http://www.jmolecularsignaling.com The latest research articles published by Journal of Molecular Signaling 2012-05-14T00:00:00Z 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 beta-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 beta-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. http://www.jmolecularsignaling.com/content/7/1/4 Salih Kocer Hsien-yu Wang Craig Malbon Journal of Molecular Signaling 2012, null:4 2012-05-14T00:00:00Z doi:10.1186/1750-2187-7-4 /content/figures/1750-2187-7-4-toc.gif Journal of Molecular Signaling 1750-2187 ${item.volume} 4 2012-05-14T00:00:00Z PDF 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. http://www.jmolecularsignaling.com/content/7/1/3 Pratima Pandey Singh Harbinder Journal of Molecular Signaling 2012, null:3 2012-01-26T00:00:00Z doi:10.1186/1750-2187-7-3 /content/figures/1750-2187-7-3-toc.gif Journal of Molecular Signaling 1750-2187 ${item.volume} 3 2012-01-26T00:00:00Z XML 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.FindingsUsing 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. http://www.jmolecularsignaling.com/content/7/1/2 Jianying Shen Ichiro Maruyama Journal of Molecular Signaling 2012, null:2 2012-01-24T00:00:00Z doi:10.1186/1750-2187-7-2 /content/figures/1750-2187-7-2-toc.gif Journal of Molecular Signaling 1750-2187 ${item.volume} 2 2012-01-24T00:00:00Z XML 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. http://www.jmolecularsignaling.com/content/7/1/1 Stephen McManus Sophie Roux Journal of Molecular Signaling 2012, null:1 2012-01-04T00:00:00Z doi:10.1186/1750-2187-7-1 /content/figures/1750-2187-7-1-toc.gif Journal of Molecular Signaling 1750-2187 ${item.volume} 1 2012-01-04T00:00:00Z XML 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. http://www.jmolecularsignaling.com/content/6/1/13 Jessica Beach Laura Nary Yasuko Hirakawa Eli Holland Rebeka Hovanessian Rheem Medh Journal of Molecular Signaling 2011, null:13 2011-10-05T00:00:00Z doi:10.1186/1750-2187-6-13 /content/figures/1750-2187-6-13-toc.gif Journal of Molecular Signaling 1750-2187 ${item.volume} 13 2011-10-05T00:00:00Z XML 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. http://www.jmolecularsignaling.com/content/6/1/12 Shingo Tanaka Kumiko Terada Tsutomu Nohno Journal of Molecular Signaling 2011, null:12 2011-10-05T00:00:00Z doi:10.1186/1750-2187-6-12 /content/figures/1750-2187-6-12-toc.gif Journal of Molecular Signaling 1750-2187 ${item.volume} 12 2011-10-05T00:00:00Z XML Background: Blood platelets undergo a carefully regulated change in shape to serve as the primary mediators of hemostasis and thrombosis. These processes manifest through platelet spreading and aggregation and are dependent on platelet actin cytoskeletal changes orchestrated by the Rho GTPase family member Rac1. To elucidate how Rac1 is regulated in platelets, we captured Rac1-interacting proteins from platelets and identified Rac1-associated proteins by mass spectrometry.FindingsHere, we demonstrate that Rac1 captures the Rac guanine nucleotide exchange factor P-Rex1 from platelet lysates. Western blotting experiments confirmed that P-Rex1 is expressed in platelets and associated with Rac1. To investigate the functional role of platelet P-Rex1, platelets from P-Rex1-/- -deficient mice were treated with platelet agonists or exposed to platelet activating surfaces of fibrinogen, collagen and thrombin. Platelets from P-Rex1-/- mice responded to platelet agonists and activating surfaces similarly to wild type platelets. Conclusions: These findings suggest that P-Rex1 is not required for Rac1-mediated platelet activation and that the GEF activities of P-Rex1 may be more specific to GPCR chemokine receptor mediated processes in immune cells and tumor cells. http://www.jmolecularsignaling.com/content/6/1/11 Joseph Aslan Alex Spencer Cassandra Loren Jiaqing Pang Heidi Welch Daniel Greenberg Owen McCarty Journal of Molecular Signaling 2011, null:11 2011-09-01T00:00:00Z doi:10.1186/1750-2187-6-11 /content/figures/1750-2187-6-11-toc.gif Journal of Molecular Signaling 1750-2187 ${item.volume} 11 2011-09-01T00:00:00Z XML Background: Post-transcriptional processing of pre-mRNA takes place in several steps and requires involvement of a number of RNA-binding proteins. How pre-mRNA processing is regulated is in large enigmatic. The catalytic (C) subunit of protein kinase A (PKA) is a serine/threonine kinase, which regulates numerous cellular processes including pre-mRNA splicing. Despite that a significant fraction of the C subunit is found in splicing factor compartments in the nucleus, there are no indications of a direct interaction between RNA and PKA. Based on this we speculate if the specificity of the C subunit in regulating pre-mRNA splicing may be mediated indirectly through other nuclear proteins. Results: Using yeast two-hybrid screening with the PKA C subunit Cbeta2 as bait, we identified the G-patch domain and KOW motifs-containing protein (GPKOW), also known as the T54 protein or MOS2 homolog, as an interaction partner for Cbeta2. We demonstrate that GPKOW, which contains one G-patch domain and two KOW motifs, is a nuclear RNA-binding protein conserved between species. GPKOW contains two sites that are phosphorylated by PKA in vitro. By RNA immunoprecipitation and site directed mutagenesis of the PKA phosphorylation sites we revealed that GPKOW binds RNA in vivo in a PKA sensitive fashion. Conclusion: GPKOW is a RNA-binding protein that binds RNA in a PKA regulated fashion. Together with our previous results demonstrating that PKA regulates pre-mRNA splicing, our results suggest that PKA phosphorylation is involved in regulating RNA processing at several steps. http://www.jmolecularsignaling.com/content/6/1/10 Anne Kristin Aksaas Anja Larsen Marie Rogne Ken Rosendal Anne-Katrine Kvissel Bjorn Skalhegg Journal of Molecular Signaling 2011, null:10 2011-08-31T00:00:00Z doi:10.1186/1750-2187-6-10 /content/figures/1750-2187-6-10-toc.gif Journal of Molecular Signaling 1750-2187 ${item.volume} 10 2011-08-31T00:00:00Z XML Background: In the present study, we describe heterodimerization between human-Somatostatin Receptor 5 (hSSTR5) and β2-Adrenergic Receptor (β2AR) and its impact on the receptor trafficking, coupling to adenylyl cyclase and signaling including mitogen activated protein kinases and calcineurin-NFAT pathways. Methods: We used co-immunoprecipitation, photobleaching- fluorescence resonance energy transfer and Fluorescence assisted cell sorting analysis to characterize heterodimerization between SSTR5 and β2AR. Results: Our results indicate that hSSTR5/β2AR exist as preformed heterodimers in the basal condition which is enhanced upon co-activation of both receptors. In contrast, the activation of individual receptors leads to the dissociation of heterodimers. Receptor coupling to adenylyl cyclase displayed predominant effect of β2AR, however, somatostatin mediated inhibition of cAMP was enhanced upon blocking β2AR. Our results indicate hSSTR5 mediated significant activation of ERK1/2 and inhibition of phospho-p38. The phospho-NFAT level was enhanced in cotransfected cells indicating the blockade of calcineurin mediated dephosphorylation of NFAT upon receptor heterodimerization. Conclusion: These data for the first time unveil a novel insight for the role of hSSTR5/β2AR in the modulation of signaling pathways which has not been addressed earlier. http://www.jmolecularsignaling.com/content/6/1/9 Rishi Somvanshi Nicole Chaudhari Xiaofan Qiu Ujendra Kumar Journal of Molecular Signaling 2011, null:9 2011-08-12T00:00:00Z doi:10.1186/1750-2187-6-9 /content/figures/1750-2187-6-9-toc.gif Journal of Molecular Signaling 1750-2187 ${item.volume} 9 2011-08-12T00:00:00Z XML 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. http://www.jmolecularsignaling.com/content/6/1/8 Shujuan Gao Hsien-yu Wang Craig Malbon Journal of Molecular Signaling 2011, null:8 2011-08-10T00:00:00Z doi:10.1186/1750-2187-6-8 /content/figures/1750-2187-6-8-toc.gif Journal of Molecular Signaling 1750-2187 ${item.volume} 8 2011-08-10T00:00:00Z XML