scholarly journals Analysis of the Yeast Kinome Reveals a Network of Regulated Protein Localization during Filamentous Growth

2008 ◽  
Vol 19 (7) ◽  
pp. 2708-2717 ◽  
Author(s):  
Nikë Bharucha ◽  
Jun Ma ◽  
Craig J. Dobry ◽  
Sarah K. Lawson ◽  
Zhifen Yang ◽  
...  
Keyword(s):  
Stress Response ◽  
Protein Kinase ◽  
Fluorescent Protein ◽  
Nuclear Translocation ◽  
Protein Localization ◽  
Yellow Fluorescent Protein ◽  
Filamentous Growth ◽  
Mitogen Activated Protein Kinase ◽  
Yeast Cells ◽  
Systematic Analysis

The subcellular distribution of kinases and other signaling proteins is regulated in response to cellular cues; however, the extent of this regulation has not been investigated for any gene set in any organism. Here, we present a systematic analysis of protein kinases in the budding yeast, screening for differential localization during filamentous growth. Filamentous growth is an important stress response involving mitogen-activated protein kinase and cAMP-dependent protein kinase signaling modules, wherein yeast cells form interconnected and elongated chains. Because standard strains of yeast are nonfilamentous, we constructed a unique set of 125 kinase-yellow fluorescent protein chimeras in the filamentous Σ1278b strain for this study. In total, we identified six cytoplasmic kinases (Bcy1p, Fus3p, Ksp1p, Kss1p, Sks1p, and Tpk2p) that localize predominantly to the nucleus during filamentous growth. These kinases form part of an interdependent, localization-based regulatory network: deletion of each individual kinase, or loss of kinase activity, disrupts the nuclear translocation of at least two other kinases. In particular, this study highlights a previously unknown function for the kinase Ksp1p, indicating the essentiality of its nuclear translocation during yeast filamentous growth. Thus, the localization of Ksp1p and the other kinases identified here is tightly controlled during filamentous growth, representing an overlooked regulatory component of this stress response.

scholarly journals The Mitogen-Activated Protein Kinase BcSak1 of Botrytis cinerea Is Required for Pathogenic Development and Has Broad Regulatory Functions Beyond Stress Response

2012 ◽  
Vol 25 (6) ◽  
pp. 802-816 ◽  
Author(s):  
Jens Heller ◽  
Nadja Ruhnke ◽  
José Juan Espino ◽  
Michelli Massaroli ◽  
Isidro Gonzalez Collado ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Stress Response ◽  
Protein Kinase ◽  
Osmotic Stress ◽  
Botrytis Cinerea ◽  
Fluorescent Protein ◽  
Mitogen Activated Protein Kinase ◽  
In Planta ◽  
Mitogen Activated Protein

The mitogen-activated protein kinase (MAPK) BcSak1 of Botrytis cinerea is activated upon exposure to H2O2 and, hence, might be involved in coping with oxidative stress during infection. However, beside osmotic and oxidative stress sensitivity, Δbcsak1 mutants have a pleiotropic phenotype, as they do not produce conidia and are unable to penetrate unwounded host tissue. In this study, the role of BcSak1 was investigated in the stress response and during infection of French beans by Botrytis cinerea. Using a macroarray approach, it was shown that BcSak1 is only marginally involved in the specific oxidative stress response. In fact, the induction of several genes after oxidative stress treatment is BcSak1-dependent, but most of these genes are also induced under conditions of osmotic stress. The majority of genes regulated by BcSak1 are not involved in the stress response at all. Using a translational fusion of BcSak1 to green fluorescent protein, it was shown clearly that the localization of this MAPK depends on the type of stress being applied; it associates rapidly to the nucleus only under osmotic stress. Therefore, a model is proposed in which BcSak1 acts in the cytosol by activation of one or more transcription factors under oxidative stress and, at the same time, it reacts to osmotic stress by migrating to the nucleus. Interestingly, the MAPK is also involved in the regulation of secondary metabolism, as the major phytotoxins secreted by this fungus are reduced in the Δbcsak1 deletion mutant. Experiments done in planta underlined the essential role of BcSak1 in the early stages of infection, when it translocates to the nucleus and then changes to cytosolic distribution during hyphal growth within the tissue.

scholarly journals Large-Scale Analysis of Yeast Filamentous Growth by Systematic Gene Disruption and Overexpression

2008 ◽  
Vol 19 (1) ◽  
pp. 284-296 ◽  
Author(s):  
Rui Jin ◽  
Craig J. Dobry ◽  
Phillip J. McCown ◽  
Anuj Kumar
Keyword(s):  
Protein Kinase ◽  
Gene Disruption ◽  
Large Scale ◽  
Fluorescent Protein ◽  
Filamentous Growth ◽  
Mitogen Activated Protein Kinase ◽  
Yeast Saccharomyces Cerevisiae ◽  
Filamentous Form ◽  
Transposon Insertion ◽  
Yeast Genes

Under certain conditions of nutrient stress, the budding yeast Saccharomyces cerevisiae initiates a striking developmental transition to a filamentous form of growth, resembling developmental transitions required for virulence in closely related pathogenic fungi. In yeast, filamentous growth involves known mitogen-activated protein kinase and protein kinase A signaling modules, but the full scope of this extensive filamentous response has not been delineated. Accordingly, we have undertaken the first systematic gene disruption and overexpression analysis of yeast filamentous growth. Standard laboratory strains of yeast are nonfilamentous; thus, we constructed a unique set of reagents in the filamentous Σ1278b strain, encompassing 3627 integrated transposon insertion alleles and 2043 overexpression constructs. Collectively, we analyzed 4528 yeast genes with these reagents and identified 487 genes conferring mutant filamentous phenotypes upon transposon insertion and/or gene overexpression. Using a fluorescent protein reporter integrated at the MUC1 locus, we further assayed each filamentous growth mutant for aberrant protein levels of the key flocculence factor Muc1p. Our results indicate a variety of genes and pathways affecting filamentous growth. In total, this filamentous growth gene set represents a wealth of yeast biology, highlighting 84 genes of uncharacterized function and an underappreciated role for the mitochondrial retrograde signaling pathway as an inhibitor of filamentous growth.

scholarly journals Candida albicans SNO1 and SNZ1 expressed in stationary-phase planktonic yeast cells and base of biofilm

Microbiology ◽  
2006 ◽  
Vol 152 (7) ◽  
pp. 2031-2038 ◽  
Author(s):  
Priya Uppuluri ◽  
Bhaskarjyoti Sarmah ◽  
W. LaJean Chaffin
Keyword(s):  
Candida Albicans ◽  
Stationary Phase ◽  
Fluorescent Protein ◽  
Protein Localization ◽  
Expression Patterns ◽  
Yellow Fluorescent Protein ◽  
Growth Conditions ◽  
Yeast Cells ◽  
Yeast Culture

The Candida albicans homologues of the most studied Saccharomyces cerevisiae stationary-phase genes, SNO1 and SNZ1, were used to test the hypothesis that, within a biofilm, some cells reach stationary phase within continuously fed, as well as static, C. albicans biofilms grown on dental acrylic. The authors first studied the expression patterns of these two genes in planktonic growth conditions. Using real-time RT-PCR (RT-RTPCR), increased peak expression of both SNZ1 and SNO1 was observed at 5 and 6 days, respectively, in C. albicans grown in suspension culture. SNZ1–yellow fluorescent protein (YFP) and SNO1–YFP were constructed to study expression at the cellular level and protein localization in C. albicans. Snz1p–YFP and Sno1p–YFP localized to the cytoplasm with maximum expression (>90 %) at 5 and 6 days, respectively, in planktonic conditions. When yeast growth was reinitiated, loss of fluorescence began immediately. Germ tubes and hyphae were non-fluorescent. Pseudohyphae began appearing at 9 days in planktonic yeast culture and expressed each protein by 11 days; however, the cells budding from pseudohyphae were not fluorescent. Biofilm was formed in vitro under either static or continuously fed conditions. Increased expression of the two genes was shown by RT-RTPCR, beginning by day 3 and increasing through to day 15 (continuously fed biofilm). Only the bottommost layer of acrylic-adhered cells in the biofilm showed 25 and 40 % fluorescence at 6 and 15 days, respectively. These observations suggest that only a few cells in C. albicans biofilms express genes associated with the planktonic stationary phase and that these are found at the bottom of the biofilm adhered to the surface.

scholarly journals Disparate Subcellular Localization Patterns of Pseudomonas aeruginosa Type IV Pilus ATPases Involved in Twitching Motility

2005 ◽  
Vol 187 (3) ◽  
pp. 829-839 ◽  
Author(s):  
Poney Chiang ◽  
Marc Habash ◽  
Lori L. Burrows
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Fluorescent Protein ◽  
Protein Localization ◽  
Yellow Fluorescent Protein ◽  
Distribution Patterns ◽  
Opportunistic Pathogen ◽  
Twitching Motility ◽  
Polar Localization ◽  
Type Iv ◽  
And Function

ABSTRACT The opportunistic pathogen Pseudomonas aeruginosa expresses polar type IV pili (TFP), which are responsible for adhesion to various materials and twitching motility on surfaces. Twitching occurs by alternate extension and retraction of TFP, which arise from assembly and disassembly of pilin subunits at the base of the pilus. The ATPase PilB promotes pilin assembly, while the ATPase PilT or PilU or both promote pilin dissociation. Fluorescent fusions to two of the three ATPases (PilT and PilU) were functional, as shown by complementation of the corresponding mutants. PilB and PilT fusions localized to both poles, while PilU fusions localized only to the piliated pole. To identify the portion of the ATPases required for localization, sequential C-terminal deletions of PilT and PilU were generated. The conserved His and Walker B boxes were dispensable for polar localization but were required for twitching motility, showing that localization and function could be uncoupled. Truncated fusions that retained polar localization maintained their distinctive distribution patterns. To dissect the cellular factors involved in establishing polarity, fusion protein localization was monitored with a panel of TFP mutants. The localization of yellow fluorescent protein (YFP)-PilT and YFP-PilU was independent of the subunit PilA, other TFP ATPases, and TFP-associated proteins previously shown to be associated with the membrane or exhibiting polar localization. In contrast, YFP-PilB exhibited diffuse cytoplasmic localization in a pilC mutant, suggesting that PilC is required for polar localization of PilB. Finally, localization studies performed with fluorescent ATPase chimeras of PilT and PilU demonstrated that information responsible for the characteristic localization patterns of the ATPases likely resides in their N termini.

scholarly journals Anti-Inflammatory Activity of Populus deltoides Leaf Extract via Modulating NF-κB and p38/JNK Pathways

2018 ◽  
Vol 19 (12) ◽  
pp. 3746 ◽  
Author(s):  
Ye Jeong ◽  
Mi-Young Lee
Keyword(s):  
Nitric Oxide ◽  
Protein Kinase ◽  
Leaf Extract ◽  
Populus Deltoides ◽  
Nuclear Translocation ◽  
Mapk Signaling ◽  
Mitogen Activated Protein Kinase ◽  
Inflammatory Activity ◽  
No Production ◽  
Anti Inflammatory

Populus deltoides, known as eastern cottonwood, has been commonly used as a medicinal plant. The aim of the present study was to investigate the mechanism underlying the anti-inflammatory activity of P. deltoides leaf extract (PLE). PLE effectively inhibited the expression of inducible nitric oxide synthase (iNOS) and nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells, but not that of cyclooxygenase-2 (COX-2) and prostaglandin E2. Proinflammatory tumor necrosis factor alpha (TNF-α) levels were also reduced by the extract. PLE inhibited the phosphorylation of nuclear factor-kappa B (NF-κB) and inhibitor of Kappa Bα (IκBα), and blunted LPS-triggered enhanced nuclear translocation of NF-κB p65. In mitogen-activated protein kinase (MAPK) signaling, PLE effectively decreased the phosphorylation of p38 and c-Jun N-terminal protein kinase (JNK), but not of extracellular signal-regulated kinase 1/2 (ERK1/2). Taken together, these results suggest that anti-inflammatory activity of P. deltoides leaf extract might be driven by iNOS and NO inhibition mediated by modulation of the NF-κB and p38/JNK signaling pathways.

scholarly journals Both Binding and Activation of p38 Mitogen-Activated Protein Kinase (MAPK) Play Essential Roles in Regulation of the Nucleocytoplasmic Distribution of MAPK-Activated Protein Kinase 5 by Cellular Stress

2002 ◽  
Vol 22 (20) ◽  
pp. 6931-6945 ◽  
Author(s):  
Ole Morten Seternes ◽  
Bjarne Johansen ◽  
Beate Hegge ◽  
Mona Johannessen ◽  
Stephen M. Keyse ◽  
...  
Keyword(s):  
Protein Kinase ◽  
P38 Mapk ◽  
Nuclear Export ◽  
Fluorescent Protein ◽  
Mapk Pathway ◽  
Nuclear Export Signal ◽  
Mitogen Activated Protein Kinase ◽  
Protein Fusion ◽  
Translational Machinery ◽  
Mitogen Activated Protein

ABSTRACT The p38 mitogen-activated protein kinase (MAPK) pathway is an important mediator of cellular responses to environmental stress. Targets of p38 include transcription factors, components of the translational machinery, and downstream serine/threonine kinases, including MAPK-activated protein kinase 5 (MK5). Here we have used enhanced green fluorescent protein fusion proteins to analyze the subcellular localization of MK5. Although this protein is predominantly nuclear in unstimulated cells, MK5 shuttles between the nucleus and the cytoplasm. Furthermore, we have shown that the C-terminal domain of MK5 contains both a functional nuclear localization signal (NLS) and a leucine-rich nuclear export signal (NES), indicating that the subcellular distribution of this kinase reflects the relative activities of these two signals. In support of this, we have shown that stress-induced activation of the p38 MAPK stimulates the chromosomal region maintenance 1 protein-dependent nuclear export of MK5. This is regulated by both binding of p38 MAPK to MK5, which masks the functional NLS, and stress-induced phosphorylation of MK5 by p38 MAPK, which either activates or unmasks the NES. These properties may define the ability of MK5 to differentially phosphorylate both nuclear and cytoplasmic targets or alternatively reflect a mechanism whereby signals initiated by activation of MK5 in the nucleus may be transmitted to the cytoplasm.

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