Effects of isoproterenol and forskolin on tension, cyclic AMP levels, and cyclic AMP dependent protein kinase activity in bovine coronary artery

The effects of isoproterenol and forskolin on tension, cyclic AMP levels, and cyclic AMP dependent protein kinase activity were compared in helical strips of bovine coronary artery. Elevation of cyclic AMP and activation of the protein kinase appeared to be well correlated with relaxation of potassium-contracted arteries by isoproterenol. Forskolin, at 1 microM or higher concentrations, also markedly elevated cyclic AMP levels, activated the kinase, and relaxed the arteries. However, a lower concentration of forskolin (0.1 microM) caused significant increases in both cyclic AMP levels and cyclic AMP dependent protein kinase activity, but did not relax the muscles. Relaxation caused by isoproterenol was accompanied by an apparent translocation of cyclic AMP dependent protein kinase activity from the soluble to the particulate fraction in these preparations. A similar shift in the distribution of the kinase was caused by various concentrations of forskolin, irrespective of whether the arteries were relaxed or not. In contrast to previous results in other tissues, low concentrations of forskolin (less than or equal to 1 microM), which themselves markedly elevated cyclic AMP levels in the arteries, did not potentiate the effects of isoproterenol on cyclic AMP levels or tension in these preparations. These results suggest that either cyclic AMP is not solely responsible for the relaxation caused by these agents, or some form of functional compartmentalization of cyclic AMP and cyclic AMP dependent protein kinase exists in this tissue.     

Phosphorylated guanine nucleotide exchange factor C3G,induced by pervanadate and Src family kinases localizes to the Golgi and subcortical actin cytoskeleton

Background  

The guanine nucleotide exchange factor C3G (RapGEF1) along with its effector proteins participates in signaling pathways that regulate eukaryotic cell proliferation, adhesion, apoptosis and embryonic development. It activates Rap1, Rap2 and R-Ras members of the Ras family of GTPases. C3G is activated upon phosphorylation at tyrosine 504 and therefore, determining the localization of phosphorylated C3G would provide an insight into its site of action in the cellular context.     

A physical map for the Amborella trichopoda genome sheds light on the evolution of angiosperm genome structure

Background

Recent phylogenetic analyses have identified Amborella trichopoda, an understory tree species endemic to the forests of New Caledonia, as sister to a clade including all other known flowering plant species. The Amborella genome is a unique reference for understanding the evolution of angiosperm genomes because it can serve as an outgroup to root comparative analyses. A physical map, BAC end sequences and sample shotgun sequences provide a first view of the 870 Mbp Amborella genome.

Results

Analysis of Amborella BAC ends sequenced from each contig suggests that the density of long terminal repeat retrotransposons is negatively correlated with that of protein coding genes. Syntenic, presumably ancestral, gene blocks were identified in comparisons of the Amborella BAC contigs and the sequenced Arabidopsis thaliana, Populus trichocarpa, Vitis vinifera and Oryza sativa genomes. Parsimony mapping of the loss of synteny corroborates previous analyses suggesting that the rate of structural change has been more rapid on lineages leading to Arabidopsis and Oryza compared with lineages leading to Populus and Vitis. The gamma paleohexiploidy event identified in the Arabidopsis, Populus and Vitis genomes is shown to have occurred after the divergence of all other known angiosperms from the lineage leading to Amborella.

Conclusions

When placed in the context of a physical map, BAC end sequences representing just 5.4% of the Amborella genome have facilitated reconstruction of gene blocks that existed in the last common ancestor of all flowering plants. The Amborella genome is an invaluable reference for inferences concerning the ancestral angiosperm and subsequent genome evolution.     

TC-PTP dephosphorylates the guanine nucleotide exchange factor C3G (RapGEF1) and negatively regulates differentiation of human neuroblastoma cells

The guanine nucleotide exchange factor, C3G (RapGEF1), functions in multiple signaling pathways involved in cell adhesion, proliferation, apoptosis and actin reorganization. C3G is regulated by tyrosine phosphorylation on Y504, known to be mediated by c-Abl and Src family kinases. In the present study we explored the possibility of cellular phospho-C3G (pC3G) being a substrate of the intracellular T-cell protein tyrosine phosphatase TC-PTP (PTPN2) using the human neuroblastoma cell line, IMR-32. In vivo and in vitro binding assays demonstrated interaction between C3G and TC-PTP. Interaction is mediated through the Crk-binding region of C3G and C-terminal noncatalytic residues of TC-PTP. C3G interacted better with a substrate trap mutant of TC48 and this complex formation was inhibited by vanadate. Endogenous pC3G colocalized with catalytically inactive mutant TC48 in the Golgi. Expression of TC48 abrogated pervanadate and c-Src induced phosphorylation of C3G without affecting total cellular phospho-tyrosine. Insulin-like growth factor treatment of c-Src expressing cells resulted in dephosphorylation of C3G dependent on the activity of endogenous TC48. TC48 expression inhibited forskolin induced tyrosine phosphorylation of C3G and neurite outgrowth in IMR-32 cells. Our results identify a novel Golgi localized substrate of TC48 and delineate a role for TC48 in dephosphorylation of substrates required during differentiation of human neuroblastoma cells.     

Rapid, repeated, and clustered loss of duplicate genes in allopolyploid plant populations of independent origin

The predictability of evolution is debatable, with recent evidence suggesting that outcomes may be constrained by gene interaction networks [1]. Whole-genome duplication (WGD; polyploidization-ubiquitous in plant evolution [2]) provides the opportunity to evaluate the predictability of genome reduction, a pervasive feature of evolution [3, 4]. Repeated patterns of genome reduction appear to have occurred via duplicated gene (homeolog) loss in divergent species following ancient WGD [5-9], with evidence for preferential retention of duplicates in certain gene classes [8-10]. The speed at which these patterns arise is unknown. We examined presence/absence of 70 homeologous loci in 59 Tragopogon miscellus plants from five natural populations of independent origin; this allotetraploid arose ~80 years ago via hybridization between diploid parents and WGD [11]. Genes were repeatedly retained or lost in clusters, and the gene ontology categories of the missing genes correspond to those lost after ancient WGD in the same family (Asteraceae; sunflower family) [6] and with gene dosage sensitivity [8]. These results provide evidence that the outcomes of WGD are predictable, even in 40 generations, perhaps due to the connectivity of gene products [8, 10, 12]. The high frequency of single-allele losses detected and low frequency of changes fixed within populations provide evidence for ongoing evolution.     

The guanine nucleotide exchange factor, C3G regulates differentiation and survival of human neuroblastoma cells

Neuronal differentiation involving neurite growth is dependent on environmental cues which are relayed by signalling pathways to actin cytoskeletal remodelling. C3G, the exchange factor for Rap1, functions in pathways leading to actin reorganization and filopodia formation, processes required during neurite growth. In the present study, we have analyzed the function of C3G, in regulating neuronal cell survival and plasticity. Human neuroblastoma cells, IMR-32 induced to differentiate by serum starvation or by treatment with nerve growth factor (NGF) or forskolin showed enhanced C3G protein levels. Transient over-expression of C3G stimulated neurite growth and also increased responsiveness to NGF and serum deprivation induced differentiation. C3G-induced neurite growth was dependent on both its catalytic and N-terminal regulatory domains, and on the functions of Cdc42 and Rap1. Knockdown of C3G using small hairpin RNA inhibited forskolin and NGF-induced morphological differentiation of IMR-32 cells. Forskolin-induced differentiation was dependent on catalytic activity of C3G. Forskolin and NGF treatment resulted in phosphorylation of C3G at Tyr504 predominantly in the Golgi. C3G expression induced the cell cycle inhibitor p21 and C3G knockdown enhanced cell death in response to serum starvation. These findings demonstrate a novel function for C3G in regulating survival and differentiation of human neuroblastoma cells.     

Signalling to actin: role of C3G, a multitasking guanine-nucleotide-exchange factor

C3G (Crk SH3-domain-binding guanine-nucleotide-releasing factor) is a ubiquitously expressed member of a class of molecules called GEFs (guanine-nucleotide-exchange factor) that activate small GTPases and is involved in pathways triggered by a variety of signals. It is essential for mammalian embryonic development and many cellular functions in adult tissues. C3G participates in regulating functions that require cytoskeletal remodelling such as adhesion, migration, maintenance of cell junctions, neurite growth and vesicle traffic. C3G is spatially and temporally regulated to act on Ras family GTPases Rap1, Rap2, R-Ras, TC21 and Rho family member TC10. Increased C3G protein levels are associated with differentiation of various cell types, indicating an important role for C3G in cellular differentiation. In signalling pathways, C3G serves functions dependent on catalytic activity as well as protein interaction and can therefore integrate signals necessary for the execution of more than one cellular function. This review summarizes our current knowledge of the biology of C3G with emphasis on its role as a transducer of signals to the actin cytoskeleton. Deregulated C3G may also contribute to pathogenesis of human disorders and therefore could be a potential therapeutic target.     

Effects of prostaglandin E1, isoproterenol and forskolin on cyclic AMP levels and tension in rabbit aortic rings

The role of cyclic AMP in the control of vascular smooth muscle tone was studied by monitoring the effects of prostaglandin E1 (PGE1), isoproterenol and forskolin on cyclic AMP levels and tension in rabbit aortic rings. PGE1, isoproterenol and forskolin all increased cyclic AMP levels in rabbit aortic rings. Isoproterenol and forskolin relaxed phenylephrine-contracted aortic rings, but PGE1 contracted the rings in the presence or absence of phenylephrine. Isoproterenol relaxed these PGE1-contracted aortic rings without further change in total cyclic AMP levels, which were already elevated by the PGE1 alone. Pretreatment with forskolin potentiated the effects of PGE1 on cyclic AMP levels. PGE1 caused contractions in muscles partially relaxed by forskolin, even though very large increases in cyclic AMP levels (30 fold) were produced by PGE1 in the presence of forskolin. Isoproterenol was able to relax these forskolin-treated, PGE1-contracted muscles with no further increase in cyclic AMP levels. Thus, there does not appear to be a good correlation between total tissue levels of cyclic AMP and tension in these experiments. Our results suggest that, if cyclic AMP is responsible for relaxation of smooth muscle, some form of functional compartmentalization of cyclic AMP must exist in this tissue.