To study the importance of phosphorylation for p53transactivation function, we generated mutations ateach of its known phosphorylated serine amino acids.Mutations of murine p53 serine residues individually toeither alanine or glutamic acid at positions 7, 9, 12, 18,37, 312, and 389 resulted in equivalent levels of transcriptionalactivation in standard transient transfectionexperiments. However, when p53 transcriptional activitywas measured in cells that attain G1 arrest uponcontact inhibition, wild-type p53 was inactive, and onlyalteration at serine 389 to glutamic acid resulted in afunctional p53 protein. This Ser 3 Glu mutant also hasan increased ability to bind DNA. Elimination of thephosphorylation site by substitution of an alanineamino acid resulted in loss of transcriptional activity.We also demonstrated that specific phosphorylation ofp53 at serine 389 is induced by cyclin E overexpressionin high-density cells. Our data establish for the first timethat phosphorylation of p53 at serine 389 is important inactivating its function in vivo.