scholarly journals PKCε, Via its Regulatory Domain and Independently of its Catalytic Domain, Induces Neurite-like Processes in Neuroblastoma Cells

1999 ◽  
Vol 145 (4) ◽  
pp. 713-726 ◽  
Author(s):  
Ruth Zeidman ◽  
Bjarne Löfgren ◽  
Sven Påhlman ◽  
Christer Larsson
Keyword(s):  
Neurite Outgrowth ◽  
Fluorescent Protein ◽  
Catalytic Domain ◽  
Neuroblastoma Cells ◽  
Dominant Negative ◽  
Regulatory Domain ◽  
Process Formation ◽  
Synaptic Markers ◽  
Green Fluorescent ◽  
Necessary And Sufficient

To investigate the role of protein kinase C (PKC) isoforms in regulation of neurite outgrowth, PKCα, βII, δ, and ε fused to enhanced green fluorescent protein (EGFP) were transiently overexpressed in neuroblastoma cells. Overexpression of PKCε–EGFP induced cell processes whereas the other isoforms did not. The effect of PKCε–EGFP was not suppressed by the PKC inhibitor GF109203X. Instead, process formation was more pronounced when the regulatory domain was introduced. Overexpression of various fragments from PKCε regulatory domain revealed that a region encompassing the pseudosubstrate, the two C1 domains, and parts of the V3 region were necessary and sufficient for induction of processes. By deleting the second C1 domain from this construct, a dominant-negative protein was generated which suppressed processes induced by full-length PKCε and neurites induced during retinoic acid- and growth factor–induced differentiation. As with neurites in differentiated neuroblastoma cells, processes induced by the PKCε– PSC1V3 protein contained α-tubulin, neurofilament-160, and F-actin, but the PKCε–PSC1V3-induced processes lacked the synaptic markers synaptophysin and neuropeptide Y.  These data suggest that PKCε, through its regulatory domain, can induce immature neurite-like processes via a mechanism that appears to be of importance for neurite outgrowth during neuronal differentiation.

scholarly journals The catalytic domain limits the translocation of protein kinase Calpha in response to increases in Ca2+ and diacylglycerol

2003 ◽  
Vol 370 (3) ◽  
pp. 901-912 ◽  
Author(s):  
Arathi RAGHUNATH ◽  
Mia LING ◽  
Christer LARSSON
Keyword(s):  
Protein Kinase ◽  
Fluorescent Protein ◽  
Catalytic Domain ◽  
Fluorescent Proteins ◽  
Neuroblastoma Cells ◽  
Full Length ◽  
C2 Domain ◽  
Regulatory Domain ◽  
Green Fluorescent ◽  
C1 Domains

Translocation of protein kinase C (PKC) α, βII, Δ and ∊ fused to enhanced green fluorescent protein (EGFP) was studied in living neuroblastoma cells by confocal microscopy. Exposure to carbachol elicited transient translocation of PKCα-EGFP and βII-EGFP in most of the cells, PKCΔ-EGFP in a few cells and induced sustained translocation of PKC∊-EGFP. To monitor levels of Ca2+ and diacylglycerol and the translocation of PKC in the same cell, the Ca2+-sensitive C2 domain, diacylglycerol-sensitive C1 domains and full-length PKC were fused to red, cyan and yellow fluorescent proteins respectively. PKCα was translocated a few seconds after the C2 domain, which represents an increase in Ca2+. This delay was insensitive to removal of the pseudosubstrate in PKCα, but the isolated regulatory domain translocated simultaneously with the C2 domain. Translocation of PKC∊ coincided with the increase in diacylglycerol. Ionomycin induced translocation of PKCα and the C2 domain, whereas 1,2-dioctanoylglycerol caused translocation of the C1 domains and PKC∊, but not PKCα. Experiments with individual C1 domains showed that treatment with carbachol or phorbol 12,13-dibutyrate elicited translocation of PKCαC1a, PKC∊C1a and PKC∊C1b, whereas PKCαC1b was largely insensitive to these agents. In contrast with full-length PKCα, the regulatory domain of PKCα and pseudosubstrate-devoid PKCα responded to the carbachol-stimulated increase in diacylglycerol.

Determinants of the enzymatic activity and the subcellular localization of aspartate N-acetyltransferase

2011 ◽  
Vol 441 (1) ◽  
pp. 105-112 ◽  
Author(s):  
Gaëlle Tahay ◽  
Elsa Wiame ◽  
Donatienne Tyteca ◽  
Pierre J. Courtoy ◽  
Emile Van Schaftingen
Keyword(s):  
Catalytic Activity ◽  
Subcellular Localization ◽  
Fluorescent Protein ◽  
Catalytic Domain ◽  
Kinetic Properties ◽  
Conserved Residues ◽  
Membrane Bound ◽  
Green Fluorescent ◽  
Necessary And Sufficient ◽  
Membrane Region

Aspartate N-acetyltransferase (NAT8L, N-acetyltransferase 8-like), the enzyme that synthesizes N-acetylaspartate, is membrane-bound and is at least partially associated with the ER (endoplasmic reticulum). The aim of the present study was to determine which regions of the protein are important for its catalytic activity and its subcellular localization. Transfection of truncated forms of NAT8L into HEK (human embryonic kidney)-293T cells indicated that the 68 N-terminal residues (regions 1 and 2) have no importance for the catalytic activity and the subcellular localization of this enzyme, which was exclusively associated with the ER. Mutation of conserved residues that precede (Arg81 and Glu101, in region 3) or follow (Asp168 and Arg220, in region 5) the putative membrane region (region 4) markedly affected the kinetic properties, suggesting that regions 3 and 5 form the catalytic domain and that the membrane region has a loop structure. Evidence is provided for the membrane region comprising α-helices and the catalytic site being cytosolic. Transfection of chimaeric proteins in which GFP (green fluorescent protein) was fused to different regions of NAT8L indicated that the membrane region (region 4) is necessary and sufficient to target NAT8L to the ER. Thus NAT8L is targeted to the ER membrane by a hydrophobic loop that connects two regions of the catalytic domain.

scholarly journals The PWWP Domain of Dnmt3a and Dnmt3b Is Required for Directing DNA Methylation to the Major Satellite Repeats at Pericentric Heterochromatin

2004 ◽  
Vol 24 (20) ◽  
pp. 9048-9058 ◽  
Author(s):  
Taiping Chen ◽  
Naomi Tsujimoto ◽  
En Li
Keyword(s):  
Dna Methylation ◽  
Fluorescent Protein ◽  
Catalytic Domain ◽  
Regulatory Region ◽  
Pericentric Heterochromatin ◽  
Pwwp Domain ◽  
Major Satellite ◽  
Satellite Repeats ◽  
Associated Proteins ◽  
Green Fluorescent

ABSTRACT Dnmt3a and Dnmt3b are responsible for the establishment of DNA methylation patterns during development. These proteins contain, in addition to a C-terminal catalytic domain, a unique N-terminal regulatory region that harbors conserved domains, including a PWWP domain. The PWWP domain, characterized by the presence of a highly conserved proline-tryptophan-tryptophan-proline motif, is a module of 100 to 150 amino acids found in many chromatin-associated proteins. However, the function of the PWWP domain remains largely unknown. In this study, we provide evidence that the PWWP domains of Dnmt3a and Dnmt3b are involved in functional specialization of these enzymes. We show that both endogenous and green fluorescent protein-tagged Dnmt3a and Dnmt3b are particularly concentrated in pericentric heterochromatin. Mutagenesis analysis indicates that their PWWP domains are required for their association with pericentric heterochromatin. Disruption of the PWWP domain abolishes the ability of Dnmt3a and Dnmt3b to methylate the major satellite repeats at pericentric heterochromatin. Furthermore, we demonstrate that the Dnmt3a PWWP domain has little DNA-binding ability, in contrast to the Dnmt3b PWWP domain, which binds DNA nonspecifically. Collectively, our results suggest that the PWWP domains of Dnmt3a and Dnmt3b are essential for targeting these enzymes to pericentric heterochromatin, probably via a mechanism other than protein-DNA interactions.

scholarly journals Differential Localization of Rho Gtpases in Live Cells

2001 ◽  
Vol 152 (1) ◽  
pp. 111-126 ◽  
Author(s):  
David Michaelson ◽  
Joseph Silletti ◽  
Gretchen Murphy ◽  
Peter D'Eustachio ◽  
Mark Rush ◽  
...  
Keyword(s):  
Rho Gtpases ◽  
Fluorescent Protein ◽  
Essential Feature ◽  
Hypervariable Region ◽  
Dominant Negative ◽  
Live Cells ◽  
Rho Proteins ◽  
Guanine Nucleotide Dissociation Inhibitor ◽  
Membrane Compartment ◽  
Green Fluorescent

Determinants of membrane targeting of Rho proteins were investigated in live cells with green fluorescent fusion proteins expressed with or without Rho-guanine nucleotide dissociation inhibitor (GDI)α. The hypervariable region determined to which membrane compartment each protein was targeted. Targeting was regulated by binding to RhoGDIα in the case of RhoA, Rac1, Rac2, and Cdc42hs but not RhoB or TC10. Although RhoB localized to the plasma membrane (PM), Golgi, and motile peri-Golgi vesicles, TC10 localized to PMs and endosomes. Inhibition of palmitoylation mislocalized H-Ras, RhoB, and TC10 to the endoplasmic reticulum. Although overexpressed Cdc42hs and Rac2 were observed predominantly on endomembrane, Rac1 was predominantly at the PM. RhoA was cytosolic even when expressed at levels in vast excess of RhoGDIα. Oncogenic Dbl stimulated translocation of green fluorescent protein (GFP)-Rac1, GFP-Cdc42hs, and GFP-RhoA to lamellipodia. RhoGDI binding to GFP-Cdc42hs was not affected by substituting farnesylation for geranylgeranylation. A palmitoylation site inserted into RhoA blocked RhoGDIα binding. Mutations that render RhoA, Cdc42hs, or Rac1, either constitutively active or dominant negative abrogated binding to RhoGDIα and redirected expression to both PMs and internal membranes. Thus, despite the common essential feature of the CAAX (prenylation, AAX tripeptide proteolysis, and carboxyl methylation) motif, the subcellular localizations of Rho GTPases, like their functions, are diverse and dynamic.

scholarly journals Recycling of Golgi-resident Glycosyltransferases through the ER Reveals a Novel Pathway and Provides an Explanation for Nocodazole-induced Golgi Scattering

1998 ◽  
Vol 143 (6) ◽  
pp. 1505-1521 ◽  
Author(s):  
Brian Storrie ◽  
Jamie White ◽  
Sabine Röttger ◽  
Ernst H.K. Stelzer ◽  
Tatsuo Suganuma ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Time Lapse ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Protein Synthesis Inhibitors ◽  
Microtubule Depolymerization ◽  
Golgi Stack ◽  
Gradual Accumulation ◽  
Green Fluorescent ◽  
Er Export

During microtubule depolymerization, the central, juxtanuclear Golgi apparatus scatters to multiple peripheral sites. We have tested here whether such scattering is due to a fragmentation process and subsequent outward tracking of Golgi units or if peripheral Golgi elements reform through a novel recycling pathway. To mark the Golgi in HeLa cells, we stably expressed the Golgi stack enzyme N-acetylgalactosaminyltransferase-2 (GalNAc-T2) fused to the green fluorescent protein (GFP) or to an 11–amino acid epitope, VSV-G (VSV), and the trans/TGN enzyme β1,4-galactosyltransferase (GalT) fused to GFP. After nocodazole addition, time-lapse microscopy of GalNAc-T2–GFP and GalT–GFP revealed that scattered Golgi elements appeared abruptly and that no Golgi fragments tracked outward from the compact, juxtanuclear Golgi complex. Once formed, the scattered structures were relatively stable in fluorescence intensity for tens of minutes. During the entire process of dispersal, immunogold labeling for GalNAc-T2–VSV and GalT showed that these were continuously concentrated over stacked Golgi cisternae and tubulovesicular Golgi structures similar to untreated cells, suggesting that polarized Golgi stacks reform rapidly at scattered sites. In fluorescence recovery after photobleaching over a narrow (FRAP) or wide area (FRAP-W) experiments, peripheral Golgi stacks continuously exchanged resident proteins with each other through what appeared to be an ER intermediate. That Golgi enzymes cycle through the ER was confirmed by microinjecting the dominant-negative mutant of Sar1 (Sar1pdn) blocking ER export. Sar1pdn was either microinjected into untreated or nocodazole-treated cells in the presence of protein synthesis inhibitors. In both cases, this caused a gradual accumulation of GalNAc-T2–VSV in the ER. Few to no peripheral Golgi elements were seen in the nocodazole-treated cells microinjected with Sar1pdn. In conclusion, we have shown that Golgi-resident glycosylation enzymes recycle through the ER and that this novel pathway is the likely explanation for the nocodazole-induced Golgi scattering observed in interphase cells.

scholarly journals Phosphatidylinositol 3-Kinase, Cdc42, and Rac1 Act Downstream of Ras in Integrin-Dependent Neurite Outgrowth in N1E-115 Neuroblastoma Cells

2000 ◽  
Vol 20 (1) ◽  
pp. 158-172 ◽  
Author(s):  
Shula Sarner ◽  
Robert Kozma ◽  
Sohail Ahmed ◽  
Louis Lim
Keyword(s):  
Neurite Outgrowth ◽  
Kinase Activity ◽  
Neuroblastoma Cells ◽  
Dominant Negative ◽  
Serum Starvation ◽  
Phosphatidylinositol 3 Kinase ◽  
Serum Factors ◽  
Rac1 Activity ◽  
Neurite Formation ◽  
Phosphatidylinositol 3

ABSTRACT Ras and Rho family GTPases have been ascribed important roles in signalling pathways determining cellular morphology and growth. Here we investigated the roles of the GTPases Ras, Cdc42, Rac1, and Rho and that of phosphatidylinositol 3-kinase (PI 3-kinase) in the pathway leading from serum starvation to neurite outgrowth in N1E-115 neuroblastoma cells. Serum-starved cells grown on a laminin matrix exhibited integrin-dependent neurite outgrowth. Expression of dominant negative mutants of Ras, PI 3-kinase, Cdc42, or Rac1 all blocked this neurite outgrowth, while constitutively activated mutants of Ras, PI 3-kinase, or Cdc42 were each sufficient to promote outgrowth even in the presence of serum. A RasH40C;G12V double mutant which binds preferentially to PI 3-kinase also promoted neurite formation. Activated RasG12V-induced outgrowth required PI 3-kinase activity, but activated PI 3-kinase-induced outgrowth did not require Ras activity. Although activated Rac1 by itself did not induce neurites, neurite outgrowth induced by activated Cdc42G12Vwas Rac1 dependent. Cdc42G12V-induced neurites appeared to lose their normal polarization, almost doubling the average number of neurites produced by a single cell. Outgrowth induced by activated Ras or PI 3-kinase required both Cdc42 and Rac1 activity, but Cdc42G12V-induced outgrowth did not need Ras or PI 3-kinase activity. Active RhoG14V reduced outgrowth promoted by RasG12V. Finally, expression of dominant negative Jun N-terminal kinase or extracellular signal-regulated kinase did not inhibit outgrowth, suggesting these pathways are not essential for this process. Our results suggest a hierarchy of signalling where Ras signals through PI 3-kinase to Cdc42 and Rac1 activation (and Rho inactivation), culminating in neurite outgrowth. Thus, in the absence of serum factors, Ras may initiate cell cycle arrest and terminal differentiation in N1E-115 neuroblastoma cells.

scholarly journals Heterologous downregulation of vasopressin type 2 receptor is induced by transferrin

2013 ◽  
Vol 304 (5) ◽  
pp. F553-F564 ◽  
Author(s):  
Richard Bouley ◽  
Paula Nunes ◽  
Billy Andriopoulos ◽  
Margaret McLaughlin ◽  
Matthew J. Webber ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Dominant Negative ◽  
Target Cells ◽  
Intracellular Camp ◽  
Coated Pits ◽  
Parathyroid Hormone Receptor ◽  
Body Fluid Homeostasis ◽  
Green Fluorescent ◽  
G Protein Coupled

Vasopressin (VP) binds to the vasopressin type 2 receptor (V2R) to trigger physiological effects including body fluid homeostasis and blood pressure regulation. Signaling is terminated by receptor downregulation involving clathrin-mediated endocytosis and V2R degradation. We report here that both native and epitope-tagged V2R are internalized from the plasma membrane of LLC-PK1 kidney epithelial cells in the presence of another ligand, transferrin (Tf). The presence of iron-saturated Tf (holo-Tf; 4 h) reduced V2R binding sites at the cell surface by up to 33% while iron-free (apo-Tf) had no effect. However, no change in green fluorescent protein-tagged V2R distribution was observed in the presence of bovine serum albumin, atrial natriuretic peptide, or ANG II. Conversely, holo-Tf did not induce the internalization of another G protein-coupled receptor, the parathyroid hormone receptor. In contrast to the effect of VP, Tf did not increase intracellular cAMP or modify aquaporin-2 distribution in these cells, although addition of VP and Tf together augmented VP-induced V2R internalization. Tf receptor coimmunoprecipitated with V2R, suggesting that they interact closely, which may explain the additive effect of VP and Tf on V2R endocytosis. Furthermore, Tf-induced V2R internalization was abolished in cells expressing a dominant negative dynamin (K44A) mutant, indicating the involvement of clathrin-coated pits. We conclude that Tf can induce heterologous downregulation of the V2R and this might desensitize VP target cells without activating downstream V2R signaling events. It also provides new insights into urine-concentrating defects observed in rat models of hemochromatosis.

Smooth muscle cell-specific transcription is regulated by nuclear localization of the myocardin-related transcription factors

2007 ◽  
Vol 292 (2) ◽  
pp. H1170-H1180 ◽  
Author(s):  
Jeremiah S. Hinson ◽  
Matthew D. Medlin ◽  
Kashelle Lockman ◽  
Joan M. Taylor ◽  
Christopher P. Mack
Keyword(s):  
Smooth Muscle ◽  
Transcription Factors ◽  
Nuclear Localization ◽  
Transforming Growth Factor ◽  
Serum Response Factor ◽  
Fluorescent Protein ◽  
Nuclear Translocation ◽  
Dominant Negative ◽  
Specific Gene ◽  
Green Fluorescent

On the basis of our previous studies on RhoA signaling in smooth muscle cells (SMC), we hypothesized that RhoA-mediated nuclear translocalization of the myocardin-related transcription factors (MRTFs) was important for regulating SMC phenotype. MRTF-A protein and MRTF-B message were detected in aortic SMC and in many adult mouse organs that contain a large SMC component. Both MRTFs upregulated SMC-specific promoter activity as well as endogenous SM22α expression in multipotential 10T1/2 cells, although to a lesser extent than myocardin. We used enhanced green fluorescent protein (EGFP) fusion proteins to demonstrate that the myocardin factors have dramatically different localization patterns and that the stimulation of SMC-specific transcription by certain RhoA-dependent agonists was likely mediated by increased nuclear translocation of the MRTFs. Importantly, a dominant-negative form of MRTF-A (ΔB1/B2) that traps endogenous MRTFs in the cytoplasm inhibited the SM α-actin, SM22α, and SM myosin heavy chain promoters in SMC and attenuated the effects of sphingosine 1-phosphate and transforming growth factor (TGF)-β on SMC-specific transcription. Our data confirmed the importance of the NH2-terminal RPEL domains for regulating MRTF localization, but our analysis of MRTF-A/myocardin chimeras and myocardin RPEL2 mutations indicated that the myocardin B1/B2 region can override this signal. Gel shift assays demonstrated that myocardin factor activity correlated well with ternary complex formation at the SM α-actin CArGs and that MRTF-serum response factor interactions were partially dependent on CArG sequence. Taken together, our results indicate that the MRTFs regulate SMC-specific gene expression in at least some SMC subtypes and that regulation of MRTF nuclear localization may be important for the effects of selected agonists on SMC phenotype.

scholarly journals Regulation of Neurite Outgrowth in N1E-115 Cells through PDZ-Mediated Recruitment of Diacylglycerol Kinase ζ

2005 ◽  
Vol 25 (16) ◽  
pp. 7289-7302 ◽  
Author(s):  
Yury Yakubchyk ◽  
Hanan Abramovici ◽  
Jean-Christian Maillet ◽  
Elias Daher ◽  
Christopher Obagi ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Neurite Outgrowth ◽  
Cortical Neurons ◽  
Neuroblastoma Cells ◽  
Diacylglycerol Kinase ◽  
Dominant Negative ◽  
Adult Brain ◽  
Binding Motif ◽  
Kinase C

ABSTRACT Syntrophins are scaffold proteins that regulate the subcellular localization of diacylglycerol kinase ζ (DGK-ζ), an enzyme that phosphorylates the lipid second-messenger diacylglycerol to yield phosphatidic acid. DGK-ζ and syntrophins are abundantly expressed in neurons of the developing and adult brain, but their function is unclear. Here, we show that they are present in cell bodies, neurites, and growth cones of cultured cortical neurons and differentiated N1E-115 neuroblastoma cells. Overexpression of DGK-ζ in N1E-115 cells induced neurite formation in the presence of serum, which normally prevents neurite outgrowth. This effect was independent of DGK-ζ kinase activity but dependent on a functional C-terminal PDZ-binding motif, which specifically interacts with syntrophin PDZ domains. DGK-ζ mutants with a blocked C terminus acted as dominant-negative inhibitors of outgrowth from serum-deprived N1E-115 cells and cortical neurons. Several lines of evidence suggest DGK-ζ promotes neurite outgrowth through association with the GTPase Rac1. DGK-ζ colocalized with Rac1 in neuronal processes and DGK-ζ-induced outgrowth was inhibited by dominant-negative Rac1. Moreover, DGK-ζ directly interacts with Rac1 through a binding site located within its C1 domains. Together with syntrophin, these proteins form a tertiary complex in N1E-115 cells. A DGK-ζ mutant that mimics phosphorylation of the MARCKS domain was unable to bind an activated Rac1 mutant (Rac1V12) and phorbol myristate acetate-induced protein kinase C activation inhibited the interaction of DGK-ζ with Rac1V12, suggesting protein kinase C-mediated phosphorylation of the MARCKS domain negatively regulates DGK-ζ binding to active Rac1. Collectively, these findings suggest DGK-ζ, syntrophin, and Rac1 form a regulated signaling complex that controls polarized outgrowth in neuronal cells.

scholarly journals The sorting sequence of the peroxisomal integral membrane protein PMP47 is contained within a short hydrophilic loop.

1996 ◽  
Vol 133 (2) ◽  
pp. 269-280 ◽  
Author(s):  
J M Dyer ◽  
J A McNew ◽  
J M Goodman
Keyword(s):  
Amino Acids ◽  
Fluorescent Protein ◽  
Membrane Surface ◽  
Integral Membrane Protein ◽  
The Matrix ◽  
Peroxisomal Targeting ◽  
Targeting Signal ◽  
Green Fluorescent ◽  
Necessary And Sufficient ◽  
Hydrophilic Loop

No targeting sequence for peroxisomal integral membrane proteins has yet been identified. We have previously shown that a region of 67 amino acids is necessary to target Pmp47, a protein that spans the membrane six times, to peroxisomes. This region comprises two membrane spans and the intervening loop. We now demonstrate that the 20 amino acid loop, which is predicted to face the matrix, is both necessary and sufficient for peroxisomal targeting. Sufficiency was demonstrated with both chloramphenicol acetyltransferase and green fluorescent protein as carriers. There is a cluster of basic amino acids in the middle of the loop that we predict protrudes from the membrane surface into the matrix by a flanking stem structure. We show that the targeting signal is composed of this basic cluster and a block of amino acids immediately down-stream from it.

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