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9 results on '"Marshall, Maria Nieves Martinez"'

1. Analysis of the roles of phosphatidylinositol-4,5-bisphosphate and individual subunits in assembly, localization, and function of Saccharomyces cerevisiae target of rapamycin complex 2

Author

Marshall, Maria Nieves Martinez, Emmerstorfer-Augustin, Anita, Leskoske, Kristin L, Zhang, Lydia H, Li, Biyun, and Thorner, Jeremy

Subjects
Underpinning research, 1.1 Normal biological development and functioning, Generic health relevance, Armadillo Domain Proteins, Carrier Proteins, Cell Membrane, Mechanistic Target of Rapamycin Complex 2, Phosphatidylinositol 4, 5-Diphosphate, Protein Domains, Protein Subunits, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Biological Sciences, Medical and Health Sciences, Developmental Biology
Abstract

Eukaryotic cell survival requires maintenance of plasma membrane (PM) homeostasis in response to environmental insults and changes in lipid metabolism. In yeast, a key regulator of PM homeostasis is target of rapamycin (TOR) complex 2 (TORC2), a multiprotein complex containing the evolutionarily conserved TOR protein kinase isoform Tor2. PM localization is essential for TORC2 function. One core TORC2 subunit (Avo1) and two TORC2--associated regulators (Slm1 and Slm2) contain pleckstrin homology (PH) domains that exhibit specificity for binding phosphatidylinositol-4,5-bisphosphate (PtdIns4,5P2). To investigate the roles of PtdIns4,5P2 and constituent subunits of TORC2, we used auxin-inducible degradation to systematically eliminate these factors and then examined localization, association, and function of the remaining TORC2 components. We found that PtdIns4,5P2 depletion significantly reduced TORC2 activity, yet did not prevent PM localization or disassembly of TORC2. Moreover, truncated Avo1 (lacking its C-terminal PH domain) was still recruited to the PM and supported growth. Even when all three PH-containing proteins were absent, the remaining TORC2 subunits were PM-bound. Revealingly, Avo3 localized to the PM independent of both Avo1 and Tor2, whereas both Tor2 and Avo1 required Avo3 for their PM anchoring. Our findings provide new mechanistic information about TORC2 and pinpoint Avo3 as pivotal for TORC2 PM localization and assembly in vivo.

Published
2019

2. Regulation of plasma membrane homeostasis: Dissecting TORC2 signaling

Author

Thorner, Jeremy, Locke, Melissa N, Marshall, Maria Nieves Martinez, Emmerstorfer-Augustin, Anita, Leskoske, Kristin L, and Roelants, Francoise M

Subjects
Biochemistry & Molecular Biology, Biochemistry and Cell Biology, Physiology, Medical Physiology
Published
2019

3. The Stress-Sensing TORC2 Complex Activates Yeast AGC-Family Protein Kinase Ypk1 at Multiple Novel Sites

Author

Leskoske, Kristin L, Roelants, Françoise M, Marshall, Maria Nieves Martinez, Hill, Jennifer M, and Thorner, Jeremy

Subjects
Biochemistry and Cell Biology, Biological Sciences, 1.1 Normal biological development and functioning, Underpinning research, Generic health relevance, Amino Acid Substitution, Enzyme Stability, Gene Expression Regulation, Fungal, Mechanistic Target of Rapamycin Complex 2, Phosphorylation, Protein Processing, Post-Translational, Protein Serine-Threonine Kinases, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Serine, Stress, Physiological, Threonine, phosphorylation, TORC2, Ypk1, regulation, mutants, stress response, homeostasis, Genetics, Developmental Biology, Biochemistry and cell biology
Abstract

Yeast (Saccharomyces cerevisiae) target of rapamycin (TOR) complex 2 (TORC2) is a multi-subunit plasma membrane-associated protein kinase and vital growth regulator. Its essential functions are exerted via phosphorylation and stimulation of downstream protein kinase Ypk1 (and its paralog Ypk2). Ypk1 phosphorylates multiple substrates to regulate plasma membrane lipid and protein composition. Ypk1 function requires phosphorylation of Thr504 in its activation loop by eisosome-associated Pkh1 (and its paralog Pkh2). For cell survival under certain stresses, however, Ypk1 activity requires further stimulation by TORC2-mediated phosphorylation at C-terminal sites, dubbed the "turn" (Ser644) and "hydrophobic" (Thr662) motifs. Here we show that four additional C-terminal sites are phosphorylated in a TORC2-dependent manner, collectively defining a minimal consensus. We found that the newly identified sites are as important for Ypk1 activity, stability, and biological function as Ser644 and Thr662. Ala substitutions at the four new sites abrogated the ability of Ypk1 to rescue the phenotypes of Ypk1 deficiency, whereas Glu substitutions had no ill effect. Combining the Ala substitutions with an N-terminal mutation (D242A), which has been demonstrated to bypass the need for TORC2-mediated phosphorylation, restored the ability to complement a Ypk1-deficient cell. These findings provide new insights about the molecular basis for TORC2-dependent activation of Ypk1.

Published
2017

5. The role of the LAMMER kinase Kns1 and the calcium/calmodulin-dependent kinase Cmk2 in the adaptation of Saccharomyces cerevisiae to alkaline pH stress

Author

Marshall, Maria Nieves Martinez, Saumweber, Harald, Sommer, Thomas, and Heinemann, Udo

Subjects
LAMMER kinase, LAMMER-Kinase, ddc:570, Calcium/Calmodulin-abhängige Proteinkinase II, calcium/calmodulin-dependent protein kinase II, 32 Biologie, Cmk2, 570 Biowissenschaften, Biologie, WL 5190, alkaline pH stress, Kns1, alkalische pH-stress, Saccharomyces cerevisiae
Abstract

Die LAMMER-Kinasen sind Dual-Spezifität-Proteinkinasen, die durch das namensgebende einzigartige LAMMER-Motiv gekennzeichnet sind. Sie sind evolutionär hoch konserviert und in den meisten Eukaryonten vorhanden. Die vorliegende Arbeit stellt die erste funktionelle Charakterisierung eines bisher kaum erforschten Vertreters der LAMMER-Proteinkinase Familie Kns1 aus der Bäckerhefe dar. Phänotypische Analysen belegten eine entscheidende Rolle für Kns1 in der Regulation der Toleranz gegenüber basischem pH-Stress. Das Entfernen des KNS1 Gens führte zu einer gesteigerten Empfindlichkeit der Zellen gegenüber basischen Wachstumsbedingungen. Weitere Analysen zeigten, dass Kns1 neben der katalytischen Aktivität auch nicht-katalytischen Mechanismen zur Förderung des Zellwachstums unter alkalischem pH-Stress nutzt. Die Reinigung des Kns1 Proteins in voller Länge aus E. coli ermöglichte die Identifizierung von neun in vitro-Autophosphorylierungsstellen mittels Massenspektrometrie. Die Mutation von Thr562, eine Autophosphorylierungsstelle innerhalb des LAMMER-Motivs, zu Alanin ergab in vitro eine Kinase mit intrinsischer katalytischer Aktivität, die sich jedoch in vivo hauptsächlich wie die katalytisch inaktive Kns1-Mutante verhielt. Die Calcium/Calmodulin-abhängige Proteinkinase II Cmk2, die konstitutiv autokatalytische Eigenschaften besitzt, wurde früher als mögliches in vitro Substrat von Kns1 vorgeschlagen. In dieser Arbeit beweise ich durch Verwendung einer katalytisch inaktiven Cmk2-Mutante als Substrat, dass Kns1 Cmk2 in vitro phosphoryliert. Darüber hinaus zeige ich, dass Cmk2 die basische pH-Toleranz der Zellen beschränkt. Gestützt durch genetische Hinweise agieren beide Proteine gemeinsam bei der Regulation der alkalischen Stresstoleranz, wobei Kns1 möglicherweise Cmk2 herabreguliert. Zusammenfassend beschreibt diese Arbeit eine neue und entscheidende Rolle von Kns1 und Cmk2 bei der Anpassung der Hefe an alkalisches Milieu. The LAMMER protein kinases, termed after a unique signature motif found in their catalytic domains, are an evolutionary conserved family of dual-specificity kinases that are present in most eukaryotes. Here I report the first functional characterization of one of the most unexplored members of the LAMMER family, the budding yeast Kns1. Phenotypic analysis uncovered a crucial role for Kns1 in the control of the yeast tolerance to high pH stress. Deletion of the KNS1 gene conferred high sensitivity to alkaline pH, whereas its overexpression increased tolerance to this stress. Further analysis established that Kns1 promotes growth under alkaline pH stress using not only its catalytic activity but also non-catalytic mechanisms. Large-scale purification of full-length Kns1 from E. coli allowed for the identification of nine in vitro autophosphorylation sites on Kns1 by mass spectrometry. Mutation of the threonine residue at position 562, an autophosphorylation site located within the LAMMER motif, to a non-phosphorylatable residue yielded a kinase that preserves intrinsic catalytic activity in vitro but mostly behaves like the catalytically inactive mutant in vivo. This finding showed the physiological importance of autophosphorylation site Thr562 in the regulation of Kns1 function. The protein Cmk2, a calcium/calmodulin-dependent protein kinase II with autocatalytic properties, has been previously proposed as a possible in vitro substrate for Kns1. Here I demonstrate that Kns1 phosphorylates Cmk2 in vitro using a catalytically inactive Cmk2 mutant as substrate and show that Cmk2 restricts alkaline tolerance. Genetic evidence suggested that both proteins act in concert on a common pathway, in which Kns1 may downregulate Cmk2 to confer alkaline tolerance. In conclusion, this thesis describes a novel and crucial role for Kns1 and its in vitro substrate Cmk2 in the adaptation of yeast to alkaline stress.

Published
2013

6. The TORC2-Dependent Signaling Network in the Yeast Saccharomyces cerevisiae.

Author

Roelants, Françoise M., Leskoske, Kristin L., Marshall, Maria Nieves Martinez, Locke, Melissa N., and Thorner, Jeremy

Subjects
SACCHAROMYCES cerevisiae, TOR proteins, CELL communication
Abstract

To grow, eukaryotic cells must expand by inserting glycerolipids, sphingolipids, sterols, and proteins into their plasma membrane, and maintain the proper levels and bilayer distribution. A fungal cell must coordinate growth with enlargement of its cell wall. In Saccharomyces cerevisiae, a plasma membrane-localized protein kinase complex, Target of Rapamicin (TOR) complex-2 (TORC2) (mammalian ortholog is mTORC2), serves as a sensor and master regulator of these plasma membrane- and cell wall-associated events by directly phosphorylating and thereby stimulating the activity of two types of effector protein kinases: Ypk1 (mammalian ortholog is SGK1), along with a paralog (Ypk2); and, Pkc1 (mammalian ortholog is PKN2/PRK2). Ypk1 is a central regulator of pathways and processes required for plasma membrane lipid and protein homeostasis, and requires phosphorylation on its T-loop by eisosome-associated protein kinase Pkh1 (mammalian ortholog is PDK1) and a paralog (Pkh2). For cell survival under various stresses, Ypk1 function requires TORC2-mediated phosphorylation at multiple sites near its C terminus. Pkc1 controls diverse processes, especially cell wall synthesis and integrity. Pkc1 is also regulated by Pkh1- and TORC2-dependent phosphorylation, but, in addition, by interaction with Rho1-GTP and lipids phosphatidylserine (PtdSer) and diacylglycerol (DAG). We also describe here what is currently known about the downstream substrates modulated by Ypk1-mediated and Pkc1-mediated phosphorylation. [ABSTRACT FROM AUTHOR]

Published
2017
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7. The role of the LAMMER kinase Kns1 and the calcium/calmodulin-dependent kinase Cmk2 in the adaptation of Saccharomyces cerevisiae to alkaline pH stress

Author

Saumweber, Harald, Sommer, Thomas, Heinemann, Udo, Marshall, Maria Nieves Martinez, Saumweber, Harald, Sommer, Thomas, Heinemann, Udo, and Marshall, Maria Nieves Martinez

Abstract

Die LAMMER-Kinasen sind Dual-Spezifität-Proteinkinasen, die durch das namensgebende einzigartige LAMMER-Motiv gekennzeichnet sind. Sie sind evolutionär hoch konserviert und in den meisten Eukaryonten vorhanden. Die vorliegende Arbeit stellt die erste funktionelle Charakterisierung eines bisher kaum erforschten Vertreters der LAMMER-Proteinkinase Familie Kns1 aus der Bäckerhefe dar. Phänotypische Analysen belegten eine entscheidende Rolle für Kns1 in der Regulation der Toleranz gegenüber basischem pH-Stress. Das Entfernen des KNS1 Gens führte zu einer gesteigerten Empfindlichkeit der Zellen gegenüber basischen Wachstumsbedingungen. Weitere Analysen zeigten, dass Kns1 neben der katalytischen Aktivität auch nicht-katalytischen Mechanismen zur Förderung des Zellwachstums unter alkalischem pH-Stress nutzt. Die Reinigung des Kns1 Proteins in voller Länge aus E. coli ermöglichte die Identifizierung von neun in vitro-Autophosphorylierungsstellen mittels Massenspektrometrie. Die Mutation von Thr562, eine Autophosphorylierungsstelle innerhalb des LAMMER-Motivs, zu Alanin ergab in vitro eine Kinase mit intrinsischer katalytischer Aktivität, die sich jedoch in vivo hauptsächlich wie die katalytisch inaktive Kns1-Mutante verhielt. Die Calcium/Calmodulin-abhängige Proteinkinase II Cmk2, die konstitutiv autokatalytische Eigenschaften besitzt, wurde früher als mögliches in vitro Substrat von Kns1 vorgeschlagen. In dieser Arbeit beweise ich durch Verwendung einer katalytisch inaktiven Cmk2-Mutante als Substrat, dass Kns1 Cmk2 in vitro phosphoryliert. Darüber hinaus zeige ich, dass Cmk2 die basische pH-Toleranz der Zellen beschränkt. Gestützt durch genetische Hinweise agieren beide Proteine gemeinsam bei der Regulation der alkalischen Stresstoleranz, wobei Kns1 möglicherweise Cmk2 herabreguliert. Zusammenfassend beschreibt diese Arbeit eine neue und entscheidende Rolle von Kns1 und Cmk2 bei der Anpassung der Hefe an alkalisches Milieu., The LAMMER protein kinases, termed after a unique signature motif found in their catalytic domains, are an evolutionary conserved family of dual-specificity kinases that are present in most eukaryotes. Here I report the first functional characterization of one of the most unexplored members of the LAMMER family, the budding yeast Kns1. Phenotypic analysis uncovered a crucial role for Kns1 in the control of the yeast tolerance to high pH stress. Deletion of the KNS1 gene conferred high sensitivity to alkaline pH, whereas its overexpression increased tolerance to this stress. Further analysis established that Kns1 promotes growth under alkaline pH stress using not only its catalytic activity but also non-catalytic mechanisms. Large-scale purification of full-length Kns1 from E. coli allowed for the identification of nine in vitro autophosphorylation sites on Kns1 by mass spectrometry. Mutation of the threonine residue at position 562, an autophosphorylation site located within the LAMMER motif, to a non-phosphorylatable residue yielded a kinase that preserves intrinsic catalytic activity in vitro but mostly behaves like the catalytically inactive mutant in vivo. This finding showed the physiological importance of autophosphorylation site Thr562 in the regulation of Kns1 function. The protein Cmk2, a calcium/calmodulin-dependent protein kinase II with autocatalytic properties, has been previously proposed as a possible in vitro substrate for Kns1. Here I demonstrate that Kns1 phosphorylates Cmk2 in vitro using a catalytically inactive Cmk2 mutant as substrate and show that Cmk2 restricts alkaline tolerance. Genetic evidence suggested that both proteins act in concert on a common pathway, in which Kns1 may downregulate Cmk2 to confer alkaline tolerance. In conclusion, this thesis describes a novel and crucial role for Kns1 and its in vitro substrate Cmk2 in the adaptation of yeast to alkaline stress.

Published
2013

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