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:10.1101/632547

Cited by 16 publications

(27 citation statements)

References 130 publications

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“…Not surprisingly, we found the number of genes regulated by calcineurin 8 to be broader than that dependent upon NFAT (this study), indicating that in addition to NFAT, calcineurin likely acts through other effectors to enforce leukemia inducing potential in T-ALL. In line with this hypothesis, several new Cn interacting proteins were recently identified, the inactivation of which could synergize with Cn inhibition to impair T-ALL expansion 38,39 . We also noticed a number of genes specifically deregulated upon Nfat deletion, but not upon CnB1 deletion, indicating that NFAT activity can be critically regulated by upstream signaling pathways in addition to their Cn-mediated nuclear translocation.…”

Section: Discussionmentioning

confidence: 81%

NFAT Transcription Factors are Essential and Redundant Actors for Leukemia Initiating Potential in T-cell Acute Lymphoblastic Leukemia

Catherinet

Passaro

Gachet

et al. 2020

Preprint

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T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy with few available targeted therapies. We previously reported that the phosphatase calcineurin (Cn) is required for LIC (leukemia Initiating Capacity) potential of T-ALL pointing to Cn as an interesting therapeutic target. Calcineurin inhibitors have however unwanted side effect. NFAT transcription factors play crucial roles downstream of calcineurin during thymocyte development, T cell differentiation, activation and anergy. Here we elucidate NFAT functional relevance in T-ALL. Using murine T-ALL models in which Nfat genes can be inactivated either singly or in combination, we show that NFATs are required for T-ALL LIC potential and essential to survival, proliferation and migration of T-ALL cells. We also demonstrate that Nfat genes are functionally redundant in T-ALL and identified a node of genes commonly deregulated upon Cn or NFAT inactivation, which may serve as future candidate targets for T-ALL.

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Section: Discussionmentioning

confidence: 81%

NFAT Transcription Factors are Essential and Redundant Actors for Leukemia Initiating Potential in T-cell Acute Lymphoblastic Leukemia

Catherinet

Passaro

Gachet

et al. 2020

Preprint

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“…In recent years, phage display screens of signaling domains against a 16-mer peptide library derived from the human proteome (Pro-PD) have been used to define SLiM specificity profiles and predict novel interaction partners (43)(44)(45)(46). In this work, we used MassTitr to screen more than 400,000 36-residue segments of the human proteome against the cytoskeleton regulator ENAH and learned that examining such long fragments provides important insights into the role of SLiM sequence context.…”

Section: Discussionmentioning

confidence: 99%

Native proline-rich motifs exploit sequence context to target actin-remodeling Ena/VASP proteins

Hwang

Grant

Ilunga

et al. 2021

Preprint

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Protein interactions between intrinsically disordered, short linear motifs (SLiMs) and modular recognition domains are critical elements in many signal transduction pathways. Yet for most interactions, it is still unclear how low-complexity SLiMs discriminate between highly conserved but biologically distinct SLiM-binding proteins. The Ena/VASP family of proteins pose one such specificity problem. Paralogs ENAH, VASP, and EVL bind to a 5-residue SLiM prevalent in the proteome and perform distinct cellular functions despite sharing high sequence and structural similarity. To interrogate how the sequence context of SLiMs impacts Ena/VASP interactions, we performed an unbiased proteomic screen against the ENAH EVH1 domain. We discovered unexpected ways in which local and distal sequence elements flanking native SLiMs modulate binding. Particularly notable is a peptide from PCARE that achieves paralog specificity by stabilizing a unique conformation adopted only by ENAH. A PCARE-derived peptide can selectively recruit ENAH and block its activity in cells, establishing it as a valuable reagent to disentangle the roles of Ena/VASP paralogs and a potential agent for modulating ENAH function in breast cancer. Guided by our analyses of native interactions, we designed the tightest known ENAH EVH1 binder with a dissociation constant of 50 nM and 400-600-fold selectivity over EVL and VASP. Our work demonstrates that sequence context plays a prominent role in dictating SLiM interactions and can inform the design of custom molecules that target SLiM-based interactions.

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“…ProP-PD has previously successfully been used to determine binding specificities of protein domains with single binding pockets [20,[42][43][44], as well as two pockets [45]. Similarly the computational peptide docking was previously shown to successfully predict the structure of protein-peptide complexes even in cases where the binding site is unknown [21].…”

Section: Discussionmentioning

confidence: 99%

Short linear motif based interactions and dynamics of the ezrin, radixin, moesin and merlin FERM domains

Ali

Khramushin

Yadav

et al. 2020

Preprint

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The ERMs (ezrin, radixin and moesin) and the closely related merlin (NF2) participate in signaling events at the cell cortex through interactions mediated by their conserved FERM domain. We systematically investigated the FERM domain mediated interactions with short linear motifs (SLiMs) by screening the FERM domains againsts a phage peptidome representing intrinsically disordered regions of the human proteome. We uncovered a diverse set of interacting partners with similar but distinct binding motifs (FYDF, xYxV, FY(D/E)L and LQE(I/L) that bind to distinct binding pockets. We validated interactions between moesin and merlin FERM domains and full-length FAM83G, HIF1A, LATS1, NOP53, PAK6, RRBP1 and ZNF622 through pull-down experiments. Using biophysical binding assays, we determined affinities of, and uncovered allosteric interdependencies between, different binding partners, suggesting that the FERM domain acts as a switchable interaction hub. Using Rosetta FlexPepDock computational peptide docking protocols, we investigated the energy landscapes of identified interactions, which provide a detailed molecular understanding of the binding of the distinct binding motifs, as well as possible allosteric interconnections. This study demonstrates how experimental and computational approaches together can unravel a complex system of protein-peptide interactions that includes a family of proteins with multiple binding sites that interact with similar but distinct binding motifs.HighlightsWe screened the human disorderome for motif-containing partners of the FERM domainsWe expand the ERM and merlin interactomes of the ERMs and merlinWe identify four distinct motif classes that bind the ERM and merlin FERM domains: FYDF, xYxV, FY(D/E)L and LQE(I/L)In-vitro and in-silico data suggest that the FYDF motif binds to the F3a site and that xYxV motif binds to the F3b siteIn-silico modelling sheds light on the underlying conformational changes responsible for ligand interdependenciesAbstract Figure

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Systematic discovery of Short Linear Motifs decodes calcineurin phosphatase signaling

Cited by 16 publications

References 130 publications

NFAT Transcription Factors are Essential and Redundant Actors for Leukemia Initiating Potential in T-cell Acute Lymphoblastic Leukemia

NFAT Transcription Factors are Essential and Redundant Actors for Leukemia Initiating Potential in T-cell Acute Lymphoblastic Leukemia

Native proline-rich motifs exploit sequence context to target actin-remodeling Ena/VASP proteins

Short linear motif based interactions and dynamics of the ezrin, radixin, moesin and merlin FERM domains

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