A novel motif in calcimembrin/C16orf74 dictates multimeric dephosphorylation by calcineurin.

Calcineurin (CN), the only Ca 2+ -calmodulin activated protein phosphatase, dephosphorylates substrates within membrane-associated Ca 2+ microdomains. CN binds to substrates and regulators via short linear motifs (SLIMs), PxIxIT and LxVP. PxIxIT binding to CN is Ca 2+ independent and affects its distribution, while LxVP associates only with the active enzyme and promotes catalysis. 31 human proteins contain one or more composite 'LxVPxIxIT' motifs, whose functional properties have not been examined. Here we report studies of calcimembrin/C16orf74 (CLMB), a largely uncharacterized protein containing a composite motif that binds and directs CN to membranes. We demonstrate that CLMB associates with membranes via N-myristoylation and dynamic S-acylation and is dephosphorylated by CN on Thr44. The LxVP and PxIxIT portions of the CLMB composite sequence, together with Thr44 phosphorylation, confer high affinity PxIxIT-mediated binding to CN (KD∼8.9 nM) via an extended, 33 LxVPxIxITxx(p)T 44 sequence. This binding promotes CLMB-based targeting of CN to membranes, but also protects Thr44 from dephosphorylation. Thus, we propose that CN dephosphorylates CLMB in multimeric complexes, where one CLMB molecule recruits CN to membranes via PxIxIT binding, allowing others to engage through their LxVP motif for dephosphorylation. This unique mechanism makes dephosphorylation sensitive to CLMB:CN ratios and is supported by in vivo and in vitro analyses. CLMB overexpression is associated with poor prognoses for several cancers, suggesting that it promotes oncogenesis by shaping CN signaling.

Calcineurin associates with centrosomes and regulates cilia length maintenance.

Calcineurin, or protein phosphatase 2B (PP2B), the Ca2+ and calmodulin-activated phosphatase and target of immunosuppressants, has many substrates and functions that remain uncharacterized. By combining rapid proximity-dependent labeling with cell cycle synchronization, we mapped the spatial distribution of calcineurin in different cell cycle stages. While calcineurin-proximal proteins did not vary significantly between interphase and mitosis, calcineurin consistently associated with multiple centrosomal and/or ciliary proteins. These include POC5, which binds centrins in a Ca2+-dependent manner and is a component of the luminal scaffold that stabilizes centrioles. We show that POC5 contains a calcineurin substrate motif (PxIxIT type) that mediates calcineurin binding in vivo and in vitro. Using indirect immunofluorescence and ultrastructure expansion microscopy, we demonstrate that calcineurin colocalizes with POC5 at the centriole, and further show that calcineurin inhibitors alter POC5 distribution within the centriole lumen. Our discovery that calcineurin directly associates with centriolar proteins highlights a role for Ca2+ and calcineurin signaling at these organelles. Calcineurin inhibition promotes elongation of primary cilia without affecting ciliogenesis. Thus, Ca2+ signaling within cilia includes previously unknown functions for calcineurin in maintenance of cilia length, a process that is frequently disrupted in ciliopathies.

A cellular atlas of calcineurin signaling.

Intracellular Ca2+ signals are temporally controlled and spatially restricted. Signaling occurs adjacent to sites of Ca2+ entry and/or release, where Ca2+-dependent effectors and their substrates co-localize to form signaling microdomains. Here we review signaling by calcineurin, the Ca2+/calmodulin regulated protein phosphatase and target of immunosuppressant drugs, Cyclosporin A and FK506. Although well known for its activation of the adaptive immune response via NFAT dephosphorylation, systematic mapping of human calcineurin substrates and regulators reveals unexpected roles for this versatile phosphatase throughout the cell. We discuss calcineurin function, with an emphasis on where signaling occurs and mechanisms that target calcineurin and its substrates to signaling microdomains, especially binding of cognate short linear peptide motifs (SLiMs). Calcineurin is ubiquitously expressed and regulates events at the plasma membrane, other intracellular membranes, mitochondria, the nuclear pore complex and centrosomes/cilia. Based on our expanding knowledge of localized CN actions, we describe a cellular atlas of Ca2+/calcineurin signaling.

Palmitoylation targets the calcineurin phosphatase to the phosphatidylinositol 4-kinase complex at the plasma membrane.

Calcineurin, the conserved protein phosphatase and target of immunosuppressants, is a critical mediator of Ca2+ signaling. Here, to discover calcineurin-regulated processes we examined an understudied isoform, CNAß1. We show that unlike canonical cytosolic calcineurin, CNAß1 localizes to the plasma membrane and Golgi due to palmitoylation of its divergent C-terminal tail, which is reversed by the ABHD17A depalmitoylase. Palmitoylation targets CNAß1 to a distinct set of membrane-associated interactors including the phosphatidylinositol 4-kinase (PI4KA) complex containing EFR3B, PI4KA, TTC7B and FAM126A. Hydrogen-deuterium exchange reveals multiple calcineurin-PI4KA complex contacts, including a calcineurin-binding peptide motif in the disordered tail of FAM126A, which we establish as a calcineurin substrate. Calcineurin inhibitors decrease PI4P production during Gq-coupled GPCR signaling, suggesting that calcineurin dephosphorylates and promotes PI4KA complex activity. In sum, this work discovers a calcineurin-regulated signaling pathway which highlights the PI4KA complex as a regulatory target and reveals that dynamic palmitoylation confers unique localization, substrate specificity and regulation to CNAß1.

MRBLE-pep Measurements Reveal Accurate Binding Affinities for B56, a PP2A Regulatory Subunit.

Signal transduction pathways rely on dynamic interactions between protein globular domains and short linear motifs (SLiMs). The weak affinities of these interactions are essential to allow fast rewiring of signaling pathways and downstream responses but also pose technical challenges for interaction detection and measurement. We recently developed a technique (MRBLE-pep) that leverages spectrally encoded hydrogel beads to measure binding affinities between a single protein of interest and 48 different peptide sequences in a single small volume. In prior work, we applied it to map the binding specificity landscape between calcineurin and the PxIxIT SLiM (Nguyen, H. Q. et al. Elife 2019, 8). Here, using peptide sequences known to bind the PP2A regulatory subunit B56α, we systematically compare affinities measured by MRBLE-pep or isothermal calorimetry (ITC) and confirm that MRBLE-pep accurately quantifies relative affinity over a wide dynamic range while using a fraction of the material required for traditional methods such as ITC.

Cell Biology: Deciphering the ABCs of SLiMs in G1-CDK Signaling.

A new study uses an elegant in vivo assay to comprehensively characterize the LP docking motif, which determines G1-CDK substrate specificity in fungi. The authors show that LP-cyclin docking strength determines the timing of Sic1 degradation, a key cell cycle event.

Systematic Discovery of Short Linear Motifs Decodes Calcineurin Phosphatase Signaling.

Short linear motifs (SLiMs) drive dynamic protein-protein interactions essential for signaling, but sequence degeneracy and low binding affinities make them difficult to identify. We harnessed unbiased systematic approaches for SLiM discovery to elucidate the regulatory network of calcineurin (CN)/PP2B, the Ca2+-activated phosphatase that recognizes LxVP and PxIxIT motifs. In vitro proteome-wide detection of CN-binding peptides, in vivo SLiM-dependent proximity labeling, and in silico modeling of motif determinants uncovered unanticipated CN interactors, including NOTCH1, which we establish as a CN substrate. Unexpectedly, CN shows SLiM-dependent proximity to centrosomal and nuclear pore complex (NPC) proteins-structures where Ca2+ signaling is largely uncharacterized. CN dephosphorylates human and yeast NPC proteins and promotes accumulation of a nuclear transport reporter, suggesting conserved NPC regulation by CN. The CN network assembled here provides a resource to investigate Ca2+ and CN signaling and demonstrates synergy between experimental and computational methods, establishing a blueprint for examining SLiM-based networks.

A calcineurin-Hoxb13 axis regulates growth mode of mammalian cardiomyocytes.

A major factor in the progression to heart failure in humans is the inability of the adult heart to repair itself after injury. We recently demonstrated that the early postnatal mammalian heart is capable of regeneration following injury through proliferation of preexisting cardiomyocytes1,2 and that Meis1, a three amino acid loop extension (TALE) family homeodomain transcription factor, translocates to cardiomyocyte nuclei shortly after birth and mediates postnatal cell cycle arrest3. Here we report that Hoxb13 acts as a cofactor of Meis1 in postnatal cardiomyocytes. Cardiomyocyte-specific deletion of Hoxb13 can extend the postnatal window of cardiomyocyte proliferation and reactivate the cardiomyocyte cell cycle in the adult heart. Moreover, adult Meis1-Hoxb13 double-knockout hearts display widespread cardiomyocyte mitosis, sarcomere disassembly and improved left ventricular systolic function following myocardial infarction, as demonstrated by echocardiography and magnetic resonance imaging. Chromatin immunoprecipitation with sequencing demonstrates that Meis1 and Hoxb13 act cooperatively to regulate cardiomyocyte maturation and cell cycle. Finally, we show that the calcium-activated protein phosphatase calcineurin dephosphorylates Hoxb13 at serine-204, resulting in its nuclear localization and cell cycle arrest. These results demonstrate that Meis1 and Hoxb13 act cooperatively to regulate cardiomyocyte maturation and proliferation and provide mechanistic insights into the link between hyperplastic and hypertrophic growth of cardiomyocytes.

Protocol for Peptide Synthesis on Spectrally Encoded Beads for MRBLE-pep Assays.

Every living cell relies on signal transduction pathways comprised of protein-protein interactions (PPIs). In many cases, these PPIs are between a folded protein domain and a short linear motif (SLiM) within an unstructured region of a protein. As a result of this small interaction interface (3-10 amino acids), the affinities of SLiM-mediated interactions are typically weak (K ds of ~1-10 µM), allowing physiologically relevant changes in cellular concentrations of either protein partner to dictate changes in occupancy and thereby transmit cellular signals. However, these weak affinities also render detection and quantitative measurement of these interactions challenging and labor intensive. To address this, we recently developed MRBLE-pep, a technology that employs peptide libraries synthesized on spectrally encoded hydrogel beads to allow multiplexed affinity measurements between a protein and many different peptides in parallel. This approach dramatically reduces both the amount of protein and peptide as well as the time required to measure protein-peptide affinities compared to traditional methods. Here, we provide a detailed protocol describing how to: (1) functionalize polyethylene glycol diacrylate (PEG-DA) MRBLE beads with free amine groups, (2) synthesize peptide libraries on functionalized MRBLEs, (3) validate synthesized peptide sequences via MALDI mass spectrometry and quantify evenness of peptide coverage on MRBLEs, (4) use MRBLE-bound peptide libraries in multiplexed protein binding assays, and (5) analyze binding data to determine binding affinities. We anticipate that this protocol should prove useful for other researchers seeking to use MRBLE-pep in their own laboratories as well as for researchers broadly interested in solid-phase peptide synthesis and protein-protein binding assay development.

Identifying New Substrates and Functions for an Old Enzyme: Calcineurin.

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 Ca2+ and calmodulin. This ubiquitously expressed phosphatase controls Ca2+-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.

Quantitative mapping of protein-peptide affinity landscapes using spectrally encoded beads.

Transient, regulated binding of globular protein domains to Short Linear Motifs (SLiMs) in disordered regions of other proteins drives cellular signaling. Mapping the energy landscapes of these interactions is essential for deciphering and perturbing signaling networks but is challenging due to their weak affinities. We present a powerful technology (MRBLE-pep) that simultaneously quantifies protein binding to a library of peptides directly synthesized on beads containing unique spectral codes. Using MRBLE-pep, we systematically probe binding of calcineurin (CN), a conserved protein phosphatase essential for the immune response and target of immunosuppressants, to the PxIxIT SLiM. We discover that flanking residues and post-translational modifications critically contribute to PxIxIT-CN affinity and identify CN-binding peptides based on multiple scaffolds with a wide range of affinities. The quantitative biophysical data provided by this approach will improve computational modeling efforts, elucidate a broad range of weak protein-SLiM interactions, and revolutionize our understanding of signaling networks.

The unique C terminus of the calcineurin isoform CNAß1 confers non-canonical regulation of enzyme activity by Ca2+ and calmodulin.

Calcineurin, the conserved Ca2+/calmodulin-regulated phosphatase and target of immunosuppressants, plays important roles in the circulatory, nervous, and immune systems. Calcineurin activity strictly depends on Ca2+ and Ca2+-bound calmodulin (Ca2+/CaM) to relieve autoinhibition of the catalytic subunit (CNA) by its C terminus. The C terminus contains two regulatory domains, the autoinhibitory domain (AID) and calmodulin-binding domain (CBD), which block the catalytic center and a conserved substrate-binding groove, respectively. However, this mechanism cannot apply to CNAß1, an atypical CNA isoform generated by alternative 3'-end processing, whose divergent C terminus shares the CBD common to all isoforms, but lacks the AID. We present the first biochemical characterization of CNAß1, which is ubiquitously expressed and conserved in vertebrates. We identify a distinct C-terminal autoinhibitory four-residue sequence in CNAß1, 462LAVP465, which competitively inhibits substrate dephosphorylation. In vitro and cell-based assays revealed that the CNAß1-containing holoenzyme, CNß1, is autoinhibited at a single site by either of two inhibitory regions, CBD and LAVP, which block substrate access to the substrate-binding groove. We found that the autoinhibitory segment (AIS), located within the CBD, is progressively removed by Ca2+ and Ca2+/CaM, whereas LAVP remains engaged. This regulatory strategy conferred higher basal and Ca2+-dependent activity to CNß1, decreasing its dependence on CaM, but also limited maximal enzyme activity through persistence of LAVP-mediated autoinhibiton during Ca2+/CaM stimulation. These regulatory properties may underlie observed differences between the biological activities of CNß1 and canonical CNß2. Our insights lay the groundwork for further studies of CNß1, whose physiological substrates are currently unknown.

Calcineurin, the Ca2+-dependent phosphatase, regulates Rga2, a Cdc42 GTPase-activating protein, to modulate pheromone signaling.

Calcineurin, the conserved Ca2+/calmodulin-activated phosphatase, is required for viability during prolonged exposure to pheromone and acts through multiple substrates to down-regulate yeast pheromone signaling. Calcineurin regulates Dig2 and Rod1/Art4 to inhibit mating-induced gene expression and activate receptor internalization, respectively. Recent systematic approaches identified Rga2, a GTPase-activating protein (GAP) for the Cdc42 Rho-type GTPase, as a calcineurin substrate. Here we establish a physiological context for this regulation and show that calcineurin dephosphorylates and positively regulates Rga2 during pheromone signaling. Mating factor activates the Fus3/MAPK kinase, whose substrates induce gene expression, cell cycle arrest, and formation of the mating projection. Our studies demonstrate that Fus3 also phosphorylates Rga2 at inhibitory S/TP sites, which are targeted by Cdks during the cell cycle, and that calcineurin opposes Fus3 to activate Rga2 and decrease Cdc42 signaling. Yeast expressing an Rga2 mutant that is defective for regulation by calcineurin display increased gene expression in response to pheromone. This work is the first to identify cross-talk between Ca2+/calcineurin and Cdc42 signaling and to demonstrate modulation of Cdc42 activity through a GAP during mating.

Using yeast to determine the functional consequences of mutations in the human p53 tumor suppressor gene: An introductory course-based undergraduate research experience in molecular and cell biology.

The opportunity to engage in scientific research is an important, but often neglected, component of undergraduate training in biology. We describe the curriculum for an innovative, course-based undergraduate research experience (CURE) appropriate for a large, introductory cell and molecular biology laboratory class that leverages students' high level of interest in cancer. The course is highly collaborative and emphasizes the analysis and interpretation of original scientific data. During the course, students work in teams to characterize a collection of mutations in the human p53 tumor suppressor gene via expression and analysis in yeast. Initially, student pairs use both qualitative and quantitative assays to assess the ability of their p53 mutant to activate expression of reporter genes, and they localize their mutation within the p53 structure. Through facilitated discussion, students suggest possible molecular explanations for the transactivation defects displayed by their p53 mutants and propose experiments to test these hypotheses that they execute during the second part of the course. They use a western blot to determine whether mutant p53 levels are reduced, a DNA-binding assay to test whether recognition of any of three p53 target sequences is compromised, and fluorescence microscopy to assay nuclear localization. Students studying the same p53 mutant periodically convene to discuss and interpret their combined data. The course culminates in a poster session during which students present their findings to peers, instructors, and the greater biosciences community. Based on our experience, we provide recommendations for the development of similar large introductory lab courses. © 2016 by The International Union of Biochemistry and Molecular Biology, 45(2):161-178, 2017.

Short linear motifs - ex nihilo evolution of protein regulation.

Short sequence motifs are ubiquitous across the three major types of biomolecules: hundreds of classes and thousands of instances of DNA regulatory elements, RNA motifs and protein short linear motifs (SLiMs) have been characterised. The increase in complexity of transcriptional, post-transcriptional and post-translational regulation in higher Eukaryotes has coincided with a significant expansion of motif use. But how did the eukaryotic cell acquire such a vast repertoire of motifs? In this review, we curate the available literature on protein motif evolution and discuss the evidence that suggests SLiMs can be acquired by mutations, insertions and deletions in disordered regions. We propose a mechanism of ex nihilo SLiM evolution - the evolution of a novel SLiM from "nothing" - adding a functional module to a previously non-functional region of protein sequence. In our model, hundreds of motif-binding domains in higher eukaryotic proteins connect simple motif specificities with useful functions to create a large functional motif space. Accessible peptides that match the specificity of these motif-binding domains are continuously created and destroyed by mutations in rapidly evolving disordered regions, creating a dynamic supply of new interactions that may have advantageous phenotypic novelty. This provides a reservoir of diversity to modify existing interaction networks. Evolutionary pressures will act on these motifs to retain beneficial instances. However, most will be lost on an evolutionary timescale as negative selection and genetic drift act on deleterious and neutral motifs respectively. In light of the parallels between the presented model and the evolution of motifs in the regulatory segments of genes and (pre-)mRNAs, we suggest our understanding of regulatory networks would benefit from the creation of a shared model describing the evolution of transcriptional, post-transcriptional and post-translational regulation.

Hcm1 integrates signals from Cdk1 and calcineurin to control cell proliferation.

Cyclin-dependent kinase (Cdk1) orchestrates progression through the cell cycle by coordinating the activities of cell-cycle regulators. Although phosphatases that oppose Cdk1 are likely to be necessary to establish dynamic phosphorylation, specific phosphatases that target most Cdk1 substrates have not been identified. In budding yeast, the transcription factor Hcm1 activates expression of genes that regulate chromosome segregation and is critical for maintaining genome stability. Previously we found that Hcm1 activity and degradation are stimulated by Cdk1 phosphorylation of distinct clusters of sites. Here we show that, upon exposure to environmental stress, the phosphatase calcineurin inhibits Hcm1 by specifically removing activating phosphorylations and that this regulation is important for cells to delay proliferation when they encounter stress. Our work identifies a mechanism by which proliferative signals from Cdk1 are removed in response to stress and suggests that Hcm1 functions as a rheostat that integrates stimulatory and inhibitory signals to control cell proliferation.

A high-enrollment course-based undergraduate research experience improves student conceptions of scientific thinking and ability to interpret data.

We present an innovative course-based undergraduate research experience curriculum focused on the characterization of single point mutations in p53, a tumor suppressor gene that is mutated in more than 50% of human cancers. This course is required of all introductory biology students, so all biology majors engage in a research project as part of their training. Using a set of open-ended written prompts, we found that the course shifts student conceptions of what it means to think like a scientist from novice to more expert-like. Students at the end of the course identified experimental repetition, data analysis, and collaboration as important elements of thinking like a scientist. Course exams revealed that students showed gains in their ability to analyze and interpret data. These data indicate that this course-embedded research experience has a positive impact on the development of students' conceptions and practice of scientific thinking.

Calcineurin regulates the yeast synaptojanin Inp53/Sjl3 during membrane stress.

During hyperosmotic shock, Saccharomyces cerevisiae adjusts to physiological challenges, including large plasma membrane invaginations generated by rapid cell shrinkage. Calcineurin, the Ca(2+)/calmodulin-dependent phosphatase, is normally cytosolic but concentrates in puncta and at sites of polarized growth during intense osmotic stress; inhibition of calcineurin-activated gene expression suggests that restricting its access to substrates tunes calcineurin signaling specificity. Hyperosmotic shock promotes calcineurin binding to and dephosphorylation of the PI(4,5)P2 phosphatase synaptojanin/Inp53/Sjl3 and causes dramatic calcineurin-dependent reorganization of PI(4,5)P2-enriched membrane domains. Inp53 normally promotes sorting at the trans-Golgi network but localizes to cortical actin patches in osmotically stressed cells. By activating Inp53, calcineurin repolarizes the actin cytoskeleton and maintains normal plasma membrane morphology in synaptojanin-limited cells. In response to hyperosmotic shock and calcineurin-dependent regulation, Inp53 shifts from associating predominantly with clathrin to interacting with endocytic proteins Sla1, Bzz1, and Bsp1, suggesting that Inp53 mediates stress-specific endocytic events. This response has physiological and molecular similarities to calcineurin-regulated activity-dependent bulk endocytosis in neurons, which retrieves a bolus of plasma membrane deposited by synaptic vesicle fusion. We propose that activation of Ca(2+)/calcineurin and PI(4,5)P2 signaling to regulate endocytosis is a fundamental and conserved response to excess membrane in eukaryotic cells.

The calcineurin signaling network evolves via conserved kinase-phosphatase modules that transcend substrate identity.

To define a functional network for calcineurin, the conserved Ca(2+)/calmodulin-regulated phosphatase, we systematically identified its substrates in S. cerevisiae using phosphoproteomics and bioinformatics, followed by copurification and dephosphorylation assays. This study establishes new calcineurin functions and reveals mechanisms that shape calcineurin network evolution. Analyses of closely related yeasts show that many proteins were recently recruited to the network by acquiring a calcineurin-recognition motif. Calcineurin substrates in yeast and mammals are distinct due to network rewiring but, surprisingly, are phosphorylated by similar kinases. We postulate that corecognition of conserved substrate features, including phosphorylation and docking motifs, preserves calcineurin-kinase opposition during evolution. One example we document is a composite docking site that confers substrate recognition by both calcineurin and MAPK. We propose that conserved kinase-phosphatase pairs define the architecture of signaling networks and allow other connections between kinases and phosphatases to develop that establish common regulatory motifs in signaling networks.