No abstract

Epac belongs to a new family of proteins that can directly mediate the action of the intracellular second messenger cAMP by activating a downstream small GTPase Rap1. The Epac/Rap1 pathway represents a novel cAMP-signaling cascade that is independent of the cAMP-dependent protein kinase (PKA). In this study, we have used fluorescence microscopy to probe the intracellular targeting of Epac during different stages of the cell division cycle and the structural features that are important for Epac localization. Our results suggest Epac, endogenous or expressed as a green fluorescent protein fusion protein, is mainly localized to the nuclear membrane and mitochondria during interphase in COS-7 cells. Deletion mutagenesis analysis reveals that whereas the DEP domain is responsible for membrane association, the mitochondrial-targeting sequence is located at the N terminus. Although Epac predominantly exhibits perinuclear localization in interphase, the subcellular localization of Epac is cell cycle-dependent. Epac disassociates from the nuclear membrane and localizes to the mitotic spindle and centrosomes in metaphase. At the end of the cell cycle, Epac is observed to reassociate with the nuclear envelope and concentrate around the contractile ring. Furthermore, overexpression of Epac in COS-7 cells leads to an increase in multinuclear cell populations. These results suggest that Epac may play an important role in mitosis.Cyclic adenosine 3Ј,5Ј-monophosphate (cAMP) is the first intracellular second messenger to be discovered and plays an important role in mediating the actions of extracellular signals (1). cAMP has been implicated in regulating a myriad of cellular functions such as metabolism, proliferation, differentiation, secretion, and gene expression. For many years, the consensus was that cAMP-mediated signaling in eukaryotic cells existed as a linear pathway that involves the sequential activation of a series of signaling molecules. The cAMP signaling pathway consists of both plasma membrane and intracellular components. Upon binding of ligand, the membrane receptor at the cell surface transduces an extracellular signal across the cell membrane via stimulatory or inhibitory heterotrimeric G-proteins that interact with the membrane-bound adenylyl cyclase to regulate cAMP production inside the cell. It was believed until recently that all known effects of cAMP in mammalian cells, with the exception of cyclic nucleotide-gated channels in photoreceptor cells and olfactory sensory neurons (2), were mediated intracellularly by cAMP-dependent protein kinase (PKA). The effects of cAMP on different cellular functions are often described as cell type-specific and dependent on biological response (3-10). These differential effects of cAMP on various cell functions suggest that more than one cAMP receptor may exist in mammalian cells. The search for a new cAMP receptor has recently revealed a novel class of signaling molecules, Epacs (exchange proteins directly activated by cAMP) or cAMP-GEFs (cAMP-regulated guanine nucleo...

Alphaviruses are arthropod-borne viruses (arboviruses) that include a number of important human and animal pathogens. Their replication proceeds in the cytoplasm of infected cells and does not directly depend on nuclei. Alphaviruses encode only four nonstructural proteins that are required for the replication of viral genome and transcription of the subgenomic RNA. However, the replicative enzyme complexes (RCs) appear to include cellular proteins and assemble on cellular organelles. We have developed a set of recombinant Sindbis (SIN) viruses with green fluorescent protein (GFP) insertions in one of the nonstructural proteins, nsP3, to further understand the RCs' genesis and structure. We studied the assembly of nsP3/GFP-containing protein complexes at different stages of infection and isolated a combination of cellular proteins that are associated with SIN nsP3. We demonstrated the following. (i) SIN nsP3 can tolerate the insertion of GFP into different fragments of the coding sequence; the designed recombinant viruses are viable, and their replication leads to the assembly of nsP3/GFP chimeric proteins into gradually developing, higher-order structures differently organized at early and late times postinfection. (ii) At late times postinfection, nsP3 is assembled into complexes of similar sizes, which appear to be bound to cytoskeleton filaments and can aggregate into larger structures. (iii) Protein complexes that are associated with nsP3/GFP contain a high concentration of cytoskeleton proteins, chaperones, elongation factor 1A, heterogeneous nuclear ribonucleoproteins, 14-3-3 proteins, and some of the ribosomal proteins. These proteins are proposed to be essential for SIN RC formation and/or functioning.Alphaviruses are a widely distributed group of significant human and animal pathogens. Some of them, including Venezuelan, eastern, and western equine encephalitis viruses, cause serious febrile illness and encephalitis (26). Others cause diseases with mild symptoms that usually include rash, fever, and arthritis (19). Alphavirus structural and nonstructural proteins (nsPs) demonstrate an obvious homology, suggesting that replication of their genomes, interactions with host cell biology, and formation of viral particles have much in common (52).The alphavirus genome is a single-stranded RNA of positive polarity and almost 12 kb in length that mimics the structure of cellular mRNAs. It contains both a 5Ј methylguanylate cap and a 3Ј polyadenylate tail (27, 51). These features allow the translation of viral proteins by host cell machinery directly from the genome RNA. The 5Ј two-thirds of the genome is translated into nonstructural proteins that comprise the viral components of the replicative enzyme complex (RC) that is required for replication of the viral genome and transcription of the subgenomic RNA. The subgenomic RNA corresponds to the 3Ј third of the genome. It is synthesized from the subgenomic promoter and translated into viral structural proteins, which are dispensable for RNA replication. The RNAs...

Infectious and inflammatory stimuli induce expression of gene networks controlling proinflammatory and innate immune responses. One important arm of the inflammatory response is mediated by monocyte-derived tumor necrosis factor (TNF), a cytokine that activates gene expression programs in adjacent epithelial cells to propagate mucosal inflammation (42, 51). Here, the spectrum of functional activities and the magnitude and timing of TNF-induced gene expression are important determinants of tissue homeostasis. A central mediator of epithelial genomic response is nuclear factor-B (NF-B), an inducible transcription factor that controls the expression of proinflammatory genes and whose dysregulation has also been implicated in the pathogenesis of inflammatory, neoplastic, and autoimmune diseases (4,6,17).In most non-B-lymphoid cells, NF-B is sequestered as an inactive complex in the cytoplasm by the inhibitors of B (IBs). The mechanism by which NF-B is activated by TNF, referred to as the "canonical" activation pathway, has been extensively investigated (22). The canonical pathway is activated by ligands of the TNF superfamily including TNF alpha, the prototypical macrophage-derived cytokine that binds to the ubiquitously expressed TNF receptor 1 (TNFRI) (50). TNFinduced TNFRI trimerization induces the recruitment of cytoplasmic signal adapters, including the TNF receptor-associated death domain (TRADD), TNF receptor-associated factor 2 (TRAF2) and TRAF6, receptor-interacting protein (RIP), mitogen/extracellular signal-regulated kinase kinase 3, and others (23)(24)(25). This activated submembranous TNFRI complex transiently recruits the IB kinase (IKK) complex, resulting in the phosphorylation of the catalytic IKK␣ and -␤ kinases followed by its cytosolic release (15,43). In the cytosol, activated IKK phosphorylates serine (Ser) residues 32 and 36 on the NH2 terminal regulatory domain of IB␣, targeting IB␣ for proteolytic destruction by ubiquitination and calpain pathways

] i oscillations by increasing the frequency, base line, and duration of Ca 2؉ spiking. In addition, ATP stimulated gonadotropin secretion and enhanced agonist-induced gonadotropin release. ATP was found to be secreted by pituitary cells during agonist stimulation and was promptly degraded by ectonucleotidase to adenosine. These observations indicate that ATP represents a paracrine/autocrine factor in the regulation of Ca 2؉ signaling and secretion in gonadotrophs, and that these actions are mediated by P 2 receptor channels.