Tumor suppressor p53-dependent stress response pathways play an important role in cell fate determination. In this study, we have found that glucose depletion promotes the phosphorylation of AMP-activated protein kinase catalytic subunit ␣ (AMPK␣) in association with a significant up-regulation of p53, thereby inducing p53-dependent apoptosis in vivo and in vitro. Thymocytes prepared from glucose-depleted wild-type mice but not from p53-deficient mice underwent apoptosis, which was accompanied by a remarkable phosphorylation of AMPK␣ and a significant induction of p53 as well as pro-apoptotic Bax. Similar results were also obtained in human osteosarcoma-derived U2OS cells bearing wild-type p53 following glucose starvation. Of note, glucose deprivation led to a significant accumulation of p53 phosphorylated at Ser-46, but not at Ser-15 and Ser-20, and a transcriptional induction of p53 as well as proapoptotic p53 AIP1. Small interference RNA-mediated knockdown of p53 caused an inhibition of apoptosis following glucose depletion. Additionally, apoptosis triggered by glucose deprivation was markedly impaired by small interference RNA-mediated depletion of AMPK␣. Under our experimental conditions, down-regulation of AMPK␣ caused an attenuation of p53 accumulation and its phosphorylation at Ser-46. In support of these observations, enforced expression of AMPK␣ led to apoptosis and resulted in an induction of p53 at protein and mRNA levels. Furthermore, p53 promoter region responded to AMPK␣ and glucose deprivation as judged by luciferase reporter assay. Taken together, our present findings suggest that AMPK-dependent transcriptional induction and phosphorylation of p53 at Ser-46 play a crucial role in the induction of apoptosis under carbon source depletion. AMP-activated protein kinase (AMPK)3 was originally identified as an enzyme that has an ability to inhibit hydroxymethylglutaryl-CoA reductase (1) and also regulate acetyl-CoA carboxylase by reversible phosphorylation (2). Subsequent studies demonstrated that AMPK is widely expressed and exists as a heterotrimeric complex, which consists of a catalytic subunit (␣) and two regulatory subunits (␤ and ␥). The mammalian genome contains seven AMPK genes encoding two ␣ (␣1 and ␣2), two ␤ (␤1 and ␤2), and three ␥ (␥1, ␥2, and ␥3) isoforms (3-5). The catalytic ␣ subunit is composed of three functional domains, including an NH 2 -terminal Ser/Thr protein kinase domain, a central auto-inhibitory region, and a COOH-terminal regulatory subunit-binding domain. AMPK acts as an intracellular energy sensor by monitoring cellular energy levels. For example, AMPK becomes activated by the tumor suppressor LKB1 complex-mediated phosphorylation at Thr-172 in response to certain energy-depleting stresses such as glucose deprivation, hypoxia, and oxidative stress, which increase the intracellular AMP:ATP ratio (6 -10). AMPK can also be activated allosterically in the AMP:ATP ratio (11). Upon activation, AMPK down-regulates the ATP consuming metabolic pathways and activates the energy-g...

In response to DNA damage, p73 plays a critical role in cell fate determination. In this study, we have found that Plk1 (pololike kinase 1) associates with p73, phosphorylates p73 at Thr-27, and thereby inhibits its pro-apoptotic activity. During cisplatinmediated apoptosis in COS7 cells in which the endogenous p53 is inactivated by SV40 large T antigen, p73 was induced to accumulate in association with a significant down-regulation of Plk1. Consistent with these observations, Plk1 reduced the stability of the endogenous p73. Immunoprecipitation and in vitro pulldown assay demonstrated that p73 binds to the kinase domain of Plk1 through its NH 2 -terminal region. Luciferase reporter assay and reverse transcription-PCR analysis revealed that Plk1 is able to block the p73-mediated transcriptional activation. Of note, kinase-deficient Plk1 mutant (Plk1(K82M)) retained an ability to interact with p73; however, it failed to inactivate the p73-mediated transcriptional activation, suggesting that kinase activity of Plk1 is required for the inhibition of p73. Indeed, in vitro kinase assay indicated that p73 is phosphorylated at Thr-27 by Plk1. Furthermore, small interference RNA-mediated knockdown of the endogenous Plk1 in p53-deficient H1299 cells resulted in a significant increase in the number of cells with sub-G 1 DNA content accompanied by the upregulation of p73 and pro-apoptotic p53 AIP1 as well as the proteolytic cleavage of poly(ADP-ribose) polymerase. Thus, our present results suggest that Plk1-mediated dysfunction of p73 is one of the novel molecular mechanisms to inhibit the p53-independent apoptosis, and the inhibition of Plk1 might provide an attractive therapeutic strategy for cancer treatment.p73 is one of newly identified p53 tumor suppressor gene family members (p53, p73, and p63) that encodes a nuclear transcription factor (1-3). Like the other p53 family members, p73 encodes multiple isoforms, including TA (transactivating), with distinct COOH-terminal extensions arising from alternative splicing events and ⌬N (nontransactivating) variants generated by alternative promoter usage (2-4). ⌬Np73 has an oncogenic potential (5) and displays a dominant-negative behavior toward TAp73 as well as wild-type p53 (6). TAp73 transactivates overlapping set of p53-target genes implicated in the induction of cell cycle arrest and/or apoptosis, and plays an important role in the regulation of DNA damage response, which is closely linked to its DNA binding activity. The initial studies demonstrated that TAp73 does not induce enough to accumulate in response to DNA damage arising from UV exposure or actinomycin D treatment (1); however, it has been shown that, in response to certain subset of DNA-damaging agents, TAp73 accumulates in the cell nucleus and exerts its pro-apoptotic function (7).Accumulating evidence suggests that TAp73 is regulated by post-translational modifications such as phosphorylation and acetylation. For example, TAp73 is stabilized in response to DNA damage such as cisplatin (CDDP) 2 treatment or e...

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Plk3, one of Polo-like kinase family members, is involved in the regulation of cell cycle progression and DNA damage response. In this study, we found that Plk3 inhibits pro-apoptotic activity of p73 through physical interaction and phosphorylation. During cisplatin (CDDP)-mediated apoptosis, Plk3 was transcriptionally induced, whereas its protein level was kept at basal level, suggesting that Plk3 might rapidly degrade in response to CDDP. Immunoprecipitation and in vitro pull-down experiments demonstrated that Plk3 interacts with p73. Luciferase reporter assays and RT-PCR experiments revealed that Plk3 inhibits p73-mediated transcriptional activity. Consistent with these results, pro-apoptotic activity of p73 was blocked by Plk3. Additionally, Plk3 decreased the stability of p73. Intriguingly, kinase-deficient Plk3 failed to inhibit p73 function, indicating that kinase activity of Plk3 is required for Plk3-mediated inhibition of p73. Indeed, in vitro kinase reaction showed that NH(2)-terminal portion of p73 is phosphorylated by Plk3. In accordance with these observations, knocking down of Plk3 increased the stability of p73 and promoted CDDP-mediated apoptosis in association with up-regulation of p73. Collectively, our present findings suggest that Plk3 plays an important role in the regulation of cell fate determination in response to DNA damage through the inhibition of p73.