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...

Thiamin diphosphate, a key coenzyme in sugar metabolism, is comprised of the thiazolium and 4-aminopyrimidine aromatic rings, but only recently has participation of the 4-aminopyrimidine moiety in catalysis gained wider acceptance. We report the use of electronic spectroscopy to identify the various tautomeric forms of the 4-aminopyrimidine ring on four thiamin diphosphate enzymes, all of which decarboxylate pyruvate: the E1 component of human pyruvate dehydrogenase complex, the E1 subunit of Escherichia coli pyruvate dehydrogenase complex, yeast pyruvate decarboxylase, and pyruvate oxidase from Lactobacillus plantarum. It is shown that, according to circular dichroism spectroscopy, both the 1,4-iminopyrimidine and the 4-aminopyrimidine tautomers coexist on the E1 component of human pyruvate dehydrogenase complex and pyruvate oxidase. Because both tautomers are seen simultaneously, these two enzymes provide excellent evidence for nonidentical active centers (asymmetry) in solution in these multimeric enzymes. Asymmetry of active centers can also be induced upon addition of acetylphosphinate, an excellent electrostatic pyruvate mimic, which participates in an enzyme-catalyzed addition to form a stable adduct, resembling the common predecarboxylation thiamin-bound intermediate, which exists in its 1,4-iminopyrimidine form. The identification of the 1,4-iminopyrimidine tautomer on four enzymes is almost certainly applicable to all thiamin diphosphate enzymes: this tautomer is the intramolecular trigger to generate the reactive ylide/carbene at the thiazolium C2 position in the first fundamental step of thiamin catalysis.1Ј,4Ј-iminothiamin diphospate ͉ 2-oxoacid decarboxylases ͉ active site asymmetry T he hypothesis that the 4Ј-aminopyrimidine (AP) ring of the coenzyme thiamin diphosphate (ThDP) (1, 2) actively participates in the reaction of enzymes that use it was suggested some years ago (3, 4). The hypothesis gained wider acceptance with the observation of two highly conserved features noted in the first structures of ThDP-dependent enzymes (5-7): (i) a distance shorter than 3.5 Å between the N4Ј atom of the AP and the C2 atom of the thiazolium ring, creating the possibility for intramolecular proton abstraction to generate the C2 carbanion/ ylide, identified by Breslow (8) as the nucleophile that initiates the sequence of reactions involving multiple ThDP-bound covalent intermediates [exemplified with yeast pyruvate decarboxylase (YPDC) (EC 4.1.1.1) in Scheme 1] and (ii) a glutamate residue within short hydrogen bonding distance of the N1Ј atom, poised to catalyze the tautomerization shown on the left side of Scheme 1. The tautomerization reaction requires three forms of the AP ring of which two are neutral, the AP and 1Ј,4Ј-iminopyrimidine (IP), but these forms must interconvert via the positively charged, N1Ј-protonated 4Ј-aminopyrimidinium ion (APH ϩ ). Our goal is to establish which form of the AP ring is present on each intermediate on the pathway in Scheme 1, and indeed in all ThDP enzymes. We here report...

Activity of the mammalian pyruvate dehydrogenase complex (PDC) is regulated by phosphorylation-dephosphorylation of three serine residues (designated site 1, Ser-264; site 2, Ser-271; site 3, Ser-203) in the ␣ subunit of the pyruvate dehydrogenase (E1) component. Substitutions of the phosphorylation sites were generated by site-directed mutagenesis. Glutamate (S1E) and aspartate (S1D) substitutions at site 1 resulted in the complete loss of PDC activity; however, these mutants were variably active in the decarboxylation and 2,6-dichlorophenolindophenol assays. S1Q had only 3% of wild-type PDC activity. The apparent K m values for pyruvate increased for the mutants of site 1 when determined in the 2,6-dichlorophenolindophenol assay. The substitutions at sites 2 and 3 caused only moderate reductions in activity in the three assays. S3E had a 27-fold increase in the apparent K m for thiamine pyrophosphate and 8-fold increase in the K i for pyrophosphate. Site 3 was almost completely protected from phosphorylation by thiamine pyrophosphate. The results show that the size rather than negative charge of the substituted amino acid residue affects the active site of E1 and that modification of each of the three serine residues affect the active site in a site-specific manner for its ability to bind the cofactor and substrates.The mammalian pyruvate dehydrogenase complex (PDC) 1 plays an important role in defining the fate of three-carbon compounds derived largely from carbohydrates and to some extent from amino acids. PDC comprises multiple copies of three catalytic enzymes, namely pyruvate dehydrogenase (E1), dihydrolipoamide acetyltransferase (E2), and dihydrolipoamide dehydrogenase (E3); in addition, the complex also contains a binding protein referred to as E3-binding protein (E3BP), and two regulatory enzymes, namely pyruvate dehydrogenase kinase (PDK) and phosphopyruvate dehydrogenase phosphatase. E1 carries out the decarboxylation of pyruvic acid and the reductive acetylation of lipoyl moieties covalently linked to E2. E2 then catalyzes the transfer of acetyl groups to CoA, forming acetyl-CoA and fully reduced lipoyl moieties. E3 reoxidizes the reduced lipoyl groups of E2 and transfers the electrons to NAD ϩ , forming NADH. Sixty subunits of mammalian E2 and 12 subunits of E3BP form the inner core structure, whereas 20 -30 tetrameric E1 (␣ 2 ␤ 2 ) components bind this core and 6 E3 homodimers bind to the E3BP subunits (1, 2).Regulation of mammalian PDC activity is accomplished in large part by phosphorylation (resulting in inactivation) of the E1 component by a family of pyruvate dehydrogenase kinases (PDK 1-4 isozymes) and dephosphorylation (leading to activation) of phosphorylated E1 by a set of specific phosphatases (phosphopyruvate dehydrogenase phosphatase 1-2 isozymes) (1, 3-6). The ␣ subunit of the E1 component has three phosphorylation sites, named site 1 (Ser-264), site 2 (Ser-271), and site 3 (Ser-203), and phosphorylation of any one of these three sites results in inactivation (7-9). In vivo inactivati...

Mammalian pyruvate dehydrogenase (alpha 2 beta 2) (E1) is regulated by phosphorylation-dephosphorylation, catalyzed by the E1-kinase and the phospho-E1-phosphatase. Using site-directed mutagenesis of the three phosphorylation sites (sites 1, 2, and 3) on E1 alpha, several human E1 mutants were made with single, double, and triple mutations by changing Ser to Ala. Mutation at site 1 but not at sites 2 and/or 3 decreased E1 specific activity and also increased Km values for thiamin pyrophosphate and pyruvate. Sites 1, 2, and 3 in the E1 mutants were phosphorylated either individually or in the presence of the other sites by the dihydrolipoamide acetyltransferase-protein X-E1 kinase indicating a site-independent mechanism of phosphorylation. Phosphorylation of each site resulted in complete inactivation of the E1. However, the rates of phosphorylation and inactivation were site-specific. Sites 1, 2, and 3 were dephosphorylated either individually or in the presence of the other sites by the phospho-E1-phosphatase resulting in complete reactivation of the E1. The rates of dephosphorylation and reactivation were similar for sites 1, 2, and 3, indicating a random dephosphorylation mechanism.