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

The p53 tumor suppressor protein regulates centrosome duplication through m ultiple pathways, and p21(Waf1/Cip1) (Waf1), a major target of p53's transa ctivation 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 centro some duplication in a transactivation-independent manner, different 'hot sp ot' mutants may regulate centrosome duplication differently. To test this, we examined the effect of two 'hot spot' mutants (R175H and R249S) for thei r centrosome duplication regulatory activities. We found that R175H lost th e ability to regulate centrosome duplication, while R249S partially retaine d it. Moreover, R249S associates with both unduplicated and duplicated cent rosomes similar to wild-type p53, while R175H only associates with duplicat ed, 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 3 15 to Ala (A) and Asp (D), both of which retain the transactivation functio n. We found that S315D retained a complete centrosome duplication activity, while S315A only partially retained it. Moreover, S315D associates with bo th unduplicated and duplicated centrosomes, while S315A associates with onl y duplicated, but not unduplicated centrosomes. Thus, p53 controls the cent rosome duplication cycle both in transactivation-dependent and transactivat ion-independent manners, and the ability to bind to unduplicated centrosome s, which is controlled by phosphorylation on Ser 315, may be important for the overall p53-mediated regulation of centrosome duplication.