Martha S. Cyert - US grants

9357017 Cyert This is an NSF Young Investigator Award to Dr. Martha S. Cyert. The overall objective of her research is to elucidate the physiological functions and substrates of the phosphoprotein phosphatase, calcineurin in the budding yeast, Saccharomyces cerevisciae. A major focus of her research will be to examine the role and mechanism of action of calcineurin in the process of adaptation to pheromone and recovery from pheromone-induced growth arrest. She will also examine the possible involvement of calcineurin in regulation of the cell cycle. Her experimental approaches include techniques of molecular genetics and biochemistry. ***

DESCRIPTION: Calcineurin (or PP2B), a conserved protein phosphatase that is activated by Ca2+ and calmodulin, participates in signal transduction by effecting Ca2+-dependent changes in the phosphorylation state of cellular proteins. Calcineurin is a critical regulator of mammalian T-cells, and inhibitors of this phosphatase (FK506, cyclosporinA) are powerful immunosuppressants. Our goal is to understand the functions of Ca2+-dependent signaling in yeast, with particular emphasis on the role of calcineurin. Our studies of yeast should provide insight into the mechanisms and functions of Ca2+-dependent signaling, especially calcineurin-mediated signaling, in all eukaryotic cells. The mating response provides an opportunity to examine the role of Ca2+-dependent signaling in yeast. Addition of pheromone to haploid yeast cells produces a rise in cytosolic Ca2+, and activation of calcineurin and calmodulin-stimulated kinase (CaM kinase) by this Ca2+ signal is required for viability. One essential function of calcineurin is to activate the Crz1p transcription factor, although the critical targets of calcineurin/CRZ1-dependent transcriptional regulation have not yet been identified. Calcineurin also performs additional, as yet uncharacterized, essential functions in pheromone-treated cells. The role of CaM kinase in the pheromone response is not understood. The proposed experiments will examine the functions of calcineurin and CaM kinase in cells exposed to pheromone. We will: 1) Characterize the mechanism of pheromone-induced transcription mediated by calcineurin and Crz1p. The mode of Crz1p regulation by calcineurin will be elucidated and additional gene products that participate in the calcineurin/CRZ1 response pathway will be identified. 2) Identify gene products that modify the requirement for Ca2+-dependent signaling during incubation with pheromone. Components of both calcineurin and CaM kinase-dependent responses will be studied. 3) Identify targets of pheromone-induced calcineurin-dependent transcriptional regulation and determine the contribution of these genes to cell viability. Microarray technology will be employed to examine all 6000 yeast genes for calcineurin-dependent changes in gene expression. 4) Identify substrates of calcineurin by identifying gene products that physically interact with calcineurin in vivo and by testing specific candidate proteins in vitro for calcineurin-dependent dephosphorylation.

DESCRIPTION (provided by applicant): Calcineurin (CN) is a highly conserved ser/thr protein phosphatase that is regulated by Ca2+ and calmodulin, and thus mediates Ca2+-dependent changes in phosphorylation in vivo. In humans, CN regulates immune cell function, promotes development of muscle, vascular and pancreatic tissues, and modulates learning and memory in the brain. CN inhibitors, FK506 and cyclosporin A, are in wide clinical use as immunosupressants, and perturbation of CN-dependent signaling is associated with many pathophyisological conditions including Down's syndrome, heart disease, schizophrenia, cancer, and diabetes. Identification of CN substrates is key to understanding and modulating this wide range of physiological activities. This proposal seeks to identify the functions and substrates of CN in yeast. Previous studies demonstrated that CN promotes yeast cell survival during environmental stress, and identified several substrates required for this response. However, additional functions of CN, for which substrates have not yet been identified, are also indicated. Now approaches for global identification of CN substrates in yeast are proposed. This simple eukaryote offers many experimental advantages;these studies will provide insights into CN-dependent signaling that are relevant to all cells and will pave the way for global identification of phosphatase substrates in other organisms. The specific aims of the proposed work are to: 1) Identify CN substrates comprehensively, using two complementary, proteomic approaches. First, proteins dephosphorylated by CN will be identified with a novel in vitro assay that uses phosphorylated protein microarrays. Second, phosphopeptides that are enriched in extracts of CN-deficient cells will be identified by label-free, quantitative mass spectrometry. Functional characterization of the resulting collection of substrates will expand our understanding of CN regulatory activities. Biochemical and bioinformatic analyses of these substrates will further define sequences involved in CN substrate recognition. 2) Identify critical regions of CN by identifying mutations that alter its activity and interactions with substrates in vivo. These mutants will provide new tools for investigating CN function and substrate specificity in many organisms. 3) Identify the role of CN-interacting proteins in CN signaling pathways. Characterization of CN substrates is essential for defining its physiological functions. The role of CN substrates, Slm1 and Slm2, in TOR-dependent activation of Ypk1/2 kinases during heat stress, will be investigated, and the interaction of CN with Whi3, an RNA binding protein that regulates entry into the cell cycle will be examined. Preliminary observations suggest roles for CN and Whi3 in yeast stress granules, which form in response to glucose starvation and contain non-translating mRNAs complexed with initiation factors. PUBLIC HEALTH RELEVANCE: This project examines the functions of a highly conserved protein, calcineurin. Calcineurin regulates many processes in humans in response to changes in Ca2+, and alterations in calcineurin activity occur in immune disorders, diabetes, cancer, heart disease and schizophrenia. This research studies, at the biochemical level, the functions of calcineurin in a simple, experimentally tractable organism, i.e. yeast, and will serve as a model for understanding the actions of calcineurin in human cells.

calcium ion; calcineurin; intermolecular interaction; biomedical resource;

protein protein interaction; calcineurin; phosphoproteins; posttranslational modifications; phosphorylation; biomedical resource;

protein protein interaction; calcineurin; biomedical resource;

protein structure function; protein kinase C; biomedical resource;

[unreadable] DESCRIPTION (provided by applicant): In mammals, calcineurin, the Ca2+/calmodulin dependent protein phosphatase, regulates immune cell activity, promotes heart and blood vessel development, mediates cardiac muscle response to stress, and modulates learning and memory in the brain. Calcineurin inhibitors, FK506 and cyclosporin A, are used clinically as immunosupressants, and, in animal models, reduce cardiac hypertrophy. As a key effector of Ca2+/calmodulin dependent signaling, detailed analysis of calcineurin function has the potential to impact many aspects of human health and development. In S. cerevisiae, calcineurin promotes survival during environmental stress, and in response to cell wall damage. A major role of calcineurin is to dephosphorylate and activate the Crz1p transcription factor, using mechanism analogous to calcineurin regulation of the mammalian transcription factor, NFAT. Additional calcineurin-mediated events that promote yeast survival during environmental stress are less well characterized, and are the focus of this application. This research aims to identify comprehensively the functions and components of calcineurin-dependent signaling pathways. We address these questions using S. cerevisiae, because of many experimental advantages offered by this simple eukaryotic organism, but strive to establish general principles that apply to calcineurin-dependent signaling in all cells. Previously, we exploited a particular feature of calcineurin signaling, i.e. the requirement for calcineurin to interact directly with its substrates via a docking site that is distinct from residues that are dephosphorylated, to identify several new components of calcineurin-mediated signaling pathways including 3 novel substrates: Slm1p, Slm2p and Hph1p. Here we propose to characterize further the mechanism by which calcineurin interacts with its substrates, which is evolutionary conserved. We will also apply genetic, genomic, and proteomic approaches to the identification of additional substrates and regulators of calcineurin. Specifically we will 1) Characterize calcineurin-docking sites in substrates and examine the impact of calcineurin-substrate affinity on signaling. 2) Examine the effect of specific point mutations in calcineurin on substrate interaction. 3) Identify the role of calcineurin interacting proteins in calcineurin signaling pathways. 4) Identify additional calcineurin substrates using novel proteomic and genomic screening methods. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Our program will direct predoctoral training in cellular and molecular biology at Stanford University. Exceptional students will be provided with a common training experience that includes 1. Formal course work from a menu of both foundational and specialized courses taught collaboratively by the training faculty;2. Seminars and journal clubs;and 3. Exciting thesis research opportunities with exceptional faculty preceptors. These elements are woven together to provide all trainees with a common training experience. In addition to the common program elements mentioned above, there are 3 programmatic components exclusively for our trainees. We have initiated a CBM Research Symposium and a CMS Science/Social. These "enrichment" components will enhance program identity and cohesiveness. The Program Directors and the Executive Committee to assure that all committee meetings, course requirements, and exams are completed on schedule and that thesis progress is satisfactory. They will carefully monitor student progress and compliance with programmatic schedules. We expect that our students will continue to be highly successful in careers in biomedical research and many will become leaders. RELEVANCE: The objectives of this program are to attract the best students, to train them in cutting edge biomedical research through work with excellent faculty, and to produce tomorrow's leaders in biomedical research.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Hph1 and Hph2 are homologous integral endoplasmic reticulum (ER) membrane proteins required for Saccharomyces cerevisiae survival under environmental stress conditions. To investigate the molecular functions of Hph1 and Hph2, we carried out a split-ubiquitin-membrane-based yeast two-hybrid screen and identified their interactions with Sec71, a subunit of the Sec63/Sec62 complex, which mediates posttranslational translocation of proteins into the ER. Hph1 and Hph2 likely function in posttranslational translocation, as they interact with other Sec63/Sec62 complex subunits, i.e., Sec72, Sec62, and Sec63. hph1 hph2 cells display reduced vacuole acidification;increased instability of Vph1, a subunit of vacuolar proton ATPase (V-ATPase);and growth defects similar to those of mutants lacking V-ATPase activity. sec71 cells exhibit similar phenotypes, indicating that Hph1/Hph2 and the Sec63/Sec62 complex function during V-ATPase biogenesis. Hph1/Hph2 and the Sec63/Sec62 complex may act together in this process, as vacuolar acidification and Vph1 stability are compromised to the same extent in hph1 hph2 and hph1 hph2 sec71 cells. In contrast, loss of Pkr1, an ER protein that promotes posttranslocation assembly of Vph1 with V-ATPase subunits, further exacerbates hph1 hph2 phenotypes, suggesting that Hph1 and Hph2 function independently of Pkr1-mediated V-ATPase assembly. We propose that Hph1 and Hph2 aid Sec63/Sec62-mediated translocation of specific proteins, including factors that promote efficient biogenesis of V-ATPase, to support yeast cell survival during environmental stress.

? DESCRIPTION (provided by applicant): The absence of experimental and computational tools for global identification of phosphatase substrates leaves a major gap in our understanding of cellular regulatory networks and prevents systems-level analyses of phosphatase signaling. The proposal addresses this knowledge gap by systematically identifying targets of calcineurin (CN), the ubiquitous Ca2+/calmodulin-dependent protein phosphatase and target of immunosuppressants, FK506 and cyclosporin A. Only 25 substrates are currently attributed to CN in mammals--the same as in yeast, whose proteome is ~tenfold smaller, and chronic inhibition of CN in patient's cause's side-effects by disrupting unidentified regulatory events. This suggests that in humans, the majority of CN substrates, and thus its regulatory functions, remain to be identified. Recent insights into CN substrate recognition drive our approach: CN binds to short linear motifs (SLiMs), termed PxIxIT and LxVP, which can occur hundreds of residues away substrate dephosphorylation sites. Mutating these motifs or preventing their binding to conserved surfaces on CN, i.e. with FK506, CysA, or viral inhibitor A238L, blocks dephosphorylation. Building upon our previous success establishing the CN signaling network in yeast, we are determining human CN substrates by systematically identifying CN- binding peptides in the proteome, which is challenging due to their low affinities and degenerate sequences. This work aims to 1) systematically discover human PxIxIT and LxVP-type sequences by synergizing experimental and computational methods. CN-binding sequences are directly selected from phage display libraries that contain all disordered regions of the human proteome, where SLiMs reside. PxIxITs are also identified in silico by leveraging their characteristic structural features and using a novel method to predict binding to the conserved PxIxIT-docking surface on CN. Candidate sequences are validated for CN-binding in vitro, and in Aim 2 their parent proteins are tested for interaction with and/or dephosphorylation by CN in cultured animal cells. In Aim 3 we characterize the functions and binding mode of a newly discovered CN-binding motif, YLxxLF, which may identify a distinct class of CN- interacting proteins and provide avenues for selective disruption of CN signaling events. This systems- level analysis of CN signaling in humans will ultimately identify intersections with other regulatory networks that can be exploited therapeutically, such as providing strategies to ameliorate immunosuppressant side effects. Furthermore, this work will provide fundamental insights into how phosphatases achieve specificity and will create a critical new resource for researchers studying Ca2+ or phosphorylation-dependent regulation of protein function.

Project Summary Comprehensive mapping and monitoring of signaling pathways are essential for achieving the goals of precision medicine. Calcineurin (CN), the serine/threonine protein phosphatase and target of immunosuppressants, FK506 and CysA, is a critical mediator of Ca2+-dependent signaling with multiple functions relevant to human health. However, many CN-regulated substrates and processes remain to be elucidated. This proposal focuses on CNb1, a CN isoform with unique properties and functions that is conserved in vertebrates and broadly expressed in human tissues, but significantly under-studied. By addressing fundamental gaps in knowledge about CNb1, this research will discover novel CN-regulated signaling pathways, elucidate roles for CNb1 in healthy and diseased cells, and ultimately identify methods to therapeutically manipulate the enzyme. Our studies show that its unique C-tail, generated by alternative 3? end mRNA processing, confers distinct regulation and localization to CNb1: In vitro, maximal phosphatase activity of CNb1 in the presence of Ca2+/calmodulin is significantly lower than that of canonical CNb2, due to a unique C-terminal auto-inhibitory sequence that occludes an essential substrate binding site. In vivo, CNb1 localizes to membrane compartments, including the plasma membrane and Golgi, in contrast to canonical CN isoforms, which are primarily cytosolic. We show that lipidation of conserved cysteines in the CNAb1 C-tail promotes membrane association and that palmitoylation of CNAb1 is dynamic, suggesting a novel mechanism for regulating its distribution and activity in cells that will be examined in Aim 1. CNb1 does not dephosphorylate NFAT transcription factors and its substrates are currently unknown. Our central hypothesis is that by mapping CNb1-regulated signaling pathways we will uncover unique functions for CN at membranes and provide critical new insights into CN regulation in a broad range of tissues and processes. Our preliminary data, which suggests that CNb1 regulates synthesis of phosphatidylinositol 4-phosphate (PI4P) at the PM during GPCR signaling by dephosphorylating FAM126A, whose genetic disruption gives rise to hypomyelination and congenital cataracts (HCC), supports this hypothesis, which is further tested in Aim 2. We also propose innovative approaches, coupling TurboID for proximity labeling over fast time frames with computational identification of CN-binding peptides, to systematically discover additional CNb1 substrates and thus map this enzymes?s unique signaling network in Aim 3. We anticipate that this knowledge will have therapeutic applications for pathologies to which CN signaling contributes, and will create a critical new resource for researchers studying Ca2+- or phosphorylation-dependent signaling.

Project Summary All eukaryotes use phosphorylation-based signaling networks, composed of protein kinases and phosphatases, to regulate cellular processes. While global information about kinase signaling has exploded, knowledge of phosphatases has lagged behind. The lack of systematic, unbiased approaches to analyze phosphatase signaling leaves a major gap in our understanding of cellular regulatory networks. For >25 years, my research on calcineurin, the conserved Ca2+/calmodulin-regulated phosphatase, in S. cerevisiae and humans has directly addressed this issue. Calcineurin is ubiquitously expressed and plays critical regulatory roles in the cardiovascular, nervous and immune systems (1); however, only ~70 proteins are currently established as calcineurin substrates (2). Calcineurin dephosphorylates the Nuclear Factor of Activated T-cells (NFAT) transcription factors to activate the adaptive immune response (3), and calcineurin inhibitors, FK506 and cyclosporin A, are commonly prescribed as immunosuppressants (4). However, these drugs cause unwanted effects, including hypertension, diabetes, and seizures by inhibiting calcineurin in non-immune tissues (5), highlighting the need to map human calcineurin signaling pathways systematically. My work elucidates calcineurin signaling through novel approaches based on the discovery of short linear motifs (SLiMs): short degenerate peptide sequences found within regions of intrinsic disorder that determine specific, low- affinity interactions that are essential for signaling (6). Using experimental and in silico SLiM-based methods, we recently defined the human calcineurin signaling network (2). This work uncovered conserved regulation of nuclear pore complexes by calcineurin and showed unexpected calcineurin proximity to centrosomes (2). Future efforts will elucidate Ca2+ and calcineurin signaling at these compartments using a combination of methods that include phosphoproteomics and proximity labeling with faster-labeling biotin ligases (miniTurbo (33), Split TurboID). Fluorescent sensors will be used to probe Ca2+ signals at these locations in vivo. Calcineurin functions at membranes will be analyzed by focusing on CNb1, a little-studied isoform with distinct localization and regulation conferred by its unique, lipidated C-terminal sequences (10). Regulation of CNb1 activity via dynamic palmitoylation will be examined, and CNb1-specific signaling pathways, including regulation of PI4P synthesis, will be established to achieve a comprehensive and mechanistic understanding of this enzyme. Finally, the novel technology, MRBLE-PEP (Microspheres Ratiometric Barcode Lanthenide Encoding coupled to PEPtides) (11), will be developed for quantitative analysis and discovery of SLiMs. Overall this research aims to map human calcineurin signaling pathways systematically and to advance our understanding of SLiM-based signaling more broadly.