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Biomedical subjects

R Wetzker

Publications and source records attributed to R Wetzker.

At least 19 recordsLinked to original sources
PubMed2003

Inhibition of signal transduction in EGF-dependent epithelial cell lines.

The epidermal growth factor (EGF) receptor plays a pivotal role in growth regulation of epidermal keratinocytes. Its expression and function can be markedly altered during malignant transformation in squamous cell carcinoma. The present study investigated the potential of growth inhibition by signal-transduction inhibitors in EGF-dependent epithelial cell lines in vitro. Benign HaCaT keratinocytes and malignant A431 cells were grown in vitro and exposed to various concentrations of a panel of eleven kinase and phosphodiesterase inhibitors. Cell growth was measured after 24 h and 48 h using fluorescence labeling with Hoechst 33342 and propidium iodide. Significant growth inhibition was achieved with all inhibitors when applied to HaCaT cells. The strongest growth inhibition was achieved with inhibitors H-7, A3 and diacylglycerol kinase inhibitors I and II. A431 cells were inhibited significantly by H-7, A3 and H-9. Selected signal-transduction inhibitors such as A3, H-7 and H-9 acting on intracellular kinases are capable of suppressing growth of EGF-dependent benign and malignant epithelial cell lines in vitro. They might be of future potential in the treatment of epithelial cancer but further studies are necessary.

Benzimidazoles↗
PubMed2001

Phosphoinositide 3-kinase-gamma induces Xenopus oocyte maturation via lipid kinase activity.

Type-I phosphoinositide 3-kinases (PI3Ks) were characterized as a group of intracellular signalling proteins expressing both protein and lipid kinase activities. Recent studies implicate PI3Ks as mediators of oocyte maturation, but the molecular mechanisms are poorly defined. Here we used the Xenopus oocyte expression system as a model to investigate a possible contribution of the gamma-isoform of PI3K (PI3Kgamma) in the different pathways leading to cell-cycle progression by monitoring the time course of germinal vesicle breakdown (GVBD). Expression of a constitutive active PI3Kgamma (PI3Kgamma-CAAX) induced GVBD and increased the levels of phosphorylated Akt/protein kinase B and mitogen-activated protein kinase (MAPK). Furthermore, PI3Kgamma-CAAX accelerated progesterone-induced GVBD, but had no effect on GVBD induced by insulin. The effects of PI3Kgamma-CAAX could be suppressed by pre-incubation of the oocytes with LY294002, PD98059 or roscovitine, inhibitors of PI3K, MEK (MAPK/extracellular-signal-regulated protein kinase kinase) and cdc2/cyclin B kinase, respectively. Mutants of PI3Kgamma-CAAX, in which either lipid kinase or both lipid and protein kinase activities were altered or eliminated, did not induce significant GVBD. Our data demonstrate that expression of PI3Kgamma in Xenopus oocytes accelerates their progesterone-induced maturation and that lipid kinase activity is required to induce this effect.

Animals↗
PubMed2001

Anionic phospholipids are involved in membrane targeting of PI 3-kinase gamma.

Phosphoinositide 3-kinases (PI 3-kinases) have critical roles in diverse cellular signaling processes and in protein trafficking. In contrast to the class I PI 3-kinases alpha, beta, and delta which bind via src homology 2 (SH2) domains of adaptor proteins to tyrosine kinase receptors, the mechanism of recruitment of the PI 3-kinase gamma to membranes is unknown. We report in vitro experiments using immobilized proteins and small unilamellar vesicles which suggest an involvement of anionic phospholipids in membrane association of PI 3-kinase gamma. Furthermore we provide evidence that the enzyme displays beside the catalytic center a phospholipid binding domain which is essential for enzyme activity.

Animals↗
PubMed2001

The deletion of CaVPS34 in the human pathogenic yeast Candida albicans causes defects in vesicle-mediated protein sorting and nuclear segregation.

A Candida albicans null mutant of the phosphatidylinositol (PI) 3-kinase gene (CaVPS34) involved in virulence was examined by different microscopical techniques. We observed that vacuoles of the Cavps34 null mutant were considerably enlarged and electron-transparent. An interesting result obtained by transmission electron microscopy analysis of Cavps34 mutant cells was the aberrant patch-like accumulation of vesicles, which were localized in the periplasm close to the plasma membrane. We assume that the vesicles result from missorted prevacuolar compartments. In contrast to the accumulations of the specific endocytic dye FM4-64 in the vacuole membrane in C. albicans wild-type strains (ring staining pattern), the Cavps34 mutant strain showed a staining of punctuate structures, possibly multivesicular bodies (MVB), that are scattered all over the cell. This defect indicates a late block in endocytic vesicle transport. Measurement of the total activity of carboxypeptidase Y revealed significantly lower activity in Cavps34 mutant cells. This may indicate that carboxypeptidase Y is not properly activated as a result of mislocalization due to the lack of Vps34p. The deletion of the CaVPS34 gene caused disturbance of normal nuclear migration, which suggests that in the Cavps34 mutant the cell-size mediated control process of cell division is affected.

Acids↗
PubMed2001

Limits for the detection of (poly-)phosphoinositides by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS).

Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been recently established as a powerful tool for the analysis of biomolecules. Here, MALDI-TOF MS was used for the detection of (poly-)phosphoinositides (PPI). PPI possess higher molecular weights than other phospholipids and a high phosphorylation-dependent negative charge. Both features affect the MALDI detection limits expressed as the minimum of analyte on the sample plate resulting in a signal-to-noise-ratio of S/N = 5. Using 2,5-dihydroxybenzoic acid (DHB) as matrix the detection limit for phosphatidylinositol (PI) is seven times higher than for phosphatidylcholine (PC) and further increases with increasing phosphorylation or in mixtures with other well-detectable phospholipids. For phosphatidylinositol-tris-phosphate (PIP3) in a mixture with PC, the limit is about 20 times higher than for PI. The consequences for the experimental conditions are discussed. It is advisable to pre-separate PPI from biological lipid mixtures prior to the application of MALDI-TOF MS.

Phosphatidylinositol Phosphates↗
PubMed2000

A permissive function of phosphoinositide 3-kinase in Ras activation mediated by inhibition of GTPase-activating proteins.

The activation status of the guanosine triphosphate (GTP)-binding protein Ras is dictated by the relative intensities of two opposing reactions: the formation of active Ras-GTP complexes, promoted by guanine-nucleotide exchange factors (GEFs), and their conversion to inactive Ras-GDP as a result of the deactivating action of GTPase-activating proteins (GAPs). The relevance of phosphoinositide 3-kinase (PI 3-kinase) to these processes is still unclear. We have investigated the regulation of Ras activation by PI 3-kinase in the myelomonocytic U937 cell line. These cells exhibited basal levels of Ras-GTP, which were suppressed by two PI 3-kinase inhibitors and a dominant-negative PI 3-kinase. In addition, PI 3-kinase inhibition aborted Ras activation by all stimuli tested, including foetal calf serum (FCS) and phorbol 12-myristate 13-acetate (TPA). Significantly, TPA does not activate PI 3-kinase in U937 cells, indicating that PI 3-kinase has a permissive rather than an intermediary role in Ras activation. Investigation of the mechanism of PI 3-kinase action revealed that inhibition of PI 3-kinase does not affect nucleotide exchange on Ras but abrogates Ras-GTP accumulation through an increase in GAP activity. These findings establish blockage of GAP action as the mechanism underlying a permissive function of PI 3-kinase in Ras activation.

GTPase-Activating Proteins↗
PubMed2000

A phosphatidylinositol 3-kinase of Candida albicans: molecular cloning and characterization.

A phosphatidylinositol (PI) 3-kinase gene (CaVPS34) of the human pathogenic yeast Candida albicans was cloned by a PCR-based homology approach. The open reading frame encodes a 1020 amino acid protein with a calculated molecular weight of 118 kDa and a relative isoelectric point of 6.9. It shares 47% sequence identity with Saccharomyces cerevisiae Vps34p. Southern pattern indicated that CaVPS34 is probably present as a single copy gene per haploid genome in C. albicans. We localized the CaVPS34 gene on chromosome 1. Under all conditions tested a major CaVPS34 transcript of approximately 3. 5 kb could be detected. CaVPS34 mRNA levels increased during exponential growth up to 12-fold followed by a decline upon entry into stationary phase. The size of a 6xHis tag-CaVps34p fusion protein purified from Escherichia coli is in agreement with the calculated molecular mass of CaVps34p. It exhibits in vitro PI 3-kinase activity and produces only phosphatidylinositol 3-phosphate. The CaVPS34 gene under the control of its own promoter were not able to complement the temperature-sensitive growth of S. cerevisiae vps34. However, overexpression of CaVPS34 was sufficient to rescue the temperature-sensitive vps34 phenotype, suggesting a functional conservation in C. albicans.

Amino Acid Sequence↗
PubMed2000

Selective protein kinase inhibitors block head-specific differentiation in hydra.

Several studies have suggested that morphogenesis and patterning in hydra are regulated through pathways involving protein kinase C (PKC). Nevertheless, the complete signal system for regeneration in hydra is still not completely understood. Using inhibitors of different signalling pathways we are dissecting this system. We found that sphingosine (2 microM), staurosporine (0.1 microM), PP1/AGL1872 (1 microM) and H7 (25 microM) were able to inhibit head but not foot regeneration. The inhibition was reversible. When the inhibitor was replaced with hydra medium the animals continue their regeneration in a normal way. The exception was PP1/AGL1872, in this case the animals regenerated only one or two tentacles. These results imply that head and foot regeneration are independent processes and they are not directly related as has been proposed. Sphingosine and PP1/AGL1872 inhibit the transcription of ks1, an early regeneration gene, at 24 and 48 h of treatment. Sphingosine 2 microM arrested the cells on the G1 phase of the cell cycle, but 1 microM of PP1/AGL1872 did not. The regeneration was not affected if the animals were exposed to inhibitors of human growth factor receptors. We propose that head regeneration in hydra may be regulated at least by two pathways, one going through PKC and the other through Src. The first pathway could be related to cellular proliferation and the second one to cellular differentiation.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed2000

Internalization of extraintestinal Escherichia coli O18 strains by epithelial cells is modulated by EGF, insulin, and effectors of transmembrane signal transduction.

Adhesion to and internalization into host cells is an essential step in the pathogenesis of various bacterial infections. Here we investigated the effects of growth factors on the internalization of Escherichia coli O18 strains isolated from patients with urinary tract infection (UTI) by human epithelial cells. A dramatic increase in the uptake of Escherichia coli was observed after treatment of epithelial cells with epidermal growth factor (EGF) and to a lower extent with insulin. EGF-dependent internalization can be suppressed by tyrosine kinase inhibitors suggesting an involvement of the receptor tyrosine kinases in the regulation of the endocytotic process. Inhibitors of phospholipase A2, lipoxygenase, and cyclooxygenase significantly decreased internalization of bacteria induced by EGF. Finally, the specific inhibitor of PI 3-kinases Wortmannin was shown to suppress completely the EGF-independent internalization. The data of this analysis indicate the involvement of several signaling paths in bacterial internalization of uropathogenic Escherichia coli O18 strains and contribute to the comprehension of the pathogenesis of recurrent UTI.

Bacterial Adhesion↗
PubMed2000

Different signaling pathways are involved in CCK(B) receptor-mediated MAP kinase activation in COS-7 cells.

Recently, the involvement of the MAP kinase ERK in mitogenic signaling of cholecystokininB (CCK(B)) receptors has been shown. However, the intracellular effector systems involved in this signaling pathway are poorly defined. In this study, we used COS-7 cells transiently transfected with the human CCK(B) receptor to investigate cholecystokinin-induced MAP kinase activation. CCK-8 induced activation of ERK2 which is associated with its phosphorylation and localization in the nucleus. The CCK-8-dependent ERK stimulation is sensitive to wortmannin an inhibitor of phosphoinositide 3-kinases (PI3Ks) indicating the involvement of PI3K activity. To identify the PI3K species involved in mitogenic signaling of the CCK(B) receptor several dominant-negative mutants of PI3K regulatory and catalytic subunits were transiently expressed. Surprisingly, different catalytically inactive mutants of the G protein-sensitive PI3Kgamma did not affect ERK stimulation induced by CCK, whereas a dominant-negative mutant of the regulatory p85 subunit induced significant inhibition of CCK-dependent ERK activity. These results indicate an involvement of PI3K class 1A species alpha, beta or/and delta in signal transduction via CCK(B) receptors. In addition, protein kinase C (PKC)-dependent signaling pathways contribute to CCK(B)-mediated MAP kinase signaling as shown by inhibition of CCK-8-induced ERK activation by the PKC inhibitor bisindolylmaleimide.

Animals↗
PubMed1999

An epidermal growth factor receptor/Gab1 signaling pathway is required for activation of phosphoinositide 3-kinase by lysophosphatidic acid.

Phosphoinositide 3-kinase (PI3K) has been shown to play an essential role in G protein-induced signaling even in non-myeloid cells where few agonists of G protein-coupled receptors are known to activate PI3K. We have identified adherent cell lines where lysophosphatidic acid (LPA) strongly and rapidly activates the accumulation of PI3K lipid products. The process is not modified by expression of a kinase-dead mutant of the Gbetagamma-responsive PI3K p110gamma. In contrast, it is inhibited by genistein or expression of a dominant negative mutant of p85 and potentiated by overexpressing wild-type p110alpha or -beta but not -gamma. By using a specific chemical inhibitor of the epidermal growth factor receptor (EGFR) and expression of a dominant negative mutant, we have observed that recruitment of p85/p110 PI3Ks occurs through transactivation of the EGFR by LPA and downstream mobilization of the docking protein Gab1 that associates with p85 upon LPA stimulation. Finally, we show that LPA cannot activate PI3K in cell lines lacking the EGFR/Gab1 pathway, including cells that transactivate the PDGF receptor. Altogether, these results demonstrate that activation of PI3K by LPA is conditioned by the ability of LPA to transactivate an EGFR/Gab1 signaling pathway.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Farnesylation of Ras is important for the interaction with phosphoinositide 3-kinase gamma.

The correct functioning of Ras proteins requires post-translational modification of the GTP hydrolases (GTPases). These modifications provide hydrophobic moieties that lead to the attachment of Ras to the inner side of the plasma membrane. In this study we investigated the role of Ras processing in the interaction with various putative Ras-effector proteins. We describe a specific, GTP-independent interaction between post-translationally modified Ha- and Ki-Ras4B and the G-protein responsive phosphoinositide 3-kinase p110gamma. Our data demonstrate that post-translational processing increases markedly the binding of Ras to p110gamma in vitro and in Sf9 cells, whereas the interaction with p110alpha is unaffected under the same conditions. Using in vitro farnesylated Ras, we show that farnesylation of Ras is sufficient to produce this effect. The complex of p110gamma and farnesylated RasGTP exhibits a reduced dissociation rate leading to the efficient shielding of the GTPase from GTPase activating protein (GAP) action. Moreover, Ras processing affects the dissociation rate of the RasGTP complex with the Ras binding domain (RBD) of Raf-1, indicating that processing induces alterations in the conformation of RasGTP. The results suggest a direct interaction between a moiety present only on fully processed or farnesylated Ras and the putative target protein p110gamma.

Animals↗
PubMed1999

Bradykinin B(2) receptor-mediated mitogen-activated protein kinase activation in COS-7 cells requires dual signaling via both protein kinase C pathway and epidermal growth factor receptor transactivation.

The signaling routes linking G-protein-coupled receptors to mitogen-activated protein kinase (MAPK) may involve tyrosine kinases, phosphoinositide 3-kinase gamma (PI3Kgamma), and protein kinase C (PKC). To characterize the mitogenic pathway of bradykinin (BK), COS-7 cells were transiently cotransfected with the human bradykinin B(2) receptor and hemagglutinin-tagged MAPK. We demonstrate that BK-induced activation of MAPK is mediated via the alpha subunits of a G(q/11) protein. Both activation of Raf-1 and activation of MAPK in response to BK were blocked by inhibitors of PKC as well as of the epidermal growth factor (EGF) receptor. Furthermore, in PKC-depleted COS-7 cells, the effect of BK on MAPK was clearly reduced. Inhibition of PI3-Kgamma or Src kinase failed to diminish MAPK activation by BK. BK-induced translocation and overexpression of PKC isoforms as well as coexpression of inactive or constitutively active mutants of different PKC isozymes provided evidence for a role of the diacylglycerol-sensitive PKCs alpha and epsilon in BK signaling toward MAPK. In addition to PKC activation, BK also induced tyrosine phosphorylation of EGF receptor (transactivation) in COS-7 cells. Inhibition of PKC did not alter BK-induced transactivation, and blockade of EGF receptor did not affect BK-stimulated phosphatidylinositol turnover or BK-induced PKC translocation, suggesting that PKC acts neither upstream nor downstream of the EGF receptor. Comparison of the kinetics of PKC activation and EGF receptor transactivation in response to BK also suggests simultaneous rather than consecutive signaling. We conclude that in COS-7 cells, BK activates MAPK via a permanent dual signaling pathway involving the independent activation of the PKC isoforms alpha and epsilon and transactivation of the EGF receptor. The two branches of this pathway may converge at the level of the Ras-Raf complex.

Androstadienes↗
PubMed1999

Differential regulation of lipid and protein kinase activities of phosphoinositide 3-kinase gamma in vitro.

G protein sensitive phosphoinositide 3-kinase gamma (PI3Kgamma) has been characterised as a pleiotropic signalling protein expressing lipid kinase and protein kinase activities. Whereas the regulation of the lipid kinase activity has been investigated in detail, the regulatory features of PI3Kgamma protein kinase activity are unknown. Here we report that Gbetagamma subunits of heterotrimeric G proteins induce a biphasic response of PI3Kgamma autophosphorylation in vitro, which contrasts the regulatory effects of the G proteins on PI3Kgamma lipid kinase activity. In addition to autophosphorylation PI3Kgamma is able to catalyse transphosphorylation of the adapter protein p101 and the protein kinase MEK-1. In the presence of the p101, Gbetagamma affects PI3Kgamma protein kinase activities in a complex manner. In summary, the differential regulatory effects of heterotrimeric G proteins on PI3Kgamma lipid and protein kinase activities in vitro reflect the functional diversity of the enzyme observed in vivo.

GTP-Binding Protein beta Subunits↗
PubMed1999

Retinoic acid induces selective expression of phosphoinositide 3-kinase gamma in myelomonocytic U937 cells.

Retinoic acid provokes growth inhibition and differentiation of the human leukemic cell line U937 to macrophage-like cells. We report that treatment of U937 cells with all-trans retinoic acid (ATRA), but not the phorbol ester 12-O-tetradecanoylphorbol-13-acetate, results in an increased gene expression of the phosphoinositide 3-kinase (PI3K) species PI3Kgamma. PI3Kgamma expression was transcriptionally elevated, indicating that the PI3Kgamma gene may be a direct target for ATRA. In contrast to its effect on PI3Kgamma expression, ATRA did neither affect the levels of the PI3K species beta and delta nor the adapter proteins p85 and p101. Enhanced expression by ATRA of PI3Kgamma correlated with an increase of PI3K lipid kinase activity. Additionally, ATRA induced significant and lasting stimulation of mitogen-activated protein kinase/Erk2 activity. This effect was sensitive to the PI3K inhibitors wortmannin or LY294002 and, therefore, attributed to the upregulation of PI3Kgamma expression. Our findings suggest that sustained MAPK activation via PI3Kgamma precedes ATRA -dependent differentiation or growth inhibition.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1998

A novel mitogenic signaling pathway of bradykinin in the human colon carcinoma cell line SW-480 involves sequential activation of a Gq/11 protein, phosphatidylinositol 3-kinase beta, and protein kinase Cepsilon.

The signaling routes connecting G protein-coupled receptors to the mitogen-activated protein kinase (MAPK) pathway reveal a high degree of complexity and cell specificity. In the human colon carcinoma cell line SW-480, we detected a mitogenic effect of bradykinin (BK) that is mediated via a pertussis toxin-insensitive G protein of the Gq/11 family and that involves activation of MAPK. Both BK-induced stimulation of DNA synthesis and activation of MAPK in response to BK were abolished by two different inhibitors of phosphatidylinositol 3-kinase (PI3K), wortmannin and LY 294002, as well as by two different inhibitors of protein kinase C (PKC), bisindolylmaleimide and Ro 31-8220. Stimulation of SW-480 cells by BK led to increased formation of PI3K lipid products (phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3, 4-bisphosphate) and to enhanced translocation of the PKCepsilon isoform from the cytosol to the membrane. Both effects of BK were inhibited by wortmannin, too. Using subtype-specific antibodies, only the PI3K subunits p110beta and p85, but not p110alpha and p110gamma, were detected in SW-480 cells. Finally, p110beta was found to be co-immunoprecipitated with PKCepsilon. Our data suggest that in SW-480 cells, (i) dimeric PI3Kbeta is activated via a Gq/11 protein; (ii) PKCepsilon is a downstream target of PI3Kbeta mediating the mitogenic signal to the MAPK pathway; and (iii) PKCepsilon associates with the p110 subunit of PI3Kbeta. Thus, these results add a novel possibility to the emerging picture of multiple pathways linking G protein-coupled receptors to MAPK.

Bradykinin↗
PubMed1998

Bifurcation of lipid and protein kinase signals of PI3Kgamma to the protein kinases PKB and MAPK.

Phosphoinositide 3-kinases (PI3Ks) activate protein kinase PKB (also termed Akt), and PI3Kgamma activated by heterotrimeric guanosine triphosphate-binding protein can stimulate mitogen-activated protein kinase (MAPK). Exchange of a putative lipid substrate-binding site generated PI3Kgamma proteins with altered or aborted lipid but retained protein kinase activity. Transiently expressed, PI3Kgamma hybrids exhibited wortmannin-sensitive activation of MAPK, whereas a catalytically inactive PI3Kgamma did not. Membrane-targeted PI3Kgamma constitutively produced phosphatidylinositol 3,4, 3,4,5-trisphosphate and activated PKB but not MAPK. Moreover, stimulation of MAPK in response to lysophosphatidic acid was blocked by catalytically inactive PI3Kgamma but not by hybrid PI3Kgammas. Thus, two major signals emerge from PI3Kgamma: phosphoinositides that target PKB and protein phosphorylation that activates MAPK.

Amino Acid Sequence↗