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Biomedical subjects

B Margolis

Publications and source records attributed to B Margolis.

At least 19 recordsLinked to original sources
PubMed2000

Modulation of amyloid precursor protein metabolism by X11alpha /Mint-1. A deletion analysis of protein-protein interaction domains.

Modulation of amyloid precursor protein (APP) metabolism plays a pivotal role in the pathogenesis of Alzheimer's disease. The phosphotyrosine-binding/protein interaction (PTB/PI) domain of X11alpha, a neuronal cytosolic adaptor protein, binds to the YENPTY sequence in the cytoplasmic carboxyl terminus of APP. This interaction prolongs the half-life of APP and inhibits Abeta40 and Abeta42 secretion. X11alpha/Mint-1 has multiple protein-protein interaction domains, a Munc-18 interaction domain (MID), a Cask/Lin-2 interaction domain (CID), a PTB/PI domain, and two PDZ domains. These X11alpha protein interaction domains may modulate its effect on APP processing. To test this hypothesis, we performed a deletion analysis of X11alpha effects on metabolism of APP(695) Swedish (K595N/M596L) (APP(sw)) by transient cotransfection of HEK 293 cells with: 1) X11alpha (X11alpha-wt, N-MID-CID-PTB-PDZ-PDZ-C), 2) amino-terminal deletion (X11alpha-DeltaN, PTB-PDZ-PDZ), 3) carboxyl-terminal deletion (X11alpha-DeltaPDZ, MID-CID-PTB), or 4) deletion of both termini (PTB domain only, PTB). The carboxyl terminus of X11alpha was required for stabilization of APP(sw) in cells. In contrast, the amino terminus of X11alpha was required to stimulate APPs secretion. X11alpha, X11alpha-DeltaN, and X11alpha-PTB, but not X11alpha-DeltaPDZ, were effective inhibitors of Abeta40 and Abeta42 secretion. These results suggest that additional protein interaction domains of X11alpha modulate various aspects of APP metabolism.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Molecular cloning and characterization of Pals, proteins associated with mLin-7.

In Caenorhabditis elegans, three PDZ domain proteins, Lin-2, Lin-7, and Lin-10, are necessary for the proper targeting of the Let-23 growth factor receptor to the basolateral surface of epithelial cells. It has been demonstrated that homologues of Lin-2, Lin-7, and Lin-10 form a heterotrimeric complex in mammalian brain. Using Far Western overlay assay, we have identified additional proteins that can bind to the amino terminus of mLin-7 and cloned the genes encoding these proteins using bacterial expression cloning. We call these proteins Pals, for proteins associated with Lin-7. These proteins, which include mammalian Lin-2, contain a conserved mLin-7 binding domain in addition to guanylate kinase, PDZ (postsynaptic density 95/discs large/zona occludens-1), and Src homology 3 domains. Using site-directed mutagenesis, we have identified the conserved residues among these proteins crucial for mLin-7 binding. Two of these proteins, Pals1 and Pals2, are newly described. Pals1 consists of 675 amino acids and maps to mouse chromosome 12. Pals2 was found to exist in two splice forms of 539 and 553 amino acids and maps to mouse chromosome 6. Like mLin-2, Pals1 and Pals2 localize to the lateral membrane in Madin-Darby canine kidney cells. Pals proteins represent a new subfamily of membrane-associated guanylate kinases that allow for multiple targeting complexes containing mLin-7.

Amino Acid Sequence↗
PubMed2000

ERBIN: a basolateral PDZ protein that interacts with the mammalian ERBB2/HER2 receptor.

The ERBB receptors have a crucial role in morphogenesis and oncogenesis. We have identified a new PDZ protein we named ERBIN (ERBB2 interacting protein) that acts as an adaptor for the receptor ERBB2/HER2 in epithelia. ERBIN contains 16 leucine-rich repeats (LRRs) in its amino terminus and a PDZ (PSD-95/DLG/ZO-1) domain at its carboxy terminus, and belongs to a new PDZ protein family. The PDZ domain directly and specifically interacts with ERBB2/HER2. ERBIN and ERBB2/HER2 colocalize to the lateral membrane of human intestinal epithelial cells. The ERBIN-binding site in ERBB2/HER2 has a critical role in restricting this receptor to the basolateral membrane of epithelial cells, as mutation of the ERBIN-binding site leads to the mislocalization of the receptor in these cells. We suggest that ERBIN acts in the localization and signalling of ERBB2/HER2 in epithelia.

Adaptor Proteins, Signal Transducing↗
PubMed2000

mLin-7 is localized to the basolateral surface of renal epithelia via its NH(2) terminus.

In Caenorhabditis elegans, the basolateral localization of the Let-23 growth factor receptor tyrosine kinase requires the expression of three genes: lin-2, lin-7, and lin-10. Mammalian homologs of these three genes have been identified, and a complex of their protein products exists in mammalian neurons. In this paper, we examine the interaction of these mammalian proteins in renal epithelia. Coprecipitation experiments demonstrated that mLin-2/CASK binds to mLin-7, and immunofluorescent labeling showed that these proteins colocalized at the basolateral surface of Madin-Darby canine kidney cells and renal epithelia. Although labeling intensity varied markedly among different renal epithelial cells, those cells strongly expressing mLin-7 also showed intense mLin-2/CASK labeling. We have also demonstrated that mLin-2/CASK binding requires amino acids 12-32 of mLin-7 and have shown that this region of mLin-7 is also necessary for the targeting of mLin-7 to the basolateral surface. Furthermore, the overexpression of mLin-2/CASK mutants in Madin-Darby canine kidney cells caused endogenous mLin-7 to mislocalize. In summary, the NH(2) terminus of mLin-7 is crucial for its basolateral localization, likely through its interaction with mLin-2/CASK.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Molecular events implicated in brain tumor angiogenesis and invasion.

We have conducted studies designed to help elucidate the molecular mechanisms involved in brain tumor invasion and angiogenesis, which are critical in the growth of malignant tumors of the central nervous system. A variety of molecular factors have been implicated in these processes. Here we focus on three that are of particular importance in the progression of brain tumors. Angiopoietins are involved in the regulation of vascular development. Hypoxia inducible factor-1 is a transcription factor that up-regulates genes, including genes encoding vascular endothelial growth factor under hypoxic conditions. Focal adhesion kinase is associated with infiltration of tumor cells and angiogenesis.

Brain Neoplasms↗
PubMed1999

Interaction of c-Jun amino-terminal kinase interacting protein-1 with p190 rhoGEF and its localization in differentiated neurons.

c-Jun amino-terminal kinase (JNK) interacting protein-1 (JIP-1) was originally identified as a cytoplasmic inhibitor of JNK. More recently, JIP-1 was proposed to function as a scaffold protein by complexing specific components of the JNK signaling pathway, namely JNK, mitogen-activated protein kinase kinase 7, and mixed lineage kinase 3. We have identified the human homologue of JIP-1 that contains a phosphotyrosine binding (PTB) domain in addition to a JNK binding domain and an Src homology 3 domain. To identify binding targets for the hJIP-1 PTB domain, a mouse embryo cDNA library was screened using the yeast two-hybrid system. One clone encoded a 191-amino acid region of the neuronal protein rhoGEF, an exchange factor for rhoA. Overexpression of rhoGEF promotes cytoskeletal rearrangement and cell rounding in NIE-115 neuronal cells. The interaction of JIP-1 with rhoGEF was confirmed by coimmunoprecipitation of these proteins from lysates of transiently transfected HEK 293 cells. Using glutathione S-transferase rhoGEF fusion proteins containing deletion or point mutations, we identified a putative PTB binding site within rhoGEF. This binding site does not contain tyrosine, indicating that the JIP PTB domain, like that of Xll alpha and Numb, binds independently of phosphotyrosine. Several forms of endogenous JIP-1 protein can be detected in neuronal cell lines. Indirect immunofluorescence analysis localized endogenous JIP-1 to the tip of the neurites in differentiated NIE-115 and PC12 cells. The interaction of JIP-1 with rhoGEF and its subcellular localization suggests that JIP-1 may function to specifically localize a signaling complex in neuronal cells.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Molecular analysis of the X11-mLin-2/CASK complex in brain.

A heterotrimeric complex containing Lin-10/X11alpha, Lin-2/CASK, and Lin-7 is evolutionarily conserved from worms to mammals. In Caenorhabditis elegans, it localizes Let-23, a receptor tyrosine kinase, to the basolateral side of vulval epithelium, a step crucial for proper vulva development. In mammals, the complex may also participate in receptor targeting in neurons. Accordingly, phosphotyrosine binding (PTB) and postsynaptic density-95/Discs large/Zona Occludens-1 domains found in X11alpha and mLin-2/CASK bind to cell-surface proteins, including amyloid precursor protein, neurexins, and syndecans. In this paper, we have further analyzed the X11alpha-mLin-2/CASK association that is mediated by a novel protein-protein interaction. We show that the mLin-2/CASK calmodulin kinase II (CKII) domain directly binds to a 63 amino acids peptide located between the Munc-18-1 binding site and the PTB domain in X11alpha. Ca2+/calmodulin association with mLin-2/CASK does not modify the X11alpha-mLin-2 interaction. A region containing the mLin-2/CASK guanylate kinase domain also interacts with X11alpha but with a lower affinity than the CKII domain. Immunostaining of X11alpha in the brain shows that the protein is expressed in areas shown previously to be positive for mLin-2/CASK staining. Together, our data demonstrate that the X11alpha-mLin-2 complex contacts many partners, creating a macrocomplex suitable for receptor targeting at the neuronal plasma membrane.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Role of tyrosine residues and protein interaction domains of SHC adaptor in VEGF receptor 3 signaling.

The VEGFR3/FLT4 receptor, which is involved in vasculogenesis and angiogenesis, binds and phosphorylates SHC proteins on tyrosine residues. SHC contains two phosphotyrosine interaction domains: a PTB (Phosphotyrosine Binding) and a SH2 (Src Homology 2) domain. Previous studies have shown that SHC proteins are phosphorylated on Y239/Y240 and Y313 (Y317 in humans) by tyrosine kinases such as the EGF and IL3 receptors. We have investigated which of the SHC tyrosine residues are targeted by the VEGFR3/ FLT4 kinase and the role of the SHC PTB and SH2 domains in this process. Our results show that Y239/ Y240 and Y313 are simultaneously phosphorylated by the kinase, creating GRB2 binding sites. Mutation of SHC PTB, but not SH2, domain interferes with the SHC phosphorylation by VEGFR3/FLT4. Soft agar assay experiments revealed that the VEGFR3/FLT4 transforming capacity is increased by the mutation of Y239/Y240 to phenylalanines in SHC, suggesting that these two residues mediate an inhibitory signal for cell growth. Mutation of the two phosphorylation sites increases this effect, suggesting that they have a synergistic role.

Cell Line↗
PubMed1999

The PTB Domain: The Name Doesn't Say It All.

The phosphotyrosine-binding domain is a recently described protein-protein interaction domain which, despite its name, is involved in both phosphotyrosine-dependent and -independent interactions. Proteins with this domain are involved in diverse cellular functions, ranging from receptor signaling to protein targeting.

Journal Article↗
PubMed1999

The function of PTB domain proteins.

Phosphotyrosine binding (PTB) domains have been identified in a large number of proteins. In proteins like Shc and IRS-1, the PTB domain binds in a phosphotyrosine-dependent fashion to peptides that form a b turn. In these proteins, PTB domains play an important role in signal transduction by growth factor receptors. However, in several other proteins, the PTB domains have been found to participate in phosphotyrosine-independent interactions. The X11 family of proteins contains a PTB domain that binds peptides in a phosphotyrosine-independent fashion. The homologue of X11 in C. elegans is the lin-10 gene, a gene crucial for receptor targeting to the basolateral surface of body wall epithelia. The X11/Lin-10 proteins are found in a complex with two other proteins, Lin-2 and Lin-7, which have also been implicated in basolateral targeting in worm epithelia. This protein complex is also likely to be important in the targeting of cell surface proteins in mammalian neurons and epithelia. The ability of the PTB domain to bind peptides in a phosphotyrosine-dependent and -independent fashion allows this domain to be involved in diverse cellular functions.

Animals↗
PubMed1999

Asterriquinones produced by Aspergillus candidus inhibit binding of the Grb-2 adapter to phosphorylated EGF receptor tyrosine kinase.

Five new asterriquinone analogs (2-4, 6, 7), together with previously identified neoasterriquinone (1) and isoasterriquinone (5), were isolated from a fermentation broth of the fungus Aspergillus candidus and purified by HSCCC (high speed counter current chromatography) followed by HPLC. The structures were determined by 1D and 2D NMR and MS/MS techniques. All seven showed inhibitory activity against the binding of a recombinant protein containing the SH2 protein domain of Grb-2 to the tyrosine phosphorylated form of the EGF receptor tyrosine kinase. Some of these asterriquinones exhibited specific inhibition of Grb-2 binding compared to Grb-7 and PLC-gamma.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Insulin receptor substrate 2 and Shc play different roles in insulin-like growth factor I signaling.

The major substrates for the type I insulin-like growth factor (IGF-I) receptor are Shc and insulin receptor substrate (IRS) proteins. In the current study, we report that IGF-I induces a sustained tyrosine phosphorylation of Shc and its association with Grb2 in SH-SY5Y human neuroblastoma cells. The time course of Shc tyrosine phosphorylation parallels the time course of IGF-I-stimulated activation of extracellular signal-regulated kinase (ERK). Transfection of SH-SY5Y cells with a p52 Shc mutant decreases Shc tyrosine phosphorylation and Shc-Grb2 association. This results in the inhibition of IGF-I-mediated ERK tyrosine phosphorylation and neurite outgrowth. In contrast, IGF-I induces a transient tyrosine phosphorylation of IRS-2 and an association of IRS-2 with Grb2. The time course of IRS-2 tyrosine phosphorylation and IRS-2-Grb2 and IRS-2-p85 association closely resembles the time course of IGF-I-mediated membrane ruffling. Treating cells with the phosphatidylinositol 3'-kinase inhibitors wortmannin and LY294002 blocks IGF-I-induced membrane ruffling. The ERK kinase inhibitor PD98059, as well as transfection with the p52 Shc mutant, has no effect on IGF-I-mediated membrane ruffling. Immunolocalization studies show IRS-2 and Grb2, but not Shc, concentrated at the tip of the extending growth cone where membrane ruffling is most active. Collectively, these results suggest that the association of Shc with Grb2 is essential for IGF-I-mediated neurite outgrowth, whereas the IRS-2-Grb2-phosphatidylinositol 3'-kinase complex may regulate growth cone extension and membrane ruffling.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Interaction of cytosolic adaptor proteins with neuronal apolipoprotein E receptors and the amyloid precursor protein.

Apolipoprotein E, alpha2-macroglobulin, and amyloid precursor protein (APP) are involved in the development of Alzheimer's disease. All three proteins are ligands for the low density lipoprotein (LDL) receptor-related protein (LRP), an abundant neuronal surface receptor that has also been genetically linked to Alzheimer's disease. The cytoplasmic tails of LRP and other members of the LDL receptor gene family contain NPxY motifs that are required for receptor endocytosis. To investigate whether these receptors may have functions that go beyond ligand internalization, e.g. possible roles in cellular signaling, we searched for proteins that might interact with the cytoplasmic tails of the receptors. A family of adaptor proteins containing protein interaction domains that can interact with NPxY motifs has previously been described. Using yeast 2-hybrid and protein coprecipitation approaches in vitro, we show that the neuronal adaptor proteins FE65 and mammalian Disabled bind to the cytoplasmic tails of LRP, LDL receptor, and APP, where they can potentially serve as molecular scaffolds for the assembly of cytosolic multiprotein complexes. FE65 contains two distinct protein interaction domains that interact with LRP and APP, respectively, raising the possibility that LRP can modulate the intracellular trafficking of APP. Tyrosine-phosphorylated mammalian Disabled can recruit nonreceptor tyrosine kinases, such as src and abl, to the cytoplasmic tails of the receptors to which it binds, suggesting a molecular pathway by which receptor/ligand interaction on the cell surface could generate an intracellular signal.

Amino Acid Sequence↗
PubMed1998

Identification of an evolutionarily conserved heterotrimeric protein complex involved in protein targeting.

In Caenorhabditis elegans, lin-2, lin-7, and lin-10 genetically interact to control the trafficking of the Let-23 growth factor receptor to the basolateral surface of body epithelia. The human homologue of the lin-10 gene has recently been identified as a member of the X11 gene family. The X11 proteins contain one phosphotyrosine binding (PTB) and two PSD-95.Dlg.ZO-1 (PDZ) domains as well as an extended amino terminus. We have previously shown that the PTB domain of X11alpha (also known as Mint1) can bind to the amyloid precursor protein (APP) in a phosphotyrosine-independent fashion and can markedly inhibit the processing of APP to the amyloid beta (Abeta) peptide. Here, we report that X11alpha directly binds to the mammalian homologue of Lin-2 (mLin-2), also known as CASK. This binding is mediated by direct interaction between the Calmodulin Kinase II (CKII)-like domain of mLin-2 and the amino terminus of X11alpha. Furthermore, we can detect direct interactions between mLin-2 and mammalian Lin-7 (mLin-7). In mouse brain, we have identified a heterotrimeric complex that contains mLin-2, mLin-7, and X11alpha and that is likely important for the localization of proteins in polarized cells. This complex may play an important role in the trafficking and processing of APP in neurons.

Adaptor Proteins, Signal Transducing↗
PubMed1998

The X11alpha protein slows cellular amyloid precursor protein processing and reduces Abeta40 and Abeta42 secretion.

Constitutive amyloid precursor protein (APP) metabolism results in the generation of soluble APP (APPs) and Abeta peptides, including Abeta40 and Abeta42-the major component of amyloid plaques in Alzheimer's disease brain. The phosphotyrosine binding (PTB) domain of X11 binds to a peptide containing a YENPTY motif found in the carboxyl terminus of APP. We have cloned the full-length X11 gene now referred to as X11alpha. Coexpression of X11alpha with APP results in comparatively greater levels of cellular APP and less APPs, Abeta40, and Abeta42 recovered in conditioned medium of transiently transfected HEK 293 cells. These effects are impaired by a single missense mutation of either APP (Y682G within the YENPTY motif) or X11alpha (F608V within the PTB domain), which diminishes their interaction, thus demonstrating specificity. The inhibitory effect of X11alpha on Abeta40 and Abeta42 secretion is amplified by coexpression with the Swedish mutation of APP (K595N/M596L), which promotes its amyloidogenic processing. Pulse-chase analysis demonstrates that X11alpha prolongs the half-life of APP from approximately 2 h to approximately 4 h. The effects of X11alpha on cellular APP and APPs recovery were confirmed in a 293 cell line stably transfected with APP. The specific binding of the PTB domain of X11alpha to the YENPTY motif-containing peptide of APP appears to slow cellular APP processing and thus reduces recovery of its soluble fragments APPs, Abeta40, and Abeta42 in conditioned medium of transfected HEK 293 cells. X11alpha may be involved in APP trafficking and metabolism in neurons and thus may be implicated in amyloidogenesis in normal aging and Alzheimer's disease brain.

Alzheimer Disease↗
PubMed1998

Functional analysis of the Numb phosphotyrosine-binding domain using site-directed mutagenesis.

The Numb protein is involved in cell fate determination during Drosophila neural development. Numb has a protein domain homologous to the phosphotyrosine-binding domain (PTB) in the adaptor protein Shc. In Shc, this domain interacts with specific phosphotyrosine containing motifs on receptor tyrosine kinases and other signaling molecules. Residues N-terminal to the phosphotyrosine are also crucial for phosphopeptide binding to the Shc PTB domain. Several amino acid residues in Shc have been implicated by site-directed mutagenesis to be critical for Shc binding to receptor tyrosine kinases. We have generated homologous mutations in Numb to test whether, in vivo, these changes affect Numb function during Drosophila sensory organ development. Two independent amino acid changes that interfere with Shc binding to phosphotyrosine residues do not affect Numb activity in vivo. In contrast, a mutation shown to abrogate the ability of the Shc PTB domain to bind residues upstream of the phosphotyrosine virtually eliminates Numb function. Similar results were observed in vitro by examining the binding of the Numb PTB domain to proteins from Schneider S2 cells. Our data confirm the importance of the PTB domain for Numb function but strongly suggest that the Numb PTB domain is not involved in phosphotyrosine-dependent interactions.

Amino Acid Sequence↗