Regulation of p53 levels by the E1B 55-kilodalton protein and E4orf6 in adenovirus-infected cells.

The adenovirus type 5 243R E1A protein induces p53-dependent apoptosis in the absence of the 19- and 55-kDa E1B polypeptides. This effect appears to result from an accumulation of p53 protein and is unrelated to expression of E1B products. We now report that in the presence of the E1B 55-kDa polypeptide, the 289R E1A protein does not induce such p53 accumulation and, in fact, is able to block that induced by E1A 243R. This inhibition also requires the 289R-dependent transactivation of E4orf6 expression. E4orf6 is known to form complexes with the E1B 55-kDa protein and to function both in the transport and stabilization of viral mRNA and in shutoff of host cell protein synthesis. We demonstrated that the block in p53 accumulation is not due to the generalized shutoff of host cell metabolism. Rather, it appears to result from a mechanism targeted specifically to p53, most likely involving a decrease in the stability of p53 protein. The E1B 55-kDa protein is known to interact with both E4orf6 and p53, and as demonstrated recently by others, we showed that E4orf6 also binds directly to p53. Thus, multiple interactions between all three proteins may regulate p53 stability, resulting in the maintenance of low levels of p53 following virus infection.     

Involvement of three intracellular messenger systems, protein kinase C, calcium ion and cyclic AMP, in the regulation of c-fos gene expression in Swiss 3T3 cells

In quiescent cultures of Swiss 3T3 cells, platelet-derived growth factor or fibroblast growth factor known to induce both protein kinase C activation and Ca2+ mobilization raised c-fos mRNA. This action of the growth factors was mimicked by the specific activators for protein kinase C, such as phorbol esters and a membrane-permeable synthetic diacylglycerol, and also by the Ca2+ ionophores, such as A23187 and ionomycin. Prostaglandin E1 known to elevate cyclic AMP also raised c-fos mRNA, and this action was mimicked by 8-bromo-cyclic AMP, dibutyryl cyclic AMP and forskolin. These results suggest that expression of the c-fos gene is regulated by three different intracellular messenger systems, protein kinase C, Ca2+ and cyclic AMP, in Swiss 3T3 cells.     

Stabilities and interrelations of multiple species of human adenovirus type 5 early region 1 proteins in infected and transformed cells.

Early region 1A (E1A) of human adenovirus type 5 (Ad5) produces two mRNAs coding for phosphoproteins of 289 and 243 residues (289R and 243R). Each of these products has been shown to migrate on sodium dodecyl sulfate gels as two major and two minor species. In the present study, the stabilities of E1A polypeptides, as well as those of some other early Ad5 proteins, were studied in infected KB cells that were pulse-labeled with [35S]methionine and then chased in the presence or absence of cycloheximide. The E1B 58,000- and 19,000-molecular-weight proteins (58K and 19K proteins; 496R and 176R) as well as the E2A 72K DNA-binding protein were relatively stable over the 4-h chase period; turnover was less than 30%. The E1A species were considerably more unstable, with an overall half-life of about 60 min. Interestingly, it was found that when cycloheximide was present during the chase, E1A proteins were much more stable, and the half-life increased to about 240 min. Analysis of the stabilities of individual E1A species indicated that the products of the 1.1-kilobase mRNA (289R) had half lives (about 55 min) somewhat shorter than those (about 90 min) of the 0.9-kilobase mRNA products (243R). In addition, the faster-migrating species produced from each mRNA (molecular weights, 48,500 and 45,000) had significantly shorter half-lives than did the slower-migrating species (52,000 and 50,000). In the presence of cycloheximide, the faster-migrating species were still quite short-lived, but the half-lives of the 52K and 50K species were considerably increased. An examination of the kinetics of turnover of the various E1A species suggested that the faster-migrating forms may be precursors to the slower-migrating ones. Somewhat similar stabilities were also found for the various E1A species in Ad5-transformed 293 cells.     

The role of cyclic AMP in the thermosensitive lesion of the formation of closed covalent circular Rts 1 DNA.

The formation of closed covalent circular DNA of R factor, Rts 1, does not take place at non-permissive temperature, 42°C, in $\text{E. coli}$ 20SO. However, when Rts 1 was placed in mutants having a low level of cyclic AMP or lacking cyclic AMP receptor protein, the thermosensitive lesion is overcome. Addition of cyclic AMP caused inhibition of the formation of ccc DNA in mutants with low cyclic AMP level, but not in mutants lacking cyclic AMP receptor protein.     

Cyclic AMP and mitogen-activated protein kinases are required for glutamate-dependent cyclic AMP response element binding protein and Elk-1 phosphorylation in the dorsal striatum in vivo

Dopaminergic and glutamatergic signalling cascades are integrated in striatal medium spiny neurones by cyclic AMP response-element binding protein and Elk-1 phosphorylation. Phosphorylated cyclic AMP response-element binding protein and phosphorylated Elk-1 contribute to c-fos expression by binding to the calcium and cyclic AMP response-element and the serum response element, respectively, in the c-fos promoter. The role of cyclic AMP and mitogen-activated protein kinase signalling cascades in glutamate-induced cyclic AMP response-element binding protein and Elk-1 phosphorylation and Fos expression was investigated using semiquantitative immunocytochemistry in vivo. Intracerebroventricular infusion of the sodium channel blocker, tetrodotoxin, decreased the glutamate-induced increase in phosphorylated cyclic AMP response-element binding protein, phosphorylated Elk-1, and Fos immunoreactivity. Intracerebroventricular infusion of the mitogen-activated and extracellular signal-regulated kinase inhibitor, PD98059, the p38 mitogen-activated protein kinase inhibitor, SB203580, or the cyclic AMP inhibitor, Rp-8-Br-cAMPS, decreased glutamate-induced phosphorylated cyclic AMP response-element binding protein, phosphorylated Elk-1, and Fos immunoreactivity. Simultaneous infusion of glutamate and Sp-8-Br-cAMPS, a cyclic AMP analogue, augmented induction of Fos immunoreactivity but not phosphorylated cyclic AMP response-element binding protein or phosphorylated Elk-1 immunoreactivity. These data indicate that cyclic AMP and mitogen-activated protein kinase signalling cascades are necessary for glutamate to induce cyclic AMP response-element binding protein and Elk-1 phosphorylation and Fos expression in the striatum. Furthermore, neuronal activity plays an important role in glutamate-induced signalling cascades in vivo.     

Involvement of two intracellular messenger systems, protein kinase C and cyclic AMP, in the regulation of c-fos gene expression in human promyelocytic leukemia (HL-60) cells

Incubation of human promyelocytic leukemia (HL-60) cells with 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a protein kinase C-activating phorbol ester, caused a marked increase in c-fos mRNA in a dose-dependent manner. Phorbol-12,13-dibutyrate and 1-oleoyl-2-acetyl-glycerol, other protein kinase C-activating agents, were also active in this capacity. 4 alpha-Phorbol-12,13-didecanoate, known to be inactive for protein kinase C, was ineffective. 8-Bromo-cyclic AMP (8-Br-cAMP) also increased c-fos mRNA in a dose-dependent manner. This action of 8-Br-cAMP was mimicked by prostaglandin E2, which is known to raise the cyclic AMP level in HL-60 cells. c-fos mRNA increased within 15 min and reached a maximal level 45 min after the stimulation of the cells by TPA or 8-Br-cAMP. The simultaneous stimulation of the cells by TPA and 8-Br-cAMP at the respective doses giving maximal elevation of c-fos mRNA increased this mRNA in an additive manner. These results suggest that in HL-60 cells expression of the c-fos gene is regulated independently by two different intracellular messenger systems, protein kinase C and cyclic AMP.     

Helicobacter pylori and mitogen-activated protein kinases mediate activator protein-1 (AP-1) subcomponent protein expression and DNA-binding activity in gastric epithelial cells

Emerging evidence has suggested a critical role for activator protein-1 (AP)-1 in regulating various cellular functions. The goal of this study was to investigate the effects of Helicobacter pylori and mitogen-activated protein kinases (MAPK) on AP-1 subcomponents expression and AP-1 DNA-binding activity in gastric epithelial cells. We found that H. pylori infection resulted in a time- and dose-dependent increase in the expression of the proteins c-Jun, JunB, JunD, Fra-1, and c-Fos, which make up the major AP-1 DNA-binding proteins in AGS and MKN45 cells, while the expression levels of Fra-2 and FosB remained unchanged. Helicobacter pylori infection and MAPK inhibition altered AP-1 subcomponent protein expression and AP-1 DNA-binding activity, but did not change the overall subcomponent composition. Different clinical isolates of H. pylori showed various abilities to induce AP-1 DNA binding. Mutation of cagA, cagPAI, or vacA, and the nonphosphorylateable CagA mutant (cagA(EPISA)) resulted in less H. pylori-induced AP-1 DNA-binding activity, while mutation of the H. pylori flagella had no effect. extracellular signal-related kinase (ERK), p38, and c-Jun N-terminal kinase (JNK) each selectively regulated AP-1 subcomponent expression and DNA-binding activity. These results provide more insight into how H. pylori and MAPK modulate AP-1 subcomponents in gastric epithelial cells to alter the expression of downstream target genes and affect cellular functions.