Identification of a PDZ protein, PIST, as a binding partner for Rho effector Rhotekin: biochemical and cell-biological characterization of Rhotekin-PIST interaction.
ABSTRACT: Among various effector proteins for the small GTPase Rho, the function(s) of Rhotekin is (are) almost unknown. We have identified PIST [PDZ (PSD-95, Discs-large and ZO-1) domain protein interacting specifically with TC10 (a Rho-family small GTPase)] as a binding partner for Rhotekin, using yeast two-hybrid screening. Rhotekin was found to associate with PIST in vitro and in both polarized and non-polarized MDCK (Madin-Darby canine kidney) cells. The C-terminal SPV (Ser-Pro-Val) motif of Rhotekin exhibited binding to the PDZ domain of PIST. The binding was markedly inhibited by an activated version of Rho and partially by that of Rac or Cdc42 in COS7 cells. In contrast, TC10 had no effects on the binding. Immunofluorescence analyses revealed the co-localization of PIST and Rhotekin at the Golgi apparatus in non-polarized fibroblast-like MDCK cells and AJs (adherens junctions) in the fully polarized cells. PIST and Rhotekin are recruited from the cytosol to AJs as the cell becomes polarized. Expression of constitutively active Rho or prevention of Rhotekin-PIST interaction induced diffuse cytoplasmic distribution of Rhotekin in polarized MDCK cells. These results suggest that there is (1) Rho-dependent regulation of Rhotekin-PIST interaction, (2) involvement of PIST in the recruitment of Rhotekin to AJs and (3) a possible role(s) for these two proteins in cell-polarity development and/or maintenance.
Project description:Rho signaling is increasingly recognized to contribute to invasion and metastasis. In this study, we discovered that metastasis-associated protein S100A4 interacts with the Rho-binding domain (RBD) of Rhotekin, thus connecting S100A4 to the Rho pathway. Glutathione S-transferase pull-down and immunoprecipitation assays demonstrated that S100A4 specifically and directly binds to Rhotekin RBD, but not the other Rho effector RBDs. S100A4 binding to Rhotekin is calcium-dependent and uses residues distinct from those bound by active Rho. Interestingly, we found that S100A4 and Rhotekin can form a complex with active RhoA. Using RNA interference, we determined that suppression of both S100A4 and Rhotekin leads to loss of Rho-dependent membrane ruffling in response to epidermal growth factor, an increase in contractile F-actin 'stress' fibers and blocks invasive growth in three-dimensional culture. Accordingly, our data suggest that interaction of S100A4 and Rhotekin permits S100A4 to complex with RhoA and switch Rho function from stress fiber formation to membrane ruffling to confer an invasive phenotype.
Project description:PIST [PDZ (PSD-95, Discs-large and ZO-1) protein interacting specifically with TC10] functions as a regulator of membrane trafficking with Rab6A. Recently, the involvement of the fusion of PIST with ROS1 in cancer development has been identified. In this study, the coiled-coil domain of PIST, which is the domain responsible for interaction with Rab6A and fusion with ROS1, corresponding to amino acids 29-133, was overexpressed in Escherichia coli using engineered C-terminal His tags. The coiled-coil domain of PIST was then purified to homogeneity and crystallized at 293 K. Finally, X-ray diffraction data were collected to a resolution of 4.0 Å from a crystal belonging to the hexagonal space group P6(2)22 or P6(4)22, with unit-cell parameters a = b = 85.19, c = 240.09 Å, ? = 120.00°.
Project description:Vinexin is a SH3 domain-containing adaptor protein that has diverse roles in cell adhesion, signal transduction, gene regulation and stress granule assembly. In this study, we found that vinexin localizes at the midbody during cell division and facilitates cytokinesis. Knockdown of vinexin in HeLa cells delayed the mitotic cell cycle progression and increased the time of cell abscission and the failure to resolve the cytoplasmic bridge. Midbody-localized vinexin is essential for recruiting rhotekin to this structure for cytokinesis because overexpression of a vinexin mutant without a rhotekin-binding motif or knockdown of rhotekin also impaired cytokinetic abscission and increased the number of cells arrested at the midbody stage. Aberrant expression of vinexin and rhotekin in various cancers has been implicated to promote metastasis because of their functions in cell adhesion and signaling. Our findings reveal a novel role of vinexin and rhotekin in cytokinetic abscission and provide another perspective of how both molecules may affect oncogenic transformation via this fundamental cell cycle process.
Project description:PSD-95/discs large/ZO-1 (PDZ) domain proteins integrate many G-protein coupled receptors (GPCRs) into membrane associated signalling complexes. Additional PDZ proteins are involved in intracellular receptor trafficking. We show that three PDZ proteins (SNX27, PIST and NHERF1/3) regulate the mouse somatostatin receptor subtype 5 (SSTR5). Whereas the PDZ ligand motif of SSTR5 is not necessary for plasma membrane targeting or internalization, it protects the SSTR5 from postendocytic degradation. Under conditions of lysosomal inhibition, recycling of the SSTR5 to the plasma membrane does not depend on the PDZ ligand. However, recycling of the wild type receptor carrying the PDZ binding motif depends on SNX27 which interacts and colocalizes with the receptor in endosomal compartments. PIST, implicated in lysosomal targeting of some membrane proteins, does not lead to degradation of the SSTR5. Instead, overexpressed PIST retains the SSTR5 at the Golgi. NHERF family members release SSTR5 from retention by PIST, allowing for plasma membrane insertion. Our data suggest that PDZ proteins act sequentially on the GPCR at different stages of its subcellular trafficking.
Project description:Although crucial for their correct function, the mechanisms controlling surface expression of ion channels are poorly understood. In the case of the voltage-gated potassium channel KV10.1, this is determinant not only for its physiological function in brain, but also for its pathophysiology in tumors and possible use as a therapeutic target. The Golgi resident protein PIST binds several membrane proteins, thereby modulating their expression. Here we describe a PDZ domain-mediated interaction of KV10.1 and PIST, which enhances surface levels of KV10.1. The functional, but not the physical interaction of both proteins is dependent on the coiled-coil and PDZ domains of PIST; insertion of eight amino acids in the coiled-coil domain to render the neural form of PIST (nPIST) and the corresponding short isoform in an as-of-yet unknown form abolishes the effect. In addition, two new isoforms of PIST (sPIST and nsPIST) lacking nearly the complete PDZ domain were cloned and shown to be ubiquitously expressed. PIST and KV10.1 co-precipitate from native and expression systems. nPIST also showed interaction, but did not alter the functional expression of the channel. We could not document physical interaction between KV10.1 and sPIST, but it reduced KV10.1 functional expression in a dominant-negative manner. nsPIST showed weak physical interaction and no functional effect on KV10.1. We propose these isoforms to work as modulators of PIST function via regulating the binding on interaction partners.
Project description:PDZ domains represent one group of the major structural units that mediate protein interactions in intercellular contact, signal transduction and assembly of biological machineries. Tax-interacting protein (TIP)-1 protein is composed of a single PDZ domain that distinguishes TIP-1 from other PDZ domain proteins that more often contain multiple protein domains and function as scaffolds for protein complex assembly. However, the biological functions of TIP-1, especially in cell transformation and tumor progression, are still controversial as observed in a variety of cell types. In this study, we have identified ARHGEF7, a guanine nucleotide exchange factor for Rho GTPases, as one novel TIP-1-interacting protein in human glioblastoma cells. We found that the presence of TIP-1 protein is essential to the intracellular redistribution of ARHGEF7 and rhotekin, one Rho effector and the spatiotemporally coordinated activation of Rho GTPases (RhoA, Cdc42 and Rac1) in migrating glioblastoma cells. TIP-1 knockdown resulted in both aberrant localization of ARHGEF7 and rhotekin, as well as abnormal activation of Rho GTPases that was accompanied with impaired motility of glioblastoma cells. Furthermore, TIP-1 knockdown suppressed tumor cell dispersal in orthotopic glioblastoma murine models. We also observed high levels of TIP-1 expression in human glioblastoma specimens, and the elevated TIP-1 levels are associated with advanced staging and poor prognosis in glioma patients. Although more studies are needed to further dissect the mechanism(s) by which TIP-1 modulates the intracellular redistribution and activation of Rho GTPases, this study suggests that TIP-1 holds potential as both a prognostic biomarker and a therapeutic target of malignant gliomas.
Project description:GOPC (FIG/PIST/CAL) is a PDZ-domain scaffolding protein that regulates the trafficking of a wide array of proteins, including small GTPases, receptors and cell surface molecules such as cadherin 23 and cystic fibrosis transmembrane regulator. In Madin-Darby canine kidney (MDCK) cells, we find that GOPC localizes to the trans-Golgi network (TGN) but not to the cis- or trans-Golgi cisternae. Colocalization occurs with the early endosome Rab GTPase Rab5 and a TGN/endosome marker Rab14 but not with Rab11, a marker of recycling endosomes. No localization of GOPC was detected to the lateral membranes or tight junctions. Knockdown of GOPC in MDCK cells results in decreased transepithelial resistance and increased paracellular flux. This might be attributable to the compromised trafficking of tight junction components from the TGN, as GOPC-knockdown cells have decreased lateral labeling of the tight junction protein claudin-1 and decreased protein levels of claudin-2. GOPC might mediate the trafficking of newly synthesized tight junction proteins from the TGN to the cell surface or the recycling of these proteins from specialized endosomal compartments.
Project description:The influenza A virus NS1 protein contains a conserved 4-amino-acid-residue PDZ-ligand binding motif (PBM) at the carboxyl terminus that can function as a virulence determinant by targeting cellular PDZ proteins. The NS1 proteins from avian and human viral isolates have consensus PBM sequences ESEV and RSKV, respectively. Currently circulating highly pathogenic H5N1 viruses contain the ESEV PBM which specifically associates with the PDZ proteins Scribble, Dlg1, MAGI-1, MAGI-2, and MAGI-3. In this study, we found NS1 proteins from viral isolates that contain the PBM sequence RSKV, KSEV, or EPEV are unable to associate with these PDZ proteins. Other results showed that the ESEV PBM mediates an indirect association with PDZ protein, Lin7C, via an interaction with Dlg1. Infection with a virus that expresses a NS1 protein with the ESEV PBM results in colocalization of NS1, Scribble, and Dlg1 within perinuclear puncta and mislocalization of plasma membrane-associated Lin7C to the cytoplasm. Infection of polarized MDCK cells with the ESEV virus additionally results in functional disruption of the tight junction (TJ) as measured by altered localization of TJ markers ZO-1 and Occludin, decreased transepithelial electrical resistance, and increased fluorescein isothiocyanate (FITC)-inulin diffusion across the polarized cell monolayer. A similar effect on the TJ was observed in MDCK cells depleted for either Scribble or Dlg1 by small interfering RNA (siRNA). These findings indicate that ESEV PBM-mediated binding of NS1 to Scribble and Dlg1 functions to disrupt the cellular TJ and that this effect likely contributes to the severe disease associated with highly pathogenic H5N1 influenza A viruses.
Project description:Epithelial polarization involves the segregation of apical and basolateral membrane domains, which are stabilized and maintained by tight junctions and membrane traffic. We report that unlike most apical and basolateral proteins in MDCK cells, which separate only after junctions have formed, the apical marker gp135 signifies an early level of polarized membrane organization established already in single cells. We identified gp135 as the dog orthologue of podocalyxin. With a series of domain mutants we show that the COOH-terminal PSD-95/Dlg/ZO-1 (PDZ)-binding motif is targeting podocalyxin to the free surface of single cells as well as to a subdomain of the terminally polarized apical membrane. This special localization of podocalyxin is shared by the cytoplasmic PDZ-protein Na+/H+ exchanger regulatory factor (NHERF)-2. Depleting podocalyxin by RNA interference caused defects in epithelial polarization. Together, our data suggest that podocalyxin and NHERF-2 function in epithelial polarization by contributing to an early apical scaffold based on PDZ domain-mediated interactions.
Project description:The Crumbs (Crb) complex, formed by Crb, PALS1, and PATJ, is evolutionarily conserved in metazoans and acts as a master cell-growth and -polarity regulator at the apical membranes in polarized epithelia. Crb intracellular functions, including its direct binding to PALS1, are mediated by Crb's highly conserved 37-residue cytoplasmic tail. However, the mechanistic basis governing the highly specific Crb-PALS1 complex formation is unclear, as reported interaction between the Crb tail (Crb-CT) and PALS1 PSD-95/DLG/ZO-1 (PDZ) domain is weak and promiscuous. Here we have discovered that the PDZ-Src homolgy 3 (SH3)-Guanylate kinase (GK) tandem of PALS1 binds to Crb-CT with a dissociation constant of 70 nM, which is ? 100-fold stronger than the PALS1 PDZ-Crb-CT interaction. The crystal structure of the PALS1 PDZ-SH3-GK-Crb-CT complex reveals that PDZ-SH3-GK forms a structural supramodule with all three domains contributing to the tight binding to Crb. Mutations disrupting the tertiary interactions of the PDZ-SH3-GK supramodule weaken the PALS1-Crb interaction and compromise PALS1-mediated polarity establishment in Madin-Darby canine kidney (MDCK) cysts. We further show that specific target binding of other members of membrane-associated guanylate kinases (MAGUKs) (e.g., CASK binding to neurexin) also requires the presence of their PDZ-SH3-GK tandems.