p53-Induced Growth Arrest Is Regulated by the Mitochondrial SirT3 Deacetylase
Figure 2
Deletion of p53 identifies the novel mitochrondria -associated senescence domain (MASD) between amino acids 64 and 209 of p53.
(A) Various deletions of FLAG –tagged p53 were used to determine involvement in mitochrondria –associated senescence programs in EJ-p53 cells. The following abbreviations were used to characterize individual domains within p53 as, AD1, activation domain 1; AD2, activation domain 2; PRD, proline rich domain; DBD-NLS-TD-NES, combined DNA binding domain-nuclear localization signal-transactivation domain-nuclear export signal; BD, basic domain. Following the transient transfection of individual p53 constructs into the uninduced EJ-p53 cells, expression of p53 protein levels were normalized versus cell number to measure the level of SA -β galactosidase activity by staining with Xgal. ELISA analysis was then performed using antisera against human prohibitin (Research Diagnostics, Inc.). Colormetric analysis was then used to measure the amount of SA-β galactosidase activity ELISA was performed to measure prohibitin levels (fg/ml lysate) and normalized by the amount of immunoprecipitated FLAG-tagged p53 protein used as input from the ELISA assay. (B) Immunoblot analysis was then performed with anti –prohibitin nitrocellulose filter was reused to immunoblot with an anti- β actin polyclonal antisera (Sigma-Aldrich). (C) Electron micrograph (10,000X) of EJ carcinoma cells transfected with the different FLAG-tagged and truncated variants of human p53 and stained with the anti-FLAG monoclonal antibody (Sigma-Aldrich). Region corresponding to the outline of the mitochrondria is indicated. (D) Interaction of Sirt3 with the MASD region of p53. Using FLAG-tagged variants of the deleted p53 cDNAs expressed by transient transfections of p53 shown (left) were used to identify specific interactions with endogenous Sirt3 by immunoprecipitation with M2 agarose (Sigma-Aldrich) followed by standard immunoblotting protocols.