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Hsien-yu Wang

Publications and source records attributed to Hsien-yu Wang.

18 recordsLinked to original sources
PubMed2006

Suppression of cyclic GMP-dependent protein kinase is essential to the Wnt/cGMP/Ca2+ pathway.

Novel downstream effectors sensing changes in intracellular concentrations of Ca2+ and cyclic GMP in response to activation of the Wnt/Frizzled-2 pathway were sought. Activation of Frizzled-2 suppressed protein kinase G activity while activating NF-AT-dependent transcription. Each of these responses was abolished by pertussis toxin and by knock-down of the expression of either Galphat2 or Galphao. Activation of NF-AT-dependent transcription in response to Wnt5a stimulation was suppressed by activation of protein kinase G and by buffering intracellular Ca2+. Elevation of intracellular cyclic GMP either by inhibition of cyclic GMP phosphodiesterase or by addition of 8-bromocyclic GMP was shown to activate protein kinase G, to block Ca2+ mobilization, as well as to markedly attenuate activation of NF-AT-dependent transcription in response to Wnt5a stimulation. Chemical inhibition of protein kinase G by Rp-8-pCPT-cGMP, conversely, was shown to provoke increased NF-AT gene transcription and Ca2+ mobilization in the absence of Wnt stimulation. Protein kinase G is shown to be a critical downstream effector of the noncanonical Wnt-Frizzled-2/cGMP/Ca2+ pathway.

Animals↗
PubMed2006

Regulation of AKAP-membrane interactions by calcium.

The AKAP gravin is a scaffold for protein kinases, phosphatases, and adaptor molecules obligate for resensitization and recycling of beta2-adrenergic receptors. Gravin binds to the receptor through well characterized protein-protein interactions. These interactions are facilitated approximately 1000-fold when gravin is anchored to the cytoplasmic leaflet of the plasma membrane. Although the N-terminal region (approximately 550 residues) is highly negatively charged and probably natively unfolded, it could anchor gravin to the inner leaflet through hydrophobic insertion of its N-terminal myristate and electrostatic binding of three short positively charged domains (PCDs). Loss of the site of N-myristoylation was found to affect neither AKAP macroscopic localization nor AKAP function. Synthetic peptides corresponding to PCD1-3 bound in vitro to unilamellar phospholipid vesicles with high affinity, a binding reversed by calmodulin in the presence of Ca2+. In vivo gravin localization is regulated by intracellular Ca2+, a function mapping to the N terminus of the protein harboring PCD1, PCD2, and PCD3. Mutation of any two PCDs eliminates membrane association of the non-myristoylated gravin, the sensitivity to Ca2+/calmodulin, and the ability of this scaffold to catalyze receptor resensitization and recycling.

A Kinase Anchor Proteins↗
PubMed2006

Casein kinase 2 Is activated and essential for Wnt/beta-catenin signaling.

Wnt/beta-catenin signaling is essential to early development. Activation of Frizzled-1 by Wnts induces nuclear accumulation of beta-catenin and activation of Lef/Tcf-dependent gene expression. Casein kinase 2 has been shown to affect Wnt/beta-catenin signaling. How casein kinase 2 exerts an influence in Wnt signaling is not clear; casein kinase 2 has been reported to be constitutively active (i.e. not regulated). Herein we show to the contrary that casein kinase 2 activity is rapidly and transiently increased in response to Wnt3a stimulation and is essential for Wnt/beta-catenin signaling. Chemical inhibition of casein kinase 2 or suppression of its expression blocks Frizzled-1 activation of Lef/Tcf-sensitive gene expression. Treatment with pertussis toxin or knock down of Galpha(q) or Galpha(o) blocks Wnt stimulation of casein kinase 2 activation, as does suppression of the phosphoprotein Dishevelled, demonstrating that casein kinase 2 is downstream of heterotrimeric G proteins and Dishevelled. Expression of a constitutively active mutant of either Galpha(q) or Galpha(o) stimulates casein kinase 2 activation and Lef/Tcf-sensitive gene expression. Thus, casein kinase 2 is shown to be regulated by Wnt3a and essential to stimulation of the Frizzled-1/beta-catenin/Lef-Tcf pathway.

Adaptor Proteins, Signal Transducing↗
PubMed2006

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.

Amino Acid Sequence↗
PubMed2006

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.

Animals↗
PubMed2004

G alpha 13 signals via p115RhoGEF cascades regulating JNK1 and primitive endoderm formation.

The heterotrimeric G-protein G(13) mediates the formation of primitive endoderm from mouse P19 embryonal carcinoma cells in response to retinoic acid, signaling to the level of activation of c-Jun N-terminal kinase. The signal linkage map from MEKK1/MEKK4 to MEK1/MKK4 to JNK is obligate in this G alpha(13)-mediated pathway, whereas that between G alpha(13) and MEKKs is not known. The overall pathway to primitive endoderm formation was shown to be inhibited by treatment with Clostridium botulinum C3 exotoxin, a specific inactivator of RhoA family members. Constitutively active G alpha(13) was found to activate RhoA as well as Cdc42 and Rac1 in these cells. Although constitutively active Cdc42, Rac1, and RhoA all can activate JNK1, only the RhoA mutant was able to promote formation of primitive endoderm, mimicking expression of the constitutively activated G alpha(13). Expression of the constitutively active mutant form of p115RhoGEF (guanine nucleotide exchange factor) was found to activate RhoA and JNK1 activities. Expression of the dominant negative p115RhoGEF was able to inhibit activation of both RhoA and JNK1 in response to either retinoic acid or the expression of a constitutively activated mutant of G alpha(13). Expression of the dominant negative mutants of RhoA as well as those of either Cdc42 or Rac1, but not Ras, attenuated G alpha(13)-stimulated as well as retinoic acid-stimulated activation of all three of these small molecular weight GTPases, suggesting complex interrelationships among the three GTPases in this pathway. The formation of primitive endoderm in response to retinoic acid also could be blocked by expression of dominant negative mutants of RhoA, Cdc42, or Rac1. Thus, the signal propagated from G alpha(13) to JNK requires activation of p115RhoGEF cascades, including p115RhoGEF itself, RhoA, Cdc42, and Rac1. In a concerted effort, RhoA in tandem with Cdc42 and Rac1 activates the MEKK1/4, MEK1/MKK4, and JNK cascade, thereby stimulating formation of primitive endoderm.

Animals↗
PubMed2004

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.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed2004

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.

Amino Acid Sequence↗
PubMed2004

Trafficking of beta2-adrenergic receptors: insulin and beta-agonists regulate internalization by distinct cytoskeletal pathways.

Insulin and beta-adrenergic agonists stimulate a rapid phosphorylation and sequestration of the beta2-adrenergic receptors (beta2ARs). Although the expectation was that a common pathway would be involved in the trafficking of the beta2AR in response to either hormone, studies reported herein show the existence of unique cytoskeletal requirements for internalization/recycling of G-protein-coupled receptors, such as the beta2AR. Treatment of human epidermoid carcinoma A431 cells with nocodazole, which binds tubulin monomer in vivo and catalyzes the depolymerization of microtubules, effectively blocks beta-adrenergic agonist-induced, but not insulin-induced, sequestration of beta2ARs. Treatment with latrunculin-A, an agent that sequesters actin monomer and leads to loss of actin filaments, had no effect on the ability of beta-adrenergic agonists to stimulate internalization of beta2ARs, but blocked the ability of insulin to stimulate counterregulation of beta2ARs via internalization. Although nocodazole had no effect on insulin-stimulated sequestration of beta2ARs, the recycling of the internalized receptors to the cell membrane was sensitive to depolymerization of microtubules by this agent. Latrunculin-A, by contrast, blocks the recycling of beta2ARs internalized in response to beta-agonist, while attenuating recycling of receptors internalized in response to insulin stimulation. These data show the existence of unique cytoskeletal requirements for G-protein-coupled-receptor trafficking in response to agonist compared with a counterregulatory hormone, and for sequestration versus recycling of the receptors to the cell membrane.

Adrenergic beta-Agonists↗
PubMed2004

Probing receptor structure/function with chimeric G-protein-coupled receptors.

Owing its name to an image borrowed from Greek mythology, a chimera is seen to represent a new entity created as a composite from existing creatures or, in this case, molecules. Making use of various combinations of three basic domains of the receptors (i.e., exofacial, transmembrane, and cytoplasmic segments) that couple agonist binding into activation of effectors through heterotrimeric G-proteins, molecular pharmacology has probed the basic organization, structure/function relationships of this superfamily of heptahelical receptors. Chimeric G-protein-coupled receptors obviate the need for a particular agonist ligand when the ligand is resistant to purification or, in the case of orphan receptors, is not known. Chimeric receptors created from distant members of the heptahelical receptors enable new strategies in understanding how these receptors transduce agonist binding into receptor activation and may be able to offer insights into the evolution of G-protein-coupled receptors from yeast to humans.

Animals↗
PubMed2003

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.

Adult↗
PubMed2002

Signaling of rat Frizzled-2 through phosphodiesterase and cyclic GMP.

The Frizzled-2 receptor (Rfz2) from rat binds Wnt proteins and can signal by activating calcium release from intracellular stores. We show that wild-type Rfz2 and a chimeric receptor consisting of the extracellular and transmembrane portions of the beta2-adrenergic receptor with cytoplasmic domains of Rfz2 also signaled through modulation of cyclic guanosine 3',5'-monophosphate (cGMP). Activation of either receptor led to a decline in the intracellular concentration of cGMP, a process that was inhibited in cells treated with pertussis toxin, reduced by suppression of the expression of the heterotrimeric GTP-binding protein (G protein) transducin, and suppressed through inhibition of cGMP-specific phosphodiesterase (PDE) activity. Moreover, PDE inhibitors blocked Rfz2-induced calcium transients in zebrafish embryos. Thus, Frizzled-2 appears to couple to PDEs and calcium transients through G proteins.

Amino Acid Sequence↗
PubMed2002

Activation of the beta-catenin/Lef-Tcf pathway is obligate for formation of primitive endoderm by mouse F9 totipotent teratocarcinoma cells in response to retinoic acid.

The morphogen retinoic acid promotes the formation of primitive endoderm in mouse F9 teratocarcinoma cells as does the stimulation of the Frizzled-1 pathway. We investigated whether the beta-catenin/Lef-Tcf-sensitive transcriptional pathway activated by Frizzled-1 plays a role in the retinoic acid-induced pathway to primitive endoderm formation. An analysis of Lef-Tcf-sensitive transcription reveals increased transcription at 1 and 4 h post-treatment with retinoic acid. The stimulation of Lef-Tcf-sensitive transcription as well as the formation of primitive endoderm was accompanied by the stabilization of beta-catenin as observed in activation of the Frizzled-1 pathway. Transient transfection of F9 cells with an expression vector harboring a dominant-negative mutant of Tcf4 resulted in the attenuation of both the increase in Lef-Tcf-sensitive transcription and formation of primitive endoderm in response to the morphogen. Clones stably transfected to express the dominant-negative Tcf4 displayed a block in retinoic acid-induced activation of Lef-Tcf-sensitive transcription and primitive endoderm formation. These data reveal the obligate role of the beta-catenin/Lef-Tcf transcriptional pathway in the action of the morphogen retinoic acid.

Animals↗
PubMed2002

Akt mediates sequestration of the beta(2)-adrenergic receptor in response to insulin.

The counterregulation of catecholamine action by insulin includes insulin-stimulated sequestration of the beta(2)-adrenergic receptor. Herein we examined the signaling downstream of insulin receptor activation, focusing upon the role of 1-phosphatidylinositol 3-kinase and the serine-threonine protein kinase Akt (also known as protein kinase B) in the internalization of beta(2)-adrenergic receptors. Inhibition of 1-phosphatidylinositol 3-kinase by LY294002 blocks insulin-induced sequestration of the beta(2)-adrenergic receptor, implicating Akt in downstream signaling to the beta(2)-adrenergic receptor. Phosphorylation studies of the C-terminal cytoplasmic domain of the beta(2)-adrenergic receptor by Akt in vitro identified Ser(345) and Ser(346) within a consensus motif for Akt phosphorylation. Double mutation (i.e. S345A/S346A) within this motif abolishes insulin counterregulation of beta-adrenergic stimulation of cyclic AMP accumulation as well as insulin-stimulated sequestration. Furthermore, expression of constitutively activated Akt (T308D/S473D) mimics insulin action on cyclic AMP responses and beta(2)-adrenergic receptor internalization. Expression of the dominant-negative version of Akt (K179A/T308A/S473A), in contrast, abolishes both insulin counterregulation of the cyclic AMP response as well as insulin-stimulated sequestration of the beta(2)-adrenergic receptor. The action of the serine-threonine protein kinase Akt in insulin counterregulation mirrors the central role of protein kinase A in beta-agonist-induced desensitization.

Amino Acid Sequence↗
PubMed2002

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.

3T3 Cells↗
PubMed2001

Expression of Galpha 13 (Q226L) induces P19 stem cells to primitive endoderm via MEKK1, 2, or 4.

Galpha13 mediates the ability of the morphogen retinoic acid to promote primitive endoderm formation from mouse P19 embryonal carcinoma stem cells, a process that includes the obligate activation of Jun N-terminal kinase. Expression of the constitutively activated (Q226L) GTPase-deficient form of Galpha13 mimics retinoic acid and was used to investigate the signaling upstream of primitive endoderm formation. Jun N-terminal kinase 1 activity, MEK1,2, MKK4, and MEKK1 were constitutively activated in clones stably transfected to express Q226L Galpha13. Dominant negative forms of MEKK1 and MEKK4 were expressed stably in the clones harboring Q226L Galpha13. Expression of dominant negative versions of either MEKK1 or MEKK4 effectively blocks both the activation of Jun N-terminal kinase as well as the formation of primitive endoderm. Depletion of MEKK1, -2, or -4 by antisense oligodeoxynucleotides suppressed signaling from Q226L Galpha13 to JNK1 and primitive endoderm formation. We demonstrate that the signal linkage map from Galpha13 activation to primitive endoderm formation in these stem cells requires activation at three levels of the mitogen-activated protein kinase cascade: MEKK1, -2, or -4 for MAP kinase kinase kinase; MKK4 and/or MEK1 for MAP kinase kinase; and JNK1 for MAP kinase.

3T3 Cells↗