Protein kinases display minimal interpositional dependence on substrate sequence: potential implications for the evolution of signalling networks

Abstract

Characterization of in vitro substrates of protein kinases by peptide library screening provides a wealth of information on the substrate specificity of kinases for amino acids at particular positions relative to the site of phosphorylation, but provides no information concerning interdependence among positions. High-throughput techniques have recently made it feasible to identify large numbers of in vivo kinase substrates. We used data from experiments on the kinases ATM/ATR and CDK1, and curated CK2 substrates to evaluate the prevalence of interactions between substrate positions within a motif and the utility of these interactions in predicting kinase substrates. Among these data, evidence of interpositional sequence dependencies is strikingly rare, and what dependency exists does little to aid in the prediction of novel kinase substrates. Significant increases in the ability of models to predict kinase -substrate specificity beyond position-independent models must come largely from inclusion of elements of biological and cellular context, rather than further analysis of substrate sequences alone. Our results suggest that, evolutionarily, kinase substrate fitness exists in a smooth energetic landscape. Taken with results from others indicating that phosphopeptide-binding domains do exhibit interpositional dependence, our data suggest that incorporation of new substrate molecules into phospho-signalling networks may be rate-limited by the evolution of suitability for binding by phosphopeptide-binding domains.

Comprehensive substrate specificity profiling of the human Nek kinome reveals unexpected signaling outputs

“…Additionally, the sequence surrounding the phosphorylation site also affects substrate positioning, and can therefore affect phospho-acceptor site specificity. As an example of this interdependence, we have previously noted that the preference of the kinases ATM/ATR for phosphorylating serine-glutamine versus threonine-glutamine motifs appears to depend on the residues found at other positions in the motif (Joughin et al, 2012). A similar observation of interdependence between phospho-acceptor residue identity and specific amino acids in other positions within the motif has been shown for the dual-specificity kinase CK2 (Marin et al, 1999).…”

Ancestral resurrection reveals evolutionary mechanisms of kinase plasticity

“…Computational analyses have suggested that kinases display little inter-positional dependence in their primary peptide specificity and that each substrate peptide amino acid interacts with the kinase active site more or less independently ( Joughin et al, 2012 ). Our observation that +1 specificity is dependent on the identity of the phosphoacceptor represents an exception to this model.…”

Regulation and Function of Phosphorylation on VP8, the Major Tegument Protein of Bovine Herpesvirus 1

“…Generally, CK2 has more than one active site on substrates, and overall about 75% of the active sites match the CK2 motif S/T-X-X-D/E (30). Within the VP8 sequence, 10 threonines and 19 serines match the motif.…”