Vitamin K3 inhibits FtsZ assembly, disrupts the Z-ring in Streptococcus pneumoniae and displays anti-pneumococcal activity.

The respiratory pathogen, Streptococcus pneumoniae has acquired multiple-drug resistance over the years. An attractive strategy to combat pneumococcal infection is to target cell division to inhibit the proliferation of S. pneumoniae. This work presents Vitamin K3 as a potential anti-pneumococcal drug that targets FtsZ, the master coordinator of bacterial cell division. Vitamin K3 strongly inhibited S. pneumoniae proliferation with a minimum inhibitory concentration (MIC) and a minimum bactericidal concentration (MBC) of 6 µg/ml. Vitamin K3 disrupted the Z-ring localization in both S. pneumoniae and Bacillus subtilis within 30 min of treatment, while the membrane integrity and nucleoid segregation remain unchanged. Several complementary experiments showed that Vitamin K3 inhibits the assembly of purified S. pneumoniae FtsZ (SpnFtsZ) and induces conformational changes in the protein. Interestingly, Vitamin K3 interfered with GTP binding onto FtsZ and increased the GTPase activity of FtsZ polymers. The intrinsic tryptophan fluorescence of SpnFtsZ revealed that Vitamin K3 delays the nucleation of FtsZ polymers and reduces the rate of polymerization. In the presence of a non-hydrolyzable analog of GTP, Vitamin K3 did not show inhibition of FtsZ polymerization. These results indicated that Vitamin K3 induces conformational changes in FtsZ that increase GTP hydrolysis and thereby, destabilize the FtsZ polymers. Together, our data provide evidence that Vitamin K3 derives its potent anti-pneumococcal activity by inhibiting FtsZ assembly.

Permeant fluorescent probes visualize the activation of SARM1 and uncover an anti-neurodegenerative drug candidate.

SARM1 regulates axonal degeneration through its NAD-metabolizing activity and is a drug target for neurodegenerative disorders. We designed and synthesized fluorescent conjugates of styryl derivative with pyridine to serve as substrates of SARM1, which exhibited large red shifts after conversion. With the conjugates, SARM1 activation was visualized in live cells following elevation of endogenous NMN or treatment with a cell-permeant NMN-analog. In neurons, imaging documented mouse SARM1 activation preceded vincristine-induced axonal degeneration by hours. Library screening identified a derivative of nisoldipine (NSDP) as a covalent inhibitor of SARM1 that reacted with the cysteines, especially Cys311 in its ARM domain and blocked its NMN-activation, protecting axons from degeneration. The Cryo-EM structure showed that SARM1 was locked into an inactive conformation by the inhibitor, uncovering a potential neuroprotective mechanism of dihydropyridines.

Celastrol inhibits ezrin-mediated migration of hepatocellular carcinoma cells.

Progression of hepatocellular carcinoma involves multiple genetic and epigenetic alterations that promote cancer invasion and metastasis. Our recent study revealed that hyperphosphorylation of ezrin promotes intrahepatic metastasis in vivo and cell migration in vitro. Celastrol is a natural product from traditional Chinese medicine which has been used in treating liver cancer. However, the mechanism of action underlying celastrol treatment was less clear. Here we show that ROCK2 is a novel target of celastrol and inhibition of ROCK2 suppresses elicited ezrin activation and liver cancer cell migration. Using cell monolayer wound healing, we carried out a phenotype-based screen of natural products and discovered the efficacy of celastrol in inhibiting cell migration. The molecular target of celastrol was identified as ROCK2 using celastrol affinity pull-down assay. Our molecular docking analyses indicated celastrol binds to the active site of ROCK2 kinase. Mechanistically, celastrol inhibits the ROCK2-mediated phosphorylation of ezrin at Thr567 which harnesses liver cancer cell migration. Our findings suggest that targeting ROCK2-ezrin signaling is a potential therapeutic niche for celastrol-based intervention of cancer progression in hepatocellular carcinoma.

Target Based Virtual Screening of New Leads Inhibitor against Bacterial Cell Division Protein FtsZ for the Discovery of Antibacterial Agents.

BACKGROUND: Staphylococus epidermidis coagulase negative and gram positive streptococci have emerged as major nosocomial pathogens associated with the infection of implanted medical devices and dandruff on human scalp. S. epidermidis filamenting temperature-sensitive mutant Z (FtsZ) gene encoded FtsZ protein that assembles at future bacterial cell division site that forms Z-ring structure. FtsZ is a tubulin homolog protein with low sequence similarity; this makes it possible to inhibit bacterial FtsZ protein without affecting the eukaryote cell division. OBJECTIVE: In the present study, phytochemicals of Cinnamomum zeylanicum, Punica granatum and Glycyrrhiza glabra were virtually screened for their antibacterial activity against Staphylococcus epidermidis cell division protein, FtsZ. METHODS: Molecular docking method was used to investigate new lead inhibitor against bacterial cell division protein FtsZ. SwissADME and ProTox tool were used to evaluate the toxicity of the lead molecule. RESULTS: Molecular docking based screening confirmed that among 122 phytochemicals, ß- sitosterol and glabrol showed the highest inhibitory activity against FtsZ. SwissADME tool showed ß-sitosterol and glabrol as the ideal antibacterial agents. CONCLUSION: Structure based drug design strategy has been broadly used to optimize antimicrobial activity of small molecule/ligand against large protein receptor of disease, causing pathogens which gives a major breakthrough in pharmaceuticals industries. The molecular docking and SwissADME tool showed that ß-sitosterol and glabrol may be developed to be potential topical and sublingual antibacterial agents, respectively.

Pocket detection and interaction-weighted ligand-similarity search yields novel high-affinity binders for Myocilin-OLF, a protein implicated in glaucoma.

Traditional structure and ligand based virtual screening approaches rely on the availability of structural and ligand binding information. To overcome this limitation, hybrid approaches were developed that relied on extraction of ligand binding information from proteins sharing similar folds and hence, evolutionarily relationship. However, they cannot target a chosen pocket in a protein. To address this, a pocket centric virtual ligand screening approach is required. Here, we employ a new, iterative implementation of a pocket and ligand-similarity based approach to virtual ligand screening to predict small molecule binders for the olfactomedin domain of human myocilin implicated in glaucoma. Small-molecule binders of the protein might prevent the aggregation of the protein, commonly seen during glaucoma. First round experimental assessment of the predictions using differential scanning fluorimetry with myoc-OLF yielded 7 hits with a success rate of 12.7%; the best hit had an apparent dissociation constant of 99nM. By matching to the key functional groups of the best ligand that were likely involved in binding, the affinity of the best hit was improved by almost 10,000 fold from the high nanomolar to the low picomolar range. Thus, this study provides preliminary validation of the methodology on a medically important glaucoma associated protein.

Virtual screening of potential inhibitor against FtsZ protein from Staphylococcus aureus.

The gram-positive bacterium Staphylococcus aureus, responsible for a wide variety of diseases in human involve all organ systems ranging from localized skin infections to life-threatening systemic infections. FtsZ, the key protein of bacterial cell division was selected as a potent anti bacterial target. In order to identify the new compounds structure based screening process was carried out. An enrichment study was performed to select a suitable scoring function and to retrieve potential candidates against FtsZ from a large chemical database. The docking score and docking energy values were compared and their atomic interaction was also evaluated. Furthermore molecular dynamics simulation were also been performed to check the stability and the amino acids interacted towards the FtsZ. Finally we selected C ID 16284, 25916, 15894, 13403 as better lead compounds. From these results, we conclude that our insilico results will provide a framework for the detailed in vitro and in vivo studies about the FtsZ protein activity in drug development process.

Dimethyl sulphoxide and Ca2+ stimulate assembly of Vibrio cholerae FtsZ.

We cloned, overexpressed and purified Vibrio cholerae FtsZ protein for the first time. We used several complementary techniques to probe and compare the comparative assembly properties of recombinant Vibrio cholerae FtsZ (VcFtsZ) and Escherichia coli FtsZ (EcFtsZ). We observed that VcFtsZ polymerized at a slower rate than EcFtsZ and interestingly its polymerization was highly dependent on the presence of Ca(2+) ion. Furthermore, DMSO specifically modulated the polymerization of VcFtsZ, promoted polymer bundling and increased the stability of the VcFtsZ protofilaments. Whereas DMSO showed no significant stimulatory effect on the assembly and bundling of EcFtsZ. Transmission electron microscopy experiments demonstrated that in presence of 8% DMSO the average thickness of the VcFtsZ polymers were increased significantly. DMSO specifically stabilized the VcFtsZ polymers against dilution induced disassembly and it reduced the GTPase activity of VcFtsZ. These results collectively suggested that despite lot of sequence homology, the assembly of VcFtsZ and EcFtsZ are differently regulated processes. We expect to use this knowledge of assembly properties of VcFtsZ for screening of small molecules against VcFtsZ for development of anti-cholera agent.

Ligands for glaucoma-associated myocilin discovered by a generic binding assay.

Mutations in the olfactomedin domain of myocilin (myoc-OLF) are the strongest link to inherited primary open angle glaucoma. In this recently identified protein misfolding disorder, aggregation-prone disease variants of myocilin hasten glaucoma-associated elevation of intraocular pressure, leading to vision loss. Despite its well-documented pathogenic role, myocilin remains a domain of unknown structure or function. Here we report the first small-molecule ligands that bind to the native state of myoc-OLF. To discover these molecules, we designed a general label-free, mix-and-measure, high throughput chemical assay for restabilization (CARS), which is likely readily adaptable to discover ligands for other proteins. Of the 14 hit molecules identified from screening myoc-OLF against the Sigma-Aldrich Library of Pharmacologically Active Compounds using CARS, surface plasmon resonance binding studies reveal three are stoichiometric ligand scaffolds with low micromolar affinity. Two compounds, GW5074 and apigenin, inhibit myoc-OLF amyloid formation in vitro. Structure-activity relationship-based soluble derivatives reduce aggregation in vitro as well as enhance secretion of full-length mutant myocilin in a cell culture model. Our compounds set the stage for a new chemical probe approach to clarify the biological function of wild-type myocilin and represent lead therapeutic compounds for diminishing intracellular sequestration of toxic mutant myocilin.

Proteomics of vitamin B12 processing.

The causes of cobalamin (B12, Cbl) deficiency are multifactorial. Whether nutritional due to poor dietary intake, or functional due to impairments in absorption or intracellular processing and trafficking events, the major symptoms of Cbl deficiency include megaloblastic anemia, neurological deterioration and in extreme cases, failure to thrive and death. The common biomarkers of Cbl deficiency (hyperhomocysteinemia and methylmalonic acidemia) are extremely valuable diagnostic indicators of the condition, but little is known about the changes that occur at the protein level. A mechanistic explanation bridging the physiological changes associated with functional B12 deficiency with its intracellular processers and carriers is lacking. In this article, we will cover the effects of B12 deficiency in a cblC-disrupted background (also referred to as MMACHC) as a model of functional Cbl deficiency. As will be shown, major protein changes involve the cytoskeleton, the neurological system as well as signaling and detoxification pathways. Supplementation of cultured MMACHC-mutant cells with hydroxocobalamin (HOCbl) failed to restore these variants to the normal phenotype, suggesting that a defective Cbl processing pathway produces irreversible changes at the protein level.

Effect of supplementation with an 80:20 cis9,trans11 conjugated linoleic acid blend on the human platelet proteome.

SCOPE: The dietary fatty acid cis9,trans11 conjugated linoleic acid (cis9,trans11 CLA) has been shown to modify the function of endothelial cells, monocytes, and platelets, all of which are involved in the development of atherosclerosis. Potential mechanisms for the platelet effects have not been assessed previously. In this study, we assessed how supplementation of the diet with an 80:20 cis9,trans11 CLA blend affects the platelet proteome. METHODS AND RESULTS: In a double-blind, randomized, placebo-controlled, parallel-group trial, 40 overweight but apparently healthy adults received either 4 g per day of cis9,trans11 CLA-enriched oil or placebo oil, consisting of palm oil and soybean oil, for 3 months. Total platelet proteins were extracted from washed platelets, separated using two-dimensional gel electrophoresis and differentially regulated protein spots were identified by LC-ESI-MS/MS. Supplementation with the CLA blend, compared with placebo, resulted in significant alterations in levels of 46 spots (p < 0.05), of which 40 were identified. Network analysis revealed that the majority of these proteins participate in regulation of the cytoskeleton and platelet structure, as well as receptor action, signaling, and focal adhesion. CONCLUSION: The platelet proteomics approach revealed novel insights into regulation of cellular biomarkers of atherogenic and thrombotic pathways by an 80:20 cis9,trans11 CLA blend.

Carnosic acid protects neuronal HT22 Cells through activation of the antioxidant-responsive element in free carboxylic acid- and catechol hydroxyl moieties-dependent manners.

In a previous study, we found that carnosic acid (CA) protected cortical neurons by activating the Keap1/Nrf2 pathway, which activation was initiated by S-alkylation of the critical cysteine thiol of the Keap1 protein by the "electrophilic"quinone-type of CA [T. Satoh, K. Kosaka, K. Itoh, A. Kobayashi, M. Yamamoto, Y. Shimojo, C. Kitajima, J. Cui, J. Kamins, S. Okamoto, T. Shirasawa, S.A. Lipton, Carnosic acid, a catechol-type electrophilic compound, protects neurons both in vitro and in vivo through activation of the Keap1/Nrf2 pathway via S-alkylation of targeted cysteines on Keap1. J Neurochem., in press]. In the present study, we used HT22 cells, a neuronal cell line, to test CA derivatives that might be more suitable for in vivo use, as an electrophile like CA might react with other molecules prior to reaching its intended target. CA and carnosol protected the HT22 cells against oxidative glutamate toxicity. CA activated the transcriptional antioxidant-responsive element of phase-2 genes including hemeoxygenase-1, NADPH-dependent quinone oxidoreductase, and gamma-glutamyl cysteine ligase, all of which provide neuroprotection by regulating cellular redox. This finding was confirmed by the result that CA significantly increased the level of glutathione. We synthesized a series of its analogues in which CA was esterified at its catechol hydroxyl moieties to prevent the oxidation from the catechol to quinone form or esterified at those moieties and its carbonic acid to stop the conversion from CA to carnosol. In both cases, the conversion and oxidation cannot occur until the alkyl groups are removed by an intracellular esterase. Thus, the most potent active form as the activator of the Keap1/Nrf2 pathway, the quinone-type CA, will be produced inside the cells. However, neither chemical modulation potentiated the neuroprotective effects, possibly because of increased lipophilicity. These results suggest that the neuroprotective effects of CA critically require both free carboxylic acid and catechol hydroxyl moieties. Thus, the hydrophilicity of CA might be a critical feature for its neuroprotective effects.

Binding of the intracellular Fas ligand (FasL) domain to the adaptor protein PSTPIP results in a cytoplasmic localization of FasL.

The tumor necrosis factor family member Fas ligand (FasL) induces apoptosis in Fas receptor-expressing target cells and is an important cytotoxic effector molecule used by CTL- and NK-cells. In these hematopoietic cells, newly synthesized FasL is stored in specialized secretory lysosomes and only delivered to the cell surface upon activation and target cell recognition. FasL contains an 80-amino acid-long cytoplasmic tail, which includes a proline-rich domain as a bona fide Src homology 3 domain-binding site. This proline-rich domain has been implicated in FasL sorting to secretory lysosomes, and it may also be important for reverse signaling via FasL, which has been described to influence T-cell activation. Here we report the identification of the Src homology 3 domain-containing adaptor protein PSTPIP as a FasL-interacting partner, which binds to the proline-rich domain. PSTPIP co-expression leads to an increased intracellular localization of Fas ligand, thereby regulating extracellular availability and cytotoxic activity of the molecule. In addition, we demonstrate recruitment of the tyrosine phosphatase PTP-PEST by PSTPIP into FasL.PSTPIP.PTP-PEST complexes which may contribute to FasL reverse signaling.

Morphological and oxidative alterations on Sertoli cells cytoskeleton due to retinol-induced reactive oxygen species.

Retinol (vitamin A) is involved in several cellular processes, like cell division, differentiation, transformation and apoptosis. Although it has been shown that retinol is a limitant factor for all these processes, the precise mechanisms by which retinol acts are still unknown. In the present study we hypothesised that alterations in the cytoskeleton of Sertoli cells induced by retinol supplementation could indicate an adaptive maintenance of its functions, since it plays an important role in the transformation process that we observed. Previous results demonstrated that Sertoli cells treated with retinol showed an oxidative imbalance, that leads the cell to two phenotypes: apoptosis or transformation. Our group has identified characteristics of Sertoli cells transformed by retinol which results in normal cell functions modification. In the present study the actin filament fluorescence assay and the deformation coefficient showed a modification in the morphology induced by retinol. We also observed an oxidative alteration in isolated cytoskeleton proteins and did not show alterations when these proteins are analyzed by electrophoreses. Our results showed an increase in mitochondria superoxide production and a decrease in nitric oxide levels. All results were partially or completely reverted by co-treatment of the antioxidant Trolox. These findings suggest that the cytoskeleton components suffer individual alterations in different levels and that these alterations generate a global phenotype modification and that these processes are probably ROS dependent. We believe that the results from this study indicate an adaptation of the cytoskeleton to oxidative imbalance since there was not a loss of its function.

Sall1, a causative gene for Townes-Brocks syndrome, enhances the canonical Wnt signaling by localizing to heterochromatin.

The Spalt (sal) gene family plays an important role in regulating developmental processes of many organisms. Mutations of human SALL1 cause the autosomal dominant disorder, Townes-Brocks syndrome (TBS), and result in ear, limb, anal, renal, and heart anomalies. Targeted deletion of mouse Sall1 results in kidney agenesis or severe dysgenesis. Molecular mechanisms of Sall1, however, have remained largely unknown. Here we report that Sall1 synergistically activates canonical Wnt signaling. The transcriptional activity of Sall1 is related to its nuclear localization to punctate nuclear foci (pericentromeric heterochromatin), but not to its localization or association with beta-catenin, the nuclear component of Wnt signaling. In contrast, the RNA interference of Sall1 reduces reporter activities of canonical Wnt signaling. The N-terminal truncated Sall1, produced by mutations often found in TBS, disturbs localization of native Sall1 to heterochromatin, and also down-regulates the synergistic transcriptional enhancement for Wnt signal by native Sall1. Thus, we propose a new mechanism for Wnt signaling activation, that is the heterochromatin localization of Sall1.

Co-assembly of envoplakin and periplakin into oligomers and Ca(2+)-dependent vesicle binding: implications for cornified cell envelope formation in stratified squamous epithelia.

Plakin family members envoplakin and periplakin have been shown to be part of the cornified cell envelope in terminally differentiating stratified squamous epithelia. In the present study, purified recombinant human envoplakin and periplakin were used to investigate their properties and interactions. We found that envoplakin was insoluble at physiological conditions in vitro, and co-assembly with periplakin was required for its solubility. Envoplakin and periplakin formed soluble complexes with equimolar stoichiometry. Chemical cross-linking revealed that the major soluble form of all periplakin constructs and of envoplakin/periplakin rod domains was a dimer, although co-assembly of the full-length proteins resulted in formation of higher order oligomers. Electron microscopy of rotary-shadowed periplakin demonstrated thin flexible molecules with an average contour length of 88 nm for the rod-plus-tail fragment, and immunolabeling EM confirmed the molecule as a parallel, in-register, dimer. Both periplakin and envoplakin/periplakin oligomers were able to bind synthetic lipid vesicles whose composition mimicked the cytoplasmic side of the plasma membrane of eukaryotic cells. This binding was dependent on anionic phospholipids and Ca(2+). These findings raise the possibility that envoplakin and periplakin bind to the plasma membrane upon elevation of intracellular [Ca(2+)] in differentiating keratinocytes, where they serve as a scaffold for cornified cell envelope assembly.

Assembly and trafficking of a multiprotein ROMK (Kir 1.1) channel complex by PDZ interactions.

The ROMK subtypes of inward rectifier K+ channels (Kir 1.1, KCNJ1) mediate potassium secretion and regulate NaCl reabsorption in the kidney. In the present study, the role of the PDZ binding motif in ROMK function is explored. Here we identify the Na/H exchange regulatory factors, NHERF-1 and NHERF-2, as PDZ domain interaction partners of the ROMK channel. Characterization of the basis and consequences of NHERF association with ROMK reveals a PDZ interaction-dependent trafficking process and a coupling mechanism for linking ROMK to a channel modifier protein, the cystic fibrosis transmembrane regulator (CFTR). As measured by antibody binding of external epitope-tagged forms of Kir 1.1 in intact cells, NHERF-1 or NHERF-2 coexpression increased cell surface expression of ROMK. Channel interaction with NHERF proteins and effects of NHERF on ROMK localization were dependent on the presence of the PDZ domain binding motif in ROMK. Both NHERF proteins contain two PDZ domains; recombinant protein-protein binding assays and yeast-two-hybrid studies revealed that ROMK preferentially associates with the second PDZ domain of NHERF-1 and with the first PDZ domain of NHERF-2, precisely opposite of what has been reported for CFTR. Consistent with the scaffolding capacity of the NHERF proteins, coexpression of NHERF-2 with ROMK and CFTR dramatically increases the amount of ROMK protein that coimmunopurifies and functionally interacts with CFTR. Thus NHERF facilitates assembly of a ternary complex containing ROMK and CFTR. These observations raise the possibility that PDZ-based interactions may underscore physiological regulation and membrane targeting of ROMK in the kidney.

Tyrosine phosphorylation of plakoglobin causes contrary effects on its association with desmosomes and adherens junction components and modulates beta-catenin-mediated transcription.

Plakoglobin is a protein closely related to beta-catenin that links desmosomal cadherins to intermediate filaments. Plakoglobin can also substitute for beta-catenin in adherens junctions, providing a connection between E-cadherin and alpha-catenin. Association of beta-catenin with E-cadherin and alpha-catenin is regulated by phosphorylation of specific tyrosine residues; modification of beta-catenin Tyr654 and Tyr142 decreases binding to E-cadherin and alpha-catenin, respectively. We show here that plakoglobin can also be phosphorylated on tyrosine residues, but unlike beta-catenin, this modification is not always associated with disrupted association with junctional components. Protein tyrosine kinases present distinct specificities on beta-catenin and plakoglobin, and phosphorylation of beta-catenin-equivalent Tyr residues of plakoglobin affects its interaction with components of desmosomes or adherens junctions differently. For instance, Src, which mainly phosphorylates Tyr86 in beta-catenin, modifies Tyr643 in plakoglobin, decreasing the interaction with E-cadherin and alpha-catenin and increasing the interaction with the alpha-catenin-equivalent protein in desmosomes, desmoplakin. The tyrosine kinase Fer, which modifies beta-catenin Tyr142, lessening its association with alpha-catenin, phosphorylates plakoglobin Tyr549 and exerts the contrary effect: it raises the binding of plakoglobin to alpha-catenin. These results suggest that tyrosine kinases like Src or Fer modulate desmosomes and adherens junctions differently. Our results also indicate that phosphorylation of Tyr549 and the increased binding of plakoglobin to components of adherens junctions can contribute to the upregulation of the transcriptional activity of the beta-catenin-Tcf-4 complex observed in many epithelial tumor cells.

Selective interactions between helix VIII of the human mu-opioid receptors and the C terminus of periplakin disrupt G protein activation.

Analysis of interactions between the C-terminal tail of the MOP-1 and MOP-1A variants of the human mu-opioid receptor with proteins derived from a human brain cDNA library resulted in identification of the actin and intermediate filament-binding protein periplakin. Mapping of this interaction indicated that the predicted fourth intracellular loop/helix VIII of the receptor interacts with the C-terminal rod and linker region of periplakin. Periplakin is widely expressed in the central nervous system of both man and rat and demonstrated an overlapping but not identical distribution with mu-opioid (MOP) receptors. Co-expression of periplakin with MOP-1 or a MOP-1-eYFP fusion construct in HEK293 cells did not interfere with agonist-mediated internalization of the receptor. When co-expressed with a MOP-1-Gi1 alpha fusion protein periplakin significantly reduced the capacity of the agonist to stimulate binding of 35S-labeled guanosine 5'-3-O-(thio)triphosphate ([35S]GTP gamma S) to the receptor-associated G protein. By contrast, periplakin did not interfere with agonist-stimulation of [35S]GTP gamma S binding to either an alpha 2A-adrenoreceptor-Gi1 alpha fusion protein or a beta2-adrenoreceptor-Gs alpha fusion protein, indicating its selectivity of function. This represents the first example of an opioid receptor-interacting protein that functions to disrupt agonist-mediated G protein activation.

Plasma membrane Ca2+ ATPase isoform 2b interacts preferentially with Na+/H+ exchanger regulatory factor 2 in apical plasma membranes.

Spatial and temporal regulation of Ca(2+) signaling require the assembly of multiprotein complexes linking molecules involved in Ca(2+) influx, sensing, buffering, and extrusion. Recent evidence indicates that plasma membrane Ca(2+) ATPases (PMCAs) participate in the control of local Ca(2+) fluxes, but the mechanism of multiprotein complex formation of specific PMCAs is poorly understood. Using the PMCA2b COOH-terminal tail as bait in a yeast two-hybrid screen, we identified the PSD-95, Dlg, ZO-1 (PDZ) domain-containing Na(+)/H(+) exchanger regulatory factor-2 (NHERF2) as an interacting partner. Protein pull-down and coimmunoprecipitation experiments using recombinant PMCA2b and PMCA4b as well as NHERF1 and NHERF2 showed that the interaction of PMCA2b with NHERF2 was specific and selective. PMCA4b did not interact with either of the NHERFs, and PMCA2b selectively preferred NHERF2 over NHERF1. Green fluorescent protein-tagged PMCA2b was expressed at the apical membrane in Madin-Darby canine kidney epithelial cells, where it colocalized with apically targeted NHERF2. Our study identifies NHERF2 as the first specific PDZ partner for PMCA2b not shared with PMCA4b, and demonstrates that PMCA splice forms differing only minimally in their COOH-terminal residues interact with unique PDZ proteins. NHERFs have been implicated in the targeting, retention and regulation of membrane proteins including the beta(2)-adrenergic receptor, cystic fibrosis transmembrane conductance regulator, and Trp4 Ca(2+) channel, and NHERF2 is now shown to also interact with PMCA2b. This interaction may allow the functional assembly of PMCA2b in a multiprotein Ca(2+) signaling complex, facilitating integrated cross-talk between local Ca(2+) influx and efflux.

Proof-of-principle: oncogenic beta-catenin is a valid molecular target for the development of pharmacological inhibitors.

Activation of beta-catenin is a critical step in the pathogenesis of many common human cancers and is the initiating event in adenocarcinoma of the colon. Because activation of beta-catenin provides a gain-of-function, it is tempting to speculate that specific pharmacological inhibition of activated beta-catenin might reverse the tumorigenic properties of human cancer cells and therefore form the basis of an effective anticancer strategy. In an effort to provide proof-of-principle for such a strategy, we used a novel clonal growth assay based on human somatic cell gene targeting to determine whether activated beta-catenin remains a necessary oncogenic stimulus in advanced human cancer cells. Using this approach, we demonstrate that beta-catenin is a necessary oncogene in human SW48 and DLD1 colon cancer cells but not in HCT116 cells. These data indicate that activated beta-catenin can remain a critical oncogenic stimulus throughout the progression of human colon cancer and suggest that the small molecule inhibitors of activated beta-catenin currently under development will be effective anticancer therapeutics in a subset of malignant colon cancers.