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.
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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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.).
The authors declare no competing financial interests.
- 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.
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.
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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