THE IN-VITRO PHOSPHORYLATION OF P53 BY CALCIUM-DEPENDENT PROTEIN-KINASE-C - CHARACTERIZATION OF A PROTEIN-KINASE-C-BINDING SITE ON P53

We show that, in vitro, Ca2+-dependent protein kinase C (PKC) phosphor ylates recombinant murine p53 protein on several residues contained wi thin a conserved basic region of 25 amino acids, located in the C-term inal part of the protein. Accordingly, synthetic p53-(357-381)-peptide is phosphorylated by PKC at multiple Ser and Thr residues, including Ser360, Thr365, Ser370 and Thr377. We also establish that p53-(357-381 )-peptide at micromolar concentrations has the ability to stimulate se quence-specific DNA binding by p53. That stimulation is lost upon phos phorylation by PKC. To further characterise the mechanisms that regula te PKC-dependent phosphorylation of p53-(357-381)-peptide, the phospho rylation of recombinant p53 and p53-(357-381)-peptide by PKC were comp ared. The results suggest that phosphorylation of full-length p53 on t he C-terminal PKC sites is highly dependent on the accessibility of th e phosphorylation sites and that a domain on p53 distinct from p53-(35 7-381)-peptide is involved in binding PKC. Accordingly, we have identi fied a conserved 27-amino-acid peptide, p53-(320-346)-peptide, within the C-terminal region of p53 and adjacent to residues 357-381 that int eracts with PKC in vitro. The interaction between p53-(320-346)-peptid e and PKC inhibits PKC autophosphorylation and the phosphorylation of substrates, including p53-(357-381)-peptide, neurogranin and histone H 1. Conventional Ca2+-dependent PKC alpha, beta and gamma and the catal ytic fragment of PKC (PKM) were nearly equally susceptible to inhibiti on by p53-(320-346)-peptide. The Ca2+-independent PKC delta was much l ess sensitive to inhibition. The significance of these findings for un derstanding the in vivo phosphorylation of p53 by PKC are discussed.