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Protein-tyrosine kinase 2-beta activates Interferon Regulatory Factor 5 to control inflammation

ABSTRACT: Interferon regulatory factor 5 (IRF5) plays a distinct role in the regulation of immune responses in health and disease and is a genetic risk factor several autoimmune conditions, including systemic lupus erythematosus, diabetes, arthritis and inflammatory bowel disease. IRF5 function as a transcription factor requires post-translational modifications of the protein such as phosphorylation. Though several upstream signalling regulators of IRF5 have been identified, there is as yet little understanding of how they control IRF5 activity or whether other unknown kinases are involved. In this study, we systematically searched for IRF5 kinases using a reporter-based method for screening a library of 365 inhibitors of protein kinases. Based on the pool of the identified IRF5-inhibiting compounds we have compiled a list of putative IRF5 kinases, which we tested in subsequent in vitro assays. Protein-tyrosine kinase 2-beta (Ptk2b or PYK2) was one of the top hits, capable of binding to and phosphorylating mouse IRF5 at tyrosine (Y) 171. We have shown that IRF5 recruitment to target genes and IRF5-dependent transcription was impaired in cells treated with highly selective PYK2 inhibitors or in PYK2-deficient cells. Moreover, expression of pro-inflammatory cytokines was blocked in the supernatants of human biopsies from inflamed colon of patients with ulcerative colitis and treated ex vivo with a PYK2 inhibitor. Thus, we have identified a major role for PYK2 in regulating the inflammatory response and mapped its activity to the IRF5 innate sensing pathways.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Cell Culture, Macrophage

DISEASE(S): Acute Leukemia

SUBMITTER: Roman Fischer

LAB HEAD: Roman Fischer

PROVIDER: PXD014033 | Pride | 2021-10-14

REPOSITORIES: Pride

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			Action	DRS
	FL983_MSQ1448_20180913_HannahAlmuttaqi_3.mgf	Mgf
	FL983_MSQ1448_20180913_HannahAlmuttaqi_3.pride.mgf.gz	Mgf
	FL983_MSQ1448_20180913_HannahAlmuttaqi_3.raw	Raw
	FL983_MSQ1448_20180913_HannahAlmuttaqi_4.mgf	Mgf
	FL983_MSQ1448_20180913_HannahAlmuttaqi_4.pride.mgf.gz	Mgf

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Project description:Background Annexin-1 (ANXA1) plays pivotal roles in regulating various physiological processes including inflammation, proliferation and apoptosis, and deregulations of ANXA1 functions have been associated with tumorigenesis and metastasis events in several cancers. Though ANXA1 levels correlate with breast cancer disease status and outcome, its distinct functional involvement in breast cancer initiation and progression remains unclear. We hypothesized ANXA1-responsive kinase signaling alteration and associated phosphorylation signaling underlie early events in breast cancer initiation events and hence profiled ANXA1-dependent phosphorylation changes in mammary gland epithelial cells. Methods Quantitative phosphoproteomics analysis of mammary gland epithelial cells derived from ANXA1-heterozygous and ANXA1-deficient mice was carried out using SILAC-based mass spectrometry. Kinase and signaling changes underlying ANXA1 perturbations were derived by upstream kinase prediction and integrated network analysis of altered proteins and phosphoproteins. Results We identified a total of 8,110 unique phosphorylation sites, of which 582 phosphorylation sites on 372 proteins showed ANXA1-responsive changes. A majority of these phosphorylation changes occurred on proteins associated with cytoskeletal reorganization spanning the focal adhesion, stress fibers, and also the microtubule network pressing on new roles for ANXA1 in regulating microtubule dynamics. Comparative analysis of regulated global proteome and phosphoproteome highlighted key differences in translational and post-translational effects of ANXA1, and stressed on a close-coordinated rewiring of the cell adhesion network. Kinase prediction analysis suggested activity modulation of CAMK2, PAK, ERK, and IKK kinases upon loss of ANXA1. Integrative analysis revealed regulation of WNT and also Hippo signaling pathways in ANXA1-deficient mammary epithelial cells, wherein there is a downregulation of transcriptional effects of TEAD suggestive of ANXA1-responsive transcriptional rewiring. Conclusions The phosphoproteome landscape uncovered several novel perspectives for ANXA1 in mammary gland biology and stressed on its involvement in key signaling pathways modulating cell adhesion and migration that could contribute to breast cancer initiation.

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Protein-tyrosine kinase 2-beta activates Interferon Regulatory Factor 5 to control inflammation

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