The tumor suppressor p53 is a canonical inducer of cellular senescence (irreversible loss of proliferative potential and senescent morphology). p53 can also cause reversible arrest without senescent morphology, which has usually been interpreted as failure of p53 to induce senescence. Here we demonstrate that p53-induced quiescence actually results from suppression of senescence by p53. In previous studies, suppression of senescence by p53 was masked by p53-induced cell cycle arrest. Here, we separated these two activities by inducing senescence through overexpression of p21 and then testing the effect of p53 on senescence. We found that in p21-arrested cells, p53 converted senescence into quiescence. Suppression of senescence by p53 required its transactivation function. Like rapamycin, which is known to suppress senescence, p53 inhibited the mTOR pathway. We suggest that, while inducing cell cycle arrest, p53 may simultaneously suppress the senescence program, thus causing quiescence and that suppression of senescence and induction of cell cycle arrest are distinct functions of p53. Thus, in spite of its ability to induce cell cycle arrest, p53 can act as a suppressor of cellular senescence.nduction of p53 can cause apoptosis, reversible cell cycle arrest, and cellular senescence (1-5). In contrast to reversible cell cycle arrest (quiescence), cellular senescence is defined by irreversible loss of proliferative potential, acquisition of characteristic morphology (large, flattened cells), and expression of specific biomarkers (e.g., senescence-associated β-galactosidase, SA-β-Gal) (6). Since p53 appeared to induce senescence in some situations, observations of p53-induced quiescence have usually been interpreted as failure of p53 to activate the senescence program, which remains poorly understood. We recently reported that in two cell lines in which ectopic expression of p21 caused senescence, activation of endogenous p21 by endogenous p53 caused quiescence (7). The simplest conventional explanation for this result is that p53 failed to activate p21 to the degree required for induction of senescence, although it was sufficient for induction of quiescence. However, an alternative possibility is that p53 acts as a suppressor of the senescence program. This model leads to the testable prediction that induction of p53 would suppress p21-mediated senescence and convert it into quiescence. Here we demonstrate that this model is indeed correct, which indicates that, despite its ability to induce cell cycle arrest, p53 is a suppressor, not an inducer, of cellular senescence. Retrospectively, this result is not completely unexpected. First, it is known that p53 inhibits the mTOR (mammalian target of rapamycin) pathway (8-12). Second, it is known that inhibition of mTOR by rapamycin converts senescence into quiescence (13-15). In turn, this predicts that p53, like rapamycin, may suppress senescence. Here we confirmed this prediction. ResultsThe p53 Activator Nutlin-3a Suppresses p21-Induced Senescence. As previously show...

Transient induction of p53 can cause reversible quiescence and irreversible senescence. Using nutlin-3a (a small molecule that activates p53 without causing DNA damage), we have previously identified cell lines in which nutlin-3a caused quiescence. Importantly, nutlin-3a caused quiescence by actively suppressing the senescence program (while still causing cell cycle arrest). Noteworthy, in these cells nutlin-3a inhibited the mTOR (mammalian Target of Rapamycin) pathway, which is known to be involved in the senescence program. Here we showed that shRNA-mediated knockdown of TSC2, a negative regulator of mTOR, partially converted quiescence into senescence in these nutlin-arrested cells. In accord, in melanoma cell lines and mouse embryo fibroblasts, which easily undergo senescence in response to p53 activation, nutlin-3a failed to inhibit mTOR. In these senescence-prone cells, the mTOR inhibitor rapamycin converted nutlin-3a-induced senescence into quiescence. We conclude that status of the mTOR pathway can determine, at least in part, the choice between senescence and quiescence in p53-arrested cells.

Activity of the mammalian pyruvate dehydrogenase complex is regulated by phosphorylation-dephosphorylation of three specific serine residues (site 1, Ser-264; site 2, Ser-271; site 3, Ser-203) of the ␣ subunit of the pyruvate dehydrogenase (E1) component. Phosphorylation is carried out by four pyruvate dehydrogenase kinase (PDK) isoenzymes. Specificity of the four mammalian PDKs toward the three phosphorylation sites of E1 was investigated using the recombinant E1 mutant proteins with only one functional phosphorylation site present. All four PDKs phosphorylated site 1 and site 2, however, with different rates in phosphate buffer (for site 1, PDK2 > PDK4ϷPDK1 > PDK3; for site 2, PDK3 > PDK4 > PDK2 > PDK1). Site 3 was phosphorylated by PDK1 only. The maximum activation by dihydrolipoamide acetyltransferase was demonstrated by PDK3. In the free form, all PDKs phosphorylated site 1, and PDK4 had the highest activity toward site 2. The activity of the four PDKs was stimulated to a different extent by the reduction and acetylation state of the lipoyl moieties of dihydrolipoamide acetyltransferase with the maximum stimulation of PDK2. Substitution of the site 1 serine with glutamate, which mimics phosphorylation-dependent inactivation of E1, did not affect phosphorylation of site 2 by four PDKs and of site 3 by PDK1. Site specificity for phosphorylation of four PDKs with unique tissue distribution could contribute to the tissue-specific regulation of the pyruvate dehydrogenase complex in normal and pathophysiological states.

The PDC (pyruvate dehydrogenase complex) plays a central role in the maintenance of glucose homoeostasis in mammals. The carbon flux through the PDC is meticulously controlled by elaborate mechanisms involving post-translational (short-term) phosphorylation/dephosphorylation and transcriptional (long-term) controls. The former regulatory mechanism involving multiple phosphorylation sites and tissue-specific distribution of the dedicated kinases and phosphatases is not only dependent on the interactions among the catalytic and regulatory components of the complex but also sensitive to the intramitochondrial redox state and metabolite levels as indicators of the energy status. Furthermore, differential transcriptional controls of the regulatory components of PDC further add to the complexity needed for long-term tuning of PDC activity for the maintenance of glucose homoeostasis during normal and disease states.

The derivative of vitamin B1, thiamin pyrophosphate, is a cofactor of enzymes performing catalysis in pathways of energy production. In ␣ 2 ␤ 2 -heterotetrameric human pyruvate dehydrogenase, this cofactor is used to cleave the C ␣ ؊C(‫؍‬O) bond of pyruvate followed by reductive acetyl transfer to lipoyl-dihydrolipoamide acetyltransferase. The dynamic nonequivalence of two, otherwise chemically equivalent, catalytic sites has not yet been understood. To understand the mechanism of action of this enzyme, we determined the crystal structure of the holo-form of human pyruvate dehydrogenase at 1.95-Å resolution. We propose a model for the flip-flop action of this enzyme through a concerted ϳ2-Å shuttlelike motion of its heterodimers. Similarity of thiamin pyrophosphate binding in human pyruvate dehydrogenase with functionally related enzymes suggests that this newly defined shuttle-like motion of domains is common to the family of thiamin pyrophosphate-dependent enzymes.The thiamin pyrophosphate (TPP) 1 -dependent enzymes perform a wide range of catalytic functions in the pathways of energy production, including decarboxylation of ␣-keto acids followed by transketolation. The enzymes that have been structurally characterized so far, 2-oxoisovalerate dehydrogenase from Pseudomonas putida (1), human branched-chain ␣-ketoacid dehydrogenase (2), bacterial pyruvate dehydrogenase (3), transketolase (4), pyruvate decarboxylase (5), benzoylformate decarboxylase (6), acetohydroxyacid synthase (7), pyruvate oxidase (8), and pyruvate:ferredoxin oxidoreductase (9), have shown a common mechanism of TPP activation by (i) forming the ionic N-H⅐⅐⅐O Ϫ hydrogen bonding between the N1Ј atom of the aminopyrimidine ring of the coenzyme and an intrinsic ␥-carboxylate group of glutamate and (ii) imposing an "active" V-conformation that brings the N4Ј atom of the aminopyrimidine to the distance required for the intramolecular C-H⅐⅐⅐N hydrogen-bonding with the thiazolium C2 atom (Fig. 1). Within these two hydrogen bonds that rapidly exchange protons, protonation of the N1Ј atom of the aminopyrimidine system is strictly connected with the deprotonation of the 4Ј-amino group in that system and eventually abstraction of the proton from C2 and formation of the reactive 4Ј-amino-C2-carbanion (Fig. 1a) (10). This reactive C2 atom of TPP is the nucleophile that attacks the carbonyl carbon of different substrates used in the family of TPP-dependent enzymes. Within pyruvate dehydrogenase (E1), the first catalytic component enzyme of pyruvate dehydrogenase complex (PDC), this substrate is pyruvate (S 1 ). The cleavage of the central C ␣ -C(ϭO) bond of this substrate proceeds from induction of the intermediate, 4Ј-imino-2-(2-hydroxypropionyl)thiamin pyrophosphate, i.e. lactyl-TPP (LTPP) (Fig. 1b), followed by conversion to 4Ј-imino-2-(1-hydroxyethyl) thiamin pyrophosphate (HETPP) with release of carbon dioxide (P 1 ) (Fig. 1c). The fate of this active C2-␣-carbanion/enamine HETPP differs among various TPP-dependent enzymes depending on the nature of t...