Identification of adenovirus 12-encoded E1A tumor antigens synthesized in infected and transformed mammalian cells and in Escherichia coli

Abstract

COOH (peptide 204), targeted to the common C-terminus of human adenovirus 12 (Adl2) tumor antigens encoded by the ElA 13S mRNA and 12S mRNA, has been synthesized. Antibody prepared in rabbits against peptide 204 immunoprecipitated two proteins of apparent Mr 47,000 and 45,000 from extracts of [35S]methionine-labeled Adl2-early infected KB cells and a 47,000 protein from extracts of the Adl2-transformed hamster cell line, HE C19. Immunoprecipitation analysis of infected and transformed cells labeled with 32P, showed that both major Adl2 EIA T antigens are phosphoproteins. Immunofluorescence microscopy of Adl2-early infected KB cells with antipeptide antibody showed the site of ElA protein concentration to be predominantly nuclear. ElA proteins were detected by imtnunofluorescence at 4 to 6 h postinfection and continued to increase until at least 18 h postinfection. Antipeptide 204 antibody was used to analyze the proteins synthesized in Escherichia coli cells transformed by plasmids containing cDNA copies of the Adl2 EIA 13S mRNA or 12S mRNA under the control of the tac promoter (D.

Phosphorylation in vitro of Escherichia coli-produced 235R and 266R tumor antigens encoded by human adenovirus type 12 early transformation region 1A

“…1A, lane 2) and 266R (Fig. 1A, lane 3) T antigens produced in E. coli incorporate [35S]methionine and were detected by immunoprecipitation (21,22). However, neither T antigen was phosphorylated in E. coli, as shown by the absence of immunoprecipitable 32P-labeled proteins (Fig.…”

“…ElA T antigens were immunoprecipitated from the labeled cell extracts with antipeptide 204 antibody, which is targeted to the common C terminus of the Adl2 ElA T antigens (22). Two phosphorylated species of apparent Mrs of 45,000 and 47,000 were detected in 32Pi-labeled Adl2-infected KB cells (Fig.…”

“…Immunoprecipitation of phosphorylated Adl2 T antigens. Phosphorylated T antigens were immunoprecipitated from kinase reaction mixtures or from extracts of 32Pi-labeled infected cells by using 10 ,ul of antipeptide 204 antibody (22). Immunoprecipitates were processed and analyzed by SDSpolyacrylamide gel electrophoresis and autoradiography (17).…”

“…A potential drawback of producing biologically interesting proteins in E. coli is that posttranslational modifications unique to eucaryotic cells may be required for activity in vitro. The adenovirus EIA T antigens are posttranslationally phosphorylated (13,22,34), as are other tumor virus transformation proteins. Phosphorylation can play an important role in the activity of regulatory proteins (for a review, see reference 19).…”

Posttranslational modification at the N terminus of the human adenovirus type 12 E1A 235R tumor antigen

“…The nature of the posttranslational modifications that generate different ElA T antigen forms is unknown. Although the ElA T antigens are phosphorylated (9,17,18,25), it is not known whether differential phosphorylation contributes to the diversity of T antigen species; i.e., multiple-T-antigen species are translated from each mRNA in vitro (14).…”