Uncharged tRNA activates GCN2 by displacing the protein kinase moiety from a bipartite tRNA-binding domain

Protein kinase GCN2 regulates translation in amino acid-starved cells by phosphorylating elF2. GCN2 contains a regulatory domain related to histidyl-tRNA synthetase (HisRS) postulated to bind multiple deacylated tRNAs as a general sensor of starvation. In accordance with this model, GCN2 bound several deacylated tRNAs with similar affinities, and aminoacylation of tRNAphe weakened its interaction with GCN2. Unexpectedly, the C-terminal ribosome binding segment of GCN2 (C-term) was required in addition to the HisRS domain for strong tRNA binding. A combined HisRS+ C-term segment bound to the isolated protein kinase (PK) domain in vitro, and tRNA impeded this interaction. An activating mutation (GCN2c-E803V) that weakens PK-C-term association greatly enhanced tRNA binding by GCN2. These results provide strong evidence that tRNA stimulates the GCN2 kinase moiety by preventing an inhibitory interaction with the bipartite tRNA binding domain.     

Functional analysis of B and T lymphocyte attenuator engagement on CD4+ and CD8+ T cells

T cell activation can be profoundly altered by coinhibitory and costimulatory molecules. B and T lymphocyte attenuator (BTLA) is a recently identified inhibitory Ig superfamily cell surface protein found on lymphocytes and APC. In this study we analyze the effects of an agonistic anti-BTLA mAb, PK18, on TCR-mediated T cell activation. Unlike many other allele-specific anti-BTLA mAb we have generated, PK18 inhibits anti-CD3-mediated CD4+ T cell proliferation. This inhibition is not dependent on regulatory T cells, nor does the Ab induce apoptosis. Inhibition of T cell proliferation correlates with a profound reduction in IL-2 secretion, although this is not the sole cause of the block of cell proliferation. In contrast, PK18 has no effect on induction of the early activation marker CD69. PK18 also significantly inhibits, but does not ablate, IL-2 secretion in the presence of costimulation as well as reduces T cell proliferation under limiting conditions of activation in the presence of costimulation. Similarly, PK18 inhibits Ag-specific T cell responses in culture. Interestingly, PK18 is capable of delivering an inhibitory signal as late as 16 h after the initiation of T cell activation. CD8+ T cells are significantly less sensitive to the inhibitory effects of PK18. Overall, BTLA adds to the growing list of cell surface proteins that are potential targets to down-modulate T cell function.     

The tRNA-binding moiety in GCN2 contains a dimerization domain that interacts with the kinase domain and is required for tRNA binding and kinase activation

GCN2 stimulates translation of GCN4 mRNA in amino acid-starved cells by phosphorylating translation initiation factor 2. GCN2 is activated by binding of uncharged tRNA to a domain related to histidyl-tRNA synthetase (HisRS). The HisRS-like region contains two dimerization domains (HisRS-N and HisRS-C) required for GCN2 function in vivo but dispensable for dimerization by full-length GCN2. Residues corresponding to amino acids at the dimer interface of Escherichia coli HisRS were required for dimerization of recombinant HisRS-N and for tRNA binding by full-length GCN2, suggesting that HisRS-N dimerization promotes tRNA binding and kinase activation. HisRS-N also interacted with the protein kinase (PK) domain, and a deletion impairing this interaction destroyed GCN2 function without reducing tRNA binding; thus, HisRS-N-PK interaction appears to stimulate PK function. The C-terminal domain of GCN2 (C-term) interacted with the PK domain in a manner disrupted by an activating PK mutation (E803V). These results suggest that the C-term is an autoinhibitory domain, counteracted by tRNA binding. We conclude that multiple domain interactions, positive and negative, mediate the activation of GCN2 by uncharged tRNA.     

Glyconeogenic pathway in isolated skeletal muscles of rats

Although the conversion of lactate to glycogen (glyconeogenesis) in muscle was demonstrated a long time ago, the biochemical reactions responsible for this process are still a controversial matter. In the present study, advantage was taken from the specific inhibition induced by phenylalanine on muscle pyruvate kinase (PK) to investigate the role of reverse PK activity in muscle glyconeogenesis. Addition of phenylalanine to the incubation medium of a preparation of isolated, intact skeletal muscles that maintain metabolic activity for several hours reduced by 50% the rate of incorporation of [14C]lactate or [14C]bicarbonate into muscle glycogen. Muscle extracts presented high levels of maximal activity of PK in the reverse direction, which was completely blocked in the presence of phenylalanine. In contrast, mercaptopicolinic acid, an inhibitor of phosphoenolpyruvate carboxykinase (PEPCK), did not affect the incorporation of 14C from either lactate or bicarbonate into muscle glycogen. Maximal PEPCK activity was much lower in muscle extracts than in gluconeogenic or glyceroneogenic tissues and was suppressed in the presence of mercaptopicolinic acid. The data suggest that a reversal of the metabolic flux through the reaction catalyzed by PK contributes to the accumulation of lactate-derived glycogen that occurs in skeletal muscle under certain physiological conditions.     

Reversible phosphorylation control of skeletal muscle pyruvate kinase and phosphofructokinase during estivation in the spadefoot toad,Scaphiopus couchii

Both pyruvate kinase (PK) and phosphofructokinase (PFK) occur in two different forms, separable by isoelectric focusing (IEF), in skeletal muscle of the spadefoot toad Scaphiopus couchii. During estivation (aerobic dormancy) the proportions of the two forms changed compared with controls; in both cases the amount of enzyme in Peak I (pI = 5.3-5.4) decreased whereas activity in Peak II (isoelectric point = 6.2-6.4) increased. In vitro incubation of crude muscle extracts with 32P-ATP under conditions that promoted the activity of cAMP-dependent protein kinase led to strong radiolabeling associated with Peak I, but not Peak II, and reverse phase HPLC confirmed that 32P was associated with the subunits of both PK and PFK found in Peak I. Specific radiolabeling of Peak I PK and PFK by protein kinase A was further confirmed using immunoprecipitation. In total, this information allowed identification of the Peaks I and II enzymes as the phosphorylated and dephosphorylated forms, respectively, and the effect of estivation was to increase the proportion of dephosphorylated PK and PFK in muscle. Analysis of the kinetic properties of partially purified PK and PFK revealed significant kinetic differences between the two forms of each enzyme. For PK, the Peak II (low phosphate) enzyme showed a 1.6-fold higher Km for phosphoenolpyruvate and a 2.4-fold higher Ka for fructose-1,6-bisphosphate than did the Peak I (high phosphate) form. These kinetic properties suggest that Peak II PK is the less active form, and coupled with the shift to predominantly the Peak II form during estivation (87% Peak II vs. 13% Peak I), are consistent with a suppression of PK activity in estivating muscle, as part of the overall metabolic rate depression of the estivating state. A similar shift to predominantly the Peak II, low phosphate, form of PFK (75% Peak II, 25% Peak I) in muscle of estivating animals is also consistent with metabolic suppression since phosphorylation of vertebrate skeletal muscle PFK is typically stimulated during exercise to enhance enzyme binding to myofibrils in active muscle. Peak II PFK also showed reduced sensitivity to inhibition by Mg:ATP (I50 50% higher) compared with the Peak I form suggesting that the enzyme in estivating muscle is less tightly regulated by cellular adenylate status than in awake toads. The data indicate that reversible phosphorylation control over the activity states of enzymes of intermediary metabolism is an important mechanism for regulating transitions between dormant and active states in estivating species.     

Evidence for defective transfer ribonucleic acid in polymyopathic hamsters and its inhibitory effect on protein synthesis

1. Different reaction steps involved in protein synthesis were studied in skeletal muscles from control and myopathic hamsters. 2. There was no difference between partially purified aminoacyl-tRNA synthetases from myopathic and control animals in yield or catalytic activity, as tested with exogenous deacylated tRNA. 3. However, isolated deacylated tRNA from myopathic muscle was aminoacylated by these synthetases to a lesser extent than that derived from control muscle. 4. Addition of deacylated tRNA isolated from control muscle improved the performance of pH5 enzymes from myopathic muscle in polypeptide synthesis on homologous polyribosomes; tRNA isolated from myopathic animals did not. 5. Preparation of extracts from both types of animals in the presence of the ribonuclease-absorbent bentonite led to an increased capacity of endogenous tRNA to accept amino acids in pH5 enzymes prepared from normal and abnormal tissue, but the difference between the two systems remained the same. 6. Total tRNA nucleotidyltransferase activity, tested with twice-pyrophosphorolysed rat liver tRNA, was identical in both extracts. 7. Added tRNA nucleotidyltransferase incorporated more AMP and CMP into endogenous tRNA with the pH5 enzyme from myopathic muscle than with that from control muscle. 8. Preincubation of deacylated tRNA from myopathic muscle with ATP, CTP and tRNA nucleotidyltransferase more than doubled its subsequent aminoacyl-acceptor activity, and halved the extent of the defect relative to aminoacylation of control tRNA similarly treated. Endogenous tRNA in pH5 enzyme preparations behaved likewise. 9. It is suggested that a 3'-exonuclease in myopathic muscles attacks tRNA molecules in such a way that some of them remain substrates for tRNA nucleotidyltransferase, which may incorporate into RNA not only AMP and CMP, but also GMP. 10. Cell-free protein synthesis in preparations from myopathic hamster muscles is limited by the supply of intact tRNA molecules.     

Concurrent interactions of heme and FLU with Glu tRNA reductase (HEMA1), the target of metabolic feedback inhibition of tetrapyrrole biosynthesis, in dark- and light-grown Arabidopsis plants

The regulation of tetrapyrrole biosynthesis in higher plants has been attributed to metabolic feedback inhibition of Glu tRNA reductase by heme. Recently, another negative regulator of tetrapyrrole biosynthesis has been discovered, the FLU protein. During an extensive second site screen of mutagenized flu seedlings a suppressor of flu, ulf3, was identified that is allelic to hy1 and encodes a heme oxygenase. Increased levels of heme in the hy1 mutant have been implicated with inhibiting Glu tRNA reductase and suppressing the synthesis of delta-aminolevulinic acid (ALA) and Pchlide accumulation. When combined with hy1 or ulf3 upregulation of ALA synthesis and overaccumulation of protochlorophyllide in the flu mutants were severely suppressed supporting the notion that heme antagonizes the effect of the flu mutation by inhibiting Glu tRNA reductase independently of FLU. The coiled-coil domain at the C-terminal end of Glu tRNA reductase interacts with FLU, whereas the N-terminal site of Glu tRNA reductase that is necessary for the inhibition of the enzyme by heme is not required for this interaction. The interaction with FLU is specific for the Glu tRNA reductase encoded by HEMA1 that is expressed in photosynthetically active tissues. FLU seems to be part of a second regulatory circuit that controls chlorophyll biosynthesis by interacting directly with Glu tRNA reductase not only in etiolated seedlings but also in light-adapted green plants.     

Characterization of high affinity binding sites for charybdotoxin in sarcolemmal membranes from bovine aortic smooth muscle. Evidence for a direct association with the high conductance calcium-activated potassium channel

Charybdotoxin (ChTX), a peptidyl inhibitor of the high conductance Ca2+-activated K+ channel (PK,Ca), has been radiolabeled to high specific activity with 125I, and resulting derivatives have been well separated. The monoiodotyrosine adduct blocks PK,Ca in vascular smooth muscle with slightly reduced potency compared with the native peptide under defined experimental conditions. [125I]ChTX, representing this derivative, binds specifically and reversibly to a single class of sites in sarcolemmal membrane vesicles prepared from bovine aortic smooth muscle. These sites display a Kd of 100 pM for the iodinated toxin, as determined by either equilibrium or kinetic binding analyses. Binding site density is about 500 fmol/mg of protein in isolated membranes. The addition of low digitonin concentrations to disrupt the vesicle permeability barrier increases the maximum receptor concentration to 1.5 pmol/mg of protein, correlating with the observations that ChTX binds only at the external pore of PK,Ca and that the membrane preparation is of mixed polarity. Competition studies with ChTX yield a Ki of about 20 pM for native toxin. Binding of [125I]ChTX is modulated by ionic strength as well as by metal ions that are known to interact with PK,Ca. Moreover, tetraethylammonium ion, which blocks PK,Ca with moderately high affinity when applied at the external membrane surface, inhibits [125I]ChTX binding in an apparently competitive fashion with a Ki similar to that found for channel inhibition. In marked contrast, agents that do not inhibit PK,Ca in smooth muscle (e.g. tetrabutylammonium ion, other toxins homologous with ChTX, and pharmacological agents that modulate the activity of dissimilar ion channels) have no effect on [125I]ChTX binding in this tissue. Taken together, these results suggest that the binding sites for ChTX which are present in vascular smooth muscle are directly associated with PK,Ca, thus identifying [125I]ChTX as a useful probe for elucidating the biochemical properties of these channels.     

Three of four pseudoknots in tmRNA are interchangeable and are substitutable with single-stranded RNAs

A novel translation, trans-translation, is facilitated by a highly structured RNA molecule, tmRNA. This molecule has two structural domains, a tRNA domain and an mRNA domain, the latter including four pseudoknot structures (PK1 to PK4). Here, we show that replacement of each of these pseudoknots, except PK1, in Escherichia coli tmRNA with a single stranded RNA did not seriously affect the functions as an alanine tRNA and as an mRNA. Furthermore, these three pseudoknots were interchangeable with only small losses of the two functions. These findings suggest that neither PK2, PK3 nor PK4 interacts in a functional manner with ribosome during the trans-translation process. Together with an earlier study showing the significance of PK1, it is concluded that among the four pseudoknots, PK1 is the most functional.     

Hsp90 (heat shock protein 90) inhibitor occupancy is a direct determinant of client protein degradation and tumor growth arrest in vivo

Several Hsp90 (heat shock protein 90) inhibitors are currently under clinical evaluation as anticancer agents. However, the correlation between the duration and magnitude of Hsp90 inhibition and the downstream effects on client protein degradation and cancer cell growth inhibition has not been thoroughly investigated. To investigate the relationship between Hsp90 inhibition and cellular effects, we developed a method that measures drug occupancy on Hsp90 after treatment with the Hsp90 inhibitor IPI-504 in living cells and in tumor xenografts. In cells, we find the level of Hsp90 occupancy to be directly correlated with cell growth inhibition. At the molecular level, the relationship between Hsp90 occupancy and Hsp90 client protein degradation was examined for different client proteins. For sensitive Hsp90 clients (e.g. HER2 (human epidermal growth factor receptor 2), client protein levels directly mirror Hsp90 occupancy at all time points after IPI-504 administration. For insensitive client proteins, we find that protein abundance matches Hsp90 occupancy only after prolonged incubation with drug. Additionally, we investigate the correlation between plasma pharmacokinetics (PK), tumor PK, pharmacodynamics (PD) (client protein degradation), tumor growth inhibition, and Hsp90 occupancy in a xenograft model of human cancer. Our results indicate Hsp90 occupancy to be a better predictor of PD than either plasma PK or tumor PK. In the nonsmall cell lung cancer xenograft model studied, a linear correlation between Hsp90 occupancy and tumor growth inhibition was found. This novel binding assay was evaluated both in vitro and in vivo and could be used as a pharmacodynamic readout in the clinic.     

Lung-selective impairment of cytochrome P-450-dependent monooxygenases and cellular injury by 1,1-dichloroethylene in mice

The calcium/phospholipid-dependent protein kinase (PKC) and the H4 protease-activated protein kinase (H4PK) from lymphosarcoma cells were separated by CM Sephadex chromatography. PKC activity was increased 10-fold in the presence of calcium and phosphatidylserine, but no activation by Mg+2-ATP preincubation or inhibition by NaF was observed. In contrast, H4PK activity was increased 8-fold by preincubation with Mg+2ATP and NaF completely inhibited this enzyme. Activators and inhibitors of PKC did not affect H4PK activity. The substrate specificity of the H4PK and PKC also differed substantially. On the basis of these data it is concluded that PKC and H4PK are not related enzymes.     

Model to assess muscle protein turnover: domain of validity using amino acyl-tRNA vs. surrogate measures of precursor pool

Current models to measure protein turnover across muscle bed are based on many surrogate measures of amino acyl-tRNA. We measured muscle protein turnover based on tracer-to-tracee ratios of the stable isotopes of leucine, phenylalanine, and ketoisocaproate (KIC) in artery and vein and muscle amino acyl-tRNA and muscle tissue fluid (TF) in 26 healthy subjects. A three-compartment model calculation based on arteriovenous and tRNA measurements was first performed and its domain of validity assessed. The results were then compared with those using simpler approaches based on surrogate measures of tRNA such as those of TF and KIC and a one-compartment model based on arteriovenous amino acids. In 96% of cases, the model using tRNA was applicable, but only in a lower percentage of cases were the results using surrogate measures applicable. Protein breakdown, protein synthesis, and shunting of amino acids from artery to vein were consistently underestimated, and fluxes of amino acid from artery to intracellular compartment and from intracellular compartment to vein were overestimated, when surrogate measures were used. The one-compartment model also underestimated protein breakdown and synthesis. Measurements using tissue fluid gave results closer to those based on tRNA. In conclusion, a three-compartment model using arteriovenous samples and amino acyl-tRNA provides measurements of muscle protein turnover of acceptable precision in 96% of cases. The precision was unacceptable in a substantial percentage of cases, and the accuracy of the estimation of protein fluxes was significantly affected when surrogate measures were used.     

Purification, catalytic and regulatory properties of Rana ridibunda erythrocyte pyruvate kinase

Pyruvate kinase of Rana ridibunda erythrocytes is one of the regulatory enzymes of glycolysis. PK was purified about 7800-fold. The purified enzyme showed on SDS-electrophoresis three protein bands with an apparent molecular weight of between 60 and 65 kD. The enzyme is subject to activation by FDP and to inhibition by ATP. It showed Km values for PEP and ADP of 0.095 and 0.98 mM respectively. It was activated by K+, Mg2+ and Ca2+ ions whereas it was inhibited by Na+ ions. The role of PK of Rana ridibunda erythrocytes, as a key and rate controlling enzyme of the glycolytic flux is discussed.     

Studies on metabolic properties in the Northern Krill, Meganyctiphanes norvegica (Crustacea, euphausiacea): influence of nutrition and season on pyruvate kinase

The specific activity and the kinetic properties of partly purified pyruvate kinase (PK) (EC 2.7.1.40) from the Northern Krill, Meganyctiphanes norvegica, were investigated in relation to varying food resources. In order to evaluate the effect of starvation on the total energy metabolism, the respiration rates of fed and unfed krill were determined. The FPLC-elution profile of PK displayed two distinct peaks - PK I and II. The first isoform represented 80% of the total PK activity in the organism, and 20% was contributed by the second isoform. PK I was inhibited by ATP but was not influenced by fructose-1,6-bisphosphate (FBP). In contrast, PK II showed ATP inhibition and up to 2.5-fold increased activity by addition of 17 micromol.l(-1) FBP. The Michaelis-Menten constants of both isoforms were 2-10-fold higher for ADP than for phosphoenolpyruvate (PEP). Alanine showed no regulatory effect on PK I and II. In specimens starved for 7 days oxygen consumption decreased by 20%. Neither the feeding experiments nor the animals captured in the field during low and high productive seasons indicate that PK properties of M. norvegica are modified in relation to food supply. Accordingly, alternative mechanisms are involved in the depression of the metabolic rate in terms of oxygen consumption.     

Staurosporine Activates a 60,000 Mr Protein Kinase in Bovine Chromaffin Cells That Phosphorylates Myelin Basic Protein In Vitro

Abstract: Bovine chromaffin cells contain a family of renaturable protein kinases. One of these, a 60,000 M_r kinase (PK60) that phosphorylated myelin basic protein in vitro, was activated fourfold when cells were treated with the protein kinase inhibitor Staurosporine. Because staurosporine inhibits protein kinase C, the role of this kinase in the regulation of PK60 activity was investigated. Fifty nanomolar Staurosporine produced half-maximal inhibition of protein kinase C activity in chromaffin cells, whereas ∼225 n M Staurosporine was required to induce half-maximal activation of PK60. Other protein kinase C inhibitors, H-7 and K-252a, did not mimic the effect of Staurosporine on PK60 activity. Chromaffin cells have three protein kinase C isoforms: α, ε, and ζ. Prolonged treatment with phorbol esters depleted the cells of protein kinase C α and ε, but not ζ. Neither activation nor depletion of protein kinase C affected the basal activity of PK60. Moreover, Staurosporine activated PK60 in cells depleted of protein kinase C α and e; thus, Staurosporine appeared to activate PK60 by a mechanism that does not require these protein kinase C isoforms. Incubation of cell extracts with Staurosporine in vitro did not activate PK60. Incubation of these extracts with adenosine 5'-O-(3-thiotriphosphate), however, caused a twofold activation of PK60. Although this suggests that PK60 activity is regulated by phosphorylation, the mechanism by which Staurosporine activates PK60 is not known. Staurosporine has been reported to promote neurite outgrowth from chromaffin cells. The role of PK60 in mediating the effects of Staurosporine on chromaffin cell function remains to be determined.     

Regulatory effects of S-100 protein and parvalbumin on protein kinases and phosphoprotein phosphatases from brain and skeletal muscle

Summary In the eluted fractions of histone-treated crude extracts separated by Sephadex G-200 filtration, multiple protein kinase (PK) activities, including three from brain and two from skeletal muscle, were augmented by both S-100 protein and parvalbumin on the phosphorylation of endogenous substrates. One additional PK activity suppressed by both S-100 and parvalbumin was also found in muscle. In comparison, phosphoprotein phosphatases (PPase), which were also prepared by the same procedure of initial step of histone-treatment followed by the steps of Bio-Gel P-6DG for brain and DNA-cellulose for muscle, were all activated by S-100 while inhibited by parvalbumin and phosphatidylserine.     

Carbohydrate metabolism in temporal and persistent hypoglycemic chickens induced by insulin infusion

In order to elucidate the regulatory mechanism of blood glucose concentrations specific to chickens, carbohydrate metabolism in the liver, muscle and kidney and metabolite concentrations in the blood were investigated in chickens with acute and persistent hypoglycemia. Acute and persistent hypoglycemia were experimentally induced by a single injection of insulin (8 U/kg BW) or by continuous infusion of insulin (22.5 U/kg BW/day) for 4 days. Non-esterified fatty acid (NEFA) concentration in plasma and D-3-hydroxybutyrate (3HB) concentrations in liver and muscle increased in the acute hypoglycemia. Plasma NEFA concentration and 3HB concentration in the blood and liver were not changed at day 3 of persistent hypoglycemia, while 3HB concentration in the muscle was decreased. Phosphofructokinase (PFK) activity in the liver tended to increase but PFK and pyruvate kinase (PK) activities were unchanged in acute hypoglycemia. In persistent hypoglycemia, increase of hepatic PFK activity at day 1 in which it was reversed at day 3, and a small increase of muscle PK activity were observed, while PK and phosphoenolpyruvate carboxykinase (PEPCK) activities in the liver and kidney were not significantly changed. These results show that in the persistent hypoglycemic chickens, hepatic glycolysis transiently increases, which is followed by a small decrease, while glycolysis in muscles and gluconeogenesis in the liver and kidney are not significantly changed.     

Smooth muscle calponin. Inhibition of actomyosin MgATPase and regulation by phosphorylation

Calponin isolated from chicken gizzard smooth muscle inhibits the actin-activated MgATPase activity of smooth muscle myosin in a reconstituted system composed of contractile and regulatory proteins. ATPase inhibition is not due to inhibition of myosin phosphorylation since, at calponin concentrations sufficient to cause maximal ATPase inhibition, myosin phosphorylation was unaffected. Furthermore, calponin inhibited the actin-activated MgATPase of fully phosphorylated or thiophosphorylated myosin. Although calponin is a Ca2(+)-binding protein, inhibition did not require Ca2+. Furthermore, although calponin also binds to tropomyosin, ATPase inhibition was not dependent on the presence of tropomyosin. Calponin was phosphorylated in vitro by protein kinase C and Ca2+/calmodulin-dependent protein kinase II, but not by cAMP- or cGMP-dependent protein kinases, or myosin light chain kinase. Phosphorylation of calponin by either kinase resulted in loss of its ability to inhibit the actomyosin ATPase. The phosphorylated protein retained calmodulin and tropomyosin binding capabilities, but actin binding was greatly reduced. The calponin-actin interaction, therefore, appears to be responsible for inhibition of the actomyosin ATPase. These observations suggest that calponin may be involved in regulating actin-myosin interaction and, therefore, the contractile state of smooth muscle. Calponin function in turn is regulated by Ca2(+)-dependent phosphorylation.