The ATF‐2 transcription factor can mediate adenovirus E1A‐inducible transcriptional activation. Deletion analysis has indicated that the N‐terminal region of ATF‐2 is essential for this response. Furthermore, the N‐terminus can activate transcription in the absence of E1A when fused to a heterologous DNA binding domain. However, in the intact protein this activation domain is masked. In this report we show that residues in the N‐terminus required for activation are also required for mediating E1A stimulation. In particular two threonine residues at positions 69 and 71 are essential. These residues are phosphorylated in vivo and can be efficiently phosphorylated in vitro by the JNK/SAPK subgroup of the MAPK family. ATF‐2 can bind to a UV‐inducible kinase through a region in the N‐terminus that is distinct from the sites of phosphorylation; this binding region is both necessary for phosphorylation by JNK/SAPK in vitro and for transcriptional activation in vivo. The activity of the N‐terminus is stimulated by UV irradiation which stimulates the signalling pathway leading to JNK/SAPK. Finally, although ATF‐2 binds to the E1A protein, the N‐terminal activation domain is not required for this interaction. The results show that ATF‐2, like other members of the ATF/CREB family of DNA binding proteins is regulated by specific signalling pathways.
The mechanism by which the binding of epidermal growth factor (EGF) to specific cell surface receptors induces a range of biological responses remains poorly understood. An important part of the study of signal transduction in this system involves the production of sufficient native and mutant EGF receptor species for X‐ray crystallographic and spectroscopic analysis. Baculovirus vectors containing the cDNA encoding the human EGF receptor protein have here been utilized to infect insect cells. This results in expression of a 155‐kb transmembrane protein which is recognized by four antibodies against different regions of the human EGF receptor. Studies with tunicamycin, monensen and endoglycosidase H show the difference in size between the recombinant and the native receptor is due to alterations in glycocsylation. Studies of [125I] EGF binding shows a Kd of 2 X 10(‐9) M in intact infected insect cells which falls to 2 X 10(‐7) M upon detergent solubilization. The recombinant protein exhibits an EGF‐stimulated tyrosine protein kinase activity and an analysis of tryptic peptides shows that the phosphate acceptor sites are similar to those of the EGF receptor isolated from A431 cells. These observations indicate that functional EGF receptor can be expressed in insect cells, and furthermore, this system can be used for large‐scale production.
SUMMARYEight different hybridoma cell lines producing monoclonal antibodies against the major antigens of human adenovirus type 5 have been obtained. They were selected by screening initial hybridomas by the fluorescent antibody technique followed by radioimmune precipitation and they reacted with hexon, penton, fibre and 100K polypeptides. Five apparently different epitopes against the hexon antigen were detected showing a spectrum of activity against the hexons of other serotypes, suggesting that the hexon contained a variety of subgroup specificities as well as the previously described group and type specificities.
An improved method for the isolation of baculovirus recombinants is described. The method involves the replication and maintenance of the baculovirus genome in the yeast Saccharomyces cerevisiae which was accomplished by the isolation of a baculovirus recombinant containing yeast ARS and CEN sequences ensuring stable replication in yeast and a URA3 selectable marker. The viral DNA maintained its ability to replicate in insect cells. An efficient and rapid selection system was set up, to isolate viral recombinants in yeast; DNA from selected yeast colonies was transfected into insect cells to obtain recombinant virus. We demonstrate the utility of this system by isolating recombinant viruses that express two different members of the CREB/ATF family of transcription factors.
The baculovirus expression system has been successfully used to overproduce a number of different protein products. In this report we describe the construction of a recombinant baculovirus containing the adenovirus E1A 13s cDNA sequence. Infection of insect cells with this virus results in the production of phosphorylated E1A protein. The phosphorylation pattern appears to be similar to the complex pattern associated with E1A protein synthesis in mammalian cells. Purified baculovirus generated E1A protein activated transcription of specific poIIII promoters both in microinjected Xenopus laevis oocytes and in HeLa cell in vitro transcription extracts. The protein also stimulates in vitro transcription of the poIIII transcribed VA1 gene.
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