To study the importance of phosphorylation for p53 transactivation function, we generated mutations at each of its known phosphorylated serine amino acids. Mutations of murine p53 serine residues individually to either alanine or glutamic acid at positions 7, 9, 12, 18, 37, 312, and 389 resulted in equivalent levels of transcriptional activation in standard transient transfection experiments. However, when p53 transcriptional activity was measured in cells that attain G 1 arrest upon contact inhibition, wild-type p53 was inactive, and only alteration at serine 389 to glutamic acid resulted in a functional p53 protein. This Ser 3 Glu mutant also has an increased ability to bind DNA. Elimination of the phosphorylation site by substitution of an alanine amino acid resulted in loss of transcriptional activity. We also demonstrated that specific phosphorylation of p53 at serine 389 is induced by cyclin E overexpression in high-density cells. Our data establish for the first time that phosphorylation of p53 at serine 389 is important in activating its function in vivo.The p53 tumor suppressor is a transcriptional activator that regulates several cellular processes including cell cycle control, apoptosis, and DNA repair (1, 2). The wild-type p53 protein has been shown to bind to specific DNA sequences in the promoters or introns of several genes, including p21 WAF1/cip1 , mdm2, gadd45, bax, and cyclin G, activating their transcription (3-8).The majority of inactivating mutations in the p53 gene disrupt the DNA-binding domain and hence the transcriptional activation function of p53 (9, 10).The role of p53 in the cell cycle has been characterized in some detail. Activation of the p21 WAF1/cip1 gene by p53 results in growth suppression of cultured cells (4). p21WAF1/cip1 was also independently identified as an inhibitor of the cell cycle regulators cyclin/cyclin-dependent kinases (Cdk) 1 (11). p21 WAF1/cip1 seems to be a universal inhibitor of the cyclin-Cdk complexes, although it inhibits the cyclin E-Cdk2 complex most potently (11). The binding of cyclin E to Cdk2 activates its kinase function at the G 1 /S stage of the cell cycle (12, 13).The p53 protein is phosphorylated at several serine amino acids. A cluster of phosphoserines has been identified in vivo in the transactivation domain of murine p53 at amino acids 7, 9, 18, and 37 (14). In vitro studies showed that purified casein kinase I can phosphorylate serines 7, 9, and 12 (15). Purified DNA-activated protein kinase has also been shown to phosphorylate serines 7 and 18 in murine p53 and serines 15 and 37 in human p53 (16). Experiments examining the role of p53 phosphorylated amino-terminal amino acids in transcriptional activation indicated that mutations of these serines did not alter p53 function (17,18). Other data indicated a weak effect of phosphorylation at serine 15 (19, 20). Thus, the phosphorylation of amino-terminal serines has little if any effect on p53 function.Two phosphoserines in the C-terminal domain, serines 312 and 389 in murine p53, have been...

The ability of p53 to suppress transformation correlates with its ability to activate transcription. To

The wild-type p53 protein is a potent growth suppressor when overexpressed in vitro. It functions as a transcriptional activator and causes growth arrest at the G1/S stage of the cell cycle. We monitored p53 transactivation as an indicator of p53 function throughout the cell cycle. We first demonstrate that cells which exhibited contact inhibition of growth lacked p53 transactivation function at high cell density. Since these cells were noncycling, we examined whether the ectopic expression of any cyclin could override contact inhibition of growth and restore p53 transactivation function. The transfection of cyclin E at high cell density stimulated the progression of cells through the cell cycle and restored p53 transactivation function. The transcriptional activity of p53 induced by cyclin E was regulated at the level of DNA binding. Cells that did not show contact inhibition of growth had a functional p53 regardless of cell density. Thus, contact inhibition of cell growth corresponded to a lack of p53 transactivation function and the overexpression of cyclin E in these contact-inhibited cells stimulated cell cycle progression and resulted in p53 transcriptional activity.

The role of DNA topoisomerase II in multifactorial resistance to antineoplastic agents is reviewed. We have previously observed that in Adriamycin (ADR) resistant P388 murine leukemia cells, DNA topoisomerase II enzyme content and cleavage and catalytic activities were all reduced and correlated with drug sensitivity. A subsequent study provided evidence for an allelic mutation of the gene for DNA topoisomerase II as a possible molecular mechanism underlying the enzyme alterations. To ascertain how universal were these observations, a study was undertaken of DNA topoisomerase II (topo II) in other cell lines resistant either to ADR or another topo-II-interactive drug, mitoxantrone. In ADR-resistant Chinese hamster ovary (CHO) cells, topo II cleavage and catalytic activities and the gene product were all reduced; however, only cleavage activity correlated with drug sensitivity. No differences were noted between ADR-sensitive and -resistant CHO cells by Northern or Southern blot analysis, raising the possibility that the enzyme in resistant cells may be regulated at a posttranscriptional level. Findings on a gel retardation or immunoblot band depletion assay showed that the enzyme in CHO/ADR-1 cells failed to bind to the DNA-drug-enzyme complex, suggesting a qualitative as well as quantitative enzyme alteration in those cells. Mitoxantrone-resistant HeLa cells (Mito-1) displayed not only a lower level of cleavage activity but also of enzyme content and catalytic activity, relative to the parental drug-sensitive HeLa cells. As with the CHO cells, no differences were noted between mitoxantrone-sensitive and -resistant HeLa cells on Northern and Southern blot analyses, suggesting that enzyme regulation in these resistant cells may also be at a posttranscriptional level. There was no evidence of enzyme binding to DNA-drug-enzyme complex in resistant HeLa/Mito-1 cells, once again suggesting the presence of a qualitative enzyme alteration. The findings in both ADR-resistant CHO cells and mitoxantrone-resistant HeLa cells do not exclude the possibility that subtle changes in the topoisomerase II gene, such as point mutations, may account for these enzyme changes. The apparent qualitative changes observed in enzyme may result from posttranslational modifications such as phosphorylation.

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