scholarly journals The COOH-Terminal Domain of Wild-Type Cot Regulates Its Stability and Kinase Specific Activity

2003 ◽  
Vol 23 (20) ◽  
pp. 7377-7390 ◽  
Author(s):  
Maria Luisa Gándara ◽  
Pilar López ◽  
Raquel Hernando ◽  
José G. Castaño ◽  
Susana Alemany
Keyword(s):  
Half Life ◽  
Kinase Activity ◽  
Fluorescent Protein ◽  
Specific Activity ◽  
Yellow Fluorescent Protein ◽  
Proteasome Inhibitors ◽  
Fold Increase ◽  
Mitogen Activated Protein Kinase ◽  
Wild Type ◽  
Terminal Domain

ABSTRACT Cot, initially identified as an oncogene in a truncated form, is a mitogen-activated protein kinase kinase kinase implicated in cellular activation and proliferation. Here, we show that this truncation of Cot results in a 10-fold increase in its overall kinase activity through two different mechanisms. Truncated Cot protein exhibits a lower turnover rate (half-life, 95 min) than wild-type Cot (half-life, 35 min). The degradation of wild-type and truncated Cot can be specifically inhibited by proteasome inhibitors in situ. The 20S proteasome also degrades wild-type Cot more efficiently than the truncated protein. Furthermore, the amino acid 435 to 457 region within the wild-type Cot COOH-terminal domain confers instability when transferred to the yellow fluorescent protein and targets this fusion protein to degradation via the proteasome. On the other hand, the kinase specific activity of wild-type Cot is 3.8-fold lower than that of truncated Cot, and it appears that the last 43 amino acids of the wild-type Cot COOH-terminal domain are those responsible for this inhibition of kinase activity. In conclusion, these data demonstrate that the oncogenic activity of truncated Cot is the result of its prolonged half-life and its higher kinase specific activity with respect to wild-type Cot.

scholarly journals Probing the domain structure of FtsZ by random truncation and insertion of GFP

Microbiology ◽  
2005 ◽  
Vol 151 (12) ◽  
pp. 4033-4043 ◽  
Author(s):  
Masaki Osawa ◽  
Harold P. Erickson
Keyword(s):  
Fluorescent Protein ◽  
Dynamic Instability ◽  
Yellow Fluorescent Protein ◽  
Dominant Negative ◽  
Wild Type ◽  
Negative Effects ◽  
Terminal Domain ◽  
Z Ring ◽  
Green Fluorescent

Random transposon-mediated mutagenesis has been used to create truncations and insertions of green fluorescent protein (GFP), and Venus-yellow fluorescent protein (YFP), in Escherichia coli FtsZ. Sixteen unique insertions were obtained, and one of them, in the poorly conserved C-terminal spacer, was functional for cell division with the Venus-YFP insert. The insertion of enhanced GFP (eGFP) at this same site was not functional; Venus-YFP was found to be superior to eGFP in other respects too. Testing the constructs for dominant negative effects led to the following general conclusion. The N-terminal domain, aa 1–195, is an independently folding domain that can poison Z-ring function when expressed without a functional C-terminal domain. The effects were weak, requiring expression of the mutant at 3–5 times the level of wild-type FtsZ. The C-terminal domain, aa 195–383, was also independently folding, but had no activity in vivo. The differential activity of the N- and C-terminal domains suggests that FtsZ protofilament assembly is directional, with subunits adding primarily at the bottom of the protofilament. Directional assembly could occur by either a treadmilling or a dynamic instability mechanism.

scholarly journals Apparent lack of physical or functional interaction between CaV1.1 and its distal C terminus

2015 ◽  
Vol 145 (4) ◽  
pp. 303-314 ◽  
Author(s):  
Joshua D. Ohrtman ◽  
Christin F. Romberg ◽  
Ong Moua ◽  
Roger A. Bannister ◽  
S. Rock Levinson ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Channel Activity ◽  
Functional Interaction ◽  
Adult Skeletal Muscle ◽  
Apparent Lack ◽  
Terminal Domain ◽  
C Terminus ◽  
Decay Times

CaV1.1 acts as both the voltage sensor that triggers excitation–contraction coupling in skeletal muscle and as an L-type Ca2+ channel. It has been proposed that, after its posttranslational cleavage, the distal C terminus of CaV1.1 remains noncovalently associated with proximal CaV1.1, and that tethering of protein kinase A to the distal C terminus is required for depolarization-induced potentiation of L-type Ca2+ current in skeletal muscle. Here, we report that association of the distal C terminus with proximal CaV1.1 cannot be detected by either immunoprecipitation of mouse skeletal muscle or by colocalized fluorescence after expression in adult skeletal muscle fibers of a CaV1.1 construct labeled with yellow fluorescent protein (YFP) and cyan fluorescent protein on the N and C termini, respectively. We found that L-type Ca2+ channel activity was similar after expression of constructs that either did (YFP-CaV1.11860) or did not (YFP-CaV1.11666) contain coding sequence for the distal C-terminal domain in dysgenic myotubes null for endogenous CaV1.1. Furthermore, in response to strong (up to 90 mV) or long-lasting prepulses (up to 200 ms), tail current amplitudes and decay times were equally increased in dysgenic myotubes expressing either YFP-CaV1.11860 or YFP-CaV1.11666, suggesting that the distal C-terminal domain was not required for depolarization-induced potentiation. Thus, our experiments do not support the existence of either biochemical or functional interactions between proximal CaV1.1 and the distal C terminus.

Plasma membrane delivery of the gastric H,K-ATPase: the role of β-subunit glycosylation

2003 ◽  
Vol 285 (4) ◽  
pp. C968-C976 ◽  
Author(s):  
O. Vagin ◽  
S. Denevich ◽  
G. Sachs
Keyword(s):  
Plasma Membrane ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Glycosylation Site ◽  
Β Subunit ◽  
Wild Type ◽  
Α Subunit ◽  
Hek 293 ◽  
Glycosylation Sites ◽  
293 Cells

The factors determining trafficking of the gastric H,K-ATPase to the apical membrane remain elusive. To identify such determinants in the gastric H,K-ATPase, fusion proteins of yellow fluorescent protein (YFP) and the gastric H,K-ATPase β-subunit (YFP-β) and cyan fluorescent protein (CFP) and the gastric H,K-ATPase α-subunit (CFP-α) were expressed in HEK-293 cells. Then plasma membrane delivery of wild-type CFP-α, wild-type YFP-β, and YFP-β mutants lacking one or two of the seven β-subunit glycosylation sites was determined using confocal microscopy and surface biotinylation. Expression of the wild-type YFP-β resulted in the plasma membrane localization of the protein, whereas the expressed CFP-α was retained intracellularly. When coexpressed, both CFP-α and YFP-β were delivered to the plasma membrane. Removing each of the seven glycosylation sites, except the second one, from the extracellular loop of YFP-β prevented plasma membrane delivery of the protein. Only the mutant lacking the second glycosylation site (Asn103Gln) was localized both intracellularly and on the plasma membrane. A double mutant lacking the first (Asn99Gln) and the second (Asn103Gln) glycosylation sites displayed intracellular accumulation of the protein. Therefore, six of the seven glycosylation sites in the β-subunit are essential for the plasma membrane delivery of the β-subunit of the gastric H,K-ATPase, whereas the second glycosylation site (Asn103), which is not conserved among the β-subunits from different species, is not critical for plasma delivery of the protein.

scholarly journals The mitogen-activated protein kinase 4-phosphorylated heat shock factor A4A regulates responses to combined salt and heat stresses

2019 ◽  
Vol 70 (18) ◽  
pp. 4903-4918 ◽  
Author(s):  
Norbert Andrási ◽  
Gábor Rigó ◽  
Laura Zsigmond ◽  
Imma Pérez-Salamó ◽  
Csaba Papdi ◽  
...  
Keyword(s):  
Heat Shock ◽  
Map Kinases ◽  
Target Genes ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Phosphorylation Site ◽  
Heat Shock Factor ◽  
Mitogen Activated Protein Kinase ◽  
Heat Shock Factors ◽  
Mitogen Activated Protein

Abstract Heat shock factors regulate responses to high temperature, salinity, water deprivation, or heavy metals. Their function in combinations of stresses is, however, not known. Arabidopsis HEAT SHOCK FACTOR A4A (HSFA4A) was previously reported to regulate responses to salt and oxidative stresses. Here we show, that the HSFA4A gene is induced by salt, elevated temperature, and a combination of these conditions. Fast translocation of HSFA4A tagged with yellow fluorescent protein from cytosol to nuclei takes place in salt-treated cells. HSFA4A can be phosphorylated not only by mitogen-activated protein (MAP) kinases MPK3 and MPK6 but also by MPK4, and Ser309 is the dominant MAP kinase phosphorylation site. In vivo data suggest that HSFA4A can be the substrate of other kinases as well. Changing Ser309 to Asp or Ala alters intramolecular multimerization. Chromatin immunoprecipitation assays confirmed binding of HSFA4A to promoters of target genes encoding the small heat shock protein HSP17.6A and transcription factors WRKY30 and ZAT12. HSFA4A overexpression enhanced tolerance to individually and simultaneously applied heat and salt stresses through reduction of oxidative damage. Our results suggest that this heat shock factor is a component of a complex stress regulatory pathway, connecting upstream signals mediated by MAP kinases MPK3/6 and MPK4 with transcription regulation of a set of stress-induced target genes.

Design and Validation of Genes Encoding Hyperstable β-Globin mRNAs.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4059-4059
Author(s):  
Osheiza Abdulmalik ◽  
J. Eric Russell
Keyword(s):  
Sickle Cell Disease ◽  
Mrna Stability ◽  
Half Life ◽  
Fold Increase ◽  
Globin Genes ◽  
Wild Type ◽  
Cell Disease ◽  
Globin Mrna ◽  
Stem Loop ◽  
The Stability

Abstract 4059 Poster Board III-994 Transgenic approaches to β thalassemia and sickle cell disease require viral vectors that express high levels of therapeutic β-like globin proteins. We recently proposed that the overall expression of these transgenes would likely be improved by structural modifications that prolong the cytoplasmic half-lives of their encoded mRNAs. Relevant experiments from our laboratory have previously linked the constitutively high stability of β-globin mRNA to a region of its 3'UTR that appears to interact with at least two distinct cytoplasmic mRNA-stabilizing factors, and is predicted to form an imperfect stem-loop (SL) structure. Based upon these findings, we conducted enzymatic secondary-structure mapping studies of the β-globin 3'UTR, unequivocally validating the existence of the predicted functional stem-loop element. We subsequently reasoned that the constitutive half-life of β-globin mRNA might be prolonged by the insertion of multiple SL motifs into its 3'UTR, resulting in increased levels of the mRNA–and its encoded β-globin product–in terminally differentiating erythroid cells. To test this hypothesis, we constructed full-length β-globin genes containing either wild-type 3'UTRs, or variant 3'UTRs that had been modified to contain either two or three tandem SL motifs. Each gene was identically linked to a tetracycline-suppressible promoter, permitting pulse-chase mRNA stability analyses to be conducted in vivo in intact cultured cells. Erythroid-phenotype K562 cells were transiently transfected with SL-variant and control wild-type β-globin genes, exposed to tetracycline, and levels of β-globin mRNA determined by qRT-PCR at defined intervals using tet-indifferent β-actin mRNA as internal control. Relative to wild-type β-globin mRNA, SL-duplicate β-globin mRNAs displayed a position-dependent two-fold increase in cytoplasmic half-life; SL-triplicate β-globin mRNAs did not exhibit any additional stability. These experiments confirm the existence of a defined SL structure within the β-globin 3'UTR, and demonstrate that duplication of this motif can substantially increase the stability of β-globin mRNA. We subsequently designed a series of experiments to elucidate post-transcriptional processes involved in mRNA hyperstability. These studies required the construction of HeLa cells that stably express either wild-type β-globin mRNA (11 subclones) or SL-duplicate β-globin mRNAs (10 subclones). Preliminary analyses indicate an approximate 1.5-fold increase in the median steady-state expression of SL-duplicate genes, consistent with a prolongation in the half-life of its encoded mRNA. While formal mRNA stability studies are not yet complete, early data appear to replicate results from experiments conducted in transiently transfected cells. We have also initiated structural studies to link differences in the stability of SL-variant β-globin mRNA to alterations in its poly(A) tail. Using an RNase H-based strategy, we identified a previously unknown poly(A)-site heterogeneity–of undetermined significance–affecting both wild-type and SL-duplicate β-globin mRNAs. Finally, we recently isolated fifty-four K562 subclones expressing SL-duplicate or control β-globin mRNAs; parallel analyses of these cells will permit the cell-specificity of β-globin SL-directed mRNA stabilization to be investigated in detail. Results from each of these studies will be immediately applicable to the design of high-efficiency therapeutic transgenes for β thalassemia and sickle-cell disease. Disclosures: No relevant conflicts of interest to declare.

scholarly journals High-Throughput Growth Assay for Toxoplasma gondii Using Yellow Fluorescent Protein

2003 ◽  
Vol 47 (1) ◽  
pp. 309-316 ◽  
Author(s):  
Marc-Jan Gubbels ◽  
Catherine Li ◽  
Boris Striepen
Keyword(s):  
Toxoplasma Gondii ◽  
High Throughput ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Host Cells ◽  
Rapid Screening ◽  
Published Data ◽  
Parasite Line ◽  
Wild Type ◽  
Growth Assay

ABSTRACT A high-throughput growth assay for the protozoan parasite Toxoplasma gondii was developed based on a highly fluorescent transgenic parasite line. These parasites are stably transfected with a tandem yellow fluorescent protein (YFP) and are 1,000 times more fluorescent than the wild type. Parasites were inoculated in optical-bottom 384-well culture plates containing a confluent monolayer of host cells, and growth was monitored by using a fluorescence plate reader. The signal was linearly correlated with parasite numbers over a wide array. Direct comparison of the YFP growth assay with the β-galactosidase growth assay by using parasites expressing both reporters demonstrated that the assays' sensitivities were comparable but that the accuracy of the YFP assay was higher, especially at higher numbers of parasites per well. Determination of the 50%-inhibitory concentrations of three known growth-inhibiting drugs (cytochalasin D, pyrimethamine, and clindamycin) resulted in values comparable to published data. The delayed parasite death kinetics of clindamycin could be measured without modification of the assay, making this assay very versatile. Additionally, the temperature-dependent effect of pyrimethamine was assayed in both wild-type and engineered drug-resistant parasites. Lastly, the development of mycophenolic acid resistance after transfection of a resistance gene in T. gondii was followed. In conclusion, the YFP growth assay limits pipetting steps to a minimum, is highly versatile and amendable to automation, and should enable rapid screening of compounds to fulfill the need for more efficient and less toxic antiparasitic drugs.

scholarly journals A Cell Number Counting Factor Regulates Akt/Protein Kinase B To Regulate Dictyostelium discoideum Group Size

2004 ◽  
Vol 3 (5) ◽  
pp. 1176-1184 ◽  
Author(s):  
Tong Gao ◽  
David Knecht ◽  
Lei Tang ◽  
R. Diane Hatton ◽  
Richard H. Gomer
Keyword(s):  
Protein Kinase ◽  
Dictyostelium Discoideum ◽  
Group Size ◽  
Kinase Activity ◽  
Fluorescent Protein ◽  
Protein Kinase B ◽  
Cell Number ◽  
Pleckstrin Homology Domain ◽  
Wild Type ◽  
High Concentration

ABSTRACT Little is known about how individual cells can organize themselves to form structures of a given size. During development, Dictyostelium discoideum aggregates in dendritic streams and forms groups of ∼20,000 cells. D. discoideum regulates group size by secreting and simultaneously sensing a multiprotein complex called counting factor (CF). If there are too many cells in a stream, the associated high concentration of CF will decrease cell-cell adhesion and increase cell motility, causing aggregation streams to break up. The pulses of cyclic AMP (cAMP) that mediate aggregation cause a transient translocation of Akt/protein kinase B (Akt/PKB) to the leading edge of the plasma membrane and a concomitant activation of the kinase activity, which in turn stimulates motility. We found that countin− cells (which lack bioactive CF) and wild-type cells starved in the presence of anticountin antibodies (which block CF activity) showed a decreased level of cAMP-stimulated Akt/PKB membrane translocation and kinase activity compared to parental wild-type cells. Recombinant countin has the bioactivity of CF, and a 1-min treatment of cells with recombinant countin potentiated Akt/PKB translocation to membranes and Akt/PKB activity. Western blotting of total cell lysates indicated that countin does not affect the total level of Akt/PKB. Fluorescence microscopy of cells expressing an Akt/PKB pleckstrin homology domain-green fluorescent protein (PH-GFP) fusion protein indicated that recombinant countin and anti-countin antibodies do not obviously alter the distribution of Akt/PKB PH-GFP when it translocates to the membrane. Our data indicate that CF increases motility by potentiating the cAMP-stimulated activation and translocation of Akt/PKB.

scholarly journals Phosphoprotein Enriched in Astrocytes-15 kDa Expression Inhibits Astrocyte Migration by a Protein Kinase Cδ-dependent Mechanism

2006 ◽  
Vol 17 (12) ◽  
pp. 5141-5152 ◽  
Author(s):  
François Renault-Mihara ◽  
Frédéric Beuvon ◽  
Xavier Iturrioz ◽  
Brigitte Canton ◽  
Sophie De Bouard ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Fluorescent Protein ◽  
Constitutive Expression ◽  
Mitogen Activated Protein Kinase ◽  
Wild Type ◽  
Bryostatin 1 ◽  
Calcium Calmodulin Dependent ◽  
Mitogen Activated Protein ◽  
Green Fluorescent

Phosphoprotein enriched in astrocytes-15 kDa (PEA-15), a phosphoprotein enriched in astrocytes, inhibits both apoptosis and proliferation in normal and cancerous cells. Here, analysis of PEA-15 expression in glioblastoma organotypic cultures revealed low levels of PEA-15 in tumor cells migrating away from the explants, regardless of the expression levels in the originating explants. Because glioblastomas are highly invasive primary brain tumors that can originate from astrocytes, we explored the involvement of PEA-15 in the control of astrocyte migration. PEA-15−/− astrocytes presented an enhanced motility in vitro compared with their wild-type counterparts. Accordingly, NIH-3T3 cells transfected by green fluorescent protein-PEA-15 displayed a reduced migration. Reexpression of PEA-15 restored PEA-15−/− astrocyte motility to wild-type levels. Pharmacological manipulations excluded a participation of extracellular signal-regulated kinase/mitogen-activated protein kinase, phosphatidylinositol 3-kinase/Akt, and calcium/calmodulin-dependent protein kinase II in this effect of PEA-15. In contrast, treatment by bisindolylmaleimide, Gö6976, and rottlerin, and chronic application of phorbol 12-myristate 13-acetate and/or bryostatin-1 indicated that PKCδ mediated PEA-15 inhibition of astrocyte migration. PEA-15−/− astrocytes constitutively expressed a 40-kDa form of PKCδ that was down-regulated upon PEA-15 reexpression. Together, these data reveal a new function for PEA-15 in the inhibitory control of astrocyte motility through a PKCδ-dependent pathway involving the constitutive expression of a catalytic fragment of PKCδ.

Integrin αE(CD103)β7 influences cellular shape and motility in a ligand-dependent fashion

Blood ◽  
2008 ◽  
Vol 112 (3) ◽  
pp. 619-625 ◽  
Author(s):  
Stephanie Schlickum ◽  
Helga Sennefelder ◽  
Mike Friedrich ◽  
Gregory Harms ◽  
Martin J. Lohse ◽  
...  
Keyword(s):  
T Cells ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Time Lapse ◽  
Current Data ◽  
Immune Functions ◽  
Wild Type ◽  
Deficient Mice ◽  
E Cadherin

Abstract While the extravasation cascade of lymphocytes is well characterized, data on their intraepithelial positioning and morphology are scant. However, the latter process is presumably crucial for many immune functions. Integrin αE(CD103)β7 has previously been implicated in epithelial retention of some T cells through binding to E-cadherin. Our current data suggest that αE(CD103)β7 also determines shape and motility of some lymphocytes. Time-lapse microscopy showed that wild-type αE(CD103)β7 conferred the ability to form cell protrusions/filopodia and to move in an amoeboid fashion on E-cadherin, an activity that was abrogated by αE(CD103)β7-directed antibodies or cytochalasin D. The αE-dependent motility was further increased (P < .001) when point-mutated αE(CD103) locked in a constitutively active conformation was expressed. Moreover, different yellow fluorescent protein–coupled αE(CD103) species demonstrated that the number and length of filopodia extended toward purified E-cadherin, cocultured keratinocytes, cryostat-cut skin sections, or epidermal sheets depended on functional αE(CD103). The in vivo relevance of these findings was demonstrated by wild-type dendritic epidermal T cells (DETCs), which showed significantly more dendrites and spanned larger epidermal areas as compared with DETCs of αE(CD103)-deficient mice (P < .001). Thus, integrin αE(CD103)β7 is not only involved in epithelial retention, but also in shaping and proper intraepithelial morphogenesis of some leukocytes.

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