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1.
Biochem Biophys Res Commun ; 378(3): 360-5, 2009 Jan 16.
Article in English | MEDLINE | ID: mdl-19013433

ABSTRACT

In this work, we describe how the Erbin PDZ domain interacts with Smad3, a transductor of the Transforming Growth Factor-beta (TGFbeta) pathway, via its MH2 domain. This interaction was described as important for TGFbeta signaling as it could potentially repress the transcriptional activity of the growth factor. In order to clarify our preliminary experimental observations pointing this interaction, we built a 3D model of the Erbin PDZ/Smad3 MH2 complex and checked its stability using molecular dynamics simulations. This model pointed out charged residues in Smad3 and Erbin which could be important for the interaction. By introducing point mutations of these residues within the proposed binding domains, we experimentally confirmed that arginine 279, glutamic acid 246 in Smad3 and glutamic acid 1321 in Erbin are important for the binding. These data suggest a possible novel interface of binding in the Erbin PDZ domain and reveal an unconventional mode of interaction for a PDZ domain and its ligand.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , PDZ Domains , Smad3 Protein/metabolism , Adaptor Proteins, Signal Transducing/genetics , Amino Acid Sequence , Arginine/genetics , Arginine/metabolism , Cell Line , Glutamic Acid/genetics , Glutamic Acid/metabolism , Humans , Protein Binding , Protein Interaction Mapping , Smad3 Protein/genetics , Transforming Growth Factor beta/metabolism
2.
Hum Mol Genet ; 17(22): 3552-65, 2008 Nov 15.
Article in English | MEDLINE | ID: mdl-18716323

ABSTRACT

Genetic studies have highlighted the key role of Scrib in the development of Metazoans. Deficiency in Scrib impairs many aspects of cell polarity and cell movement although the mechanisms involved remain unclear. In mammals, Scrib belongs to a protein complex containing betaPIX, an exchange factor for Rac/Cdc42, and GIT1, a GTPase activating protein for ARF6 implicated in receptor recycling and exocytosis. Here we show that the Scrib complex associates with PAK, a serine-threonine kinase family crucial for cell migration. PAK colocalizes with members of the Scrib complex at the leading edge of heregulin-treated T47D breast cancer cells. We demonstrate that the Scrib complex is required for epithelial cells and primary mouse embryonic fibroblasts to efficiently respond to chemoattractant cues. In Scrib-deficient cells, the pool of cortical PAK is decreased, thereby precluding its proper activation by Rac. Loss of Scrib also impairs the polarized distribution of active Rac at the leading edge and compromises the regulated activation of the GTPase in T47D cells and mouse embryonic fibroblasts. These data underscore the role of Scrib in cell migration and show the strong impact of Scrib in the function of PAK and Rac, two key molecules implicated in this process.


Subject(s)
Cell Movement/physiology , Intracellular Signaling Peptides and Proteins/metabolism , Membrane Proteins/metabolism , Tumor Suppressor Proteins/metabolism , p21-Activated Kinases/metabolism , Animals , Cell Line, Tumor , Cells, Cultured , Chemotaxis , Fibroblasts , Guanine Nucleotide Exchange Factors/metabolism , Humans , Mice , Microscopy, Fluorescence , Neuregulin-1/metabolism , RNA, Small Interfering , Rho Guanine Nucleotide Exchange Factors , cdc42 GTP-Binding Protein/metabolism , rac GTP-Binding Proteins/metabolism
3.
J Biol Chem ; 282(20): 15197-207, 2007 May 18.
Article in English | MEDLINE | ID: mdl-17355968

ABSTRACT

NOD2 plays an important role in the innate immunity of the intestinal tract. By sensing the muramyl dipeptide (MDP), a bacterial wall component, NOD2 triggers the NF-kappaB signaling pathway and promotes the release of proinflammatory cytokines such as interleukin-8. Mutations in Nod2 (1007FS, R702W, G908R) impinge on NOD2 functions and are associated with the pathogenesis of Crohn disease, a chronic inflammatory bowel disease. Although NOD2 is usually described as a cytosolic receptor for MDP, the protein is also localized at the plasma membrane, and the 1007FS mutation delocalizes NOD2 to the cytoplasm (Barnich, N., Aguirre, J. E., Reinecker, H. C., Xavier, R., and Podolsky, D. K. (2005) J. Cell Biol. 170, 21-26; McDonald, C., Chen, F. F., Ollendorff, V., Ogura, Y., Marchetto, S., Lecine, P., Borg, J. P., and Nunez, G. (2005) J. Biol. Chem. 280, 40301-40309). In this study, we demonstrate that membrane-bound versions of NOD2 and Crohn disease-associated mutants R702W and G908R are capable of responding to MDP and activating the NF-kappaB pathway from this location. In contrast, the 1007FS mutant remains unable to respond to MDP from the plasma membrane. We also show that NOD2 promotes the membrane recruitment of RICK, a serine-threonine kinase involved in NF-kappaB activation downstream of NOD2. Furthermore, the artificial attachment of RICK at the plasma membrane provokes a constitutive and strong activation of the NF-kappaB pathway and secretion of interleukin-8 showing that optimal RICK activity depends upon its subcellular localization. Finally, we show that endogenous RICK localizes at the plasma membrane in the THP1 cell line. Thus, our data suggest that NOD2 is responsible for the membrane recruitment of RICK to induce a regulated NF-kappaB signaling and production of proinflammatory cytokines.


Subject(s)
Acetylmuramyl-Alanyl-Isoglutamine/metabolism , Crohn Disease/metabolism , Membrane Proteins/metabolism , Nod2 Signaling Adaptor Protein/metabolism , Receptor-Interacting Protein Serine-Threonine Kinase 2/metabolism , Signal Transduction , Animals , Caco-2 Cells , Cell Membrane/metabolism , Cell Membrane/pathology , Crohn Disease/genetics , Crohn Disease/pathology , Dogs , Humans , Inflammation/genetics , Inflammation/metabolism , Inflammation/pathology , Interleukin-8/metabolism , Membrane Proteins/genetics , Mutation, Missense , NF-kappa B/metabolism , Nod2 Signaling Adaptor Protein/genetics , Protein Transport/genetics , Signal Transduction/genetics
4.
Curr Biol ; 14(11): 987-95, 2004 Jun 08.
Article in English | MEDLINE | ID: mdl-15182672

ABSTRACT

Drosophila Scribble is implicated in the development of normal synapse structure and epithelial tissues, but it remains unclear how it plays a role and which process it controls. The mammalian homolog of Scribble, hScrib, has a primary structure and subcellular localization similar to that of its fly homolog, but its function remains unknown. Here we have used tandem mass spectrometry to identify major components of the hScrib network. We show that it includes betaPIX (also called Cool-1), a guanine nucleotide exchange factor (GEF), and its partner GIT1 (also called p95-APP1), a GTPase activating protein (GAP). betaPIX directly binds to the hScrib PDZ domains, and the hScrib/betaPIX complex is efficiently recovered in epithelial and neuronal cells and tissues. In cerebellar granule cell cultures, hScrib and betaPIX are both partially localized at neuronal presynaptic compartments. Furthermore, we show that hScrib is required to anchor betaPIX at the cell cortex and that dominant-negative betaPIX or hScrib proteins can each inhibit Ca2+-dependent exocytosis in neuroendocrine PC12 cells, demonstrating a functional relationship between these proteins. These data reveal the existence of a tight hScrib/betaPIX interaction and suggest that this complex potentially plays a role in neuronal transmission.


Subject(s)
Cell Cycle Proteins/metabolism , GTPase-Activating Proteins/metabolism , Guanine Nucleotide Exchange Factors/metabolism , Membrane Proteins/metabolism , Phosphoproteins/metabolism , Adaptor Proteins, Signal Transducing , Cell Cycle Proteins/pharmacology , Cell Membrane/metabolism , Cells, Cultured , Chromatography, High Pressure Liquid , DNA, Complementary/genetics , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Exocytosis/drug effects , Guanine Nucleotide Exchange Factors/pharmacology , Humans , Mass Spectrometry , Membrane Proteins/pharmacology , Precipitin Tests , Presynaptic Terminals/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Rho Guanine Nucleotide Exchange Factors , Tumor Suppressor Proteins , Two-Hybrid System Techniques
5.
BMC Cell Biol ; 5: 20, 2004 May 16.
Article in English | MEDLINE | ID: mdl-15144564

ABSTRACT

BACKGROUND: The Transforming Growth Factor-beta (TGF-beta) regulates myriad cellular events by signaling through members of the Smad family signal transducers. As a key signal transducer of TGF-beta, Smad3 exhibits the property of receptor-activated transcriptional modulator and also the novel ability of regulating the proteasomal degradation of two Smad3 interacting proteins, SnoN and HEF1. It has been shown that Smad3 recruits two types of Ub E3 ligases, Smurf2 and the Anaphase Promoting Complex (APC), to mediate SnoN ubiquitination, thereby enhancing SnoN degradation. The molecular mechanisms underlying Smad3-regulated HEF1 degradation are not well understood. Furthermore, it is not clear how Smad3 recruits the APC complex. RESULTS: We detected physical interaction between Smad3 and an APC component APC10, as well as the interaction between HEF1 and CDH1, which is the substrate-interacting component within APC. Detailed domain mapping studies revealed distinct subdomains within the MH2 domain of Smad3 for binding to APC10 and HEF1 and suggests the formation of a complex of these four proteins (Smad3, HEF1, APC10 and CDH1). In addition, the protein levels of HEF1 are subjected to the regulation of overexpressed APC10 and CDH1. CONCLUSIONS: Our data suggests that Smad3 may recruit the APC complex via a direct interaction with the APC subunit APC10 to regulate the ubiquitination and degradation of its interactor HEF1, which is recognized as an ubiquitination substrate by the CDH1 subunit of the APC complex.


Subject(s)
DNA-Binding Proteins/metabolism , Phosphoproteins/metabolism , Proteasome Endopeptidase Complex/metabolism , Trans-Activators/metabolism , Ubiquitin-Protein Ligase Complexes/metabolism , Activin Receptors, Type I/metabolism , Adaptor Proteins, Signal Transducing , Anaphase-Promoting Complex-Cyclosome , Binding Sites/genetics , Cell Line , DNA-Binding Proteins/genetics , Humans , Mutation , Phosphoproteins/genetics , Plasmids/genetics , Protein Binding , Protein Serine-Threonine Kinases , Protein Subunits/genetics , Protein Subunits/metabolism , Receptor, Transforming Growth Factor-beta Type I , Receptors, Transforming Growth Factor beta/metabolism , Smad3 Protein , Trans-Activators/genetics , Transfection , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta/metabolism , Ubiquitin-Protein Ligase Complexes/genetics
6.
Sci STKE ; 2003(179): RE7, 2003 Apr 22.
Article in English | MEDLINE | ID: mdl-12709532

ABSTRACT

Protein-protein interactions are key elements in building functional protein complexes. Among the plethora of domains identified during the last 10 years, PDZ domains are one of the most commonly found protein-protein interaction domains in organisms from bacteria to humans. Although they may be the sole protein interaction domain within a cytoplasmic protein, they are most often found in combination with other protein interaction domains (for instance, SH3, PTB, WW) participating in complexes that facilitate signaling or determine the localization of receptors. Diversity of PDZ-containing protein function is provided by the large number of PDZ proteins that Mother Nature has distributed in the genome and implicates this protein family in the wiring of a huge number of molecules in molecular networks from the plasma membrane to the nucleus. Although at first sight their binding specificity appeared rather monotonous, involving only binding to the carboxyl-terminus of various proteins, it is now recognized that PDZ domains interact with greater versatility through PDZ-PDZ domain interaction; they bind to internal peptide sequences and even to lipids. Furthermore, PDZ domain-mediated interactions can sometimes be modulated in a dynamic way through target phosphorylation. In this review, we attempt to describe the structural basis of PDZ domain recognition and to give some functional insights into their role in the scaffolding of protein complexes implicated in normal and pathological biological processes.


Subject(s)
Proteins/classification , Proteins/physiology , Animals , Humans , Protein Binding/physiology , Protein Structure, Tertiary/physiology , Proteins/chemistry , Repetitive Sequences, Amino Acid/physiology
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