Difference in the centrosome duplication regulatory activity among p53 'hot spot' mutants: potential role of Ser 315 phosphorylation-dependent centrosome binding of p53.

Abstract

The p53 tumor suppressor protein regulates centrosome duplication through multiple pathways, and p21(Waf1/Cip1) (Waf1), a major target of p53's transactivation function, has been shown to be one of the effectors. However, it had been unclear whether the p53's Waf1-independent centrosome duplication regulatory pathways require its transactivation function. In human cancers, specific residues of p53 are mutated at a high frequency. These 'hot spot' mutations abrogate p53's transactivation function. If p53 regulates centrosome duplication in a transactivation-independent manner, different 'hot spot' mutants may regulate centrosome duplication differently. To test this, we examined the effect of two 'hot spot' mutants (R175H and R249S) for their centrosome duplication regulatory activities. We found that R175H lost the ability to regulate centrosome duplication, while R249S partially retained it. Moreover, R249S associates with both unduplicated and duplicated centrosomes similar to wild-type p53, while R175H only associates with duplicated, but not unduplicated centrosomes. Since cyclin-dependent kinase 2 (CDK2) triggers initiation of centrosome duplication, and p53 is phosphorylated on Ser 315 by CDK2, we examined the p53 mutants with a replacement of Ser 315 to Ala (A) and Asp (D), both of which retain the transactivation function. We found that S315D retained a complete centrosome duplication activity, while S315A only partially retained it. Moreover, S315D associates with both unduplicated and duplicated centrosomes, while S315A associates with only duplicated, but not unduplicated centrosomes. Thus, p53 controls the centrosome duplication cycle both in transactivation-dependent and transactivation-independent manners, and the ability to bind to unduplicated centrosomes, which is controlled by phosphorylation on Ser 315, may be important for the overall p53-mediated regulation of centrosome duplication.