Identification of mNET1 as a Candidate Ligand for the First PDZ Domain of MAGI-1

doi:10.1006/bbrc.2001.4880

Biochemical and Biophysical Research Communications

Volume 283, Issue 4, 18 May 2001, Pages 969-975

https://doi.org/10.1006/bbrc.2001.4880 Get rights and content

Abstract

This paper reports the identification of a Rho family nucleotide exchange factor termed mNET1 as a candidate-interacting partner for the first PDZ domain of MAGI-1, a membrane-associated guanylate kinase with inverted arrangement of protein–protein interacting modules. mNET1 was identified in a yeast two-hybrid screen and has a consensus tripeptide for PDZ domain binding at its extreme carboxy-terminus. In addition to this sequence, a cluster of basic residues located near the carboxy terminus is essential for the binding. The interaction of the first PDZ domain of MAGI-1with mNET1 was documented using a variety of biochemical methods.

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At epithelial and endothelial junctions, MAGI-1 interacts with Ig-like adhesion molecules such as JAM-4 [254] and ESAM [255], and with β-catenin [239], whereas MAGI-3 interacts with the receptor tyrosine protein phosphatase RPTP-β [245]. Through these interactions, and also by recruiting the GEFs PDZGEF-1/RapGEP and mNET1 [256,257], MAGI-family proteins help stabilize epithelial and endothelial cell-cell junctions. In fact, MAGI protein cleavage by caspases is an important step for cell-cell detachment in apoptosis [258].
Tight and adherens junctions are specialized sites of cell-cell interaction in epithelia and endothelia, and are involved in barrier, adhesion, and signaling functions. These functions are orchestrated by a highly organized meshwork of macromolecules in the membrane and cytoplasmic compartments. In this review, we discuss the structural organization and functions of the major cytoplasmic scaffolding and adaptor proteins of vertebrate apical junctions (ZO proteins, afadin, PLEKHA7, cingulin, paracingulin, polarity complex proteins, and a few others), focusing on their interactions with cytoskeletal and signaling proteins. Furthermore, we discuss recent results highlighting how mechanical tension, protein-protein interactions and post-translational modifications regulate the conformation and function of scaffolding proteins, and how spontaneous phase separation into biomolecular condensates contributes to apical junction assembly. Using a sequence-based algorithm, a large fraction of cytoplasmic proteins of apical junctions are predicted to be phase separating proteins (PSPs), suggesting that formation of biomolecular condensates is a general mechanism to organize cell-cell contacts by clustering proteins.
The C. elegans MAGI-1 protein is a novel component of cell junctions that is required for junctional compartmentalization
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Cell junctions are essential to maintain polarity and tissue integrity. Epithelial cell junctions are composed of distinct sub-compartments that together ensure the strong adhesion between neighboring cells. In Caenorhabditis elegans epithelia, the apical junction (CeAJ) forms a single electron-dense structure, but at the molecular level it is composed of two sub-compartments that function redundantly and localize independently as two distinct but adjacent circumferential rings on the lateral plasma membrane. While investigating the role of the multi PDZ-domain containing protein MAGI-1 during C. elegans epidermal morphogenesis, we found that MAGI-1 localizes apical to both the Cadherin/Catenin (CCC) and AJM-1/DLG-1 (DAC) containing sub-domains. Removal of MAGI-1 function causes a loss of junctional compartmentalization along the lateral membrane and reduces the overall robustness of cell-cell adhesion mediated by either type of cell junctions. Our results suggest that MAGI-1 functions as an “organizer” that ensures the correct segregation of different cell adhesion complexes into distinct domains along the lateral plasma membrane. Thus, the formation of stable junctions requires the proper distribution of the CCC and DAC adhesion protein complexes along the lateral plasma membrane.
Interaction of the RhoA exchange factor Net1 with discs large homolog 1 protects it from proteasome-mediated degradation and potentiates Net 1 activity
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Citation Excerpt :
These data also indicate that endogenous Net1 is an unstable protein in actively growing MCF7 cells. The PDZ domain-containing scaffolding protein Magi-1b was previously identified as a Net1-interacting protein in yeast two-hybrid assays (24). To determine whether PDZ domain-containing proteins other than Dlg1 could stabilize Net1, we examined whether Magi-1b associated with Net1A in breast cancer cells and if co-expression of Magi-1b extended the half-life of Net1A in these cells.
Net1 is a nuclear Rho guanine nucleotide exchange factor that is specific for the RhoA subfamily of small G proteins. Truncated forms of Net1 are transforming in NIH3T3 cells, and this activity requires cytoplasmic localization of Net1 as well as the presence of a COOH-terminal PDZ binding site. We have previously shown that Net1 interacts with PDZ domain-containing proteins within the Discs Large (Dlg) family and relocalizes them to the nucleus. In the present work, we demonstrate that Net1 binds directly to the first two PDZ domains of Dlg1 and that both PDZ domains are required for maximal interaction in cells. Furthermore, we show that Net1 is an unstable protein in MCF7 breast epithelial cells and that interaction with Dlg1 significantly enhances Net1 stability. Stabilization by Dlg1 significantly increases the ability of Net1 to stimulate RhoA activation in cells. The stability of endogenous Net1 is strongly enhanced by cell-cell contact, and this correlates with a dramatic increase in the interaction between Net1 and Dlg1. Importantly, disruption of E-cadherin-mediated cell contacts, either by depletion of external calcium or by treatment with transforming growth factor β, leads to a rapid loss of the interaction between Net1 and Dlg1 and a subsequent increase in the ubiquitylation of Net1. These results indicate that Net1 requires interaction with PDZ domain proteins, such as Dlg1, to protect it from proteasome-mediated degradation and to maximally stimulate RhoA and that this interaction is regulated by cell-cell contact.
Structural organization of the tight junctions
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Citation Excerpt :
Type I PDZ domains (like the domains of MAGI-1) contain a critical histidine residue in the second α-helix and bind proteins ending with the consensus motif Ser/Thr-X-ΦCOOH (X, any residue; Φ, hydrophobic residue). At variance, type II PDZ domains (like the domains of ZO-1) contain a hydrophobic residue in the second α-helix and bind proteins ending with the consensus motif Phe/Tyr-X-ΦCOOH[95]. Thus, JAM-A, which ends in Phe-Leu-Val, binds ZO-1 (precisely, the type II PDZ-2 and PDZ-3 domains) [39,40], but not MAGI-1.
Tight junctions are the most apical organelle of the apical junctional complex and are primarily involved in the regulation of paracellular permeability and membrane polarity. Extensive research in the past two decades has identified not only the individual molecules of the tight junctions but also their mutual interactions, which are the focus of the present review article. While a complete map of the interactions among the tight junction molecules is probably far from being complete, the available evidence already allows outlining the general molecular architecture of the tight junctions. Here, with the aim of gaining deeper mechanistic understanding of tight junction assembly, regulation and function, we have subdivided the known molecular interactions into four major clusters that are centered on cell surface, polarity, cytoskeletal and signaling molecules.
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This reiterated use of PDZ-binding motifs in Rho-GEFs might have originated as a way to facilitate the evolution of new cellular functions within cells, by creating novel connections between existing proteins and pathways. Several interactions between Rho-GEFs and PDZ-domain proteins have been recently reported (Table 1, Figure 1) [7–13]. In the examples studied, these interactions have a role in targeting the Rho-GEF to a specific location in the cell, restricting the nucleotide exchange activity and subsequent GTPase activation both spatially and temporally.
The activation of Rho GTPases is mediated by guanine-nucleotide exchange factors (GEFs), which catalyze the exchange of GDP for GTP. Rho-GEFs are a very diverse family, with >70 members in humans. Bioinformatics analysis of the human Rho-GEFs shows that ∼40% contain a putative PDZ-binding motif at the C-terminus. PDZ domains are protein–protein interaction domains that act as scaffolds to concentrate signaling molecules at specialized regions in the cell. We propose that the interaction between Rho-GEFs and PDZ-domain proteins is a general mechanism that controls Rho-GEF targeting and activation, helping to restrict and concentrate the exchange activity to appropriate subcellular destinations. Here, we summarize recent data that highlight the importance of these interactions in Rho-GEF regulation.
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¹: Current address: Department of Molecular Genetics, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, TX 75390. Fax: (214) 648-8804. E-mail: [email protected].

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Regular ArticleIdentification of mNET1 as a Candidate Ligand for the First PDZ Domain of MAGI-1

Abstract

Molecular architecture of tight junctions

Annu. Rev. Physiol.

MAGI-1 interacts with beta-catenin and is associated with cell-cell adhesion structures

Biochem. Biophys. Res. Commun.

Evidence for regulation of the PTEN tumor suppressor by a membrane-localized multi-PDZ domain containing scaffold protein MAGI-2

Proc. Natl. Acad. Sci. USA

Atrophin-1, the DRPLA gene product, interacts with two families of WW domain-containing proteins

Mol. Cell. Neurosci.

Interaction of the tumor suppressor PTEN/MMAC with a PDZ domain of MAGI3, a novel membrane-associated guanylate kinase

J. Biol. Chem.

MAGI-1, a membrane-associated guanylate kinase with a unique arrangement of protein-protein interaction domains

J. Biol. Chem.

Activation of RhoA and SAPK/JNK signaling pathways by the RhoA-specific exchange factor mNET1

EMBO J.

Cellular Interactions in Development: A Practical Approach

CASK: A novel dlg/PSD95 homolog with an N-terminal calmodulin-dependent protein kinase domain identified byinteraction with neurexins

J. Neurosci.

Ras–Raf interaction: Two-hybrid analysis

Methods Enzymol.

Eukaryotic proteins expressed in Escherichia coli: An improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase

Anal. Biochem.

Mammalian Ras interacts directly with the serine/threonine kinase Raf

Cell

Recognition of unique carboxyl-terminal motifs by distinct PDZ domains

Science

Crystal structures of a complexed and peptide-free membrane protein-binding domain: Molecular basis of peptide recognition by PDZ

Cell

Identification of a novel, putative Rho-specific GDP/GTP exchange factor and a RhoA-binding protein: Control of neuronal morphology

J. Cell Biol.

Analysis of RhoA-binding proteins reveals an interaction domain conserved in heterotrimeric G protein beta subunits and the yeast response regulator protein Skn7

J. Biol. Chem.

Regular Article
Identification of mNET1 as a Candidate Ligand for the First PDZ Domain of MAGI-1