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

Calcineurin (CN), the only Ca2+/calmodulin-activated protein phosphatase and target of immunosuppressant drugs, dephosphorylates substrates within membrane-associated Ca2+ microdomains. CN binds to substrates and regulators via short linear motifs (SLIMs), PxIxIT and LxVP, which have distinct docking sites on CN. PxIxIT binding to CN is Ca2+-independent and affects its distribution, while LxVP associates only with the active enzyme and promotes catalysis.

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.

A cellular atlas of calcineurin signaling.

Intracellular Ca signals are temporally controlled and spatially restricted. Signaling occurs adjacent to sites of Ca entry and/or release, where Ca-dependent effectors and their substrates co-localize to form signaling microdomains. Here we review signaling by calcineurin, the Ca/calmodulin regulated protein phosphatase and target of immunosuppressant drugs, Cyclosporin A and FK506.

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.

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 Ca signaling. Here, to discover calcineurin-regulated processes we examined an understudied isoform, CNAβ1. We show that unlike canonical cytosolic calcineurin, CNAβ1 localizes to the plasma membrane and Golgi due to palmitoylation of its divergent C-terminal tail, which is reversed by the ABHD17A depalmitoylase.

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 ( s 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.

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 Ca-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.

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 cardiomyocytes 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 arrest. Here we report that Hoxb13 acts as a cofactor of Meis1 in postnatal cardiomyocytes.

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 Ca and calmodulin.

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.

The unique C terminus of the calcineurin isoform CNAβ1 confers non-canonical regulation of enzyme activity by Ca and calmodulin.

Calcineurin, the conserved Ca/calmodulin-regulated phosphatase and target of immunosuppressants, plays important roles in the circulatory, nervous, and immune systems. Calcineurin activity strictly depends on Ca and Ca-bound calmodulin (Ca/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.

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

Calcineurin, the conserved Ca/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.

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.

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.

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.

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.

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.

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.

Specific α-arrestins negatively regulate Saccharomyces cerevisiae pheromone response by down-modulating the G-protein-coupled receptor Ste2.

G-protein-coupled receptors (GPCRs) are integral membrane proteins that initiate responses to extracellular stimuli by mediating ligand-dependent activation of cognate heterotrimeric G proteins. In yeast, occupancy of GPCR Ste2 by peptide pheromone α-factor initiates signaling by releasing a stimulatory Gβγ complex (Ste4-Ste18) from its inhibitory Gα subunit (Gpa1). Prolonged pathway stimulation is detrimental, and feedback mechanisms have evolved that act at the receptor level to limit the duration of signaling and stimulate recovery from pheromone-induced G1 arrest, including upregulation of the expression of an α-factor-degrading protease (Bar1), a regulator of G-protein signaling protein (Sst2) that stimulates Gpa1-GTP hydrolysis, and Gpa1 itself.

Whi3, an S. cerevisiae RNA-binding protein, is a component of stress granules that regulates levels of its target mRNAs.

RNA binding proteins (RBPs) are vital to the regulation of mRNA transcripts, and can alter mRNA localization, degradation, translation, and storage. Whi3 was originally identified in a screen for small cell size mutants, and has since been characterized as an RBP. The identification of Whi3-interacting mRNAs involved in mediating cellular responses to stress suggested that Whi3 might be involved in stress-responsive RNA processing.

A calcineurin-dependent switch controls the trafficking function of α-arrestin Aly1/Art6.

Proper regulation of plasma membrane protein endocytosis by external stimuli is required for cell growth and survival. In yeast, excess levels of certain nutrients induce endocytosis of the cognate permeases to prevent toxic accumulation of metabolites. The α-arrestins, a family of trafficking adaptors, stimulate ubiquitin-dependent and clathrin-mediated endocytosis by interacting with both a client permease and the ubiquitin ligase Rsp5.

The molecular mechanism of substrate engagement and immunosuppressant inhibition of calcineurin.

Ser/thr phosphatases dephosphorylate their targets with high specificity, yet the structural and sequence determinants of phosphosite recognition are poorly understood. Calcineurin (CN) is a conserved Ca(2+)/calmodulin-dependent ser/thr phosphatase and the target of immunosuppressants, FK506 and cyclosporin A (CSA). To investigate CN substrate recognition we used X-ray crystallography, biochemistry, modeling, and in vivo experiments to study A238L, a viral protein inhibitor of CN.

Regulation of cation balance in Saccharomyces cerevisiae.

All living organisms require nutrient minerals for growth and have developed mechanisms to acquire, utilize, and store nutrient minerals effectively. In the aqueous cellular environment, these elements exist as charged ions that, together with protons and hydroxide ions, facilitate biochemical reactions and establish the electrochemical gradients across membranes that drive cellular processes such as transport and ATP synthesis. Metal ions serve as essential enzyme cofactors and perform both structural and signaling roles within cells.