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A J Verkleij

Publications and source records attributed to A J Verkleij.

At least 19 recordsLinked to original sources
PubMed2000

Hydrogen peroxide inhibits epidermal growth factor receptor internalization in human fibroblasts.

Several cellular signal transduction cascades are affected by oxidative stress. In this study, the effect of hydrogen peroxide (H2O2) on the endocytosis of the epidermal growth factor (EGF) receptor was investigated. Exposure of HER14 cells to H2O2 resulted in a concentration-dependent inhibition of EGF receptor internalization. Binding studies demonstrated that this H2O2-induced inhibition in internalization was not due to altered binding of EGF to its receptor. Addition of H2O2 at different time points during internalization showed that EGF receptor internalization was rapidly reduced, suggesting that one of the first steps in the internalization process is inhibited. In addition, H2O2 inhibited the internalization of a different receptor, the chicken hepatic lectin receptor, in a concentration-dependent manner as well. Treatment of cells with another inducer of oxidative stress, cumene hydroperoxide, also resulted in a decreased internalization. Finally, we showed that H2O2 inhibited EGF-induced mono-ubiquitination of the EGF receptor pathway substrate clone 15, a process that normally occurs during EGF receptor endocytosis. These results clearly show that oxidative stress interferes with EGF signaling.

3T3 Cells↗
PubMed2000

The cell wall architecture of Candida albicans wild-type cells and cell wall-defective mutants.

In Candida albicans wild-type cells, the beta1, 6-glucanase-extractable glycosylphosphatidylinositol (GPI)-dependent cell wall proteins (CWPs) account for about 88% of all covalently linked CWPs. Approximately 90% of these GPI-CWPs, including Als1p and Als3p, are attached via beta1,6-glucan to beta1,3-glucan. The remaining GPI-CWPs are linked through beta1,6-glucan to chitin. The beta1,6-glucanase-resistant protein fraction is small and consists of Pir-related CWPs, which are attached to beta1,3-glucan through an alkali-labile linkage. Immunogold labelling and Western analysis, using an antiserum directed against Saccharomyces cerevisiae Pir2p/Hsp150, point to the localization of at least two differentially expressed Pir2 homologues in the cell wall of C. albicans. In mnn9Delta and pmt1Delta mutant strains, which are defective in N- and O-glycosylation of proteins respectively, we observed enhanced chitin levels together with an increased coupling of GPI-CWPs through beta1,6-glucan to chitin. In these cells, the level of Pir-CWPs was slightly upregulated. A slightly increased incorporation of Pir proteins was also observed in a beta1, 6-glucan-deficient hemizygous kre6Delta mutant. Taken together, these observations show that C. albicans follows the same basic rules as S. cerevisiae in constructing a cell wall and indicate that a cell wall salvage mechanism is activated when Candida cells are confronted with cell wall weakening.

Aminoglycosides↗
PubMed1999

Integrin signaling at the M/G1 transition induces expression of cyclin E.

The activities of the mammalian G1 cyclins, cyclin D and cyclin E, during cell cycle progression (G1/S) are believed to be regulated by cell attachment and the presence of growth factors. In order to study the importance of cell attachment and concomitant integrin signaling on the expression of G1 cyclins during the natural adhesion process from mitosis to interphase, protein expression was monitored in cells that were synchronized by mitotic shake off. Here we show that in Chinese hamster ovary (CHO) and neuroblastoma (N2A) cells, expression of cyclin E at the M/G1 transition is regulated by both growth factors and cell attachment, while expression of cyclin D seems to be entirely dependent on the presence of serum. Expression of cyclin E appears to be correlated with the phosphorylation of the retinoblastoma protein, suggesting a link with the activity of the cyclin D/cdk4 complex. Expression of the cdk inhibitors p21(cip1/Waf1) and p27(Kip1) is not changed upon serum depletion or detachment of cells during early G1, suggesting no direct role for these CKIs in the regulation of cyclin activity. Although inhibition of cyclin E/cdk2 kinase activity has been reported previously, this is the first time that cyclin E expression is shown to be dependent on cell attachment.

Animals↗
PubMed1999

Protein kinase Czeta is a negative regulator of protein kinase B activity.

Protein kinase B (PKB), also known as Akt or RAC-PK, is a serine/threonine kinase that can be activated by growth factors via phosphatidylinositol 3-kinase. In this article we show that PKCzeta but not PKCalpha and PKCdelta can co-immunoprecipitate PKB from CHO cell lysates. Association of PKB with PKCzeta was also found in COS-1 cells transiently expressing PKB and PKCzeta, and moreover we found that this association is mediated by the AH domain of PKB. Stimulation of COS-1 cells with platelet-derived growth factor (PDGF) resulted in a decrease in the PKB-PKCzeta interaction. The use of kinase-inactive mutants of both kinases revealed that dissociation of the complex depends upon PKB activity. Analysis of the activities of the interacting kinases showed that PDGF-induced activation of PKCzeta was not affected by co-expression of PKB. However, both PDGF- and p110-CAAX-induced activation of PKB were significantly abolished in cells co-expressing PKCzeta. In contrast, co-expression of a kinase-dead PKCzeta mutant showed an increased induction of PKB activity upon PDGF treatment. Downstream signaling of PKB, such as the inhibition of glycogen synthase kinase-3, was also reduced by co-expression of PKCzeta. A clear inhibitory effect of PKCzeta was found on the constitutively active double PKB mutant (T308D/S473D). In summary, our results demonstrate that PKB interacts with PKCzeta in vivo and that PKCzeta acts as a negative regulator of PKB.

Amino Acid Sequence↗
PubMed1999

Nuclear translocation of mitogen-activated protein kinase p42MAPK during the ongoing cell cycle.

Mitogen-activated protein (MAP) kinases are serine/threonine protein kinases that are activated rapidly in cells stimulated by various extracellular signals. With stimulation of quiescent cells by growth factors, activated p42/p44 MAP kinases rapidly translocate to the nucleus, where they induce immediate early gene transcription. The MAP kinase signal transduction pathway represents an important mechanism by which growth factors regulate cellular events such as cell cycle progression or cell growth. In the present study, p42MAPK (ERK2) was studied during the ongoing cell cycle of Chinese hamster ovary cells synchronized by mitotic shake-off. We show that protein expression of p42MAPK increased in mid-G1 and that MAP kinase is phosphorylated during G1, as visualized by a gel-mobility shift and by the use of phosphospecific antibodies. This phosphorylation appeared to occur in the cytoplasm rather than at the plasma-membrane. In addition, phosphorylated p42MAPK was found to translocate to the nucleus during late/mid-G1. Treatment of cells with MEK inhibitor PD098059 prevented the phosphorylation and nuclear translocation of MAP kinase and DNA synthesis. Thus, nuclear translocation of p42MAPK is not restricted to the G0/G1 transition but occurs in every cell cycle and seems to be required for cell cycle progression.

Animals↗
PubMed1999

Membrane fusion and the lamellar-to-inverted-hexagonal phase transition in cardiolipin vesicle systems induced by divalent cations.

The polymorphic phase behavior of bovine heart cardiolipin (CL) in the presence of different divalent cations and the kinetics of CL vesicle fusion induced by these cations have been investigated. (31)P-NMR measurements of equilibrium cation-CL complexes showed the lamellar-to-hexagonal (L(alpha)-H(II)) transition temperature (T(H)) to be 20-25 degrees C for the Sr(2+) and Ba(2+) complexes, whereas in the presence of Ca(2+) or Mg(2+) the T(H) was below 0 degrees C. In the presence of Sr(2+) or Ba(2+), CL large unilamellar vesicles (LUVs) (0.1 microm diameter) showed kinetics of destabilization, as assessed by determination of the release of an aqueous fluorescent dye, which strongly correlated with the L(alpha)-H(II) transition of the final complex: at temperatures above the T(H), fast and extensive leakage, mediated by vesicle-vesicle contact, was observed. On the other hand, mixing of vesicle contents was limited and of a highly transient nature. A different behavior was observed with Ca(2+) or Mg(2+): in the temperature range of 0-50 degrees C, where the H(II) configuration is the thermodynamically favored phase, relatively nonleaky fusion of the vesicles occurred. Furthermore, with increasing temperature the rate and extent of leakage decreased, with a concomitant increase in fusion. Fluorescence measurements, involving incorporation of N-NBD-phosphatidylethanolamine in the vesicle bilayer, demonstrated a relative delay in the L(alpha)-H(II) phase transition of the CL vesicle system in the presence of Ca(2+). Freeze-fracture electron microscopy of CL LUV interaction products revealed the exclusive formation of H(II) tubes in the case of Sr(2+), whereas with Ca(2+) large fused vesicles next to H(II) tubes were seen. The extent of binding of Ca(2+) to CL in the lamellar phase, saturating at a binding ratio of 0.35 Ca(2+) per CL, was close to that observed for Sr(2+) and Ba(2+). It is concluded that CL LUVs in the presence of Ca(2+) undergo a transition that favors nonleaky fusion of the vesicles over rapid collapse into H(II) structures, despite the fact that the equilibrium Ca(2+)-CL complex is in the H(II) phase. On the other hand, in the presence of Sr(2+) or Ba(2+) at temperatures above the T(H) of the respective cation-CL complexes, CL LUVs rapidly convert to H(II) structures with a concomitant loss of vesicular integrity. This suggests that the nature of the final cation-lipid complex does not primarily determine whether CL vesicles exposed to the cation will initially undergo a nonleaky fusion event or collapse into nonvesicular structures.

4-Chloro-7-nitrobenzofurazan↗
PubMed1999

Function of trehalose and glycogen in cell cycle progression and cell viability in Saccharomyces cerevisiae.

Trehalose and glycogen accumulate in Saccharomyces cerevisiae when growth conditions deteriorate. It has been suggested that aside from functioning as storage factors and stress protectants, these carbohydrates may be required for cell cycle progression at low growth rates under carbon limitation. By using a mutant unable to synthesize trehalose and glycogen, we have investigated this requirement of trehalose and glycogen under carbon-limited conditions in continuous cultures. Trehalose and glycogen levels increased with decreasing growth rates in the wild-type strain, whereas no trehalose or glycogen was detected in the mutant. However, the mutant was still able to grow and divide at low growth rates with doubling times similar to those for the wild-type strain, indicating that trehalose and glycogen are not essential for cell cycle progression. Nevertheless, upon a slight increase of extracellular carbohydrates, the wild-type strain degraded its reserve carbohydrates and was able to enter a cell division cycle faster than the mutant. In addition, wild-type cells survived much longer than the mutant cells when extracellular carbon was exhausted. Thus, trehalose and glycogen have a dual role under these conditions, serving as storage factors during carbon starvation and providing quickly a higher carbon and ATP flux when conditions improve. Interestingly, the CO2 production rate and hence the ATP flux were higher in the mutant than in the wild-type strain at low growth rates. The possibility that the mutant strain requires this steady higher glycolytic flux at low growth rates for passage through Start is discussed.

Biomass↗
PubMed1999

Localization of synthesis of beta1,6-glucan in Saccharomyces cerevisiae.

Beta1,6-Glucan is a key component of the yeast cell wall, interconnecting cell wall proteins, beta1,3-glucan, and chitin. It has been postulated that the synthesis of beta1,6-glucan begins in the endoplasmic reticulum with the formation of protein-bound primer structures and that these primer structures are extended in the Golgi complex by two putative glucosyltransferases that are functionally redundant, Kre6 and Skn1. This is followed by maturation steps at the cell surface and by coupling to other cell wall macromolecules. We have reinvestigated the role of Kre6 and Skn1 in the biogenesis of beta1,6-glucan. Using hydrophobic cluster analysis, we found that Kre6 and Skn1 show significant similarities to family 16 glycoside hydrolases but not to nucleotide diphospho-sugar glycosyltransferases, indicating that they are glucosyl hydrolases or transglucosylases instead of genuine glucosyltransferases. Next, using immunogold labeling, we tried to visualize intracellular beta1,6-glucan in cryofixed sec1-1 cells which had accumulated secretory vesicles at the restrictive temperature. No intracellular labeling was observed, but the cell surface was heavily labeled. Consistent with this, we could detect substantial amounts of beta1,6-glucan in isolated plasma membrane-derived microsomes but not in post-Golgi secretory vesicles. Taken together, our data indicate that the synthesis of beta1, 6-glucan takes place largely at the cell surface. An alternative function for Kre6 and Skn1 is discussed.

Amino Acid Sequence↗
PubMed1998

Actin polymerization is required for negative feedback regulation of epidermal growth factor-induced signal transduction.

Epidermal growth factor (EGF) induces rapid actin filament assembly in the membrane skeleton of a variety of cells. To investigate the significance of this process for signal transduction, actin polymerization is inhibited by dihydrocytochalasin B (CB). CB almost completely abolishes EGF-induced actin polymerization, as assessed by quantitative confocal laser scanning microscopy. Under these conditions, EGF induces enhanced EGF receptor (EGFR) tyrosine kinase activity, as well as superinduction of the c-fos proto-oncogene. These data suggest that EGF-induced actin polymerization may be important for negative feedback regulation of signal transduction by the EGFR. The phosphorylation of Thr654 by protein kinase C (PKC) is a well-characterized negative feedback control mechanism for signal transduction by the EGFR tyrosine kinase. A synthetic peptide, corresponding to the regions flanking Thr654 of the EGFR, is used to analyze EGF stimulated PKC activity by incorporation of 32P into the peptide. Cotreatment of cells with CB and EGF results in a complete loss of EGF-induced phosphorylation of the peptide. These data suggest that actin polymerization is obligatory for negative feedback regulation of the EGFR tyrosine kinase through the C-kinase pathway.

Actins↗
PubMed1998

Glucose repression in Saccharomyces cerevisiae is related to the glucose concentration rather than the glucose flux.

Glucose plays an important regulatory role in the yeast Saccharomyces cerevisiae, which is mostly reflected at the transcriptional level by glucose repression. The signal that initiates glucose repression is unknown, but data indicate that it is located at or above the level of glucose 6-phosphate, suggesting the involvement of either the intracellular or extracellular glucose concentration or the glucose flux in triggering glucose repression. We have investigated the role of the glucose flux and the extracellular glucose concentration in glucose repression by growing the cells in continuous culture under nitrogen limitation. By a step-wise increase in the glucose feed concentration, the glucose flux and extracellular glucose concentrations were modulated in an accurate way. Furthermore, the glucose flux and glucose concentrations were modulated independently of each other by increasing the dilution rate or by the use of fructose as a substrate. Using these approaches we demonstrate that glucose repression is related to the extracellular (or intracellular) glucose concentration rather than the glucose flux. At external glucose concentrations lower than 14 mM, glucose repression of SUC2 gene transcription was not triggered, whereas glucose repression of this gene was activated when the glucose concentration exceeded 18 mM. A comparable effect was observed for the glucose-repressible carbon source fructose.

Dose-Response Relationship, Drug↗
PubMed1998

Pre-embedding immunolabeling for electron microscopy: an evaluation of permeabilization methods and markers.

For scarce antigens or antigens which are embedded in a dense macromolecular structure, on-section labeling, the first method of choice, is not always successful. Often, the antigen can be localized by immunofluorescence microscopy, usually by a pre-embedding labeling method. Most of these methods lead to loss of ultrastructural details and, hence, labeling at electron microscope resolution does not add essential information. The scope of this paper is to compare five permeabilization methods for pre-embedding labelling for electron microscopy. We aim for a method that is easy to use and suitable for routine investigations. For our ongoing work, special attention is given to labeling of the cell nucleus. Accessibility of cytoplasmic and nuclear antigens is monitored with a set of different marker antibodies. From this investigation, we suggest that prefixation with formaldehyde/glutaraldehyde is necessary to stabilize the ultrastructure before using a detergent (Triton X-100 or Brij 58) to permeabilize or remove the membranes. The experimental conditions for labeling should be checked first with fluorescence or fluorescence-gold markers by fluorescence microscopy. Then either ultrasmall gold particles (with or without fluorochrome) with silver enhancement or, if the ultrasmall gold particles are obstructed, peroxidase markers are advised. The most promising technique to localize scarce antigens with good contrast is the combination of a pre-embedding peroxidase/tyramide-FITC or -biotin labeling followed by an on-section colloidal gold detection.

Cell Nucleus↗
PubMed1998

Impaired secretion of a hydrophobic cutinase by Saccharomyces cerevisiae correlates with an increased association with immunoglobulin heavy-chain binding protein (BiP).

This study focuses on the different efficiencies of secretion of two fungal cutinases by Saccharomyces cerevisiae, a wild-type cutinase (CY000) and a hydrophobic mutant cutinase (CY028). Both cutinases are placed under control of the GAL7 promoter, by which the expression levels can be regulated. Wild-type cutinase was secreted at up to 25 mg per g (dry weight), while CY028 was secreted at a level of 2 mg per g (dry weight); this difference is nearly independent of the expression level. Pulse-chase experiments revealed that whereas CY000 cutinase is secreted, CY028 is irreversibly retained in the cell. Immunogold labelling followed by electron microscopy revealed colocalization of CY028 with immunoglobulin heavy-chain binding protein (BiP) in the endoplasmic reticulum (ER). The increase of wild-type cutinase expression did not result in higher levels of the molecular chaperone BiP, but BiP levels are raised by increased induction of the hydrophobic mutant cutinase. Immunoprecipitation studies showed that in contrast to the wild-type cutinase, the hydrophobic mutant cutinase interacts with BiP. These results indicate that the introduction of two exposed hydrophobic patches in cutinase results in a higher affinity for BiP which might cause the retention of this mutant cutinase in the ER.

Blotting, Western↗
PubMed1997

Identification of an intracellular domain of the EGF receptor required for high-affinity binding of EGF.

Although all EGF receptors in EGF receptor-expressing cells are molecularly identical, they can be subdivided in two different classes that have either a high or a low affinity for EGF. Specifically the high-affinity class is associated with filamentous actin. To determine whether the interaction of the EGF receptor with actin induces its high-affinity state, we studied EGF-binding properties of an EGF receptor mutant that lacks the actin-binding site. Interestingly, we found that cells expressing this mutant receptor still display both high- and low-affinity classes of EGF receptors, indicating that the actin-binding domain does not determine the high-affinity binding state. By further mutational analysis we identified a receptor domain, within the tyrosine kinase domain, that regulates the affinity for EGF.

3T3 Cells↗
PubMed1997

Epidermal growth factor induces ubiquitination of Eps15.

Epidermal growth factor (EGF) receptor pathway substrate clone 15 (Eps15) has been described as a 142-kDa EGF receptor substrate. It has been shown to bind to the EGF receptor, adaptor protein-2, and clathrin and is present at clathrin-coated pits and vesicles. Upon stimulation of cells with EGF or transforming growth factor alpha, Eps15 becomes rapidly and transiently phosphorylated on tyrosine residues. This phosphorylation coincides with an increase of 8 kDa in molecular mass. Here we show that this increase in molecular mass is not due to tyrosine phosphorylation. Instead, we found both by Western blotting and protein sequencing that this EGF-induced increase in molecular mass is the result of monoubiquitination. Eps15 ubiquitination but not tyrosine phosphorylation was inhibited under conditions that blocked EGF-induced internalization of the EGF receptor. Our results establish ubiquitination as a second form of EGF-stimulated covalent modification of Eps15.

3T3 Cells↗
PubMed1997

A freeze-fracture study of the membrane morphology of phosphatidylethanolamine-deficient Escherichia coli cells.

Freeze-fracture electron microscopy was applied to study membrane morphology in a phosphatidylethanolamine-deficient E. coli strain. For growth, this strain requires millimolar concentrations of specific divalent cations like Mg2+ or Ca2+. These cations bring the bilayer to nonbilayer phase transition temperature of the lipids back to wild type levels by shifting the phase preference of cardiolipin in the membrane towards the inverted hexagonal (H(II)) phase. Under growth conditions, these cells show a bilayer based membrane with an intramembrane particle distribution as in wild type cells. Upon lowering the temperature, smooth areas are observed corresponding to gel state lipid bilayer domains. Ca2+ was used to manipulate the phase behavior of the membrane lipids in situ. Exposing the cells to Ca2+ up to 100 mM at 42 degrees C did not result in the appearance of nonbilayer structures, despite the fact that in total lipid extracts under these conditions the hexagonal H(II) phase was observed. However, the addition of a Ca2+ ionophore, which leads to exposure to Ca2+ of both faces of the plasma membrane, gives rise to formation of H(II) phase, stacked bilayer domains and blebbing upon addition of 50 mM CaCl2 at 42 degrees C. We conclude that the asymmetrical localization of divalent cations in the periplasm of this strain allows them to be functionally effective while membrane stability is maintained.

ATP-Binding Cassette Transporters↗
PubMed1997

Association and colocalization of Eps15 with adaptor protein-2 and clathrin.

Eps15 has been identified as a substrate of the EGF receptor tyrosine kinase. In this report, we show that activation of the EGF receptor by either EGF or TGF-alpha results in phosphorylation of Eps15. Stimulation of cells with PDGF or insulin did not lead to Eps15 phosphorylation, suggesting that phosphorylation of Eps15 is a receptor-specific process. We demonstrate that Eps15 is constitutively associated with both alpha-adaptin and clathrin. Upon EGF stimulation, Eps15 and alpha-adaptin are recruited to the EGF receptor. Using a truncated EGF receptor mutant, we demonstrate that the regulatory domain of the cytoplasmic tail of the EGF receptor is essential for the binding of Eps15. Fractionation studies reveal that Eps15 is present in cell fractions enriched for plasma membrane and endosomal membranes. Immunofluorescence studies show that Eps15 colocalizes with adaptor protein-2 (AP-2) and partially with clathrin. No colocalization of Eps15 was observed with the early endosomal markers rab4 and rab5. These observations indicate that Eps15 is present in coated pits and coated vesicles of the clathrin-mediated endocytic pathway, but not in early endosomes. Neither AP-2 nor clathrin are required for the binding of Eps15 to coated pits or coated vesicles, since in membranes lacking AP-2 and clathrin, Eps15 still shows the same staining pattern. These findings suggest that Eps15 may play a critical role in the recruitment of active EGF receptors into coated pit regions before endocytosis of ligand-occupied EGF receptors.

3T3 Cells↗
PubMed1997

Ultrastructural co-localization of calmodulin and B-50/growth-associated protein-43 at the plasma membrane of proximal unmyelinated axon shafts studied in the model of the regenerating rat sciatic nerve.

Calmodulin and de-phosphorylated B-50/growth-associated protein-43 (GAP-43) have been shown to bind in vitro in a molecular complex, but evidence for an in situ association in the nervous system does not exist. Previously, we have reported that, in the model of the regenerating rat sciatic nerve, the B-50/GAP-43 immunoreactivity is increased and concentrated at the axolemma of unmyelinated axons located proximal to the site of injury and axon outgrowth. To explore a putative function of B-50/GAP-43, namely, the capacity of binding calmodulin to the plasma membrane, we examined the ultrastructural distribution of calmodulin in the proximal unmyelinated axon shafts of this model, using double immunolabelling and detection by fluorescent or gold probes conjugated to second antibodies. Immunofluorescence showed that seven days post-sciatic nerve crush the calmodulin immunoreactivity, similar to B-50/GAP-43 immunoreactivity, was intense in unmyelinated axon shafts located proximal to the site of injury of the regenerating nerve. Ultrastructurally, calmodulin was located at the axolemma of these regenerating unmyelinated axon shafts and inside the axoplasm, where it was associated with vesicles and microtubules. The plasma membrane labelling (approximately 69%) was significantly higher than the axoplasmic labelling. Over 60% of the plasma membrane-associated calmodulin co-localized with B-50/GAP-43 in a non-random distribution. Since normally calmodulin is largely present in the cytoplasm, these data suggest that calmodulin has been concentrated at the plasma membrane of unmyelinated axons, most probably by B-50/GAP-43. If the concentrating effect is due to B-50/GAP-43, then there is a possibility that these proteins may be present as a molecular complex in situ. The physiological significance could be that this association regulates the local availability of both B-50/GAP-43 and calmodulin for other interactions.

Animals↗