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 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.

Dishevelled3 is a novel arginine methyl transferase substrate.

Dishevelled, a phosphoprotein scaffold, is a central component in all the Wnt-sensitive signaling pathways. In the present study, we report that Dishevelled is post-translationally modified, both in vitro and in vivo, via arginine methylation. We also show protein arginine methyl transferases 1 and 7 as the key enzymes catalyzing Dishevelled methylation.

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/β-catenin pathway specifically, Dvl3 polymerizes to form dynamic protein aggregates, so-called "signalsomes", which propagate signals from the Wnt receptor Frizzled to downstream elements.

"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.

Wnt3a-stimulated LRP6 phosphorylation is dependent upon arginine methylation of G3BP2.

Wnt signaling is initiated upon binding of Wnt proteins to Frizzled proteins and their co-receptors LRP5 and 6. The signal is then propagated to several downstream effectors, mediated by the phosphoprotein scaffold, dishevelled. We report a novel role for arginine methylation in regulating Wnt3a-stimulated LRP6 phosphorylation.

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.

Arginine methylation of G3BP1 in response to Wnt3a regulates β-catenin mRNA.

Wnt/β-catenin signaling is essential for normal mammalian development. Wnt3a activates the Wnt/β-catenin pathway through stabilization of β-catenin; a process in which the phosphoprotein Dishevelled figures prominently. Protein arginine methylation in signaling complexes containing Dishevelled was investigated.

AKAP5 and AKAP12 Form Homo-oligomers.

Background: A-kinase-anchoring proteins, AKAPs, constitute a family of scaffolds that play an essential role in catalyzing the spatial-temporal, dynamic interactions of protein kinase A, protein kinase C, tyrosine kinases, G-protein-coupled receptors and ion channels. We studied AKAP5 (AKAP79; MW ~47 kDa) and AKAP12 (gravin, SSECKS; MW ~191 kDa) to probe if these AKAP scaffolds oligomerize.

Results: In gel analysis and sodium-dodecyl sulfate denaturation, AKAP12 behaved with a MW of a homo-dimer.

Wnt signalling: the case of the 'missing' G-protein.

Wnt signalling remains a hot topic for cell signalling sleuthhounds. The trail of signalling downstream of the seven-transmembrane segment Frizzleds, which bind Wnt ligands, is replete of clues [e.g.

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.

Dishevelled-3 C-terminal His single amino acid repeats are obligate for Wnt5a activation of non-canonical signaling.

Background: The Wnt non-canonical pathway (Wnt5a > Frizzled-2 > cyclic GMP phosphodiesterase/Ca2+-mobilization pathway regulates the activation of NF-AT) is mediated by three mammalian Dishevelleds (Dvl1, Dvl2, and Dvl3) and the role of the C-terminal region unique to Dvl3 was interrogated.

Results: Dvl1, Dvl2, and Dvl3 are expressed at varying levels in mouse totipotent F9 embryonal teratocarcinoma cells. The expression of each endogenous Dvl isoform, as defined by knock-down with siRNA, was obligate for Wnt5a to activate NF-AT-sensitive transcription.

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.

AKAR2-AKAP12 fusion protein "biosenses" dynamic phosphorylation and localization of a GPCR-based scaffold.

Background: The cAMP-dependent protein kinase A (PKA) plays a pivotal role in virtually all cells, there being a multitude of important target molecules that are substrates for PKA in cell signaling. The spatial-temporal dynamics of PKA activation in living cells has been made accessible by the development of clever biosensors that yield a FRET signal in response to the phosphorylation by PKA. AKAR2 is genetically encoded fluorescent probe that acts as a biosensor for PKA activation.

Dishevelled-KSRP complex regulates Wnt signaling through post-transcriptional stabilization of beta-catenin mRNA.

Canonical Wnt/beta-catenin signaling is crucial during embryonic development. Upon Wnt stimulation, Dishevelled proteins relay the signal from upstream Frizzled receptors to downstream effectors. By using affinity purification followed by ion-trap mass spectrometry we identified K-homology splicing regulator protein (KSRP) as a novel Dishevelled-interacting protein.

Dishevelled-2 docks and activates Src in a Wnt-dependent manner.

Wnt3a activates the ;canonical' signaling pathway, stimulating the nuclear accumulation of beta-catenin and activation of Lef/Tcf-sensitive transcription of developmentally important genes. Using totipotent mouse F9 teratocarcinoma cells expressing frizzled-1 (Fz1), we investigated roles of tyrosine kinase activity in Wnt/beta-catenin signaling. Treatment with either genistein or Src family kinase inhibitor PP2 attenuates Wnt3a-stimulated Lef/Tcf transcription activation and primitive endoderm formation.

Mitogen-activated protein kinases and Wnt/beta-catenin signaling: Molecular conversations among signaling pathways.

Wnt/beta-catenin canonical pathway is critical for normal embryonic development; mutations and aberrant expression of specific components of this pathway can be oncogenic. Mitogen-activated protein kinase (MAPK) pathways, prominent in intracellular signaling, have been shown to have unique and provocative roles that impact the Wnt/beta-catenin signaling. We discuss recent insights that implicate the three major pathways of the MAPK network, i.

G-protein-coupled receptor-associated A-kinase anchoring proteins AKAP5 and AKAP12: differential signaling to MAPK and GPCR recycling.

Background: A-kinase Anchoring Protein AKAP5 and AKAP12 both dock to the beta2-adrenergic receptor, the former constitutively, the latter dynamically in response to activation of the receptor with agonist.

Results: In the current work we analyze the ability of each AKAP to contribute to two downstream signaling events, the activation of mitogen-activate protein kinase and the resensitization/recycling of the internalized, desensitized beta2-adrenergic receptor to the cell membrane. Although both AKAP share a large number of docking partners in common (e.

G-protein-coupled receptor-associated A-kinase anchoring proteins AKAP5 and AKAP12: differential trafficking and distribution.

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. AKAP5 and AKAP12 are multivalent (with respect to protein kinases and phosphatases) and display the ability to associate with the prototypic member of G protein-coupled receptors, the beta(2)-adrenergic receptor. We probed the relative abundance, subcellular distribution and localization of AKAP5 and AKAP12 in human embryonic kidney HEK293 and epidermoid carcinoma A431 cells.

p38 mitogen-activated protein kinase regulates canonical Wnt-beta-catenin signaling by inactivation of GSK3beta.

The Wnt-beta-catenin canonical signaling pathway is crucial for normal embryonic development, and aberrant expression of components of this pathway results in oncogenesis. Upon scanning for the mitogen-activated protein kinase (MAPK) pathways that might intersect with the canonical Wnt-beta-catenin signaling pathway in response to Wnt3a, we observed a strong activation of p38 MAPK in mouse F9 teratocarcinoma cells. Wnt3a-induced p38 MAPK activation was sensitive to siRNAs against Galpha(q) or Galpha(s), but not against either Galpha(o) or Galpha(11).

Wnt and Frizzled RNA expression in human mesenchymal and embryonic (H7) stem cells.

Background: Wnt signals are important for embryonic stem cells renewal, growth and differentiation. Although 19 Wnt, 10 Frizzled genes have been identified in mammals, their expression patterns in stem cells were largely unknown.

Results: We conducted RNA expression profiling for the Wnt ligands, their cellular receptors "Frizzleds" and co-receptors LRP5/6 in human embryonic stem cells (H7), human bone marrow mesenchymal cells, as well as mouse totipotent F9 teratocarcinoma embryonal cells.

Altered gene expression in early atherosclerosis is blocked by low level apolipoprotein E.

Background: Mice deficient in apolipoprotein E (apoE(-/-)) develop atherosclerosis. The possible linkage between expression of adhesion molecules/cofactors and atherosclerosis was probed at the level of mRNA and protein expression. The hypothesis of a linkage between changes of adhesion molecules/cofactors and atherosclerosis was tested further by suppression of aortic lesion formation in apoE(-/-) mice by expression of very low levels of transgenic apolipoprotein E.

OSTM1 regulates beta-catenin/Lef1 interaction and is required for Wnt/beta-catenin signaling.

The Wnt/beta-catenin signaling pathway controls key aspects of embryonic development and adult tissue homeostasis, including the formation and maintenance of bone. Recently, mutations in the OSTM1 gene were found to be the cause of severe autosomal recessive osteopetrosis in both the mouse and humans. This disorder is characterized by increased bone mass resulting from a defect in osteoclast maturation.

G alpha o mediates WNT-JNK signaling through dishevelled 1 and 3, RhoA family members, and MEKK 1 and 4 in mammalian cells.

In Drosophila, activation of Jun N-terminal Kinase (JNK) mediated by Frizzled and Dishevelled leads to signaling linked to planar cell polarity. A biochemical delineation of WNT-JNK planar cell polarity was sought in mammalian cells, making use of totipotent mouse F9 teratocarcinoma cells that respond to WNT3a via Frizzled-1. The canonical WNT-beta-catenin signaling pathway requires both G alpha o and G alpha q heterotrimeric G-proteins, whereas we show that WNT-JNK signaling requires only G alpha o protein.

Phosphoprotein phosphatase-2A docks to Dishevelled and counterregulates Wnt3a/beta-catenin signaling.

Background: Wnt3a stimulates cellular trafficking of key signaling elements (e.g., Axin, Dishevelled-2, beta-catenin, and glycogen synthase kinase-3beta) and primitive endoderm formation in mouse F9 embryonic teratocarcinoma cells.