Protein kinase CK2-dependent regulation of p53 function: Evidence that thephosphorylation status of the serine 386 (CK2) site of p53 is constitutiveand stable

The p53 tumour suppressor protein is regulated by several mechanisms includ ing multisite phosphorylation. One of the protein kinases which has an esta blished role in regulating p53 function is the protein kinase CK2. The regu lation by CK2 occurs both through interaction of p53 with CK2. itself(the r egulatory beta subunit) and phosphorylation at the penultimate residue of p 53, serine 386 (murine p53). Strikingly, this phosphorylation event control s several independent functions of p53 including site-specific DNA binding, strand renaturation, transcriptional repression and the anti-proliferative function of p53. However, CK2 is a constitutively-active enzyme and theref ore the mechanism by which the phosphorylation of p53 at serine 386 is itse lf regulated, or indeed the question as to whether phosphorylation of this site is regulated at all, remains unresolved. In this paper we provide evid ence that serine 386 is highly resistant to dephosphorylation in cultured c ells, even though this site can be dephosphorylated in vitro by recombinant protein phosphatase 1. These data suggest that, once phosphorylated at the CK2 site, a p53 molecule remains in this modified form throughout its life span. To address the issue of whether the level of serine 386 phosphorylati on may be regulated through controlling the subcellular compartmentalisatio n of p53 and CK2, we examined the subcellular localisation of p53 and CK2 a lpha in C57MG cells and Rat-1 fibroblasts by immunofluorescence staining. B oth proteins were present in the cytoplasm and enriched in the nucleus, wit h minor variations in the intensity of subcellular location over the course of the cell cycle. Similarly, activation of p53 by UV irradiation or DNA d amage-inducing drugs had no effect on either the localisation or levels of CK2 alpha, even although significant nuclear p53 accumulation was observed. A striking observation arising from these studies was the intense staining of CK2 alpha with the centrosomes, suggesting a potentially important role for this kinase in microtubule formation and/or chromosomal segregation.