Carl W. Anderson

The ATM protein, encoded by the gene responsible for the human genetic disorder ataxia telangiectasia (A-T), regulates several cellular responses to DNA breaks. ATM shares a phosphoinositide 3-kinase-related domain with several proteins, some of them protein kinases. A wortmannin-sensitive protein kinase activity was associated with endogenous or recombinant ATM and was abolished by structural ATM mutations. In vitro substrates included the translation repressor PHAS-I and the p53 protein. ATM phosphorylated p53 in vitro on a single residue, serine-15, which is phosphorylated in vivo in response to DNA damage. This activity was markedly enhanced within minutes after treatment of cells with a radiomimetic drug; the total amount of ATM remained unchanged. Various damage-induced responses may be activated by enhancement of the protein kinase activity of ATM.

Activation of p53-mediated transcription is a critical cellular response to DNA damage. p53 stability and site-specific DNA-binding activity and, therefore, transcriptional activity, are modulated by post-translational modifications including phosphorylation and acetylation. Here we show that p53 is acetylated in vitro at separate sites by two different histone acetyltransferases (HATs), the coactivators p300 and PCAF. p300 acetylates Lys-382 in the carboxy-terminal region of p53, whereas PCAF acetylates Lys-320 in the nuclear localization signal. Acetylations at either site enhance sequence-specific DNA binding. Using a polyclonal antisera specific for p53 that is phosphorylated or acetylated at specific residues, we show that Lys-382 of human p53 becomes acetylated and Ser-33 and Ser-37 become phosphorylated in vivo after exposing cells to UV light or ionizing radiation. In vitro, amino-terminal p53 peptides phosphorylated at Ser-33 and/or at Ser-37 differentially inhibited p53 acetylation by each HAT. These results suggest that DNA damage enhances p53 activity as a transcription factor in part through carboxy-terminal acetylation that, in turn, is directed by amino-terminal phosphorylation.

In unstressed cells, the tumor suppressor protein p53 is present in a latent state and is maintained at low levels through targeted degradation. A variety of genotoxic stresses initiate signaling pathways that transiently stabilize the p53 protein, cause it to accumulate in the nucleus, and activate it as a transcription factor. Activation leads either to growth arrest at the G1/S or G2/M transitions of the cell cycle or to apoptosis. Recent studies point to roles for multiple post-translational modifications in mediating these events in response to genotoxic stresses through several potentially interacting but distinct pathways. The approximately 100 amino-acid N-terminal and approximately 90 amino-acid C-terminal domains are highly modified by post-translational modifications. The N-terminus is heavily phosphorylated while the C-terminus contains phosphorylated, acetylated and sumoylated residues. Antibodies that recognize p53 only when it has been modified at specific sites have been developed, and studies with these reagents show that most known post-translational modifications are induced when cells are exposed to genotoxic stresses. These recent results, coupled with biochemical and genetic studies, suggest that N-terminal phosphorylations are important for stabilizing p53 and are crucial for acetylation of C-terminal sites, which in combination lead to the full p53-mediated response to genotoxic stresses. Modifications to the C-terminus inhibit the ability of this domain to negatively regulate sequence-specific DNA binding; additionally, they modulate the stability, the oligomerization state, the nuclear import/export process and the degree of ubiquitination of p53.

Analysis of (35)S-methionine-labeled extracts of adenovirus 2-infected KB cells revealed 22 virus-induced polypeptide components. Most proteins of the virion were easily detected in extracts of whole cells labeled for short periods between 15 and 30 h after infection; however, several virion components were conspicuously absent. Radioactivity appeared in two of these virion components during a chase in nonradioactive medium, and this appearance was paralleled by a decrease in the radioactivity associated with two nonvirion adenovirus-induced proteins, results which imply precursor-product relationships for these components. Comparison of one of the chasable adenovirus-induced components (designated P-VII; mass of 20,000 daltons) and the major core protein (VII; mass of 18,500 daltons) of the virion showed that they have four common methionine-containing tryptic peptides; P-VII has an additional methionine residue which is not found in the major core protein. We propose that at least two of the adenovirus 2 virion components are derived by the cleavage of higher molecular weight precursor polypeptides.