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  • Review Article
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Mechanisms of specificity in protein phosphorylation

Nature Reviews Molecular Cell Biology volume 8, pages 530–541 (2007)Cite this article

An Erratum to this article was published on 01 August 2007

Key Points

  • Protein phosphorylation is the most common type of post-translational modification, and essentially affects every basic cellular process.

  • A typical protein kinase recognizes between one and a few hundred bona fide phosphorylation sites in a background of ∼700,000 potentially phosphorylatable residues. Protein kinases have evolved a range of mechanisms to ensure specificity in targeting residues for phosphorylation (see below).

  • One mechanism that ensures specific phosphorylation is the depth of the kinase catalytic cleft, which allows kinases to discriminate between tyrosine and serine/threonine residues.

  • A second mechanism involves local interactions near the phosphorylation site, which direct many protein kinases to substrates with particular consensus sequences.

  • A third mechanism for phosphorylation specificity uses docking sites, which are separated from the catalytic site of the kinase and the phosphorylation site of the substrate, and might provide enforced proximity and allosteric regulation of a correct kinase–substrate pair.

  • Another mechanism is localization, which restricts kinases to a subset of substrates and increases local kinase concentrations.

  • A further mechanism to ensure the specific targeting of residues for phosphorylation uses scaffolds, which form dynamic ternary complexes with kinases and substrates and might contribute to kinase specificity in several ways.

  • Competition also ensures that the correct residues are targeted for phosphorylation. This mechanism can suppress the phosphorylation of off-target substrates and can add thresholds and temporal ordering to phosphorylation responses.

  • Finally, there are the mechanisms of multisite phosphorylation and kinetic proofreading, which minimize the consequences of aberrant phosphorylations.

Abstract

A typical protein kinase must recognize between one and a few hundred bona fide phosphorylation sites in a background of ∼700,000 potentially phosphorylatable residues. Multiple mechanisms have evolved that contribute to this exquisite specificity, including the structure of the catalytic site, local and distal interactions between the kinase and substrate, the formation of complexes with scaffolding and adaptor proteins that spatially regulate the kinase, systems-level competition between substrates, and error-correction mechanisms. The responsibility for the recognition of substrates by protein kinases appears to be distributed among a large number of independent, imperfect specificity mechanisms.

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Figure 1: Protein kinases share a common mechanism and fold.
Figure 2: The depth of the catalytic cleft determines phosphorylation site (P-site) amino-acid specificity.
Figure 3: Local interactions are important in establishing specificity.
Figure 4: Distal docking sites have an important role in substrate recognition.
Figure 5: Scaffolds help to provide specificity.
Figure 6: Substrate competition for phosphorylation.

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References

  1. Manning, G., Whyte, D. B., Martinez, R., Hunter, T. & Sudarsanam, S. The protein kinase complement of the human genome. Science 298, 1912–1934 (2002). This study catalogues and classifies the complete complement of protein kinases in the human genome.

    CAS  PubMed  Google Scholar 

  2. Manning, G., Plowman, G. D., Hunter, T. & Sudarsanam, S. Evolution of protein kinase signaling from yeast to man. Trends Biochem. Sci. 27, 514–520 (2002).

    CAS  PubMed  Google Scholar 

  3. Caenepeel, S., Charydczak, G., Sudarsanam, S., Hunter, T. & Manning, G. The mouse kinome: discovery and comparative genomics of all mouse protein kinases. Proc. Natl Acad. Sci. USA 101, 11707–11712 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Zhu, H. et al. Analysis of yeast protein kinases using protein chips. Nature Genet. 26, 283–289 (2000).

    CAS  PubMed  Google Scholar 

  5. Pinna, L. A. & Ruzzene, M. How do protein kinases recognize their substrates? Biochim. Biophys. Acta 1314, 191–225 (1996).

    CAS  PubMed  Google Scholar 

  6. Cohen, P. The regulation of protein function by multisite phosphorylation — a 25 year update. Trends Biochem. Sci. 25, 596–601 (2000).

    CAS  PubMed  Google Scholar 

  7. Echols, N. et al. Comprehensive analysis of amino acid and nucleotide composition in eukaryotic genomes, comparing genes and pseudogenes. Nucleic Acids Res. 30, 2515–2523 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Ptacek, J. et al. Global analysis of protein phosphorylation in yeast. Nature 438, 679–684 (2005). The authors test the ability of 75% of all yeast kinases to phosphorylate 4,400 yeast proteins that were spotted on high-density protein arrays.

    CAS  PubMed  Google Scholar 

  9. Ubersax, J. A. et al. Targets of the cyclin-dependent kinase Cdk1. Nature 425, 859–864 (2003). This study screens a yeast proteomic library for proteins that are directly phosphorylated by Cdk1 in whole-cell extracts and identifies∼200 substrates.

    CAS  PubMed  Google Scholar 

  10. Hanks, S. K., Quinn, A. M. & Hunter, T. The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Science 241, 42–52 (1988).

    CAS  PubMed  Google Scholar 

  11. Hanks, S. K. & Hunter, T. Protein kinases 6. The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification. FASEB J. 9, 576–596 (1995).

    CAS  PubMed  Google Scholar 

  12. Knighton, D. R. et al. Crystal structure of the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase. Science 253, 407–414 (1991). This first crystal structure of the cAMP-dependent protein kinase PKA is the touchstone for all subsequent structural studies of kinases.

    CAS  PubMed  Google Scholar 

  13. Knighton, D. R. et al. 2.0 Å refined crystal structure of the catalytic subunit of cAMP-dependent protein kinase complexed with a peptide inhibitor and detergent. Acta Crystallogr. D 49, 357–361 (1993).

    CAS  PubMed  Google Scholar 

  14. Bossemeyer, D., Engh, R. A., Kinzel, V., Ponstingl, H. & Huber, R. Phosphotransferase and substrate binding mechanism of the cAMP-dependent protein kinase catalytic subunit from porcine heart as deduced from the 2.0 Å structure of the complex with Mn2+ adenylyl imidodiphosphate and inhibitor peptide PKI(5–24). EMBO J. 12, 849–859 (1993).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. De Bondt, H. L. et al. Crystal structure of cyclin-dependent kinase 2. Nature 363, 595–602 (1993).

    CAS  PubMed  Google Scholar 

  16. Zhang, F., Strand, A., Robbins, D., Cobb, M. H. & Goldsmith, E. J. Atomic structure of the MAP kinase ERK2 at 2.3 A resolution. Nature 367, 704–711 (1994).

    CAS  PubMed  Google Scholar 

  17. Jeffrey, P. D. et al. Mechanism of CDK activation revealed by the structure of a cyclin A–CDK2 complex. Nature 376, 313–320 (1995).

    Article  CAS  PubMed  Google Scholar 

  18. Xu, W., Harrison, S. C. & Eck, M. J. Three-dimensional structure of the tyrosine kinase c-Src. Nature 385, 595–602 (1997).

    CAS  PubMed  Google Scholar 

  19. Lowe, E. D. et al. The crystal structure of a phosphorylase kinase peptide substrate complex: kinase substrate recognition. EMBO J. 16, 6646–6658 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Faux, M. C. & Scott, J. D. More on target with protein phosphorylation: conferring specificity by location. Trends Biochem. Sci. 21, 312–315 (1996).

    CAS  PubMed  Google Scholar 

  21. Sim, A. T. & Scott, J. D. Targeting of PKA, PKC and protein phosphatases to cellular microdomains. Cell Calcium 26, 209–217 (1999).

    CAS  PubMed  Google Scholar 

  22. Sharrocks, A. D., Yang, S. H. & Galanis, A. Docking domains and substrate-specificity determination for MAP kinases. Trends Biochem. Sci. 25, 448–453 (2000).

    CAS  PubMed  Google Scholar 

  23. Adams, J. A. Kinetic and catalytic mechanisms of protein kinases. Chem. Rev. 101, 2271–2290 (2001). A terrific, chemically-orientated review that includes a detailed discussion of the kinetic and catalytic mechanisms of protein kinases.

    CAS  PubMed  Google Scholar 

  24. Biondi, R. M. & Nebreda, A. R. Signalling specificity of Ser/Thr protein kinases through docking-site-mediated interactions. Biochem. J. 372, 1–13 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Bhattacharyya, R. P., Remenyi, A., Yeh, B. J. & Lim, W. A. Domains, motifs, and scaffolds: the role of modular interactions in the evolution and wiring of cell signaling circuits. Annu. Rev. Biochem. 75, 655–680 (2006).

    CAS  PubMed  Google Scholar 

  26. Remenyi, A., Good, M. C. & Lim, W. A. Docking interactions in protein kinase and phosphatase networks. Curr. Opin. Struct. Biol. 16, 676–685 (2006).

    CAS  PubMed  Google Scholar 

  27. Shi, Z., Resing, K. A. & Ahn, N. G. Networks for the allosteric control of protein kinases. Curr. Opin. Struct. Biol. 16, 686–692 (2006).

    CAS  PubMed  Google Scholar 

  28. Hubbard, S. R. Crystal structure of the activated insulin receptor tyrosine kinase in complex with peptide substrate and ATP analog. EMBO J. 16, 5572–5581 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Brown, N. R., Noble, M. E., Endicott, J. A. & Johnson, L. N. The structural basis for specificity of substrate and recruitment peptides for cyclin-dependent kinases. Nature Cell Biol. 1, 438–443 (1999).

    CAS  PubMed  Google Scholar 

  30. Jia, Z., Barford, D., Flint, A. J. & Tonks, N. K. Structural basis for phosphotyrosine peptide recognition by protein tyrosine phosphatase 1B. Science 268, 1754–1758 (1995).

    CAS  PubMed  Google Scholar 

  31. Yang, J. et al. Structural basis for substrate specificity of protein-tyrosine phosphatase SHP-1. J. Biol. Chem. 275, 4066–4071 (2000).

    CAS  PubMed  Google Scholar 

  32. Andersen, J. N. et al. Structural and evolutionary relationships among protein tyrosine phosphatase domains. Mol. Cell. Biol 21, 7117–7136 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Kemp, B. E., Graves, D. J., Benjamini, E. & Krebs, E. G. Role of multiple basic residues in determining the substrate specificity of cyclic AMP-dependent protein kinase. J. Biol. Chem. 252, 4888–4894 (1977).

    CAS  PubMed  Google Scholar 

  34. Hunter, T. Synthetic peptide substrates for a tyrosine protein kinase. J. Biol. Chem. 257, 4843–4848 (1982).

    CAS  PubMed  Google Scholar 

  35. Foulkes, J. G., Chow, M., Gorka, C., Frackelton, A. R. Jr & Baltimore, D. Purification and characterization of a protein-tyrosine kinase encoded by the Abelson murine leukemia virus. J. Biol. Chem. 260, 8070–8077 (1985).

    CAS  PubMed  Google Scholar 

  36. Kemp, B. E., Bylund, D. B., Huang, T. S. & Krebs, E. G. Substrate specificity of the cyclic AMP-dependent protein kinase. Proc. Natl Acad. Sci. USA 72, 3448–3452 (1975).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Daile, P., Carnegie, P. R. & Young, J. D. Synthetic substrate for cyclic AMP-dependent protein kinase. Nature 257, 416–418 (1975).

    CAS  PubMed  Google Scholar 

  38. Pearson, R. B. & Kemp, B. E. Protein kinase phosphorylation site sequences and consensus specificity motifs: tabulations. Methods Enzymol. 200, 62–81 (1991).

    CAS  PubMed  Google Scholar 

  39. Songyang, Z. et al. Use of an oriented peptide library to determine the optimal substrates of protein kinases. Curr. Biol. 4, 973–982 (1994). The authors developed a technique for determining the substrate specificity and consensus phosphorylation sites of protein kinases using an orientated library of >2.5 billion peptide substrates.

    CAS  PubMed  Google Scholar 

  40. Hutti, J. E. et al. A rapid method for determining protein kinase phosphorylation specificity. Nature Methods 1, 27–29 (2004).

    CAS  PubMed  Google Scholar 

  41. Olsen, J. V. et al. Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. Cell 127, 635–648 (2006).

    CAS  PubMed  Google Scholar 

  42. Yaffe, M. B. et al. A motif-based profile scanning approach for genome-wide prediction of signaling pathways. Nature Biotech. 19, 348–353 (2001).

    CAS  Google Scholar 

  43. Holmes, J. K. & Solomon, M. J. The role of Thr160 phosphorylation of Cdk2 in substrate recognition. Eur. J. Biochem. 268, 4647–4652 (2001).

    CAS  PubMed  Google Scholar 

  44. Zheng, J. et al. 2.2 Å refined crystal structure of the catalytic subunit of cAMP-dependent protein kinase complexed with MnATP and a peptide inhibitor. Acta Crystallogr. D 49, 362–365 (1993).

    CAS  PubMed  Google Scholar 

  45. Taylor, S. S. et al. Dynamics of signaling by PKA. Biochim. Biophys. Acta 1754, 25–37 (2005).

    CAS  PubMed  Google Scholar 

  46. Holland, P. M. & Cooper, J. A. Protein modification: docking sites for kinases. Curr. Biol. 9, R329–R331 (1999).

    CAS  PubMed  Google Scholar 

  47. Kallunki, T. et al. JNK2 contains a specificity-determining region responsible for efficient c-Jun binding and phosphorylation. Genes Dev. 8, 2996–3007 (1994).

    CAS  PubMed  Google Scholar 

  48. Bardwell, L., Cook, J. G., Chang, E. C., Cairns, B. R. & Thorner, J. Signaling in the yeast pheromone response pathway: specific and high-affinity interaction of the mitogen-activated protein (MAP) kinases Kss1 and Fus3 with the upstream MAP kinase kinase Ste7. Mol. Cell. Biol. 16, 3637–3650 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Bardwell, L. & Thorner, J. A conserved motif at the amino termini of MEKs might mediate high-affinity interaction with the cognate MAPKs. Trends Biochem. Sci. 21, 373–374 (1996).

    CAS  PubMed  Google Scholar 

  50. Williams, D. D., Marin, O., Pinna, L. A. & Proud, C. G. Phosphorylated seryl and threonyl, but not tyrosyl, residues are efficient specificity determinants for GSK-3β and Shaggy. FEBS Lett. 448, 86–90 (1999).

    CAS  PubMed  Google Scholar 

  51. Biondi, R. M. et al. Identification of a pocket in the PDK1 kinase domain that interacts with PIF and the C-terminal residues of PKA. EMBO J. 19, 979–988 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Adams, P. D. et al. Identification of a cyclin–cdk2 recognition motif present in substrates and p21-like cyclin-dependent kinase inhibitors. Mol. Cell. Biol. 16, 6623–6633 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Schulman, B. A., Lindstrom, D. L. & Harlow, E. Substrate recruitment to cyclin-dependent kinase 2 by a multipurpose docking site on cyclin A. Proc. Natl Acad. Sci. USA 96, 10453–10458 (1998).

    Google Scholar 

  54. Chen, Y. G. et al. Determinants of specificity in TGF-β signal transduction. Genes Dev. 12, 2144–2152 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Lo, R. S., Chen, Y. G., Shi, Y., Pavletich, N. P. & Massague, J. The L3 loop: a structural motif determining specific interactions between SMAD proteins and TGF-β receptors. EMBO J. 17, 996–1005 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. Tanoue, T., Adachi, M., Moriguchi, T. & Nishida, E. A conserved docking motif in MAP kinases common to substrates, activators and regulators. Nature Cell Biol. 2, 110–116 (2000).

    CAS  PubMed  Google Scholar 

  57. Gupta, S., Campbell, D., Derijard, B. & Davis, R. J. Transcription factor ATF2 regulation by the JNK signal transduction pathway. Science 267, 389–393 (1995).

    CAS  PubMed  Google Scholar 

  58. Livingstone, C., Patel, G. & Jones, N. ATF-2 contains a phosphorylation-dependent transcriptional activation domain. EMBO J. 14, 1785–1797 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. Yang, S. H., Yates, P. R., Whitmarsh, A. J., Davis, R. J. & Sharrocks, A. D. The Elk-1 ETS-domain transcription factor contains a mitogen-activated protein kinase targeting motif. Mol. Cell. Biol. 18, 710–720 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  60. Yang, S. H., Galanis, A. & Sharrocks, A. D. Targeting of p38 mitogen-activated protein kinases to MEF2 transcription factors. Mol. Cell. Biol. 19, 4028–4038 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  61. Lee, T. et al. Docking motif interactions in MAP kinases revealed by hydrogen exchange mass spectrometry. Mol. Cell 14, 43–55 (2004). This study used hydrogen-exchange mass spectroscopy to identify conformational changes in MAPK on binding of DEF-domain and D-domain peptides.

    CAS  PubMed  Google Scholar 

  62. Remenyi, A., Good, M. C., Bhattacharyya, R. P. & Lim, W. A. The role of docking interactions in mediating signaling input, output, and discrimination in the yeast MAPK network. Mol. Cell 20, 951–962 (2005). Structural analysis of MAPK bound to three different binding partners, which demonstrates how different D-domain peptides can selectively bind different MAPKs through a common docking groove and have different effects on kinase activity.

    CAS  PubMed  Google Scholar 

  63. Jacobs, D., Glossip, D., Xing, H., Muslin, A. J. & Kornfeld, K. Multiple docking sites on substrate proteins form a modular system that mediates recognition by ERK MAP kinase. Genes Dev. 13, 163–175 (1999). This paper identified two important ERK docking sites through the analysis of lin-1 mutations in Caenorhabditis elegans.

    CAS  PubMed  PubMed Central  Google Scholar 

  64. Jacobs, D., Beitel, G. J., Clark, S. G., Horvitz, H. R. & Kornfeld, K. Gain-of-function mutations in the Caenorhabditis elegans lin-1 ETS gene identify a C-terminal regulatory domain phosphorylated by ERK MAP kinase. Genetics 149, 1809–1822 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  65. MacKenzie, S. J., Baillie, G. S., McPhee, I., Bolger, G. B. & Houslay, M. D. ERK2 mitogen-activated protein kinase binding, phosphorylation, and regulation of the PDE4D cAMP-specific phosphodiesterases. The involvement of COOH-terminal docking sites and NH2-terminal UCR regions. J. Biol. Chem. 275, 16609–16617 (2000).

    CAS  PubMed  Google Scholar 

  66. Dimitri, C. A., Dowdle, W., MacKeigan, J. P., Blenis, J. & Murphy, L. O. Spatially separate docking sites on ERK2 regulate distinct signaling events in vivo. Curr. Biol. 15, 1319–1324 (2005).

    CAS  PubMed  Google Scholar 

  67. Hubbard, S. R. & Till, J. H. Protein tyrosine kinase structure and function. Annu. Rev. Biochem. 69, 373–398 (2000).

    CAS  PubMed  Google Scholar 

  68. Deshaies, R. J. & Ferrell, J. E. Jr . Multisite phosphorylation and the countdown to S phase. Cell 107, 819–822 (2001).

    CAS  PubMed  Google Scholar 

  69. Chang, C. I., Xu, B. E., Akella, R., Cobb, M. H. & Goldsmith, E. J. Crystal structures of MAP kinase p38 complexed to the docking sites on its nuclear substrate MEF2A and activator MKK3b. Mol. Cell 9, 1241–1249 (2002).

    CAS  PubMed  Google Scholar 

  70. Heo, Y. S. et al. Structural basis for the selective inhibition of JNK1 by the scaffolding protein JIP1 and SP600125. EMBO J. 23, 2185–2195 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  71. Miller, M. E. & Cross, F. R. Cyclin specificity: how many wheels do you need on a unicycle? J. Cell Sci. 114, 1811–1820 (2001).

    CAS  PubMed  Google Scholar 

  72. Cheng, K. Y. et al. The role of the phospho-CDK2/cyclin A recruitment site in substrate recognition. J. Biol. Chem. 281, 23167–23179 (2006).

    CAS  PubMed  Google Scholar 

  73. Archambault, V., Buchler, N. E., Wilmes, G. M., Jacobson, M. D. & Cross, F. R. Two-faced cyclins with eyes on the targets. Cell Cycle 4, 125–130 (2005).

    CAS  PubMed  Google Scholar 

  74. Cross, F. R., Yuste-Rojas, M., Gray, S. & Jacobson, M. D. Specialization and targeting of B-type cyclins. Mol. Cell 4, 11–19 (1999).

    CAS  PubMed  Google Scholar 

  75. Wilmes, G. M. et al. Interaction of the S-phase cyclin Clb5 with an 'RXL' docking sequence in the initiator protein Orc6 provides an origin-localized replication control switch. Genes Dev. 18, 981–991 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  76. Takeda, D. Y., Wohlschlegel, J. A. & Dutta, A. A bipartite substrate recognition motif for cyclin-dependent kinases. J. Biol. Chem. 276, 1993–1997 (2001).

    CAS  PubMed  Google Scholar 

  77. Arvai, A. S., Bourne, Y., Hickey, M. J. & Tainer, J. A. Crystal structure of the human cell cycle protein CksHs1: single domain fold with similarity to kinase N-lobe domain. J. Mol. Biol. 249, 835–842 (1995).

    CAS  PubMed  Google Scholar 

  78. Bourne, Y. et al. Crystal structure and mutational analysis of the human CDK2 kinase complex with cell cycle-regulatory protein CksHs1. Cell 84, 863–874 (1996).

    CAS  PubMed  Google Scholar 

  79. Patra, D. & Dunphy, W. G. Xe-p9, a Xenopus Suc1/Cks protein, is essential for the Cdc2-dependent phosphorylation of the anaphase-promoting complex at mitosis. Genes Dev. 12, 2549–2559 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  80. Tang, Y. & Reed, S. I. The Cdk-associated protein Cks1 functions both in G1 and G2 in Saccharomyces cerevisiae. Genes Dev. 7, 822–832 (1993).

    CAS  PubMed  Google Scholar 

  81. Elia, A. E., Cantley, L. C. & Yaffe, M. B. Proteomic screen finds pSer/pThr-binding domain localizing Plk1 to mitotic substrates. Science 299, 1228–1231 (2003).

    CAS  PubMed  Google Scholar 

  82. Elia, A. E. et al. The molecular basis for phosphodependent substrate targeting and regulation of Plks by the polo-box domain. Cell 115, 83–95 (2003). References 81 and 82 discover and explain the binding of polo-box domains to phosphorylated peptides.

    CAS  PubMed  Google Scholar 

  83. Fiol, C. J., Mahrenholz, A. M., Wang, Y., Roeske, R. W. & Roach, P. J. Formation of protein kinase recognition sites by covalent modification of the substrate. Molecular mechanism for the synergistic action of casein kinase II and glycogen synthase kinase 3. J. Biol. Chem. 262, 14042–14048 (1987).

    CAS  PubMed  Google Scholar 

  84. Dajani, R. et al. Crystal structure of glycogen synthase kinase 3β: structural basis for phosphate-primed substrate specificity and autoinhibition. Cell 105, 721–732 (2001).

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  86. ter Haar, E. et al. Structure of GSK3β reveals a primed phosphorylation mechanism. Nature Struct. Biol. 8, 593–596 (2001).

    CAS  PubMed  Google Scholar 

  87. Frodin, M., Jensen, C. J., Merienne, K. & Gammeltoft, S. A phosphoserine-regulated docking site in the protein kinase RSK2 that recruits and activates PDK1. EMBO J. 19, 2924–2934 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  88. Biondi, R. M. et al. High resolution crystal structure of the human PDK1 catalytic domain defines the regulatory phosphopeptide docking site. EMBO J. 21, 4219–4228 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  89. Jackman, M., Firth, M. & Pines, J. Human cyclins B1 and B2 are localized to strikingly different structures: B1 to microtubules, B2 primarily to the Golgi apparatus. EMBO J. 14, 1646–1654 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  90. Hagting, A., Jackman, M., Simpson, K. & Pines, J. Translocation of cyclin B1 to the nucleus at prophase requires a phosphorylation-dependent nuclear import signal. Curr. Biol. 9, 680–689 (1999).

    CAS  PubMed  Google Scholar 

  91. Jackman, M., Lindon, C., Nigg, E. A. & Pines, J. Active cyclin B1–Cdk1 first appears on centrosomes in prophase. Nature Cell Biol. 5, 143–148 (2003).

    CAS  PubMed  Google Scholar 

  92. Draviam, V. M., Orrechia, S., Lowe, M., Pardi, R. & Pines, J. The localization of human cyclins B1 and B2 determines CDK1 substrate specificity and neither enzyme requires MEK to disassemble the Golgi apparatus. J. Cell Biol. 152, 945–958 (2001). References 89–92 provide compelling evidence for the importance of localization in CDK function.

    CAS  PubMed  PubMed Central  Google Scholar 

  93. Moore, J. D., Kirk, J. A. & Hunt, T. Unmasking the S-phase-promoting potential of cyclin B1. Science 300, 987–990 (2003).

    CAS  PubMed  Google Scholar 

  94. Miller, M. E. & Cross, F. R. Distinct subcellular localization patterns contribute to functional specificity of the Cln2 and Cln3 cyclins of Saccharomyces cerevisiae. Mol. Cell. Biol. 20, 542–555 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  95. Vaudry, D., Stork, P. J., Lazarovici, P. & Eiden, L. E. Signaling pathways for PC12 cell differentiation: making the right connections. Science 296, 1648–1649 (2002).

    CAS  PubMed  Google Scholar 

  96. Robinson, M. J., Stippec, S. A., Goldsmith, E., White, M. A. & Cobb, M. H. A constitutively active and nuclear form of the MAP kinase ERK2 is sufficient for neurite outgrowth and cell transformation. Curr. Biol. 8, 1141–1150 (1998).

    CAS  PubMed  Google Scholar 

  97. Disatnik, M. H., Buraggi, G. & Mochly-Rosen, D. Localization of protein kinase C isozymes in cardiac myocytes. Exp. Cell Res. 210, 287–297 (1994).

    CAS  PubMed  Google Scholar 

  98. Mochly-Rosen, D., Khaner, H. & Lopez, J. Identification of intracellular receptor proteins for activated protein kinase C. Proc. Natl Acad. Sci. USA 88, 3997–4000 (1991).

    CAS  PubMed  PubMed Central  Google Scholar 

  99. Wong, W. & Scott, J. D. AKAP signalling complexes: focal points in space and time. Nature Rev. Mol. Cell Biol. 5, 959–970 (2004).

    CAS  Google Scholar 

  100. Brown, G. C. & Kholodenko, B. N. Spatial gradients of cellular phospho-proteins. FEBS Lett. 457, 452–454 (1999).

    CAS  PubMed  Google Scholar 

  101. Pawson, T. & Scott, J. D. Signaling through scaffold, anchoring, and adaptor proteins. Science 278, 2075–2080 (1997).

    CAS  PubMed  Google Scholar 

  102. van Drogen, F., Stucke, V. M., Jorritsma, G. & Peter, M. MAP kinase dynamics in response to pheromones in budding yeast. Nature Cell Biol. 3, 1051–1059 (2001).

    CAS  PubMed  Google Scholar 

  103. Schwartz, M. A. & Madhani, H. D. Principles of MAP kinase signaling specificity in Saccharomyces cerevisiae. Annu. Rev. Genet. 38, 725–748 (2004).

    CAS  PubMed  Google Scholar 

  104. Park, S. H., Zarrinpar, A. & Lim, W. A. Rewiring MAP kinase pathways using alternative scaffold assembly mechanisms. Science 299, 1061–1064 (2003). This study demonstrates that the scaffold Ste5 tethers kinase–substrate pairs in space, and that changing the proteins to which Ste5 binds can produce non-natural input–output properties.

    CAS  PubMed  Google Scholar 

  105. Howard, P. L., Chia, M. C., Del Rizzo, S., Liu, F. F. & Pawson, T. Redirecting tyrosine kinase signaling to an apoptotic caspase pathway through chimeric adaptor proteins. Proc. Natl Acad. Sci. USA 100, 11267–11272 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  106. Feliciello, A., Gottesman, M. E. & Avvedimento, E. V. The biological functions of A-kinase anchor proteins. J. Mol. Biol. 308, 99–114 (2001).

    CAS  PubMed  Google Scholar 

  107. Sierralta, J. & Mendoza, C. PDZ-containing proteins: alternative splicing as a source of functional diversity. Brain Res. Brain Res. Rev. 47, 105–115 (2004).

    CAS  PubMed  Google Scholar 

  108. Muller, J., Cacace, A. M., Lyons, W. E., McGill, C. B. & Morrison, D. K. Identification of B-KSR1, a novel brain-specific isoform of KSR1 that functions in neuronal signaling. Mol. Cell. Biol. 20, 5529–5539 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  109. Bhattacharyya, R. P. et al. The Ste5 scaffold allosterically modulates signaling output of the yeast mating pathway. Science 311, 822–826 (2006). This study demonstrates that scaffolds are not passive participants in signalling cascades but can allosterically activate binding partners.

    CAS  PubMed  Google Scholar 

  110. Coghlan, V. M. et al. Association of protein kinase A and protein phosphatase 2B with a common anchoring protein. Science 267, 108–111 (1995).

    CAS  PubMed  Google Scholar 

  111. Klauck, T. M. et al. Coordination of three signaling enzymes by AKAP79, a mammalian scaffold protein. Science 271, 1589–1592 (1996).

    CAS  PubMed  Google Scholar 

  112. Dodge, K. L. et al. mAKAP assembles a protein kinase A/PDE4 phosphodiesterase cAMP signaling module. EMBO J. 20, 1921–1930 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  113. Whitmarsh, A. J., Cavanagh, J., Tournier, C., Yasuda, J. & Davis, R. J. A mammalian scaffold complex that selectively mediates MAP kinase activation. Science 281, 1671–1674 (1998).

    CAS  PubMed  Google Scholar 

  114. Yasuda, J., Whitmarsh, A. J., Cavanagh, J., Sharma, M. & Davis, R. J. The JIP group of mitogen-activated protein kinase scaffold proteins. Mol. Cell. Biol. 19, 7245–7254 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  115. Willoughby, E. A., Perkins, G. R., Collins, M. K. & Whitmarsh, A. J. The JNK-interacting protein-1 scaffold protein targets MAPK phosphatase-7 to dephosphorylate JNK. J. Biol. Chem. 278, 10731–10736 (2003).

    CAS  PubMed  Google Scholar 

  116. Willoughby, E. A. & Collins, M. K. Dynamic interaction between the dual specificity phosphatase MKP7 and the JNK3 scaffold protein β-arrestin 2. J. Biol. Chem. 280, 25651–25658 (2005).

    CAS  PubMed  Google Scholar 

  117. Loog, M. & Morgan, D. O. Cyclin specificity in the phosphorylation of cyclin-dependent kinase substrates. Nature 434, 104–108 (2005). This study shows how the intrinsic biochemical properties of different cyclins help to promote the correct timing of CDK substrate phosphorylation during the cell cycle.

    CAS  PubMed  Google Scholar 

  118. Kim, S. Y. & Ferrell, J. E. Jr . Substrate competition as a source of ultrasensitivity in the inactivation of Wee1. Cell 128, 1133–1145 (2007).

    CAS  PubMed  Google Scholar 

  119. Hopfield, J. J. Kinetic proofreading: a new mechanism for reducing errors in biosynthetic processes requiring high specificity. Proc. Natl Acad. Sci. USA 71, 4135–4139 (1974). This important study describes how kinetic proofreading, driven by phosphate hydrolysis, can increase the specificity of biological processes beyond the level available from free-energy differences in intermediates or kinetic barriers.

    CAS  PubMed  PubMed Central  Google Scholar 

  120. Swain, P. S. & Siggia, E. D. The role of proofreading in signal transduction specificity. Biophys. J. 82, 2928–2933 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  121. Ferrell, J. E. Jr . & Bhatt, R. R. Mechanistic studies of the dual phosphorylation of mitogen-activated protein kinase. J. Biol. Chem. 272, 19008–19016 (1997).

    CAS  PubMed  Google Scholar 

  122. Breitkreutz, A., Boucher, L. & Tyers, M. MAPK specificity in the yeast pheromone response independent of transcriptional activation. Curr. Biol. 11, 1266–1271 (2001).

    CAS  PubMed  Google Scholar 

  123. Sabbagh, W. Jr, Flatauer, L. J., Bardwell, A. J. & Bardwell, L. Specificity of MAP kinase signaling in yeast differentiation involves transient versus sustained MAPK activation. Mol. Cell 8, 683–691 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  124. Bao, M. Z., Schwartz, M. A., Cantin, G. T., Yates, J. R. & Madhani, H. D. Pheromone-dependent destruction of the Tec1 transcription factor is required for MAP kinase signaling specificity in yeast. Cell 119, 991–1000 (2004).

    CAS  PubMed  Google Scholar 

  125. Chou, S., Huang, L. & Liu, H. Fus3-regulated Tec1 degradation through SCFCdc4 determines MAPK signaling specificity during mating in yeast. Cell 119, 981–990 (2004).

    CAS  PubMed  Google Scholar 

  126. McClean, M. N., Mody, A., Broach, J. R. & Ramanathan, S. Cross-talk and decision making in MAP kinase pathways. Nature Genet. 39, 409–414 (2007). References 124–126 demonstrate that input signals will sometimes leak along pathways with shared signalling components; the studies also identify additional mechanisms that ensure that these leaks do not lead to aberrant outputs.

    CAS  PubMed  Google Scholar 

  127. Pinna, L. A. & Donella-Deana, A. Phosphorylated synthetic peptides as tools for studying protein phosphatases. Biochim. Biophys. Acta 1222, 415–431 (1994).

    CAS  PubMed  Google Scholar 

  128. Hunter, T. & Sefton, B. M. Transforming gene product of Rous sarcoma virus phosphorylates tyrosine. Proc. Natl Acad. Sci. USA 77, 1311–1315 (1980).

    CAS  PubMed  PubMed Central  Google Scholar 

  129. Alonso, A. et al. Protein tyrosine phosphatases in the human genome. Cell 117, 699–711 (2004).

    CAS  PubMed  Google Scholar 

  130. Arena, S., Benvenuti, S. & Bardelli, A. Genetic analysis of the kinome and phosphatome in cancer. Cell Mol. Life Sci. 62, 2092–2099 (2005).

    CAS  PubMed  Google Scholar 

  131. Moorhead, G. B., Trinkle-Mulcahy, L. & Ulke-Lemee, A. Emerging roles of nuclear protein phosphatases. Nature Rev. Mol. Cell Biol. 8, 234–244 (2007).

    CAS  Google Scholar 

  132. Cohen, P. T. Protein phosphatase 1 — targeted in many directions. J. Cell Sci. 115, 241–256 (2002).

    CAS  PubMed  Google Scholar 

  133. Gray, C. H., Good, V. M., Tonks, N. K. & Barford, D. The structure of the cell cycle protein Cdc14 reveals a proline-directed protein phosphatase. EMBO J. 22, 3524–3535 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  134. DeLano, W. L. The PyMOL molecular graphics system [online] (2002).

    Google Scholar 

  135. Xu, W., Doshi, A., Lei, M., Eck, M. J. & Harrison, S. C. Crystal structures of c-Src reveal features of its autoinhibitory mechanism. Mol. Cell 3, 629–638 (1999).

    CAS  PubMed  Google Scholar 

  136. Songyang, Z. et al. A structural basis for substrate specificities of protein Ser/Thr kinases: primary sequence preference of casein kinases I and II, NIMA, phosphorylase kinase, calmodulin-dependent kinase II, CDK5, and Erk1. Mol. Cell. Biol. 16, 6486–6493 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  137. Flotow, H. et al. Phosphate groups as substrate determinants for casein kinase I action. J. Biol. Chem. 265, 14264–14269 (1990).

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  139. Fiol, C. J., Wang, A., Roeske, R. W. & Roach, P. J. Ordered multisite protein phosphorylation. Analysis of glycogen synthase kinase 3 action using model peptide substrates. J. Biol. Chem. 265, 6061–6065 (1990).

    CAS  PubMed  Google Scholar 

  140. Till, J. H., Chan, P. M. & Miller, W. T. Engineering the substrate specificity of the Abl tyrosine kinase. J. Biol. Chem. 274, 4995–5003 (1999).

    CAS  PubMed  Google Scholar 

  141. Songyang, Z. et al. Catalytic specificity of protein-tyrosine kinases is critical for selective signalling. Nature 373, 536–539 (1995).

    CAS  PubMed  Google Scholar 

  142. Obata, T. et al. Peptide and protein library screening defines optimal substrate motifs for AKT/PKB. J. Biol. Chem. 275, 36108–36115 (2000).

    CAS  PubMed  Google Scholar 

  143. Friedmann, M., Nissen, M. S., Hoover, D. S., Reeves, R. & Magnuson, N. S. Characterization of the proto-oncogene pim-1: kinase activity and substrate recognition sequence. Arch. Biochem. Biophys. 298, 594–601 (1992).

    CAS  PubMed  Google Scholar 

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Acknowledgements

We thank S. Pearlman and Z. Serber for bioinformatics help; Z.S., N. Breaux and members of the Ferrell laboratory for critical comments on the manuscript; and M. Laub, D. Morgan, S. Taylor and J. Thorner for discussions. We apologize to those whose work we could not discuss owing to space and reference limits. Our work in this area is supported by a grant from the National Institutes of Health and a Helen Hay Whitney Postdoctoral Fellowship (to J.A.U.).

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  1. Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, 94305-5174, California, USA

    Jeffrey A. Ubersax & James E. Ferrell Jr

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Correspondence to Jeffrey A. Ubersax.

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DATABASES

Protein Data Bank

1ATP

1IR3

1IRK

1QMZ

2B9J

2CCI

2PHK

FURTHER INFORMATION

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Glossary

Phosphorylation site

(P-site). By convention, residues that are situated N-terminally of the P-site residue are numbered P–1, P–2, P–3 and so on, whereas residues that are situated C-terminally of the P-site are numbered P+1, P+2, P+3 and so on.

Mitogen-activated protein kinase

(MAPK). A member of a family of protein kinases that are activated in response to diverse mitogens, stresses and developmental signals. MAPKs are the terminal components of three-kinase cascades.

Cyclin-dependent kinase

(CDK). A Ser/Thr-specific kinase that depends on the binding of a cyclin for full activity. CDKs are essential for cell-cycle progression.

Insulin receptor

The heteromeric tyrosine kinase receptor for the anabolic hormone insulin.

Allostery

The regulation of protein activity through phosphorylation, or through the binding of a small molecule or protein, at a site distinct from the active site. Communication between the allosteric site and the active site usually occurs through a conformational change.

cAMP-dependent protein kinase

A Ser/Thr-specific protein kinase that is activated by the cAMP-induced dissociation of a regulatory subunit.

Edman degradation

A method of sequencing proteins in which the N-terminal residue is chemically labelled, cleaved from the peptide and then identified chromatographically. The process can be repeated to obtain the sequence of the first ∼10–50 amino acids in the protein or peptide.

Glycogen synthase kinase-3

A Ser/Thr kinase that is important for insulin and Wnt signalling. It was initially identified in studies of metabolic regulation and also has roles in development.

D domain

A distal docking site that is ubiquitous in mitogen-activated protein kinase substrates. The sequence of the D domain usually conforms to an (R/K)1–2-(X)2–6-Φ-X-Φ pattern, where Φ is a hydrophobic residue.

DEF domain

A distal docking site that is located ten amino acids downstream of the phosphorylation site, and is found in many, but not all, mitogen-activated protein kinase substrates.

Src

A non-receptor Tyr kinase proto-oncogene product. Src is normally kept inactive by intramolecular interactions between its kinase domain and its SH2 and SH3 domains, and can be activated by interaction with other SH2- and SH3-domain-binding proteins.

RXL motif

A distal docking site that is found in substrates of cyclin-dependent kinases (CDKs). The RXL motif interacts with the hydrophobic patch that is found on the cyclin partner of the CDK.

Polo-like kinase

A conserved Ser/Thr kinase that is involved in mitotic progression. Polo-like kinases are activated by binding to peptide epitopes (often phosphoepitopes).

Protein kinase C

(PKC). Classical PKC isoforms are activated by the presence of two second messengers: membrane-associated diacylglycerol and cytosolic calcium.

AND gate

A basic logic circuit in which two inputs together yield a high output, but either input alone yields no output.

Ultrasensitive response

A response to an increasing stimulus that is described by a sigmoidal dose-response curve. Low levels of stimulus produce a poor response but, as the stimulus level increases, there is an abrupt increase in the response to near-maximal levels.

Steady state

A condition that is reached when the concentrations of reactants and products in a complex system do not change with time.

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Ubersax, J., Ferrell Jr, J. Mechanisms of specificity in protein phosphorylation. Nat Rev Mol Cell Biol 8, 530–541 (2007). https://doi.org/10.1038/nrm2203

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