Substrate affinity and specificity can

Substrate affinity and specificity can be enhanced by docking interactions, in which regions distal to the site of nigericin bind to grooves, pockets, or surfaces outside of the kinase catalytic cleft 1, 2, 5, 6. Like catalytic site interactions, docking interactions can involve recognition of short linear sequence motifs, for example through peptide-binding modules such as SH2 and SH3 domains. Docking interactions in some cases mediate processivity, facilitating phosphorylation of multiple distinct sites on a substrate. Kinases can also be recruited to their substrates through indirect interactions mediated by adaptor and scaffold proteins. Adaptor proteins that bind to both kinase and substrate promote phosphorylation through induced proximity. In addition, adaptor proteins can direct kinases to their substrates by controlling their subcellular localization independently of direct interactions with substrates. Scaffolds, which form stable complexes with multiple proteins, often serve as hubs for kinase regulation [2]. Kinases are frequently organized into cascades, and in these cases scaffolds can be important for channeling upstream kinases to activate specific downstream kinases. Scaffolds can also promote substrate specificity by localizing an active pool of the kinase in proximity to its substrates and, in some cases, causing conformational changes in substrates that promote phosphorylation [2]. The mechanisms of substrate targeting illustrated above are not mutually exclusive, and authentic kinase–substrate pairs likely require multiple interactions to achieve efficient phosphorylation in vivo. Recent insight into the biochemical and structural principles underlying these mechanisms has provided a more complete picture of how kinases interact with their substrates, moving beyond classical concepts involving recognition of simple consensus sequences. This detailed understanding has in turn influenced models of how kinases function within complex biological systems.
Recognition of Phosphorylation Site Sequence Motifs Early biochemical studies suggested that protein kinases phosphorylated substrates in the context of specific sequence motifs. In cocrystal structures, peptide substrates generally bind to the kinase in an extended conformation (Figure 2A). The peptide makes β-sheet-like hydrogen bonding interactions with a portion of the kinase activation loop, a conformationally flexible region important for regulation. Residues within the catalytic cleft define its shape and biophysical characteristics 4, 5 to determine phosphorylation site specificity, which varies substantially among kinases. Examples of both classical and more recently established kinase consensus sequences are provided in Table 1. Recent structural and biochemical studies have provided new insight into phosphorylation site specificity, including how kinases recognize the phosphoacceptor residue itself (Figure 2A). For example, it has been long appreciated that some STKs have substantial preference for either Ser or Thr as the phosphoacceptor residue. Chen et al. identified the residue immediately downstream of the conserved DFG motif found in the kinase activation loop (DFG+1) as a major determinant of Ser–Thr phosphoacceptor specificity [7]. Large hydrophobic DFG+1 residues promote Ser phosphorylation, whereas smaller β-branched residues confer specificity for Thr. Phosphoacceptor identity did not appear to affect substrate binding to the kinase. Rather, in kinase–peptide cocrystal structures, the DFG+1 residue appeared to position the phosphoacceptor residue in either a productive or nonproductive conformation for catalysis. Intriguingly, a separate study found that phosphorylation of protein kinase C (PKC)-δ could alter its Ser–Thr phosphoacceptor specificity. Phosphorylation of the kinase at a site (Ser359) within the Gly-rich loop, a conserved region proximal to the ATP binding site [8] (Figure 2A), conferred a strong preference for a Ser phosphoacceptor, while the dephosphorylated form was nonselective. As a phosphorylatable residue is present at the analogous position in ∼30% of human kinases, phosphorylation at this site may be a common mechanism for dynamic regulation of kinase phosphoacceptor specificity.