A Cell-Signaling Network Temporally Resolves Specific versus Promiscuous Phosphorylation

Kanshin, Evgeny; Bergeron-Sandoval, Louis-Philippe; Işık, Semra; Thibault, Pierre; Michnick, Stephen W.

doi:10.1016/j.celrep.2015.01.052

Cited by 86 publications

(143 citation statements)

References 65 publications

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“…Phosphorylation of the MAPK consensus sites in the Ste50 IDR results in attenuation of signaling by dissociation of the signaling complex from the membrane (33). Phosphoproteomic studies also indicate phosphorylation of a subset of these sites in standard growth conditions (19,(38)(39)(40)(41)(42)(43), which we refer to as basal phosphorylation. To test the function of this region in S. cerevisiae, we therefore made an unphosphorylatable mutant, where each consensus site was mutated to alanine (referred to as 5A mutant) (Materials and Methods).…”

Section: Significancementioning

confidence: 99%

“…We calculated the basal net charge of each IDR by considering its net charge (sum of positive and negatively charged residues) including basal phosphorylation at up to two SP sites, as mass spectrometry studies have found that up to two serines are phosphorylated in this IDR under basal conditions in S. cerevisiae (as reported in refs. 19,[38][39][40][41][42][43]. Therefore, if the IDR has two or more SP sites, we assume that two of these serines are phosphorylated under basal conditions and add a charge of -4 (-2 for each phosphorylation site) to the net charge of the IDR (see Materials and Methods for details).…”

Section: Basal Net Charge Of Diverged Sequences Is Correlated With Fumentioning

confidence: 99%

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Selection maintains signaling function of a highly diverged intrinsically disordered region

Zarin

Tsai

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Intrinsically disordered regions (IDRs) are characterized by their lack of stable secondary or tertiary structure and comprise a large part of the eukaryotic proteome. Although these regions play a variety of signaling and regulatory roles, they appear to be rapidly evolving at the primary sequence level. To understand the functional implications of this rapid evolution, we focused on a highly diverged IDR in Saccharomyces cerevisiae that is involved in regulating multiple conserved MAPK pathways. We hypothesized that under stabilizing selection, the functional output of orthologous IDRs could be maintained, such that diverse genotypes could lead to similar function and fitness. Consistent with the stabilizing selection hypothesis, we find that diverged, orthologous IDRs can mostly recapitulate wildtype function and fitness in S. cerevisiae. We also find that the electrostatic charge of the IDR is correlated with signaling output and, using phylogenetic comparative methods, find evidence for selection maintaining this quantitative molecular trait despite underlying genotypic divergence.C urrent predictions suggest that close to 40% of all proteins in eukaryotic organisms are either entirely disordered or contain sizeable regions that are disordered, meaning they do not autonomously fold into defined secondary or tertiary structures (1, 2). These intrinsically disordered regions (IDRs) are thought to have important implications for protein function (3, 4) and are known to play regulatory roles, often through short linear motifs (SLiMs) that control protein-protein interactions, localization, degradation, and posttranslational modifications (5, 6). Although proteome-wide studies have provided in silico evidence for conservation of length (7) and composition (8) in some IDRs, reports of increased rates of insertions and deletions (9-13) and amino acid substitutions (14) in IDRs are indicative of their rapid evolution compared with ordered regions. In addition, although some SLiMs are indeed conserved in IDRs (15-17), others appear in clusters where precise position and number are not conserved (18)(19)(20). Although it is reasonable to assume that conservation of sequence in IDRs is indicative of functional conservation of SLiMs, it is more difficult to interpret the functional consequences of IDRs that are highly diverged at the sequence level: These may represent either nonfunctional sequences evolving in the absence of constraint or weakly constrained functional elements that are gained or lost in a compensatory manner [undergoing evolutionary turnover (as described in refs. 18, 21)], such that they are not conserved at the amino acid sequence level.Like IDRs, noncoding DNA often shows relatively rapid evolution and weak constraints at the sequence level (22). Interestingly, IDRs show other parallels with noncoding DNA (18,23,24). For example, nonconserved clusters of phosphorylation sites in IDRs are reminiscent of nonconserved transcription factor binding sites in enhancers. Although these enhancers and the bi...

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Section: Significancementioning

confidence: 99%

Section: Basal Net Charge Of Diverged Sequences Is Correlated With Fumentioning

confidence: 99%

Selection maintains signaling function of a highly diverged intrinsically disordered region

Zarin

Tsai

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…http://dx.doi.org/10.1101/209676 doi: bioRxiv preprint first posted online response to NaCl, an osmotic stressor, measuring phosphorylation changes for 60 seconds poststimulation at uniform 5 second intervals (Kanshin et al, 2015). They identified 1,596 phosphorylated proteins, including 1,401 dynamic phosphopeptides on 784 proteins based on their fold changes in the salt stress time series with respect to a control (Table S13).…”

Section: Tps Generates High-quality Pathway Models From Lower Temporamentioning

confidence: 99%

“…As proteomic technologies continue to improve in terms of depth of coverage Zhou et al, 2013) and temporal resolution (Humphrey et al, 2015;Kanshin et al, 2015;Reddy et al, 2016), the need to systematically interpret these data will likewise grow. TPS enables reasoning with temporal phosphorylation changes and physical protein interactions to define what drives the vast protein modifications that are not represented by existing knowledge in pathway databases.…”

Section: Future Directions In Pathway Synthesismentioning

confidence: 99%

“…Future versions of TPS could incorporate perturbation data that links kinase inhibition or deletion to phosphorylation changes that are far downstream from the kinase. For instance, both temporal (Kanshin et al, 2015) and kinase perturbation (MacGilvray et al, 2017;Romanov et al, 2017) phosphoproteomic data are available for the yeast osmotic stress response. Modeling multiple related conditions (e.g., different ligand stimuli and inhibitor perturbations) could allow TPS to learn not only the signs of interactions but also the logic employed when multiple incoming signals influence a protein.…”

Section: Future Directions In Pathway Synthesismentioning

confidence: 99%

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Synthesizing Signaling Pathways from Temporal Phosphoproteomic Data

Köksal

Beck

Cronin

et al. 2017

Preprint

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Advances in proteomics reveal that pathway databases fail to capture the majority of cellular signaling activity. Our mass spectrometry study of the dynamic epidermal growth factor (EGF) response demonstrates that over 89% of significantly (de)phosphorylated proteins are excluded from individual EGF signaling maps, and 63% are absent from all annotated pathways. We present a computational method, the Temporal Pathway Synthesizer (TPS), to discover missing pathway elements by modeling temporal phosphoproteomic data. TPS uses constraint solving to exhaustively explore all possible structures for a signaling pathway, eliminating structures that are inconsistent with protein-protein interactions or the observed phosphorylation event timing. Applied to our EGF response data, TPS connects 83% of the responding proteins to receptors and signaling proteins in EGF pathway maps. Inhibiting predicted active kinases supports the TPS pathway model. The TPS algorithm is broadly applicable and also recovers an accurate model of the yeast osmotic stress response.

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