Carboxyl-terminal binding protein (CtBP) is a transcriptional corepressor originally identified through its ability to interact with adenovirus E1A. The finding that CtBP-E1A interactions were regulated by the nicotinamide adeninine dinucleotides NAD ؉ and NADH raised the possibility that CtBP could serve as a nuclear redox sensor. This model requires differential binding affinities of NAD ؉ and NADH, which has been controversial. The structure of CtBP determined by x-ray diffraction revealed a tryptophan residue adjacent to the proposed nicotinamide adenine dinucleotide binding site. We find that this tryptophan residue shows strong fluorescence resonance energy transfer to bound NADH. In this report, we take advantage of these findings to measure the dissociation constants for CtBP with NADH and NAD ؉ . The affinity of NADH was determined by using fluorescence resonance energy transfer. The binding of NADH to protein is associated with an enhanced intensity of NADH fluorescence and a blue shift in its maximum. NAD ؉ affinity was estimated by measuring the loss of the fluorescence blue shift as NADH dissociates on addition of NAD ؉ . Our studies show a >100-fold higher affinity of NADH than NAD ؉ , consistent with the proposed function of CtBP as a nuclear redox sensor. Moreover, the concentrations of NADH and NAD ؉ required for half-maximal binding are approximately the same as their concentrations in the nuclear compartment. These findings support the possibility that changes in nuclear nicotinamide adenine dinucleotides could regulate the functions of CtBP in cell differentiation, development, or transformation.A n emerging theme in gene regulation is the dependence of transcriptional coregulators on molecules linked to cellular respiration. Acetyl-CoA is required by the histone acetyltransferase coactivators (1) and is perhaps the most central of all intermediary metabolites, bridging the major catabolic and anabolic processes to the Kreb's cycle, where its two carbons are converted to the respiratory product, CO 2 . ATP is important for essentially all cellular processes requiring energy, including the chromatin remodeling proteins involved in modifying nucleosomal structure (2). The pervasive involvement of acetyl-CoA and ATP in cellular processes generally obscures recognition of their specific contributions to transcriptional regulation, however. In addition, the relatively small changes in acetyl-CoA and ATP that occur during metabolism may not be suitable for regulating the activities of the relevant enzymes.The breakdown of carbon sources is also associated with the reduction of the nicotinamide adenine dinucleotide NAD ϩ to NADH. NADH serves as an electron carrier that transports reducing equivalents to the electron transport chain, where ATP is synthesized. The synthesis of ATP involves oxidative phosphorylation, wherein NADH is oxidized to NAD ϩ and molecular oxygen is reduced to water. These roles of NAD ϩ and NADH provide additional, albeit somewhat indirect, connections between energy homeostas...

The PPM family of Ser/Thr protein phosphatases have recently been shown to down-regulate the stress response pathways in eukaryotes. Within the stress pathway, key signaling kinases, which are activated by protein phosphorylation, have been proposed as the in vivo substrates of PP2C, the prototypical member of the PPM family. Although it is known that these phosphatases require metal cations for activity, the molecular details of these important reactions have not been established. Therefore, here we report a detailed biochemical study to elucidate the kinetic and chemical mechanism of PP2C␣. Steady-state kinetic and product inhibition studies revealed that PP2C␣ employs an ordered sequential mechanism, where the metal cations bind before phosphorylated substrate, and phosphate is the last product to be released. The metal-dependent activity of PP2C (as reflected in k cat and k cat /K m ), indicated that Fe 2؉ was 1000-fold better than Mg 2؉. The pH rate profiles revealed two ionizations critical for catalytic activity. An enzyme ionization with a pK a value of 7 must be unprotonated for catalysis, and an enzyme ionization with a pK a of 9 must be protonated for substrate binding. Brö nsted analysis of substrate leaving group pK a indicated that phosphomonoester hydrolysis is rate-limiting at pH 7.0, but not at pH 8.5 where a common step independent of the nature of the substrate and alcohol product limits turnover (k cat ). Rapid reaction kinetics between phosphomonoester and PP2C yielded exponential "bursts" of product formation, consistent with phosphate release being the slow catalytic step at pH 8.5. Dephosphorylation of synthetic phosphopeptides corresponding to several protein kinases revealed that PP2C displays a strong preference for diphosphorylated peptides in which the phosphorylated residues are in close proximity. Protein phosphatases (PP)1 catalyze the dephosphorylation of proteins containing phosphoserine/phosphothreonine and are divided into two distinct gene families, designated PPP and PPM (1). Although both PP families require divalent cations for activity, the PPM family is often distinguished by its Mg 2ϩ and Mn 2ϩ dependence. PP2C is the defining member of the PPM family. PP2C homologues have been identified in bacteria, plants, yeast, and mammals and appear to have a conserved role in negatively regulating stress response. PP2C was shown to be a negative regulator of two mitogen-activated protein kinase (MAPK) pathways involved in stress response, the p38 and c-Jun N-terminal kinase pathways. Like other MAPK pathways, these consist of a MAPK, a MAPK kinase (MAPKK), and a MAPKK kinase (MAPKKK) (2). MAPK is phosphorylated on conserved threonine and tyrosine residues by the activated MAPKK. The MAPKK is activated by phosphorylation on conserved threonine and/or serine residues by the MAPKKK. The stress response pathways are activated by proinflammatory cytokines, osmotic shock, oxidative stress, UV irradiation, and heat shock (3, 4). PP2C is thought to directly dephosphorylate and inactivate...

CtBP (carboxyl-terminal binding protein) participates in regulating cellular development and differentiation by associating with a diverse array of transcriptional repressors. Most of these interactions occur through a consensus CtBP-binding motif, PXDLS, in the repressor proteins. We previously showed that the CtBP-binding motif in E1A is flanked by a Lys residue and suggested that acetylation of this residue by the p300/CBPassociated factor P/CAF disrupts the CtBP interaction. In this study, we show that the interaction between CtBP and the nuclear hormone receptor corepressor RIP140 is regulated similarly, in this case by p300/CBP itself. CtBP was shown to interact with RIP140 in vitro and in vivo through a sequence, PIDLSCK, in the amino-terminal third of the RIP140 protein. Acetylation of the Lys residue in this motif, demonstrated in vivo by using an acetylated RIP140-specific antibody, dramatically reduced CtBP binding. Mutation of the Lys residue to Gln resulted in a decrease in CtBP binding in vivo and a loss of transcriptional repression. We suggest that p300/CBP-mediated acetylation disrupts the RIP140-CtBP complex and derepresses nuclear hormone receptor-regulated genes. Disruption of repressor-CtBP interactions by acetylation may be a general mode of gene activation.