Prevalence and significance of the commonest phosphorylated motifs in the human proteome: a global analysis


Protein phosphorylation is the most frequent post-translational modification by which the properties of eukaryotic proteins can be reversibly modified. In humans, over 500 protein kinases generate a huge phosphoproteome including more than 200,000 individual phosphosites, a figure which is still continuously increasing. The in vivo selectivity of protein kinases is the outcome of a multifaceted and finely tuned process where numerous factors play an integrated role. To gain information about the actual contribution to this process of local features that reflect the interaction of the protein targets with the catalytic site of the kinases, the prevalence of the commonest motifs determining the consensus sequence of Ser/Thr-specific kinases has been examined in the whole human phosphoproteome and in the phosphoproteomes generated by a panel of the 47 most pleiotropic protein kinases. Our analysis shows that: (1) most phosphosites do conform to at least one of the motifs considered, with a substantial proportion conforming to two or more of them; (2) some motifs, with special reference to the one recognized by protein kinase CK2 (pS/pT-x-x-E/D) are very promiscuous, being abundantly represented also at the phosphosites of all the other protein kinases considered; (3) by contrast, other phosphorylated motifs, notably pS/pT-P, pS/pT-Q and pS-x-E, are more discriminatory and selective, being nearly absent in the phosphosites that are not attributable to certain categories of kinases. The information provided will prove helpful to make reliable inferences based on the manual inspection of individual phosphosites.

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  1. 1.

    Kennelly PJ, Krebs EG (1991) Consensus sequences as substrate specificity determinants for protein kinases and protein phosphatases. J Biol Chem 266:15555–15558

    CAS  PubMed  Google Scholar 

  2. 2.

    Kemp BE, Pearson RB (1990) Protein kinase recognition sequence motifs. Trends Biochem Sci 15:342–346

    CAS  Article  Google Scholar 

  3. 3.

    Pinna LA, Ruzzene M (1996) How do protein kinases recognize their substrates? Biochim Biophys Acta 1314:191–255

    CAS  Article  Google Scholar 

  4. 4.

    Manning G, Whyte DB, Martinez R, Hunter T, Sudarsanam S (2002) The protein kinase complement of the human genome. Science 298:1912–1934.

    CAS  Article  PubMed  Google Scholar 

  5. 5.

    Ubersax JA, Ferrell JE Jr (2007) Mechanisms of specificity in protein phosphorylation. Nat Rev Mol Cell Biol 8:530–541.

    CAS  Article  PubMed  Google Scholar 

  6. 6.

    Yaffe MB, Leparc GG, Lai J, Obata T, Volinia S, Cantley LC (2001) A motif-based profile scanning approach for genome-wide prediction of signaling pathways. Nat Biotechnol 19:348–353.

    CAS  Article  PubMed  Google Scholar 

  7. 7.

    Obenauer JC, Cantley LC, Yaffe MB (2003) Scansite 2.0: Proteome-wide prediction of cell signaling interactions using short sequence motifs. Nucleic Acids Res 31:3635–3641

    CAS  Article  Google Scholar 

  8. 8.

    Blom N, Sicheritz-Pontén T, Gupta R, Gammeltoft S, Brunak S (2004) Prediction of post-translational glycosylation and phosphorylation of proteins from the amino acid sequence. Proteomics 4:1633–1649.

    CAS  Article  PubMed  Google Scholar 

  9. 9.

    Huang HD, Lee TY, Tzeng SW, Horng JT (2005) KinasePhos: a web tool for identifying protein kinase-specific phosphorylation sites. Nucleic Acids Res 33:W226–W229.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Kobe B, Kampmann T, Forwood JK, Listwan P, Brinkworth RI (2005) Substrate specificity of protein kinases and computational prediction of substrates. Biochim Biophys Acta 1754:200–209.

    CAS  Article  PubMed  Google Scholar 

  11. 11.

    Hornbeck PV, Zhang B, Murray B, Kornhauser JM, Latham V, Skrzypek E (2015) PhosphoSitePlus, 2014: mutations, PTMs and recalibrations. Nucleic Acids Res 43:D512–D520.

    CAS  Article  PubMed  Google Scholar 

  12. 12.

    Crooks GE, Hon G, Chandonia JM, Brenner SE (2004) WebLogo: a sequence logo generator. Genome Res 14:1188–1190.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  13. 13.

    Schneider TD, Stephens RM (1990) Sequence logos: a new way to display consensus sequences. Nucleic Acids Res 18:6097–6100

    CAS  Article  Google Scholar 

  14. 14.

    Vacic V, Iakoucheva LM, Radivojac P (2006) Two sample logo: a graphical representation of the differences between two sets of sequence alignments. Bioinformatics 22:1536–1537

    CAS  Article  Google Scholar 

  15. 15.

    Tagliabracci VS, Wiley SE, Guo X, Kinch LN, Durrant E, Wen J, Xiao J, Cui J, Nguyen KB, Engel JL, Coon JJ, Grishin N, Pinna LA, Pagliarini DJ, Dixon JE (2015) A single kinase generates the majority of the secreted phosphoproteome. Cell 161:1619–1632.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  16. 16.

    Chen C, Ha BH, Thévenin AF, Lou HJ, Zhang R, Yip KY, Peterson JR, Gerstein M, Kim PM, Filippakopoulos P, Knapp S, Boggon TJ, Turk BE (2014) Identification of a major determinant for serine-threonine kinase phosphoacceptor specificity. Mol Cell 53:140–147.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  17. 17.

    Venerando A, Ruzzene M, Pinna LA (2014) Casein kinase: the triple meaning of a misnomer. Biochem J 460:141–156.

    CAS  Article  PubMed  Google Scholar 

  18. 18.

    Meggio F, Pinna LA (2003) One-thousand-and-one substrates of protein kinase CK2? FASEB J 17:349–368.

    CAS  Article  PubMed  Google Scholar 

  19. 19.

    Franchin C, Borgo C, Cesaro L, Zaramella S, Vilardell J, Salvi M, Arrigoni G, Pinna LA (2017) 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.

    CAS  Article  PubMed  Google Scholar 

  20. 20.

    Roach PJ (1991) Multisite and hierarchal protein phosphorylation. J Biol Chem 266:14139–14142

    CAS  PubMed  Google Scholar 

  21. 21.

    Marin O, Bustos VH, Cesaro L, Meggio F, Pagano MA, Antonelli M, Allende CC, Pinna LA, Allende JE (2003) A noncanonical sequence phosphorylated by casein kinase 1 in beta-catenin may play a role in casein kinase 1 targeting of important signaling proteins. Proc Natl Acad Sci USA 100:10193–10200

    CAS  Article  Google Scholar 

  22. 22.

    Skurat AV, Roach PJ (1995) Phosphorylation of sites 3a and 3b (Ser640 and Ser644) in the control of rabbit muscle glycogen synthase. J Biol Chem 270:12491–12497

    CAS  Article  Google Scholar 

  23. 23.

    Frame S, Cohen P, Biondi RM (2001) A common phosphate binding site explains the unique substrate specificity of GSK3 and its inactivation by phosphorylation. Mol Cell 7:1321–1327

    CAS  Article  Google Scholar 

  24. 24.

    Cesaro L, Pinna LA (2015) The generation of phosphoserine stretches in phosphoproteins: mechanism and significance. Mol Biosyst 11:2666–2679.

    CAS  Article  PubMed  Google Scholar 

  25. 25.

    Songyang Z, Blechner S, Hoagland N, Hoekstra MF, Piwnica-Worms H, Cantley LC (1994) Use of an oriented peptide library to determine the optimal substrates of protein kinases. Curr Biol 4:973–982

    CAS  Article  Google Scholar 

  26. 26.

    Sarno S, Vaglio P, Meggio F, Issinger OG, Pinna LA (1996) Protein kinase CK2 mutants defective in substrate recognition. Purification and kinetic analysis. J Biol Chem 271:10595–10601

    CAS  Article  Google Scholar 

  27. 27.

    Sarno S, Vaglio P, Marin O, Issinger OG, Ruffato K, Pinna LA (1997) Mutational analysis of residues implicated in the interaction between protein kinase CK2 and peptide substrates. Biochemistry 36:11717–11724

    CAS  Article  Google Scholar 

  28. 28.

    Kim ST, Lim DS, Canman CE, Kastan MB (1999) Substrate specificities and identification of putative substrates of ATM kinase family members. J Biol Chem 274:37538–37543

    CAS  Article  Google Scholar 

  29. 29.

    Leighton IA, Dalby KN, Caudwell FB, Cohen PTW, Cohen P (1995) Comparison of the specificities of p70 S6 kinase and MAPKAP kinase-1 identifies a relatively specific substrate for p70 S6 kinase: the N-terminal kinase domain of MAPKAP kinase-1 is essential for peptide phosphorylation. FEBS Lett 375:289–293.

    CAS  Article  PubMed  Google Scholar 

  30. 30.

    Perich JW, Meggio F, Reynolds EC, Marin O, Pinna LA (1992) Role of Phosphorylated aminoacyl residues in generating atypical consensus sequences which are recognized by casein kinase-2 but not by casein kinase-1. Biochemistry 31:5893–5897.

    CAS  Article  PubMed  Google Scholar 

  31. 31.

    Lasa-Benito M, Marin O, Meggio F, Pinna LA (1996) Golgi apparatus mammary gland casein kinase: monitoring by a specific peptide substrate and definition of specificity determinants. FEBS Lett 382:149–152

    CAS  Article  Google Scholar 

  32. 32.

    Tagliabracci VS, Engel JL, Wen J, Wiley SE, Worby CA, Kinch LN, Xiao J, Grishin NV, Dixon JE (2012) Secreted kinase phosphorylates extracellular proteins that regulate biomineralization. Science 336:1150–1153.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Salvi M, Trashi E, Marin O, Negro A, Sarno S, Pinna LA (2012) Superiority of PLK-2 as α-synuclein phosphorylating agent relies on unique specificity determinants. Biochem Biophys Res Commun 418:156–160.

    CAS  Article  PubMed  Google Scholar 

  34. 34.

    Salvi M, Trashi E, Cozza G, Franchin C, Arrigoni G, Pinna LA (2012) Investigation on PLK2 and PLK3 substrate recognition. Biochim Biophys Acta 1824:1366–1373.

    CAS  Article  PubMed  Google Scholar 

  35. 35.

    Onorato JJ, Palczewski K, Regan JW, Caron MG, Lefkowitz RJ, Benovic JL (1991) Role of acidic amino acids in peptide substrates of the beta-adrenergic receptor kinase and rhodopsin kinase. Biochemistry 30:5118–5125

    CAS  Article  Google Scholar 

  36. 36.

    Chartier M, Chénard T, Barker J, Najmanovich R (2013) Kinome render: a stand-alone and web-accessible tool to annotate the human protein kinome tree. PeerJ 1:e126.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

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This work was supported by a grant from AIRC (Italian Association for Cancer Research), Project IG 18756.

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Correspondence to Lorenzo A. Pinna.

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Supplementary file1 Table S1. Summary of the number of phospho-sites notoriously generated by individual protein kinases (drawn from PhosphoSitePlus) denoting how many of these conform to each of the commonest phosphomotifs. (XLS 235 kb)


Supplementary file2 Table S2. Phosphosites generated by GSK3 conforming only to the motifs pS/pT-P and pS/pT-x-x-x-pS/pT, or to both or to neither of these are separately listed. Constructed with data from PhosphoSitePlus. Phosphosites generated by both isoforms of GSK3 have been collectively considered. Lower case letters denote phosphorylated residues. (XLS 38 kb)


Supplementary file3 Table S3. Classification of the 47 most pleiotropic protein kinases according to the presence in their phosphosites of basic, acidic, phosphorylated, pS/pT-P and pS/pT-Q signatures. (DOCX 13 kb)

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Cesaro, L., Pinna, L.A. Prevalence and significance of the commonest phosphorylated motifs in the human proteome: a global analysis. Cell. Mol. Life Sci. 77, 5281–5298 (2020).

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  • Consensus sequences
  • Specificity determinants
  • Protein kinase specificity
  • Phosphosites
  • CK2
  • GSK3