Dr. Martha Cyert directs a research lab that studies Ca2+-dependent signal transduction, focusing on calcineurin, the highly conserved Ca2+/calmodulin-regulated protein phosphatase that plays critical roles in muscle, immune and neural cells. Dr. Cyert pioneered studies of yeast calcineurin, where her work elucidated conserved aspects of substrate recognition and mechanisms by which the signaling network evolves. Her studies on human calcineurin uncovered the mechanism by which immunosuppressant drugs, FK506 and cyclosporine A, inhibit this enzyme. Current research focuses on establishing the human calcineurin signaling network using both experimental and computational approaches to systematically identify calcineurin targets . Professor Cyert is also an active educator. She received the Dean’s Award for Outstanding Teaching, developed an innovative, inquiry-based, introductory laboratory course that utilizes S.cerevisiae, and a summer transition program for incoming freshman from under resourced schools. She was Senior Associate Vice Provost for Undergraduate Education from 2010-13, serves on the Education committee of the American Society for Cell Biology, and has taught in Cell Biology workshops sponsored by the ASCB in Africa. She directs an NIH-funded graduate training program in Cell and Molecular Biology and is a standing member of the TWD-A study section at NIH. Dr. Cyert is a member of the Stanford Cardiovascular and Bio-X Institutes and served on the Public Affairs Advisory Council for ASBMB. She has been awarded fellowships from the American Cancer Society, the Life Sciences Research Foundation and the Lucille P. Markey Charitable Trust, and was named by Stanford University as a Terman Fellow, a Gabilan Fellow, and as the Thomas W. and Susan B. Ford University Fellow in Undergraduate Education.
Senior Associate Vice Provost for Undergraduate Education, Stanfiord University (2010 - 2013)
Director, Graduate training program in Cell and Molecular Biology, Stanford Unviersity (2009 - Present)
Honors & Awards
Scholar, Lucille P. Markey Charitable Trust (1997)
Terman Fellow, Stanford University (2000)
Dean's Award for Outstanding Teaching, Stanford University (2004)
Thomas W. and Susan B. Ford University Fellow in Undergraduate Education, Stanford University (2012-present)
Gabilan Fellow, Stanford University (2014-present)
Boards, Advisory Committees, Professional Organizations
Public Affairs Advisory Council, Ameican Society for Biochemisty and Molecular Biology (2015 - Present)
Advisory Board, Leland Scholars Program, Stanford University (2014 - Present)
Organizer, Protein Phosphatases FASEB condference (2014 - Present)
Education Committee, American Society for Cell Biology (2010 - Present)
Organizer, Cell Biology of Yeasts Conference, Cold Spring Harbor Laboratory (2010 - Present)
Editorial Board, Molecular and Cellular Biology (2009 - Present)
Leadership Council, BioX (2004 - Present)
Organizer, International Symposium Calcium Binding Proteins (2002 - Present)
Postdoctoral Fellow, University of California, Berkeley, Biochemistry (1992)
Ph D., UCSF, Genetics (1988)
A.B., Harvard University, Biochemistry (1980)
Community and International Work
African Regional Workshops on the Cell Biology of Infectious Pathogens, Ghana
American Society for Cell Biology
Opportunities for Student Involvement
Current Research and Scholarly Interests
1. MAPPING THE HUMAN CALCINEURIN PHOSPHATASE SIGNALING NETWORK THROUGH GLOBAL IDENTIFICATION OF SHORT LINEAR MOTIFS THAT MEDIATE SUBSTRATE RECOGNITION.
Systems-level analyses of phosphorylation-based signaling networks has transformed our understanding of kinase function, but knowledge of phosphatase signaling has lagged behind, primarily because global approaches to identify phosphatase substrates are lacking. Calcineurin, the conserved Ca2+/calmodulin-dependent protein phosphatase and target of immunosuppressants, FK506 and Cyclosporin A, is ubiquitously expressed, and critically regulates Ca2+-dependent processes in the immune system, heart, and brain. However, in the literature only 27 substrates are attributed to calcineurin. Systematic identification of calcineurin targets is now feasible due to insights into its conserved mechanism of substrate recognition. Calcineurin acts on phosphosites with little primary sequence similarity; thus specificity is not encoded within regions contiguous to the phosphosite. Rather, the enzyme binds to short linear motifs (SLiMs),“PxIxIT” and “LxVP”, which can occur hundreds of residues away from dephosphorylation sites. CsA , FK506 and the viral A238L protein inhibit calcineurin by blocking SLiM binding to conserved surfaces on the enzyme. SLiMs are a growing class of sequences that localize within intrinsically disordered regions, i.e. flexible protein domains that lack a defined structure. SLiMs mediate most protein-protein interactions in cells and evolve rapidly to mediate rewiring of signaling networks, including that of calcineurin. However, degenerate sequences and low affinities for their target domains make SLiMs challenging to identify. We are using novel experimental and computational approaches to identify calcineurin-binding SLiMs systematically in the human proteome. In collaboration with Ylva Ivarsson, Uppsala Unversity, Proteome peptide Phage Display (ProP-PD) was used to directly select calcineurin-binding sequences of the PxIxIT and LxVP types from predicted disordered regions of the human proteome. We also developed a novel computational tool to predict PxIxIT sequences, which makes use of their characteristic structural features (i.e. intrinsic disorder and beta strand formation), and predicts binding to the conserved PxIxIT-docking surface on CNA, the calcineurin catalytic subunit. Sequences identified either experimentally or computationally are validated using a high throughput calcineurin-binding assay, and their parent proteins tested for co-immunoprecipitation with calcineurin in HEK-293 cells. These studies have identified a new calcineurin substrate, C16Orf74, which is a marker for invasive bladder cancer and defined PxIxIT sites in calcineurin substrates, KSR2 and amphiphysin. Furthermore, more than 50 new targets for calcineurin have been identified, including ion channels, kinases, transcription factors and receptors, that reveal points of cross-talk between calcineurin and other signaling pathways in human cells. This basic approach can be broadly applied to systematic characterization of any SLiM-based signaling network.
2. FUNCTIONAL STUDIES OF HUMAN CNAβ1 SPLICE VARIANT
Calcineurin is tightly controlled by Ca2+ and calmodulin, which activate the enzyme by relieving auto-inhibition of the active site, and revealing a critical binding pocket for "LxVP" substrate motifs. We are studying regulation of a conserved splice variant of the human CNAβ gene, CNAβ1, which promotes cardiac regeneration in vivo. The CNAβ1 C-terminus contains an LxVP sequence, which we showed auto-inhibits phosphatase activity by blocking substrate engagement. CNAβ1 has distinct enzymatic properties, and is relatively independent of calmodulin. Functional studies are identifying and characterizing unique protein partners for CNAβ1 and its substrates in cardiovascular and endocrine signaling.
- Introduction to Data Management and Analysis for Genomics Research
BIOS 264 (Win)
- Introduction to Laboratory Research in Cell and Molecular Biology
BIO 45 (Aut, Win)
- Introduction to Problem Solving in Biology
BIO 60 (Spr)
Independent Studies (9)
- Advanced Research Laboratory in Experimental Biology
BIO 199 (Aut, Win, Spr, Sum)
- Directed Reading in Biology
BIO 198 (Aut, Win, Spr, Sum)
- Directed Reading in Cancer Biology
CBIO 299 (Aut, Sum)
- Graduate Research
BIO 300 (Aut, Win, Spr, Sum)
- Graduate Research
CBIO 399 (Aut, Sum)
- Out-of-Department Advanced Research Laboratory in Experimental Biology
BIO 199X (Win, Spr, Sum)
- Out-of-Department Directed Reading
BIO 198X (Aut, Win, Spr, Sum)
- Out-of-Department Graduate Research
BIO 300X (Win, Spr, Sum)
- Teaching of Biology
BIO 290 (Aut, Win, Spr)
- Advanced Research Laboratory in Experimental Biology
- Prior Year Courses
Graduate and Fellowship Programs
Biology (School of Humanities and Sciences) (Phd Program)
- Short linear motifs - ex nihilo evolution of protein regulation CELL COMMUNICATION AND SIGNALING 2015; 13
Hcm1 integrates signals from Cdk1 and calcineurin to control cell proliferation
MOLECULAR BIOLOGY OF THE CELL
2015; 26 (20): 3570-3577
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.
View details for DOI 10.1091/mbc.E15-07-0469
View details for Web of Science ID 000363036400003
View details for PubMedID 26269584
A high-enrollment course-based undergraduate research experience improves student conceptions of scientific thinking and ability to interpret data.
CBE life sciences education
2015; 14 (2)
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.
View details for DOI 10.1187/cbe.14-05-0092
View details for PubMedID 26033869
- A High-Enrollment Course-Based Undergraduate Research Experience Improves Student Conceptions of Scientific Thinking and Ability to Interpret Data CBE-LIFE SCIENCES EDUCATION 2015; 14 (2)
Calcineurin regulates the yeast synaptojanin Inp53/Sjl3 during membrane stress
MOLECULAR BIOLOGY OF THE CELL
2015; 26 (4): 769-785
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.
View details for DOI 10.1091/mbc.E14-05-1019
View details for Web of Science ID 000351945000016
The Calcineurin Signaling Network Evolves via Conserved Kinase-Phosphatase Modules that Transcend Substrate Identity
2014; 55 (3): 422-435
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.
View details for DOI 10.1016/j.molcel.2014.05.012
View details for Web of Science ID 000340646600009
View details for PubMedID 24930733
- Specific alpha-Arrestins Negatively Regulate Saccharomyces cerevisiae Pheromone Response by Down-Modulating the G-Protein-Coupled Receptor Ste2 MOLECULAR AND CELLULAR BIOLOGY 2014; 34 (14): 2660-2681
- Whi3, an S. cerevisiae RNA-Binding Protein, Is a Component of Stress Granules That Regulates Levels of Its Target mRNAs PLOS ONE 2013; 8 (12)
A Calcineurin-dependent Switch Controls the Trafficking Function of alpha-Arrestin Aly1/Art6
JOURNAL OF BIOLOGICAL CHEMISTRY
2013; 288 (33): 24063-24080
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. However, the molecular mechanisms that control α-arrestin function are not well understood. Here, we show that α-arrestin Aly1/Art6 is a phosphoprotein that specifically interacts with and is dephosphorylated by the Ca(2+)- and calmodulin-dependent phosphoprotein phosphatase calcineurin/PP2B. Dephosphorylation of Aly1 by calcineurin at a subset of phospho-sites is required for Aly1-mediated trafficking of the aspartic acid and glutamic acid transporter Dip5 to the vacuole, but it does not alter Rsp5 binding, ubiquitinylation, or stability of Aly1. In addition, dephosphorylation of Aly1 by calcineurin does not regulate the ability of Aly1 to promote the intracellular sorting of the general amino acid permease Gap1. These results suggest that phosphorylation of Aly1 inhibits its vacuolar trafficking function and, conversely, that dephosphorylation of Aly1 by calcineurin serves as a regulatory switch to promote Aly1-mediated trafficking to the vacuole.
View details for DOI 10.1074/jbc.M113.478511
View details for Web of Science ID 000330611400043
View details for PubMedID 23824189
Regulation of Cation Balance in Saccharomyces cerevisiae
2013; 193 (3): 677-713
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. However, because these ions can also be toxic, cells have developed sophisticated homeostatic mechanisms to regulate their levels and avoid toxicity. Studies in Saccharomyces cerevisiae have characterized many of the gene products and processes responsible for acquiring, utilizing, storing, and regulating levels of these ions. Findings in this model organism have often allowed the corresponding machinery in humans to be identified and have provided insights into diseases that result from defects in ion homeostasis. This review summarizes our current understanding of how cation balance is achieved and modulated in baker's yeast. Control of intracellular pH is discussed, as well as uptake, storage, and efflux mechanisms for the alkali metal cations, Na(+) and K(+), the divalent cations, Ca(2+) and Mg(2+), and the trace metal ions, Fe(2+), Zn(2+), Cu(2+), and Mn(2+). Signal transduction pathways that are regulated by pH and Ca(2+) are reviewed, as well as the mechanisms that allow cells to maintain appropriate intracellular cation concentrations when challenged by extreme conditions, i.e., either limited availability or toxic levels in the environment.
View details for DOI 10.1534/genetics.112.147207
View details for Web of Science ID 000315920000003
View details for PubMedID 23463800
The Molecular Mechanism of Substrate Engagement and Immunosuppressant Inhibition of Calcineurin
2013; 11 (2)
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. We show that A238L competitively inhibits CN by occupying a critical substrate recognition site, while leaving the catalytic center fully accessible. Critically, the 1.7 Å structure of the A238L-CN complex reveals how CN recognizes residues in A238L that are analogous to a substrate motif, "LxVP." The structure enabled modeling of a peptide substrate bound to CN, which predicts substrate interactions beyond the catalytic center. Finally, this study establishes that "LxVP" sequences and immunosuppressants bind to the identical site on CN. Thus, FK506, CSA, and A238L all prevent "LxVP"-mediated substrate recognition by CN, highlighting the importance of this interaction for substrate dephosphorylation. Collectively, this work presents the first integrated structural model for substrate selection and dephosphorylation by CN and lays the groundwork for structure-based development of new CN inhibitors.
View details for DOI 10.1371/journal.pbio.1001492
View details for Web of Science ID 000315355500016
View details for PubMedID 23468591
Whi3, an S. cerevisiae RNA-binding protein, is a component of stress granules that regulates levels of its target mRNAs.
2013; 8 (12)
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. We show that Whi3 localizes to stress granules in response to glucose deprivation or heat shock. The kinetics and pattern of Whi3 localization in response to a range of temperatures were subtly but distinctly different from those of known components of RNA processing granules. Deletion of Whi3 resulted in an increase in the relative abundance of Whi3 target RNAs, either in the presence or absence of heat shock. Increased levels of the CLN3 mRNA in whi3Δ cells may explain their decreased cell size. Another mRNA target of Whi3 encodes the zinc-responsive transcription factor Zap1, suggesting a role for Whi3 in response to zinc stress. Indeed, we found that whi3Δ cells have enhanced sensitivity to zinc toxicity. Together our results suggest an expanded model for Whi3 function: in addition to its role as a regulator of the cell cycle, Whi3 may have a role in stress-dependent RNA processing and responses to a variety of stress conditions.
View details for DOI 10.1371/journal.pone.0084060
View details for PubMedID 24386330
Curcumin Inhibits Growth of Saccharomyces cerevisiae through Iron Chelation
2011; 10 (11): 1574-1581
Curcumin, a polyphenol derived from turmeric, is an ancient therapeutic used in India for centuries to treat a wide array of ailments. Interest in curcumin has increased recently, with ongoing clinical trials exploring curcumin as an anticancer therapy and as a protectant against neurodegenerative diseases. In vitro, curcumin chelates metal ions. However, although diverse physiological effects have been documented for this compound, curcumin's mechanism of action on mammalian cells remains unclear. This study uses yeast as a model eukaryotic system to dissect the biological activity of curcumin. We found that yeast mutants lacking genes required for iron and copper homeostasis are hypersensitive to curcumin and that iron supplementation rescues this sensitivity. Curcumin penetrates yeast cells, concentrates in the endoplasmic reticulum (ER) membranes, and reduces the intracellular iron pool. Curcumin-treated, iron-starved cultures are enriched in unbudded cells, suggesting that the G(1) phase of the cell cycle is lengthened. A delay in cell cycle progression could, in part, explain the antitumorigenic properties associated with curcumin. We also demonstrate that curcumin causes a growth lag in cultured human cells that is remediated by the addition of exogenous iron. These findings suggest that curcumin-induced iron starvation is conserved from yeast to humans and underlies curcumin's medicinal properties.
View details for DOI 10.1128/EC.05163-11
View details for Web of Science ID 000296723600022
View details for PubMedID 21908599
Hph1 and Hph2 Are Novel Components of the Sec63/Sec62 Posttranslational Translocation Complex That Aid in Vacuolar Proton ATPase Biogenesis
2011; 10 (1): 63-71
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.
View details for DOI 10.1128/EC.00241-10
View details for Web of Science ID 000286002400005
View details for PubMedID 21097665
alpha-Arrestins Aly1 and Aly2 Regulate Intracellular Trafficking in Response to Nutrient Signaling
MOLECULAR BIOLOGY OF THE CELL
2010; 21 (20): 3552-3566
Extracellular signals regulate trafficking events to reorganize proteins at the plasma membrane (PM); however, few effectors of this regulation have been identified. ?-Arrestins relay signaling cues to the trafficking machinery by controlling agonist-stimulated endocytosis of G-protein-coupled receptors. In contrast, we show that yeast ?-arrestins, Aly1 and Aly2, control intracellular sorting of Gap1, the general amino acid permease, in response to nutrients. These studies are the first to demonstrate association of ?-arrestins with clathrin and clathrin adaptor proteins (AP) and show that Aly1 and Aly2 interact directly with the ?-subunit of AP-1, Apl4. Aly2-dependent trafficking of Gap1 requires AP-1, which mediates endosome-to-Golgi transport, and the nutrient-regulated kinase, Npr1, which phosphorylates Aly2. During nitrogen starvation, Npr1 phosphorylation of Aly2 may stimulate Gap1 incorporation into AP-1/clathrin-coated vesicles to promote Gap1 trafficking from endosomes to the trans-Golgi network. Ultimately, increased Aly1-/Aly2-mediated recycling of Gap1 from endosomes results in higher Gap1 levels within cells and at the PM by diverting Gap away from trafficking pathways that lead to vacuolar degradation. This work defines a new role for arrestins in membrane trafficking and offers insight into how ?-arrestins coordinate signaling events with protein trafficking.
View details for Web of Science ID 000282870300006
View details for PubMedID 20739461
Cracking the Phosphatase Code: Docking Interactions Determine Substrate Specificity
2009; 2 (100)
Phosphoserine- and phosphothreonine-directed phosphatases display remarkable substrate specificity, yet the sites that they dephosphorylate show little similarity in amino acid sequence. Studies reveal that docking interactions are key for the recognition of substrates and regulators by two conserved phosphatases, protein phosphatase 1 (PP1) and the Ca2+-calmodulin-dependent phosphatase calcineurin. In each case, a small degenerate sequence motif in the interacting protein directs low-affinity binding to a docking surface on the phosphatase that is distinct from the active site; several such interactions combine to confer overall binding specificity. Some docking surfaces are conserved, such as a hydrophobic groove on a face opposite the active site that serves as a major recognition surface for the "RVxF" motif of proteins that interact with PP1 and the "PxIxIT" motif of substrates of calcineurin. Secondary motifs combine with this primary targeting sequence to specify phosphatase binding. A comprehensive interactome for mammalian PP1 was described, analysis of which defines several PP1-binding motifs. Studies of "LxVP," a secondary calcineurin-binding sequence, establish that this motif is a conserved feature of calcineurin substrates and that the immunosuppressants FK506 and cyclosporin A inhibit the phosphatase by interfering with LxVP-mediated docking.
View details for DOI 10.1126/scisignal.2100re9
View details for Web of Science ID 000275646400006
View details for PubMedID 19996458
A Conserved Docking Surface on Calcineurin Mediates Interaction with Substrates and Immunosuppressants
2009; 33 (5): 616-626
The phosphatase calcineurin, a target of the immunosuppressants cyclosporin A and FK506, dephosphorylates NFAT transcription factors to promote immune activation and development of the vascular and nervous systems. NFAT interacts with calcineurin through distinct binding motifs: the PxIxIT and LxVP sites. Although many calcineurin substrates contain PxIxIT motifs, the generality of LxVP-mediated interactions is unclear. We define critical residues in the LxVP motif, and we demonstrate its binding to a hydrophobic pocket at the interface of the two calcineurin subunits. Mutations in this region disrupt binding of mammalian calcineurin to NFATC1 and the interaction of yeast calcineurin with substrates including Rcn1, which contains an LxVP motif. These mutations also interfere with calcineurin-immunosuppressant binding, and an LxVP-based peptide competes with immunosuppressant-immunophilin complexes for binding to calcineurin. These studies suggest that LxVP-type sites are a common feature of calcineurin substrates, and that immunosuppressant-immunophilin complexes inhibit calcineurin by interfering with this mode of substrate recognition.
View details for DOI 10.1016/j.molcel.2009.01.030
View details for Web of Science ID 000264237800008
View details for PubMedID 19285944
- Renaming the DSCR1/Adapt78 gene family as RCAN: regulators of calcineurin FASEB JOURNAL 2007; 21 (12): 3023-3028
A conserved docking site modulates substrate affinity for calcineurin, signaling output, and in vivo function
2007; 25 (6): 889-901
Calcineurin, the conserved Ca(2+)/calmodulin-regulated protein phosphatase, mediates diverse aspects of Ca(2+)-dependent signaling. We show that substrates bind calcineurin with varying strengths and examine the impact of this affinity on signaling. We altered the calcineurin-docking site, or PxIxIT motif, in Crz1, the calcineurin-regulated transcription factor in S. cerevisiae, to decrease (Crz1(PVIAVN)) or increase (Crz1(PVIVIT)) its affinity for calcineurin. As a result, the Ca(2+)-dependent dephosphorylation and activation of Crz1(PVIAVN) are decreased, whereas Crz1(PVIVIT) is constitutively dephosphorylated and hyperactive. Surprisingly, the physiological consequences of altering calcineurin-Crz1 affinity depend on the growth conditions. Crz1(PVIVIT) improves yeast growth under several environmental stress conditions but causes a growth defect during alkaline stress, most likely by titrating calcineurin away from other substrates or regulators. Thus, calcineurin-substrate affinity determines the Ca(2+) concentration dependence and output of signaling in vivo as well as the balance between different branches of calcineurin signaling in an overall biological response.
View details for DOI 10.1016/j.molcel.2007.02.014
View details for Web of Science ID 000245513900013
View details for PubMedID 17386265
Slm1 and Slm2 are novel substrates of the calcineurin phosphatase required for heat stress-induced endocytosis of the yeast uracil permease
MOLECULAR AND CELLULAR BIOLOGY
2006; 26 (12): 4729-4745
The Ca2+/calmodulin-dependent phosphatase calcineurin promotes yeast survival during environmental stress. We identified Slm1 and Slm2 as calcineurin substrates required for sphingolipid-dependent processes. Slm1 and Slm2 bind to calcineurin via docking sites that are required for their dephosphorylation by calcineurin and are related to the PXIXIT motif identified in NFAT. In vivo, calcineurin mediates prolonged dephosphorylation of Slm1 and Slm2 during heat stress, and this response can be mimicked by exogenous addition of the sphingoid base phytosphingosine. Slm proteins also promote the growth of yeast cells in the presence of myriocin, an inhibitor of sphingolipid biosynthesis, and regulation of Slm proteins by calcineurin is required for their full activity under these conditions. During heat stress, sphingolipids signal turnover of the uracil permease, Fur4. In cells lacking Slm protein activity, stress-induced endocytosis of Fur4 is blocked, and Fur4 accumulates at the cell surface in a ubiquitinated form. Furthermore, cells expressing a version of Slm2 that cannot be dephosphorylated by calcineurin display an increased rate of Fur4 turnover during heat stress. Thus, calcineurin may modulate sphingolipid-dependent events through regulation of Slm1 and Slm2. These findings, in combination with previous work identifying Slm1 and Slm2 as targets of Mss4/phosphatidylinositol 4,5-bisphosphate and TORC2 signaling, suggest that Slm proteins integrate information from a variety of signaling pathways to coordinate the cellular response to heat stress.
View details for DOI 10.1128/MCB.01973-05
View details for Web of Science ID 000238143100029
View details for PubMedID 16738335
Mapping pathways and phenotypes by systematic gene overexpression
2006; 21 (3): 319-330
Many disease states result from gene overexpression, often in a specific genetic context. To explore gene overexpression phenotypes systematically, we assembled an array of 5280 yeast strains, each containing an inducible copy of an S. cerevisiae gene, covering >80% of the genome. Approximately 15% of the overexpressed genes (769) reduced growth rate. This gene set was enriched for cell cycle-regulated genes, signaling molecules, and transcription factors. Overexpression of most toxic genes resulted in phenotypes different from known deletion mutant phenotypes, suggesting that overexpression phenotypes usually reflect a specific regulatory imbalance rather than disruption of protein complex stoichiometry. Global overexpression effects were also assayed in the context of a cyclin-dependent kinase mutant (pho85Delta). The resultant gene set was enriched for Pho85p targets and identified the yeast calcineurin-responsive transcription factor Crz1p as a substrate. Large-scale application of this approach should provide a strategy for identifying target molecules regulated by specific signaling pathways.
View details for DOI 10.1016/j.molcel.2005.12.011
View details for Web of Science ID 000235436100003
View details for PubMedID 16455487
Genomic and proteomic comparisons between bacterial and archaeal genomes and related comparisons with the yeast and fly genomes
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2005; 102 (20): 7309-7314
Bacterial, archaeal, yeast, and fly genomes are compared with respect to predicted highly expressed (PHX) genes and several genomic properties. There is a striking difference in the status of PHX ribosomal protein (RP) genes where the archaeal genome generally encodes more RP genes and fewer PHX RPs compared with bacterial genomes. The increase in RPs in archaea and eukaryotes compared with that in bacteria may reflect a more complex set of interactions in archaea and eukaryotes in regulating translation, e.g., differences in structure requiring scaffolding of longer rRNA molecules, expanded interactions with the chaperone machinery, and, in eukaryotic interactions with endoplasmic reticulum components. The yeast genome is similar to fast-growing bacteria in PHX genes but also features several cytoskeletal genes, including actin and tropomyosin, and several signal transduction regulatory proteins from the 14.3.3 family. The most PHX genes of Drosophila encode cytoskeletal and exoskeletal proteins. We found that the preference of a microorganism for an anaerobic metabolism correlates with the number of PHX enzymes of the glycolysis pathway that well exceeds the number of PHX enzymes acting in the tricarboxylic acid cycle. Conversely, if the number of PHX enzymes of the tricarboxylic acid cycle well exceeds the PHX enzymes of glycolysis, an aerobic metabolism is preferred. Where the numbers are approximately commensurate, a facultative growth behavior prevails.
View details for DOI 10.1073/pnas.0502314102
View details for Web of Science ID 000229292200046
View details for PubMedID 15883367
Molecular analysis reveals localization of Saccharomyces cerevisiae protein kinase C to sites of polarized growth and Pkc1p targeting to the nucleus and mitotic spindle
2005; 4 (1): 36-45
The catalytic activity and intracellular localization of protein kinase C (PKC) are both highly regulated in vivo. This family of kinases contains conserved regulatory motifs, i.e., the C1, C2, and HR1 domains, which target PKC isoforms to specific subcellular compartments and restrict their activity spatially. Saccharomyces cerevisiae contains a single PKC isozyme, Pkc1p, which contains all of the regulatory motifs found in mammalian PKCs. Pkc1p localizes to sites of polarized growth, consistent with its main function in maintaining cell integrity. We dissected the molecular basis of Pkc1p localization by expressing each of its domains individually and in combinations as green fluorescent protein fusions. We find that the Rho1p-binding domains, HR1 and C1, are responsible for targeting Pkc1p to the bud tip and cell periphery, respectively. We demonstrate that Pkc1p activity is required for its normal localization to the bud neck, which also depends on the integrity of the septin ring. In addition, we show for the first time that yeast protein kinase C can accumulate in the nucleus, and we identify a nuclear exit signal as well as nuclear localization signals within the Pkc1p sequence. Thus, we propose that Pkc1p shuttles in and out of the nucleus and consequently has access to nuclear substrates. Surprisingly, we find that deletion of the HR1 domain results in Pkc1p localization to the mitotic spindle and that the C2 domain is responsible for this targeting. This novel nuclear and spindle localization of Pkc1p may provide a molecular explanation for previous observations that suggest a role for Pkc1p in regulating microtubule function.
View details for DOI 10.1128/EC.4.1.36-45.2005
View details for Web of Science ID 000226465200003
View details for PubMedID 15643058
Integration of stress responses: Modulation of calcineurin signaling in Saccharomyces cerevisiae by protein kinase A
2004; 3 (5): 1147-1153
Calcineurin is a Ca2+/calmodulin-dependent protein phosphatase required for Saccharomyces cerevisiae to adapt to a variety of environmental stresses. Once activated, calcineurin dephosphorylates the Zn-finger transcription factor Crz1p/Tcn1p, causing it to accumulate in the nucleus where it activates gene expression. Here we show that cyclic AMP-dependent protein kinase A (PKA) phosphorylates and negatively regulates Crz1p activity by inhibiting its nuclear import. Activation of PKA in vivo decreases Crz1p-dependent transcription. PKA phosphorylates Crz1p in vitro, and we identify specific residues required for this phosphorylation, all of which reside in or adjacent to the nuclear localization signal. Mutation of these residues to alanine results in increased nuclear import of Crz1p and results in higher levels of both basal and Ca2+-induced Crz1p transcriptional activity. PKA regulates the general stress response in yeast and coordinates this response with nutrient availability. In contrast, calcineurin regulates the cellular response to a restricted set of environmental insults. Thus, these studies identify a specific biochemical mechanism through which the activities of multiple stress-activated signaling pathways are integrated in vivo.
View details for DOI 10.1128/EC.3.5.1147-1153.2004
View details for Web of Science ID 000224822300008
View details for PubMedID 15470242
Hph1p and Hph2p, novel components of calcineurin-mediated stress responses in Saccharomyces cerevisiae
2004; 3 (3): 695-704
Calcineurin is a Ca2+- and calmodulin-dependent protein phosphatase that plays a key role in animal and yeast physiology. In the yeast Saccharomyces cerevisiae, calcineurin is required for survival during several environmental stresses, including high concentrations of Na+, Li+, and Mn2+ ions and alkaline pH. One role of calcineurin under these conditions is to activate gene expression through its regulation of the Crz1p transcription factor. We have identified Hph1p as a novel substrate of calcineurin. HPH1 (YOR324C) and its homolog HPH2 (YAL028W) encode tail-anchored integral membrane proteins that interact with each other in the yeast two-hybrid assay and colocalize to the endoplasmic reticulum. Hph1p and Hph2p serve redundant roles in promoting growth under conditions of high salinity, alkaline pH, and cell wall stress. Calcineurin modifies the distribution of Hph1p within the endoplasmic reticulum and is required for full Hph1p activity in vivo. Furthermore, calcineurin directly dephosphorylates Hph1p and interacts with it through a sequence motif in Hph1p, PVIAVN. This motif is related to calcineurin docking sites in other substrates, such as NFAT and Crz1p, and is required for regulation of Hph1p by calcineurin. In contrast, Hph2p neither interacts with nor is dephosphorylated by calcineurin. Ca2+-induced Crz1p-mediated transcription is unaffected in hph1delta hph2delta mutants, and genetic analyses indicate that HPH1/HPH2 and CRZ1 act in distinct pathways downstream of calcineurin. Thus, Hph1p and Hph2p are components of a novel Ca2+- and calcineurin-regulated response required to promote growth under conditions of high Na+, alkaline pH, and cell wall stress.
View details for DOI 10.1128/EC.3.3.695-704.2004
View details for Web of Science ID 000222076000012
View details for PubMedID 15189990
Calcineurin signaling in Saccharomyces cereviside: how yeast go crazy in response to stress
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
2003; 311 (4): 1143-1150
In the yeast Saccharomyces cerevisiae, Ca(2+) signaling mediated by the Ca(2+)/calmodulin dependent phosphatase, calcineurin, is required for survival during environmental stress. One role of the phosphatase under these conditions is to activate gene expression through its regulation of the Crz1p ("crazy") transcription factor. Calcineurin dephosphorylates Crz1p and causes its rapid translocation from the cytosol to the nucleus. Crz1p then activates the transcription of genes whose products promote cell survival. Recent studies concerning the regulation of Crz1p by calcineurin are discussed in this review and the mechanisms by which calcineurin controls gene expression in yeast and mammalian cells are compared.
View details for DOI 10.1016/S0006-291X(03)01552-3
View details for Web of Science ID 000186803900050
View details for PubMedID 14623300
Negative regulation of calcineurin signaling by Hrr25p, a yeast homolog of casein kinase I
GENES & DEVELOPMENT
2003; 17 (21): 2698-2708
Calcineurin is a Ca2+/calmodulin-regulated protein phosphatase required for Saccharomyces cerevisiae to respond to a variety of environmental stresses. Calcineurin promotes cell survival during stress by dephosphorylating and activating the Zn-finger transcription factor Crz1p/Tcn1p. Using a high-throughput assay, we screened 119 yeast kinases for their ability to phosphorylate Crz1p in vitro and identified the casein kinase I homolog Hrr25p. Here we show that Hrr25p negatively regulates Crz1p activity and nuclear localization in vivo. Hrr25p binds to and phosphorylates Crz1p in vitro and in vivo. Overexpression of Hrr25p decreases Crz1p-dependent transcription and antagonizes its Ca2+-induced nuclear accumulation. In the absence of Hrr25p, activation of Crz1p by Ca2+/calcineurin is potentiated. These findings represent the first identification of a negative regulator for Crz1p, and establish a novel physiological role for Hrr25p in antagonizing calcineurin signaling.
View details for DOI 10.1101/gad.1140603
View details for Web of Science ID 000186299700011
View details for PubMedID 14597664
Genome-wide analysis of gene expression regulated by the calcineurin/Crz1p signaling pathway in Saccharomyces cerevisiae
JOURNAL OF BIOLOGICAL CHEMISTRY
2002; 277 (34): 31079-31088
In Saccharomyces cerevisiae, the Ca(2+)/calmodulin-dependent protein phosphatase, calcineurin, is activated by specific environmental conditions, including exposure to Ca(2+) and Na(+), and induces gene expression by regulating the Crz1p/Tcn1p transcription factor. We used DNA microarrays to perform a comprehensive analysis of calcineurin/Crz1p-dependent gene expression following addition of Ca(2+) (200 mm) or Na(+) (0.8 m) to yeast. 163 genes exhibited increased expression that was reduced 50% or more by calcineurin inhibition. These calcineurin-dependent genes function in signaling pathways, ion/small molecule transport, cell wall maintenance, and vesicular transport, and include many open reading frames of previously unknown function. Three distinct gene classes were defined as follows: 28 genes displayed calcineurin-dependent induction in response to Ca(2+) and Na(+), 125 showed calcineurin-dependent expression following Ca(2+) but not Na(+) addition, and 10 were regulated by calcineurin in response to Na(+) but not Ca(2+). Analysis of crz1Delta cells established Crz1p as the major effector of calcineurin-regulated gene expression in yeast. We identified the Crz1p-binding site as 5'-GNGGC(G/T)CA-3' by in vitro site selection. A similar sequence, 5'-GAGGCTG-3', was identified as a common sequence motif in the upstream regions of calcineurin/ Crz1p-dependent genes. This finding is consistent with direct regulation of these genes by Crz1p.
View details for DOI 10.1074/jbc.M202718200
View details for Web of Science ID 000177579800087
View details for PubMedID 12058033
Calcineurin-dependent regulation of Crz1p nuclear export requires Msn5p and a conserved calcineurin docking site
GENES & DEVELOPMENT
2002; 16 (5): 608-619
Calcineurin, a conserved Ca(2+)/calmodulin-regulated protein phosphatase, plays a crucial role in Ca(2+) signaling in a wide variety of cell types. In Saccharomyces cerevisiae, calcineurin positively regulates transcription in response to stress by dephosphorylating the transcription factor Crz1p/Tcn1p. Dephosphorylation promotes Crz1p nuclear localization in part by increasing the efficiency of its nuclear import. In this work, we show that calcineurin-dependent dephosphorylation of Crz1p also down-regulates its nuclear export. Using a genetic approach, we identify Msn5p as the exportin for Crz1p. In addition, we define the Crz1p nuclear export signal (NES) and show that it interacts with Msn5p in a phosphorylation-dependent manner. This indicates that calcineurin regulates Crz1p nuclear export by dephosphorylating and inactivating its NES. Finally, we define a motif in Crz1p, PIISIQ, similar to the PxIxIT docking site for calcineurin on the mammalian transcription factor NFAT, that mediates the in vivo interaction between calcineurin and Crz1p and is required for calcineurin-dependent regulation of Crz1p nuclear export and activity. Therefore, in yeast as in mammals, a docking site is required to target calcineurin to its substrate such that it can dephosphorylate it efficiently.
View details for Web of Science ID 000174314200008
View details for PubMedID 11877380
Internal Ca2+ release in yeast is triggered by hypertonic shock and mediated by a TRP channel homologue
JOURNAL OF CELL BIOLOGY
2002; 156 (1): 29-34
Calcium ions, present inside all eukaryotic cells, are important second messengers in the transduction of biological signals. In mammalian cells, the release of Ca(2+) from intracellular compartments is required for signaling and involves the regulated opening of ryanodine and inositol-1,4,5-trisphosphate (IP3) receptors. However, in budding yeast, no signaling pathway has been shown to involve Ca(2+) release from internal stores, and no homologues of ryanodine or IP3 receptors exist in the genome. Here we show that hyperosmotic shock provokes a transient increase in cytosolic Ca(2+) in vivo. Vacuolar Ca(2+), which is the major intracellular Ca(2+) store in yeast, is required for this response, whereas extracellular Ca(2+) is not. We aimed to identify the channel responsible for this regulated vacuolar Ca(2+) release. Here we report that Yvc1p, a vacuolar membrane protein with homology to transient receptor potential (TRP) channels, mediates the hyperosmolarity induced Ca(2+) release. After this release, low cytosolic Ca(2+) is restored and vacuolar Ca(2+) is replenished through the activity of Vcx1p, a Ca(2+)/H(+) exchanger. These studies reveal a novel mechanism of internal Ca(2+) release and establish a new function for TRP channels.
View details for Web of Science ID 000173256300004
View details for PubMedID 11781332
Calcineurin-dependent nuclear import of the transcription factor Crz1p requires Nmd5p
JOURNAL OF CELL BIOLOGY
2001; 154 (5): 951-960
Calcineurin is a conserved Ca2+/calmodulin-specific serine-threonine protein phosphatase that mediates many Ca2+-dependent signaling events. In yeast, calcineurin dephosphorylates Crz1p, a transcription factor that binds to the calcineurin-dependent response element, a 24-bp promoter element. Calcineurin-dependent dephosphorylation of Crz1p alters Crz1p nuclear localization. This study examines the mechanism by which calcineurin regulates the nuclear localization of Crz1p in more detail. We describe the identification and characterization of a novel nuclear localization sequence (NLS) in Crz1p, which requires both basic and hydrophobic residues for activity, and show that the karyopherin Nmd5p is required for Crz1p nuclear import. We also demonstrate that the binding of Crz1p to Nmd5p is dependent upon its phosphorylation state, indicating that nuclear import of Crz1p is regulated by calcineurin. Finally, we demonstrate that residues in both the NH2- and COOH-terminal portions of Crz1p are required for regulated Crz1p binding to Nmd5p, supporting a model of NLS masking for regulating Crz1p nuclear import.
View details for Web of Science ID 000170875700005
View details for PubMedID 11535618
The eukaryotic response regulator Skn7p regulates calcineurin signaling through stabilization of Crz1p
2001; 20 (13): 3473-3483
To survive ionic, pH and pheromone stress, the yeast Saccharomyces cerevisiae activates signaling through the Ca2+-activated phosphatase calcineurin to the transcription factor Crz1p/Tcn1p. We show that the overexpression of SKN7, a response-regulator transcription factor, activates transcription from a calcineurin/Crz1p-dependent response element (CDRE). Ca2+-induced, calcineurin/Crz1p-dependent activation of several genes is reduced in skn7 mutants. Skn7p modulates CDRE-dependent transcription by affecting Crz1p protein levels. Specifically, the rate of Crz1p turnover is increased in skn7 mutants. Calcineurin, but not its phosphatase activity, is required for Skn7p-mediated Crz1p stabilization. Skn7p binds to both calcineurin and Crz1p in vitro, and we suggest that this interaction is required for Skn7p regulation of Crz1p. The DNA-binding and internal coiled-coil domains, but not the response- regulator phosphorylation of Skn7p, are necessary for Crz1p-dependent transcriptional activation and Crz1p stabilization by Skn7 in vivo. The DNA-binding domain of Skn7p is also required for binding to Crz1p and calcineurin in vitro. Thus, we propose that Skn7p protects Crz1p from degradation by binding to it and calcineurin through its DNA-binding domain.
View details for Web of Science ID 000169803700019
View details for PubMedID 11432834
- Regulation of nuclear localization during signaling JOURNAL OF BIOLOGICAL CHEMISTRY 2001; 276 (24): 20805-20808
Genetic analysis of calmodulin and its targets in Saccharomyces cerevisiae
ANNUAL REVIEW OF GENETICS
2001; 35: 647-672
Calmodulin, a small, ubiquitous Ca2+-binding protein, regulates a wide variety of proteins and processes in all eukaryotes. CMD1, the single gene encoding calmodulin in S. cerevisiae, is essential, and this review discusses studies that identified many of calmodulin's physiological targets and their functions in yeast cells. Calmodulin performs essential roles in mitosis, through its regulation of Nuf1p/Spc110p, a component of the spindle pole body, and in bud growth, by binding Myo2p, an unconventional class V myosin required for polarized secretion. Surprisingly, mutant calmodulins that fail to bind Ca2+ can perform these essential functions. Calmodulin is also required for endocytosis in yeast and participates in Ca2+-dependent, stress-activated signaling pathways through its regulation of a protein phosphatase, calcineurin, and the protein kinases, Cmk1p and Cmk2p. Thus, calmodulin performs important physiological functions in yeast cells in both its Ca2+-bound and Ca2+-free form.
View details for Web of Science ID 000172991500022
View details for PubMedID 11700296
Luv1p/Rki1p/Tcs3p/Vps54p, a yeast protein that localizes to the late Golgi and early endosome, is required for normal vacuolar morphology.
MOLECULAR BIOLOGY OF THE CELL
2000; 11 (7): 2429-2443
We have characterized LUV1/RKI1/TCS3/VPS54, a novel yeast gene required to maintain normal vacuolar morphology. The luv1 mutant was identified in a genetic screen for mutants requiring the phosphatase calcineurin for vegetative growth. luv1 mutants lack a morphologically intact vacuole and instead accumulate small vesicles that are acidified and contain the vacuolar proteins alkaline phosphatase and carboxypeptidase Y and the vacuolar membrane H(+)-ATPase. Endocytosis appears qualitatively normal in luv1 mutants, but some portion (28%) of carboxypeptidase Y is secreted. luv1 mutants are sensitive to several ions (Zn(2+), Mn(2+), and Cd(2+)) and to pH extremes. These mutants are also sensitive to hygromycin B, caffeine, and FK506, a specific inhibitor of calcineurin. Some vacuolar protein-sorting mutants display similar drug and ion sensitivities, including sensitivity to FK506. Luv1p sediments at 100,000 x g and can be solubilized by salt or carbonate, indicating that it is a peripheral membrane protein. A Green Fluorescent Protein-Luv1 fusion protein colocalizes with the dye FM 4-64 at the endosome, and hemagglutinin-tagged Luv1p colocalizes with the trans-Golgi network/endosomal protease Kex2p. Computer analysis predicts a short coiled-coil domain in Luv1p. We propose that this protein maintains traffic through or the integrity of the early endosome and that this function is required for proper vacuolar morphology.
View details for Web of Science ID 000088184800020
View details for PubMedID 10888679
Identification of a novel region critical for calcineurin function in vivo and in vitro
JOURNAL OF BIOLOGICAL CHEMISTRY
1999; 274 (26): 18543-18551
Calcineurin is a Ca2+/calmodulin-regulated protein phosphatase that plays critical functional roles in T-cell activation and other Ca2+-mediated signal transduction pathways in mammalian cells. In Saccharomyces cerevisiae, calcineurin regulates the transcription of several genes involved in maintaining ion homeostasis (PMC1, PMR1, and PMR2) and cell wall synthesis (FKS2). In this paper, we report the identification and characterization of 11 single amino acid substitutions in the yeast calcineurin catalytic subunit Cna1p. We show that six substitutions (R177G, F211S, S232F, D258V, L259P, and A262P) affect the stability of calcineurin and that two substitutions (V385D and M400R) disrupt the interaction between Cna1p and the calcineurin regulatory subunit Cnb1p. We also identify three mutations (S373P, H375L, and L379S) that are clustered between the catalytic and the calcineurin B subunit-binding domains. These mutations do not significantly affect the ability of Cna1p to interact with Cnb1p, calmodulin, or Fkb1p (FK506-binding protein). However, these residue substitutions dramatically affect calcineurin activity both in vitro and in vivo. Thus, by using a random mutagenesis approach, we have shown for the first time that the linker region of the calcineurin catalytic subunit, as defined by the Ser373, His375, and Leu379 residues, is crucial for its function as a phosphatase.
View details for Web of Science ID 000081056700055
View details for PubMedID 10373463
Yeast calcineurin regulates nuclear localization of the Crz1p transcription factor through dephosphorylation
GENES & DEVELOPMENT
1999; 13 (7): 798-803
Calcineurin, a Ca2+/calmodulin dependent protein phosphatase, regulates Ca2+-dependent processes in a wide variety of cells. In the yeast, Saccharomyces cerevisiae, calcineurin effects Ca2+-dependent changes in gene expression through regulation of the Crz1p transcription factor. We show here that calcineurin dephosphorylates Crz1p and that this results in translocation of Crz1p to the nucleus. We identify a region of Crz1p that is required for calcineurin-dependent regulation of its phosphorylation, localization, and activity, and show that this region has significant sequence simlarity to a portion of NF-AT, a family of mammalian transcription factors whose localization is also regulated by calcineurin. Thus, the mechanism of Ca2+/calcineurin-dependent signaling shows remarkable conservation between yeast and mammalian cells.
View details for Web of Science ID 000079692000005
View details for PubMedID 10197980
Importance of phenylalanine residues of yeast calmodulin for target binding and activations
JOURNAL OF BIOLOGICAL CHEMISTRY
1998; 273 (41): 26375-26382
Recent genetic studies of yeast calmodulin (yCaM) have shown that alterations of different sets of Phe residues result in distinct functional defects (Ohya, Y., and Botstein, D. (1994) Science 263, 963-966). To examine the importance of Phe residues for target binding and activation, we purified mutant yCaMs containing single or double Phe to Ala substitutions and determined their ability to bind and activate two target proteins, calcineurin and CaM-dependent protein kinase (CaMK). Binding assays using the gel overlay technique and quantitative analyses using surface plasmon resonance measurements indicated that the binding of yCaM to calcineurin is impaired by either double mutations of F16A/F19A or a single mutation of F140A, while binding to CaMK is impaired by F89A, F92A, or F140A. These same mutant yCaMs fail to activate calcineurin and CaMK, respectively, in vitro. In addition, F19A exhibited a severe defect in activation of both enzymes. F12A activated calcineurin to only 50% of the level achieved by wild-type calmodulin but fully activated CaMK. These results suggest that each target protein requires a specific and distinct subset of Phe residues in yCaM for target binding and activation.
View details for Web of Science ID 000076373300022
View details for PubMedID 9756868
Ion tolerance of Saccharomyces cerevisiae lacking the Ca2+/CaM-dependent phosphatase (Calcineurin) is improved by mutations in URE2 or PMA1
1998; 149 (2): 865-878
Calcineurin is a conserved, Ca2+/CaM-stimulated protein phosphatase required for Ca2+-dependent signaling in many cell types. In yeast, calcineurin is essential for growth in high concentrations of Na+, Li+, Mn2+, and OH-, and for maintaining viability during prolonged treatment with mating pheromone. In contrast, the growth of calcineurin-mutant yeast is better than that of wild-type cells in the presence of high concentrations of Ca2+. We identified mutations that suppress multiple growth defects of calcineurin-deficient yeast (cnb1Delta or cna1Delta cna2Delta). Mutations in URE2 suppress the sensitivity of calcineurin mutants to Na+, Li+, and Mn2+, and increase their survival during treatment with mating pheromone. ure2 mutations require both the transcription factor Gln3p and the Na+ ATPase Pmr2p to confer Na+ and Li+ tolerance. Mutations in PMA1, which encodes the yeast plasma membrane H+-ATPase, also suppress many growth defects of calcineurin mutants. pma1 mutants display growth phenotypes that are opposite to those of calcineurin mutants; they are resistant to Na+, Li+, and Mn2+, and sensitive to Ca2+. We also show that calcineurin mutants are sensitive to aminoglycoside antibiotics such as hygromycin B while pma1 mutants are more resistant than wild type. Furthermore, pma1 and calcineurin mutations have antagonistic effects on intracellular [Na+] and [Ca2+]. Finally, we show that yeast expressing a constitutively active allele of calcineurin display pma1-like phenotypes, and that membranes from these yeast have decreased levels of Pma1p activity. These studies further characterize the roles that URE2 and PMA1 play in regulating intracellular ion homeostasis.
View details for Web of Science ID 000074028400034
View details for PubMedID 9611198
Temperature-induced expression of yeast FKS2 is under the dual control of protein kinase C and calcineurin
MOLECULAR AND CELLULAR BIOLOGY
1998; 18 (2): 1013-1022
FKS1 and FKS2 are alternative subunits of the glucan synthase complex, which is responsible for synthesizing 1,3-beta-glucan chains, the major structural polymer of the Saccharomyces cerevisiae cell wall. Expression of FKS1 predominates during growth under optimal conditions. In contrast, FKS2 expression is induced by mating pheromone, high extracellular [Ca2+], growth on poor carbon sources, or in an fks1 mutant. Induction of FKS2 expression in response to pheromone, CaCl2, or loss of FKS1 function requires the Ca2+/calmodulin-dependent protein phosphatase calcineurin. Therefore, a double mutant in calcineurin (CNB1) and FKS1 is inviable due to a deficiency in FKS2 expression. To identify novel regulators of FKS2 expression, we isolated genes whose overexpression obviates the calcineurin requirement for viability of an fks1 mutant. Two components of the cell integrity signaling pathway controlled by the RHO1 G protein (MKK1 and RLM1) were identified through this screen. This signaling pathway is activated during growth at moderately high temperatures. We demonstrate that calcineurin and the cell integrity pathway function in parallel, through separable promoter elements, to induce FKS2 expression during growth at 39 degrees C. Because RHO1 also serves as a regulatory subunit of the glucan synthase, our results define a regulatory circuit through which RHO1 controls both the activity of this enzyme complex and the expression of at least one of its components. We show also that FKS2 induction during growth on poor carbon sources is a response to glucose depletion and is under the control of the SNF1 protein kinase and the MIG1 transcriptional repressor. Finally, we show that FKS2 expression is induced as cells enter stationary phase through a SNF1-, calcineurin-, and cell integrity signaling-independent pathway.
View details for Web of Science ID 000071716000037
View details for PubMedID 9447998
Calcineurin acts through the CRZ1/TCN1-encoded transcription factor to regulate gene expression in yeast
GENES & DEVELOPMENT
1997; 11 (24): 3432-3444
Calcineurin is a conserved Ca2+/calmodulin-dependent protein phosphatase that plays a critical role in Ca2+ signaling. We describe new components of a calcineurin-mediated response in yeast, the Ca2+-induced transcriptional activation of FKS2, which encodes a beta-1,3 glucan synthase. A 24-bp region of the FKS2 promoter was defined as sufficient to confer calcineurin-dependent transcriptional induction on a minimal promoter in response to Ca2+ and was named CDRE (for calcineurin-dependent response element). The product of CRZ1 (YNL027w) was identified as an activator of CDRE-driven transcription. Crz1p contains zinc finger motifs and binds specifically to the CDRE. Genetic analysis revealed that crz1Delta mutant cells exhibit several phenotypes similar to those of calcineurin mutants and that overexpression of CRZ1 in calcineurin mutants suppressed these phenotypes. These results suggest that Crz1p functions downstream of calcineurin to effect multiple calcineurin-dependent responses. Moreover, the calcineurin-dependent transcriptional induction of FKS2 in response to Ca2+, alpha-factor, and Na+ was found to require CRZ1. In addition, we found that the calcineurin-dependent transcriptional regulation of PMR2 and PMC1 required CRZ1. However, transcription of PMR2 and PMC1 was activated by only a subset of the treatments that activated FKS2 transcription. Thus, in response to multiple signals, calcineurin acts through the Crz1p transcription factor to differentially regulate the expression of several target genes in yeast.
View details for Web of Science ID 000071209500014
View details for PubMedID 9407035
An essential role of the yeast pheromone-induced Ca2+ signal is to activate calcineurin
MOLECULAR BIOLOGY OF THE CELL
1997; 8 (2): 263-277
Previous studies showed that, in wild-type (MATa) cells, alpha-factor causes an essential rise in cytosolic Ca2+. We show that calcineurin, the Ca2+/calmodulin-dependent protein phosphatase, is one target of this Ca2+ signal. Calcineurin mutants lose viability when incubated with mating pheromone, and overproduction of constitutively active (Ca(2+)-independent) calcineurin improves the viability of wild-type cells exposed to pheromone in Ca(2+)-deficient medium. Thus, one essential consequence of the pheromone-induced rise in cytosolic Ca2+ is activation of calcineurin. Although calcineurin inhibits intracellular Ca2+ sequestration in yeast cells, neither increased extracellular Ca2+ nor defects in vacuolar Ca2+ transport bypasses the requirement for calcineurin during the pheromone response. These observations suggest that the essential function of calcineurin in the pheromone response may be distinct from its modulation of intracellular Ca2+ levels. Mutants that do not undergo pheromone-induced cell cycle arrest (fus3, far1) show decreased dependence on calcineurin during treatment with pheromone. Thus, calcineurin is essential in yeast cells during prolonged exposure to pheromone and especially under conditions of pheromone-induced growth arrest. Ultrastructural examination of pheromone-treated cells indicates that vacuolar morphology is abnormal in calcineurin-deficient cells, suggesting that calcineurin may be required for maintenance of proper vacuolar structure or function during the pheromone response.
View details for Web of Science ID A1997WK47200006
View details for PubMedID 9190206
The product of HUM1, a novel yeast gene, is required for vacuolar Ca2+/H+ exchange and is related to mammalian Na+/Ca2+ exchangers
MOLECULAR AND CELLULAR BIOLOGY
1996; 16 (7): 3730-3741
Calcineurin, or PP2B, plays a critical role in mediating Ca2+-dependent signaling in many cell types. In yeast cells, this highly conserved protein phosphatase regulates aspects of ion homeostasis and cell wall synthesis. We show that calcineurin mutants are sensitive to high concentrations of Mn2+ and identify two genes, CCC1 and HUM1, that, at high dosages, increase the Mn2+ tolerance of calcineurin mutants. CCC1 was previously identified by complementation of a Ca2+-sensitive (csg1) mutant. HUM1 (for "high copy number undoes manganese") is a novel gene whose predicted protein product shows similarity to mammalian Na+/Ca2+ exchangers. hum1 mutations confer Mn2+ sensitivity in some genetic backgrounds and exacerbate the Mn2+ sensitivity of calcineurin mutants. Furthermore, disruption of HUM1 in a calcineurin mutant strain results in a Ca2+-sensitive phenotype. We investigated the effect of disrupting HUM1 in other strains with defects in Ca2+ homeostasis. The Ca2+ sensitivity of pmc1 mutants, which lack a P-type ATPase presumed to transport Ca2+ into the vacuole, is exacerbated in a hum1 mutant strain background. Also, the Ca2+ content of hum1 pmc1 cells is less than that of pmc1 cells. In contrast, the Ca2+ sensitivity of vph1 mutants, which are specifically defective in vacuolar acidification, is not significantly altered by disruption of Hum1p function. These genetic interactions suggest that Hum1p may participate in vacuolar Ca2+/H+ exchange. Therefore, we prepared vacuolar membrane vesicles from wild-type and hum1 cells and compared their Ca2+ transport properties. Vacuolar membrane vesicles from hum1 mutants lack all Ca2+/H+ antiport activity, demonstrating that Hum1p catalyzes the exchange of Ca2+ for H+ across the yeast vacuolar membrane.
View details for Web of Science ID A1996UT08600053
View details for PubMedID 8668190
Two classes of plant cDNA clones differentially complement yeast calcineurin mutants and increase salt tolerance of wild-type yeast
JOURNAL OF BIOLOGICAL CHEMISTRY
1996; 271 (22): 12859-12866
The salt-sensitive phenotype of yeast cells deficient in the phosphoprotein phosphatase, calcineurin, was used to identify genes from the higher plant Arabidopsis thaliana that complement this phenotype. cDNA clones corresponding to two different sequences, designated STO (salt tolerance) and STZ (salt tolerance zinc finger), were found to increased tolerance of calcineurin mutants and of wild-type yeast to both Li+ and Na+ ions. STZ is related to Cys2/His2-type zinc-finger proteins found in higher plants, and STO is similar to the Arabidopsis CONSTANS protein in regions that may also be zinc fingers. Although neither protein has sequence similarity to any protein phosphatase, STO was able to at least partially compensate for all tested additional phenotypic effects of calcineurin deficiency, and STZ compensated for a subset of these effects. Salt tolerance produced by STZ appeared to be partially dependent on ENA1/PMR2, a P-type ATPase required for Li+ and Na+ efflux in yeast, whereas the effect of STO on salt tolerance was independent of ENA1/PMR2. STZ and STO were found to be expressed in Arabidopsis roots and leaves, whereas only STO message was detectable in flowers. An apparent increase in the level of STZ mRNA was observed in response NaCl exposure in Arabidopsis seedlings, but the level of STO mRNA was not altered by this treatment.
View details for Web of Science ID A1996UN47400028
View details for PubMedID 8662738
CALCINEURIN, THE CA2+/CALMODULIN-DEPENDENT PROTEIN PHOSPHATASE, IS ESSENTIAL IN YEAST MUTANTS WITH CELL INTEGRITY DEFECTS AND IN MUTANTS THAT LACK A FUNCTIONAL VACUOLAR H+-ATPASE
MOLECULAR AND CELLULAR BIOLOGY
1995; 15 (8): 4103-4114
Calcineurin is a conserved Ca2+/calmodulin-dependent protein phosphatase that plays a critical role in Ca(2+)-mediated signaling in many cells. Yeast cells lacking functional calcineurin (cna1 cna2 or cnb1 mutants) display growth defects under specific environmental conditions, for example, in the presence of high concentrations of Na+, Li+, Mn2+, or OH- but are indistinguishable from wild-type cells under standard culture conditions. To characterize regulatory pathways that may overlap with calcineurin, we performed a synthetic lethal screen to identify mutants that require calcineurin on standard growth media. The characterization of one such mutant, cnd1-8, is presented. The CND1 gene was cloned, and sequence analysis predicts that it encodes a novel protein 1,876 amino acids in length with multiple membrane-spanning domains. CND1 is identical to the gene identified previously as FKS1, ETG1, and CWH53, cnd1 mutants are sensitive to FK506 and cyclosporin A and exhibit slow growth that is improved by the addition of osmotic stabilizing agents. This osmotic agent-remedial growth defect and microscopic evidence of spontaneous cell lysis in cnd1 cultures suggest that cell integrity is compromised in these mutants. Mutations in the genes for yeast protein kinase C (pkc1) and a MAP kinase (mpk1/slt2) disrupt a Ca(2+)-dependent signaling pathway required to maintain a normal cell wall and cell integrity. We show that pkc1 and mpk1/slt2 growth defects are more severe in the absence of calcineurin function and less severe in the presence of a constitutively active form of calcineurin. These observations suggest that calcineurin and protein kinase C perform independent but physiologically related functions in yeast cells. We show that several mutants that lack a functional vacuolar H(+)-ATPase (vma) require calcineurin for vegetative growth. We discuss possible roles for calcineurin in regulating intracellular ion homeostasis and in maintaining cell integrity.
View details for Web of Science ID A1995RJ77900014
View details for PubMedID 7542741