Journal of Molecular Signaling最新文献

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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.

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.

Background: The present study examines the hypothesis that Akt (protein kinase B)/mTOR (mammalian target of rapamycin) signaling is increased in hypertrophic and decreased in atrophic denervated muscle. Protein expression and phosphorylation of Akt1, Akt2, glycogen synthase kinase-3beta (GSK-3beta), eukaryotic initiation factor 4E binding protein 1 (4EBP1), 70 kD ribosomal protein S6 kinase (p70S6K1) and ribosomal protein S6 (rpS6) were examined in six-days denervated mouse anterior tibial (atrophic) and hemidiaphragm (hypertrophic) muscles.

Results: In denervated hypertrophic muscle expression of total Akt1, Akt2, GSK-3beta, p70S6K1 and rpS6 proteins increased 2-10 fold whereas total 4EBP1 protein remained unaltered. In denervated atrophic muscle Akt1 and Akt2 total protein increased 2-16 fold. A small increase in expression of total rpS6 protein was also observed with no apparent changes in levels of total GSK-3beta, 4EBP1 or p70S6K1 proteins. The level of phosphorylated proteins increased 3-13 fold for all the proteins in hypertrophic denervated muscle. No significant changes in phosphorylated Akt1 or GSK-3beta were detected in atrophic denervated muscle. The phosphorylation levels of Akt2, 4EBP1, p70S6K1 and rpS6 were increased 2-18 fold in atrophic denervated muscle.

Conclusions: The results are consistent with increased Akt/mTOR signaling in hypertrophic skeletal muscle. Decreased levels of phosphorylated Akt (S473/S474) were not observed in denervated atrophic muscle and results downstream of mTOR indicate increased protein synthesis in denervated atrophic anterior tibial muscle as well as in denervated hypertrophic hemidiaphragm muscle. Increased protein degradation, rather than decreased protein synthesis, is likely to be responsible for the loss of muscle mass in denervated atrophic muscles.

Background: Somatostatin (SST) via five Gi coupled receptors namely SSTR1-5 is known to inhibit cell proliferation by cytostatic and cytotoxic mechanisms. Heterodimerization plays a crucial role in modulating the signal transduction pathways of SSTR subtypes. In the present study, we investigated human SSTR2/SSTR3 heterodimerization, internalization, MAPK signaling, cell proliferation and apoptosis in HEK-293 cells in response to SST and specific agonists for SSTR2 and SSTR3.

Results: Although in basal conditions, SSTR2 and SSTR3 colocalize at the plasma membrane and exhibit heterodimerization, the cell surface distribution of both receptors decreased upon agonist activation and was accompanied by a parallel increase in intracellular colocalization. Receptors activation by SST and specific agonists significantly decreased cAMP levels in cotransfected cells in comparison to control. Agonist-mediated modulation of pERK1/2 was time and concentration-dependent, and pronounced in serum-deprived conditions. pERK1/2 was inhibited in response to SST; conversely receptor-specific agonist treatment caused inhibition at lower concentration and activation at higher concentration. Strikingly, ERK1/2 phosphorylation was sustained upon prolonged treatment with SST but not with receptor-specific agonists. On the other hand, SST and receptor-specific agonists modulated p38 phosphorylation time-dependently. The receptor activation in cotransfected cells exhibits Gi-dependent inhibition of cell proliferation attributed to increased PARP-1 expression and TUNEL staining, whereas induction of p21 and p27Kip1 suggests a cytostatic effect.

Conclusion: Our study provides new insights in SSTR2/SSTR3 mediated signaling which might help in better understanding of the molecular interactions involving SSTRs in tumor biology.

The canonical Wnt signaling pathway is crucial for embryonic development and adult tissue homeostasis. Activating mutations in the Wnt pathway are frequently associated with the pathogenesis of various types of cancer, particularly colon cancer. Upon Wnt stimulation, β-catenin plays a central role as a coactivator through direct interaction with Tcf/Lef transcription factors to stimulate target gene expression. We have previously shown that the evolutionarily conserved protein Chibby (Cby) physically binds to β-catenin to repress β-catenin-dependent gene activation by 1) competing with Tcf/Lef factors for binding to β-catenin and 2) facilitating nuclear export of β-catenin via interaction with 14-3-3 proteins. In this study, we employed human colon adenocarcinoma SW480 cells with high levels of endogenous β-catenin to address a potential tumor suppressor role of Cby. In SW480 stable cells expressing wild-type Cby (CbyWT), but not 14-3-3-binding- defective Cby mutant CbyS20A, a significant fraction of endogenous β-catenin was detected in the cytoplasm. Consistent with this, CbyWT-expressing cells showed low levels of β-catenin signaling activity, leading to reduced growth. Our results suggest that Cby, in collaboration with 14-3-3 proteins, can counteract oncogenic β-catenin signaling in colon cancer cells.

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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 β-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 β-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.

Dopaminergic inputs are sensed on the cell surface by the seven-transmembrane dopamine receptors that belong to a superfamily of G-protein-coupled receptors (GPCRs). Dopamine receptors are classified as D1-like or D2-like receptors based on their homology and pharmacological profiles. In addition to well established G-protein coupled mechanism of dopamine receptors in mammalian system they can also interact with other signaling pathways. In C. elegans four dopamine receptors (dop-1, dop-2, dop-3 and dop-4) have been reported and they have been implicated in a wide array of behavioral and physiological processes. We performed this study to assign the signaling pathway for DOP-2, a D2-like dopamine receptor using a split-ubiquitin based yeast two-hybrid screening of a C. elegans cDNA library with a novel dop-2 variant (DOP-2XL) as bait. Our yeast two-hybrid screening resulted in identification of gpa-14, as one of the positively interacting partners. gpa-14 is a Gα coding sequence and shows expression overlap with dop-2 in C. elegans ADE deirid neurons. In-vitro pull down assays demonstrated physical coupling between dopamine receptor DOP-2XL and GPA-14. Further, we sought to determine the DOP-2 region necessary for GPA-14 coupling. We generated truncated DOP-2XL constructs and performed pair-wise yeast two-hybrid assay with GPA-14 followed by in-vitro interaction studies and here we report that the third intracellular loop is the key domain responsible for DOP-2 and GPA-14 coupling. Our results show that the extra-long C. elegans D2-like receptor is coupled to gpa-14 that has no mammalian homolog but shows close similarity to inhibitory G-proteins. Supplementing earlier investigations, our results demonstrate the importance of an invertebrate D2-like receptor's third intracellular loop in its G-protein interaction.

Background: Neurotrophins (NTs) and their receptors play crucial roles in the development, functions and maintenance of nervous systems. It is widely believed that NT-induced dimerization of the receptors initiates the transmembrane signaling. However, it is still controversial whether the receptor molecule has a monomeric or dimeric structure on the cell surface before its ligand binding.

Findings: Using chemical cross-linking, bimolecular fluorescence complementation (BiFC) and luciferase fragment complementation (LFC) assays, in this study, we show the brain-derived neurotrophic factor (BDNF) receptor TrkB exists as a homodimer before ligand binding. We have also found by using BiFC and LFC that the dimer forms in the endoplasmic reticulum (ER), and that the receptor lacking its intracellular domain cannot form the dimeric structure.

Conclusions: Most, if not all, of the TrkB receptor has a preformed, yet inactive, homodimeric structure before BDNF binding. The intracellular domain of TrkB plays a crucial role in the spontaneous dimerization of the newly synthesized receptors, which occurs in ER. These findings provide new insight into an understanding of a molecular mechanism underlying transmembrane signaling mediated by NT receptors.

Paget's disease of bone (PDB) is a skeletal disorder characterized by focal and disorganized increases in bone turnover and overactive osteoclasts. The discovery of mutations in the SQSTM1/p62 gene in numerous patients has identified protein p62 as an important modulator of bone turnover. In both precursors and mature osteoclasts, the interaction between receptor activator of NF-κB ligand (RANKL) and its receptor RANK results in signaling cascades that ultimately activate transcription factors, particularly NF-κB and NFATc1, promoting and regulating the osteoclast differentiation, activity, and survival. As a scaffold with multiple protein-protein interaction motifs, p62 is involved in virtually all the RANKL-activated osteoclast signaling pathways, along with being implicated in numerous other cellular processes. The p62 adaptor protein is one of the functional links reported between RANKL and TRAF6-mediated NF-κB activation, and also plays a major role as a shuttling factor that targets polyubiquitinated proteins for degradation by either the autophagy or proteasome pathways. The dysregulated expression and/or activity of p62 in bone disease up-regulates osteoclast functions. This review aims to outline and summarize the role of p62 in RANKL-induced signaling pathways and in ubiquitin-mediated signaling in osteoclasts, and the impact of PDB-associated p62 mutations on these processes.

Background: Synthetic GCs serve as therapeutic agents for some lymphoid leukemias because of their ability to induce transcriptional changes via the GC receptor (GR) and trigger apoptosis. Upregulation of the BH3-only member of Bcl-2 family proteins, Bim, has been shown to be essential for GC-evoked apoptosis of leukemic lymphoblasts. Using human T cell leukemic sister clones CEM-C7-14 and CEM-C1-15, we have previously shown that the bZIP transcriptional repressor, E4BP4, is preferentially upregulated by GCs in CEM-C7-14 cells that are susceptible to GC-evoked apoptosis, but not in refractory CEM-C1-15 cells. E4BP4 is an evolutionarily conserved member of the PAR family of bZIP transcription factors related to the C. elegans death specification gene ces2.

Results: Mouse E4BP4 was ectopically expressed in CEM-C1-15 cells, resulting in sensitization to GC-evoked apoptosis in correlation with restoration of E4BP4 and Bim upregulation. shRNA mediated modest knockdown of E4BP4 in CEM-C7-14 cells resulted in concomitant reduction in Bim expression, although GC-evoked fold-induction and sensitivity to apoptosis was similar to parental cells.

Conclusion: Data presented here suggest that GC-mediated upregulation of E4BP4 facilitates Bim upregulation and apoptosis of CEM cells. Since the Bim promoter does not contain any consensus GRE or EBPRE sequences, induction of Bim may be a secondary response.

Background: Wnt/β-catenin signaling is involved in various aspects of skeletal muscle development and regeneration. In addition, Wnt3a and β-catenin are required for muscle-specific gene transcription in embryonic carcinoma cells and satellite-cell proliferation during adult skeletal muscle regeneration. Downstream targets of canonical Wnt signaling are cyclin D1 and c-myc. However both target genes are suppressed during differentiation of mouse myoblast cells, C2C12. Underlying molecular mechanisms of β-catenin signaling during myogenic differentiation remain unknown.

Results: Using C2C12 cells, we examined intracellular signaling and gene transcription during myoblast proliferation and differentiation. We confirmed that several Wnt signaling components, including Wnt9a, Sfrp2 and porcupine, were consistently upregulated in differentiating C2C12 cells. Troponin T-positive myotubes were decreased by Wnt3a overexpression, but not Wnt4. TOP/FOP reporter assays revealed that co-expression with Wnt4 reduced Wnt3a-induced luciferase activity, suggesting that Wnt4 signaling counteracted Wnt3a signaling in myoblasts. FH535, a small-molecule inhibitor of β-catenin/Tcf complex formation, reduced basal β-catenin in the cytoplasm and decreased myoblast proliferation. K252a, a protein kinase inhibitor, increased both cytosolic and membrane-bound β-catenin and enhanced myoblast fusion. Treatments with K252a or Wnt4 resulted in increased cytoplasmic vesicles containing phosphorylated β-catenin (Tyr654) during myogenic differentiation.

Conclusions: These results suggest that various Wnt ligands control subcellular β-catenin localization, which regulate myoblast proliferation and myotube formation. Wnt signaling via β-catenin likely acts as a molecular switch that regulates the transition from cell proliferation to myogenic differentiation.