Par-3-mediated junctional localization of the lipid phosphatase PTEN is required for cell polarity establishment

PDZ domain-containing scaffold protein Par-3 is the central organizer of the evolutionarily conserved cell polarity-regulatory Par-3.Par-6.atypical protein kinase C complex. The PDZ domains of Par-3 have also been implicated as potential phosphoinositide signaling integrators, since its second PDZ domain binds to phosphoinositides, and the third PDZ interacts with phosphoinositide phosphatase PTEN. However, the molecular basis of Par-3/PTEN interaction is still poorly understood. Additionally, it is not known whether the regulatory function of PTEN in cell polarity is specifically mediated by its interaction with Par-3. The structures of Par-3 PDZ3 in both its free and PTEN tail peptide-bound forms determined in this work reveal that Par-3 PDZ3 binds to PTEN with two discrete binding sites: a canonical PDZ-ligand interaction site and a distal, opposite charge-charge interaction site. This distinct target recognition mechanism confers the interaction specificity of the Par-3.PTEN complex. We show that the Par-3 PDZ3-PTEN binding is required for the enrichment of PTEN at the junctional membranes of Madin-Darby canine kidney cells. Finally, we demonstrate that the junctional membrane-localized PTEN is specifically required for the polarization of Madin-Darby canine kidney cells. These results, together with earlier data, firmly establish that Par-3 functions as a scaffold in integrating phosphoinositide signaling events during cellular polarization.     

Energetic determinants of animal cell polarity regulator Par-3 interaction with the Par complex

The animal cell polarity regulator Par-3 recruits the Par complex (consisting of Par-6 and atypical PKC, aPKC) to specific sites on the cell membrane. Although numerous physical interactions have been reported between Par-3 and the Par complex, it is unclear how each of these interactions contributes to the overall binding. Using a purified, intact Par complex and a quantitative binding assay, here, we found that the energy required for this interaction is provided by the second and third PDZ protein interaction domains of Par-3. We show that both Par-3 PDZ domains bind to the PDZ-binding motif of aPKC in the Par complex, with additional binding energy contributed from the adjacent catalytic domain of aPKC. In addition to highlighting the role of Par-3 PDZ domain interactions with the aPKC kinase domain and PDZ-binding motif in stabilizing Par-3–Par complex assembly, our results indicate that each Par-3 molecule can potentially recruit two Par complexes to the membrane during cell polarization. These results provide new insights into the energetic determinants and structural stoichiometry of the Par-3–Par complex assembly.     

Cdc42 regulates the Par-6 PDZ domain through an allosteric CRIB-PDZ transition

Regulation of protein interaction domains is required for cellular signaling dynamics. Here, we show that the PDZ protein interaction domain from the cell polarity protein Par-6 is regulated by the Rho GTPase Cdc42. Cdc42 binds to a CRIB domain adjacent to the PDZ domain, increasing the affinity of the Par-6 PDZ for its carboxy-terminal ligand by approximately 13-fold. Par-6 PDZ regulation is required for function as mutational disruption of Cdc42-Par-6 PDZ coupling leads to inactivation of Par-6 in polarized MDCK epithelial cells. Structural analysis reveals that the free PDZ domain has several deviations from the canonical PDZ conformation that account for its low ligand affinity. Regulation results from a Cdc42-induced conformational transition in the CRIB-PDZ module that causes the PDZ to assume a canonical, high-affinity PDZ conformation. The coupled CRIB and PDZ architecture of Par-6 reveals how simple binding domains can be combined to yield complex regulation.     

Internal recognition through PDZ domain plasticity in the Par-6-Pals1 complex

PDZ protein interaction domains are typically selective for C-terminal ligands, but non-C-terminal, 'internal' ligands have also been identified. The PDZ domain from the cell polarity protein Par-6 binds C-terminal ligands and an internal sequence from the protein Pals1/Stardust. The structure of the Pals1-Par-6 PDZ complex reveals that the PDZ ligand-binding site is deformed to allow for internal binding. Whereas binding of the Rho GTPase Cdc42 to a CRIB domain adjacent to the Par-6 PDZ regulates binding of C-terminal ligands, the conformational change that occurs upon binding of Pals1 renders its binding independent of Cdc42. These results suggest a mechanism by which the requirement for a C terminus can be readily bypassed by PDZ ligands and reveal a complex set of cooperative and competitive interactions in Par-6 that are likely to be important for cell polarity regulation.     

The Par-3 NTD adopts a PB1-like structure required for Par-3 oligomerization and membrane localization

The evolutionarily conserved Par-3/Par-6/aPKC complex is essential for the establishment and maintenance of polarity of a wide range of cells. Both Par-3 and Par-6 are PDZ domain containing scaffold proteins capable of binding to polarity regulatory proteins. In addition to three PDZ domains, Par-3 also contains a conserved N-terminal oligomerization domain (NTD) that is essential for proper subapical membrane localization and consequently the functions of Par-3. The molecular basis of NTD-mediated Par-3 membrane localization is poorly understood. Here, we describe the structure of a monomeric form of the Par-3 NTD. Unexpectedly, the domain adopts a PB1-like fold with both type-I and type-II structural features. The Par-3 NTD oligomerizes into helical filaments via front-to-back interactions. We further demonstrate that the NTD-mediated membrane localization of Par-3 in MDCK cells is solely attributed to its oligomerization capacity. The data presented in this study suggest that the Par-3 NTD is likely to facilitate the assembly of higher-order Par-3/Par-6/aPKC complex with increased avidities in targeting the complex to the subapical membrane domain and in binding to other polarity-regulating proteins.     

Some (dis)assembly required: partial unfolding in the Par-6 allosteric switch

Allostery is commonly described as a functional connection between two distant sites in a protein, where a binding event at one site alters affinity at the other. Here, we review the conformational dynamics that encode an allosteric switch in the PDZ domain of Par-6, which is a scaffold protein that organizes other proteins into a complex required to initiate and maintain cell polarity. NMR measurements revealed that the PDZ domain samples an evolutionarily conserved unfolding intermediate allowing rearrangement of two adjacent loop residues that control ligand binding affinity. Cdc42 binding to Par-6 creates a novel interface between the PDZ domain and the adjoining CRIB motif that stabilizes the high-affinity PDZ conformation. Thermodynamic and kinetic studies suggest that partial PDZ unfolding is an integral part of the Par-6 switching mechanism. The Par-6 CRIB-PDZ module illustrates two important structural aspects of protein evolution: the interface between adjacent domains in the same protein can give rise to allosteric regulation, and thermodynamic stability may be sacrificed to increase the sampling frequency of an unfolding intermediate required for conformational switching.     

Interactions between the PDZ domains of Bazooka (Par-3) and phosphatidic acid: in vitro characterization and role in epithelial development

Bazooka (Par-3) is a conserved polarity regulator that organizes molecular networks in a wide range of cell types. In epithelia, it functions as a plasma membrane landmark to organize the apical domain. Bazooka is a scaffold protein that interacts with proteins through its three PDZ (postsynaptic density 95, discs large, zonula occludens-1) domains and other regions. In addition, Bazooka has been shown to interact with phosphoinositides. Here we show that the Bazooka PDZ domains interact with the negatively charged phospholipid phosphatidic acid immobilized on solid substrates or in liposomes. The interaction requires multiple PDZ domains, and conserved patches of positively charged amino acid residues appear to mediate the interaction. Increasing or decreasing levels of diacylglycerol kinase or phospholipase D-enzymes that produce phosphatidic acid-reveal a role for phosphatidic acid in Bazooka embryonic epithelial activity but not its localization. Mutating residues implicated in phosphatidic acid binding revealed a possible role in Bazooka localization and function. These data implicate a closer connection between Bazooka and membrane lipids than previously recognized. Bazooka polarity landmarks may be conglomerates of proteins and plasma membrane lipids that modify each other's activities for an integrated effect on cell polarity.     

Par-3 controls tight junction assembly through the Rac exchange factor Tiam1

The par (partitioning-defective) genes express a set of conserved proteins that function in polarization and asymmetric cell division. Par-3 has multiple protein-interaction domains, and associates with Par-6 and atypical protein kinase C (aPKC). In Drosophila, Par-3 is essential for epithelial cell polarization. However, its function in mammals is unclear. Here we show that depletion of Par-3 in mammalian epithelial cells profoundly disrupts tight junction assembly. Expression of a carboxy-terminal fragment plus the third PDZ domain of Par-3 partially rescues junction assembly, but neither Par-6 nor aPKC binding is required. Unexpectedly, Rac is constitutively activated in cells lacking Par-3, and the assembly of tight junctions is efficiently restored by a dominant-negative Rac mutant. The Rac exchange factor Tiam1 (ref. 7) binds directly to the carboxy-terminal region of Par-3, and knockdown of Tiam1 enhances tight junction formation in cells lacking Par-3. These results define a critical function for Par-3 in tight junction assembly, and reveal a novel mechanism through which Par-3 engages in the spatial regulation of Rac activity and establishment of epithelial polarity.     

Cooperative phosphoinositide and peptide binding by PSD-95/discs large/ZO-1 (PDZ) domain of polychaetoid, Drosophila zonulin

PDZ domains are well known protein-protein interaction modules that, as part of multidomain proteins, assemble molecular complexes. Some PDZ domains have been reported to interact with membrane lipids, in particular phosphatidylinositol phosphates, but few studies have been aimed at elucidating the prevalence or the molecular details of such interactions. We screened 46 Drosophila PDZ domains for phosphoinositide-dependent cellular localization and discovered that the second PDZ domain of polychaetoid (Pyd PDZ2) interacts with phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) at the plasma membrane. Surface plasmon resonance binding experiments with recombinant protein established that Pyd PDZ2 interacts with phosphatidylinositol phosphates with apparent affinities in the micromolar range. Electrostatic interactions involving an extended positively charged surface of Pyd PDZ2 are crucial for the PtdIns(4,5)P(2)-dependent membrane interactions as shown by a combination of three-dimensional modeling, mutagenesis, binding, and localization studies. In vivo localization studies further suggested that both lipid and peptide binding contribute to membrane localization. We identified the transmembrane protein Crumbs as a Pyd PDZ2 ligand and probed the relation between peptide and PtdIns(4,5)P(2) binding. Contrary to the prevalent view on PDZ/peptide/lipid binding, we did not find competition between peptide and lipid ligands. Instead, preloading the protein with the 10-mer Crb3 peptide increased the apparent affinity of Pyd PDZ2 for PtdIns(4,5)P(2) 6-fold. Our results suggest that membrane localization of Pyd PDZ2 may be driven by a combination of peptide and PtdIns(4,5)P(2) binding, which raises the intriguing possibility that the domain may coordinate protein- and phospholipid-mediated signals.     

A Golgi-associated PDZ domain protein modulates cystic fibrosis transmembrane regulator plasma membrane expression.

We identified a novel cystic fibrosis transmembrane conductance regulator (CFTR)-associating, PDZ domain-containing protein, CAL (CFTR associated ligand) containing two predicted coiled-coiled domains and one PDZ domain. The PDZ domain of CAL binds to the C terminus of CFTR. Although CAL does not have any predicted transmembrane domains, CAL is associated with membranes mediated by a region containing the coiled-coil domains. CAL is located primarily at the Golgi apparatus, co-localizing with trans-Golgi markers and is sensitive to Brefeldin A treatment. Immunoprecipitation experiments suggest that CAL exists as a multimer. Overexpression of CAL reduces CFTR chloride currents in mammalian cells and decreases expression, rate of insertion and half-life of CFTR in the plasma membrane. The Na(+)/H(+) exchanger regulatory factor, NHE-RF, a subplasma membrane PDZ domain protein, restores cell surface expression of CFTR and chloride currents. In addition, NHE-RF inhibits the binding of CAL to CFTR. CAL modulates the surface expression of CFTR. CAL favors retention of CFTR within the cell, whereas NHE-RF favors surface expression by competing with CAL for the binding of CFTR. Thus, the regulation of CFTR in the plasma membrane involves the dynamic interaction between at least two PDZ domain proteins.     

Interdomain interactions in the tumor suppressor discs large regulate binding to the synaptic protein GukHolder

Multidomain scaffolding proteins are central components of many signaling pathways and are commonly found at membrane specializations. Here we have shown that multiple interdomain interactions in the scaffold Discs Large (Dlg) regulate binding to the synaptic protein GukHolder (GukH). GukH binds the Src homology 3 (SH3) and guanylate kinase-like (GK) protein interaction domains of Dlg, whereas an intramolecular interaction between the two domains inhibits association with GukH. Regulation occurs through a PDZ domain adjacent to the SH3 that allows GukH to interact with the composite SH3-GK binding site, but PDZ ligands inhibit GukH binding such that Dlg forms mutually exclusive PDZ ligand and GukH cellular complexes. The PDZ-SH3-GK module is a common feature of membrane associate guanylate kinase scaffolds such as Dlg, and these results indicate that its supramodular architecture leads to regulation of Dlg complexes.     

Nuclear speckles and nucleoli targeting by PIP2-PDZ domain interactions

PDZ (Postsynaptic density protein, Disc large, Zona occludens) domains are protein-protein interaction modules that predominate in submembranous scaffolding proteins. Recently, we showed that the PDZ domains of syntenin-1 also interact with phosphatidylinositol 4,5-bisphosphate (PIP2) and that this interaction controls the recruitment of the protein to the plasma membrane. Here we evaluate the general importance of PIP2-PDZ domain interactions. We report that most PDZ proteins bind weakly to PIP2, but that syntenin-2, the closest homolog of syntenin-1, binds with high affinity to PIP2 via its PDZ domains. Surprisingly, these domains target syntenin-2 to nuclear PIP2 pools, in nuclear speckles and nucleoli. Targeting to these sites is abolished by treatments known to affect these PIP2 pools. Mutational and domain-swapping experiments indicate that high-affinity binding to PIP2 requires both PDZ domains of syntenin-2, but that its first PDZ domain contains the nuclear PIP2 targeting determinants. Depletion of syntenin-2 disrupts the nuclear speckles-PIP2 pattern and affects cell survival and cell division. These findings show that PIP2-PDZ domain interactions can directly contribute to subnuclear assembly processes.     

Tamalin is a scaffold protein that interacts with multiple neuronal proteins in distinct modes of protein-protein association

Tamalin is a scaffold protein that comprises multiple protein-interacting domains, including a 95-kDa postsynaptic density protein (PSD-95)/discs-large/ZO-1 (PDZ) domain, a leucine-zipper region, and a carboxyl-terminal PDZ binding motif. Tamalin forms a complex with metabotropic glutamate receptors and guanine nucleotide exchange factor cytohesins and promotes intracellular trafficking and cell surface expression of group 1 metabotropic glutamate receptors. In the present study, using several different approaches we have shown that tamalin interacts with multiple neuronal proteins through its distinct protein-binding domains. The PDZ domain of tamalin binds to the PDZ binding motifs of SAP90/PSD-95-associated protein and tamalin itself, whereas the PDZ binding motif of tamalin is capable of interacting with the PDZ domain of S-SCAM. In addition, tamalin forms a complex with PSD-95 and Mint2/X11beta/X11L by mechanisms different from the PDZ-mediated interaction. Tamalin has the ability to assemble with these proteins in vivo; their protein complex with tamalin was verified by coimmunoprecipitation of rat brain lysates. Interestingly, the distinct protein-interacting domains of tamalin are evolutionarily conserved, and mRNA expression is developmentally up-regulated at the postnatal period. The results indicate that tamalin exists as a key element that forms a protein complex with multiple postsynaptic and protein-trafficking scaffold proteins.     

抑癌蛋白PTEN及PDZ蛋白对其的调节

pten基因是迄今为止发现的第1个具有双特异性磷酸酶活性的抑癌基因,该基因的编码产物PTEN蛋白,是具有蛋白与脂质磷酸酯酶活性的双特异性磷酸酯酶,作为1种重要的信号分子参与细胞增殖、分化、黏附、迁移、凋亡以及基因转录的调控. 最近,关于PTEN在信号转导中的作用以及细胞内PTEN的调节机制研究较多,尤其是PDZ蛋白对PTEN的调节作用. PTEN蛋白包括1个氨基端（N端）磷酸酯酶区域,1个与脂质结合的C2区域和1个含有PDZ结合序列的羧基端（C端）区域. PDZ结构域通过识别目标蛋白羧基端PDZ结合序列与目标蛋白相互作用,调控多种重要的细胞生理过程和信号传导途径.本文就抑癌基因pten编码产物PTEN蛋白的结构、PTEN的生物学功能和PDZ蛋白对PTEN调节的研究进展进行综述.     

The helically extended SH3 domain of the T cell adaptor protein ADAP is a novel lipid interaction domain

Adhesion and degranulation-promoting adapter protein (ADAP) is critically involved in downstream signalling events triggered by the activation of the T cell receptor. Cytokine production, proliferation and integrin clustering of T cells are dependent on ADAP function, but the molecular basis for these processes is poorly understood. We now show the hSH3 domain of ADAP to be a lipid-interaction module that binds to acidic lipids, including phosphatidylinositides. Positively charged surface patches of the domain preferentially bind to polyvalent acidic lipids such as PIP2 or PIP3 over the monovalent PS phospholipid and this interaction is dependent on the N-terminal helix of the hSH3 domain fold. Basic amino acid side-chains from the SH3 scaffold also contribute to lipid binding. In the context of T cell signalling, our findings suggest that ADAP, upon recruitment to the cell-cell junction as part of a multiprotein complex, directly interacts with phosphoinositide-enriched regions of the plasma membrane. Furthermore, the ADAP lipid interaction defines the helically extended SH3 scaffold as a novel member of membrane interaction domains.     

Identification of three novel Smad binding proteins involved in cell polarity

A yeast two-hybrid screen was utilized to identify novel Smad 3 binding proteins expressed in developing mouse orofacial tissue. Three proteins (Erbin, Par-3, and Dishevelled) were identified that share several similar structural and functional characteristics. Each contains at least one PDZ domain and all have been demonstrated to play a role in the establishment and maintenance of cell polarity. In GST (glutathione S-transferase) pull-down assays, Erbin, Par-3, and Dishevelled bound strongly to the isolated MH2 domain of Smad 3, with weaker binding to a full-length Smad 3 protein. Failure of Erbin, Par-3, and Dishevelled to bind to a Smad 3 mutant protein that was missing the MH2 domain confirms that the binding site resides within the MH2 domain. Erbin, Par-3, and Dishevelled also interacted with the MH2 domains of other Smads, suggesting broad Smad binding specificity. Dishevelled and Erbin mutant proteins, in which the PDZ domain was removed, still retained their ability to bind Smad 3, albeit with lower affinity. While transforming growth factor beta (TGFbeta) has been suggested to alter cell polarity through a Smad-independent mechanism involving activation of members of the RhoA family of GTP binding proteins, the observation that Smads can directly interact with proteins involved in cell polarity, as shown in the present report, suggests an additional means by which TGFbeta could alter cell polarity via a Smad-dependent signaling mechanism.     

NHERF1/EBP50 head-to-tail intramolecular interaction masks association with PDZ domain ligands

Loss of cell polarity is one of the initial alterations in the development of human epithelial cancers. Na(+)/H(+) exchanger regulatory factor (NHERF) homologous adaptors 1 and 2 are membrane-associated proteins composed of two amino (N)-terminal PDZ domains and an ezrin-radixin-moesin (ERM)-binding (EB) carboxyl (C)-terminal region. We describe here an intramolecular conformation of NHERF1/EBP50 (ERM-binding phosphoprotein 50) in which the C-terminal EB region binds to the PDZ2 domain. This novel head-to-tail conformation masked the interaction of both PDZ domains with PDZ domain-specific ligands, such as PTEN and beta-catenin. An EB region composite structure comprising an alpha-helix ending in a PDZ-binding motif imparted opposite effects to NHERF1 associations, mediating binding to ERM proteins and inhibiting binding of PDZ domain ligands. The PDZ domain inhibition was released by prior association of ezrin with the EB region, a condition that occurs in vivo and likely disrupts NHERF1 head-to-tail interaction. In contrast, NHERF2 did not present a regulatory mechanism for protein complex formation. Functionally, NHERF1 is required to organize complexes at the apical membranes of polarized epithelial cells. The regulation of NHERF1 interactions at the apical membrane thus appears to be a dynamic process that is important for maintaining epithelial-tissue integrity.     

hCASK and hDlg associate in epithelia, and their src homology 3 and guanylate kinase domains participate in both intramolecular and intermolecular interactions

Membrane-associated guanylate kinase (MAGUK) proteins act as molecular scaffolds organizing multiprotein complexes at specialized regions of the plasma membrane. All MAGUKs contain a Src homology 3 (SH3) domain and a region homologous to yeast guanylate kinase (GUK). We showed previously that one MAGUK protein, human CASK (hCASK), is widely expressed and associated with epithelial basolateral plasma membranes. We now report that hCASK binds another MAGUK, human discs large (hDlg). Immunofluorescence microscopy demonstrates that hCASK and hDlg colocalize at basolateral membranes of epithelial cells in small and large intestine. These proteins co-precipitate from lysates of an intestinal cell line, Caco-2. The GUK domain of hCASK binds the SH3 domain of hDlg in both yeast two-hybrid and fusion protein binding assays, and it is required for interaction with hDlg in transfected HEK293 cells. In addition, the SH3 and GUK domains of each protein participate in intramolecular binding that in vitro predominates over intermolecular binding. The SH3 and GUK domains of human p55 display the same interactions in yeast two-hybrid assays as those of hCASK. Not all SH3-GUK interactions among these MAGUKs are permissible, however, implying specificity to SH3-GUK interactions in vivo. These results suggest MAGUK scaffold assembly may be regulated through effects on intramolecular SH3-GUK binding.     

A conformational switch in the CRIB-PDZ module of Par-6

Here, we report a novel mechanism of PDZ (PSD-95/Dlg/ZO-1) domain regulation that distorts?a conserved element of PDZ ligand recognition. The polarity regulator Par-6 assembles a conserved multiprotein complex and is directly modulated by the?Rho GTPase Cdc42. Cdc42 binds the adjacent Cdc42/Rac interactive binding (CRIB) and PDZ domains of Par-6, increasing C-terminal ligand binding affinity by 10-fold. By solving structures of the isolated PDZ domain and a disulfide-stabilized CRIB-PDZ, we detected a conformational switch that controls affinity by altering the configuration of the conserved "GLGF" loop. As a result, lysine 165 is displaced from the PDZ core by an adjacent hydrophobic residue, disrupting coordination of the PDZ ligand-binding cleft. Stabilization of the CRIB:PDZ interface restores K165 to its canonical location in the binding pocket. We conclude that a unique "dipeptide switch" in the Par-6 PDZ transmits a signal for allosteric activation to the ligand-binding pocket.     

Biochemical and computational analysis of LNX1 interacting proteins

PDZ (Post-synaptic density, 95 kDa, Discs large, Zona Occludens-1) domains are protein interaction domains that bind to the carboxy-terminal amino acids of binding partners, heterodimerize with other PDZ domains, and also bind phosphoinositides. PDZ domain containing proteins are frequently involved in the assembly of multi-protein complexes and clustering of transmembrane proteins. LNX1 (Ligand of Numb, protein X 1) is a RING (Really Interesting New Gene) domain-containing E3 ubiquitin ligase that also includes four PDZ domains suggesting it functions as a scaffold for a multi-protein complex. Here we use a human protein array to identify direct LNX1 PDZ domain binding partners. Screening of 8,000 human proteins with isolated PDZ domains identified 53 potential LNX1 binding partners. We combined this set with LNX1 interacting proteins identified by other methods to assemble a list of 220 LNX1 interacting proteins. Bioinformatic analysis of this protein list was used to select interactions of interest for future studies. Using this approach we identify and confirm six novel LNX1 binding partners: KCNA4, PAK6, PLEKHG5, PKC-alpha1, TYK2 and PBK, and suggest that LNX1 functions as a signalling scaffold.