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

Abstract

The tumor (T) antigens encoded by the human adenovirus early transforming region 1A (ElA) are gene regulatory proteins whose functions can immortalize cells. We have recently described the synthesis in Escherichia coli and the purification of the complete T antigens encoded by the adenovirus type 12 (Adl2) ElA 12S mRNA (235-residue [235R] T antigen) and 13S mRNA (266R T antigen). In this study, we show that the Adl2 ElA T antigens are extensively phosphorylated in Adl2-infected mammalian cells but are not phosphorylated in E. coli. Inasmuch as posttranslational phosphorylation at specific amino acid sites may be important for biological activity, we have studied the phosphorylation of the E. coli-produced T antigens in vitro by using a kinase activity isolated from cultured human KB cells. The kinase was purified about 300-fold and appears to be a cyclic AMP-independent, Ca2+-independent protein kinase requiring only ATP and Mg2+ for activity. To determine which amino acids are phosphorylated and whether phosphorylation in vitro occurs at the same amino acid sites that are phosphorylated in vivo, the Adl2 ElA T-antigen species synthesized by infected cells were metabolically labeled with 32Pi and compared with the E. coli-produced ElA T antigens labeled in vitro with [y-32P]ATP by using the partially purified kinase. Partial V8 proteolysis analysis gave * Corresponding author.

Cyclin-dependent kinases phosphorylate the adenovirus E1A protein, enhancing its ability to bind pRb and disrupt pRb-E2F complexes

“…We also compared the relative mobilities of in vitro-and in vivo-32 P-labeled E1A and found them to be indistinguishable by SDS-PAGE (data not shown), which is consistent with previous results (65).…”

Genetic mapping of a major site of phosphorylation in adenovirus type 2 E1A proteins

“…Evidence is also presented here that this major site of ElA phosphorylation is not phosphorylated by cAMP-dependent protein kinases. Data supporting both of these conclusions come from the results of Lucher et al (33). They used a partially purified protein kinase from uninfected human KB cells to phosphorylate in vitro Adl2 ElA proteins synthesized in Escherichia coli.…”

Glycine N‐methyltransferases: A comparison of the crystal structures and kinetic properties of recombinant human, mouse and rat enzymes

“…It should be noted that these previous studies were done with crude ammonium sulfate fractions from liver, and the results reported here are for the purified recombinant enzymes expressed in E. coli . Moreover, rat liver GNMT is phosphorylated, while the recombinant enzymes expressed in E. coli probably are not 27. It is not known what effect this post‐translational modification has on the kinetics of GNMT.…”