Insights into the molecular evolution of the PDZ/LIM family and identification of a novel conserved protein motif.
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The PDZ and LIM domain-containing protein family is encoded by a diverse group of genes whose phylogeny has currently not been analyzed. In mammals, ten genes are found that encode both a PDZ- and one or several LIM-domains. These genes are: ALP, RIL, Elfin (CLP36), Mystique, Enigma (LMP-1), Enigma homologue (ENH), ZASP (Cypher, Oracle), LMO7 and the two LIM domain kinases (LIMK1 and LIMK2). As conventional alignment and phylogenetic procedures of full-length sequences fell short of elucidating the evolutionary history of these genes, we started to analyze the PDZ and LIM domain sequences themselves. Using information from most sequenced eukaryotic lineages, our phylogenetic analysis is based on full-length cDNA-, EST-derived- and genomic- PDZ and LIM domain sequences of over 25 species, ranging from yeast to humans. Plant and protozoan homologs were not found. Our phylogenetic analysis identifies a number of domain duplication and rearrangement events, and shows a single convergent event during evolution of the PDZ/LIM family. Further, we describe the separation of the ALP and Enigma subfamilies in lower vertebrates and identify a novel consensus motif, which we call 'ALP-like motif' (AM). This motif is highly-conserved between ALP subfamily proteins of diverse organisms. We used here a combinatorial approach to define the relation of the PDZ and LIM domain encoding genes and to reconstruct their phylogeny. This analysis allowed us to classify the PDZ/LIM family and to suggest a meaningful model for the molecular evolution of the diverse gene architectures found in this multi-domain family. (+info)
Syntenin: a novel PDZ domain-containing scaffolding protein associated with human melanoma metastasis.
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Syntenin is overexpressed in multiple human cancers and is newly recognized as a novel regulator in melanoma metastasis. It functions as a scaffolding protein, via its two PDZ domains interacting with multiple transmembrane and cytoplasmic partners to regulate many of the major signaling pathways involved in various cellular processes, such as cell surface receptor clustering, protein trafficking, cytoskeleton remodeling, and activation of transcription factor, and results in the increased abilities for tumor cell growth, adhesion, angiogenesis, invasion and metastasis. The present article attempts to review the structure and functions of syntenin by summarizing our current knowledge on the interacting partners and diverse signaling pathways related to syntenin, and highlight the importance of syntenin as a new potential therapeutic target for the aggressive human melanoma. (+info)
Regulation of c-Src by binding to the PDZ domain of AF-6.
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c-Src is a tightly regulated non-receptor tyrosine kinase. We describe the C-terminus of c-Src as a ligand for a PDZ (postsynaptic density 95, PSD-95; discs large, Dlg; zonula occludens-1, ZO-1) domain. The C-terminal residue Leu of c-Src is essential for binding to a PDZ domain. Mutation of this residue does not affect the intrinsic kinase activity in vitro, but interferes with c-Src regulation in cells. As a candidate PDZ protein, we analysed AF-6, a junctional adhesion protein. The AF-6 PDZ domain restricts the number of c-Src substrates, whereas knockdown of AF-6 has the opposite effect. Binding of c-Src to the AF-6 PDZ domain interferes with phosphorylation of c-Src at Tyr527 by the C-terminal kinase, and reduces c-Src autophosphorylation at Tyr416, resulting in a moderately activated c-Src kinase. Unphosphorylated Tyr527 allows binding of c-Src to AF-6. This can be overcome by overexpression of CSK or strong activation of c-Src. c-Src is recruited by AF-6 to cell-cell contact sites, suggesting that c-Src is regulated by a PDZ protein in special cellular locations. We identified a novel type of c-Src regulation by interaction with a PDZ protein. (+info)
Deletion of the PDZ motif of HPV16 E6 preventing immortalization and anchorage-independent growth in human tonsil epithelial cells.
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BACKGROUND: Human papillomavirus 16 (HPV16) has been associated with head and neck squamous cell carcinoma (HNSCC) in up to 60% of sampled specimens. METHODS: To understand better the viral genes required to transform human tonsil epithelial cells (HTEC), we isolated HTEC's and transduced them with retroviral vectors containing HPV16 E6 and E7. RESULTS: Immortalization and anchorage-independent growth of HTEC's only occurred with expression of E6 and E7 with resultant degradation of p53. However, cells expressing E6 lacking the PSD-95/disc-large/Zo-1 (PDZ) motif did not immortalize or grow anchorage independent. Telomerase activity and degradation of p53 were similar for wild-type and mutant E6. CONCLUSION: The mechanism of oncogenic transformation by E6 in HTEC's is dependent on the PDZ binding motif. Identification of pathways affected by the interaction of E6 and PDZ domain containing proteins will further our understanding of how HPV causes HNSCC and will provide potential therapeutic targets. (+info)
The PDZ domain-binding motif of the human T cell leukemia virus type 1 tax protein induces mislocalization of the tumor suppressor hScrib in T cells.
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Interactions with cellular PDZ domain-containing proteins obviously contribute to the tumorigenic potential of several viral oncoproteins. In this regard, the oncogenic potential of the human T cell leukemia virus type 1 Tax protein correlates with its binding capacity to the tumor suppressor hDlg. Recent results show that hDlg in T cells is associated to a network of scaffolding proteins including another PDZ domain-containing protein termed hScrib. Interestingly, previous studies have revealed complementary activities of both proteins in the control of epithelial cell polarity. Here, we demonstrate that Tax can bind to hScrib and that the resulting Tax/hScrib complex is present in human T cell leukemia virus type 1-infected T cells. By confocal microscopy, we show that Tax modifies the localization of hScrib in transfected COS cells as well as in infected T cell lines and targets hScrib to particular spots exhibiting a granular distribution, mainly distributed in the cytoplasm. Given that Tax sequesters hScrib to these particular structures, we postulate that Tax might inhibit hScrib activity. Providing further support to this idea, we find that transient overexpression of hScrib attenuates T cell receptor-induced NFAT activity but that the presence of Tax counteracts this negative effect on the NFAT pathway. The fact that hDlg and hScrib are both targeted by Tax underlies their importance in T cell function. (+info)
Identification of a postendocytic sorting sequence in CCR5.
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The chemokine receptor 5 (CCR5), a member of the G protein-coupled receptor family (GPCR), is used by human immunodeficiency virus type 1 (HIV-1) with a R5 tropism as an entry receptor in addition to CD4. It is a key target for an antiviral action aiming at inhibiting the HIV-1 entry process. Only few data are available today regarding the mechanism involved in the intracellular trafficking process of CCR5. Understanding how CCR5 cell surface expression is regulated is particularly important with regard to HIV-1 entry inhibition. We set out to investigate whether CCR5 molecular determinants were involved in the postendocytic recycling and degradative pathways. We constructed progressive deletion mutants of the C-terminal domain of CCR5 that we stably expressed in HEK293 cells. All of the deletion mutants were expressed at the cell surface and were functional HIV-1 receptors. The deletion mutants were internalized after stimulation, but they lost their ability to recycle to the plasma membrane. They were rerouted toward a lysosomal degradative pathway. We identified here a sequence of four amino acids, present at the extreme C terminus of CCR5, that is necessary for the recycling of the internalized receptor, independently of its phosphorylation. A detailed analysis of this sequence indicated that the four amino acids acted as a postsynaptic density 95/discs-large/zona occludens (PDZ) interacting sequence. These results show that the CCR5 cytoplasmic domain bears a sequence similar to the "recycling signals" previously identified in other GPCRs. Drugs able to disrupt the recycling pathway of CCR5 may constitute promising tools for therapeutic treatment. (+info)
Bivalent peptides as PDZ domain ligands.
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A series of multivalent peptides, with the ability to simultaneously bind two separate PDZ domain proteins, has been designed, synthesized, and tested by isothermal titration calorimetry (ITC). The monomer sequences, linked with succinate, varied in length from five to nine residues. The thermodynamic binding parameters, in conjunction with results from mass spectrometry, indicate that a ternary complex is formed in which each peptide arm binds two equivalents of the third PDZ domain (PDZ3) of the neuronal protein PSD-95. (+info)
The PDZ domain as a complex adaptive system.
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Specific protein associations define the wiring of protein interaction networks and thus control the organization and functioning of the cell as a whole. Peptide recognition by PDZ and other protein interaction domains represents one of the best-studied classes of specific protein associations. However, a mechanistic understanding of the relationship between selectivity and promiscuity commonly observed in the interactions mediated by peptide recognition modules as well as its functional meaning remain elusive. To address these questions in a comprehensive manner, two large populations of artificial and natural peptide ligands of six archetypal PDZ domains from the synaptic proteins PSD95 and SAP97 were generated by target-assisted iterative screening (TAIS) of combinatorial peptide libraries and by synthesis of proteomic fragments, correspondingly. A comparative statistical analysis of affinity-ranked artificial and natural ligands yielded a comprehensive picture of known and novel PDZ ligand specificity determinants, revealing a hitherto unappreciated combination of specificity and adaptive plasticity inherent to PDZ domain recognition. We propose a reconceptualization of the PDZ domain in terms of a complex adaptive system representing a flexible compromise between the rigid order of exquisite specificity and the chaos of unselective promiscuity, which has evolved to mediate two mutually contradictory properties required of such higher order sub-cellular organizations as synapses, cell junctions, and others--organizational structure and organizational plasticity/adaptability. The generalization of this reconceptualization in regard to other protein interaction modules and specific protein associations is consistent with the image of the cell as a complex adaptive macromolecular system as opposed to clockwork. (+info)