Biomedical subjects

J Boonstra

Publications and source records attributed to J Boonstra.

At least 55 records · Page 3Linked to original sources

PubMed1995

Epidermal growth factor (EGF) induces serine phosphorylation-dependent activation and calcium-dependent translocation of the cytosolic phospholipase A2.

Phospholipase A2 (PLA2) is a key enzyme in the release of arachidonic acid and subsequent production of eicosanoids, which play an important role in a variety of biological processes, including mitogenic signalling by epidermal growth factor (EGF). In a previous study [Spaargaren, M. et al. (1992) Biochem J. 287, 37-43] we identified the EGF-activated PLA2 as being similar to the recently cloned high-molecular-mass cytosolic phospholipase A2 (cPLA2). In the present study we demonstrate a rapid transient EGF-induced activation of this cPLA2 and an EGF-induced increase in phosphorylation of the cPLA2. The EGF-induced activation of cPLA2 is reversed upon phosphatase treatment showing phosphorylation-dependent activation of the cPLA2. No direct association of the cPLA2 to the EGF receptor was detected under conditions where such an association with phospholipase C-gamma was demonstrated. Phosphoamino acid analysis of this cPLA2 showed that EGF induced an increase in serine phosphorylation exclusively, no tyrosine phosphorylation being observed. EGF treatment of the cells resulted in a Ca(2+)-dependent translocation of the cPLA2 from the cytosol to the membrane fraction. This is due to an EGF-induced [Ca2+]i rise which is dependent on the influx of extracellular Ca2+ via voltage-independent Ca2+ channels. It is shown that the Ca(2+)-dependent association of cPLA2 to membranes does not require accessory membrane molecules.

3T3 Cells↗

PubMed1995

Epidermal growth factor induces serine phosphorylation of actin.

Stimulation of cells by epidermal growth factor induces a rapid polymerisation of actin in the cortical skeleton. Activation of the EGF-receptor leads to autophosphorylation and to phosphorylation of specific intracellular substrates. Here we show that actin is phosphorylated in vitro and in vivo upon EGF stimulation. Two-dimensional phospho-amino acid analysis shows that phosphorylation occurs on serine, not on tyrosine residues.

3T3 Cells↗

PubMed1995

The epidermal growth factor.

Epidermal growth factor (EGF) is a single polypeptide of 53 amino acid residues which is involved in the regulation of cell proliferation. Egf exerts its effects in the target cells by binding to the plasma membrane located EGF receptor. The EGF receptor is a transmembrane protein tyrosine kinase. Binding of EGF to the receptor causes activation of the kinase and subsequently receptor autophosphorylation. The autophosphorylation is essential for the interaction of the receptor with its substrates. These bind to the receptor by the so-called SH2 domains. The signal transduction pathways activated by EGF include the phosphatidylinositol pathway, leading to activation of protein kinase C and to increase in the intracellular Ca2+ concentration, and to the ras pathway leading to MAP kinase activation. Recently the cytoplasm has been implicated as playing an important role in EGF induced signal transduction. The EGF receptor has been demonstrated to be an actin-binding protein. In addition EGF causes a rapid actin depolymerisation and the formation of membrane ruffles. In particular these membrane ruffles have been shown to act as the first site of signal transduction after EGF binding, and thus may be considered as signal transduction structures. Finally evidence has been presented suggesting a positive role for EGF and/or the receptor in the nucleus.

Amino Acid Sequence↗

PubMed1995

Actin polymerization localizes to the activated epidermal growth factor receptor in the plasma membrane, independent of the cytosolic free calcium transient.

Epidermal growth factor (EGF) induces rapid actin filament assembly in the membrane skeleton of A431 cells, leading to a approximately 30% rise in cellular filamentous actin levels. EGF-induced actin polymerization depends upon EGF receptor (EGFR) tyrosine kinase activity, since the selective tyrosine kinase inhibitor AG213 abolishes EGF-induced actin polymerization. In accordance, confocal laser scanning microscopy shows that newly assembled actin filaments localize selectively to the tyrosine-phosphorylated EGFR in the plasma membrane, since actin polymerization is not observed at the internalized tyrosine-phosphorylated EGFR. Actin binding proteins (ABP's) are generally believed to regulate actin filament assembly. Ca2+ is known as one of the important regulatory factors for the activity of ABP's in vitro [15]. Therefore, we investigated the importance of the EGF-induced transient rise in [Ca2+]i for the regulation of actin polymerization in vivo. Continuous high [Ca2+]i in the millimolar range induces a prominent rise in cellular filamentous actin levels to approximately 50% over control cells. However, actin polymerization is unimpaired under conditions which effectively block the EGF-induced [Ca2+]i transient. These data demonstrate that EGF-induced actin polymerization localizes to the activated EGFR in the membrane skeleton, independent of the cytosolic free calcium transient.

Actins↗

PubMed1995

Localization of nuclear RNA by pre- and post-embedding in situ hybridization using different gold probes.

Pre-embedding and post-embedding in situ hybridization techniques were compared for the localization of RNAs in the nucleus. 28S rRNA and transcripts of the epidermal growth factor receptor (EGF-receptor) were localized with both hybridization methods. Pre-embedding hybridizations were performed on cells permeabilized with Triton X-100, whereas post-embedding hybridizations were carried out on Lowicryl K4M sections. From these studies it was concluded that, for labelling of 28S rRNA, the post-embedding in situ hybridization is preferred, whereas EGF-receptor transcripts were successfully detected only after pre-embedding in situ hybridization. Furthermore, the detection of the hybrids with ultra-small gold particles was compared to the detection with 6 nm gold particles in both pre- and post-embedding in situ hybridization studies. From our results it is concluded that the use of ultra-small gold particles results in higher label efficiency. Therefore, ultra-small gold particles are preferable to 6 nm gold particles for the detection of hybrids in high-resolution in situ hybridization experiments.

Cell Nucleus↗

PubMed1995

The concentration of ammonia regulates nitrogen metabolism in Saccharomyces cerevisiae.

Saccharomyces cerevisiae was grown in a continuous culture at a single dilution rate with input ammonia concentrations whose effects ranged from nitrogen limitation to nitrogen excess and glucose limitation. The rate of ammonia assimilation (in millimoles per gram of cells per hour) was approximately constant. Increased extracellular ammonia concentrations are correlated with increased intracellular glutamate and glutamine concentrations, increases in levels of NAD-dependent glutamate dehydrogenase activity and its mRNA (gene GDH2), and decreases in levels of NADPH-dependent glutamate dehydrogenase activity and its mRNA (gene GDH1), as well as decreases in the levels of mRNA for the amino acid permease-encoding genes GAP1 and PUT4. The governing factor of nitrogen metabolism might be the concentration of ammonia rather than its flux.

Amino Acid Transport Systems↗

PubMed1995

Epidermal growth factor induces rapid and transient association of phospholipase C-gamma 1 with EGF-receptor and filamentous actin at membrane ruffles of A431 cells.

Addition of epidermal growth factor to A431 cells results in dramatic changes in cell morphology. Initially the cells form membrane ruffles accompanied by increased actin polymerization, followed by cell rounding. Activation of the tyrosine kinase of the receptor by binding epidermal growth factor leads also to phosphorylation and activation of phospholipase C-gamma 1, a key enzyme in the phosphoinositide pathway. In this study we have investigated the localization of phospholipase C-gamma 1 during cell activation by epidermal growth factor. It is shown that addition of the growth factor to A431 cells leads to a translocation of phospholipase C-gamma 1 from the cytosol to the membrane fraction. Interestingly, this relocation is exclusively directed to the membrane ruffles. Most of the phospholipase C-gamma 1 associates to the membrane and a small fraction to the underlying skeleton. Immunocytochemical studies demonstrated that phospholipase C-gamma 1 co-localizes with the epidermal growth factor receptor and also filamentous actin at the membrane ruffles. Moreover, using anti-phosphotyrosine antibodies we found that the membrane ruffles are significantly enriched in phosphotyrosyl proteins. Between 5 and 10 minutes after stimulation the membrane ruffles disappear and also the co-localization of phospholipase C-gamma 1 with the epidermal growth factor receptor and filamentous actin. These results support the notion that activation of A431 cells by epidermal growth factor leads to the formation of a signalling complex of its receptor, phospholipase C-gamma 1 and filamentous actin which is primarily localized at membrane ruffles.

Actins↗

PubMed1994

Expression pattern of parathyroid hormone/parathyroid hormone related peptide receptor mRNA in mouse postimplantation embryos indicates involvement in multiple developmental processes.

In this paper we describe the cloning of the mouse Parathyroid Hormone/Parathyroid Hormone related Peptide Receptor (PTH/PTHrPR) cDNA and expression of its mRNA during mouse postimplantation development from day 5.5 until day 15.5 post coitum (p.c.). In support of a model from previous studies, in which parietal endoderm differentiation is regulated by the interaction of the PTH/PTHrPR and Parathyroid Hormone related Peptide (PTHrP), high levels of PTH/PTHrPR mRNA levels were detected in developing parietal endoderm from day 5.5 p.c. and onwards. In the embryo proper, PTH/PTHrPR mRNA expression was mainly detected at sites of epithelium/mesenchyme interactions, starting at day 9.5 p.c. in the epithelium of the intestine and later in the mesenchyme of the lung, the epithelium of meso- and metanephric tubuli, the dermis and at all sites where bone formation takes place. The complexity of the PTH/PTHrPR expression pattern suggests tight developmental regulation and indicates multiple roles in embryogenesis for the receptor and its ligands, not only in extraembryonic tissue but also in the formation of various organs.

Animals↗

PubMed1994

Effects of gravity on the cellular response to epidermal growth factor.

EGF and related polypeptides are involved in the regulation of cell growth and differentiation of continuously regenerating tissues, in tissue repair processes and in placental and fetal development. Their initial mode of action generally constitutes binding to specific plasma membrane localized receptors, transduction of the signal across the plasma membrane, subsequent activation of signalling pathways in the cell, and the induction of early nuclear gene expression. EGF-induced signal transmission from the plasma membrane to the nucleus has been studied in microgravity in order to gain insight in the molecular mechanisms that constitute the effects of gravity on cell growth. Exposure of human A431 cells to microgravity strongly suppresses EGF- and PMA-induced c-fos and c-jun expression. In contrast, forskolin- and A23187-induced c-fos expression and constitutive beta-2 microglobulin expression remain unaffected. This suggests that microgravity differentially modulates EGF-induced signal transduction pathways. Since both EGF and PMA are known to be activators of PKC, which is not the case for forskolin and A23187, PKC-mediated signal transduction may be a cellular target for microgravity. Inhibition of EGF-induced c-fos expression by microgravity occurs downstream of the initiation of EGF-induced signal transduction, i.e., EGF binding and EGFR redistribution. In addition to PKC signaling, actin microfilament organization appears to be sensitive to microgravity. Therefore, the inhibition of signal transduction by microgravity may be related to alterations in actin microfilament organization. The fact that early gene expression is affected by agents that alter the organization of the actin microfilament system supports this hypothesis. The decrease in c-fos and c-jun expression in microgravity may result in the decreased formation of the FOS and JUN proteins. Consequently, a short-term reduction in gene expression in microgravity may have a more dramatic effect over the long term, since both the JUN and FOS protein families are required for normal cell cycle progression. However, since more than 20 years of manned spaceflight have shown that humans can survive in microgravity for prolonged periods, it appears that cells in the human body can partly or completely overcome gravitational stress. Although some insight in the molecular basis on human cells has been obtained, future studies will be needed for a better understanding of the grounds for alterations in the cellular biochemistry due to altered gravity conditions.(ABSTRACT TRUNCATED AT 400 WORDS)

Actin Cytoskeleton↗

PubMed1993

Epidermal growth factor stimulates phosphorylation of eukaryotic initiation factor 4B, independently of protein kinase C.

We demonstrate that exposure of human epidermoid carcinoma A431 cells to epidermal growth factor (EGF) results in phosphorylation of eIF-4B within minutes after addition of EGF. The EGF-induced phosphorylation of eIF-4B is not caused by the EGF receptor tyrosine kinase itself, since no tyrosine-phosphorylated eIF-4B could be detected upon immunoprecipitation using an anti-phosphotyrosine antibody. Enhanced phosphorylation of eIF-4B was also detected upon exposure of the cells to phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC), suggesting that eIF-4B may be a substrate of PKC. However, down-regulation of PKC did not influence the EGF-induced eIF-4B phosphorylation, which indicates that eIF-4B is phosphorylated by an as yet unknown kinase, activated early in the EGF-induced signal transduction cascade.

Enzyme Activation↗

PubMed1993

Cholesterol and phosphoinositides increase affinity of the epidermal growth factor receptor.

The epidermal growth factor receptor (EGF-R) has been purified from human epidermoid carcinoma A431 cells by affinity chromatography in a single step using a monoclonal antibody (528) which competes with EGF for receptor binding. The purified EGF-R exhibits EGF inducible tyrosine kinase and autophosphorylation activity. Steady-state binding of EGF to the purified receptor revealed the presence of one class of binding sites exhibiting an apparent dissociation constant (Kd) of approx. 2 nM. When Angiotensin II was used as a receptor tyrosine kinase substrate the specific activity of the EGF induced kinase was 87 nmol/min per mg and the Km of the reaction was about 2 mM. Reconstitution of the EGF receptors into lipid vesicles was achieved by octylglucoside dialysis. Reconstitution of the receptor into pure dioleoylphosphatidylcholine (DOPC) vesicles had no effect on the EGF-binding properties in comparison to receptors in Triton X-100 micelles. Binding of EGF to the reconstituted receptor with ATP and Angiotensin II incorporated into the vesicles resulted in a five fold stimulation of the receptor kinase activity. The introduction of cholesterol, ranging from 10% to 50% (w/w), into DOPC vesicles resulted in an increase of the affinity of the receptor for its ligand. The Kd for EGF decreased from 1.8 nM in pure DOPC vesicles to 0.3 mM in DOPC/cholesterol (1:1 (w/w)) vesicles. With the introduction of small amounts (2% (w/w)) of phosphatidylinositol lipids into DOPC vesicles the Kd changed from 1.8 nM to 0.2 nM with phosphatidylinositol (PtdIns) and phosphatidylinositol 4,5-biphosphate (PtdIns4,5-P2) and to 0.1 nM in the case of phosphatidylinositol 4 phosphate (PtdIns4-P). No change in affinity was found when equal amounts of phosphatidylserine (PS) or phosphatidic acid (PA) were used.

Cholesterol↗

PubMed1993

Localisation of EGF-receptor mRNA in the nucleus of A431 cells by light microscopy.

We have localized the mRNA of the epidermal growth factor receptor (EGF-receptor) in nuclei of A431 cells by non-radioactive in situ hybridization at the light microscopical level using digoxigenin-labelled DNA probes. Both formaldehyde and glutaraldehyde fixations were tested before the hybridization was performed. Glutaraldehyde, compared with formaldehyde fixation, gives a less diffuse hybridization signal, which is easier to localize. Therefore, glutaraldehyde was used as a fixative in the hybridization experiments. It is demonstrated that the mRNA of the EGF-receptor is present in restricted domains mainly located around the nucleolus. This location of the EGF-receptor mRNA was unaltered after extraction of chromatin. Therefore it is concluded that the messenger RNA of the EGF-receptor is attached to the nuclear matrix. A possible biological role for the location of mRNA of the EGF-receptor around the nucleolus is discussed.

Animals↗

PubMed1993

Inhibition of EGF-induced signal transduction by microgravity is independent of EGF receptor redistribution in the plasma membrane of human A431 cells.

Epidermal growth factor (EGF)-induced c-fos and c-jun expression is strongly suppressed in microgravity. We investigate here whether this is due to inhibition of processes occurring during the initiation of EGF-induced signal transduction. For this purpose, EGF-induced receptor clustering is used as a marker. The lateral distribution of EGF receptors is directly visualized at an ultrastructural level by the label-fracture method. Quantification of the receptor distributions shows that EGF-induced receptor redistribution is similar under normal and microgravity conditions. This suggests that microgravity influences EGF-induced signal transduction downstream of EGF binding and EGF receptor redistribution, but upstream of early gene expression in human A431 cells.

Cell Membrane↗

PubMed1992

Characterization and identification of an epidermal-growth-factor-activated phospholipase A2.

The production of arachidonic acid (AA), which is involved in mitogenic signalling by epidermal growth factor (EGF), is most directly accomplished by the action of phospholipase A2 (PLA2). We demonstrate that EGF treatment of intact NEF cells rapidly activates a cytosolic PLA2, as measured in cell-free extracts by the release of radiolabelled AA from exogenously added 1-stearoyl-2-[1-14C]arachidonoyl phosphatidylcholine. Activation of PLA2 by EGF resulted in an enhanced Vmax. and no change in Km. The PLA2 activity was eluted in a single peak at 0.4 M-NaCl from a Mono Q anion-exchange column, and migrated with an approximate molecular mass of 70 kDa on a Superose 12 gel-filtration column. The EGF-activated PLA2 activity co-migrated with the basal PLA2 activity upon gel filtration, and persisted after partial purification, which indicates that the activation is due to a stable modification of the enzyme. The EGF-stimulated PLA2 is Ca(2+)-dependent, with maximal activity at micromolar concentrations of Ca2+, has a pH optimum at 9, associates with the particulate cell fraction in a Ca(2+)-dependent fashion, and is selective for arachidonoyl at the sn-2 position. These data demonstrate the EGF-induced activation of a PLA2, which is similar to a recently cloned high-molecular-mass AA-selective cytosolic PLA2, thus providing a link between EGF-receptor tyrosine kinase activation and AA metabolism.

Animals↗

PubMed1992

A differential location of phosphoinositide kinases, diacylglycerol kinase, and phospholipase C in the nuclear matrix.

Several enzymes involved in the phosphoinositide metabolism have been shown to be present in nuclei of rat liver and Friend cells. In this paper we demonstrate that nuclear matrices of mouse NIH 3T3-fibroblasts and rat liver cells, isolated by nuclease treatment and high salt extraction, contain phosphatidylinositol 4-kinase (PdtIns 4-kinase), phosphatidylinositol 4-phosphate 5-kinase (PtdIns(4)P 5-kinase), diacylglycerol kinase, and phospholipase C. By a selective extraction the nucleus can be dissected in the peripheral matrix (lamina-pore complex) and the internal matrix as shown by using marker antibodies. Surprisingly, PtdIns 4-kinase was found exclusively in the peripheral nuclear matrix, whereas PtdIns(4)P 5-kinase was found to be associated to internal matrix structures. Diacylglycerol kinase and phospholipase C activities were also preferentially detected in the internal matrix. These data demonstrate a differential localization of the phosphoinositide kinases in the nucleus and suggest that the phosphoinositide metabolism may play a specific role in the nucleus.

1-Phosphatidylinositol 4-Kinase↗

PubMed1992

Reduced cell-cell communication between mitotic and nonmitotic coupled cells.

The effects of mitosis on gap junctional intercellular communication (GJIC) were quantified in a clonal cell line of spontaneously immortalized rat granulosa cells (SIGC) using a fluorescence recovery after photobleaching assay. Reduction of GJIC was associated with the process of mitosis and was first apparent at the onset of prophase. Resumption of GJIC between newly divided cells and surrounding cells occurred slowly, requiring several hours following cytokinesis before reestablishment of maximal rates. Mitotic rates of GJIC in SIGC were comparable to values obtained in interphase cells partially uncoupled by 0.5 mM octanol. Limited studies of other cell lines generalized the mitotic-associated reduction of GJIC observed in SIGC. The data suggest that mitosis is one process which alters GJIC. This could be of significance when there is a change in the rate of proliferation, such as in the acquisition of immortalization, an early stage of transformation.

1-Octanol↗

PubMed1992

The epidermal growth factor receptor is associated with actin filaments.

In this paper we describe our investigations on the association of receptors for the epidermal growth factor (EGF) with the cytoskeleton of A431 cells. In order to determine which filamentous system the EGF receptors are associated to, the cytoskeletal fraction to which these receptors bind was isolated. Second, the possible colocalization of EGF receptors with different cytoskeletal elements was examined in A431 cells. By selective extractions of the A431 cytoskeletons, it is shown that more than 90% of the cytoskeleton-associated EGF receptors are removed from the cytoskeletons together with the actin filamentous system. During several cycles of poly- and depolymerization of actin isolated from A431 cells, the EGF receptor precipitates together with the actin containing filaments, indicating that EGF receptors are able to bind in vitro to actin filaments. With immunofluorescence studies we show that EGF receptors especially colocalize with actin filaments. These results demonstrate that the EGF receptor is associated specifically with actin filaments in A431 cells.

Actin Cytoskeleton↗

PubMed1992

Identification of specific gravity sensitive signal transduction pathways in human A431 carcinoma cells.

Epidermal growth factor (EGF) activates a well characterized signal transduction cascade in human A431 epidermoid carcinoma cells. The influence of gravity on EGF-induced EGF-receptor clustering and early gene expression as well as on actin polymerization and actin organization have been investigated. Different signalling pathways induced by the agents TPA, forskolin and A23187 that activate gene expression were tested for sensitivity to gravity. EGF-induced c-fos and c-jun expression were decreased in microgravity. However, constitutive beta-2 microglobulin expression remained unaltered. Under simulated weightlessness conditions EGF- and TPA-induced c-fos expression was decreased, while forskolin- and A23187-induced c-fos expression was independent of the gravity conditions. These results suggest that gravity affects specific signalling pathways. Preliminary results indicate the EGF-induced EGF-receptor clustering remained unaltered irrespective of the gravity conditions. Furthermore, the relative filamentous actin content of steady state A431 cells was enhanced under microgravity conditions and actin filament organization was altered. Under simulated weightlessness actin filament organization in steady state cells as well as in EGF-treated cells was altered as compared to the 1 G reference experiment. Interestingly the microtubule and keratin organization in untreated cells showed no difference with the normal gravity samples. This indicates that gravity may affect specific components of the signal transduction circuitry.

Calcimycin↗