Spatial Exclusivity Combined with Positive and Negative Selection of Phosphorylation Motifs Is the Basis for Context-Dependent Mitotic Signaling

Abstract

The timing and localization of events during mitosis is controlled by the regulated phosphorylation of proteins by the mitotic kinases, which include Aurora A, Aurora B, Nek2, Plk1, and the cyclin-dependent kinase complex Cdk1/cyclin B. Although mitotic kinases can have overlapping subcellular localizations, each kinase appears to phosphorylate its substrates on distinct sites. To gain insight into the relative importance of local sequence context in kinase selectivity, identify previously unknown substrates of these five mitotic kinases, and explore potential mechanisms for substrate discrimination, we determined the optimal substrate motifs of these major mitotic kinases by Positional Scanning Oriented Peptide Library Screening (PS-OPLS). We verified individual motifs with in vitro peptide kinetic studies and used structural modeling to rationalize the kinase-specific selection of key motif-determining residues at the molecular level. Cross comparisons among the phosphorylation site selectivity motifs of these kinases revealed an evolutionarily conserved mutual exclusion mechanism in which the positively and negatively selected portions of the phosphorylation motifs of mitotic kinases, together with their subcellular localizations, result in proper substrate targeting in a coordinated manner during mitosis.

Quantitative Phospho-proteomics to Investigate the Polo-like Kinase 1-Dependent Phospho-proteome

“…S5). Recently, asparagine in the -2 position was also detected by a peptide library-based approach to find PLK1 motifs (42) and in a phospho-proteomic approach by Santamaria et al (38) supporting the hypothesis this motif represent a new variant of the PLK1 consensus site. Our bioinformatic analysis thus identified two potential variations of the published PLK1 consensus motif (43).…”

“…This algorithm identifies amino acid sequences matching kinase consensus motifs based on in vitro kinase assays on oriented peptide libraries (28,39). In addition to the initial set of 28 kinase motifs (28) currently available in Scansite, the mitotic kinases PLK1, Aurora kinase A and B, as well as NEK2 were also recently characterized and included in Scansite (29). We analyzed our data set of 4070 phosphorylation sites for the existence of all Scansite motifs and could relate 44% of the identified sites to 27 Scansite kinase motifs with medium and high stringency.…”

“…Later the phosphorylation sites obtained by the Scansite analysis with high and medium thresholds were compared with the MS data. PSSMs for CDK1, AURKA, AURKB, NEK2, and PLK1 were provided by the Yaffe laboratory (29). Two-sided Fisher's exact test was performed to compare the incidence of kinase motifs in the BI 4834 sensitive, BI 4834 induced and BI 4834 not-regulated sets.…”

Cell cycle regulation by the NEK family of protein kinases

“…FxxS/T, where 'x' is any amino acid) (Lu et al, 1994). More recent studies have indicated that human NEKs have a similar preference, with both NEK2 and NEK6 preferring a hydrophobic residue, ideally phenylalanine or leucine, at the 23 position (F/ LxxS/T) (Alexander et al, 2011;Lizcano et al, 2002). However, these are not strict requirements, as phosphorylation sites that do not fall into this motif have been mapped on NEK substrates.…”

Domain Specificity of MAP3K Family Members, MLK and Tak1, for JNK Signaling inDrosophila

“…Recent work has provided some mechanistic insight. For example, within a single cell, related kinases might avoid inappropriate crosstalk by deploying nonoverlapping substrates or by compartmentalization of their function in cellular space or time (Alexander et al 2011). Considering the conserved three-tier kinase organization within the MAPK pathways, the core pathway may incorporate distinct upstream transducers, as is the case with the diversity of MAP3K proteins, to shift the outcome of signaling in response to distinct stimuli.…”