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 states depending on the cellular environment and perhaps the stage of the cell cycle. These different phosphorylation states can be mimicked in the baculovirus expression system by employing the phosphatase inhibitor okadaic acid. Hyperphosphorylation of p53, particularly of Ser313 and/or Ser309, stimulated its DNA binding activity (Fuchs, Hecker and Scheidtmann, Eur. J. Biochem. 228, 625, 1995). Here we show that hyperphosphorylation of p53 has different effects on its DNA-binding activity, depending on the phosphorylation sites and the binding motif: (i) Phosphorylation of amino-terminal sites appeared to reduce binding to the RGC consensus motif, whereas additional phosphorylation of both, Ser313 and Ser309 led to enhanced binding. (ii) Upon hyperphosphorylation, 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 directed against the carboxyl terminus, but this latter effect was superimposed by the phosphorylation state of p53. Thus, the DNA binding activity of p53 appears to be regulated in a complex way in that (i) binding to a given sequence motif may be regulated by differential phosphorylation and/or by interaction with other factors; (ii) binding to different motifs may be modulated in opposite ways. Thus, the different genes that are regulated by p53 may be differently affected by these parameters.