dra (down regulated in adenema) binds in vitro to the second PDZ domain of NHERF and E3KARP

ABSTRACT Oncoproteins from DNA tumor viruses associate with critical cellular proteins to regulate cell proliferation, survival, and differentiation.Human papillomavirus (HPV) E6 oncoproteins have been previously shown to associate with a cellular HECT domain ubiquitin ligase termed E6AP (UBE3A). Here we show that the E6-E6AP complex associates with and targets the degradation of the protein tyrosine phosphatase PTPN3 (PTPH1) in vitro and in living cells. PTPN3 is a membrane-associated tyrosine phosphatase with FERM, PDZ, and PTP domains previously implicated in regulating tyrosine phosphorylation of growth factor receptors and p97 VCP (valosin-containing protein, termed Cdc48 in Saccharomyces cerevisiae) and is mutated in a subset of colon cancers. Degradation of PTPN3 by E6 requires E6AP, the proteasome, and an interaction between the carboxy terminus of E6 and the PDZ domain of PTPN3. In transduced keratinocytes, E6 confers reduced growth factor requirements, a function that requires the PDZ ligand of E6 and that can in part be replicated by inhibiting the expression of PTPN3. This report demonstrates the potential of E6 to regulate phosphotyrosine metabolism through the targeted degradation of a tyrosine phosphatase.

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Role of synectin in lymphatic development in zebrafish and frogs

Blood ◽

10.1182/blood-2009-11-254557 ◽

2010 ◽

Vol 116 (17) ◽

pp. 3356-3366 ◽

Cited By ~ 30

Author(s):

Karlien Hermans ◽

Filip Claes ◽

Wouter Vandevelde ◽

Wei Zheng ◽

Ilse Geudens ◽

...

Keyword(s):

Endothelial Cells ◽

Thoracic Duct ◽

Pdz Domain ◽

Lymphatic Endothelial Cells ◽

Cardinal Vein ◽

Morpholino Knockdown ◽

Lymphatic Development ◽

Factor C ◽

Endothelial Growth Factor

Abstract The molecular basis of lymphangiogenesis remains incompletely characterized. Here, we document a novel role for the PDZ domain-containing scaffold protein synectin in lymphangiogenesis using genetic studies in zebrafish and tadpoles. In zebrafish, the thoracic duct arises from parachordal lymphangioblast cells, which in turn derive from secondary lymphangiogenic sprouts from the posterior cardinal vein. Morpholino knockdown of synectin in zebrafish impaired formation of the thoracic duct, due to selective defects in lymphangiogenic but not angiogenic sprouting. Synectin genetically interacted with Vegfr3 and neuropilin-2a in regulating lymphangiogenesis. Silencing of synectin in tadpoles caused lymphatic defects due to an underdevelopment and impaired migration of Prox-1+ lymphatic endothelial cells. Molecular analysis further revealed that synectin regulated Sox18-induced expression of Prox-1 and vascular endothelial growth factor C–induced migration of lymphatic endothelial cells in vitro. These findings reveal a novel role for synectin in lymphatic development.

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Several adaptor proteins promote intracellular localisation of the transporter MRP4/ABCC4 in platelets and haematopoietic cells

Thrombosis and Haemostasis ◽

10.1160/th16-01-0045 ◽

2017 ◽

Vol 117 (01) ◽

pp. 105-115 ◽

Cited By ~ 6

Author(s):

Yvonne Schaletzki ◽

Marie-Luise Kromrey ◽

Susanne Bröderdorf ◽

Elke Hammer ◽

Markus Grube ◽

...

Keyword(s):

Plasma Membrane ◽

Protein Interactions ◽

Pdz Domain ◽

Adaptor Protein ◽

Adaptor Proteins ◽

Apical Plasma Membrane ◽

Dense Granules ◽

Haematopoietic Cells ◽

And Function

SummaryThe multidrug resistance protein 4 (MRP4/ABCC4) has been identified as an important transporter for signalling molecules including cyclic nucleotides and several lipid mediators in platelets and may thus represent a novel target to interfere with platelet function. Besides its localisation in the plasma membrane, MRP4 has been also detected in the membrane of dense granules in resting platelets. In polarised cells it is localised at the basolateral or apical plasma membrane. To date, the mechanism of MRP4 trafficking has not been elucidated; protein interactions may regulate both the localisation and function of this transporter. We approached this issue by searching for interacting proteins by in vitro binding assays, followed by immunoblotting and mass spectrometry, and by visualising their co-localisation in platelets and haematopoietic cells. We identified the PDZ domain containing scaffold proteins ezrin-binding protein 50 (EBP50/NHERF1), postsynaptic density protein 95 (PSD95), and sorting nexin 27 (SNX27), but also the adaptor protein complex 3 subunit β3A (AP3B1) and the heat shock protein HSP90 as putative interaction partners of MRP4. The knockdown of SNX27, PSD95, and AP3B1 by siRNA in megakaryoblastic leuk aemia cells led to a redistribution of MRP4 from intracellular structures to the plasma membrane. Inhibition of HSP90 led to a diminished expression and retention of MRP4 in the endoplasmic reticulum. These results indicate that MRP4 localisation and function are regulated by multiple protein interactions. Changes in the adaptor proteins can hence lead to altered localisation and function of the transporter.Supplementary Material to this article is available at www.thrombosis-online.com.

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The Serine Protease HhoA from Synechocystis sp. Strain PCC 6803: Substrate Specificity and Formation of a Hexameric Complex Are Regulated by the PDZ Domain

Journal of Bacteriology ◽

10.1128/jb.00883-07 ◽

2007 ◽

Vol 189 (18) ◽

pp. 6611-6618 ◽

Cited By ~ 14

Author(s):

Pitter F. Huesgen ◽

Philipp Scholz ◽

Iwona Adamska

Keyword(s):

Membrane Proteins ◽

Substrate Specificity ◽

Proteolytic Activity ◽

Protease Activity ◽

Elevated Temperatures ◽

Critical Role ◽

Pdz Domain ◽

General Role ◽

Pcc 6803

ABSTRACT Enzymes of the ATP-independent Deg serine endopeptidase family are very flexible with regard to their substrate specificity. Some family members cleave only one substrate, while others act as general proteases on unfolded substrates. The proteolytic activity of Deg proteases is regulated by PDZ protein interaction domains. Here we characterized the HhoA protease from Synechocystis sp. strain PCC 6803 in vitro using several recombinant protein constructs. The proteolytic activity of HhoA was found to increase with temperature and basic pH and was stimulated by the addition of Mg2+ or Ca2+. We found that the single PDZ domain of HhoA played a critical role in regulating protease activity and in the assembly of a hexameric complex. Deletion of the PDZ domain strongly reduced proteolysis of a sterically challenging resorufin-labeled casein substrate, but unlabeled β-casein was still degraded. Reconstitution of the purified HhoA with total membrane proteins isolated from Synechocystis sp. wild-type strain PCC 6803 and a ΔhhoA mutant resulted in specific degradation of selected proteins at elevated temperatures. We concluded that a single PDZ domain of HhoA plays a critical role in defining the protease activity and oligomerization state, combining the functions that are attributed to two PDZ domains in the homologous DegP protease from Escherichia coli. Based on this first enzymatic study of a Deg protease from cyanobacteria, we propose a general role for HhoA in the quality control of extracytoplasmic proteins, including membrane proteins, in Synechocystis sp. strain PCC 6803.

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Folding PDZ2 domain using the Molecular Transfer Model

10.1101/037283 ◽

2016 ◽

Cited By ~ 1

Author(s):

Zhenxing Liu ◽

Govardhan Reddy ◽

Dave Thirumalai

Keyword(s):

Pdz Domain ◽

High Energy ◽

Coarse Grained ◽

Radius Of Gyration ◽

Transfer Model ◽

Self Organized ◽

Energy Profiles ◽

X Ray Scattering ◽

Molecular Transfer

A major challenge in molecular simulations is to describe denaturant-dependent folding of proteins order to make direct comparisons within vitroexperiments. We use the molecular transfer model (MTM), which is currently the only method that accomplishes this goal albeit phenomenologically, to quantitatively describe urea-dependent folding of PDZ domain, which plays a significant role in molecular recognition and signaling. Experiments show that urea-dependent unfolding rates of the PDZ2 domain exhibit a downward curvature at high urea concentrations ([C]s), which has been interpreted by invoking the presence of a sparsely populated high energy intermediate. Simulations using the MTM and a coarse-grained Self-Organized Polymer (SOP) representation of PDZ2 are used to show that the intermediate (IEQ), which has some native-like character, is present in equilibrium both in the presence and absence of urea. The free energy profiles as a function of the structural overlap order parameter show that there are two barriers separating the folded and unfolded states. Structures of the transition state ensembles, (TSE1 separating the unfolded and (IEQ) andTSE2 separatingIEQand the native state), determined using thePfoldmethod, show thatTSE1 is greatly expanded whileTSE2 is compact and native-like. Folding trajectories reveal that PDZ2 folds by parallel routes. In one pathway folding occurs exclusively throughI1, which resemblesIEQ. In a fraction of trajectories, constituting the second pathway, folding occurs through a combination ofI1and a kinetic intermediate. We establish that the radius of gyration (RUg) of the unfolded state is more compact (by ∼9%) under native conditions. Theory and simulations show that the decrease inRUgoccurs on the time scale on the order of utmost ∼20μs. The modest decrease inRUgand the rapid collapse suggest that high spatial and temporal resolution, currently beyond the scope of most small angle X-ray scattering experiments, are needed to detect compaction in finite-sized proteins. The present work further establishes that MTM is efficacious in producing nearly quantitative predictions for folding of proteins under conditions used to carry out experiments.

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Postsynaptic density protein 95 (PSD-95) is transported by KIF5 to dendritic regions

Molecular Brain ◽

10.1186/s13041-019-0520-x ◽

2019 ◽

Vol 12 (1) ◽

Cited By ~ 3

Author(s):

Ki-Seo Yoo ◽

Kina Lee ◽

Jun-Young Oh ◽

Hyoeun Lee ◽

Hyungju Park ◽

...

Keyword(s):

Pdz Domain ◽

Postsynaptic Density ◽

Scaffolding Protein ◽

Synaptic Localization ◽

Conventional Kinesin ◽

Dose Dependent ◽

Postsynaptic Density Protein

AbstractPostsynaptic density protein 95 (PSD-95) is a pivotal postsynaptic scaffolding protein in excitatory neurons. Although the transport and regulation of PSD-95 in synaptic regions is well understood, dendritic transport of PSD-95 before synaptic localization still remains to be clarified. To evaluate the role of KIF5, conventional kinesin, in the dendritic transport of PSD-95 protein, we expressed a transport defective form of KIF5A (ΔMD) that does not contain the N-terminal motor domain. Expression of ΔMD significantly decreased PSD-95 level in the dendrites. Consistently, KIF5 was associated with PSD-95 in in vitro and in vivo assays. This interaction was mediated by the C-terminal tail regions of KIF5A and the third PDZ domain of PSD-95. Additionally, the ADPDZ3 (the association domain of NMDA receptor and PDZ3 domain) expression significantly reduced the levels of PSD-95, glutamate receptor 1 (GluA1) in dendrites. The association between PSD-95 and KIF5A was dose-dependent on Staufen protein, suggesting that the Staufen plays a role as a regulatory role in the association. Taken together, our data suggest a new mechanism for dendritic transport of the AMPA receptor-PSD-95.

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

Molecular Biology of the Cell ◽

10.1091/mbc.e10-01-0008 ◽

2010 ◽

Vol 21 (9) ◽

pp. 1519-1529 ◽

Cited By ~ 43

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.

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Early Metazoan Origin and Multiple Losses of a Novel Clade of RIM Presynaptic Calcium Channel Scaffolding Protein Homologs

Genome Biology and Evolution ◽

10.1093/gbe/evaa097 ◽

2020 ◽

Vol 12 (8) ◽

pp. 1217-1239 ◽

Cited By ~ 1

Author(s):

Thomas Piekut ◽

Yuen Yan Wong ◽

Sarah E Walker ◽

Carolyn L Smith ◽

Julia Gauberg ◽

...

Keyword(s):

Lymnaea Stagnalis ◽

Pdz Domain ◽

Scaffolding Protein ◽

Tissue Type ◽

Ligand Specificity ◽

Synaptic Localization ◽

Trichoplax Adhaerens ◽

Stem Lineage ◽

Presynaptic Calcium

Abstract The precise localization of CaV2 voltage-gated calcium channels at the synapse active zone requires various interacting proteins, of which, Rab3-interacting molecule or RIM is considered particularly important. In vertebrates, RIM interacts with CaV2 channels in vitro via a PDZ domain that binds to the extreme C-termini of the channels at acidic ligand motifs of D/E-D/E/H-WC-COOH, and knockout of RIM in vertebrates and invertebrates disrupts CaV2 channel synaptic localization and synapse function. Here, we describe a previously uncharacterized clade of RIM proteins bearing domain architectures homologous to those of known RIM homologs, but with some notable differences including key amino acids associated with PDZ domain ligand specificity. This novel RIM emerged near the stem lineage of metazoans and underwent extensive losses, but is retained in select animals including the early-diverging placozoan Trichoplax adhaerens, and molluscs. RNA expression and localization studies in Trichoplax and the mollusc snail Lymnaea stagnalis indicate differential regional/tissue type expression, but overlapping expression in single isolated neurons from Lymnaea. Ctenophores, the most early-diverging animals with synapses, are unique among animals with nervous systems in that they lack the canonical RIM, bearing only the newly identified homolog. Through phylogenetic analysis, we find that CaV2 channel D/E-D/E/H-WC-COOH like PDZ ligand motifs were present in the common ancestor of cnidarians and bilaterians, and delineate some deeply conserved C-terminal structures that distinguish CaV1 from CaV2 channels, and CaV1/CaV2 from CaV3 channels.

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Phosphorylation of Connexin36 near the C-terminus switches binding affinities for PDZ-domain and 14–3–3 proteins in vitro

Scientific Reports ◽

10.1038/s41598-020-75375-0 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Stephan Tetenborg ◽

Helen Y. Wang ◽

Lena Nemitz ◽

Anne Depping ◽

Alexsandra B. Espejo ◽

...

Keyword(s):

Gap Junctions ◽

Binding Affinity ◽

Protein Microarray ◽

Pdz Domain ◽

Phosphorylation Site ◽

Binding Domain ◽

Dependent Manner ◽

C Terminus ◽

Activity Dependent

Abstract Connexin36 (Cx36) is the most abundant connexin in central nervous system neurons. It forms gap junction channels that act as electrical synapses. Similar to chemical synapses, Cx36-containing gap junctions undergo activity-dependent plasticity and complex regulation. Cx36 gap junctions represent multimolecular complexes and contain cytoskeletal, regulatory and scaffolding proteins, which regulate channel conductance, assembly and turnover. The amino acid sequence of mammalian Cx36 harbors a phosphorylation site for the Ca2+/calmodulin-dependent kinase II at serine 315. This regulatory site is homologous to the serine 298 in perch Cx35 and in close vicinity to a PDZ binding domain at the very C-terminal end of the protein. We hypothesized that this phosphorylation site may serve as a molecular switch, influencing the affinity of the PDZ binding domain for its binding partners. Protein microarray and pulldown experiments revealed that this is indeed the case: phosphorylation of serine 298 decreased the binding affinity for MUPP1, a known scaffolding partner of connexin36, and increased the binding affinity for two different 14–3–3 proteins. Although we did not find the same effect in cell culture experiments, our data suggest that phosphorylation of serine 315/298 may serve to recruit different proteins to connexin36/35-containing gap junctions in an activity-dependent manner.

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Inward rectifier K+ channel Kir2.3 is localized at the postsynaptic membrane of excitatory synapses

AJP Cell Physiology ◽

10.1152/ajpcell.00615.2001 ◽

2002 ◽

Vol 282 (6) ◽

pp. C1396-C1403 ◽

Cited By ~ 32

Author(s):

Atsushi Inanobe ◽

Akikazu Fujita ◽

Minoru Ito ◽

Hitonobu Tomoike ◽

Kiyoshi Inageda ◽

...

Keyword(s):

Membrane Potential ◽

Granule Cells ◽

Pdz Domain ◽

Postsynaptic Membrane ◽

Equilibrium Potential ◽

K Channel ◽

Resting Membrane Potential ◽

Kir Channel ◽

Anchoring Proteins

Classical inwardly rectifying K+ channels (Kir2.0) are responsible for maintaining the resting membrane potential near the K+ equilibrium potential in various cells, including neurons. Although Kir2.3 is known to be expressed abundantly in the forebrain, its precise localization has not been identified. Using an antibody specific to Kir2.3, we examined the subcellular localization of Kir2.3 in mouse brain. Kir2.3 immunoreactivity was detected in a granular pattern in restricted areas of the brain, including the olfactory bulb (OB). Immunoelectron microscopy of the OB revealed that Kir2.3 immunoreactivity was specifically clustered on the postsynaptic membrane of asymmetric synapses between granule cells and mitral/tufted cells. The immunoprecipitants for Kir2.3 obtained from brain contained PSD-95 and chapsyn-110, PDZ domain-containing anchoring proteins. In vitro binding assay further revealed that the COOH-terminal end of Kir2.3 is responsible for the association with these anchoring proteins. Therefore, the Kir channel may be involved in formation of the resting membrane potential of the spines and, thus, would affect the response of N-methyl-d-aspartic acid receptor channels at the excitatory postsynaptic membrane.

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