Elevation of the intracellular cAMP concentration ([cAMP]i ) regulates metabolism, cell proliferation, and differentiation and plays roles in memory formation and neoplastic growth. cAMP mediates its effects mainly through activation of protein kinase A (PKA) as well as Epac1 and Epac2, exchange factors activating the small GTPases Rap1 and Rap2. However, how cAMP utilizes these effectors to induce distinct biological responses is unknown. We here studied the specific roles of PKA and Epac in neuroendocrine PC12 cells. In these cells, elevation of [cAMP] i activates extracellular signal-regulated kinase (ERK) 1/2 and induces low-degree neurite outgrowth. The present study showed that specific stimulation of PKA triggered ERK1/2 activation that was considerably more transient than that observed upon simultaneous activation of both PKA and Epac. Unexpectedly, the PKA-specific cAMP analog induced cell proliferation rather than neurite outgrowth. The proliferative signaling pathway activated by the PKA-specific cAMP analog involved activation of the epidermal growth factor receptor and ERK1/2. Activation of Epac appeared to extend the duration of PKA-dependent ERK1/2 activation and converted cAMP from a proliferative into an anti-proliferative, neurite outgrowthpromoting signal. Thus, the present study showed that the outcome of cAMP signaling can depend heavily on the set of cAMP effectors activated. INTRODUCTIONThe normal development of organisms requires a tight coordination between growth and differentiation. Importantly, the regulation of this decision is altered in disease states, most notably in cancer, where growth signals overcome the physiological processes of differentiation. The differentiation of neuronal cells is important for the regular function of the brain. Thus, there is interest in understanding the molecular signaling mechanisms that regulate cell proliferation and differentiation.In a number of cells, most notably of neuronal origin, G s protein-coupled receptors (GsPCR) are key regulators of cell proliferation, differentiation, and survival and play important roles in numerous biological processes such as neoplasia, learning, and memory formation (Kandel, 2001;Spada and Lania, 2002;Wang et al., 2004). The signaling cascades downstream of GsPCRs to regulate these events include activation of adenylyl cyclase, elevation of the intracellular cAMP concentration ([cAMP] i ), and modulation of the extracellular signal-regulated kinase (ERK) 1/2 pathway (Stork, 2003).cAMP binds to and directly activates protein kinase A (PKA) as well as the cAMP binding guanine nucleotide exchange factors Epac1 and -2, which in turn stimulate the small GTPases Rap1 and Rap2. Together, PKA and Epacs appear to mediate the majority of effects of cAMP in mammalian cells (de Rooij et al., 1998;Kawasaki et al., 1998;Bos, 2003). Nevertheless, most work on cAMP-dependent pathways has been centered on the role of PKA. For instance, a role of PKA in memory formation has been verified in numerous in vitro and in vivo studies t...
The open reading frame III of Borna disease virus (BDV) codes for a protein with a mass of 16 kDa, named p16 or BDV-M. p16 was described as an N-glycosylated protein in several previous publications and therefore was termed gp18, although the amino acid sequence of p16 does not contain any regular consensus sequence for N glycosylation. We examined glycosylation of p16 and studied its membrane topology using antisera raised against peptides, which comprise the N and the C termini. Neither an N-nor a C-terminal peptide is cleaved from p16 during maturation. Neither deglycosylation of p16 by endoglycosidases nor binding of lectin to p16 was detectable. Introduction of typical N-glycosylation sites at the proposed sites of p16 failed in carbohydrate attachment. Flotation experiments with membranes of BDV-infected cells on density gradients revealed that p16 is not an integral membrane protein, since it can be dissociated from membranes. Our experimental data strongly suggest that p16 is a typical nonglycosylated matrix protein associated at the inner surface of the viral membrane, as is true for homologous proteins of other members of the Mononegavirales order.
Ddx42p is a recently characterized mammalian DEAD box protein with unknown cellular function. We found that in human cells Ddx42p physically interacts with ASPP2, a major apoptosis inducer known to enhance p53 transactivation of proapoptotic genes. The proteins interact via a domain within the carboxy-terminal part of Ddx42p and a mid-amino-terminal sequence as well as the ankyrin-SH3 region of ASPP2. Overexpression of Ddx42p interferes with apoptosis induction by ASPP2, whereas Ddx42p knockdown reduces the survival rate of cultured human cells. In addition, ASPP2 is found in cytoplasm and nucleus at low Ddx42p level, and predominantly in cytoplasm at high concentration of Ddx42p, respectively. Our results show that Ddx42p is capable of modulating ASPP2 function.
The only surface membrane glycoprotein of Borna disease virus (BDV) is synthesized as a polypeptide with a molecular mass of 57 kDa and N-glycosylated to a precursor glycoprotein (GP) of about 94 kDa. It is processed by the cellular protease furin into the C-terminal membrane-anchored subunit GP-C, also known as gp43, and a presumptive N-terminal subunit GP-N, that is highly glycosylated and has a molecular mass of about 51 kDa. However, up to now the latter remained undetected in BDV-infected material. We describe a novel approach to identify glycan masked linear antigenic epitopes. In the present study, GP-N was identi¢ed in BDV-infected cells by a combination of lectin precipitation, enzymatic deglycosylation on blot and immunochemistry using an N-terminal speci¢c antiserum. The GP-N has an apparent molecular mass of 45^50 kDa in its glycosylated form and 27 kDa in its deglycosylated form. N-glycan analysis revealed that the precursor GP contains only mannose-rich N-glycans, whereas GP-N and GP-C contain mannose-rich and complex-type N-glycans.
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