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A more quantitative view of kinase specificity suggests a
A more quantitative view of kinase specificity suggests a continuum of phosphorylation rates for the various substrates of a particular kinase (Figure 4A). Such differences in ‘substrate quality’ can arise from variations in phosphorylation site or docking sequences and may explain why the timing or sensitivity to perturbation can vary among substrates of the same kinase. This concept was illustrated recently for substrates of the mechanistic target of rapamycin (mTOR). mTOR partitions into two distinct complexes, mTORC1 and mTORC2, which, despite sharing a catalytic subunit, appear to have no common substrates. Strict substrate selection is enforced by direct interactions with unique adaptor protein components of the two complexes (raptor and mSin1, respectively) 67, 68. Among these substrates, the phosphorylation site sequence appears to dictate the phosphorylation rate [69]. Interestingly, ‘low-quality’ mTORC1 phosphorylation sites were found to be sensitive to the inhibitor rapamycin (Figure 4B). By contrast, ‘high-quality’ sites were resistant to the drug, presumably because they require only a low level of kinase activity to become fully phosphorylated. These observations rationalized a long-standing mystery as to why rapamycin only blocks phosphorylation of a subset of mTORC1 substrates. Substrate quality also dictated the sensitivity of sites to nutrient withdrawal, indicating that the substrate repertoire of a kinase can be controlled by the strength of its activating signal. Such a mechanism could explain how differential cellular responses are achieved by various levels of activation of a master kinase. Similar concepts may explain the timing of CDK substrate phosphorylation during the eukaryotic cell division cycle, which is important for the proper ordering of DNA replication and mitosis. Oscillating levels of various cyclin proteins generate a series of temporally distinct CDK–cyclin complexes, each responsible for phosphorylating key substrates at various phases of the calcium ionophore [70]. Classically cyclins act as both CDK activators and substrate adaptors, potentially explaining why different proteins are phosphorylated at different points within the cell cycle. Indeed, some cyclins directly interact with substrates through distal docking motifs and even residues close to the phosphorylation site 71, 72, 73. Overall CDK activity rises as cells transit the cell cycle due to degradation of CDK inhibitor proteins, as well as the intrinsically higher catalytic activity of late phase cyclin–CDK complexes (Figure 4C). Interestingly, the best characterized CDK substrate docking motifs are targeted by early (G1/S) phase cyclins [73]. Studies in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe suggest that the presence of these motifs on G1/S substrates compensates for the limited CDK activity in early phases of the cell cycle, allowing for efficient phosphorylation 74, 75. By contrast, ‘late’ (G2/M) CDK substrates lack these motifs, are less efficient substrates, and require the higher levels of CDK activity found late in the cell cycle. Accordingly, mutation of substrate docking sites delayed phosphorylation of early substrates [74]. As seen for ‘high-quality’ mTOR substrates, early substrates were also less sensitive than late ones to CDK inhibition in S. pombe[74]. Strikingly, selectively blocking phosphorylation of late substrates by partly inhibiting CDK activity in G2 phase led to reordering of the cell cycle, supporting a model where cell cycle progression is driven by increasing CDK activity (Figure 4C). While the timing of CDK substrate phosphorylation appears to be mediated by the presence of docking sites rather than phosphorylation site quality, in S. cerevisiae early sites tend to be enriched for Ser residues. The modest preference of the yeast CDK Cdc28 may contribute to this phenomenon, but it appears to be largely driven by the high activity of the pThr-specific phosphatase Cdc55 in both interphase and mitosis [76]. In this case low CDK activity in interphase is insufficient to balance Cdc55 activity, leading to selective accumulation of phosphorylation specifically at Ser residues on early substrates.