Biomedical subjects

R Seger

Publications and source records attributed to R Seger.

At least 37 records · Page 2Linked to original sources

PubMed2001

Lipid constituents in oligodendroglial cells alter susceptibility to H2O2-induced apoptotic cell death via ERK activation.

The present work examines the effect of membrane lipid composition on activation of extracellular signal-regulated protein kinases (ERK) and cell death following oxidative stress. When subjected to 50 microM docosahexaenoic acid (DHA, 22 : 6 n-3), cellular phospholipids of OLN 93 cells, a clonal line of oligodendroglia origin low in DHA, were enriched with this polyunsaturated fatty acid. In the presence of 1 mM N,N-dimethylethanolamine (dEa) a new phospholipid species analog was formed in lieu of phosphatidylcholine. Exposure of DHA-enriched cells to 0.5 mM H2O2, caused sustained activation of ERK up to 24 h. At this time massive apoptotic cell death was demonstrated by ladder and TUNEL techniques. H2O2-induced stress applied to dEa or DHA/dEa co-supplemented cells showed only a transient ERK activation and no cell death after 24 h. Moreover, while ERK was rapidly translocated into the nucleus in DHA-enriched cells, dEa supplements completely blocked ERK nuclear translocation. This study suggests that H2O2-induced apoptotic cell death is associated with prolonged ERK activation and nuclear translocation in DHA-enriched OLN 93 cells, while both phenomena are prevented by dEa supplements. Thus, the membrane lipid composition ultimately modulates ERK activation and translocation and therefore can promote or prevent apoptotic cell death.

Apoptosis↗

PubMed2001

In vitro phosphorylation of COOH termini of the epithelial Na(+) channel and its effects on channel activity in Xenopus oocytes.

Recent findings have suggested the involvement of protein phosphorylation in the regulation of the epithelial Na(+) channel (ENaC). This study reports the in vitro phosphorylation of the COOH termini of ENaC subunits expressed as glutathione S-transferase fusion proteins. Channel subunits were specifically phosphorylated by kinase-enriched cytosolic fractions derived from rat colon. The phosphorylation observed was not mediated by the serum- and glucocorticoid-regulated kinase sgk. For the gamma-subunit, phosphorylation occurred on a single, well-conserved threonine residue located in the immediate vicinity of the PY motif (T630). The analogous residue on beta(S620) was phosphorylated as well. The possible role of gammaT630 and betaS620 in channel function was studied in Xenopus laevis oocytes. Mutating these residues to alanine had no effect on the basal channel-mediated current. They do, however, inhibit the sgk-induced increase in channel activity but only in oocytes that were preincubated in low Na(+) and had a high basal Na(+) current. Thus mutating gammaT630 or betaS620 may limit the maximal channel activity achieved by a combination of sgk and low Na(+).

Animals↗

PubMed2000

Role of dynamin, Src, and Ras in the protein kinase C-mediated activation of ERK by gonadotropin-releasing hormone.

G-protein-coupled receptors are a large group of integral membranal receptors, which in response to ligand binding initiate diverse downstream signaling. Here we studied the gonadotropin-releasing hormone (GnRH) receptor, which uses Gq for its downstream signaling. We show that extracellular signal-regulated kinase (ERK) activation is fully dependent on protein kinase C (PKC), but only partially dependent on Src, dynamin, and Ras. Receptor tyrosine kinases, FAK, Gbetagamma, and beta-arrestin, which were implicated in some G-protein-coupled receptor signaling to MAPK cascades, do not play a role in the GnRH to ERK pathway. Our results suggest that the activation of ERK by GnRH involves two distinct signaling pathways, which converge at the level of Raf-1. The main pathway involves a direct activation of Raf-1 by PKC, and this step is partially dependent on a second pathway consisting of Ras activation, which occurs in a dynamin-dependent manner, downstream of Src.

Animals↗

PubMed2000

Fibroblast growth factor (FGF) signaling through PI 3-kinase and Akt/PKB is required for embryoid body differentiation.

The role of FGF signaling in early epithelial differentiation was investigated in ES (embryonic stem) cell derived embryoid bodies. A dominant negative fibroblast growth factor receptor (FGFR) mutation was created by stably introducing into ES cells an Fgfr2 cDNA, truncated in its enzymatic domains. These cells failed to differentiate into cystic embryoid bodies. No epithelial differentiation and cavitation morphogenesis could be observed, in the mutant, although its rate of cell proliferation remained unchanged. This phenotype was associated with a significant decrease in the activation of Akt/PKB and PLCgamma-1, as compared to the wild type, while the activation of MAPK/Erk was less affected. Requirement for PI 3-kinase signaling in embryoid body differentiation was demonstrated by specific inhibitors. Akt/PKB activation was abrogated by wortmannin in short-term experiments. In long-term cultures Ly294002 inhibited the differentiation of ES cells into embryoid bodies. Our data demonstrate that for early epithelial differentiation FGF signaling is required through the PI 3-kinase-Akt/ PKB pathway.

Animals↗

PubMed2000

ERK1b, a 46-kDa ERK isoform that is differentially regulated by MEK.

We identified a 46-kDa ERK, whose kinetics of activation was similar to that of ERK1 and ERK2 in most cell lines and conditions, but showed higher fold activation in response to osmotic shock and epidermal growth factor treatments of Ras-transformed cells. We purified and cloned this novel ERK (ERK1b), which is an alternatively spliced form of ERK1 with a 26-amino acid insertion between residues 340 and 341 of ERK1. When expressed in COS7 cells, ERK1b exhibited kinetics of activation and kinase activity similar to those of ERK1. Unlike the uniform pattern of expression of ERK1 and ERK2, ERK1b was detected only in some of the tissues examined and seems to be abundant in the rat and human heart. Interestingly, in Ras-transformed Rat1 cells, there was a 7-fold higher expression of ERK1b, which was also more responsive than ERK1 and ERK2 to various extracellular treatments. Unlike ERK1 and ERK2, ERK1b failed to interact with MEK1 as judged from its nuclear localization in resting cells overexpressing ERK1b together with MEK1 or by lack of coimmunoprecipitation of the two proteins. Thus, ERK1b is a novel 46-kDa ERK isoform, which seems to be the major ERK isoform that responds to exogenous stimulation in Ras-transformed cells probably due to its differential regulation by MEK.

Alternative Splicing↗

PubMed2000

The requirement of both extracellular regulated kinase and p38 mitogen-activated protein kinase for stimulation of cytosolic phospholipase A(2) activity by either FcgammaRIIA or FcgammaRIIIB in human neutrophils. A possible role for Pyk2 but not for the Grb2-Sos-Shc complex.

The signal transduction pathways initiated by opsonized zymosan (OZ) leading to activation of cytosolic phospholipase A(2) (cPLA(2)) in human neutrophils remain obscure. In a previous study, we showed that the activation of cPLA(2) by OZ is tyrosine kinase-dependent. The present study demonstrates that the signals initiated by OZ involve activation of tyrosine kinase Pyk2 but not the formation of the adhesion protein complex, Shc-Grb2-Sos. Stimulation of cPLA(2) activity by OZ is mediated by Fc gamma receptors (FcgammaRs) and not by complement receptors for the C3b protein. Cross-linking of FcgammaRIIA or FcgammaRIIIB induces p38 mitogen-activated protein (MAP) kinase and extracellular regulated kinase (ERK) phosphorylation. The kinetics of cPLA(2) activity stimulated by either of the FcgammaRs or by both is similar to that of p38 MAP kinase and was detected as early as 15 s after stimulation, maintained a plateau for 10 min, and decreased thereafter. ERK activation was detected also within 15 s but decreased significantly 5 min after stimulation. The MEK inhibitor, PD-098059, or the p38 MAP kinase inhibitor, SB-203580, caused a partial inhibition during the time course of cPLA(2) activity, whereas their combination caused a total inhibition. Thus, although ERK activation is significantly shorter than that of p38 MAP kinase, it is equally required for activation and maintenance of cPLA(2) activity by occupancy of a single receptor, FcgammaRIIA or FcgammaRIIIB.

Antigens, CD↗

PubMed2000

Detection of partially phosphorylated forms of ERK by monoclonal antibodies reveals spatial regulation of ERK activity by phosphatases.

When cells are stimulated by mitogens, extracellular signal-regulated kinase (ERK) is activated by phosphorylation of its regulatory threonine (Thr) and tyrosine (Tyr) residues. The inactivation of ERK may occur by phosphatase-mediated removal of the phosphates from these Tyr, Thr or both residues together. In this study, antibodies that selectively recognize all combinations of phosphorylation of the regulatory Thr and Tyr residues of ERK were developed, and used to study the inactivation of ERK upon mitogenic stimulation. We found that inactivation of ERK in the early stages of mitogenic stimulation involves separate Thr and Tyr phosphatases which operate differently in the nucleus and in the cytoplasm. Thus, ERK is differentially regulated in various subcellular compartments to secure proper length and strength of activation, which eventually determine the physiological outcome of many external signals.

Animals↗

PubMed2000

Protein truncation test of LYST reveals heterogenous mutations in patients with Chediak-Higashi syndrome.

Chediak-Higashi syndrome (CHS) is a rare autosomal recessive disorder in which an immune deficiency occurs in association with pigmentation abnormalities. Most patients who do not undergo bone marrow transplantation die of a lymphoproliferative syndrome, though some patients with CHS have a relatively milder clinical course of the disease. The large size of the LYST gene, defective in CHS, has made it difficult to screen for mutations in a large number of patients. Only 8 mutations have been identified so far, and all lead to a truncated LYST protein. We conducted protein truncation tests on this gene in 8 patients with CHS. Different LYST mutations were identified in all subjects through this approach, strengthening the observation of a high frequency of truncated LYST proteins as the genetic cause of CHS.

Adolescent↗

PubMed2000

Expression of MHC class II molecules contributes to lipopolysaccharide responsiveness.

Activation of phagocytes by lipopolysaccharide (LPS) causes synthesis and secretion of various mediators of inflammation. CD14, a glycosylphosphatidylinositol-anchored monocytic antigen serving as receptor for LPS, and members of the family of Toll-like receptors mediate cellular activation in response to LPS. Here we investigated whether expression of MHC class II molecules modified the response to LPS. Comparing LPS responsiveness of human and murine cells differing for expression of MHC class II molecules, we found that lack or a low level of expression of MHC class II molecules resulted in diminished secretion of pro-inflammatory cytokines following stimulation with LPS. Thus, expression of MHC class II molecules modifies LPS responsiveness, a finding suggesting that these molecules contribute to the pathogenesis not only of exotoxin-triggered toxic shock but also of endotoxin-triggered septic shock. Additionally to their role in antigen-specific immunity MHC class II molecules may influence the inflammatory response triggered by microbial constituents.

Animals↗

PubMed2000

Activation of MAPK cascades by G-protein-coupled receptors: the case of gonadotropin-releasing hormone receptor.

G-protein-coupled receptors (GPCRs) are a large group of integral membrane receptors that transmit signals from a diverse array of external stimuli, including neurotransmitters, hormones, phospholipids, photons, odorants and taste ligands. In response to ligand binding, the GPCRs initiate diverse downstream signaling pathways through four groups of G proteins and other interacting proteins. Key components in GPCR-induced intracellular signaling are four groups of mitogen-activated protein kinase (MAPK) cascades: extracellular signal-related kinase (ERK), Jun N-terminal kinase (JNK), p38MAPK and big MAPK (BMK). The hallmark of MAPK signaling is the stimulation-dependent nuclear translocation of the involved kinases, which regulate gene expression and the cytoplasmic acute response to mitogenic, stress-related, apoptotic and survival stimuli. A special type of GPCR is the gonadotropin-releasing hormone (GnRH) receptor, which uses primarily the Gq protein for its downstream signaling. GnRH activates all four MAPK cascades by a PKC-dependent mechanism. Common signaling molecules, including the tyrosine kinase c-SRC and the small GTPases CDC42, RAC and RAS, are implicated in various aspects of the GnRH-MAPK pathways. Thus, the activation of MAPK cascades by GnRH opens a new vista in the understanding of the transcriptional regulation of genes encoding gonadotropins. However, additional studies on cell lines and whole animals are required to understand GnRH signaling in the context of other hormones during the reproductive cycle of mouse and human.

Animals↗

PubMed2000

Gene therapy of chronic granulomatous disease.

Chronic granulomatous disease (CGD) is a primary immunodeficiency disorder which results from absence or malfunction of the respiratory burst oxidase normally expressed in neutrophils and other phagocytic leukocytes. Two-thirds of the patients are males hemizygous for mutations in the X-linked gene coding for gp91-phox. As a therapeutic approach towards the X-linked form of CGD bicistronic retroviral vectors containing the gp91-phox gene and a selectable marker gene were constructed. The ability of these vectors to restore NADPH oxidase activity was tested in a human myeloid leukemic cell line that is defective in superoxide production, as well as in primary CD34+ cells obtained from X-CGD patients. Under optimal conditions 80% of the CD34+ cells derived from bone marrow of one X-CGD patient were transduced. The level of superoxide production, in phagocytes derived from transduced cells was 68.9% of normal levels. Considering that low levels of superoxide generating activity are sufficient for normal host defense, the present experiments provide the basis for the development of a gene replacement therapy for the X-linked form of CGD.

Animals↗

PubMed2000

Bone marrow transplantation with unrelated donors: what is the probability of identifying an HLA-A/B/Cw/DRB1/B3/B5/DQB1-matched donor?

Patients transplanted with marrow from an HLA-ABDR serologically matched unrelated donor suffer from more post-transplant complications than those who are transplanted with marrow from an HLA-identical sibling. This is most likely due to either HLA-ABDR incompatibilities not resolved by standard techniques and/or HLA polymorphisms not tested for by routine tissue typing (HLA-Cw,-DQ). By resolving these incompatibilities by molecular techniques combined with the in vitro cytotoxic T lymphocyte precursor frequency (CTLpf) test, we have shown that a high degree of HLA compatibility is associated with increased patient survival. However, higher requirements for HLA matching decrease the number of available donors. We have estimated the probability of finding an HLA-A/B/Cw/DRB1/DRB3/DRB5/DQB1 compatible donor based on 104 consecutive unrelated bone marrow donor searches initiated between January 1995 and December 1997, with December 1998 as the endpoint. For 96 patients (92.3%), one or more ABDR-identical donors were listed in the Bone Marrow Donor Worldwide Registry (BMDW). After contacting the registries, we obtained at least one (mean, 5.36; range, 1-20; total, 461) blood sample for 86 patients. A highly compatible donor was identified for 33/86 patients (38.4%), after testing an average number of 4.5 donors/patients (range, 1-13). However, by accepting an HLA-DRB3 or -DQB1 or -Cw incompatibility, this number would be as high as 68.6%. Approximately half of the patients (n = 40) for whom a search had been initiated have been transplanted: 22 patients with a perfectly matched donor, 15 patients with an HLA-DRB3 or -DQB1 or -Cw mismatch and three with other mismatches. The average time needed to identify the most compatible donor was 4 months. Extremely long searches seemed to be less useful, because after testing the first seven, a more compatible donor was seldom found. These results show that even when requirements for compatibility are high, the chances of finding a donor remain considerably low.

Bone Marrow Transplantation↗

PubMed2000

Induction of intracellular signalling by cyclic glycerophosphates and their deoxy analogues.

Cyclic glycerophosphates can be formed by enzymic degradation of phospholipids. They have only recently attracted attention, and their physiological function is still obscure. In this study, we have searched for signalling functions of the natural 1,3-cyclic and 1,2-cyclic glycerophosphates, their deoxy analogues, and the phenyl esters of the 1,3-cyclic phosphates. Linear sn-glycerol 3-phosphate and glycerol 2-phosphate served as the control compounds. Each of the six-membered ring cyclic phosphates tested induced rapid intracellular tyrosine phosphorylation in CHO and NIH-3T3 cells when applied extracellularly at a concentration of 0.5-4 microM. The phosphorylated intracellular proteins had molecular masses of approximately 35 kDa, approximately 45 kDa, 60-70 kDa and approximately 120 kDa. The five-membered ring cyclic phosphates had a similar effect, but at an external concentration of 2-10 microM, while sn-glycerol 3-phosphate and glycerol 2-phosphate had no effect. The six-membered cyclic phosphates also induced rapid threonine phosphorylation in CHO cells of approximately 18-kDa, approximately 35-kDa, and approximately 38-kDa proteins. Further experiments indicated that the cyclic phosphates partition rapidly into the cell cytosol where they activate kinases, including mitogen-activated protein kinase. When their intracellular level increases, dephosphorylation presumably takes place. This pattern may account for the signalling profile of cyclic phosphates and suggests that they may take part in processes associated with cell differentiation.

3T3 Cells↗

PubMed1999

Identification of a cytoplasmic-retention sequence in ERK2.

A key step in the signaling mechanism of the mitogen-activated protein kinase/extracellular signal-responsive kinase (ERK) cascade is its translocation into the nucleus where it regulates transcription and other nuclear processes. In an attempt to characterize the subcellular localization of ERK2, we fused it to the 3'-end of the gene expressing green fluorescent protein (GFP), resulting in a GFP-ERK2 protein. The expression of this construct in CHO cells resulted in a nuclear localization of the GFP-ERK2 protein. However, coexpression of the GFP-ERK2 with its upstream activator, MEK1, resulted in a cytosolic retention of the GFP-ERK2, which was the result of its association with MEK1, and was reversed upon stimulation. We then examined the role of the C-terminal region of ERK2 in its subcellular localization. Substitution of residues 312-319 of GFP-ERK2 to alanine residues prevented the cytosolic retention of ERK2 as well as its association with MEK1, without affecting its activity. Most important for the cytosolic retention are three acidic amino acids at positions 316, 319, and 320 of ERK2. Substitution of residues 321-327 to alanines impaired the nuclear translocation of ERK2 upon mitogenic stimulation. Thus, we conclude that residues 312-320 of ERK2 are responsible for its cytosolic retention, and residues 321-327 play a role in the mechanism of ERK2 nuclear translocation.

Amino Acid Sequence↗

PubMed1999

LIS1 is a microtubule-associated phosphoprotein.

Lissencephaly, a severe brain malformation, may be caused by mutations in the LIS1 gene. LIS1 encodes a microtubule-associated protein (MAP) that is also part of the enzyme complex, platelet-activating factor acetylhydrolase. LIS1 is also found in a complex with two protein kinases; a T-cell Tat-associated kinase, which contains casein-dependent kinase (CDK) activating kinase (CAK), as well as CAK-inducing activity, and with a spleen protein-tyrosine kinase similar to the catalytic domain of p72syk. As phosphorylation is one of the ways to control cellular localization and protein-protein interactions, we investigated whether LIS1 undergoes this post-translational modification. Our results demonstrate that LIS1 is a developmentally regulated phosphoprotein. Phosphorylated LIS1 is mainly found in the MAP fraction. Phosphoamino acid analysis revealed that LIS1 is phosphorylated on serine residues. Alkaline phosphatase treatment reduced the number of visible LIS1 isoforms. In-gel assays demonstrate a 50-kDa LIS1 kinase that is enriched in microtubule-associated fractions. In vitro, LIS1 was phosphorylated by protein kinase CKII (casein kinase II), but not many other kinases that were tested. We suggest that LIS1 activity may be regulated by phosphorylation.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗

PubMed1999

Phosphorylation of insulin receptor substrate-1 (IRS-1) by protein kinase B positively regulates IRS-1 function.

Incubation of cells with insulin leads to a transient rise in Tyr phosphorylation of insulin receptor substrate (IRS) proteins, accompanied by elevation in their Ser(P)/Thr(P) content and their dissociation from the insulin receptor (IR). Wortmannin, a phosphatidylinositol 3-kinase inhibitor, selectively prevented the increase in Ser(P)/Thr(P) content of IRS-1, its dissociation from IR, and the decrease in its Tyr(P) content following 60 min of insulin treatment. Four conserved phosphorylation sites within the phosphotyrosine binding/SAIN domains of IRS-1 and IRS-2 served as in vitro substrates for protein kinase B (PKB), a Ser/Thr kinase downstream of phosphatidylinositol 3-kinase. Furthermore, PKB and IRS-1 formed stable complexes in vivo, and overexpression of PKB enhanced Ser phosphorylation of IRS-1. Overexpression of PKB did not affect the acute Tyr phosphorylation of IRS-1; however, it significantly attenuated its rate of Tyr dephosphorylation following 60 min of treatment with insulin. Accordingly, overexpression of IRS-1(4A), lacking the four potential PKB phosphorylation sites, markedly enhanced the rate of Tyr dephosphorylation of IRS-1, while inclusion of vanadate reversed this effect. These results implicate a wortmannin-sensitive Ser/Thr kinase, different from PKB, as the kinase that phosphorylates IRS-1 and acts as the feedback control regulator that turns off insulin signals by inducting the dissociation of IRS proteins from IR. In contrast, insulin-stimulated PKB-mediated phosphorylation of Ser residues within the phosphotyrosine binding/SAIN domain of IRS-1 protects IRS-1 from the rapid action of protein-tyrosine phosphatases and enables it to maintain its Tyr-phosphorylated active conformation. These findings implicate PKB as a positive regulator of IRS-1 functions.

Androstadienes↗

PubMed1999

Mitogen-activated protein kinase kinase activity is required for the G(2)/M transition of the cell cycle in mammalian fibroblasts.

The mitogen-activated protein kinase (MAPK) cascade is required for mitogenesis in somatic mammalian cells and is activated by a wide variety of oncogenic stimuli. Specific roles for this signaling module in growth were dissected by inhibiting MAPK kinase 1 (MAPKK1) activity in highly synchronized NIH 3T3 cells. In addition to the known role of this kinase in cell-cycle entry from G(0), the level of MAPKK activity was observed to affect the kinetics of progression through both the G(1) and G(2) phases of the cell cycle in NIH 3T3 cells. Ectopic expression of dominant-negative forms of MAPKK1, which was previously shown to inhibit G(0)/G(1) progression, was found to also delay progression of cells through G(2). In addition, treatment of cells with the specific MAPKK inhibitor PD 98059 during a synchronous S phase arrested the cells in the following G(2) phase. These data demonstrate a novel role for the MAPK cascade in progression from G(2) into mitosis in NIH 3T3 cells.

3T3 Cells↗

PubMed1999

GRFbeta, a novel regulator of calcium signaling, is expressed in pancreatic beta cells and brain.

By screening for genes expressed differentially in pancreatic beta cells, we have isolated a cDNA encoding GRFbeta, a novel 178-amino acid protein whose N terminus is identical to that of GRF1, a calcium-dependent guanine nucleotide exchange factor, and whose C terminus is unrelated to known proteins. We show that both GRF1 and GRFbeta are expressed selectively in beta cell lines, pancreatic islet cells and brain. Treatment of beta cell lines (betaTC1 and HIT) with calcium ionophore led to a significant elevation in activity of the Ras signal transduction pathway, as determined by phosphorylation of extracellular signal-related kinase (ERK). Transfection of beta cells with a plasmid encoding a dominant negative variant of GRF1 led to 70% reduction in ERK phosphorylation, consistent with a role for GRF1 in calcium-dependent Ras signaling in these cells. To examine the possible function of GRFbeta, cultured cells were transfected with a GRFbeta expression vector. This led to a significant reduction in both GRF1-dependent ERK phosphorylation and AP1-dependent reporter gene activity. The results suggest that GRF1 plays a role in mediating calcium-dependent signal transduction in beta cells and that GRFbeta represents a novel dominant negative modulator of Ras signaling.

Amino Acid Sequence↗