Hsien-yu Wang

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 Gαt2 or Gαo. 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.

Gsα Repression of Adipogenesis via Syk

Gsα regulates the differentiation of 3T3-L1 mouse embryonic fibroblasts to adipocytes, a process termed adipogenesis. Inducers of adipogenesis lead to a loss of Gsα and derepress differentiation to adipocytes. The broad spectrum tyrosine kinase inhibitor genistein is shown to block induction of adipogenesis, suggesting an early role of tyrosine phosphorylation in adipogenesis. Staining of phosphotyrosine identified prominent staining of a ∼70-kDa protein, hypothesized to be the tyrosine kinase Syk. Reverse transcription and polymerase chain reaction amplification established the expression of Syk mRNA in these embryonic fibroblasts. Immunoprecipitations with Syk-specific antibodies demonstrated the presence of Syk in fibroblasts and a rapid increase in the amount of phospho-Syk, peaking at 24 h post induction. Clones constitutively expressing Gsα, which can no longer be induced to differentiate, no longer display increased phospho-Syk levels in response to inducers. The linkage between Gsα and Syk was probed by immunoprecipitations revealing association of Syk with Gsα in the absence of induction. Upon induction of adipogenesis, Gsα levels decline and phospho-Syk levels as well as Syk kinase activity increase. Expression of wild-type Syk both potentiates the ability of inducers to act as well as induces adipogenesis itself. Expression of the kinase-deficient Syk had no such effects on adipogenesis. These data provide a new insight into the control of adipogenesis, suggesting that Gsα represses adipogenesis via Syk. Treatment with the inducers promotes a decline in Gsα, increases in levels of phospho-Syk, and adipogenesis.

Akt Mediates Sequestration of the β2-Adrenergic Receptor in Response to Insulin

The counterregulation of catecholamine action by insulin includes insulin-stimulated sequestration of the β2-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 β2-adrenergic receptors. Inhibition of 1-phosphatidylinositol 3-kinase by LY294002 blocks insulin-induced sequestration of the β2-adrenergic receptor, implicating Akt in downstream signaling to the β2-adrenergic receptor. Phosphorylation studies of the C-terminal cytoplasmic domain of the β2-adrenergic receptor by Akt in vitroidentified Ser345 and Ser346 within a consensus motif for Akt phosphorylation. Double mutation (i.e.S345A/S346A) within this motif abolishes insulin counterregulation of β-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 β2-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 β2-adrenergic receptor. The action of the serine-threonine protein kinase Akt in insulin counterregulation mirrors the central role of protein kinase A in β-agonist-induced desensitization.

Conditional, Tissue-specific Expression of Q205L Gαi2 in Vivo Mimics Insulin Activation of c-Jun N-terminal Kinase and p38 Kinase

Deficiency of the G-protein subunit Gαi2 impairs insulin action (Moxham, C. M., and Malbon, C. C. (1996) Nature 379, 840–844). By using the promoter for the phosphoenolpyruvate carboxykinase gene, conditional, tissue-specific expression of the constitutively active mutant form (Q205L) of Gαi2 was achieved in mice harboring the transgene. Expression of Q205L Gαi2 was detected in skeletal muscle, liver, and adipose tissue of transgenic mice. Whereas the Gαi2-deficient mice displayed blunted insulin action, the Q205L Gαi2-expressing mice displayed enhanced insulin-like effects. Glycogen synthase in skeletal muscle was found to be activated in Q205L Gαi2-expressing mice, in the absence of the administration of insulin. Analysis of members of mitogen-activated protein kinase family revealed that both c-Jun N-terminal kinase and p38 are constitutively activated in vivo in the mice that express the Q205L Gαi2. ERK1,2, in contrast, are unaffected in the Q205L Gαi2-expressing mice. Insulin, like expression of Q205L Gαi2, activates both p38 and c-Jun N-terminal kinases as well as glycogen synthase. Activation of c-Jun N-terminal and p38 kinases in vivo with anisomycin, however, was insufficient to activate glycogen synthase. Much like Gαi2 deficiency provokes insulin resistance, expression of Q205L constitutively active Gαi2 mimics insulin action in vivo, sharing with insulin the activation of two mitogen-activated protein kinase members, p38 and c-Jun N-terminal kinases.

Insulin activation of mitogen-activated protein kinases Erk1,2 is amplified via beta-adrenergic receptor expression and requires the integrity of the Tyr350 of the receptor.

Insulin activates a complex set of intracellular responses, including the activation of mitogen-activated protein kinases Erk1,2. The counterregulatory actions of insulin on catecholamine action are well known and include phosphorylation of the β2-adrenergic receptor on Tyr350, Tyr354, and Tyr364 in the C-terminal cytoplasmic domain, as well as enhanced sequestration of the β2-adrenergic receptor. Both β-adrenergic agonists and insulin provoke sequestration of β2-adrenergic receptors in a synergistic manner. In the current work, cross-talk between insulin action and β2-adrenergic receptors revealed that insulin activation of Erk1,2 was amplified via β2-adrenergic receptors. In Chinese hamster ovary cells, expression of β2-adrenergic receptors enhanced 5–10-fold the activation of Erk1,2 by insulin and prolonged the activation, the greatest enhancement occurring at 5 min post-insulin. The potentiation of insulin signaling on Erk1,2 was proportional to the level of expression of β2-adrenergic receptor. The potentiation of insulin signaling requires the integrity of Tyr350 of the β2-adrenergic receptor, a residue phosphorylated in response to insulin. β2-adrenergic receptors with a Y350F mutation failed to potentiate insulin activation of Erk1,2. Expression of the C-terminal domain of the β2-adrenergic receptor (Pro323-Leu418) in cells expressing the intact β2-adrenergic receptor acts as a dominant negative, blocking the potentiation of insulin activation of Erk1,2 via the β2-adrenergic receptor. Blockade of β2-adrenergic receptor sequestration does not alter the ability of the β2-adrenergic receptor to potentiate insulin action on Erk1,2. We propose a new paradigm in which a G-protein-linked receptor, such as the β2-adrenergic receptor, itself acts as a receptor-based scaffold via its binding site for Src homology 2 domains, facilitating signaling of the mitogen-activated protein kinase pathway by insulin.

Insulin Stimulates Phosphorylation of the β2-Adrenergic Receptor by the Insulin Receptor, Creating a Potent Feedback Inhibitor of Its Tyrosine Kinase

Insulin counterregulates catecholamine action at several levels, primarily in liver, fat, and adipose tissue. Herein we observe that expression of increased levels of β2-adrenergic receptor increasingly inhibits insulin-stimulated phosphorylation of its primary downstream substrates (IRS-1,2). In Chinese hamster ovary cells, the insulin receptor phosphorylates dominantly Tyr364 in the C-terminal cytoplasmic domain of the β-receptor. A Y364A mutant form of the β2-adrenergic, in contrast, loses it ability to inhibit insulin-stimulated phosphorylation of IRS-1,2. Upon phosphorylation, the C-terminal cytoplasmic domain of the β2-adrenergic receptor demonstrates a potent inhibitory feedback action that can block both insulin-stimulated autophosphorylation of the insulin receptor and phosphorylation of IRS-1,2 in NIH mouse 3T3-L1 adipocyte membranes. Studies in vitro with purified insulin receptor and the C-terminal cytoplasmic domain of the β2-adrenergic receptor demonstrate that the tyrosine-phosphorylated β-receptor domain is a potent counterregulatory inhibitor of the insulin receptor tyrosine kinase.

[1] G proteins controlling differentiation, growth, and development: Analysis by antisense RNA/DNA technology

Publisher Summary This chapter demonstrates that G proteins play an important role in complex biological processes, such as mitogenesis, oncogenesis, neonatal growth, and cellular differentiation. Antisense RNA/DNA technology provides a new strategy with which to explore the role(s) of specific subunits in both transmembrane signaling, as well as the more complex biological responses. The experience gained with the use of antisense RNA approaches includes oligodeoxynucleotides antisense to the mRNA of G sα , employed to probe the roles of this subunit in adipogenesis; retroviral expression vectors capable of generating constitutive expression of RNA antisense to G proteins. Specifically G iα2 , employed to study the role of this subunit in stem cell development to parietal and primitive endoderm; and antisense vectors activated on birth in transgenic animals to examine the roles of G iα2 and G qα in neonatal development and metabolism. Although not without limitations and constraints for experimental use, antisense RNA/DNA technology now provides flexible, powerful tools for defining functional consequences of eliminating one or more members of the G protein family both in vitro and in vivo . Application of these technologies permits to address the fundamental and unresolved questions about G proteins and their functions. This technology has applications relevant to the treatment of human diseases, much like gene therapy.