Wnt-dependent assembly of supermolecular Dishevelled-3-based complexes.

Dishevelled-3 (Dvl3) is a multivalent scaffold protein that is essential to Wnt signaling during development. Although Dvl-based punctae have been visualized by fluorescence microscopy; the physical nature and dynamic character of the such complexes are enigmatic. We use steric-exclusion chromatography, affinity pull-downs, proteomics and fluorescence correlation microscopy to characterize supermolecular Dvl3-based complexes of totipotent mouse F9 cells. The molecular mass of the complexes ranges from that of homodimeric Dvl3 to well-defined peaks harboring supermolecular complexes of 0.4 to 2.0 MDa. Addition of Wnt3a stimulates the formation of Dvl3-based complexes of greater molecular mass within 30 minutes. The presence of DKK1 and knockdown of Dishevelled proteins block formation of the 2 MDa Dvl3-based complexes and also block Wnt3a stimulation of the canonical pathway. Fluorescent correlation microscopy identified supermolecular Dvl3-based complexes with a molecular mass >30 MDa in live cells; these complexes were provoked to form structures with even greater molecular mass by Wnt3a. We establish for the first time the physical and functional nature of very large, supermolecular Dvl3-based complexes.

Differential mediation of the Wnt canonical pathway by mammalian Dishevelleds-1, -2, and -3.

In the Drosophila, a single copy of the phosphoprotein Dishevelled (Dsh) is found. In the genomes of higher organism (including mammals), three genes encoding isoforms of Dishevelled (Dvl1, Dvl2, and Dvl3) are present. In the fly, Dsh functions in the Wnt-sensitive stabilization of intracellular beta-catenin and activation of the Lef/Tcf-sensitive transcriptional response known as the Wnt "canonical" pathway. In the current work we explore the expression of Dishevelleds in mammalian cells and provide an estimate of the relative cellular abundance of each Dvl. In mouse F9 cells, all three Dvls are expressed. Dvl2 constitutes more than 95% of the total pool, the sum of Dvl1 and Dvl3 constituting the remainder. Similarly, Dvl2 constitutes more than 80% of the Dvl1-3 pool in mouse P19 and human HEK 293 cells. siRNA-induced knock-down of individual Dvls was performed using Wnt3a-sensitive canonical pathway in F9 cells as the read-out. Activation of the canonical signaling pathway by Wnt3a was dependent upon the presence of Dvl1, Dvl2, and Dvl3, but to a variable extent. Wnt3a-sensitive canonical transcription was suppressible, by knock-down of Dvl1, Dvl2, or Dvl3. Conversely, the overexpression of any one of the three Dvls individually was found to be capable of promoting Lef/Tcf-sensitive transcriptional activation, in the absence of Wnt3a, i.e., overexpression of Dvl1, Dvl2, or Dvl3 is Wnt3a-mimetic. Graded suppression of individual Dvl isoforms by siRNA was employed to test if the three Dvls could be distinguished from one another with regard to mediation of the canonical pathway. Canonical signaling was most sensitive to changes in the abundance of either Dvl3 or Dvl1. Changes in expression of Dvl2, the most abundant of the three isoforms, resulted in the least effect on canonical signaling. Dvl-based complexes were isolated by pull-downs from whole-cell extracts with isoform-specific antibodies and found to include all three Dvl isoforms. Rescue experiments were conducted in which depletion of either Dvl3 or Dvl1 suppresses Wnt3a activation of the canonical pathway and the ability of a Dvl isoform to rescue the response evaluated. Rescue of Wnt3a-stimulated transcriptional activation in these siRNA-treated cells occurred only by the expression of the very same Dvl isoform depleted by the siRNA. Thus, Dvls appear to function cooperatively as well as uniquely with respect to mediation of Wnt3a-stimulated canonical signaling. The least abundant (Dvl1, 3) plays the most obvious role, whereas the most abundant (Dvl2) plays the least obvious role, suggesting that individual Dvl isoforms in mammals may operate as a network with some features in common and others rather unique.

Insulin-like growth factor-I provokes functional antagonism and internalization of beta1-adrenergic receptors.

Hormones that activate receptor tyrosine kinases have been shown to regulate G protein-coupled receptors, and herein we investigate the ability of IGF-I to regulate the beta(1)-adrenergic receptor. Treating Chinese hamster ovary cells in culture with IGF-I is shown to functionally antagonize the ability of expressed beta(1)-adrenergic receptors to accumulate intracellular cAMP in response to stimulation by the beta-adrenergic agonist Iso. The attenuation of beta(1)-adrenergic action was accompanied by internalization of beta(1)-adrenergic receptors in response to IGF-I. Inhibiting either phosphatidylinositol 3-kinase or the serine/threonine protein kinase Akt blocks the ability of IGF-I to antagonize and to internalize beta(1)-adrenergic receptors. Mutation of one potential Akt substrate site Ser412Ala, but not another Ser312Ala, of the beta(1)-adrenergic receptor abolishes the ability of IGF-I to functionally antagonize and to sequester the beta(1)-adrenergic receptor. We also tested the ability of IGF-I to regulate beta(1)-adrenergic receptors and their signaling in adult canine cardiac myocytes. IGF-I attenuates the ability of beta(1)-adrenergic receptors to accumulate intracellular cAMP in response to Iso and promotes internalization of beta(1)-adrenergic receptors in these cardiac myocytes.

Structure-function analysis of Frizzleds.

Frizzleds, cell surface receptors that mediate the actions of Wnt ligands on early development, are heptahelical (based upon hydropathy analysis) and couple to heterotrimeric G proteins. The primary structure of all ten mammalian Frizzleds display many landmarks observed in virtually all G protein-coupled receptors, including an exofacial N-terminus that is N-glycosylated, the presence of seven hydrophobic transmembrane segments predicted to form alpha-helixes, and three intracellular loops as well as a cytoplasmic, C-terminal tail that harbor suspected sites for protein phosphorylation. Prediction of the G proteins to which Frizzleds mediate signaling based upon a bioinformatic analysis of the primary sequence of the intracellular domains are in good agreement with functional screens in Drosophila, zebrafish, and mouse models of development, e.g., predicting Frizzled-1 to interact with members of the Gi/Go protein family. Likewise various Wnt signaling pathways are sensitive to treatment with pertussis toxin and knock-down of specific G protein alpha-subunits. Homology among the sequences encoding the cytoplasmic domains of human Frizzleds is high and the various Frizzleds can be segregated into subsets predicted to share some common downstream signaling elements. Among different species, homologies can reveal conservation of signaling to cognate G protein partners. Additionally, cytoplasmic domains of the prototypic beta2-adrenergic receptor can be substituted with those from either Frizzled-1 or Frizzled-2 to create chimeric receptors that are activated by beta-adrenergic agonists, yet signal with high fidelity to the Wnt/beta-catenin and Wnt/Ca2+, cyclic GMP pathways, respectively, regulating key aspects of early development. The nature of Frizzled-based signaling complexes, their temporal assembly, and spatial distribution via scaffold protein remains to be elucidated, as does whether or not these Wnt receptors display agonist-induced desensitization, internalization, and re-cycling to the cell membrane.

G-protein-coupled receptors and tyrosine kinases: crossroads in cell signaling and regulation.

G-protein-coupled receptors and protein tyrosine kinases represent two prominent pathways for cellular signaling. As our knowledge of cell signaling pathways mediated by the superfamily of G-protein-coupled receptors and the smaller family of receptor tyrosine kinases expands, so does our appreciation of how these two major signaling platforms share information and modulate each other, otherwise termed "cross-talk". Cross-talk between G-protein-coupled receptors and tyrosine kinases can occur at several levels, including the receptor-to-receptor level, and at crucial downstream points (e.g. phosphatidylinositol-3-kinase, Akt/protein kinase B and the mitogen-activated protein kinase cascade). Regulation of G-protein-coupled receptors by non-receptor tyrosine kinases, such as Src family members, also operates in signaling. A broader understanding of how G-protein-coupled receptors and tyrosine kinases cross-talk reveals new insights into signaling modalities in both health and disease.

pp60Src mediates insulin-stimulated sequestration of the beta(2)-adrenergic receptor: insulin stimulates pp60Src phosphorylation and activation.

Insulin stimulates a rapid phosphorylation and sequestration of the beta(2)-adrenergic receptor. Analysis of the signaling downstream of the insulin receptor with enzyme inhibitors revealed roles for both phosphatidylinositol 3-kinase and pp60Src. Inhibition of Src with PP2, like the inhibition of phosphatidylinositol 3-kinase with LY294002 [2-(4-morpholynyl)-8-phenyl-4H-1-benzopyran-4-one], blocked the activation of Src as well as insulin-stimulated sequestration of the beta(2)-adrenergic receptor. Depletion of Src with antisense morpholinos also suppressed insulin-stimulated receptor sequestration. Src is shown to be phosphorylated/activated in response to insulin in human epidermoid carcinoma A431 cells as well as in mouse 3T3-L1 adipocytes and their derivative 3T3-F422A cells, well-known models of insulin signaling. Inhibition of Src with PP2 blocks the ability of insulin to sequester beta(2)-adrenergic receptors and the translocation of the GLUT4 glucose transporters. Insulin stimulates Src to associate with the beta(2)-adrenergic receptor/AKAP250/protein kinase A/protein kinase C signaling complex. We report a novel positioning of Src, mediating signals from insulin to phosphatidylinositol 3-kinase and to beta(2)-adrenergic receptor trafficking.

G-Protein-coupled receptor-associated A-kinase anchoring proteins: AKAP79 and AKAP250 (gravin).

A-kinase anchoring proteins (AKAPs) define an expanding group of scaffold proteins that display a signature binding site for the RI/RII subunit of protein kinase A. AKAPs are multivalent and a subset of these scaffold proteins also display the ability to associate with the prototypic member of G-protein-coupled receptors, the beta(2)-adrenergic receptor. Both AKAP79 (also known as AKAP5) and AKAP250 (also known as gravin or AKAP12) have been shown to associate with the beta(2)-adrenergic receptor, but each directs downstream signaling events in decidedly different manners. The primary structures, common and unique protein motifs are of interest. Both proteins display largely natively unfolded primary sequences that provide a necklace on which short, structured regions of sequence are found. Membrane association appears to involve both interactions with the lipid bilayer via docking to a G-protein-coupled receptor as well as interactions of short positively charged domains with the inner leaflet of the cell membrane. Gravin, unlike AKAP79, displays a canonical site at its N-terminus that is subject to N-myristoylation. AKAP79 appears to function in switching signaling pathways of the receptor from adenylylcyclase to activation of the mitogen-activated protein kinase cascade. Gravin, in contrast, is essential for the resensitization and recycling of the receptors following agonist-induced activation, desensitization, and internalization. Each AKAP provides a template that enables space-time continuum features to G-protein-coupled signaling pathways as well as a paradigm for explaining apparent compartmentalization of cell signaling.

Frizzled-4 C-terminus Distal to KTXXXW Motif is Essential for Normal Dishevelled Recruitment and Norrin-stimulated Activation of Lef/Tcf-dependent Transcriptional Activation.

The carboxy (C)-termini of G protein coupled receptors (GPCR) dictate essential functions. The KTXXXW motif C-terminus of Frizzleds (FZD) has been implicated in recruitment of Dishevelled (DVL). Through study of FZD4 and its associated ligand Norrin, we report that a minimum of three residues distal to the KTXXXW motif in the C-terminal tail of Frizzled-4 are essential for DVL recruitment and robust Lef/Tcf-dependent transcriptional activation in response to Norrin.

The 15-amino acid motif of the C terminus of the beta2-adrenergic receptor is sufficient to confer insulin-stimulated counterregulation to the beta1-adrenergic receptor.

Insulin counterregulates catecholamine action in part by inducing the sequestration of beta2-adrenergic receptors. Although similar to agonist-induced sequestration, insulin-induced internalization of beta2-adrenergic receptors operates through a distinct and better-understood cellular pathway. The effects of insulin treatment on the function and trafficking of both beta1- and beta2-adrenergic receptors were tested. The beta2-adrenergic receptors were counterregulated and internalized in response to insulin. The beta1-adrenergic receptors, in sharp contrast, are shown to be resistant to the ability of insulin to counterregulate function and induce receptor internalization. Using chimeric receptors composed of beta1-/beta2-adrenergic receptors in tandem with mutagenesis, we explored the role of the C-terminal cytoplasmic tail of the beta2-adrenergic receptors for insulin-induced counterregulation. Substitution of the C-terminal cytoplasmic tail of the beta2-adrenergic receptor on the beta1-adrenergic receptor enabled the chimeric G protein-coupled receptor to be functionally and spatially regulated by insulin. Truncation of the beta2-adrenergic receptor C-terminal cytoplasmic tail to a 15-amino acid motif harboring a potential Src homology 2-binding domain at Y350 and an Akt phosphorylation site at S345,346 was sufficient to enable receptor regulation by insulin.

AKAPs (A-kinase anchoring proteins) and molecules that compose their G-protein-coupled receptor signalling complexes.

Cell signalling mediated via GPCRs (G-protein-coupled receptors) is a major paradigm in biology, involving the assembly of receptors, G-proteins, effectors and downstream elements into complexes that approach in design 'solid-state' signalling devices. Scaffold molecules, such as the AKAPs (A-kinase anchoring proteins), were discovered more than a decade ago and represent dynamic platforms, enabling multivalent signalling. AKAP79 and AKAP250 were the first to be shown to bind to membrane-embedded GPCRs, orchestrating the interactions of various protein kinases (including tyrosine kinases), protein phosphatases (e.g. calcineurin) and cytoskeletal elements with at least one member of the superfamily of GPCRs, the prototypical beta2-adrenergic receptor. In this review, the multivalent interactions of AKAP250 with the cell membrane, receptor, cytoskeleton and constituent components are detailed, providing a working model for AKAP-based GPCR signalling complexes. Dynamic regulation of the AKAP-receptor complex is mediated by ordered protein phosphorylation.

The Intracellular Loop 2 F328S Frizzled-4 Mutation Implicated in Familial Exudative Vitreoretinopathy Impairs Dishevelled Recruitment.

Familial exudative vitreoretinopathy (FEVR) is a disease state characterized by aberrant retinal angiogenesis. Norrin-induced activation of Frizzled-4 (Fz4) has a major role in regulating beta-catenin levels in the eye that, in turn, modulate the blood retina barrier (BRB). Here we gain insight on the basis of the pathology of a FEVR implicated F328S Fz4 mutant by study. The receptor exhibits a substantially reduced ability to activate Lef/Tcf-dependent transcription. This impaired activation correlates with a decreased ability to stabilize and recruit Dishevelled-2 (Dvl2) to the cell surface. Aromaticity at position 328 of the intracellular loop 2 (iloop2) is revealed similarly as a prerequisite for Dvl2 recruitment to the Fz4. This aromaticity at 328 enables normal Norrin-induced canonical activation. The corresponding position in iloop2 of other Frizzleds likely functions in Dvl recruitment.

WNT-frizzled signaling via cyclic GMP.

Wnt-Frizzled signaling is an essential aspect of early development, regulating cell fate, polarity, differentiation, and migration. In addition to the well-known Wnt/beta-catenin pathway characterized for Frizzled-1, there are other pathways regulated by Wnts that are not mediated by Frizzled-1 and do not lead to stabilization of beta-catenin and activation of the Lef/Tcf-sensitive transcription of genes. The first of these non-canonical pathways to be identified is the Wnt/Ca++ pathway in which Frizzled-2 activation leads to release of beta/gamma subunit complexes from heterotrimeric G-proteins (presumably Go and Gt) to activate phospholipase C and other effectors to stimulate a mobilization of intracellular Ca++. More recently a second, related pathway of Wnt-Frizzled signaling has been discovered that regulates the intracellular levels of cyclic GMP. Frizzled-2, established as a member of the family of 7TMS receptors that couple by heterotrimeric G-proteins to effectors, can signal via the G-protein Gt2, transducin, a G-protein prominent in phototransduction in the eye, to cyclic GMP phosphodiesterase. The discovery of the expression of Gt2 in embryonic cells was co-incident with the demonstration that inhibitors of cyclic GMP phosphodiesterase potently blocked various features of Frizzled-2 signaling in mouse embryonic F9 cells and in zebrafish embryos. The signal linkage map from Wnt to changes in intracellular cyclic GMP and development is the focus of this review. The molecular features of how changes in intracellular cyclic GMP concentrations control development remain to be elucidated.

Overexpression of mitogen-activated protein kinase phosphatases MKP1, MKP2 in human breast cancer.

Expression and activity of c-Jun N-terminal and p38 protein kinases were explored in malignant and non-malignant tissue samples from patients with primary breast cancer. Differential expression was observed for p38 and c-Jun N-terminal protein kinases (JNK) in samples from 14 patients in whom there were sufficient malignant and non-malignant tissue to perform the entire assays. As previously noted, Erk1,2 expression and activity were increased sharply in the malignant tissue. The p38 kinase expression and activity were increased 3-fold in breast cancer. The expression of c-Jun N-terminal protein kinase JNK1, but not JNK2, was increased 2.5-fold in malignant as compared to normal breast tissue. Immunohistochemical analysis in situ with antibodies to JNK1 revealed intense staining in samples of cancerous epithelium. In spite of a 3-fold increase in expression, malignant samples displayed a 35% decrease in the activity of this pro-apoptotic protein kinase. The expression of mitogen and extracellularly-activated protein kinase kinase (MEK)2 and MEK3, upstream protein kinases of Erkl,2 and p38, respectively, was elevated 4- to 5-fold. The upstream regulator of JNK (e.g., MEK4), however, displayed normal levels of expression, providing no basis for the reduction in JNK activity observed for breast cancer. Mitogen-activated protein kinase phosphatases (MKP)1 and MKP2 were assayed and the expression was found to be increased 5-fold and 3-fold, respectively, in malignant as compared to non-malignant samples. The reduced activity of JNK1, in spite of its overexpression, appears to reflect increased MKP activity associated with primary breast cancer. Suppression of MKP activity therapeutically may enable the expression of the pro-apoptotic signals from JNK in malignant cells.

The Frizzled-1/(beta(2))-adrenergic receptor chimera: pharmacological properties of a unique G protein-linked receptor.

The frizzled gene family of Wnt receptors encodes proteins that have a seven-transmembrane-spanning motif characteristic of G-protein-coupled receptors. Using a chimeric receptor composed of the exofacial and the transmembrane, ligand-binding domain of the beta(2)-adrenergic receptor (beta2AR) fused with the corresponding cytoplasmic domains of the rat Frizzled-1 receptor (Rfz1), we created a unique chimera between distant members of the superfamily of G protein-coupled receptors. Herein, we describe the pharmacological properties of the chimera, which represents a receptor in which the exofacial and cytoplasmic domains are minimized in length. This unique chimera retains much of the pharmacological character of the native beta2AR, whereas the coupling can be ascribed to Rfz1 domains which operate via G alpha q and not G alpha s. The Rfz1 chimera demonstrates a robust agonist (isoproterenol)-induced sequestration. Since the Rfz1 cytoplasmic domains possess canonical sites for several protein kinases, we were able to investigate the effects of kinase inhibitors on Rfz1 chimera sequestration. Only the protein kinase A inhibitor KT5720, but not inhibitors of protein kinase C, calcium/calmodulin-sensitive kinase-2, casein kinase-2, and Src, inhibited agonist-induced sequestration. Expression of a dominant-negative form of beta-arrestin blocked sequestration of the beta2AR, but only reduced modestly that of the Rfz1 chimera. These data demonstrate that the Frizzled-1 chimera displays cardinal features of a G protein-coupled receptor, including agonist-induced sequestration, but appears to do so largely even in the presence of dominant-negative beta-arrestins.

Probing the stoichiometry of ß2-adrenergic receptor phosphorylation by targeted mass spectrometry.

BACKGROUND: Protein phosphorylation of G-protein-coupled receptors (GPCR) is central to the myriad of functions that these ubiquitous receptors perform in biology. Although readily addressable with the use of phospho-specific antibodies, analysis phosphorylation at the level of stoichiometry requires receptor isolation and advanced proteomics. We chose two key sites of potential phosphorylation of human beta2-adrenergic receptor (ß2AR residues S355 and S356) to ascertain the feasibility of applying targeted mass spectrometry to establishing the stoichiometry of the phosphorylation. METHOD: We stimulated HEK293 cells stably expressing Flag-tagged ß2AR-eGFP with 10 µM beta-adrenergic agonist (isoproterenol) and made use of proteomics and targeted mass spectrometry (MS) to quantify the molar ration of phosphorylation on S355 and S356 versus non-phosphorylated receptor in agonist-treated cells. RESULTS: Phosphorylation of either S355 or S356 residue occurred only for agonist-occupied ß2AR. The results demonstrated that pS356 is the dominant site of protein phosphorylation. The abundance of the p356 was 8.6-fold more than that of pS355. Calculation of the molar ratio of phosphorylated (pS355 plus pS356) versus non-phosphorylated receptor reveals that at high occupancy of the receptor only 12.4% of the ß2AR is phosphorylated at these sites. CONCLUSIONS: Application of advanced proteomics and use of the most sensitive targeted MS strategy makes possible the detection and quantification of phosphorylation of very low abundance peptide digests of ß2AR. Establishing the stoichiometry of two key sites of agonist-stimulated phosphorylation with ß2AR is an essential first-step to global analysis of the stoichiometry of GPCR phosphorylation.

Dvl3 translocates IPMK to the cell membrane in response to Wnt.

Wnt3a binds Frizzled-1 and the LRP5/6 co-receptors, ultimately activating Lef/Tcf-sensitive gene transcription in development. Inositol polyphosphate multikinase, IPMK, which possesses inositol phosphate kinase and lipid inositol kinase activities, is essential in Wnt3a regulation of its canonical pathway as well as physiologically in AMPK signaling. In the current report we show that translocation of IPMK to the cell membrane, where its substrates exist in high abundance, is obligate to its function in Wnt signaling. Translocation of IPMK to the cell membrane occurs within 5 min after Wnt3a stimulation. IPMK ducking onto Dishevelled-3 (Dvl3) requires a PDZ domain and the COOH-terminal prolyly-rich tail of Dvl3. Wnt3a-stimulates mobilization of Dvl3 to the cell membrane, translocating IPMK to the cell membrane also, to facilitate downstream signaling of Frizzled1. Deletion mutant of IPMK lacking the NH2-terminal variable region, IPMKΔN, fails to translocate to the cell membrane and to propagate canonical signaling. Targeting the IPMKΔN back to the cell membrane by addition of an isoprenylated CAAX box rescues its function in Wnt3a downstream signaling.

"Shaping" of cell signaling via AKAP-tethered PDE4D: Probing with AKAR2-AKAP5 biosensor.

BACKGROUND: PKA, a key regulator of cell signaling, phosphorylates a diverse and important array of target molecules and is spatially docked to members of the A-kinase Anchoring Protein (AKAP) family. AKAR2 is a biosensor which yields a FRET signal in vivo, when phosphorylated by PKA. AKAP5, a prominent member of the AKAP family, docks several signaling molecules including PKA, PDE4D, as well as GPCRs, and is obligate for the propagation of the activation of the mitogen-activated protein kinase cascade from GPCRs to ERK1,2. RESULTS: Using an AKAR2-AKAP5 fusion "biosensor", we investigated the spatial-temporal activation of AKAP5 undergoing phosphorylation by PKA in response to ß-adrenergic stimulation. The pattern of PKA activation reported by AKAR2-AKAP5 is a more rapid and spatially distinct from those "sensed" by AKAR2-AKAP12. Spatial-temporal restriction of activated PKA by AKAP5 was found to "shape" the signaling response. Phosphatase PDE4D tethered to AKAP5 also later reverses within 60 s elevated intracellular cyclic AMP levels stimulated by ß-adrenergic agonist. AKAP12, however, fails to attenuate the rise in cyclic AMP over this time. Fusion of the AKAP5 PDE4D-binding-domain to AKAP12 was found to accelerate a reversal of accumulation of intracellular cyclic AMP. CONCLUSION: AKAPs, which are scaffolds with tethered enzymes, can "shape" the temporal and spatial aspects of cell signaling.

Assembly of Dishevelled 3-based supermolecular complexes via phosphorylation and Axin.

BACKGROUND: Dishevelled-3 (Dvl3) is a multivalent scaffold essential to cell signaling in development. Dsh/Dvls enable a myriad of protein-protein interactions in Wnt signaling. In the canonical Wnt/ß-catenin pathway specifically, Dvl3 polymerizes to form dynamic protein aggregates, so-called "signalsomes", which propagate signals from the Wnt receptor Frizzled to downstream elements. RESULTS: Very large Dvl3-based supermolecular complexes form in response to Wnt3a. These complexes are identified by steric-exclusion chromatography, affinity pull-downs, proteomics, and fluorescence correlation microscopy (fcs). In the current work, the roles of Dvl3 phosphorylation and of Axin in the assembly of Dvl3-based supermolecular complexes in response to Wnt3a are probed in totipotent mouse F9 teratocarcinoma cells. Point mutations of phosphorylation sites of Dvl3 which interfere with Lef/Tcf-sensitive transcriptional activation by Wnt3a are shown to interfere more proximally with the assembly of Dvl3-based supermolecular complexes. Axin, a Dvl-interacting protein, plays a central role in organizing the beta-catenin destruction complex. The assembly of Dvl3-based supermolecular complexes is blocked either by depletion of Axin or by mutation of Axin sites necessary for polymerization in response to Wnt3a. CONCLUSION: These data demonstrate that Wnt3a activation of the canonical pathway requires specific phosphorylation events as well as Axin to assemble very large, Dvl3-based supermolecular complexes; these complexes are a prerequisite to activation of Lef/Tcf-sensitive transcription.

Probing the physical nature and composition of signalsomes.

BACKGROUND: Recent advances in our understanding of cell signaling have revealed assemblies of signaling components often viewed in fluorescence microscopy as very large, irregular "punctae". These punctae are often dynamic in nature, appearing to act as mobile scaffolds that function in integrating protein-protein interactions from large arrays of signaling components. The visualization of these punctae, termed "signalsomes" when applied to protein assemblies involved in cell signaling provokes the question, what is the physical nature of these structures made visible in live cells through the expression of fluorescently-tagged fusion molecules? RESULTS: Steric-exclusion chromatography on wide-bore matrices, fluorescence correlation spectroscopy, and advanced proteomics permits the analysis of several important physical properties of signalsomes. Wnt canonical signaling is essential to normal cell development and dysregulation can lead to cancers in humans. Punctae/signalsomes have been reported based upon the study of fluorescently-tagged mammalian Dishevelleds. Dishevelleds are phosphoprotein scaffolds that demonstrate dynamic character and mobility in cells stimulated with Wnt3a. Recent studies have successfully isolated Dvl3-based signalsomes from mouse totipotent embryonic teratocarcinoma F9 cells in culture and sized by application of steric exclusion chromatography (SEC), displaying large discrete Mr (0.5 and 2 MDa). Activation of the Wnt canonical ß-catenin/LEF-Tcf-sensitive transcriptional response leads to an upfield shift of >5 MDa of the Dvl3-based signalsome. Fluorescence correlation spectroscopy (fcs) is a single molecule analysis performed in live cells that experimentally measures the diffusion coefficient and permits calculation of MW of the signalsome (0.2 and 30 MDa species in vivo), which also reveal an upfield shift in MW in response to Wnt3a. Proteomics provides for molecular dissection of the composition of the signalsome isolated from untreated and Wnt3a-treated cells. CONCLUSION: Dvl3-based punctae/signalsomes made visible by fluorescent microscopy now can be interrogated by advanced physical means, defining such properties as signalsome Mr/MW, molecular composition, and intracellular locale.

AKAP12 and AKAP5 form higher-order hetero-oligomers.

BACKGROUND: The family of A-kinase-anchoring proteins, AKAPs, constitutes a group of molecular scaffolds that act to catalyze dynamic interactions of protein kinase A, protein kinase C, tyrosine kinases, G-protein-coupled receptors and ion channels. AKAP5 (MW ~47 kDa) and AKAP12 (MW ~191 kDa) homo-oligomerize, but whether or not such AKAPs can hetero-oligomerize into supermolecular scaffolds of increased complexity is unknown. RESULTS: Affinity chromatography using immobilized AKAPs as "bait" demonstrates unequivocally that AKAP5 and AKAP12 do form minimally hetero-dimers. Steric-exclusion chromatography of AKAP5 and AKAP12 mixtures revealed the existence of very large, supermolecular complexes containing both AKAPs. Docking of AKAP5 to AKAP12 was increased 4-fold by beta-adrenergic agonist stimulation. Overexpression of AKAP12 was found to potentiate AKAP5-mediated Erk1/2 activation in response to stimulation with beta-adrenergic agonist. CONCLUSION: AKAP5 and AKAP12 are capable of forming hetero-oligomeric supermolecular complexes that influence AKAP locale and function.