scholarly journals PDZ interactions regulate rapid turnover of the scaffolding protein EBP50 in microvilli

2012 ◽  
Vol 198 (2) ◽  
pp. 195-203 ◽  
Author(s):  
Damien Garbett ◽  
Anthony Bretscher
Keyword(s):  
Cell Extract ◽  
Scaffolding Protein ◽  
Scaffolding Proteins ◽  
Pdz Domains ◽  
Kinase C ◽  
Zonula Occludens ◽  
Discs Large ◽  
Specific Proteins

Scaffolding proteins containing PDZ (postsynaptic density 95/discs large/zonula occludens-1) domains are believed to provide relatively stable linkages between components of macromolecular complexes and in some cases to bridge to the actin cytoskeleton. The microvillar scaffolding protein EBP50 (ERM-binding phosphoprotein of 50 kD), consisting of two PDZ domains and an ezrin-binding site, retains specific proteins in microvilli and is necessary for microvillar biogenesis. Our analysis of the dynamics of microvillar proteins in vivo indicated that ezrin and microvillar membrane proteins had dynamics consistent with actin treadmilling and microvillar lifetimes. However, EBP50 was highly dynamic, turning over within seconds. EBP50 turnover was reduced by mutations that inactivate its PDZ domains and was enhanced by protein kinase C phosphorylation. Using a novel in vitro photoactivation fluorescence assay, the EBP50–ezrin interaction was shown to have a slow off-rate that was dramatically enhanced in a PDZ-regulated manner by addition of cell extract to near in vivo levels. Thus, the linking of relatively stable microvillar components can be mediated by surprisingly dynamic EBP50, a finding that may have important ramifications for other scaffolding proteins.

scholarly journals Regulation of GABAergic synapse formation and plasticity by GSK3β-dependent phosphorylation of gephyrin

2010 ◽  
Vol 108 (1) ◽  
pp. 379-384 ◽  
Author(s):  
Shiva K. Tyagarajan ◽  
Himanish Ghosh ◽  
Gonzalo E. Yévenes ◽  
Irina Nikonenko ◽  
Claire Ebeling ◽  
...  
Keyword(s):  
Synaptic Plasticity ◽  
Hippocampal Neurons ◽  
Glycogen Synthase ◽  
Phosphorylation Site ◽  
Scaffolding Protein ◽  
Scaffolding Proteins ◽  
Gabaergic Synapse ◽  
Gabaergic Synapses

Postsynaptic scaffolding proteins ensure efficient neurotransmission by anchoring receptors and signaling molecules in synapse-specific subcellular domains. In turn, posttranslational modifications of scaffolding proteins contribute to synaptic plasticity by remodeling the postsynaptic apparatus. Though these mechanisms are operant in glutamatergic synapses, little is known about regulation of GABAergic synapses, which mediate inhibitory transmission in the CNS. Here, we focused on gephyrin, the main scaffolding protein of GABAergic synapses. We identify a unique phosphorylation site in gephyrin, Ser270, targeted by glycogen synthase kinase 3β (GSK3β) to modulate GABAergic transmission. Abolishing Ser270 phosphorylation increased the density of gephyrin clusters and the frequency of miniature GABAergic postsynaptic currents in cultured hippocampal neurons. Enhanced, phosphorylation-dependent gephyrin clustering was also induced in vitro and in vivo with lithium chloride. Lithium is a GSK3β inhibitor used therapeutically as mood-stabilizing drug, which underscores the relevance of this posttranslational modification for synaptic plasticity. Conversely, we show that gephyrin availability for postsynaptic clustering is limited by Ca2+-dependent gephyrin cleavage by the cysteine protease calpain-1. Together, these findings identify gephyrin as synaptogenic molecule regulating GABAergic synaptic plasticity, likely contributing to the therapeutic action of lithium.

scholarly journals A Regulated Complex of the Scaffolding Proteins PDZK1 and EBP50 with Ezrin Contribute to Microvillar Organization

2010 ◽  
Vol 21 (9) ◽  
pp. 1519-1529 ◽  
Author(s):  
David P. LaLonde ◽  
Damien Garbett ◽  
Anthony Bretscher
Keyword(s):  
Epithelial Cells ◽  
Complex Formation ◽  
Ternary Complex ◽  
Apical Membrane ◽  
Pdz Domain ◽  
Scaffolding Proteins ◽  
Pdz Domains ◽  
Zona Occludens

PDZK1 and ezrin, radixin, moesin binding phosphoprotein 50 kDa (EBP50) are postsynaptic density 95/disc-large/zona occludens (PDZ)-domain–containing scaffolding proteins found in the apical microvilli of polarized epithelial cells. Binary interactions have been shown between the tail of PDZK1 and the PDZ domains of EBP50, as well as between EBP50 and the membrane–cytoskeletal linking protein ezrin. Here, we show that these molecules form a regulated ternary complex in vitro and in vivo. Complex formation is cooperative because ezrin positively influences the PDZK1/EBP50 interaction. Moreover, the interaction of PDZK1 with EBP50 is enhanced by the occupancy of EBP50's adjacent PDZ domain. The complex is further regulated by location, because PDZK1 shuttles from the nucleus in low confluence cells to microvilli in high confluence cells, and this regulates the formation of the PDZK1/EBP50/ezrin complex in vivo. Knockdown of EBP50 decreases the presence of microvilli, a phenotype that can be rescued by EBP50 re-expression or expression of a PDZK1 chimera that is directly targeted to ezrin. Thus, when appropriately located, PDZK1 can provide a function necessary for microvilli formation normally provided by EBP50. By entering into the ternary complex, PDZK1 can both enhance the scaffolding at the apical membrane as well as augment EBP50's role in microvilli formation.

scholarly journals The function and dynamics of the apical scaffolding protein E3KARP are regulated by cell-cycle phosphorylation

2015 ◽  
Vol 26 (20) ◽  
pp. 3615-3627 ◽  
Author(s):  
Cécile Sauvanet ◽  
Damien Garbett ◽  
Anthony Bretscher
Keyword(s):  
Pdz Domain ◽  
Duplication Event ◽  
Scaffolding Protein ◽  
Gene Duplication Event ◽  
Pdz Domains ◽  
Tail Region ◽  
Dynamics And Function ◽  
And Function

We examine the dynamics and function of the apical scaffolding protein E3KARP/NHERF2, which consists of two PDZ domains and a tail containing an ezrin-binding domain. The exchange rate of E3KARP is greatly enhanced during mitosis due to phosphorylation at Ser-303 in its tail region. Whereas E3KARP can substitute for the function of the closely related scaffolding protein EBP50/NHERF1 in the formation of interphase microvilli, E3KARP S303D cannot. Moreover, the S303D mutation enhances the in vivo dynamics of the E3KARP tail alone, whereas in vitro the interaction of E3KARP with active ezrin is unaffected by S303D, implicating another factor regulating dynamics in vivo. A-Raf is found to be required for S303 phosphorylation in mitotic cells. Regulation of the dynamics of EBP50 is known to be dependent on its tail region but modulated by PDZ domain occupancy, which is not the case for E3KARP. Of interest, in both cases, the mechanisms regulating dynamics involve the tails, which are the most diverged region of the paralogues and probably evolved independently after a gene duplication event that occurred early in vertebrate evolution.

scholarly journals Domain-swapped Dimerization of the Second PDZ Domain of ZO2 May Provide a Structural Basis for the Polymerization of Claudins

2007 ◽  
Vol 282 (49) ◽  
pp. 35988-35999 ◽  
Author(s):  
Jiawen Wu ◽  
Yinshan Yang ◽  
Jiahai Zhang ◽  
Peng Ji ◽  
Wenjing Du ◽  
...  
Keyword(s):  
Solution Structure ◽  
Pdz Domain ◽  
Three Dimensional ◽  
Structural Basis ◽  
Pdz Domains ◽  
Zonula Occludens ◽  
Associated Proteins ◽  
High Conservation

Zonula occludens proteins (ZOs), including ZO1/2/3, are tight junction-associated proteins. Each of them contains three PDZ domains. It has been demonstrated that ZO1 can form either homodimers or heterodimers with ZO2 or ZO3 through the second PDZ domain. However, the underlying structural basis is not well understood. In this study, the solution structure of the second PDZ domain of ZO2 (ZO2-PDZ2) was determined using NMR spectroscopy. The results revealed a novel dimerization mode for PDZ domains via three-dimensional domain swapping, which can be generalized to homodimers of ZO1-PDZ2 or ZO3-PDZ2 and heterodimers of ZO1-PDZ2/ZO2-PDZ2 or ZO1-PDZ2/ZO3-PDZ2 due to high conservation between PDZ2 domains in ZO proteins. Furthermore, GST pulldown experiments and immunoprecipitation studies demonstrated that interactions between ZO1-PDZ2 and ZO2-PDZ2 and their self-associations indeed exist both in vitro and in vivo. Chemical cross-linking and dynamic laser light scattering experiments revealed that both ZO1-PDZ2 and ZO2-PDZ2 can form oligomers in solution. This PDZ domain-mediated oligomerization of ZOs may provide a structural basis for the polymerization of claudins, namely the formation of tight junctions.

PKCβII modulates translation independently from mTOR and through RACK1

2008 ◽  
Vol 415 (1) ◽  
pp. 77-85 ◽  
Author(s):  
Stefano Grosso ◽  
Viviana Volta ◽  
Leonardo A. Sala ◽  
Marina Vietri ◽  
Pier Carlo Marchisio ◽  
...  
Keyword(s):  
Translational Control ◽  
Small Interfering Rna ◽  
Mammalian Target Of Rapamycin ◽  
Ribosomal Subunit ◽  
Scaffolding Protein ◽  
Kinase C ◽  
Interfering Rna ◽  
Stimulation Of

RACK1 (receptor for activated C kinase 1) is an abundant scaffolding protein, which binds active PKCβII (protein kinase C βII) increasing its activity in vitro. RACK1 has also been described as a component of the small ribosomal subunit, in proximity to the mRNA exit channel. In the present study we tested the hypothesis that PKCβII plays a specific role in translational control and verified whether it may associate with the ribosomal machinery. We find that specific inhibition of PKCβI/II reduces translation as well as global PKC inhibition, but without affecting phosphorylation of mTOR (mammalian target of rapamycin) targets. These results suggest that PKCβII acts as a specific PKC isoform affecting translation in an mTOR-independent fashion, possibly close to the ribosomal machinery. Using far-Western analysis, we found that PKCβII binds ribosomes in vitro. Co-immunoprecipitation studies indicate that a small but reproducible pool of PKCβII is associated with membranes containing ribosomes, suggesting that in vivo PKCβII may also physically interact with the ribosomal machinery. Polysomal profiles show that stimulation of PKC results in an increased polysomes/80S ratio, associated with a shift of PKCβII to the heavier part of the gradient. A RACK1-derived peptide that inhibits the binding of active PKCβII to RACK1 reduces the polysomes/80S ratio and methionine incorporation, suggesting that binding of PKCβII to RACK1 is important for PKC-mediated translational control. Finally, down-regulation of RACK1 by siRNA (small interfering RNA) impairs the PKC-mediated increase of translation. Taken together the results of the present study show that PKCβII can act as a specific PKC isoform regulating translation, in an mTOR-independent fashion, possibly close to the ribosomal machinery.

scholarly journals ϕX174 Procapsid Assembly: Effects of an Inhibitory External Scaffolding Protein and Resistant Coat Proteins In Vitro

2016 ◽  
Vol 91 (1) ◽  
Author(s):  
James E. Cherwa ◽  
Joshua Tyson ◽  
Gregory J. Bedwell ◽  
Dewey Brooke ◽  
Ashton G. Edwards ◽  
...  
Keyword(s):  
Coat Protein ◽  
Experimental Evolution ◽  
Viral Fitness ◽  
Scaffolding Protein ◽  
Coat Proteins ◽  
Scaffolding Proteins ◽  
Biochemical Methods ◽  
Trade Offs

ABSTRACT During ϕX174 morphogenesis, 240 copies of the external scaffolding protein D organize 12 pentameric assembly intermediates into procapsids, a reaction reconstituted in vitro. In previous studies, ϕX174 strains resistant to exogenously expressed dominant lethal D genes were experimentally evolved. Resistance was achieved by the stepwise acquisition of coat protein mutations. Once resistance was established, a stimulatory D protein mutation that greatly increased strain fitness arose. In this study, in vitro biophysical and biochemical methods were utilized to elucidate the mechanistic details and evolutionary trade-offs created by the resistance mutations. The kinetics of procapsid formation was analyzed in vitro using wild-type, inhibitory, and experimentally evolved coat and scaffolding proteins. Our data suggest that viral fitness is correlated with in vitro assembly kinetics and demonstrate that in vivo experimental evolution can be analyzed within an in vitro biophysical context. IMPORTANCE Experimental evolution is an extremely valuable tool. Comparisons between ancestral and evolved genotypes suggest hypotheses regarding adaptive mechanisms. However, it is not always possible to rigorously test these hypotheses in vivo. We applied in vitro biophysical and biochemical methods to elucidate the mechanistic details that allowed an experimentally evolved virus to become resistant to an antiviral protein and then evolve a productive use for that protein. Moreover, our results indicate that the respective roles of scaffolding and coat proteins may have been redistributed during the evolution of a two-scaffolding-protein system. In one-scaffolding-protein virus assembly systems, coat proteins promiscuously interact to form heterogeneous aberrant structures in the absence of scaffolding proteins. Thus, the scaffolding protein controls fidelity. During ϕX174 assembly, the external scaffolding protein acts like a coat protein, self-associating into large aberrant spherical structures in the absence of coat protein, whereas the coat protein appears to control fidelity.

scholarly journals HDAC inhibition induces expression of scaffolding proteins critical for tumor progression in pediatric glioma: focus on EBP50 and IRSp53

2019 ◽  
Vol 22 (4) ◽  
pp. 550-562 ◽  
Author(s):  
Caroline Capdevielle ◽  
Angélique Desplat ◽  
Justine Charpentier ◽  
Francis Sagliocco ◽  
Pierre Thiebaud ◽  
...  
Keyword(s):  
Cell Lines ◽  
Protein Localization ◽  
Biological Effects ◽  
Therapeutic Option ◽  
Hdac Inhibitors ◽  
Glioma Cells ◽  
Scaffolding Protein ◽  
Scaffolding Proteins

Abstract Background Diffuse midline glioma (DMG) is a pediatric malignancy with poor prognosis. Most children die less than one year after diagnosis. Recently, mutations in histone H3 have been identified and are believed to be oncogenic drivers. Targeting this epigenetic abnormality using histone deacetylase (HDAC) inhibitors such as panobinostat (PS) is therefore a novel therapeutic option currently evaluated in clinical trials. Methods BH3 profiling revealed engagement in an irreversible apoptotic process of glioma cells exposed to PS confirmed by annexin-V/propidium iodide staining. Using proteomic analysis of 3 DMG cell lines, we identified 2 proteins deregulated after PS treatment. We investigated biological effects of their downregulation by silencing RNA but also combinatory effects with PS treatment in vitro and in vivo using a chick embryo DMG model. Electron microscopy was used to validate protein localization. Results Scaffolding proteins EBP50 and IRSp53 were upregulated by PS treatment. Reduction of these proteins in DMG cell lines leads to blockade of proliferation and migration, invasion, and an increase of apoptosis. EBP50 was found to be expressed in cytoplasm and nucleus in DMG cells, confirming known oncogenic locations of the protein. Treatment of glioma cells with PS together with genetic or chemical inhibition of EBP50 leads to more effective reduction of cell growth in vitro and in vivo. Conclusion Our data reveal a specific relation between HDAC inhibitors and scaffolding protein deregulation which might have a potential for therapeutic intervention for cancer treatment.

scholarly journals ZO-3, a Novel Member of the MAGUK Protein Family Found at the Tight Junction, Interacts with ZO-1 and Occludin

1998 ◽  
Vol 141 (1) ◽  
pp. 199-208 ◽  
Author(s):  
Julie Haskins ◽  
Lijie Gu ◽  
Erika S. Wittchen ◽  
Jennifer Hibbard ◽  
Bruce R. Stevenson
Keyword(s):  
Amino Acid ◽  
Tight Junction ◽  
Molecular Mass ◽  
Mdck Cells ◽  
Cdna Libraries ◽  
Pdz Domains ◽  
Large Tumor ◽  
Junction Protein ◽  
Discs Large

A 130-kD protein that coimmunoprecipitates with the tight junction protein ZO-1 was bulk purified from Madin-Darby canine kidney (MDCK) cells and subjected to partial endopeptidase digestion and amino acid sequencing. A resulting 19–amino acid sequence provided the basis for screening canine cDNA libraries. Five overlapping clones contained a single open reading frame of 2,694 bp coding for a protein of 898 amino acids with a predicted molecular mass of 98,414 daltons. Sequence analysis showed that this protein contains three PSD-95/SAP90, discs-large, ZO-1 (PDZ) domains, a src homology (SH3) domain, and a region similar to guanylate kinase, making it homologous to ZO-1, ZO-2, the discs large tumor suppressor gene product of Drosophila, and other members of the MAGUK family of proteins. Like ZO-1 and ZO-2, the novel protein contains a COOH-terminal acidic domain and a basic region between the first and second PDZ domains. Unlike ZO-1 and ZO-2, this protein displays a proline-rich region between PDZ2 and PDZ3 and apparently contains no alternatively spliced domain. MDCK cells stably transfected with an epitope-tagged construct expressed the exogenous polypeptide at an apparent molecular mass of ∼130 kD. Moreover, this protein colocalized with ZO-1 at tight junctions by immunofluorescence and immunoelectron microscopy. In vitro affinity analyses demonstrated that recombinant 130-kD protein directly interacts with ZO-1 and the cytoplasmic domain of occludin, but not with ZO-2. We propose that this protein be named ZO-3.

scholarly journals Tobacco Mosaic Virus Movement Protein Functions as a Structural Microtubule-Associated Protein

2006 ◽  
Vol 80 (17) ◽  
pp. 8329-8344 ◽  
Author(s):  
Jamie Ashby ◽  
Emmanuel Boutant ◽  
Mark Seemanpillai ◽  
Adrian Sambade ◽  
Christophe Ritzenthaler ◽  
...  
Keyword(s):  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Movement Protein ◽  
Cell Extract ◽  
Transport Processes ◽  
In Vitro Assays ◽  
Protein Functions ◽  
Intercellular Spread

ABSTRACT The cell-to-cell spread of Tobacco mosaic virus infection depends on virus-encoded movement protein (MP), which is believed to form a ribonucleoprotein complex with viral RNA (vRNA) and to participate in the intercellular spread of infectious particles through plasmodesmata. Previous studies in our laboratory have provided evidence that the vRNA movement process is correlated with the ability of the MP to interact with microtubules, although the exact role of this interaction during infection is not known. Here, we have used a variety of in vivo and in vitro assays to determine that the MP functions as a genuine microtubule-associated protein that binds microtubules directly and modulates microtubule stability. We demonstrate that, unlike MP in whole-cell extract, microtubule-associated MP is not ubiquitinated, which strongly argues against the hypothesis that microtubules target the MP for degradation. In addition, we found that MP interferes with kinesin motor activity in vitro, suggesting that microtubule-associated MP may interfere with kinesin-driven transport processes during infection.

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