scholarly journals The ubiquitin E3 ligase POSH regulates calcium homeostasis through spatial control of Herp

2007 ◽  
Vol 177 (1) ◽  
pp. 51-61 ◽  
Author(s):  
Shmuel Tuvia ◽  
Daniel Taglicht ◽  
Omri Erez ◽  
Iris Alroy ◽  
Iris Alchanati ◽  
...  
Keyword(s):  
Calcium Homeostasis ◽  
Critical Role ◽  
Dominant Negative ◽  
Spatial Control ◽  
Ubiquitin E3 Ligase ◽  
Lysine Residues ◽  
Domain Protein ◽  
Golgi Network ◽  
Kinetics Of

The ubiquitin (Ub) domain protein Herp plays a crucial role in the maintenance of calcium homeostasis during endoplasmic reticulum (ER) stress. We now show that Herp is a substrate as well as an activator of the E3 Ub ligase POSH. Herp-mediated POSH activation requires the Ubl domain and exclusively promotes lysine-63–linked polyubiquitination. Confocal microscopy demonstrates that Herp resides mostly in the trans-Golgi network, but, shortly after calcium perturbation by thapsigargin (Tpg), it appears mainly in the ER. Substitution of all lysine residues within the Ubl domain abolishes lysine-63–linked polyubiquitination of Herp in vitro and calcium-induced Herp relocalization that is also abrogated by the overexpression of a dominant-negative POSHV14A. A correlation exists between the kinetics of Tpg-induced Herp relocalization and POSH-dependent polyubiquitination. Finally, the overexpression of POSH attenuates, whereas the inhibition of POSH by the expression of POSHV14A or by RNA interference enhances Tpg-induced calcium burst. Altogether, these results establish a critical role for POSH-mediated ubiquitination in the maintenance of calcium homeostasis through the spatial control of Herp.

scholarly journals Critical role of the extracellular signal–regulated kinase–MAPK pathway in osteoblast differentiation and skeletal development

2007 ◽  
Vol 176 (5) ◽  
pp. 709-718 ◽  
Author(s):  
Chunxi Ge ◽  
Guozhi Xiao ◽  
Di Jiang ◽  
Renny T. Franceschi
Keyword(s):  
Transcriptional Activity ◽  
Mapk Pathway ◽  
Skeletal Development ◽  
Critical Role ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal

The extracellular signal–regulated kinase (ERK)–mitogen-activated protein kinase (MAPK) pathway provides a major link between the cell surface and nucleus to control proliferation and differentiation. However, its in vivo role in skeletal development is unknown. A transgenic approach was used to establish a role for this pathway in bone. MAPK stimulation achieved by selective expression of constitutively active MAPK/ERK1 (MEK-SP) in osteoblasts accelerated in vitro differentiation of calvarial cells, as well as in vivo bone development, whereas dominant-negative MEK1 was inhibitory. The involvement of the RUNX2 transcription factor in this response was established in two ways: (a) RUNX2 phosphorylation and transcriptional activity were elevated in calvarial osteoblasts from TgMek-sp mice and reduced in cells from TgMek-dn mice, and (b) crossing TgMek-sp mice with Runx2+/− animals partially rescued the hypomorphic clavicles and undemineralized calvaria associated with Runx2 haploinsufficiency, whereas TgMek-dn; Runx2+/− mice had a more severe skeletal phenotype. This work establishes an important in vivo function for the ERK–MAPK pathway in bone that involves stimulation of RUNX2 phosphorylation and transcriptional activity.

scholarly journals c-Jun N-Terminal Kinase Activation of Activator Protein-1 Underlies Homologous Regulation of the Gonadotropin-Releasing Hormone Receptor Gene in αT3-1 Cells

Endocrinology ◽  
2003 ◽  
Vol 144 (3) ◽  
pp. 839-849 ◽  
Author(s):  
Buffy S. Ellsworth ◽  
Brett R. White ◽  
Ann T. Burns ◽  
Brian D. Cherrington ◽  
Annette M. Otis ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Kinase ◽  
Cell Model ◽  
Receptor Gene ◽  
Critical Role ◽  
Dominant Negative ◽  
Activator Protein 1 ◽  
Activator Protein

Reproductive function is dependent on the interaction between GnRH and its cognate receptor found on gonadotrope cells of the anterior pituitary gland. GnRH activation of the GnRH receptor (GnRHR) is a potent stimulus for increased expression of multiple genes including the gene encoding the GnRHR itself. Thus, homologous regulation of the GnRHR is an important mechanism underlying gonadotrope sensitivity to GnRH. Previously, we have found that GnRH induction of GnRHR gene expression in αT3-1 cells is partially mediated by protein kinase C activation of a canonical activator protein-1 (AP-1) element. In contrast, protein kinase A and a cAMP response element-like element have been implicated in mediating the GnRH response of the GnRHR gene using a heterologous cell model (GGH3). Herein we find that selective removal of the canonical AP-1 site leads to a loss of GnRH regulation of the GnRHR promoter in transgenic mice. Thus, an intact AP-1 element is necessary for GnRH responsiveness of the GnRHR gene both in vitro and in vivo. Based on in vitro analyses, GnRH appeared to enhance the interaction of JunD, FosB, and c-Fos at the GnRHR AP-1 element. Although enhanced binding of cFos reflected an increase in gene expression, GnRH appeared to regulate both FosB and JunD at a posttranslational level. Neither overexpression of a constitutively active Raf-kinase nor pharmacological blockade of GnRH-induced ERK activation eliminated the GnRH response of the GnRHR promoter. GnRH responsiveness was, however, lost in αT3-1 cells that stably express a dominant-negative c-Jun N-terminal kinase (JNK) kinase, suggesting a critical role for JNK in mediating GnRH regulation of the GnRHR gene. Consistent with this possibility, we find that the ability of forskolin and membrane-permeable forms of cAMP to inhibit the GnRH response of the GnRHR promoter is associated with a loss of both JNK activation and GnRH-mediated recruitment of the primary AP-1-binding components.

scholarly journals Regulation of Oxysterol-binding Protein Golgi Localization through Protein Kinase D–mediated Phosphorylation

2010 ◽  
Vol 21 (13) ◽  
pp. 2327-2337 ◽  
Author(s):  
Sokha Nhek ◽  
Mike Ngo ◽  
Xuemei Yang ◽  
Michelle M. Ng ◽  
Seth J. Field ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Binding Protein ◽  
Critical Role ◽  
Protein Kinase D ◽  
Cholesterol Depletion ◽  
Oxysterol Binding Protein ◽  
Golgi Localization ◽  
Golgi Network

Protein kinase D (PKD) plays a critical role at the trans-Golgi network by regulating the fission of transport carriers destined for the plasma membrane. Two known Golgi-localized PKD substrates, PI4-kinase IIIβ and the ceramide transfer protein CERT, mediate PKD signaling to influence vesicle trafficking to the plasma membrane and sphingomyelin synthesis, respectively. PKD is recruited and activated at the Golgi through interaction with diacylglycerol, a pool of which is generated as a by-product of sphingomyelin synthesis from ceramide. Here we identify a novel substrate of PKD at the Golgi, the oxysterol-binding protein OSBP. Using a substrate-directed phospho-specific antibody that recognizes the optimal PKD consensus motif, we show that PKD phosphorylates OSBP at Ser240 in vitro and in cells. We further show that OSBP phosphorylation occurs at the Golgi. Phosphorylation of OSBP by PKD does not modulate dimerization, sterol binding, or affinity for PI(4)P. Instead, phosphorylation attenuates OSBP Golgi localization in response to 25-hydroxycholesterol and cholesterol depletion, impairs CERT Golgi localization, and promotes Golgi fragmentation.

Alteration of adipocyte calcium homeostasis by Escherichia coli endotoxin

1985 ◽  
Vol 248 (3) ◽  
pp. R331-R338
Author(s):  
K. M. Nelson ◽  
J. A. Spitzer
Keyword(s):  
Escherichia Coli ◽  
Cell Surface ◽  
Calcium Homeostasis ◽  
Compartmental Analysis ◽  
In Vitro Exposure ◽  
Exchange Kinetics ◽  
Intracellular Binding ◽  
Kinetics Of

The present study evaluated calcium homeostasis in rat adipocytes after either in vivo or in vitro exposure to Escherichia coli endotoxin. Fat cells from endotoxin-treated rats showed an enhanced uptake of 45Ca. In an attempt to differentiate between 45Ca binding to the cell surface and intracellular 45Ca accumulation, adipocytes were exposed to 5 mM LaCl3. The amount of 45Ca remaining associated with lanthanum-treated adipocytes was taken to be located intracellularly and was increased in adipocytes from endotoxin-treated rats. The amount of 45Ca displaced by lanthanum was also increased in adipocytes from endotoxin-treated rats. This suggested that the endotoxin-induced increase of 45Ca accumulation included both cell surface and intracellular binding sites. Compartmental analysis of the exchange kinetics of cell-associated 45Ca with 40Ca in the medium indicated a 77% increase in the size of the cell surface compartment of adipocytes from endotoxin-treated rats compared with controls. In addition, endotoxin treatment altered the flux of calcium from the cells to the medium. In vitro exposure of freshly prepared adipocytes to 250 or 750 micrograms endotoxin/ml did not produce a perturbation of adipocyte calcium homeostasis. The results indicate that endotoxin induces alterations in the ability of adipocytes to regulate calcium translocations, suggesting that some metabolic and hormonal aspects of endotoxins' actions may be mediated through perturbation of cellular calcium homeostasis.

scholarly journals Activation of IκB Kinase β by Protein Kinase C Isoforms

1999 ◽  
Vol 19 (3) ◽  
pp. 2180-2188 ◽  
Author(s):  
Maria-José Lallena ◽  
María T. Diaz-Meco ◽  
Gary Bren ◽  
Carlos V. Payá ◽  
Jorge Moscat
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Critical Role ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Cell Functions ◽  
Kinase C ◽  
Iκb Kinase ◽  
Tnf Α

ABSTRACT The atypical protein kinase C (PKC) isotypes (λ/ιPKC and ζPKC) have been shown to be critically involved in important cell functions such as proliferation and survival. Previous studies have demonstrated that the atypical PKCs are stimulated by tumor necrosis factor alpha (TNF-α) and are required for the activation of NF-κB by this cytokine through a mechanism that most probably involves the phosphorylation of IκB. The inability of these PKC isotypes to directly phosphorylate IκB led to the hypothesis that ζPKC may use a putative IκB kinase to functionally inactivate IκB. Recently several groups have molecularly characterized and cloned two IκB kinases (IKKα and IKKβ) which phosphorylate the residues in the IκB molecule that serve to target it for ubiquitination and degradation. In this study we have addressed the possibility that different PKCs may control NF-κB through the activation of the IKKs. We report here that αPKC as well as the atypical PKCs bind to the IKKs in vitro and in vivo. In addition, overexpression of ζPKC positively modulates IKKβ activity but not that of IKKα, whereas the transfection of a ζPKC dominant negative mutant severely impairs the activation of IKKβ but not IKKα in TNF-α-stimulated cells. We also show that cell stimulation with phorbol 12-myristate 13-acetate activates IKKβ, which is entirely dependent on the activity of αPKC but not that of the atypical isoforms. In contrast, the inhibition of αPKC does not affect the activation of IKKβ by TNF-α. Interestingly, recombinant active ζPKC and αPKC are able to stimulate in vitro the activity of IKKβ but not that of IKKα. In addition, evidence is presented here that recombinant ζPKC directly phosphorylates IKKβ in vitro, involving Ser177 and Ser181. Collectively, these results demonstrate a critical role for the PKC isoforms in the NF-κB pathway at the level of IKKβ activation and IκB degradation.

scholarly journals TGN/EE SNARE protein SYP61 is ubiquitinated and required for carbon/nitrogen-nutrient responses in Arabidopsis

2020 ◽  
Author(s):  
Yoko Hasegawa ◽  
Thais Huarancca Reyes ◽  
Tomohiro Uemura ◽  
Akari Fujimaki ◽  
Yongming Luo ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Critical Role ◽  
Snare Protein ◽  
Post Translational Modification ◽  
Cellular Processes ◽  
Protein Receptor ◽  
Sensitive Factor ◽  
Cargo Proteins ◽  
Golgi Network

AbstractUbiquitination is a post-translational modification with reversible attachment of the small protein ubiquitin, which is involved in numerous cellular processes including membrane trafficking. For example, ubiquitination of cargo proteins is known to regulate their subcellular dynamics, and plays important roles in plant growth and stress adaptation. However, the regulatory mechanism of the trafficking machinery components remains elusive. Here, we report Arabidopsis trans-Golgi network/early endosome (TGN/EE) localized soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) protein SYP61 as a novel ubiquitination target of a membrane localized ubiquitin ligase ATL31. SYP61 is a key component of membrane trafficking in Arabidopsis. SYP61 was ubiquitinated with K63-linked chain by ATL31 in vitro and in plants. The knockdown mutants of SYP61 were hypersensitive to the disrupted carbon (C)/nitrogen (N)-nutrient stress, suggesting its critical role in plant homeostasis in response to nutrients. We also found the ubiquitination status of SYP61 is affected by C/N-nutrient availability. These results provided possibility that ubiquitination of SNARE protein has important role in plant physiology.

Disruption of Golgi structure and function in mammalian cells expressing a mutant dynamin

2000 ◽  
Vol 113 (11) ◽  
pp. 1993-2002 ◽  
Author(s):  
H. Cao ◽  
H.M. Thompson ◽  
E.W. Krueger ◽  
M.A. McNiven
Keyword(s):  
Mammalian Cells ◽  
Living Cells ◽  
Dominant Negative ◽  
Coated Vesicle ◽  
Large Numbers ◽  
Golgi Structure ◽  
And Function ◽  
Golgi Network ◽  
Mutant Cells

The large GTPase dynamin is a mechanoenzyme that participates in the scission of nascent vesicles from the plasma membrane. Recently, dynamin has been demonstrated to associate with the Golgi apparatus in mammalian cells by morphological and biochemical methods. Additional studies using a well characterized, cell-free assay have supported these findings by demonstrating a requirement for dynamin function in the formation of clathrin-coated, and non-clathrin-coated vesicles from the trans-Golgi network (TGN). In this study, we tested if dynamin participates in Golgi function in living cells through the expression of a dominant negative dynamin construct (K44A). Cells co-transfected to express this mutant dynamin and a GFP-tagged Golgi resident protein (TGN38) exhibit Golgi structures that are either compacted, vesiculated, or tubulated. Electron microscopy of these mutant cells revealed large numbers of Golgi stacks comprised of highly tubulated cisternae and an extraordinary number of coated vesicle buds. Cells expressing mutant dynamin and GFP-tagged VSVG demonstrated a marked retention (8- to 11-fold) of the nascent viral G-protein in the Golgi compared to control cells. These observations in living cells are consistent with previous morphological and in vitro studies demonstrating a role for dynamin in the formation of secretory vesicles from the TGN.

scholarly journals Retrieval of Human Cytomegalovirus Glycoprotein B from Cell Surface Is Not Required for Virus Envelopment in Astrocytoma Cells

2002 ◽  
Vol 76 (10) ◽  
pp. 5147-5155 ◽  
Author(s):  
Michael A. Jarvis ◽  
Kenneth N. Fish ◽  
Cecilia Söderberg-Naucler ◽  
Daniel N. Streblow ◽  
Heather L. Meyers ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Infectious Virus ◽  
Critical Role ◽  
Dominant Negative ◽  
Glycoprotein B ◽  
Virion Assembly ◽  
Virion Envelope ◽  
Infected Cells ◽  
Dynamin I ◽  
Golgi Network

ABSTRACT Human cytomegalovirus (HCMV) is a prototypic member of the betaherpesvirus family. The HCMV virion is composed of a large DNA genome encapsidated within a nucleocapsid, which is wrapped within an inner proteinaceous tegument and an outer lipid envelope containing viral glycoproteins. Although genome encapsidation clearly occurs in the nucleus, the subsequent steps in the virion assembly process are unclear. HCMV glycoprotein B (gB) is a major component of the virion envelope that plays a critical role in virus entry and is essential for the production of infectious virus progeny. The aim of our present study was to identify the secretory compartment to which HCMV gB was localized and to investigate the role of endocytosis in mediating gB localization and HCMV biogenesis. We show that HCMV gB is localized to the trans-Golgi network (TGN) in HCMV-infected cells and that gB contains all of the trafficking information necessary for TGN localization. Endocytosis of gB was shown to play a role in mediating TGN localization of gB and in targeting of the protein to the site of virus envelopment. However, inhibition of endocytosis with a dominant-negative dynamin I molecule did not affect the production of infectious virus. These observations indicate that, although endocytosis is involved in the trafficking of gB to the site of glycoprotein accumulation in the TGN, endocytosis of gB is not required for the production of infectious HCMV.

scholarly journals Myostatin Inhibits Myoblast Differentiation by Down-regulating MyoD Expression

2002 ◽  
Vol 277 (51) ◽  
pp. 49831-49840 ◽  
Author(s):  
Brett Langley ◽  
Mark Thomas ◽  
Amy Bishop ◽  
Mridula Sharma ◽  
Stewart Gilmour ◽  
...  
Keyword(s):  
Culture Media ◽  
Myogenic Differentiation ◽  
Critical Role ◽  
Dominant Negative ◽  
Negative Regulator ◽  
Myoblast Differentiation ◽  
Proliferation And Differentiation ◽  
Smad 3 ◽  
Low Serum

Myostatin, a negative regulator of myogenesis, is shown to function by controlling the proliferation of myoblasts. In this study we show that myostatin is an inhibitor of myoblast differentiation and that this inhibition is mediated through Smad 3.In vitro, increasing concentrations of recombinant mature myostatin reversibly blocked the myogenic differentiation of myoblasts, cultured in low serum media. Western and Northern blot analysis indicated that addition of myostatin to the low serum culture media repressed the levels of MyoD, Myf5, myogenin, and p21 leading to the inhibition of myogenic differentiation. The transient transfection of C2C12myoblasts with MyoD expressing constructs did not rescue myostatin-inhibited myogenic differentiation. Myostatin signaling specifically induced Smad 3 phosphorylation and increased Smad 3·MyoD association, suggesting that Smad 3 may mediate the myostatin signal by interfering with MyoD activity and expression. Consistent with this, the expression of dominant-negativeSmad3rescued the activity of a MyoD promoter-reporter in C2C12myoblasts treated with myostatin. Taken together, these results suggest that myostatin inhibits MyoD activity and expression via Smad 3 resulting in the failure of the myoblasts to differentiate into myotubes. Thus we propose that myostatin plays a critical role in myogenic differentiation and that the muscular hyperplasia and hypertrophy seen in animals that lack functional myostatin is because of deregulated proliferation and differentiation of myoblasts.

Method for measuring gas production kinetics

1998 ◽  
Vol 22 ◽  
pp. 209-211 ◽  
Author(s):  
D. R. Mertens ◽  
P. J. Weimer
Keyword(s):  
Kinetic Parameters ◽  
Dry Matter ◽  
Critical Role ◽  
Gas Production ◽  
Production Kinetics ◽  
Digestion Kinetics ◽  
Ruminal Digestion ◽  
Kinetics Of ◽  
Or Modelling

Methodology can play a critical rôle in the measurement of digestion kinetics, especially when the objective is to define kinetic parameters for use in formulating rations or modelling animal responses. Measurement of gas production kinetics provides the opportunity to evaluate the rate of digestion of the soluble, more rapidly fermenting fractions of foods but has the potential for being more sensitive to the in vitro procedure used. Differences among procedures that have little impact on digestion of dry matter after 48 h of incubation, may have dramatic effects on fermentation of soluble matter during the first 20 h. Our objective was to develop a method for measuring the kinetics of gas production that would minimize any detrimental effects associated with the in vitro system and provide estimates of digestion kinetics that can be used to both describe foods for ration formulation systems and provide parameters for models of ruminal digestion.

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