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Re-evaluation of protein kinase CK2 pleiotropy: new insights provided by a phosphoproteomics analysis of CK2 knockout cells

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Abstract

CK2 denotes a ubiquitous and pleiotropic protein kinase whose holoenzyme is composed of two catalytic (α and/or α′) and two regulatory β subunits. The CK2 consensus sequence, S/T-x-x-D/E/pS/pT is present in numerous phosphosites, but it is not clear how many of these are really generated by CK2. To gain information about this issue, advantage has been taken of C2C12 cells entirely deprived of both CK2 catalytic subunits by the CRISPR/Cas9 methodology. A comparative SILAC phosphoproteomics analysis reveals that, although about 30% of the quantified phosphosites do conform to the CK2 consensus, only one-third of these are substantially reduced in the CK2α/α′(−/−) cells, consistent with their generation by CK2. A parallel study with C2C12 cells deprived of the regulatory β subunit discloses a role of this subunit in determining CK2 targeting. We also find that phosphosites notoriously generated by CK2 are not fully abrogated in CK2α/α′(−/−) cells, while some phosphosites unrelated to CK2 are significantly altered. Collectively taken our data allow to conclude that the phosphoproteome generated by CK2 is not as ample and rigidly pre-determined as it was believed before. They also show that the lack of CK2 promotes phosphoproteomics perturbations attributable to kinases other than CK2.

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Acknowledgements

This work was supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC), Grant number IG 18756 (to L.A.P.). C. F. was supported by a Grant from the “Collegio Ghislieri”, Pavia. J.V. was supported by a Grant from the Fondazione per la Ricerca sulla Fibrosi Cistica (Grant #10/2016 adopted by Gruppo di Sostegno FFC di Seregno) (to M.S.). The authors wish to thank the Cassa di Risparmio di Padova e Rovigo (Cariparo) Holding for funding the acquisition of the LTQ-Orbitrap XL mass spectrometer.

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  1. Cinzia Franchin and Christian Borgo contributed equally to this work.

Authors and Affiliations

  1. Department of Biomedical Sciences, University of Padova, Via U. Bassi 58/B, Padua, Italy

    Cinzia Franchin, Christian Borgo, Silvia Zaramella, Jordi Vilardell, Mauro Salvi, Giorgio Arrigoni & Lorenzo A. Pinna

  2. Proteomics Center, University of Padova and Azienda Ospedaliera di Padova, Via G. Orus 2/B, Padua, Italy

    Cinzia Franchin, Luca Cesaro, Silvia Zaramella & Giorgio Arrigoni

  3. CNR Institute of Neurosciences, Via U. Bassi 58/B, Padua, Italy

    Lorenzo A. Pinna

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  1. Cinzia Franchin
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Correspondence to Mauro Salvi, Giorgio Arrigoni or Lorenzo A. Pinna.

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18_2017_2705_MOESM1_ESM.xlsx

Table S1. Table lists all (phospho)peptides identified in the triplex SILAC experiment (both biological replicates) of wild type vs CK2α/α’(-/-) cells. Peptide sequences, confidence level, protein group accession number, modifications, PhosphoRS site probabilities, SILAC ratios, q-values, Mascot scores, expectation values, number of missed cleavages, experimental m/z, Δppm are reported (XLSX 3273 kb)

18_2017_2705_MOESM2_ESM.xlsx

Table S2. Table lists all phosphosites that were reliably quantified in the triplex SILAC experiment (both biological replicates) of wild type vs CK2α/α’(-/-) cells. Peptide sequences, protein group accession number, SILAC ratios, fold change, and protein description are reported (XLSX 69 kb)

18_2017_2705_MOESM3_ESM.xlsx

Table S3. Table lists all (phospho)peptides identified in the triplex SILAC experiment (both biological replicates) of wild type vs CK2β(-/-) cells. Peptide sequences, confidence level, protein group accession number, modifications, PhosphoRS site probabilities, SILAC ratios, q-values, Mascot scores, expectation values, number of missed cleavages, experimental m/z, Δppm are reported (XLSX 2423 kb)

18_2017_2705_MOESM4_ESM.xlsx

Table S4. Table lists all phosphosites that were reliably quantified in the triplex SILAC experiment (both biological replicates) of wild type vs CK2β(-/-) cells. Peptide sequences, protein group accession number, SILAC ratios, fold change, and protein description are reported (XLSX 44 kb)

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Franchin, C., Borgo, C., Cesaro, L. et al. Re-evaluation of protein kinase CK2 pleiotropy: new insights provided by a phosphoproteomics analysis of CK2 knockout cells. Cell. Mol. Life Sci. 75, 2011–2026 (2018). https://doi.org/10.1007/s00018-017-2705-8

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