p53 is a nuclear transcription factor with a pro-apoptotic function. Since over 50% of human cancers carry loss of function mutations in p53 gene, p53 has been considered to be one of the classical type tumor suppressors. Mutant p53 acts as the dominant-negative inhibitor toward wild-type p53. Indeed, mutant p53 has an oncogenic potential. In some cases, malignant cancer cells bearing p53 mutations display a chemo-resistant phenotype. In response to a variety of cellular stresses such as DNA damage, p53 is induced to accumulate in cell nucleus to exert its pro-apoptotic function. Activated p53 promotes cell cycle arrest to allow DNA repair and/or apoptosis to prevent the propagation of cells with serious DNA damage through the transactivation of its target genes implicated in the induction of cell cycle arrest and/or apoptosis. Thus, the DNA-binding activity of p53 is tightly linked to its tumor suppressive function. In the present review article, we describe the regulatory mechanisms of p53 and also p53-mediated therapeutic strategies to cure malignant cancers.

Polo-like kinase 1 (Plk1) has an important role in the regulation of M phase of the cell cycle. In addition to its cell cycle-regulatory function, Plk1 has a potential role in tumorigenesis. Here we found for the first time that Plk1 physically binds to the tumor suppressor p53 in mammalian cultured cells, and inhibits its transactivation activity as well as its pro-apoptotic function. During the cisplatin-induced apoptosis in human neuroblastoma SH-SY5Y cells, the expression level of Plk1 was significantly decreased both at mRNA and protein levels, whereas cisplatin treatment caused a remarkable stabilization of p53. Systematic immunoprecipitation analyses using a series of deletion mutants of p53 revealed that a sequence-specific DNA-binding region of p53 is required and sufficient for the physical interaction with Plk1. The ectopically overexpressed Plk1 was co-localized with the endogenous p53 in mammalian cell nucleus, as shown by confocal laser microscopy. Expression of exogenous Plk1 and p53 in p53-deficient lung carcinoma H1299 cells greatly decreased the p53-mediated transcription from the p53-responsive p21 WAF1 , MDM2, and BAX promoters, whereas the kinase-deficient mutant form of Plk1 failed to reduce the transcriptional activity of p53. Consistent with the luciferase reporter analysis, Plk1 had an ability to block the p53-dependent induction of the endogenous p21 WAF1 . In addition, Plk1 inhibited the pro-apoptotic function of p53 in H1299 cells. Intriguingly, Plk1-mediated repression of p53 was attenuated with ATM. Thus, our present findings strongly suggest that p53 is a critical target of Plk1, and its function is abrogated through the physical interaction with Plk1.

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...

Alzheimer's amyloid precursor protein (APP) 1 is an integral membrane protein with a receptor-like structure (1). A principal component of parenchymal amyloid deposits in Alzheimer's disease (AD) is ␤-amyloid (A␤) (2-4), which is derived from APP by proteolytic cleavage (1, 5-11). A␤ is thought to be generated through an intracellular protein secretory pathway of APP (for a review, see Ref. 12). The short APP cytoplasmic domain consisting of 47 amino acid residues is thought to be responsible for determination of APP metabolism (13-15) and possible signal transduction from a putative extracellular ligand that has yet to be identified (for a review, see Ref. 16). Because APP is a candidate pathogenic factor of AD, elucidation of the physiological function of APP as well as the determination of the metabolic mechanism of A␤ production should increase our understanding of the pathogenesis of AD. Using a yeast two-hybrid system, we isolated cDNA of proteins that interact with the cytoplasmic domain of APP (APP COOH ) in order to elucidate the molecular mechanisms of APP metabolism and function of APP. Previous efforts to identify and isolate proteins associating with APP COOH , utilizing the yeast two-hybrid system, have resulted in the isolation of proteins carrying phosphotyrosine binding/phosphotyrosine interaction (PI) domains such as Fe65 (17), 19), and X11 (20). The X11 gene, which is located on chromosome 9, was originally isolated as a gene candidate for Friedreich ataxia (21) and its partial cDNA was identified as a clone encoding a protein that associates with APP COOH (22). The PI domain of X11 interacts with the YENPTY motif of APP COOH (20). In the present study, we isolated a complete cDNA encoding an X11-like protein (X11L) from a human adult brain cDNA library, utilizing the yeast two-hybrid system and APP COOH as a bait. A partial short cDNA encoding approximately 190 amino acids in the PI domain of X11L has already been isolated using a similar procedure (22). However, detailed characterization of X11L binding to APP and identification of the role X11L plays in the physiological function of APP have not been performed. We found that human X11L requires a sequence containing the NPXY motif of APP COOH for APP binding and that association of the PI domain with APP was suppressed by a deletion of a amino-terminal domain fused to the PI domain (PI ϩ C construct) but enhanced by a deletion of a carboxyl-terminal domain fused to the PI domain (N ϩ PI construct). Co-transfection of full-length human X11L into cells that express stably transfected human APP695 cDNA resulted in decreased secretion of A␤40 but not A␤42. However, co-transfection into cells of the cDNA lacking the carboxyl-terminal domain (N ϩ PI construct) or a cDNA encoding only the PI domain (PI construct), whose protein products preserve the ability to bind to APP COOH , did not present the ability to modulate A␤ production. The present results suggest that the amino-terminal region of the PI domain is needed to regulate binding affinity...

p73 is a p53-related tumor suppressor but is also induced by oncogene products such as E2F-1, raising a question as to whether p73 is a tumor suppressor gene or oncogene. Unlike p53, p73 has several variants, including ⌬Np73, which lacks the NH 2 -terminal transactivation domain. Although, in developing neurons, ⌬Np73 is expressed abundantly and seems to inhibit the proapoptotic function of p53, the role of p73 and ⌬Np73 and their regulatory mechanism in cell growth and differentiation are poorly understood. Here we report that p73, but not p53, directly activates the transcription of endogenous ⌬Np73 by binding to the p73-specific target element located at positions ؊76 to ؊57 within the ⌬Np73 promoter region. The activation of ⌬Np73 promoter by p63 was marginal. ⌬Np73 was associated with p73␣, p73␤, and p53, as demonstrated by immunoprecipitation assays, and inhibited their transactivation activities when we used reporters of Mdm2, Bax, or ⌬Np73 itself in SAOS-2 cells. Furthermore, induction or overexpression of ⌬Np73 promoted cell survival by competing with p53 and p73 itself. Thus, our results suggest that the negative feedback regulation of p73 by its target ⌬Np73 is a novel autoregulatory system for modulating cell survival and death.