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An atlas of the catalytically active liver and spleen kinases in chicken identified by chemoproteomics

B. Nanduri, C. R. Gresham, M. Ou, W. W. Hui, R. H. Bailey, M. J. Edelmann

Abstract

Chicken is the first agricultural animal to have a sequenced genome, but current kinase annotations of Gallus gallus are overwhelmingly limited to the predictions generated based on homology or isolated studies focused on specific kinases. Our approach used chemical probes consisting of ATP and ADP derivatives binding to specific lysine residues within the ATP-binding pocket of kinases. Collectively, we identified 188 chicken kinases and corresponding 267 peptides labeled with the ATP and ADP acyl derivatives in chicken spleen and liver. The kinases identified here are publicly available through the database, Chickspress genome browser (http://geneatlas.arl.arizona.edu/cgi-bin/GeneAtlas/chickspress_genes.cgii). Analysis of putative functions of these chicken kinases indicates that kinases identified in this study might regulate hematological system development, necrosis, apoptosis, epithelial neoplasm, and other processes. The identified tissue-specific expression atlas of active chicken kinases along with the ATP binding sites of kinases provide the basis for the development of specific drug targets for multiple chicken diseases as well as starting point for inhibitor selectivity studies in this agriculturally important species. Moreover, this study will support future studies focused on identifying the role of these kinases in chicken growth, metabolism, and disease.

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Claude Opus 5.5 · xhigh effort · 8 Nov 2026 · from the full text of version 1 (10 May 2019) · CC BY 4.0

Acyl-phosphate ATP/ADP probes with mass spectrometry identify 188 catalytically active kinases (267 labeled peptides) in chicken liver and spleen; spleen yielded 4.8- to 7-fold more summed probe-labeled spectra.

Chicken kinase annotations come mostly from homology-based predictions or studies of single kinases, and the poultry kinome peptide arrays already in use cover only a subset of immune and metabolic kinases. The authors present this activity-based chemoproteomic survey as the first systematic experimental description of active chicken kinases, and its ATP-site residues give a base for inhibitor selectivity work.

Key results

  1. Shown188 catalytically active kinases, represented by 267 peptides labeled by ATP or ADP probes, were identified across chicken liver and spleen. The data are deposited in PRIDE and shown on the Chickspress genome browser.

    183 kinases detected in liver and 188 in spleen, from two birds (two biological replicates); proteomics dataset PXD011548 in ProteomeXchange/PRIDE. · Abstract; Chemoproteomics-based identification of protein…; Sample Preparation; LC-MS/MS Analysis

  2. SuggestedSpleen carried more probe-labeled kinase signal than liver, and MET was the only kinase relatively higher in liver. Western blots for three kinases agreed with the mass spectrometry direction.

    Summed spectra of all labeled peptides were 4.8-fold (ATP probe) and 7-fold (ADP probe) higher in spleen; MET was ∼5.5-fold higher in liver; IKKa, AKT1 and PIK3CA were higher in spleen on immunoblots of three samples per tissue, each from a different bird. No statistical test is reported for these differences. · Chemoproteomics-based identification of protein…; Immunoblotting

  3. ShownKinases from the major kinase groups were detected (AGC, CMGC, CAMK, CK1, STE, tyrosine kinase (TK), TK-like (TKL), RGC, Atypical, Other), but 40-50% of chicken orthologs of human kinases were not found.

    Chicken hits vs human group sizes: AGC 32 vs 63 (equal numbers in liver and spleen), CMGC 24 vs 57, STE 27 vs 47 (over 57% of human STE kinases). Kinases were counted in each of the nineteen most represented families in each tissue. · Chemoproteomics-based identification of protein…

  4. Shown107 labeled sites fall on Lys1 and 91 on Lys2, the two conserved lysines of the kinase domain. For WNK1 and Nek6, the labeled chicken lysine lines up with known ATP-binding lysines in human, rat and mouse; the labeled SMG1 residue is not annotated as ATP-binding in those species.

    The other site classes are 34 activation loop, 21 ATP site of non-canonical kinases, 7 ATP-binding loop, 4 elsewhere in the kinase domain and 3 uncategorized. Alignments are shown for three serine/threonine kinases (WNK1, SMG1, Nek6) against blastp-identified orthologs. · Chemoproteomics-based identification of protein…; Conservation of kinase active site between…

  5. SuggestedPathway annotation based on orthologs links spleen kinases to hematological and lymphoid development and B-lymphocyte viability. It links liver kinases to hepatic lesions, hepatoma-cell apoptosis, liver necrosis and liver tumors.

    Ingenuity Pathway Analysis on human, mouse and rat orthologs, using Fisher's exact test with P ≤ 0.05. The functions are listed in Fig. 5 and Tables 1-2; the text gives no enrichment values. · Analysis of the biological functions of chicken…; Ingenuity Pathway Analysis; Tables

Methods, data and reproducibility

Chicken liver and spleen lysates were labeled with desthiobiotin ATP and ADP acyl-phosphate probes that irreversibly tag conserved lysines in the ATP pocket. Labeled tryptic peptides were enriched on streptavidin and identified by data-dependent, then targeted, LC-MS/MS (Sequest, UniRef100 Gallus). Hits were mapped to Galgal5, aligned to mammalian orthologs and annotated with Ingenuity Pathway Analysis (IPA).

  • Liver and spleen from commercial male broiler chickens (60-64 days old), collected post-mortem from a processing line; chemoproteomic LC-MS/MS · chicken (Gallus gallus) · n = two birds (two biological replicates) · PXD011548 (ProteomeXchange/PRIDE)
  • Liver and spleen samples for immunoblot validation of IKKa, AKT1 and PIK3CA · chicken (Gallus gallus) · n = three samples per tissue, each from a different bird
  • UniRef100 sequence database, taxonomy ID 9031 (Gallus), used for the Sequest search · chicken (Gallus gallus)
  • UniProt (as of April 2018) and NCBI Gene records mapped to the NCBI Galgal5 genome assembly for browser display · chicken (Gallus gallus)
Methods & tools
desthiobiotin ATP/ADP acyl-phosphate probes, Thermo LTQ ion trap mass spectrometer, Agilent 1100 micro-HPLC, Sequest, UniRef100, UniProt, NCBI BLAST (blastp), Ingenuity Pathway Analysis (Ingenuity Pathways Knowledgebase), Chickspress genome browser, ProteomeXchange / PRIDE, Western blot (Cell Signaling Technology anti-IKKa, AKT1, PIK3CA antibodies)
Data & accessions
http://geneatlas.arl.arizona.edu/cgi-bin/GeneAtlas/chickspress_genes.cgihttp://geneatlas.arl.arizona.edu/cgi-bin/GeneAtlas/chickspress_genes.cgii
Reagents & environment
Database versions are stated (UniProt as of April 2018; NCBI Galgal5 assembly); no software versions are given.
Reproducibility gaps
No code released. ActivX Biosciences did the mass spectrometry sample preparation, and the probe synthesis and LC-MS/MS protocols are cited to prior work. Per-kinase sites and abundances are only in Supplementary Table 1. The Chickspress URL is printed as both .cgi and .cgii.
Limitations (5)
  • Probe labeling reports kinases whose ATP/ADP sites are accessible under the sampling conditions. The authors note that these hits still need support from activity assays on individual kinases. · stated by the authors
  • 40-50% of chicken orthologs of human kinases were not detected. The authors attribute this to kinases being inactive under the experimental conditions, unique to the human kinome, or too low in abundance to detect. · stated by the authors
  • The validation antibodies recognize human proteins. Chicken IKKa is ∼79% identical to the human protein (the others are over 95%), so band identity was checked against predicted molecular weight. · stated by the authors
  • The mass spectrometry data come from two birds collected post-mortem from a processing line, and liver vs spleen fold differences are summed spectra with no statistical test. The immunoblot methods describe three birds, while sample collection describes two. · noted in this summary
  • The functional and disease associations come from IPA annotations of human, mouse and rat orthologs; no experiment in chicken tests them. The putative SMG1 ATP-binding residue is supported only by probe labeling. · noted in this summary

Who should read:Read this if you need an experimental list of active kinases and their probe-labeled ATP-site residues in chicken liver and spleen, for example to pick poultry kinase targets or plan inhibitor selectivity studies.

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