Biomedical subjects

D Ron

Publications and source records attributed to D Ron.

At least 37 records · Page 2Linked to original sources

PubMed2000

Dynamic interaction of BiP and ER stress transducers in the unfolded-protein response.

PERK and IRE1 are type-I transmembrane protein kinases that reside in the endoplasmic reticulum (ER) and transmit stress signals in response to perturbation of protein folding. Here we show that the lumenal domains of these two proteins are functionally interchangeable in mediating an ER stress response and that, in unstressed cells, both lumenal domains form a stable complex with the ER chaperone BiP. Perturbation of protein folding promotes reversible dissociation of BiP from the lumenal domains of PERK and IRE1. Loss of BiP correlates with the formation of high-molecular-mass complexes of activated PERK or IRE1, and overexpression of BiP attenuates their activation. These findings are consistent with a model in which BiP represses signalling through PERK and IRE1 and protein misfolding relieves this repression by effecting the release of BiP from the PERK and IRE1 lumenal domains.

Animals↗

PubMed2000

Conformational disease.

A large and diverse number of diseases are now recognized as 'conformational diseases', caused by adoption of non-native protein conformations that lead to aggregation. The recent conference, 'Alpha1-antitrypsin deficiency and other conformational diseases', held in Airlie, Virginia, USA (27-30 June, 2000) focused on some of the common pathways by which cells protect themselves from toxicity associated with protein misfolding and aggregation.

Calcium-Binding Proteins↗

PubMed2000

Upregulation of BiP and CHOP by the unfolded-protein response is independent of presenilin expression.

Presenilin 1 (PS1), a polytopic membrane protein, has a critical role in the trafficking and proteolysis of a selected set of transmembrane proteins. The vast majority of individuals affected with early onset familial Alzheimer's disease (FAD) carry missense mutations in PS1. Two studies have suggested that loss of PS1 function, or expression of FAD-linked PS1 variants, compromises the mammalian unfolded-protein response (UPR), and we sought to evaluate the potential role of PS1 in the mammalian UPR. Here we show that that neither the endoplasmic reticulum (ER) stress-induced accumulation of BiP and CHOP messenger RNA, nor the activation of ER stress kinases IRE1alpha and PERK, is compromised in cells lacking both PS1 and PS2 or in cells expressing FAD-linked PS1 variants. We also show that the levels of BiP are not significantly different in the brains of individuals with sporadic Alzheimer's disease or PS1-mediated FAD to levels in control brains. Our findings provide evidence that neither loss of PS1 and PS2 function, nor expression of PS1 variants, has a discernable impact on ER stress-mediated induction of the several established 'readouts' of the UPR pathway.

Alzheimer Disease↗

PubMed2000

Uncoupling of betaIIPKC from its targeting protein RACK1 in response to ethanol in cultured cells and mouse brain.

Protein kinase C (PKC) is involved in many neuroadaptive responses to ethanol in the nervous system. PKC activation results in translocation of the enzyme from one intracellular site to another. Compartmentalization of PKC isozymes is regulated by targeting proteins such as receptors for activated C kinase (RACKs). It is possible, therefore, that ethanol-induced changes in the function and compartmentalization of PKC isozymes could be due to changes in PKC targeting proteins. Here we study the response of the targeting protein RACK1 and its corresponding kinase betaIIPKC to ethanol, and propose a novel mechanism to explain how ethanol modulates signaling cascades. In cultured cells, ethanol induces movement of RACK1 to the nucleus without affecting the compartmentalization of betaIIPKC. Ethanol also inhibits betaIIPKC translocation in response to activation. These results suggest that ethanol inhibition of betaIIPKC translocation is due to miscompartmentalization of the targeting protein RACK1. Similar events occurred in mouse brain. In vivo exposure to ethanol caused RACK1 to localize to nuclei in specific brain regions, but did not affect the compartmentalization of betaIIPKC. Thus, some of the cellular and neuroadaptive responses to ethanol may be related to ethanol-induced movement of RACK1 to the nucleus, thereby preventing the translocation and corresponding function of betaIIPKC.

Animals↗

PubMed2000

IRE1 and efferent signaling from the endoplasmic reticulum.

Genetic analysis of the cellular adaptation to malfolded proteins in the endoplasmic reticulum (the unfolded protein response - UPR) has revealed a novel signaling pathway initiated by activation of IRE1, an ER-resident protein kinase and endonuclease. In yeast, Ire1p activates gene expression by promoting a non-conventional splicing event that converts the mRNA encoding the Hac1p transcription factor from an inefficiently translated inactive mRNA to an actively translated one. Hac1p binds to the promoters of genes encoding chaperones and other targets of the UPR and activates them. Recently, mammalian IRE1 homologues have been identified and their response to ER stress is regulated by binding to the ER chaperone BiP. The mechanisms by which mammalian IRE1 activates gene expression have not been completely characterized and mammalian HAC1 homologues have not been identified. Surprisingly, mammalian IRE1s are able to activate both JUN N-terminal kinases and an alternative ER-stress signaling pathway mediated by the transcription factor ATF6. This indicates that the mammalian UPR is more complex than that found in yeast.

Endoplasmic Reticulum↗

PubMed2000

Translocation and coamplification of loci from chromosome arms 8p and 11q in the MDA-MB-175 mammary carcinoma cell line.

Rearrangement and coamplification of the 8p12 and 11q13 chromosomal regions occurs in a significant proportion of breast cancers. It usually involves a complex hybrid structure in which the FGFR1 and CCND1 genes are amplified. We report here a different type of 8p12-11q13 rearrangement in the MDA-MB-175 mammary carcinoma cell line. This amplification contains the NRG1/HGL (from 8p12-21) and DOC4 (from 11q13) genes, encoding respectively a ligand for ERBB receptors and a stress-induced protein which is a mammalian ortholog of Drosophila Tenm/Odz. It has been shown previously (Wang et al, Oncogene 18: 5718-5721, 1999) that these two genes are rearranged and fused by a translocation event. This type of event was not found in 30 tumors tested that showed coamplification of the 8p12 and 11q13 regions.

Breast Neoplasms↗

PubMed1999

Similarities and differences between the effects of heparin and glypican-1 on the bioactivity of acidic fibroblast growth factor and the keratinocyte growth factor.

The keratinocyte growth factor (KGF or FGF-7) is unique among its family members both in its target cell specificity and its inhibition by the addition of heparin and the native heparan-sulfate proteoglycan (HSPG), glypican-1 in cells expressing endogenous HSPGs. FGF-1, which binds the FGF-7 receptor with a similar affinity as FGF-7, is stimulated by both molecules. In the present study, we investigated the modulation of FGF-7 activities by heparin and glypican-1 in HS-free background utilizing either HS-deficient cells expressing the FGF-7 receptor (designated BaF/KGFR cells) or soluble extracellular domain of the receptor. At physiological concentrations of FGF-7, heparin was required for high affinity receptor binding and for signaling in BaF/KGFR cells. In contrast, binding of FGF-7 to the soluble form of the receptor did not require heparin. However, high concentrations of heparin inhibited the binding of FGF-7 to both the cell surface and the soluble receptor, similar to the reported effect of heparin in cells expressing endogenous HSPGs. The difference in heparin dependence for high affinity interaction between the cell surface and soluble receptor may be due to other molecule(s) present on cell surfaces. Glypican-1 differed from heparin in that it stimulated FGF-1 but not FGF-7 activities in BaF/KGFR cells. Glypican-1 abrogated the stimulatory effect of heparin, and heparin reversed the inhibitory effect of glypican-1, indicating that this HSPG inhibits FGF-7 activities by acting, most likely, as a competitive inhibitor of stimulatory HSPG species for FGF-7. The regulatory effect of glypican-1 is mediated at the level of interaction with the growth factor as glypican-1 did not bind the KGFR. The effect of heparin and glypican-1 on FGF-1 and FGF-7 oligomerization was studied employing high and physiological concentrations of growth factors. We did not find a correlation between the effects of these glycosaminoglycans on FGFs biological activity and oligomerization. Altogether, our findings argue against the heparin-linked dimer presentation model as key in FGFR activation, and support the notion that HSPGs primarily affect high affinity interaction of FGFs with their receptors.

Animals↗

PubMed1999

Mutations uncouple human fibroblast growth factor (FGF)-7 biological activity and receptor binding and support broad specificity in the secondary receptor binding site of FGFs.

The fibroblast growth factor (FGF) family plays a key role in a multitude of physiological and pathological processes. The activities of FGFs are mediated by a family of tyrosine kinase receptors, designated FGFRs. The mechanism by which FGFs induce receptor activation is controversial. Despite their structural similarity, FGFs display distinct receptor binding characteristics and cell type specificity. Previous studies with FGF-2 identified a low affinity receptor binding site that is located within a loop connecting its 9th and 10th beta-strands. The corresponding residues in the other family members are highly variable, and it was proposed that the variability might confer on FGFs unique receptor binding characteristics. We studied the role of this loop in FGF-7 by both site-directed mutagenesis and loop replacement. Unlike the other members of the FGF family, FGF-7 recognizes only one FGFR isoform and is, therefore, ideal for studies of how the specificity in the FGF-FGFR interaction is conferred at the structural level. Point mutations in the loop of FGF-7 did not change receptor binding affinity but resulted in reduced mitogenic potency and reduced ability to induce receptor-mediated phosphorylation events. These results suggest that the loop of FGF-7 fulfills the role of low affinity binding site required for receptor activation. The observation that it is possible to uncouple FGF-7 receptor binding and biological activity favors a bivalent model for FGFR dimerization, and it may be clinically relevant to the design of FGF-7 antagonists. Reciprocal loop replacement between FGF-7 and FGF-2 had no effect on their known receptor binding affinities nor did it alter their known specificity in eliciting a mitogenic response. In conclusion, these results suggest that, despite the diversity in the loop structure of FGF-2 and FGF-7, the loop has a similar function in both growth factors.

3T3 Cells↗

PubMed1999

Keratin 10 gene expression during differentiation of mouse epidermis requires transcription factors C/EBP and AP-2.

The epidermis forms a vital barrier composed of stratified keratinocytes and their differentiated products. One of these products, keratin K10, is critical to epidermal integrity, because mutations in k10 lead to abnormal blistering. For the normal expression of k10, differentiation-associated transcription factors C/EBPalpha, C/EBPbeta, and AP-2 are well positioned to play an important role. Here, regulation of the k10 gene is examined in keratinocytes in the skin of normal mice and in transgenic mice carrying targeted deletions of c/ebpbeta and ap-2alpha. In cultured cells, C/EBPalpha and C/EBPbeta are each capable of activating the k10 promoter via three binding sites, identified by site-directed mutagenesis. In a given epidermal cell in vivo, however, the selection of C/EBPalpha versus C/EBPbeta for k10 regulation is determined via a third transcription factor, AP-2. This novel regulatory scheme involves: (1) unique gradients of expression for each transcription factor, i.e., C/EBPbeta and AP-2 most abundant in the lower epidermis, C/EBPalpha in the upper; (2) C/EBP-binding sites in the ap-2alpha gene promoter, through which C/EBPbeta stimulates ap-2alpha; and (3) AP-2 binding sites in the c/ebpalpha promoter, through which AP-2 represses c/ebpalpha. Promoter-analysis and gene-expression data presented herein support a regulatory model in which C/EBPbeta activates and maintains AP-2 expression in basal keratinocytes, whereas AP-2 represses C/EBPalpha in those cells. In response to differentiation signals, loss of AP-2 expression leads to derepression of the c/ebpalpha promoter and activation of k10 as cells migrate upward.

Animals↗

PubMed1999

Human 75-kDa DNA-pairing protein is identical to the pro-oncoprotein TLS/FUS and is able to promote D-loop formation.

Homologous recombination plays a fundamental role in DNA double-strand break repair. Previously, we detected two mammalian nuclear proteins of 100 and 75 kDa (POMp100 and POMp75, respectively) that are able to promote homologous DNA pairing, a key step in homologous recombination. Here we describe the identification of human (h) POMp75 as the pro-oncoprotein TLS/FUS. hPOMp75/TLS binds both single- and double-stranded DNAs and mediates annealing of complementary DNA strands. More important, it promotes the uptake of a single-stranded oligonucleotide into a homologous superhelical DNA to form a D-loop. The formation of a D-loop is an essential step in DNA double-strand break repair through recombination. DNA annealing and D-loop formation catalyzed by hPOMp75/TLS require Mg(2+) and are ATP-independent. Interestingly, the oncogenic fusion form TLS-CHOP is not able to promote DNA pairing. These data suggest a possible role for hPOMp75/TLS in maintenance of genomic integrity.

Base Pairing↗

PubMed1999

RACK1, a protein kinase C anchoring protein, coordinates the binding of activated protein kinase C and select pleckstrin homology domains in vitro.

The pleckstrin homology (PH) domain, identified in numerous signaling proteins including the beta-adrenergic receptor kinase (betaARK), was found to bind to various phospholipids as well as the beta subunit of heterotrimeric G proteins (Gbeta) [Touhara, K., et al. (1994) J. Biol. Chem. 269, 10217-10220]. Several PH domain-containing proteins are also substrates of protein kinase C (PKC). Because RACK1, an anchoring protein for activated PKC, is homologous to Gbeta (both contain seven repeats of the WD-40 motif), we determined (i) whether a direct interaction between various PH domains and RACK1 occurs and (ii) the effect of PKC on this interaction. We found that recombinant PH domains of several proteins exhibited differential binding to RACK1. Activated PKC and the PH domain of beta-spectrin or dynamin-1 concomitantly bound to RACK1. Although PH domains bind acidic phospholipids, the interaction between various PH domains and RACK1 was not dependent on the phospholipid activators of PKC, phosphatidylserine and 1, 2-diacylglycerol. Binding of these PH domains to RACK1 was also not affected by either inositol 1,4,5-triphosphate (IP(3)) or phosphatidylinositol 4,5-bisphosphate (PIP(2)). Our in vitro data suggest that RACK1 binds selective PH domains, and that PKC regulates this interaction. We propose that, in vivo, RACK1 may colocalize the kinase with its PH domain-containing substrates.

Animals↗

PubMed1999

gamma-heregulin is the product of a chromosomal translocation fusing the DOC4 and HGL/NRG1 genes in the MDA-MB-175 breast cancer cell line.

gamma-heregulin is a recently described novel isoform of the heregulin/neuregulin class of EGF-like ligands that bind to and activate receptors of the ErbB family. Deregulated signaling through the heregulin-ErbB pathway is thought to be implicated in the development of a subset of human breast cancers. gamma-heregulin has been found to be expressed in the culture supernatant of MDA-MB-175, a breast carcinoma cell line. gamma-heregulin is characterized by the presence of a large N-terminal peptide extension that is not found in other heregulin isoforms. Here we report that this unique N-terminal extension of gamma-heregulin is identical to the N-terminus of DOC4, a product of a recently identified CHOP-dependent stress-induced gene. Human DOC4 and the heregulin-encoding genes map to different chromosomes and the MDA-MB-175 cell line contains a chromosomal translocation that leads to the fusion of DOC4 and HGL, on chromosomes 11 and 8, respectively. Thus, gamma-heregulin is a product of a mutant fusion gene and not a bona fide normal isoform. We speculate that the mutation may be selected for by virtue of its ability to activate ErbB signaling through the production of an autocrine ligand.

Breast Neoplasms↗

PubMed1999

Coordinated movement of RACK1 with activated betaIIPKC.

Protein kinase C (PKC) isozymes move upon activation from one intracellular site to another. PKC-binding proteins, such as receptors for activated C kinase (RACKs), play an important role in regulating the localization and diverse functions of PKC isozymes. RACK1, the receptor for activated betaIIPKC, determines the localization and functional activity of betaIIPKC. However, the mechanism by which RACK1 localizes activated betaIIPKC is not known. Here, we provide evidence that the intracellular localization of RACK1 changes in response to PKC activation. In Chinese hamster ovary cells transfected with the dopamine D2L receptor and in NG108-15 cells, PKC activation by either phorbol ester or a dopamine D2 receptor agonist caused the movement of RACK1. Moreover, PKC activation resulted in the in situ association and movement of RACK1 and betaIIPKC to the same intracellular sites. Time course studies indicate that PKC activation induces the association of the two proteins prior to their co-movement. We further show that association of RACK1 and betaIIPKC is required for the movement of both proteins. Our results suggest that RACK1 is a PKC shuttling protein that moves betaIIPKC from one intracellular site to another.

Animals↗

PubMed1999

Algorithmic stability and sanity-check bounds for leave-one-out cross-validation.

In this article we prove sanity-check bounds for the error of the leave-one-out cross-validation estimate of the generalization error: that is, bounds showing that the worst-case error of this estimate is not much worse than that of the training error estimate. The name sanity check refers to the fact that although we often expect the leave-one-out estimate to perform considerably better than the training error estimate, we are here only seeking assurance that its performance will not be considerably worse. Perhaps surprisingly, such assurance has been given only for limited cases in the prior literature on cross-validation. Any nontrivial bound on the error of leave-one-out must rely on some notion of algorithmic stability. Previous bounds relied on the rather strong notion of hypothesis stability, whose application was primarily limited to nearest-neighbor and other local algorithms. Here we introduce the new and weaker notion of error stability and apply it to obtain sanity-check bounds for leave-one-out for other classes of learning algorithms, including training error minimization procedures and Bayesian algorithms. We also provide lower bounds demonstrating the necessity of some form of error stability for proving bounds on the error of the leave-one-out estimate, and the fact that for training error minimization algorithms, in the worst case such bounds must still depend on the Vapnik-Chervonenkis dimension of the hypothesis class.

Algorithms↗

PubMed1999

Direct interaction in T-cells between thetaPKC and the tyrosine kinase p59fyn.

The protein kinase C (PKC) family has been clearly implicated in T-cell activation as have several nonreceptor protein-tyrosine kinases associated with the T-cell receptor, including p59fyn. This report demonstrates that thetaPKC and p59fyn specifically interact in vitro, in the yeast two-hybrid system, and in T-cells. Further indications of direct interaction are that p59fyn potentiates thetaPKC catalytic activity and that thetaPKC is a substrate for tyrosine phosphorylation by p59fyn. This interaction may account for the localization of thetaPKC following T-cell activation, pharmacological disruption of which results in specific cell-signaling defects. The demonstration of a physical interaction between a PKC and a protein-tyrosine kinase expands the class of PKC-anchoring proteins (receptors for activated C kinases (RACKs)) and demonstrates a direct connection between these two major T-cell-signaling pathways.

Antibodies↗

PubMed1999

Induction of a secreted protein by the myxoid liposarcoma oncogene.

The TLS-CHOP oncoprotein, found in the majority of human myxoid liposarcomas, consists of a fusion between the transcription factor CHOP/GADD153 and the N terminus of an RNA-binding protein TLS/FUS. Clinical correlation and in vitro transformation assays indicate that the N terminus of TLS plays an important role in oncogenesis by TLS-CHOP. Until now, however, the only activity attributed to the oncoprotein is that of inhibiting the binding of transcription factors of the C/EBP class to certain adipogenic target genes, a function that TLS-CHOP shares with the nononcogenic CHOP protein. Here we report the isolation of a gene, DOL54, that is activated in primary fibroblasts by the expression of TLS-CHOP. DOL54 is expressed in the neoplastic component of human myxoid liposarcomas and increases the tumorigenicity of cells injected in nude mice. Activation of DOL54 requires an intact DNA-binding and dimerization domain in TLS-CHOP, a suitable cellular dimerization partner, and depends on the TLS N terminus. Normal adipocytic differentiation is associated with an early and transient expression of DOL54, and the gene encodes a secreted protein that is tightly associated with the cell surface or extracellular matrix. TLS-CHOP thus leads to the unscheduled expression of a gene that is normally associated with adipocytic differentiation.

Animals↗

PubMed1999

Glypican-1 is a VEGF165 binding proteoglycan that acts as an extracellular chaperone for VEGF165.

Glypican-1 is a member of a family of glycosylphosphatidylinositol anchored cell surface heparan sulfate proteoglycans implicated in the control of cellular growth and differentiation. The 165-amino acid form of vascular endothelial growth factor (VEGF165) is a mitogen for endothelial cells and a potent angiogenic factor in vivo. Heparin binds to VEGF165 and enhances its binding to VEGF receptors. However, native HSPGs that bind VEGF165 and modulate its receptor binding have not been identified. Among the glypicans, glypican-1 is the only member that is expressed in the vascular system. We have therefore examined whether glypican-1 can interact with VEGF165. Glypican-1 from rat myoblasts binds specifically to VEGF165 but not to VEGF121. The binding has an apparent dissociation constant of 3 x 10(-10) M. The binding of glypican-1 to VEGF165 is mediated by the heparan sulfate chains of glypican-1, because heparinase treatment abolishes this interaction. Only an excess of heparin or heparan sulfates but not other types of glycosaminoglycans inhibited this interaction. VEGF165 interacts specifically not only with rat myoblast glypican-1 but also with human endothelial cell-derived glypican-1. The binding of 125I-VEGF165 to heparinase-treated human vascular endothelial cells is reduced following heparinase treatment, and addition of glypican-1 restores the binding. Glypican-1 also potentiates the binding of 125I-VEGF165 to a soluble extracellular domain of the VEGF receptor KDR/flk-1. Furthermore, we show that glypican-1 acts as an extracellular chaperone that can restore the receptor binding ability of VEGF165, which has been damaged by oxidation. Taken together, these results suggest that glypican-1 may play an important role in the control of angiogenesis by regulating the activity of VEGF165, a regulation that may be critical under conditions such as wound repair, in which oxidizing agents that can impair the activity of VEGF are produced, and in situations were the concentrations of active VEGF are limiting.

Endothelial Growth Factors↗

PubMed1999

Amino acid limitation regulates CHOP expression through a specific pathway independent of the unfolded protein response.

The gene encoding CHOP (C/EBP-homologous protein) is transcriptionally activated by many stimuli and by amino acid deprivation. CHOP induction was considered to be due to an accumulation of unfolded protein into the ER (unfolded protein response (UPR)). We investigate the role of the UPR in the induction of CHOP by amino acid deprivation and show that this induction is not correlated with BiP expression (an UPR marker). Moreover, amino acid deprivation and UPR inducers regulate the CHOP promoter activity using distinct cis elements. We conclude that amino acid deprivation does not activate the UPR and regulates CHOP expression through a pathway that is independent of the UPR.

Amino Acids↗