While phosphorylation site motifs are typically described in

While CH5138303 synthesis site motifs are typically described in terms of residues that promote phosphorylation, negatively selected residues can also be an important component of substrate recognition. Such ‘forbidden’ resides can act as a filter to prevent phosphorylation of a site by the ‘wrong’ kinase, which can help establish the correct order and timing of phosphorylation events [9]. This concept has been illustrated recently in a study using a novel method in which genetically encoded peptide libraries are displayed on the surface of bacteria. Peptide-expressing bacteria were treated with a TyrK, and bacteria harboring phosphorylated substrates were labeled with a fluorophore-coupled anti-phosphotyrosine (pTyr) antibody for isolation by fluorescence-activated cell sorting. This method revealed that the TyrK ZAP70, which plays a key role in transducing signals from the T cell receptor, preferred acidic residues at multiple positions and had little tolerance for basic residues at any position [10]. This ‘electrostatic filter’ both insulates ZAP70 substrates from other kinases and prevents the kinase from activating itself through autophosphorylation. Similar analysis of the upstream TyrK LCK explained its ability to phosphorylate itself as well as ZAP70. Thus, features of substrate selectivity enforce ordered phosphorylation of kinases and the compartmentalization of substrates in the T cell activation cascade.
Roles for Secondary and Tertiary Structure in Kinase Substrates Known sites of phosphorylation on substrates are enriched in unstructured regions found outside of defined protein domains, which are likely to be more accessible to interact with kinases in the canonical, extended conformation (Figure 2A) 11, 12, 13. Recently, several kinase substrates have been observed to bind in alternative conformations, suggesting that kinases may in some cases recognize elements of secondary structure. One recent example involves Haspin, an atypical kinase lacking recognizable sequence similarity to most eukaryotic protein kinases. Kettenbach et al. analyzed the substrate specificity of Haspin using a novel type of peptide library comprising a dephosphorylated proteolytic digest of HeLa cell extract [14]. Following treatment of the peptide mixture with a kinase, phosphopeptides were purified and identified using high throughput liquid chromatography-tandem mass spectrometry. This analysis found Haspin to preferentially phosphorylate sites near peptide N termini and defined a stringent sequence motif (Table 1) conforming to the sole known Haspin site, Thr3 of histone H3. Subsequent X-ray crystallography studies revealed that Haspin adopts a canonical bilobed kinase fold, but its activation loop assumes a distinct, largely helical conformation [15]. This conformation would preclude a substrate from adopting the typical extended binding mode. In a cocrystal structure, a bound histone H3 peptide made a sharp turn downstream of the phosphoacceptor to project outward toward solvent (Figure 2B). This arrangement allows for close contact between the sidechains of three residues flanking the phosphoacceptor and specific pockets in the kinase, explaining its unusually stringent sequence specificity and its preference for N-terminal sequences. Interestingly, these flanking residues, Arg2 and Lys4, are hotspots for acetylation and methylation. Modification of these residues would likely abolish phosphorylation by Haspin, rendering its activity responsive to epigenetic signals. Several recent studies of PKC isozymes have uncovered novel modes of substrate interaction, in which residues within the substrate are selected based on their arrangement within a folded structure, rather than their position within a linear sequence. Unlike most ‘basophilic’ kinases that have strict positional selectivity, PKCs prefer basic residues at multiple positions both upstream and downstream of the phosphorylation site (Table 1). While basic residues C-terminal to the phosphorylation site appear to promote catalytic efficiency, possibly by helping to position the γ-phosphate of ATP, N-terminal basic residues contribute to substrate binding [16]. An X-ray cocrystal structure of PKCι with a fragment of its substrate Par3 has revealed a unique mode of interaction with residues located upstream of the phosphorylation site [17] (Figure 2B). A hydrophobic pocket unique to PKCι (and its closest relative PKCζ) is created by an insertion within the kinase C-terminal lobe. This pocket anchors a Phe residue at the −5 position in Par3, promoting an unusual conformation involving two β-turns in the substrate backbone. This conformation allows a basic residue positioned far upstream of the phosphorylation site to engage an acidic pocket on the kinase that typically binds to more proximal residues. In this case, a sequence that is presumably disordered in the absence of the kinase adopts a specific conformation upon binding. A similar phenomenon may explain earlier observations that more distally positioned residues can be essential for phosphorylation [18]. An alternative model, in which the kinase recognizes a substrate within the context of preformed secondary structure, has been proposed for the interaction between PKCβ and α-tubulin [19] (Figure 2C). In this model, basic residues typically found within the PKC consensus motif are instead quite distal to the phosphorylation site (∼90 residues upstream) in the primary sequence. However, in the folded α-tubulin structure, these residues are located proximal to the phosphosite (Figure 2C). This type of ‘structural consensus’ may explain other instances where a substrate phosphorylation site does not conform to the simple linear sequence motif of the kinase (see Outstanding Questions).