p53-induced growth arrest is regulated by the mitochondrial SirT3 deacetylase

Abstract

A hallmark of p53 function is to regulate a transcriptional program in response to extracellular and intracellular stress that directs cell cycle arrest, apoptosis, and cellular senescence. Independent of the role of p53 in the nucleus, some of the anti-proliferative functions of p53 reside within the mitochondria [1]. p53 can arrest cell growth in response to mitochondrial p53 in an EJ bladder carcinoma cell environment that is naïve of p53 function until induced to express p53 [2]. TP53 can independently partition with endogenous nuclear and mitochondrial proteins consistent with the ability of p53 to enact senescence. In order to address the role of p53 in navigating cellular senescence through the mitochondria, we identified SirT3 to rescue EJ/p53 cells from induced p53-mediated growth arrest. Human SirT3 function appears coupled with p53 early during the initiation of p53 expression in the mitochondria by biochemical and cellular localization analysis. Our evidence suggests that SirT3 partially abrogates p53 activity to enact growth arrest and senescence. Additionally, we identified the chaperone protein BAG-2 in averting SirT3 targeting of p53 -mediated senescence. These studies identify a complex relationship between p53, SirT3, and chaperoning factor BAG-2 that may link the salvaging and quality assurance of the p53 protein for control of cellular fate independent of transcriptional activity.

Figure 1. Analysis of p53 in subcellular fractions from senescent EJ-p53 cells.

(A) Immunoblots using 50 µg protein of total cell lysate and subcellular fractions (mitochondria and nuclear fraction obtained from a sucrose gradient) were subject to SDS-PAGE and subsequently transferred to PVDF filters (Immobilon, Millipore Corp.). Blots were then blocked with BSA and initially probed using a mouse monoclonal antibody specific for p53 (clone 1801, Oncogene Research) followed by HRP-conjugated anti-mouse IgG (Roche) and then developed using ECL kit (GE Healthcare/Amersham). Blots were reprobed using mouse monoclonal specific for the mitochondrial marker, Cox II (Molecular Probes, Invitrogen) and nuclear marker PCNA (Calbiochem) followed by HRP-conjugated anti-mouse IgG and then developed using the ECL kit (GE Healthcare/Amersham). (B) Laser confocal image of p53 immunohistochemistry and CMTMR, YOYO fluorescence in EJ-p53 cells. EJ-p53 cells were maintained in (+) tet (no p53 expression) and (-)tet 2 hours through 4days (overexpression of wt p53). p53-specific mAb(Oncogene research) and Cy5-labled anti-mouse were used to immunohistochemistry localize p53 in EJ-p53 cells. YOYO was used to stain nuclei and CMTMR was to stain mitochondria. For each image, p53 was re-colored to red, Nuclei was re-colored to blue, and mitochondria was re-colored to green. p53 localization in p53-induced senescence in EJ-p53 cells was determined by overlay.

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.

Figure 3. The NAD+ -dependent Sir2 -like mitochondria protein deacetylase, SirT3, rescues EJ-p53 from p53 –inducible growth arrest.

The human SirT3 2.9 kb cDNA identified in the retroviral screen ( Fig. 2 ) was placed in the MSCV retroviral plasmid using a bicistronic cassette with GFP (Green fluorescent protein). EJ-p53 cells were transduced with MSCV particles containing only GFP, mouse c-myc and human SirT3. Cell growth kinetics were determined by laser scanning florescence microscopy (A) and by monitoring cell populations (B). (C) [³H]-thymidine incorporation studies were assayed after 72 hours in EJ-p53 cells following transduction with indicated retroviruses followed by induction of p53 expression by tetracycline withdrawal for 72 hours. (D) SA-β galactosidase activity were determined in retroviral transduced EJ-p53 with MSCV-GFP/Sirt3 transduced into EJ-p53 cells prior and following induction of p53 by tetracycline withdrawal.

Figure 4. Association of p53 with mitochondrial proteins contains NAD+ dependent protein deacetylase activity of human SirT3.

(A). Total cell lysate from (+)tet, (-)tet 6 h and 24 h EJ-p53 cells were collected and solubilized in lysis buffer containing complete proteolysis inhibitor. Monoclonal antibody against human p53 (DO1) and then protein G-agarose beads were added to the sample. The immunoprecipitated proteins from the washed beads were separated on a 10% gel, then transfer to filters for immunoblotting. Blots were blocked and probed with anti-p53 (mouse monoclonal DO-1), polyclonal rabbit anti-mthsp 70 (HSPA9) and mouse monoclonal anti-Bcl 2 (clone 124, Dako USA) and anti- SirT3 followed by HRP-conjugated anti-goat, HRP-conjugated anti-mouse and HRP-conjugated anti-rabbit, and then developed using ECL. (B). Total cell, nuclear, and mitochondrial fractions were obtained from EJ-p53 cells after 12 hours of tetracycline withdrawal and p53 expression. Each fractionated lysate was analyzed by SDS-PAGE and immunoblotted with antibodies against p53, SirT1 and SirT3. (C) Protein deacetylase activity was measured against a synthetic peptide corresponding to the human p53 protein sequence (HLKSKKGQSTSRHKKLMFK-C*) radiolabeled with [¹⁴C]-acetylCoA (GE Healthcare) and purified acetyltransferases CBP and PCAF in vitro. Deacetylase activity was determined from mitochondrial and nuclear fractions taken from EJ-p53 cells following 12 hours after induction of p53 expression by tetracycline withdrawal. Deacetylase activity was determined from p53, Sirt1, and Sirt3 immunopreciptates taken from nuclear and mitochondrial fractions. (D) Co-localization of p53 and SirT3 was performed by transient expression of human Sirt3 tagged with the Green Fluorescent Protein (GFP) and visualized by laser confocal microscopy. Inducible expression of p53 was monitored after 6 and 24 hours post-induction through the withdrawal of tetracycline (Tet) from growth medium. Separate images at each wavelength were merged to determine the signal overlay.

Figure 5. Human BAG-2 associates with p53 and stabilizes the level of p53 in vivo.

(A) EJ-p53 cells were cultured to induce p53 expression upon withdrawal of tetracycline after 24 hours. As shown, input level of p53 and human BAG-2 are shown from total cell lysates obtained from the EJ-p53 cells in the presence (+tet) or withdrawal of tetracycline (-tet) from culture medium for EJ-p53 cells. Immunoblots were conducted with antisera against p53 and BAG-2 from 50 mg of total cell lysate. Bottom, shown is an immunoprecipitation conducted with anti -p53 followed by immunoblotting with anti –BAG-2 antibodies. (B) RNA interference (RNAi) of human BAG-2 was conducted with siRNAs (Ambion, sequence ID#137542) directed at endogenous human BAG-2 mRNA in the presence of p53 expression within EJ-p53 cells. Cells were maintained at 40% confluent growth in 5% CO₂ and transfected with 7 mg of the 21nt siRNA in 30 mm Petri dishes. The control sample is representative of a transfection using scrambled 21nt RNA mixture provided by the manufacturer. In parallel cultures, both total cell RNA and protein was recovered and used to measure mRNA and protein levels by northern hybridization using cDNA probes for human BAG-2, HPRT, and p53 and immunoblotted with the antibodies corresponding to BAG-2, HPRT, and p53, respectively. (C) Thymidine incorporation into nascent genomic DNA was measured upon introduction of siRNA targeted against BAG-2 and scrambled RNAs in EJ-p53 cells induced for the expression of p53 after 24 hours. Values reflect the relative incorporation of [³H] thymidine versus DNA content. (D) Total cellular content of acetylated and total following induction of p53 in EJ-p53 cells and infection with the MSCV –hSirt3 retrovirus or transfection with small interfering RNAs (siRNA) against human BAG2 (BAG2 RNAi). Antibody against acetylated p53 as described was used to detect acetylated species of p53. (E) As shown in panel D, Human embryonic lung fibroblasts IMR-90 cells were infected with MSCV-SirT3 and the MSCV control vector. Sub-cellular mitochondria and nuclear fractions were isolated and collected. Immunoblots confirming the presence of pan-acetylated p53, Lamin B1 and adenine nucleotide translocase (ANT) are shown from each sub-cellular fraction.