illustrates kinases that select phosphorylated Tyr as their top preferred residue at substrate position −1 (green), +1 (pink), or +2 (blue). Kinases with moderate phosphopriming preferences (where phosphorylated residues are the top preferred residues at certain substrate positions, but not overall favorites) are highlighted in yellow. The kinase TXK, as a notable exception, selects phosphorylated Thr (at position +1) as its overall preferred residue. d-e, Log-selectivity of the 78 conventional RTKs and nRTKs on PSPA substrate peptides containing Tyr sites flanked by phosphotyrosine (d) or phosphothreonine (e).
Extended Data Fig. 5
Extended Data Fig. 5. Substrate phosphopriming preferences by Tyr kinases are mediated by complementary basic residues in their catalytic domains.
a, Top, structural modelling of the interaction between FAK and substrate peptides. Spatial alignment of FAK’s kinase domain structure (PDB: 6TY4) with the EPHB2-substrate peptide complex (PDB: 3FXX) where FAK and the substrate peptide are specifically shown to illustrate the role of Lys621 in the recognition of pThr at the −1 substrate position by FAK and, bottom, the corresponding experimental validation in PSPA assays. b, Top, spatial alignment of FAK’s kinase domain structure (PDB: 6TY4) with the EPHB2-substrate peptide complex (PDB: 3FXX), as performed in a, now illustrating the close proximity between pTyr at the +2 substrate position and Lys581 and Lys583 of FAK and, bottom, the corresponding experimental validation. c, Top, structural modelling of the INSR-peptide substrate complex (PDB: 1IR3), highlighting the residues in its catalytic domain that recognize pTyr at the −1 position on its substrates and, bottom, the corresponding experimental validation with its paralog IGF1R. INSR residues Lys1112 and Arg1116 are equivalent to the IGF1R residues Arg1084 and Lys1088, respectively, in their homologous alignments . Surface electrostatics are represented with Coulombic potential values were computed in ChimeraX and represented by scale bars (kcal/mol·e). Amino acid sidechains of Tyr phosphoacceptors, residues at substrate priming positions and indicated complementary residues in kinase domain are shown in ball-and-stick representation.
Extended Data Fig. 6
Extended Data Fig. 6. Steric accommodation of a +1 pTyr residue by EGFR.
a, Structural modelling of EGFR’s recognition of +1 pTyr (PDB: 5CZH). Side chains of Ala920 on EGFR and +1 pTyr on the peptide substrate as shown in spacefill representation. Sidechain of Tyr phosphoacceptor is shown in ball-and-stick representation. b, Top, log-selectivity of the 78 conventional kinases for +1 pTyr, arranged in order of decreasing favorability. Bottom, corresponding amino acid residues that align with Ala920 of EGFR (bin size: 8 kinases in the left 8 bins; 7 kinases in the right two bins). c, Experimental validation of the importance of Ala920 in facilitating phosphorylation of +1 pTyr substrate by EGFR.
Extended Data Fig. 7
Extended Data Fig. 7. Correspondence between Tyr kinase motif-based predictions and their literature-annotated substrates.
a,b, Schema and phosphorylation site motif logos for ABL derived from PSPA experiments (a) and literature-annotated cellular substrates (b). c, Percentile-score distributions of substrates for their literature-annotated kinases. Higher number of reports correlates with more favourable percentile-scores between the reported kinase and its substrate (AUCDF = area under the cumulative distribution function). The diagrams in a and b were created using BioRender.
Extended Data Fig. 8
Extended Data Fig. 8. Correspondence between the order of RTK auto-trans-phosphorylation events and motif-based scores.
a, Illustration of FGFR1 autophosphorylation. b, Reported rates and sequential order of auto-trans-phosphorylation of five Tyr sites on FGFR1 alongside their corresponding percentile scores for FGFR1’s PSSM. Noncentral Tyr residues were treated as phosphopriming events (that is, scored as pTyr) if they preceded the central Tyr in their reported order of phosphorylation. Sites of phosphorylation are indicated in red. Priming phosphorylations are indicated in green.
Extended Data Fig. 9
Extended Data Fig. 9. Kinetics of peptide phosphorylation by JAK1 and ZAP70.
a, Sequences of Tyr substrate peptides. The peptides are modelled after JAK’s physiological substrate STAT5A Tyr694 (JAK-tide) and ZAP70’s physiological substrate LAT1 Tyr255 (ZAP-tide), with amino acid substitutions introduced at the indicated positions in green. Right, the corresponding percentile scores for each peptide based on the PSSMs of JAK1 and ZAP70. b-c, Kinetics of peptide phosphorylation by JAK1 on JAKtide substrates (b) and ZAPtide substrates (c). Best-fit lines illustrate fitting of the data points to Michaelis-Menten kinetics function using GraphPad Prism 10.1. Data shows mean values with error bars indicating the standard deviations of the data (n = 3 independent reactions). d-e, Kinetics of peptide phosphorylation by ZAP70 on JAKtide substrates (d) and ZAPtide substrates (e). Best-fit lines illustrate fitting of the data points to Michaelis-Menten kinetics function using GraphPad Prism 10.1. Data shows mean values with error bars indicating the standard deviations of the data (n = 3 independent reactions). f, Kinetic parameters for phosphorylation of the indicated peptides by JAK1 and ZAP70 in b-e. The standard errors of the linear fits are indicated (±). The corresponding experimental data for all these plots are presented in Supplementary Fig. 2.
Extended Data Fig. 10
Extended Data Fig. 10. Motif-enrichment analysis of phosphoproteomics data and motif-scoring results for a suboptimal Tyr phosphorylation site on the pyruvate dehydrogenase complex.
a, Motif-enrichment results from published datasets in cells after ligand stimulation (a), oncogenic mutation (b), or targeted inhibition (c) of Tyr kinases. a, A549 cells after 5 min treatment with 100 ng/mL EGF. b, NMuMG cells after expression of FGFR2Δ18 mutant. c, H2286 cells after treatment for 3 h with 1 μM dasatinib. Kinases indicated in bold in a-c are discussed in the main text. The enrichments in a-c were determined using one-sided exact Fisher’s tests. d, Illustration of the mitochondrial-localized regulation of the pyruvate dehydrogenase complex by the PDHKs. e, Scoring results for human pyruvate dehydrogenase E1 component subunit alpha (PDHA1) Tyr301 and homologous site on the yeast ortholog PDA1, highlighting PDHK family members. Source data
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