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.

scholarly journals Diethylstilbestrol, a Novel ANO1 Inhibitor, Exerts an Anticancer Effect on Non-Small Cell Lung Cancer via Inhibition of ANO1

2021 ◽  
Vol 22 (13) ◽  
pp. 7100
Author(s):  
Yohan Seo ◽  
Sung Baek Jeong ◽  
Joo Han Woo ◽  
Oh-Bin Kwon ◽  
Sion Lee ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Small Cell Lung Cancer ◽  
Cell Lung Cancer ◽  
High Throughput Screening ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Small Cell ◽  
Channel Activity ◽  
Small Cell Lung ◽  
Protein Levels

Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer-related mortality; thus, therapeutic targets continue to be developed. Anoctamin1 (ANO1), a novel drug target considered for the treatment of NSCLC, is a Ca2+-activated chloride channel (CaCC) overexpressed in various carcinomas. It plays an important role in the development of cancer; however, the role of ANO1 in NSCLC is unclear. In this study, diethylstilbestrol (DES) was identified as a selective ANO1 inhibitor using high-throughput screening. We found that DES inhibited yellow fluorescent protein (YFP) fluorescence reduction caused by ANO1 activation but did not inhibit cystic fibrosis transmembrane conductance regulator channel activity or P2Y activation-related cytosolic Ca2+ levels. Additionally, electrophysiological analyses showed that DES significantly reduced ANO1 channel activity, but it more potently reduced ANO1 protein levels. DES also inhibited the viability and migration of PC9 cells via the reduction in ANO1, phospho-ERK1/2, and phospho-EGFR levels. Moreover, DES induced apoptosis by increasing caspase-3 activity and PARP-1 cleavage in PC9 cells, but it did not affect the viability of hepatocytes. These results suggest that ANO1 is a crucial target in the treatment of NSCLC, and DES may be developed as a potential anti-NSCLC therapeutic agent.

Different interactions of cardiac and skeletal muscle ryanodine receptors with FK-506 binding protein isoforms

1997 ◽  
Vol 272 (5) ◽  
pp. C1726-C1733 ◽  
Author(s):  
S. Barg ◽  
J. A. Copello ◽  
S. Fleischer
Keyword(s):  
Skeletal Muscle ◽  
Lipid Bilayers ◽  
Single Channel ◽  
Ryanodine Receptors ◽  
Protein Isoforms ◽  
Channel Activity ◽  
Functional Interaction ◽  
Fk 506 ◽  
Receptor Isoforms ◽  
Functional Consequences

In the present study, we compare functional consequences of dissociation and reconstitution of binding proteins FKBP12 and FKBP12.6 with ryanodine receptors from cardiac (RyR2) and skeletal muscle (RyR1). The skeletal muscle RyR1 channel became activated on removal of endogenously bound FKBP12, consistent with previous reports. Both FKBP12 and FKBP12.6 rebind to FKBP-depleted RyR1 and restore its quiescent channel behavior by altering ligand sensitivity, as studied by single-channel recordings in planar lipid bilayers, and macroscopic behavior of the channels (ryanodine binding and net energized Ca2- uptake). By contrast, removal of FKBP12.6 from the cardiac RyR2 did not modulate the function of the channel using the same types of assays as for RyR1. FKBP12 or FKBP12.6 had no effect on channel activity of FKBP12.6-depleted cardiac RyR2, although FKBP12.6 rebinds. Our studies reveal important differences between the two ryanodine receptor isoforms with respect to their functional interaction with FKBP12 and FKBP12.6.

scholarly journals Conventional Kinesin Mediates Microtubule-Microtubule Interactions In Vivo

2006 ◽  
Vol 17 (2) ◽  
pp. 907-916 ◽  
Author(s):  
Anne Straube ◽  
Gerd Hause ◽  
Gero Fink ◽  
Gero Steinberg
Keyword(s):  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Binding Activity ◽  
C Terminus ◽  
Cross Bridges ◽  
Conventional Kinesin ◽  
Motor Head

Conventional kinesin is a ubiquitous organelle transporter that moves cargo toward the plus-ends of microtubules. In addition, several in vitro studies indicated a role of conventional kinesin in cross-bridging and sliding microtubules, but in vivo evidence for such a role is missing. In this study, we show that conventional kinesin mediates microtubule-microtubule interactions in the model fungus Ustilago maydis. Live cell imaging and ultrastructural analysis of various mutants in Kin1 revealed that this kinesin-1 motor is required for efficient microtubule bundling and participates in microtubule bending in vivo. High levels of Kin1 led to increased microtubule bending, whereas a rigor-mutation in the motor head suppressed all microtubule motility and promoted strong microtubule bundling, indicating that kinesin can form cross-bridges between microtubules in living cells. This effect required a conserved region in the C terminus of Kin1, which was shown to bind microtubules in vitro. In addition, a fusion protein of yellow fluorescent protein and the Kin1tail localized to microtubule bundles, further supporting the idea that a conserved microtubule binding activity in the tail of conventional kinesins mediates microtubule-microtubule interactions in vivo.

scholarly journals Endocytic Down-Regulation of ErbB2 Is Stimulated by Cleavage of Its C-Terminus

2007 ◽  
Vol 18 (9) ◽  
pp. 3656-3666 ◽  
Author(s):  
Mads Lerdrup ◽  
Silas Bruun ◽  
Michael V. Grandal ◽  
Kirstine Roepstorff ◽  
Malene M. Kristensen ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Retention Mechanism ◽  
Full Length ◽  
Dependent Manner ◽  
Lysosomal Degradation ◽  
Down Regulation ◽  
C Terminus ◽  
Early Endosomes

High ErbB2 levels are associated with cancer, and impaired endocytosis of ErbB2 could contribute to its overexpression. Therefore, knowledge about the mechanisms underlying endocytic down-regulation of ErbB2 is warranted. The C-terminus of ErbB2 can be cleaved after various stimuli, and after inhibition of HSP90 with geldanamycin this cleavage is accompanied by proteasome-dependent endocytosis of ErbB2. However, it is unknown whether C-terminal cleavage is linked to endocytosis. To study ErbB2 cleavage and endocytic trafficking, we fused yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP) to the N- and C-terminus of ErbB2, respectively (YFP-ErbB2-CFP). After geldanamycin stimulation YFP-ErbB2-CFP became cleaved in nonapoptotic cells in a proteasome-dependent manner, and a markedly larger relative amount of cleaved YFP-ErbB2-CFP was observed in early endosomes than in the plasma membrane. Furthermore, cleavage took place at the plasma membrane, and cleaved ErbB2 was internalized and degraded far more efficiently than full-length ErbB2. Concordantly, a C-terminally truncated ErbB2 was also readily endocytosed and degraded in lysosomes compared with full-length ErbB2. Altogether, we suggest that geldanamycin leads to C-terminal cleavage of ErbB2, which releases the receptor from a retention mechanism and causes endocytosis and lysosomal degradation of ErbB2.

scholarly journals Foxo1 nucleo-cytoplasmic distribution and unidirectional nuclear influx are the same in nuclei in a single skeletal muscle fiber but vary between fibers

2018 ◽  
Vol 314 (3) ◽  
pp. C334-C348 ◽  
Author(s):  
Yewei Liu ◽  
Sarah J. Russell ◽  
Martin F. Schneider
Keyword(s):  
Skeletal Muscle ◽  
Fluorescent Protein ◽  
Muscle Fibers ◽  
Culture Conditions ◽  
Control Culture ◽  
Adult Skeletal Muscle ◽  
Large Variability ◽  
Skeletal Muscle Fibers ◽  
Foxo Transcription Factors ◽  
Green Fluorescent

Foxo transcription factors promote protein breakdown and atrophy of skeletal muscle fibers. Foxo transcriptional effectiveness is largely determined by phosphorylation-dependent nucleo-cytoplasmic shuttling. Imaging Foxo1-green fluorescent protein (GFP) over time in 124 nuclei in 68 multinucleated adult skeletal muscle fibers under control culture conditions reveals large variability between fibers in Foxo1-GFP nucleo-cytoplasmic concentration ratio (N/C) and in the apparent rate coefficient ( kI′) for Foxo1-GFP unidirectional nuclear influx (measured with efflux blocked by leptomycin B). Pairs of values of N/C or of kI′ from different nuclei in the same fiber were essentially the same, but only weakly correlated in nuclei from different fibers in the same culture well. Thus, fiber to fiber variability of cellular factors, but not extracellular factors, determines Foxo1 distribution. Over all nuclei, N/C and kI′ were closely proportional, indicating that kI′ is the major determinant of Foxo1 distribution. IGF-I activation of Foxo kinase Akt reduces variability by decreasing kI′ and N/C in all fibers. However, inhibiting Akt did not drive kI′ uniformly high, indicating other pathways in Foxo1 regulation.

scholarly journals Sexual Pheromone Induces Diffusion of the Pheromone-Homologous Polypeptide in the Extracellular Matrix of Volvox carteri

2007 ◽  
Vol 6 (11) ◽  
pp. 2157-2162 ◽  
Author(s):  
Koichi Ishida
Keyword(s):  
Extracellular Matrix ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Sexual Pheromone ◽  
Volvox Carteri ◽  
Terminal Domain ◽  
Induction Signal ◽  
C Terminus ◽  
Green Fluorescent

ABSTRACT The C-terminal domain of pherophorin II is homologous to the sexual pheromone of Volvox carteri and is released from other domains during sexual induction. Green fluorescent protein fused to the C terminus of pherophorin II was located at the extracellular matrix directly surrounding the gonidium, the final target of the sexual-induction signal.

scholarly journals The C-terminal Domain Is the Primary Determinant of Histone H1 Binding to Chromatinin Vivo

2004 ◽  
Vol 279 (19) ◽  
pp. 20028-20034 ◽  
Author(s):  
Michael J. Hendzel ◽  
Melody A. Lever ◽  
Ellen Crawford ◽  
John P. H. Th'ng
Keyword(s):  
Amino Acid ◽  
Fluorescent Protein ◽  
Point Mutations ◽  
Histone H1 ◽  
Single Amino Acid ◽  
Kinetic Measurements ◽  
Terminal Domain ◽  
C Terminus ◽  
Green Fluorescent

We have used a combination of kinetic measurements and targeted mutations to show that the C-terminal domain is required for high-affinity binding of histone H1 to chromatin, and phosphorylations can disrupt binding by affecting the secondary structure of the C terminus. By measuring the fluorescence recovery after photo-bleaching profiles of green fluorescent protein-histone H1 proteins in living cells, we find that the deletion of the N terminus only modestly reduces binding affinity. Deletion of the C terminus, however, almost completely eliminates histone H1.1 binding. Specific mutations of the C-terminal domain identified Thr-152 and Ser-183 as novel regulatory switches that control the binding of histone H1.1in vivo. It is remarkable that the single amino acid substitution of Thr-152 with glutamic acid was almost as effective as the truncation of the C terminus to amino acid 151 in destabilizing histone H1.1 bindingin vivo. We found that modifications to the C terminus can affect histone H1 binding dramatically but have little or no influence on the charge distribution or the overall net charge of this domain. A comparison of individual point mutations and deletion mutants, when reviewed collectively, cannot be reconciled with simple charge-dependent mechanisms of C-terminal domain function of linker histones.

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.

The C-terminus of the transmembrane mucin MUC17 binds to the scaffold protein PDZK1 that stably localizes it to the enterocyte apical membrane in the small intestine

2008 ◽  
Vol 410 (2) ◽  
pp. 283-289 ◽  
Author(s):  
Emily K. Malmberg ◽  
Thaher Pelaseyed ◽  
Åsa C. Petersson ◽  
Ursula E. Seidler ◽  
Hugo De Jonge ◽  
...  
Keyword(s):  
Glutathione Transferase ◽  
Fluorescent Protein ◽  
Apical Membrane ◽  
Yellow Fluorescent Protein ◽  
Pdz Domain ◽  
Scaffold Protein ◽  
Wild Type ◽  
C Terminus ◽  
In The Wild ◽  
Membrane Bound

The membrane-bound mucins have a heavily O-glycosylated extracellular domain, a single-pass membrane domain and a short cytoplasmic tail. Three of the membrane-bound mucins, MUC3, MUC12 and MUC17, are clustered on chromosome 7 and found in the gastrointestinal tract. These mucins have C-terminal sequences typical of PDZ-domain-binding proteins. To identify PDZ proteins that are able to interact with the mucins, we screened PDZ domain arrays using YFP (yellow fluorescent protein)-tagged proteins. MUC17 exhibited a strong binding to PDZK1 (PDZ domain containing 1), whereas the binding to NHERF1 (Na+/H+-exchanger regulatory factor 1) was weak. Furthermore, we showed weak binding of MUC12 to PDZK1, NHERF1 and NHERF2. GST (glutathione transferase) pull-down experiments confirmed that the C-terminal tail of MUC17 co-precipitates with the scaffold protein PDZK1 as identified by MS. This was mediated through the C-terminal PDZ-interaction site in MUC17, which was capable of binding to three of the four PDZ domains in PDZK1. Immunostaining of wild-type or Pdzk1−/− mouse jejunum with an antiserum against Muc3(17), the mouse orthologue of human MUC17, revealed strong brush-border membrane staining in the wild-type mice compared with an intracellular Muc3(17) staining in the Pdzk1−/− mice. This suggests that Pdzk1 plays a specific role in stabilizing Muc3(17) in the apical membrane of small intestinal enterocytes.

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.

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