Priming phosphorylation can also generate a binding site for
Priming alpha msh can also generate a binding site for some protein-interaction domains and adaptor proteins that recruit kinases to phosphorylate more distal sites . Well-characterized phosphoprotein-interacting domains include pTyr-binding SH2 domains present in many nonreceptor TyrKs and the pSer/pThr-interacting polobox domain found in polo-like kinases. These domains typically bind to phosphoproteins in the context of linear motifs unrelated to known kinase phosphorylation motifs. As a consequence, many different kinases can theoretically generate an interaction site for a given phosphobinding domain, allowing for flexibility in crosstalk between pathways. By contrast, two adaptor proteins were recently found to interact with phosphopeptide sequences similar to known kinase phosphorylation site motifs. One such protein, the cyclin-dependent kinase (CDK) adaptor Cks1, binds phosphopeptides within a sequence context (ϕ-x-pT-P) that overlaps the CDK phosphorylation site consensus (S/T-P-x-K/R) . Cks1 can thereby act as a processivity factor, allowing a single CDK phosphorylation site to prime for subsequent phosphorylation at multiple sites on the same substrate . Another example involves MOB1 adaptor proteins, which are obligate activators of LATS family kinases that act in the tumor suppressive Hippo pathway 46, 47. Interestingly, the phosphopeptide binding specificity of MOB1 conforms to the phosphorylation site consensus of MST1 and 2, kinases which activate LATS through direct phosphorylation . In this case, MST1/2 autophosphorylation generates MOB1 binding sites, recruiting LATS to its upstream kinase to facilitate its activation . In this way, a limited level of MST1/2 activity would be sufficient to activate the Hippo pathway without off-target phosphorylation of other proteins.
Multisite phosphorylation often arises from a single kinase acting on multiple sites within a substrate in a nonprocessive manner . In some cases, sites are phosphorylated sequentially, with the order dictated by the relative catalytic efficiency of the kinase for each site . When this occurs, high-efficiency sites that are phosphorylated first can act as decoys to inhibit phosphorylation of low-efficiency sites. This phenomenon has been argued to be a source of switch-like responses to graded stimuli at saturating levels of substrate . A recent study used time-resolved NMR analysis to investigate the dynamics of multisite phosphorylation of the transcription factor Elk-1 by ERK MAPK . Competition between two ERK docking motifs promoted different rates of phosphorylation at eight sites within the Elk-1 activation domain. Intriguingly, while rapidly phosphorylated sites promoted transcriptional activation by Elk-1, sites phosphorylated more slowly led to inactivation. These results demonstrate that multisite phosphorylation dynamics can provide a mechanism for signal attenuation in the absence of counteracting phosphatases, which may facilitate differential timing of the various signaling outputs of a kinase.
Some Substrates Are Better Than Others: Consequences of Differential Substrate Quality Classically protein kinases were viewed as having stringent consensus sequences that dictated, in a binary manner, whether or not a specific site would be a substrate . In a number of cases, new substrates for kinases have been discovered based on the presence of such consensus sequences, but these efforts are generally challenging due to motif degeneracy and redundancy. The presence of both a phosphorylation site and docking site sequence motif appears to be predictive of true substrates of some kinases . Substrate prediction can also be improved by considering the contribution of residues other than those strictly required for phosphorylation. Peptide library approaches 14, 53, 54, 55, 56 have facilitated comprehensive quantitative analysis of substrate specificity, in which the impact on phosphorylation rate of each of the 20 amino acids can be assessed for multiple positions within the peptide. Computational tools can use such quantified data to identify candidate substrates predicted to be phosphorylated most efficiently by the kinase 57, 58. This approach can take advantage of subtle differences between related kinases to identify their unique substrates 59, 60, 61, 62, 63. As substrates harboring suboptimal sites (discussed below) will escape detection, other approaches such as chemical genetic tagging  and time-resolved phosphoproteomics analysis 65, 66 have emerged to globally identify direct kinase substrates.