Systematic Discovery of Short Linear Motifs Decodes Calcineurin Phosphatase Signaling

Wigington, Callie P.; Roy, Jagoree; Damle, Nikhil P; Yadav, Vikash Kumar; Blikstad, Cecilia; Resch, Eduard; Wong, Cassandra; Mackay, Douglas R.; Wang, Jennifer T.; Krystkowiak, Izabella; Bradburn, Devin A.; Tsekitsidou, Eirini; Hong, Su Hyun; Kaderali, Malika Amyn; Xu, Shou-Ling; Stearns, Tim; Gingras, Anne‐Claude; Ullman, Katharine S.; Ivarsson, Ylva; Davey, Norman E.; Cyert, Martha S.

doi:10.1016/j.molcel.2020.06.029

Cited by 62 publications

(72 citation statements)

References 123 publications

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“…However, yeast two-hybrid analyses are also prone to false positives, and can be applied only to a subset of the proteome (Mrowka et al 2001;Stynen et al 2012). Recently developed methods that capture transient interactions such as proximity-dependent biotinylation coupled with mass spec (PDB-MS) offer a new strategy to identify dynamic calcineurin-substrate interactions, especially in combination with calcineurin mutants whose interaction with either PxIxIT or LxVP motifs is compromised (Gingras et al 2019;Wigington et al 2019). Mutations in the conserved catalytic site have also been used to "trap" phosphatase-substrate interactions and successfully identify substrates for PPI, a strategy that has yet to be applied to calcineurin (Wu et al 2018).…”

Section: Interaction-based Approachesmentioning

confidence: 99%

“…Currently, however, fewer than 15 examples each of human PxIxITs or LxVPs are in the literature, and each set includes related motifs from four NFAT isoforms. Proteomic peptide phage display (ProP-PD), which uses a phage library expressing tiled 16-mers from all predicted disordered regions in the human proteome, is an ideal method to experimentally identify SLiM instances (Sundell and Ivarsson 2014), and was used to discover 20 additional PxIxIT and 25 additional LxVP sequences in the human proteome (Wigington et al 2019). These human peptides directly identified new calcineurin substrates, including Notch1 and Nup153, and allowed construction and testing of robust PSSMs for PxIxIT and LxVP, containing 12 and 7 positions, respectively, that were used for motif discovery in the proteome (Wigington et al 2019).…”

Section: In Silico Strategies For Slim Identificationmentioning

confidence: 99%

“…Proteomic peptide phage display (ProP-PD), which uses a phage library expressing tiled 16-mers from all predicted disordered regions in the human proteome, is an ideal method to experimentally identify SLiM instances (Sundell and Ivarsson 2014), and was used to discover 20 additional PxIxIT and 25 additional LxVP sequences in the human proteome (Wigington et al 2019). These human peptides directly identified new calcineurin substrates, including Notch1 and Nup153, and allowed construction and testing of robust PSSMs for PxIxIT and LxVP, containing 12 and 7 positions, respectively, that were used for motif discovery in the proteome (Wigington et al 2019). A major strength of in silico SLiM-based strategies is the ability to identify putative calcineurin targets comprehensively (i.e., without experimental limitations such as protein level and accessibility or tissue/cell-typespecific expression).…”

Section: In Silico Strategies For Slim Identificationmentioning

confidence: 99%

“…A major strength of in silico SLiM-based strategies is the ability to identify putative calcineurin targets comprehensively (i.e., without experimental limitations such as protein level and accessibility or tissue/cell-typespecific expression). Using this approach, we discovered that calcineurin regulates transport through the nuclear pore by dephosphorylating multiple nucleoporins (Wigington et al 2019). Overall, these computational methods generate a large "parts list," and provide a starting point for investigations into new functions for Ca 2+ and calcineurin signaling.…”

Section: In Silico Strategies For Slim Identificationmentioning

confidence: 99%

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Identifying New Substrates and Functions for an Old Enzyme: Calcineurin

Roy

Cyert

2019

Cold Spring Harb Perspect Biol

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Biological processes are dynamically regulated by signaling networks composed of protein kinases and phosphatases. Calcineurin, or PP3, is a conserved phosphoserine/phosphothreonine-specific protein phosphatase and member of the PPP family of phosphatases. Calcineurin is unique, however, in its activation by Ca 2+ and calmodulin. This ubiquitously expressed phosphatase controls Ca 2+ -dependent processes in all human tissues, but is best known for driving the adaptive immune response by dephosphorylating the nuclear factor of the activated T-cells (NFAT) family of transcription factors. Therefore, calcineurin inhibitors, FK506 (tacrolimus), and cyclosporin A serve as immunosuppressants. We describe some of the adverse effects associated with calcineurin inhibitors that result from inhibition of calcineurin in nonimmune tissues, illustrating the many functions of this enzyme that have yet to be elucidated. In fact, calcineurin has essential roles beyond the immune system, from yeast to humans, but since its discovery more than 30 years ago, only a small number of direct calcineurin substrates have been shown (∼75 proteins). This is because of limitations in current methods for identification of phosphatase substrates. Here we discuss recent insights into mechanisms of calcineurin activation and substrate recognition that have been critical in the development of novel approaches for identifying its targets systematically. Rather than comprehensively reviewing known functions of calcineurin, we highlight new approaches to substrate identification for this critical regulator that may reveal molecular mechanisms underlying toxicities caused by calcineurin inhibitor-based immunosuppression.

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Section: Interaction-based Approachesmentioning

confidence: 99%

Section: In Silico Strategies For Slim Identificationmentioning

confidence: 99%

Section: In Silico Strategies For Slim Identificationmentioning

confidence: 99%

Section: In Silico Strategies For Slim Identificationmentioning

confidence: 99%

See 2 more Smart Citations

Identifying New Substrates and Functions for an Old Enzyme: Calcineurin

Roy

Cyert

2019

Cold Spring Harb Perspect Biol

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“…Therefore, we hypothesized that the PxIxIT strength of the Saccharomyces clade is higher than its sister clade that includes C. albicans and that this increased strength is needed for pulsing. To test this, we calculated the PxIxIT strength of Crz1 IDRs from 40 fungi of the Saccharomyces clade and the sister clade by using a Position Specific Scoring Matrix (PSSM (51,52), see methods) to predict PxIxIT strength (Supplementary Figure 5A, R 2 = 0.74 between the measured affinity (Kd) and the predicted PxIxIT strength of experimentally confirmed PxIxITs ( 52)).…”

Section: Pxixit Strength In a Specific Part Of The Crz1 Idr Increased In The Saccharomycetacea Cladementioning

confidence: 99%

A functionally divergent intrinsically disordered region underlying the conservation of stochastic signaling

Hsu

Strome

Lash

et al. 2021

Preprint

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Stochastic signaling dynamics expand living cells’ information processing capabilities. An increasing number of studies report that regulators encode information in their pulsatile dynamics. The evolutionary mechanisms that lead to complex signaling dynamics remain uncharacterized, perhaps because key interactions of signaling proteins are encoded in intrinsically disordered regions (IDRs), whose evolution is difficult to analyze. Here we focused on the stochastic pulsing dynamics of Crz1, a transcription factor in fungi downstream of the widely conserved calcium signaling pathway. We find that Crz1 IDRs from anciently diverged fungi can all respond transiently to calcium stress; however, only Crz1 IDRs from the Saccharomyces clade support pulsatility, encode extra information, and rescue fitness, while the Crz1 IDRs from distantly related fungi do none of the three. On the other hand, we find that Crz1 pulsing is conserved in the distantly related fungi, consistent with the evolutionary model of stabilizing selection. Further, we show that a calcineurin docking site in a specific part of the IDRs appears to be sufficient for pulsing and show evidence for a beneficial increase in the relative calcineurin affinity of this docking site. We propose that evolutionary flexibility of functionally divergent IDRs underlies the conservation of stochastic signaling by stabilizing selection.

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The Myotubularin Related Proteins and the Untapped Interaction Potential of Their Disordered C‐Terminal Regions

Saar,

Lennartsson,

Weidner

et al. 2024

Proteins

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Intrinsically disordered regions (IDRs) of proteins remain understudied with enigmatic sequence features relevant to their functions. Members of the myotubularin‐related protein (MTMR) family contain uncharacterized IDRs. After decades of research on their phosphatase activity, recent work on the C‐terminal IDRs of MTMR7 revealed new interactions and important new functions beyond the phosphatase function. Here we take a broader look at the C‐terminal domains (CTDs) of 14 human MTMRs and use bioinformatic tools and biophysical methods to ask which other functions may be probable in this protein family. The predictions show that the CTDs are disordered and carry short linear motifs (SLiMs) important for targeting of MTMRs to defined subcellular compartments and implicating them in signaling, phase separation, interaction with diverse proteins, including transcription factors and are of relevance for cancer research and neuroscience. We also present experimental methods to study the CTDs and use them to characterize the coiled coil (CC) domains of MTMR7 and MTMR9. We show homo‐ and hetero‐oligomerization with preference for MTMR7‐CC to form dimers, while MTMR9‐CC forms trimers. We relate the results to sequence features and make predictions for the structural landscape of other MTMRs. Our work gives a broad insight into the so far unrecognized features and SLiMs in MTMR‐CTDs, and provides the basis for more in‐depth experimental research on this diverse protein family and understudied IDRs in proteins in general.

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Systematic Discovery of Short Linear Motifs Decodes Calcineurin Phosphatase Signaling

Cited by 62 publications

References 123 publications

Identifying New Substrates and Functions for an Old Enzyme: Calcineurin

Identifying New Substrates and Functions for an Old Enzyme: Calcineurin

A functionally divergent intrinsically disordered region underlying the conservation of stochastic signaling

The Myotubularin Related Proteins and the Untapped Interaction Potential of Their Disordered C‐Terminal Regions

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