Insulin-stimulated trafficking of ENaC in renal cells requires PI 3-kinase activity

2003 ◽  
Vol 284 (6) ◽  
pp. C1645-C1653 ◽  
Author(s):  
Bonnie L. Blazer-Yost ◽  
Michail A. Esterman ◽  
Chris J. Vlahos
Keyword(s):  
Kinase Activity ◽  
Fluorescent Protein ◽  
Apical Cell ◽  
Parental Cell ◽  
Parental Cell Line ◽  
Insulin Stimulation ◽  
Α Subunit ◽  
Clonal Lines ◽  
Green Fluorescent ◽  
Apical Membranes

αENaC-EGFP (enhanced green fluorescent protein-tagged α-subunit of the epithelial Na+ channel) stably transfected clonal lines derived from the A6 parental cell line were used to study the physical mechanisms of insulin-stimulated Na+ transport. Within 1 min of insulin stimulation, ENaC migrates from a diffuse cytoplasmic localization to the apical and lateral membranes. Concurrently, after insulin stimulation, phosphatidylinositol 3-kinase (PI 3-kinase) is colocalized with ENaC on the lateral but not apical membrane. An inhibitor of PI 3-kinase, LY-294002, does not inhibit ENaC/PI 3-kinase colocalization but does alter the intracellular site of the colocalization, preventing the translocation of ENaC to the lateral and apical membranes. These data show that insulin stimulation causes the migration of ENaC to the lateral and apical cell membranes and that this trafficking is dependent on PI 3-kinase activity.

scholarly journals Specific Modification of Aged Proteasomes Revealed by Tag-Exchangeable Knock-In Mice

2018 ◽  
Vol 39 (1) ◽  
Author(s):  
Takuya Tomita ◽  
Shoshiro Hirayama ◽  
Yasuyuki Sakurai ◽  
Yuki Ohte ◽  
Hidehito Yoshihara ◽  
...  
Keyword(s):  
Cell Function ◽  
Fluorescent Protein ◽  
Embryonic Fibroblasts ◽  
Α Subunit ◽  
Biochemical Alterations ◽  
Cellular Processes ◽  
Responsible Gene ◽  
Intracellular Protein Degradation ◽  
Green Fluorescent

ABSTRACT The proteasome is the proteolytic machinery at the center of regulated intracellular protein degradation and participates in various cellular processes. Maintaining the quality of the proteasome is therefore important for proper cell function. It is unclear, however, how proteasomes change over time and how aged proteasomes are disposed. Here, we show that the proteasome undergoes specific biochemical alterations as it ages. We generated Rpn11-Flag/enhanced green fluorescent protein (EGFP) tag-exchangeable knock-in mice and established a method for selective purification of old proteasomes in terms of their molecular age at the time after synthesis. The half-life of proteasomes in mouse embryonic fibroblasts isolated from these knock-in mice was about 16 h. Using this tool, we found increased association of Txnl1, Usp14, and actin with the proteasome and specific phosphorylation of Rpn3 at Ser 6 in 3-day-old proteasomes. We also identified CSNK2A2 encoding the catalytic α′ subunit of casein kinase II (CK2α′) as a responsible gene that regulates the phosphorylation and turnover of old proteasomes. These findings will provide a basis for understanding the mechanism of molecular aging of the proteasome.

scholarly journals Molecular signatures of cell migration in C. elegans Q neuroblasts

2009 ◽  
Vol 185 (1) ◽  
pp. 77-85 ◽  
Author(s):  
Guangshuo Ou ◽  
Ronald D. Vale
Keyword(s):  
Cell Migration ◽  
Fluorescent Protein ◽  
Cell Movement ◽  
Live Animal ◽  
Α Subunit ◽  
C Elegans ◽  
Protein Levels ◽  
Neuroblast Migration ◽  
Green Fluorescent

Metazoan cell movement has been studied extensively in vitro, but cell migration in living animals is much less well understood. In this report, we have studied the Caenorhabditis elegans Q neuroblast lineage during larval development, developing live animal imaging methods for following neuroblast migration with single cell resolution. We find that each of the Q descendants migrates at different speeds and for distinct distances. By quantitative green fluorescent protein imaging, we find that Q descendants that migrate faster and longer than their sisters up-regulate protein levels of MIG-2, a Rho family guanosine triphosphatase, and/or down-regulate INA-1, an integrin α subunit, during migration. We also show that Q neuroblasts bearing mutations in either MIG-2 or INA-1 migrate at reduced speeds. The migration defect of the mig-2 mutants, but not ina-1, appears to result from a lack of persistent polarization in the direction of cell migration. Thus, MIG-2 and INA-1 function distinctly to control Q neuroblast migration in living C. elegans.

scholarly journals Pak1 Protein Kinase Regulates Activation and Nuclear Localization of Snf1-Gal83 Protein Kinase

2004 ◽  
Vol 24 (18) ◽  
pp. 8255-8263 ◽  
Author(s):  
Kristina Hedbacker ◽  
Seung-Pyo Hong ◽  
Marian Carlson
Keyword(s):  
Protein Kinase ◽  
Nuclear Localization ◽  
Kinase Activity ◽  
Fluorescent Protein ◽  
Protein Kinase Activity ◽  
Activation Loop ◽  
Glucose Limitation ◽  
Subunit Isoforms ◽  
Green Fluorescent ◽  
Amp Activated Protein Kinase

ABSTRACT Three kinases, Pak1, Tos3, and Elm1, activate Snf1 protein kinase in Saccharomyces cerevisiae. This cascade is conserved in mammals, where LKB1 activates AMP-activated protein kinase. We address the specificity of the activating kinases for the three forms of Snf1 protein kinase containing the β-subunit isoforms Gal83, Sip1, and Sip2. Pak1 is the most important kinase for activating Snf1-Gal83 in response to glucose limitation, but Elm1 also has a significant role; moreover, both Pak1 and Elm1 affect Snf1-Sip2. These findings exclude the possibility of a one-to-one correspondence between the activating kinases and the Snf1 complexes. We further identify a second, unexpected role for Pak1 in regulating Snf1-Gal83: the catalytic activity of Pak1 is required for the nuclear enrichment of Snf1-Gal83 in response to carbon stress. The nuclear enrichment of Snf1 fused to green fluorescent protein (GFP) depends on both Gal83 and Pak1 and is abolished by a mutation of the activation loop threonine; in contrast, the nuclear enrichment of Gal83-GFP occurs in a snf1Δ mutant and depends on Pak1 only when Snf1 is present. Snf1-Gal83 is the only form of the kinase that localizes to the nucleus. These findings, that Pak1 both activates Snf1-Gal83 and controls its nuclear localization, implicate Pak1 in regulating nuclear Snf1 protein kinase activity.

scholarly journals Activation of the erythropoietin receptor by the Friend spleen focus- forming virus gp55 glycoprotein induces constitutive protein tyrosine phosphorylation

Blood ◽  
1992 ◽  
Vol 80 (12) ◽  
pp. 3070-3078 ◽  
Author(s):  
MO Showers ◽  
JF Moreau ◽  
D Linnekin ◽  
B Druker ◽  
AD D'Andrea
Keyword(s):  
Growth Factor ◽  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Cell Lines ◽  
Kinase Activity ◽  
Parental Cell ◽  
Erythropoietin Receptor ◽  
Parental Cell Line ◽  
Tyrosine Kinase Activity ◽  
Murine Cell

The erythropoietin receptor (EPO-R) can be activated to signal cell growth by binding either EPO or gp55, the Friend spleen focus-forming virus (SFFV) glycoprotein. EPO binding induces tyrosine kinase activity and rapid tyrosine phosphorylation of several cellular substrates. To test for gp55-induced tyrosine kinase activity, we performed immunoblots on two murine cell lines that stably express EPO-R and gp55. Stimulation of the parental cell line, Ba/F3, with murine interleukin-3 (IL-3) resulted in rapid, dose-dependent tyrosine phosphorylation of a 97-Kd substrate. Stimulation with IL-3 or EPO of the Ba/F3 cells expressing the recombinant EPO-R (Ba/F3-EPO-R) resulted in tyrosine phosphorylation of the same p97 substrate. These latter cells, when transformed to growth factor-independence by the Friend gp55 glycoprotein, exhibited constitutive tyrosine phosphorylation of the 97-Kd substrate. Other growth factor-independent Ba/F3 subclones, transformed with either the oncoprotein, v-abl, or with a constitutively activated EPO-R, also had constitutive phosphorylation of a 97-Kd substrate. In CTLL-2-EPO-R cells, a T-lymphocyte line stably transfected with the EPO-R, the 97-Kd substrate was tyrosine- phosphorylated in response to IL-2 or EPO. The 97-Kd protein was constitutively phosphorylated in CTLL-2-EPO-R-gp55 cells. In conclusion, a 97-Kd protein found in two murine cell lines is tyrosine-phosphorylated in response to multiple growth factors and viral oncoproteins, and appears to be a central phosphoprotein in signal transduction.

scholarly journals Analysis of the hypoxia-sensing pathway in Drosophila melanogaster

2005 ◽  
Vol 393 (2) ◽  
pp. 471-480 ◽  
Author(s):  
Nathalie Arquier ◽  
Paul Vigne ◽  
Eric Duplan ◽  
Tien Hsu ◽  
Pascal P. Therond ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Fusion Protein ◽  
Gene Transcription ◽  
Fluorescent Protein ◽  
Hypoxia Inducible Factor ◽  
Glutathione S Transferase ◽  
Reporter Protein ◽  
Α Subunit ◽  
Von Hippel Lindau ◽  
Green Fluorescent

The mechanism by which hypoxia induces gene transcription involves the inhibition of HIF-1α (hypoxia-inducible factor-1 α subunit) PHD (prolyl hydroxylase) activity, which prevents the VHL (von Hippel-Lindau)-dependent targeting of HIF-1α to the ubiquitin/proteasome pathway. HIF-1α thus accumulates and promotes gene transcription. In the present study, first we provide direct biochemical evidence for the presence of a conserved hypoxic signalling pathway in Drosophila melanogaster. An assay for 2-oxoglutarate-dependent dioxygenases was developed using Drosophila embryonic and larval homogenates as a source of enzyme. Drosophila PHD has a low substrate specificity and hydroxylates key proline residues in the ODD (oxygen-dependent degradation) domains of human HIF-1α and Similar, the Drosophila homologue of HIF-1α. The enzyme promotes human and Drosophila [35S]VHL binding to GST (glutathione S-transferase)–ODD-domain fusion protein. Hydroxylation is enhanced by proteasomal inhibitors and was ascertained using an anti-hydroxyproline antibody. Secondly, by using transgenic flies expressing a fusion protein that combined an ODD domain and the green fluorescent protein (ODD–GFP), we analysed the hypoxic cascade in different embryonic and larval tissues. Hypoxic accumulation of the reporter protein was observed in the whole tracheal tree, but not in the ectoderm. Hypoxic stabilization of ODD–GFP in the ectoderm was restored by inducing VHL expression in these cells. These results show that Drosophila tissues exhibit different sensitivities to hypoxia.

scholarly journals Regulation of ZAP-70 Intracellular Localization: Visualization with the Green Fluorescent Protein

1997 ◽  
Vol 186 (10) ◽  
pp. 1713-1724 ◽  
Author(s):  
Joanne Sloan-Lancaster ◽  
Weiguo Zhang ◽  
John Presley ◽  
Brandi L. Williams ◽  
Robert T. Abraham ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
T Cell Receptor ◽  
Kinase Activity ◽  
Fluorescent Protein ◽  
Intracellular Localization ◽  
Cell Receptor ◽  
Cellular Activation ◽  
Intracellular Location ◽  
Green Fluorescent ◽  
Biologic Function

To investigate the cellular dynamics of ZAP-70, we have studied the distribution and regulation of its intracellular location using a ZAP-70 green fluorescent protein chimera. Initial experiments in epithelial cells indicated that ZAP-70 is diffusely located throughout the quiescent cell, and accumulates at the plasma membrane upon cellular activation, a phenotype enhanced by the coexpression of Lck and the initiation of ZAP-70 kinase activity. Subsequent studies in T cells confirmed this phenotype. Intriguingly, a large amount of ZAP-70, both chimeric and endogenous, resides in the nucleus of quiescent and activated cells. Nuclear ZAP-70 becomes tyrosine phosphorylated upon stimulation via the T cell receptor, indicating that it may have an important biologic function.

scholarly journals Binding of Drosophila Orc Proteins to Anaphase Chromosomes Requires Cessation of Mitotic Cyclin-Dependent Kinase Activity

2008 ◽  
Vol 29 (1) ◽  
pp. 140-149 ◽  
Author(s):  
Tina Baldinger ◽  
Manfred Gossen
Keyword(s):  
Origin Recognition Complex ◽  
Kinase Activity ◽  
Fluorescent Protein ◽  
Initial Step ◽  
Dynamic Imaging ◽  
Cyclin Dependent Kinase ◽  
Metaphase Chromosomes ◽  
Mitotic Cyclin ◽  
Green Fluorescent

ABSTRACT The initial step in the acquisition of replication competence by eukaryotic chromosomes is the binding of the multisubunit origin recognition complex, ORC. We describe a transgenic Drosophila model which enables dynamic imaging of a green fluorescent protein (GFP)-tagged Drosophila melanogaster ORC subunit, DmOrc2-GFP. It is functional in genetic complementation, expressed at physiological levels, and participates quantitatively in complex formation. This fusion protein is therefore able to depict both the holocomplex DmOrc1-6 and the core complex DmOrc2-6 formed by the Drosophila initiator proteins. Its localization can be monitored in vivo along the cell cycle and development. DmOrc2-GFP is not detected on metaphase chromosomes but binds rapidly to anaphase chromatin in Drosophila embryos. Expression of either stable cyclin A, B, or B3 prevents this reassociation, suggesting that cessation of mitotic cyclin-dependent kinase activity is essential for binding of the DmOrc proteins to chromosomes.

scholarly journals Three Distinct Mechanisms for Translocation and Activation of the δ Subspecies of Protein Kinase C

1998 ◽  
Vol 18 (9) ◽  
pp. 5263-5271 ◽  
Author(s):  
Shiho Ohmori ◽  
Yasuhito Shirai ◽  
Norio Sakai ◽  
Motoko Fujii ◽  
Hiroaki Konishi ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Fusion Protein ◽  
Kinase Activity ◽  
Fluorescent Protein ◽  
Simultaneous Treatment ◽  
Kinase C ◽  
Green Fluorescent ◽  
Number Of Cells

ABSTRACT We expressed δ subspecies of protein kinase C (δ-PKC) fused with green fluorescent protein (GFP) in CHO-K1 cells and observed the movement of this fusion protein in living cells after three different stimulations. The δ-PKC–GFP fusion protein had enzymological characteristics very similar to those of the native δ-PKC and was present throughout the cytoplasm in CHO-K1 cells. ATP at 1 mM caused a transient translocation of δ-PKC–GFP to the plasma membrane approximately 30 s after the stimulation and a sequent retranslocation to the cytoplasm within 3 min. A tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA; 1 μM), induced a slower translocation of δ-PKC–GFP, and the translocation was unidirectional. Concomitantly, the kinase activity of δ-PKC–GFP was increased by these two stimulations, when the kinase activity of the immunoprecipitated δ-PKC–GFP was measured in vitro in the absence of PKC activators such as phosphatidylserine and diacylglycerol. Hydrogen peroxide (H2O2; 5 mM) failed to translocate δ-PKC–GFP but increased its kinase activity more than threefold. δ-PKC–GFP was strongly tyrosine phosphorylated when treated with H2O2 but was tyrosine phosphorylated not at all by ATP stimulation and only slightly by TPA treatment. Both TPA and ATP induced the translocation of δ-PKC–GFP even after treatment with H2O2. Simultaneous treatment with TPA and H2O2 further activated δ-PKC–GFP up to more than fivefold. TPA treatment of cells overexpressing δ-PKC–GFP led to an increase in the number of cells in G2/M phase and of dikaryons, while stimulation with H2O2 increased the number of cells in S phase and induced no significant change in cell morphology. These results indicate that at least three different mechanisms are involved in the translocation and activation of δ-PKC.

scholarly journals Activation of the erythropoietin receptor by the Friend spleen focus- forming virus gp55 glycoprotein induces constitutive protein tyrosine phosphorylation

Blood ◽  
1992 ◽  
Vol 80 (12) ◽  
pp. 3070-3078 ◽  
Author(s):  
MO Showers ◽  
JF Moreau ◽  
D Linnekin ◽  
B Druker ◽  
AD D'Andrea
Keyword(s):  
Growth Factor ◽  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Cell Lines ◽  
Kinase Activity ◽  
Parental Cell ◽  
Erythropoietin Receptor ◽  
Parental Cell Line ◽  
Tyrosine Kinase Activity ◽  
Murine Cell

Abstract The erythropoietin receptor (EPO-R) can be activated to signal cell growth by binding either EPO or gp55, the Friend spleen focus-forming virus (SFFV) glycoprotein. EPO binding induces tyrosine kinase activity and rapid tyrosine phosphorylation of several cellular substrates. To test for gp55-induced tyrosine kinase activity, we performed immunoblots on two murine cell lines that stably express EPO-R and gp55. Stimulation of the parental cell line, Ba/F3, with murine interleukin-3 (IL-3) resulted in rapid, dose-dependent tyrosine phosphorylation of a 97-Kd substrate. Stimulation with IL-3 or EPO of the Ba/F3 cells expressing the recombinant EPO-R (Ba/F3-EPO-R) resulted in tyrosine phosphorylation of the same p97 substrate. These latter cells, when transformed to growth factor-independence by the Friend gp55 glycoprotein, exhibited constitutive tyrosine phosphorylation of the 97-Kd substrate. Other growth factor-independent Ba/F3 subclones, transformed with either the oncoprotein, v-abl, or with a constitutively activated EPO-R, also had constitutive phosphorylation of a 97-Kd substrate. In CTLL-2-EPO-R cells, a T-lymphocyte line stably transfected with the EPO-R, the 97-Kd substrate was tyrosine- phosphorylated in response to IL-2 or EPO. The 97-Kd protein was constitutively phosphorylated in CTLL-2-EPO-R-gp55 cells. In conclusion, a 97-Kd protein found in two murine cell lines is tyrosine-phosphorylated in response to multiple growth factors and viral oncoproteins, and appears to be a central phosphoprotein in signal transduction.

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