COMPLEX REGULATION OF THE DNA-BINDING ACTIVITY OF P53 BY PHOSPHORYLATION - DIFFERENTIAL-EFFECTS OF INDIVIDUAL PHOSPHORYLATION SITES ON THE INTERACTION WITH DIFFERENT BINDING MOTIFS

The tumor suppressor protein p53 exists in different phosphorylation s tates depending on the cellular environment and perhaps the stage of t he cell cycle. These different phosphorylation states can be mimicked in the baculovirus expression system by employing the phosphatase inhi bitor okadaic acid. Hyperphosphorylation of p53, particularly of Ser31 3 and/or Ser309, stimulated its DNA binding activity (Fuchs, Hecker an d Scheidtmann, Eur. J. Biochem. 228, 625, 1995). Here we show that hyp erphosphorylation of p53 has different effects on its DNA-binding acti vity, depending on the phosphorylation sites and the binding motif: (i ) Phosphorylation of amino-terminal sites appeared to reduce binding t o the RGC consensus motif, whereas additional phosphorylation of both, Ser313 and Ser309 led to enhanced binding. (ii) Upon hyperphosphoryla tion, binding to the RGC motif was enhanced whereas binding to the p53 response element of the bax1 gene promoter was diminished. (iii) DNA binding was also greatly enhanced by antibodies Pab 122 and 421 direct ed against the carboxyl terminus, but this latter effect was superimpo sed by the phosphorylation state of p53. Thus, the DNA binding activit y of p53 appears to be regulated in a complex way in that (i) binding to a given sequence motif may be regulated by differential phosphoryla tion and/or by interaction with other factors; (ii) binding to differe nt motifs may be modulated in opposite ways. Thus, the different genes that are regulated by p53 may be differently affected by these parame ters.