SUMMARY Many genes that affect replicative lifespan (RLS) in the budding yeast Saccharomyces cerevisiae also affect aging in other organisms such as C. elegans and M. musculus. We performed a systematic analysis of yeast RLS in a set of 4,698 viable single-gene deletion strains. Multiple functional gene clusters were identified, and full genome-to-genome comparison demonstrated a significant conservation in longevity pathways between yeast and C. elegans. Among the mechanisms of aging identified, deletion of tRNA exporter LOS1 robustly extended lifespan. Dietary restriction (DR) and inhibition of mechanistic Target of Rapamycin (mTOR) exclude Los1 from the nucleus in a Rad53-dependent manner. Moreover, lifespan extension from deletion of LOS1 is non-additive with DR or mTOR inhibition, and results in Gcn4 transcription factor activation. Thus, the DNA damage response and mTOR converge on Los1-mediated nuclear tRNA export to regulate Gcn4 activity and aging.

Once a backwater in medical sciences, aging research has emerged and now threatens to take the forefront. This dramatic change of stature is driven from three major events. First and foremost, the world is rapidly getting old. Never before have we lived in a demographic environment like today and the trends will continue such that 20% percent of the global population of 9 billion will be over the age of 60 by 2050. Given current trends of sharply increasing chronic disease incidence, economic disaster from the impending silver tsunami may be ahead. A second major driver on the rise is the dramatic progress that aging research has made using invertebrate models such as worms, flies and yeast. Genetic approaches using these organisms have led to hundreds of aging genes and, perhaps surprisingly, strong evidence of evolutionary conservation among longevity pathways between disparate species, including mammals. Current studies suggest that this conservation may extend to humans. Finally, small molecules such as rapamycin and resveratrol have been identified that slow aging in model organisms, although only rapamycin to date impacts longevity in mice. The potential now exists to delay human aging, whether it is through known classes of small molecules or a plethora of emerging ones. But how can a drug that slows aging become approved and make it to market when aging is not defined as a disease. Here, we discuss the strategies to translate discoveries from aging research into drugs. Will aging research lead to novel therapies toward chronic disease, prevention of disease or be targeted directly at extending lifespan?

Many components of cellular signaling pathways are sensitive to regulation by oxidation and reduction. Previously, we described the inactivation of cAMP-dependent protein kinase (PKA) by direct oxidation of a reactive cysteine in the activation loop of the kinase. In the present study, we demonstrate that in HeLa cells PKA activity follows a biphasic response to thiol oxidation. Under mild oxidizing conditions, or short exposure to oxidants, forskolin-stimulated PKA activity is enhanced. This enhancement was blocked by sulfhydryl reducing agents, demonstrating a reversible mode of activation. In contrast, forskolin-stimulated PKA activity is inhibited by more severe oxidizing conditions. Mild oxidation enhanced PKA activity stimulated by forskolin, isoproterenol, or the cell-permeable analog, 8-bromo-cAMP. When cells were lysed in the presence of serine/threonine phosphatase inhibitor, NaF, the PKA-enhancing effect of oxidation was blunted. These results suggest oxidation of a PKA-counteracting phosphatase may be inhibited, thus enhancing the apparent kinase activity. Using an in vivo PKA activity reporter, we demonstrated that mild oxidation does indeed prolong the PKA signal induced by isoproterenol by inhibiting counteracting phosphatase activity. The results of this study demonstrate in live cells a unique synergistic mechanism whereby the PKA signaling pathway is enhanced in an apparent biphasic manner. cAMP-dependent protein kinase (PKA or cAPK)2 is a ubiquitously expressed kinase, critical for diverse cellular functions (1). Regulation of the kinase is primarily achieved by the production and degradation of the second messenger cAMP in response to extracellular stimuli, including hormones and neurotransmitters (2). Binding of these ligands to G s -coupled receptors initiates the activation of adenylate cyclase and production of cAMP from ATP. PKA normally exists as an inactive heterotetramer composed of two regulatory subunits and two catalytic subunits (3-5). Binding of cAMP to the regulatory subunits induces the release of the active catalytic subunit, initiating the phosphorylation of numerous downstream substrates, including ion channels at the plasma membrane and sarcoplasmic reticulum (1, 6), the transcription factor cAMPresponse element-binding protein in the nucleus (1, 7-9), and phosphorylase kinase in the cytosol (1, 10). The PKA signal persists until cAMP is hydrolyzed by phosphodiesterases (2) or the free catalytic subunit is inhibited by the heat-stable inhibitor PKI (11). Upon inactivation of PKA by the removal of cAMP, the phosphorylated substrate will persist until reversed by phosphatase activity.Reactive oxygen species are capable of modulating the response of numerous cell signaling pathways (12-15), including that of PKA (16 -21). Oxidants can potentially regulate the PKA pathway at multiple points, including the production and degradation of cAMP, and the phosphorylation and dephosphorylation of substrates. At the level of cAMP production, biochemical evidence has demonstrated that hy...

Summary PKA holoenzymes contain two catalytic (C) and a regulatory (R) subunit dimer where the two R-subunits are linked by an N-terminal Dimerization/Docking (D/D) domain. Cooperative binding of four cAMP molecules induces major structural changes in the R-subunits that cause kinase activation. While cooperativity exists between the two tandem cAMP binding domains, additional levels of cooperativity are associated with the tetramer. This allostery cannot be appreciated by studying heterodimers formed between C-subunit and deletion mutants of R that lack the D/D domain. Of critical importance is the flexible linker in the R-subunit that contains the Inhibitor Site (IS) that mimics the PKA substrate sequence and binds to the active site of the C-subunit. Two flexible linkers connect the IS to the D/D domain (N-Linker) and to the cAMP binding domains (C-Linker). In the cAMP-bound conformation IS and the C-Linker are disordered but become structured at the R:C interface when RIα binds to the C-subunit. The overall conformation of the tetramer, however, is mediated in large part by the N-Linker. To probe the function of the N-Linker in RIα and specifically to determine how the N-Linker contributes to assembly of the tetrameric holoenzyme, we engineered a monomeric form of RIα that contains most of the N-Linker, RIα(73-244), and crystallized a holoenzyme complex. Our previous RIα constructs began with the inhibitor site. RIα(73-244):C holoenzyme does not form stable dimers in solution without the dimerization domain; however, part of the extended linker is now ordered by interactions with a symmetry-related-dimer in the crystal. This complex of two symmetry-related dimers forms a tetramer that not only reveals novel mechanisms for allosteric regulation but also has many features that are consistent with known properties of the full-length holoenzyme. A model of the tetrameric RIα holoenzyme, based on this structure, is also consistent with small angle X-ray and neutron scattering data reported earlier. The model has been validated with new SAXS data and with a mutant of RIα that is localized to a novel interface that is unique to the tetramer.

The mechanism of PKAc-dependent NF-κB activation and subsequent translocation into the nucleus is not well defined. Previously, we showed that A kinase interacting protein 1 (AKIP1) was important for binding and retaining PKAc in the nucleus. Since then, other groups have demonstrated that AKIP1 binds the p65 subunit of NF-κB and regulates its transcriptional activity through the phosphorylation at Ser 276 by PKAc. However, little is known about the formation and activation of the PKAc/AKIP1/p65 complex and the rate at which it enters the nucleus. Initially, we found that the AKIP1 isoform (AKIP 1A) simultaneously binds PKAc and p65 in resting and serum starved cells. Using peptide arrays, we refined the region of AKIP 1A binding on PKAc and mapped the non-overlapping regions on AKIP 1A where PKAc and p65 bind. A peptide to the amino-terminus of PKAc (CAT 1-29) was generated to specifically disrupt the interaction between AKIP 1A and PKAc to study nuclear import of the complex. The rate of p65 nuclear translocation was monitored in the presence or absence of overexpressed AKIP 1A and/or (CAT 1-29). Enhanced nuclear translocation of p65 was observed in the presence of overexpressed AKIP1 and/or CAT 1-29 in cells stimulated with TNFα, and this correlated with decreased phosphorylation of serine 276. To determine whether PKAc phosphorylation of p65 in the cytosol regulated nuclear translocation, serine 276 was mutated to alanine or aspartic acid. Accelerated nuclear accumulation of p65 was observed in the alanine mutant, while the aspartic acid mutation displayed slowed nuclear translocation kinetics. In addition, enhanced nuclear translocation of p65 was observed when PKAc was knocked-down by siRNA. Taken together, these results suggest that AKIP 1A acts to scaffold PKAc to NF-κB in the cytosol by protecting the phosphorylation site and thereby regulating the rate of nuclear translocation of p65.

Protein kinase A holoenzyme is comprised of two catalytic (C) and two regulatory (R) subunits which keep the enzyme in an inhibited state before activation by cyclic-AMP. The C-subunit folds into a conserved bi-lobal core flanked by N-and C-terminal tails. We report here characterization of a C-tail loss-offunction mutant, C F327A , and a related suppressor mutant, C F327A/K285P . Phe-327 is the only residue outside the kinase core that binds to the adenine ring of ATP, whereas Lys-285 is ϳ45 Å away and lies in an AGC kinase-specific insert. The two mutations were previously identified from a yeast genetic screen, where the F327A mutation was unable to complement cell growth but mutation of K285P in the same allele rescued cell viability. We show that C F327A exhibits significant reduction in catalytic efficiency, which likely explains the observed loss-offunction phenotype. Interestingly, the additional K285P mutation does not restore kinase activity but reduces the inhibitory interaction of the double mutant with RII subunits. The additional K285P mutation, thus, helps to keep a low but uninhibited PKA activity that is sufficient for cell viability. The crystal structure of C F327A/K285P further reveals that recruitment of Phe-327 to the ATP binding pocket not only contributes to the hydrophobic pocket, as previously thought, but also recruits its flanking C-tail region to the kinase core, thereby concertedly positioning the glycine-rich loop and ATP for phosphoryl transfer. The study exemplifies two different ways for regulating cAMPdependent protein kinase activity through non-conserved residues and sheds light on the structural and functional diversity of the kinase family.Cyclic-AMP-dependent protein kinase (PKA) 3 is involved in a wide range of cellular functions such as metabolism, transport, cell cycle, and gene regulation. The PKA tetrameric holoenzyme is comprised of two catalytic (C) subunits that possess kinase activity and two inhibitory regulatory (R) subunits that each have two tandem cyclic AMP (cAMP) binding domains (1). In the absence of cAMP, the R-subunit binds to the C-subunit and blocks substrate access so as to inhibit kinase activity. Binding of cAMP to the R-subunits releases the inhibitory interactions and unleashes the C-subunit, allowing it to perform substrate phosphorylation. The R-subunit in yeast Saccharomyces cerevisiae is Bcy1p (2).As the first kinase structure solved in 1991 (3, 4), PKA C-subunit has been a paradigm of protein kinases and is best studied in terms of its structure-function relationship and kinetic properties (5). The conserved kinase core (residues 40 -300) of the C-subunit folds into an N-terminal lobe (N-lobe) and a C-terminal lobe (C-lobe) with the ATP binding site formed in between. The N-lobe is comprised of a twisted ␤ sheet formed by five ␤-strands and two helices (␣B and ␣C). One of the structural features in the N-lobe is the glycine-rich loop between the ␤1 and ␤2 strands. This loop senses nucleotide binding in the active site and exhibits different...

Localization of protein kinase A (PKA) via A-kinase-anchoring proteins (AKAPs) is important for cAMP responsiveness in many cellular systems, and evidence suggests that AKAPs play an important role in cardiac signaling. To test the importance of AKAP-mediated targeting of PKA on cardiac function, we designed a cell-permeable peptide, which we termed trans-activator of transcription (TAT)-AKAD for TAT-conjugated A-kinase-anchoring disruptor, using the PKA binding region of AKAP10 and tested the effects of this peptide in isolated cardiac myocytes and in Langendorff-perfused mouse hearts. We initially validated TAT-AKAD as a PKA localization inhibitor in cardiac myocytes by the use of confocal microscopy and cellular fractionation to show that treatment with the peptide disrupts type I and type II PKA regulatory subunits. Knockdown of PKA activity was demonstrated by decrease in phosphorylation of phospholamban and troponin I after ␤-adrenergic stimulation in isolated myocytes. Treatment with TAT-AKAD reduced myocyte shortening and rates of contraction and relaxation. Injection of TAT-AKAD (1 M), but not scrambled control peptide, into the coronary circulation of isolated perfused hearts rapidly (<1 min) and reversibly decreased heart rate and peak left ventricular developed pressure. TAT-AKAD also had a pronounced effect on developed pressure (؊dP/dt), consistent with a delayed relaxation of the heart. The effects of TAT-AKAD on heart rate and contractility persisted in hearts pretreated with isoproterenol. Disruption of PKA localization with TAT-AKAD thus had negative effects on chronotropy, inotropy, and lusitropy, thereby indicating a key role for AKAP-targeted PKA in control of heart rate and contractile function.

Dual-specificity AKAPs bind to type I (RI) and type II (RII) regulatory subunits of cAMPdependent protein kinase A (PKA), potentially recruiting distinct cAMP responsive holoenzymes to a given intracellular location. To understand the molecular basis for this "dual" functionality, we have examined the pH-dependence, the salt-dependence, and the kinetics of binding of the A-kinase binding (AKB) domain of D-AKAP2 to the regulatory subunit isoforms of PKA. Using fluorescence anisotropy, we have found that a 27-residue peptide corresponding to the AKB domain of D-AKAP2 bound 25-fold more tightly to RIIR than to RIR. The higher affinity for RIIR was the result of a slower off-rate as determined by surface plasmon resonance. The high-affinity interaction for RIR and RIIR was pH-independent from pH 7.4 to 5.0. At pH 4.0, both isoforms had a reduction in binding affinity. Additionally, binding of the AKB domain to RIR was independent of solution ionic strength, whereas RIIR had an increased binding affinity at higher ionic strength. This suggests that the relative energetic contribution of the charge stabilization is different for the two isoforms. This prediction was confirmed by mutagenesis in which acidic mutations, primarily of E10 and D23, in the AKB domain affected binding to RIR but not to RIIR. These isoform-specific differences provide a foundation for developing isoformspecific peptide inhibitors of PKA anchoring by dual-specificity AKAPs, which can be used to evaluate the physiological significance of dual-specificity modes of PKA anchoring.