scholarly journals PICK1 is implicated in organelle motility in an Arp2/3 complex–independent manner

2015 ◽  
Vol 26 (7) ◽  
pp. 1308-1322 ◽  
Author(s):  
Yadaiah Madasu ◽  
Changsong Yang ◽  
Malgorzata Boczkowska ◽  
Kelley A. Bethoney ◽  
Adam Zwolak ◽  
...  
Keyword(s):  
Structural Model ◽  
Pdz Domain ◽  
Dependent Manner ◽  
Pdz Domains ◽  
Receptor Binding Site ◽  
Independent Manner ◽  
Bar Domain ◽  
Motility Factor ◽  
Organelle Motility ◽  
Golgi Network

PICK1 is a modular scaffold implicated in synaptic receptor trafficking. It features a PDZ domain, a BAR domain, and an acidic C-terminal tail (ACT). Analysis by small- angle x-ray scattering suggests a structural model that places the receptor-binding site of the PDZ domain and membrane-binding surfaces of the BAR and PDZ domains adjacent to each other on the concave side of the banana-shaped PICK1 dimer. In the model, the ACT of one subunit of the dimer interacts with the PDZ and BAR domains of the other subunit, possibly accounting for autoinhibition. Consistently, full-length PICK1 shows diffuse cytoplasmic localization, but it clusters on vesicle-like structures that colocalize with the trans-Golgi network marker TGN38 upon deletion of either the ACT or PDZ domain. This localization is driven by the BAR domain. Live-cell imaging further reveals that PICK1-associated vesicles undergo fast, nondirectional motility in an F-actin–dependent manner, but deleting the ACT dramatically reduces vesicle speed. Thus the ACT links PICK1-associated vesicles to a motility factor, likely myosin, but, contrary to previous reports, PICK1 neither binds nor inhibits Arp2/3 complex.

scholarly journals Small-molecule inhibitors of the PDZ domain of Dishevelled proteins interrupt Wnt signalling

2021 ◽  
Author(s):  
Nestor Kamdem ◽  
Yvette Roske ◽  
Dmytro Kovalskyy ◽  
Maxim O. Platonov ◽  
Oleksii Balinskyi ◽  
...  
Keyword(s):  
Small Molecule ◽  
Small Molecule Inhibitors ◽  
Pdz Domain ◽  
Wnt Signalling ◽  
Dependent Manner ◽  
Pdz Domains ◽  
X Ray Crystallography ◽  
Structure Activity ◽  
Dose Dependent ◽  
Anthranilic Acid Derivatives

Abstract. Dishevelled (Dvl) proteins are important regulators of the Wnt signalling pathway, interacting through their PDZ domains with the Wnt receptor Frizzled. Blocking the Dvl PDZ/Frizzled interaction represents a potential approach for cancer treatment, which stimulated the identification of small molecule inhibitors, among them the anti-inflammatory drug Sulindac and Ky-02327. Aiming to develop tighter binding compounds without side effects, we investigated structure-activity relationships of sulfonamides. X-ray crystallography showed high complementarity of anthranilic acid derivatives in the GLGF loop cavity and space for ligand growth towards the PDZ surface. Our best binding compound inhibits Wnt signalling in a dose-dependent manner as demonstrated by TOP-GFP assays (IC50 ~50 µM), and Western blotting of β-catenin levels. Real-time PCR showed reduction in the expression of Wnt-specific genes. Our compound interacted with Dvl-1 PDZ (Kd = 2.4 µM) stronger than Ky-02327 and may be developed into a lead compound interfering with the Wnt pathway.

scholarly journals The scaffolding protein EBP50 regulates microvillar assembly in a phosphorylation-dependent manner

2010 ◽  
Vol 191 (2) ◽  
pp. 397-413 ◽  
Author(s):  
Damien Garbett ◽  
David P. LaLonde ◽  
Anthony Bretscher
Keyword(s):  
Biochemical Analysis ◽  
Pdz Domain ◽  
Critical Factor ◽  
Mitotic Cell ◽  
Scaffolding Protein ◽  
Apical Surface ◽  
Dependent Manner ◽  
Pdz Domains ◽  
Pkc Activation

The mechanisms by which epithelial cells regulate the presence of microvilli on their apical surface are largely unknown. A potential regulator is EBP50/NHERF1 (ERM-binding phosphoprotein of 50 kD/Na+-H+ exchanger regulatory factor), a microvillar scaffolding protein with two PDZ domains followed by a C-terminal ezrin-binding domain. Using RNAi and expression of RNAi-resistant EBP50 mutants we systematically show that EBP50 is necessary for microvillar assembly and requires that EBP50 has both a functional first PDZ domain and an ezrin-binding site. Expression of mutants mimicking Cdc2 or PKC phosphorylation are nonfunctional in microvillar assembly. Biochemical analysis reveals that these mutants are defective in PDZ1 accessibility when PDZ2 is occupied, and can be rendered functional in vivo by additional mutation of PDZ2. EBP50 is not necessary for mitotic cell microvilli, and PKC activation causes a rearrangement of microvilli on cells due to phosphorylation-dependent loss of EBP50 function. Thus, EBP50 is a critical factor that regulates microvilli assembly and whose activity is regulated by signaling pathways and occupation of its PDZ2 domain.

scholarly journals The electrostatic allostery could be the trigger for the changes in dynamics for the PDZ domain of PICK1

2020 ◽  
Author(s):  
Amy O. Stevens ◽  
Yi He
Keyword(s):  
Surface Membrane ◽  
Pdz Domain ◽  
Binding Pocket ◽  
Pdz Domains ◽  
Interaction Domain ◽  
Bar Domain ◽  
Protein Protein Interaction ◽  
Loop Region ◽  
Key Factor ◽  
Dynamics Simulations

ABSTRACTThe PDZ domain is a highly abundant protein-protein interaction domain that exists in many signaling proteins, such as PICK1. Despite the highly conserved structure of the PDZ family, the PDZ family has an extremely low sequence identity, making each PDZ domain unique. PICK1 is the only protein in the human genome that is comprised of a PDZ domain and a BAR domain. PICK1 regulates surface membrane proteins and has been identified as an integral player in drug addiction. Like many PDZ-containing proteins, PICK1 is positively regulated by its PDZ domain and has thus drawn attention to be a potential drug target to curb the effects of substance abuse. The goal of this study is to use all-atom molecular dynamics simulations and the electrostatic analysis program, DelPhi, to better understand the unique interactions and dynamic changes in the PICK1 PDZ domain upon complex formation. Our results demonstrated that the PICK1 PDZ domain shares similar canonical PDZ-ligand hydrogen bonding networks and fluctuations of the carboxylate-binding loop to other PDZ domains. Furthermore, our results are unique to the PICK1 PDZ domain as we reveal that the binding of ligand opens up the binding pocket and, at the same time, reduces the fluctuations of both the central part of the binding pocket and the short loop region between the αA-helix and βC-strand. More importantly, the binding of ligand resulted in charge redistribution at the binding pocket region as well as the N- and C-termini of the PDZ domain that are not a part of the binding pocket. These results suggest that the electrostatic allostery resulted from ligand binding could be the key factor leading to the changes in dynamics which may be associated with the activation of PICK1. Based on these results, an effective drug to target PDZ domain must not only stably bind to the PICK1 PDZ domain but also prevent the electrostatic allostery of the PDZ domain.

scholarly journals Structural basis of the human Scribble-Vangl2 association in health and disease

2020 ◽  
Author(s):  
Jing Yuan How ◽  
Rebecca K. Stephens ◽  
Krystle Y.B. Lim ◽  
Patrick O. Humbert ◽  
Marc Kvansakul
Keyword(s):  
Cell Polarity ◽  
Neural Tube ◽  
Neural Tube Defect ◽  
Peptide Mapping ◽  
Pdz Domain ◽  
Structural Basis ◽  
Binding Motif ◽  
Dependent Manner ◽  
Loss Of Function ◽  
Pdz Domains

AbstractScribble is a critical cell polarity regulator that has been shown to work as either an oncogene or tumor suppressor in a context dependent manner, and also impacts cell migration, tissue architecture and immunity. Mutations in Scribble lead to neural tube defects in mice and humans, which has been attributed to a loss of interaction with the planar cell polarity regulator Vangl2. We show that the Scribble PDZ domains 1, 2 and 3 are able to interact with the C-terminal PDZ binding motif of Vangl2 and have now determined crystal structures of these Scribble PDZ domains bound to the Vangl2 peptide. Mapping of mammalian neural tube defect mutations reveal that mutations located distal to the canonical PDZ domain ligand binding groove can not only ablate binding to Vangl2 but also disrupt binding to multiple other signaling regulators. Our findings suggest that PDZ-associated neural tube defect mutations in Scribble may not simply act in a Vangl2 dependent manner but as broad-spectrum loss of function mutants by disrupting the global Scribble-mediated interaction network.

scholarly journals A novel adhesive complex at the base of intestinal microvilli

2021 ◽  
Author(s):  
Christian Hartmann ◽  
Eva-Maria Thüring ◽  
Birgitta E. Michels ◽  
Denise Pajonczyk ◽  
Sophia Leußink ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Apical Membrane ◽  
Intestinal Epithelial Cells ◽  
Pdz Domain ◽  
Basal Region ◽  
Dependent Manner ◽  
Intestinal Epithelial ◽  
Pdz Domains ◽  
Ternary Complex Formation ◽  
Adhesion Complex

AbstractIntestinal epithelial cells form dense arrays of microvilli at the apical membrane to enhance their functional capacity. Microvilli contain a protocadherin-based intermicrovillar adhesion complex localized at their tips which regulates microvillar length and packaging. Here, we identify a second adhesive complex in microvilli of intestinal epithelial cells. This complex is localized at the basal region of microvilli and consists of the adhesion molecule TMIGD1, the phosphoprotein EBP50 and the F-actin – plasma membrane cross-linking protein ezrin. Ternary complex formation requires unmasking of the EBP50 PDZ domains by ezrin binding and is strongly enhanced upon mutating Ser162 located in PDZ domain 2 of EBP50. Dephosphorylation of EBP50 at S162 is mediated by PP1α, a serine/threonine phosphatase localized at the microvillar base and involved in ezrin phosphocycling. Importantly, the binding of EBP50 to TMIGD1 enhances the dynamic turnover of EBP50 at microvilli in a Ser162 phosphorylation-dependent manner. We identify an adhesive complex at the microvillar base and propose a potential mechanism that regulates microvillar dynamics in enterocytes.

scholarly journals Sorting nexin 27 regulates basal and stimulated brush border trafficking of NHE3

2015 ◽  
Vol 26 (11) ◽  
pp. 2030-2043 ◽  
Author(s):  
Varsha Singh ◽  
Jianbo Yang ◽  
Boyoung Cha ◽  
Tiane-e Chen ◽  
Rafiquel Sarker ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Pdz Domain ◽  
Surface Expression ◽  
Dependent Manner ◽  
Intestinal Epithelial ◽  
Pdz Domains ◽  
Sorting Nexin ◽  
C Terminus ◽  
Cargo Proteins

Sorting nexin 27 (SNX27) contains a PDZ domain that is phylogenetically related to the PDZ domains of the NHERF proteins. Studies on nonepithelial cells have shown that this protein is located in endosomes, where it regulates trafficking of cargo proteins in a PDZ domain–dependent manner. However, the role of SNX27 in trafficking of cargo proteins in epithelial cells has not been adequately explored. Here we show that SNX27 directly interacts with NHE3 (C-terminus) primarily through the SNX27 PDZ domain. A combination of knockdown and reconstitution experiments with wild type and a PDZ domain mutant (GYGF → GAGA) of SNX27 demonstrate that the PDZ domain of SNX27 is required to maintain basal NHE3 activity and surface expression of NHE3 in polarized epithelial cells. Biotinylation-based recycling and degradation studies in intestinal epithelial cells show that SNX27 is required for the exocytosis (not endocytosis) of NHE3 from early endosome to plasma membrane. SNX27 is also required to regulate the retention of NHE3 on the plasma membrane. The findings of the present study extend our understanding of PDZ-mediated recycling of cargo proteins from endosome to plasma membrane in epithelial cells.

scholarly journals Structural basis of the human Scribble–Vangl2 association in health and disease

2021 ◽  
Vol 478 (7) ◽  
pp. 1321-1332
Author(s):  
Jing Yuan How ◽  
Rebecca K. Stephens ◽  
Krystle Y.B. Lim ◽  
Patrick O. Humbert ◽  
Marc Kvansakul
Keyword(s):  
Cell Polarity ◽  
Neural Tube ◽  
Neural Tube Defect ◽  
Peptide Mapping ◽  
Pdz Domain ◽  
Structural Basis ◽  
Binding Motif ◽  
Dependent Manner ◽  
Loss Of Function ◽  
Pdz Domains

Scribble is a critical cell polarity regulator that has been shown to work as either an oncogene or tumor suppressor in a context dependent manner, and also impacts cell migration, tissue architecture and immunity. Mutations in Scribble lead to neural tube defects in mice and humans, which has been attributed to a loss of interaction with the planar cell polarity regulator Vangl2. We show that the Scribble PDZ domains 1, 2 and 3 are able to interact with the C-terminal PDZ binding motif of Vangl2 and have now determined crystal structures of these Scribble PDZ domains bound to the Vangl2 peptide. Mapping of mammalian neural tube defect mutations reveal that mutations located distal to the canonical PDZ domain ligand binding groove can not only ablate binding to Vangl2 but also disrupt binding to multiple other signaling regulators. Our findings suggest that PDZ-associated neural tube defect mutations in Scribble may not simply act in a Vangl2 dependent manner but as broad-spectrum loss of function mutants by disrupting the global Scribble-mediated interaction network.

scholarly journals Small-molecule inhibitors of the PDZ domain of Dishevelled proteins interrupt Wnt signalling

2021 ◽  
Vol 2 (1) ◽  
pp. 355-374
Author(s):  
Nestor Kamdem ◽  
Yvette Roske ◽  
Dmytro Kovalskyy ◽  
Maxim O. Platonov ◽  
Oleksii Balinskyi ◽  
...  
Keyword(s):  
Small Molecule ◽  
Small Molecule Inhibitors ◽  
Pdz Domain ◽  
Wnt Signalling ◽  
Dependent Manner ◽  
Pdz Domains ◽  
X Ray Crystallography ◽  
Structure Activity ◽  
Dose Dependent ◽  
Anthranilic Acid Derivatives

Abstract. Dishevelled (Dvl) proteins are important regulators of the Wnt signalling pathway, interacting through their PDZ domains with the Wnt receptor Frizzled. Blocking the Dvl PDZ–Frizzled interaction represents a potential approach for cancer treatment, which stimulated the identification of small-molecule inhibitors, among them the anti-inflammatory drug Sulindac and Ky-02327. Aiming to develop tighter binding compounds without side effects, we investigated structure–activity relationships of sulfonamides. X-ray crystallography showed high complementarity of anthranilic acid derivatives in the GLGF loop cavity and space for ligand growth towards the PDZ surface. Our best binding compound inhibits Wnt signalling in a dose-dependent manner as demonstrated by TOP-GFP assays (IC50∼50 µM) and Western blotting of β-catenin levels. Real-time PCR showed reduction in the expression of Wnt-specific genes. Our compound interacted with Dvl-1 PDZ (KD=2.4 µM) stronger than Ky-02327 and may be developed into a lead compound interfering with the Wnt pathway.

scholarly journals Embryonal Carcinoma P19 Cells Produce Erythropoietin Constitutively But Express Lactate Dehydrogenase in an Oxygen-Dependent Manner

Blood ◽  
1998 ◽  
Vol 91 (4) ◽  
pp. 1185-1195 ◽  
Author(s):  
Taiho Kambe ◽  
Junko Tada ◽  
Mariko Chikuma ◽  
Seiji Masuda ◽  
Masaya Nagao ◽  
...  
Keyword(s):  
Lactate Dehydrogenase ◽  
Binding Site ◽  
Embryonal Carcinoma ◽  
Hypoxia Inducible Factor ◽  
Embryonic Stem ◽  
Gene Promoter ◽  
Dependent Manner ◽  
P19 Cells ◽  
Independent Manner ◽  
Hep3b Cells

Abstract Embryonic stem cells and embryonal carcinoma P19 cells produce erythropoietin (Epo) in an oxygen-independent manner, although lactate dehydrogenase A (LDHA) is hypoxia-inducible. To explore this paradox, we studied the operation of cis-acting sequences from these genes in P19 and Hep3B cells. The Epo gene promoter and 3′ enhancer from P19 cells conveyed hypoxia-inducible responses in Hep3B cells but not in P19 cells. Together with DNA sequencing and the normal transcription start site of P19 Epo gene, this excluded the possibility that the noninducibility of Epo gene in P19 cells was due to mutation in these sequences or unusual initiation of transcription. In contrast, reporter constructs containing LDHA enhancer and promoter were hypoxia inducible in P19 and Hep3B cells, and mutation of a hypoxia- inducible factor 1 (HIF-1) binding site abolished the hypoxic inducibility in both cells, indicating that HIF-1 activation operates normally in P19 cells. Neither forced expression of hepatocyte nuclear factor 4 in P19 cells nor deletion of its binding site from the Epo enhancer was effective in restoring Epo enhancer function. P19 cells may lack an unidentified regulator(s) required for interaction of the Epo enhancer with Epo and LDHA promoters.

scholarly journals Specific release of membrane-bound annexin II and cortical cytoskeletal elements by sequestration of membrane cholesterol.

1997 ◽  
Vol 8 (3) ◽  
pp. 533-545 ◽  
Author(s):  
T Harder ◽  
R Kellner ◽  
R G Parton ◽  
J Gruenberg
Keyword(s):  
Actin Cytoskeleton ◽  
Cytoskeletal Proteins ◽  
Annexin Ii ◽  
Dependent Manner ◽  
Cortical Actin ◽  
Independent Manner ◽  
Low Concentrations ◽  
Early Endosomes ◽  
Associated Proteins ◽  
Cytoskeletal Elements

Annexin II is an abundant protein which is present in the cytosol and on the cytoplasmic face of plasma membrane and early endosomes. It is generally believed that this association occurs via Ca(2+)-dependent binding to lipids, a mechanism typical for the annexin protein family. Although previous studies have shown that annexin II is involved in early endosome dynamics and organization, the precise biological role of the protein is unknown. In this study, we found that approximately 50% of the total cellular annexin was associated with membranes in a Ca(2+)-independent manner. This binding was extremely tight, since it resisted high salt and, to some extent, high pH treatments. We found, however, that membrane-associated annexin II could be quantitatively released by low concentrations of the cholesterol-sequestering agents filipin and digitonin. Both treatments released an identical and limited set of proteins but had no effects on other membrane-associated proteins. Among the released proteins, we identified, in addition to annexin II itself, the cortical cytoskeletal proteins alpha-actinin, ezrin and moesin, and membrane-associated actin. Our biochemical and immunological observations indicate that these proteins are part of a complex containing annexin II and that stability of the complex is sensitive to cholesterol sequestering agents. Since annexin II is tightly membrane-associated in a cholesterol-dependent manner, and since it seems to interact physically with elements of the cortical actin cytoskeleton, we propose that the protein serves as interface between membranes containing high amounts of cholesterol and the actin cytoskeleton.

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