MEK2 regulates ribonucleotide reductase activity through functional interaction with ribonucleotide reductase small subunit p53R2

The p53R2 protein, a newly identified member of the ribonucleotide reductase family that provides nucleotides for DNA damage repair, is directly regulated by p53. We show that p53R2 is also regulated by a MEK2 (ERK kinase 2/MAP kinase kinase 2)-dependent pathway. Increased MEK1/2 phosphorylation by serum stimulation coincided with an increase in the RNR activity in U2OS and H1299 cells. The inhibition of MEK2 activity, either by treatment with a MEK inhibitor or by transfection with MEK2 siRNA, dramatically decreased the serum-stimulated RNR activity. Moreover, p53R2 siRNA, but not R2 siRNA, significantly inhibits serum-stimulated RNR activity, indicating that p53R2 is specifically regulated by a MEK2-dependent pathway. Co-immunoprecipitation analyses revealed that the MEK2 segment comprising amino acids 65–171 is critical for p53R2–MEK2 interaction, and the binding domain of MEK2 is required for MEK2-mediated increased RNR activity. Phosphorylation of MEK1/2 was greatly augmented by ionizing radiation, and RNR activity was concurrently increased. Ionizing radiation-induced RNR activity was markedly attenuated by transfection of MEK2 or p53R2 siRNA, but not R2 siRNA. These data show that MEK2 is an endogenous regulator of p53R2 and suggest that MEK2 may associate with p53R2 and upregulate its activity.     

MEK2 regulates ribonucleotide reductase activity through functional interaction with ribonucleotide reductase small subunit p53R2

The p53R2 protein, a newly identified member of the ribonucleotide reductase family that provides nucleotides for DNA damage repair, is directly regulated by p53. We show that p53R2 is also regulated by a MEK2 (ERK kinase 2/MAP kinase kinase 2)-dependent pathway. Increased MEK1/2 phosphorylation by serum stimulation coincided with an increase in the RNR activity in U2OS and H1299 cells. The inhibition of MEK2 activity, either by treatment with a MEK inhibitor or by transfection with MEK2 siRNA, dramatically decreased the serum-stimulated RNR activity. Moreover, p53R2 siRNA, but not R2 siRNA, significantly inhibits serum-stimulated RNR activity, indicating that p53R2 is specifically regulated by a MEK2-dependent pathway. Co-immunoprecipitation analyses revealed that the MEK2 segment comprising amino acids 65–171 is critical for p53R2–MEK2 interaction, and the binding domain of MEK2 is required for MEK2-mediated increased RNR activity. Phosphorylation of MEK1/2 was greatly augmented by ionizing radiation, and RNR activity was concurrently increased. Ionizing radiation-induced RNR activity was markedly attenuated by transfection of MEK2 or p53R2 siRNA, but not R2 siRNA. These data show that MEK2 is an endogenous regulator of p53R2 and suggest that MEK2 may associate with p53R2 and upregulate its activity.     

Respiration-dependent removal of exogenous H2O2 in brain mitochondria: inhibition by Ca2+

In brain mitochondria, state 4 respiration supported by the NAD-linked substrates glutamate/malate in the presence of EGTA promotes a high rate of exogenous H2O2 removal. Omitting EGTA decreases the H2O2 removal rate by almost 80%. The decrease depends on the influx of contaminating Ca2+, being prevented by the Ca2+ uniporter inhibitor ruthenium red. Arsenite is also an inhibitor (maximal effect approximately 40%, IC50, 12 microm). The H2O2 removal rate (EGTA present) is decreased by 20% during state 3 respiration and by 60-70% in fully uncoupled conditions. H2O2 removal in mitochondria is largely dependent on glutathione peroxidase and glutathione reductase. Both enzyme activities, as studied in disrupted mitochondria, are inhibited by Ca2+. Glutathione reductase is decreased by 70% with an IC50 of about 0.9 microm, and glutathione peroxidase is decreased by 38% with a similar IC50. The highest Ca2+ effect with glutathione reductase is observed in the presence of low concentrations of H2O2. With succinate as substrate, the removal is 50% less than with glutamate/malate. This appears to depend on succinate-supported production of H2O2 by reverse electron flow at NADH dehydrogenase competing with exogenous H2O2 for removal. Succinate-dependent H2O2 is inhibited by rotenone, decreased DeltaPsi, as described previously, and by ruthenium red and glutamate/malate. These agents also increase the measured rate of exogenous H2O2 removal with succinate. Succinate-dependent H2O2 generation is also inhibited by contaminating Ca2+. Therefore, Ca2+ acts as an inhibitor of both H2O2 removal and the succinate-supported H2O2 production. It is concluded that mitochondria function as intracellular Ca2+-modulated peroxide sinks.     

Horseradish peroxidase-catalyzed conversion of iodine to iodide in presence of EDTA and H2O2

EDTA (4 mM) blocks the oxidation of iodide to I-3 (increase of extinction at 353 nm) by H2O2 catalyzed by horseradish peroxidase, which is reversed by the addition of an equimolar concentration of Zn2+. Addition of suboptimal concentration of EDTA (2 mM) not only decreases the rate of forward reaction of I-3 formation but also causes loss of extinction of the same when I-3 is generated. The loss of extinction of I-3 is proportional to the enzyme concentration and is blocked by azide, the inhibitor of the peroxidase. EDTA also causes bleaching of nonenzymatically formed I-3 (from iodide and H2O2) only in the presence of horseradish peroxidase, and the effect is reversed by the equimolar concentration of Zn2+. Both the bleaching of I-3 by EDTA and reversal of EDTA effect by Zn2+ are sensitive to azide. The decrease of extinction of I-3 (formed by dissolving iodine in KI solution) is dependent on EDTA, H2O2, and horseradish peroxidase. Molecular iodine is also bleached but at a slower rate than I-3. Evidence is presented to show that this bleaching of I-3 is due to enzymatic conversion of I-3 to iodide in presence of EDTA and H2O2 and this involves pseudocatalatic degradation of H2O2 to O2.     

Evidence that the Loss of the Voltage-Dependent Mg2+ Block at the N-Methyl-D-Aspartate Receptor Underlies Receptor Activation During Inhibition of Neuronal Metabolism

Both phosphointermediate- and vacuolar-type (P- and V-type, respectively) ATPase activities found in cholinergic synaptic vesicles isolated from electric organ are immunoprecipitated by a monoclonal antibody to the SV2 epitope characteristic of synaptic vesicles. The two activities can be distinguished by assay in the absence and presence of vanadate, an inhibitor of the P-type ATPase. Each ATPase has two overlapping activity maxima between pH 5.5 and 9.5 and is inhibited by fluoride and fluorescein isothiocyanate. The P-type ATPase hydrolyzes ATP and dATP best among common nucleotides, and activity is supported well by Mg2+, Mn2+, or Co2+ but not by Ca2+, Cd2+, or Zn2+. It is stimulated by hyposmotic lysis, detergent solubilization, and some mitochondrial uncouplers. Kinetic analysis revealed two Michaelis constants for MgATP of 28 microM and 3.1 mM, and the native enzyme is proposed to be a dimer of 110-kDa subunits. The V-type ATPase hydrolyzes all common nucleoside triphosphates, and Mg2+, Ca2+, Cd2+, Mn2+, and Zn2+ all support activity effectively. Active transport of acetylcholine (ACh) also is supported by various nucleoside triphosphates in the presence of Ca2+ or Mg2+, and the Km for MgATP is 170 microM. The V-type ATPase is stimulated by mitochondrial uncouplers, but only at concentrations significantly above those required to inhibit ACh active uptake. Kinetic analysis of the V-type ATPase revealed two Michaelis constants for MgATP of approximately 26 microM and 2.0 mM. The V-type ATPase and ACh active transport were inhibited by 84 and 160 pmol of bafilomycin A1/mg of vesicle protein, respectively, from which it is estimated that only one or two V-type ATPase proton pumps are present per synaptic vesicle. The presence of presumably contaminating Na+,K(+)-ATPase in the synaptic vesicle preparation is demonstrated.     

Reduction of ferryl- and metmyoglobin to ferrous myoglobin by menadione-glutathione conjugate. Spectrophotometric studies under aerobic and anaerobic conditions

Both metmyoglobin (MbIII) and ferrylmyoglobin (MbIV) are reduced by the menadiol-glutathione conjugate (GS-Q2-) to oxymyoglobin (MbIIO2) or deoxymyoglobin (MbII), depending whether the assay is carried out under aerobic or anaerobic conditions, respectively. Under aerobic conditions, the reduction of MbIII to MbIIO2 by GS-Q2- is associated with O2 consumption. The latter process is accounted for by (a) the autoxidation of the conjugate yielding H2O2 and (b) the rapid binding of O2 to MbII to yield MbIIO2. The ratio [O2]consumed/[MbIIO2]formed is approximately 1.5 at the time when MbIIO2 formation is maximal (at about 0.8 min). This ratio, higher than the unit, indicates that there is more than one O2-consuming reaction in this experimental model. The ratio of initial rates of O2 consumption and MbIIO2 formation is close to the unit [(-dO2/dt)/(+ dMbIIO2/dt) = 1.1]. The formation of H2O2 originating during the autoxidation of the GS-Q2- is substantially lower in the presence of MbIII, probably due to the heterolytic cleavage of the O--O bond of the peroxide by the hemoprotein. Although the latter reaction should yield MbIV, this species is not observed in the absorption spectrum, probably due to its rapid reduction by GS-Q2-. MbIV is reduced to MbIIO2 by the GS-Q2-. Whether this reaction takes place in one-electron transfer steps, that is, the sequence: MbIV----MbIII----MbIIO2 is difficult to evaluate by absorption spectral analysis, due to the rapid rate of the [MbIV----MbIIO2] transition. Under anaerobic conditions, the reduction of either MbIII or MbIV by GS-Q2- yields MbII as a stable molecular product. Anaerobic conditions prevent any further interaction of MbII with intermediates of O2 reduction derived from GS-Q2- autoxidation.     

The bivalent-cation dependence of phosphatidylinositol synthesis in a cell-free system from lymphocytes.

The bivalent-cation requirements of two enzymes involved in phosphatidylinositol synthesis were defined for pig lymphocyte membranes using a citric acid buffer. CTP:phosphatidic acid cytidylyltransferase (EC 2.7.7.41) is activated by free Mn2+ concentrations above 20nM and by free Mg2+ concentrations above 10 microM. When activated by Mg2+, the enzyme is weakly inhibited by Ca2+ (Ki greater than 250 microM), but Ca2+ has no effect when Mn2+ is used to stimulate CDP-diacylglycerol synthesis. The synthesis of phosphatidylinositol from phosphatidic acid is also stimulated by Mn2+ and Mg2+ concentrations similar to those above and is inhibited by free Ca2+ concentrations above 500nM, probably by its action on CDP-diacylglycerol:inositol 3-phosphatidyltransferase (EC 2.7.8.11). Taken together, these studies suggest that under physiological conditions phosphatidylinositol synthesis is activated by Mg2+ and it is possible that it is further regulated by the free concentrations of Ca2+ and/or Mn2+.     

Iron regulatory protein 2 turnover through a nonproteasomal pathway

Iron regulatory protein 2 (IRP2) controls the synthesis of many proteins involved in iron metabolism, and the level of IRP2 itself is regulated by varying the rate of its degradation. The proteasome is known to mediate degradation, with specificity conferred by an iron-sensing E3 ligase. Most studies on the degradation of IRP2 have employed cells overexpressing IRP2 and also rendered iron deficient to further increase IRP2 levels. We utilized a sensitive, quantitative assay for IRP2, which allowed study of endogenous IRP2 degradation in HEK293A cells under more physiologic conditions. We found that under these conditions, the proteasome plays only a minor role in the degradation of IRP2, with almost all the IRP2 being degraded by a nonproteasomal pathway. This new pathway is calcium-dependent but is not mediated by calpain. Elevating the cellular level of IRP2 by inducing iron deficiency or by transfection causes the proteasomal pathway to account for the major fraction of IRP2 degradation. We conclude that under physiological, iron-sufficient conditions, the steady-state level of IRP2 in HEK293A cells is regulated by the nonproteasomal pathway.     

Functional Approach to the Catalytic Site of the Sarcoplasmic Reticulum Ca2+-ATPase: Binding and Hydrolysis of ATP in the Absence of Ca2+

Isolated sarcoplasmic reticulum vesicles in the presence of Mg(2+) and absence of Ca(2+) retain significant ATP hydrolytic activity that can be attributed to the Ca(2+)-ATPase protein. At neutral pH and the presence of 5 mM Mg(2+), the dependence of the hydrolysis rate on a linear ATP concentration scale can be fitted by a single hyperbolic function. MgATP hydrolysis is inhibited by either free Mg(2+) or free ATP. The rate of ATP hydrolysis is not perturbed by vanadate, whereas the rate of p-nitrophenyl phosphate hydrolysis is not altered by a nonhydrolyzable ATP analog. ATP binding affinity at neutral pH and in a Ca(2+)-free medium is increased by Mg(2+) but decreased by vanadate when Mg(2+) is present. It is suggested that MgATP hydrolysis in the absence of Ca(2+) requires some optimal adjustment of the enzyme cytoplasmic domains. The Ca(2+)-independent activity is operative at basal levels of cytoplasmic Ca(2+) or when the Ca(2+) binding transition is impeded.     

Interrelationship between oxygen consumption,superoxide anion and hydrogen peroxide formation in phagocytosing guinea pig polymorphonuclear leucocytes

The paper presents an experimental procedure for a simultaneous assay of oxygen consumption, O2- release and H2O2 accumulation at a very early stage of the respiratory burst that is induced by phagocytosis in guinea pig polymorphonuclear leucocytes. The main findings are as follows: (a) The oxygen consumption that is measurable does not correspond to all oxygen that is reduced. The relationship between the actual oxygen consumed and the amount that is reduced depends on the fate of the intermediate products O2- and H2O2. (b) O2- is measurable extracellularly by the reduction of cytochrome c. When cytochrome c oxidizes the extracellular O2-, molecular oxygen is formed. This fact is shown by a decrease of oxygen consumption. The molar ratio between the O2- detected and the oxygen given back is 1. (c) The amount of O2- released from the cells accounts for only a small part of oxygen actually reduced. (d) H2O2 is detectable only in the presence of NaN3. In this condition almost all oxygen consumed is recovered in the form of H2O2. The molar ratio O2/H2O2 is near unity. The amount of H2O2 derived from dismutation of O2- released is only an aliquot of the total H2O2 accumulated. Thus, most of H2O2 is derived from intracellular sources. (e) In the absence of inhibitors of H2O2 degrading reactions, no detectable accumulation of peroxide occurs. Under these conditions, the main part of H2O2 formed is degraded in almost equal amount by catalase and myeloperoxidase, while only a small aliquot is degraded by NaN3 insensitive reactions.     

Dependence on Ca2+ and tropomyosin of the actin-activated ATPase activity of phosphorylated gizzard myosin in the presence of low concentrations of Mg2+

Ca2+ and tropomyosin are required for activation of ATPase activity of phosphorylated gizzard myosin by gizzard actin at less than 1 mM Mg2+, relatively low Ca2+ concentrations (1 microM), producing half-maximal activation. At higher concentrations, Mg2+ will replace Ca2+, 4 mM Mg2+ increasing activity to the same extent as does Ca2+ and abolishing the Ca2+ dependence. Above about 1 mM Mg2+, tropomyosin is no longer required for activation by actin, activity being dependent on Ca2+ between 1 and 4 mM Mg2+, but independent of [Ca2+] above 4 mM Mg2+. Phosphorylation of the 20,000-Da light chain of gizzard myosin is required for activation of ATPase activity by actin from chicken gizzard or rabbit skeletal muscle at all concentrations of Mg2+ employed. The effect of adding or removing Ca2+ is fully reversible and cannot be attributed either to irreversible inactivation of actin or myosin or to dephosphorylation. After preincubating in the absence of Ca2+, activity is restored either by adding micromolar concentrations of this cation or by raising the concentration of Mg2+ to 8 mM. Similarly, the inhibition found in the absence of tropomyosin is fully reversed by subsequent addition of this protein. Replacing gizzard actin with skeletal actin alters the pattern of activation by Ca2+ at concentrations of Mg2+ less than 1 mM. Full activation is obtained with or without Ca2+ in the presence of tropomyosin, while in its absence Ca2+ is required but produces only partial activation. Without tropomyosin, the range of Mg2+ concentrations over which activity is Ca2+-dependent is restricted to lower values with skeletal than with gizzard actin. The activity of skeletal muscle myosin is activated by the gizzard actin-tropomyosin complex without Ca2+, although Ca2+ slightly increases activity. The Ca2+ sensitivity of reconstituted gizzard actomyosin is partially retained by hybrid actomyosin containing gizzard myosin and skeletal actin, but less Ca2+ dependence is retained in the hybrid containing skeletal myosin and gizzard actin.     

A possible role of thromboxane A2 (TXA2) and prostacyclin (PGI2) in circulation.

Recently two local hormones, thromboxane A2 (TXA2) and prostacyclin (PGI2) have been discovered. These hormones are labile metabolites of arachidonic acid. TXA2 is generated by blood platelets, while PGI2 is produced by vascular endothelium. TXA2 is a potent vasoconstrictor. It also initiates the release reaction, followed by platelet aggregation. PGI2 is a vasodilator, especially potent in coronary circulation. It also inhibits platelet aggregation by virtue of stimulation of platelet adenyl cyclase. Common precursors for both hormones are cyclic endoperoxides PGG2 and PGH2, being formed by cyclooxygenation of arachidonic acid. This last enzymic reaction is more efficient in platelets than in vascular endothelium, and therefore the generation of PGI2 by vasuclar wall is accelerated by an interaction between platelets and endothelial cells. During this interaction platelets supply the endothelial PGI2 synthetase with their cyclic endoperoxides. The newly formed PGI2 repels the platelets from the intima. When PGI2 synthetase is irreversibly inactivated by low concentration of lipid peroxides, then the platelets are not rejected but stick to the endothelium, generate TXA2 and mature thrombi are formed. A balance between formation and release of PGI2, TXA2 and/or cyclic endoperoxides in circulation is of utmost importance for the control of intra-arterial thrombi formation and possibly plays a role in the pathogenesis of atherosclerosis.     

Infection of SARS-CoV-2 causes severe pathological changes in mouse testis

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has affected more than 600 million people worldwide. Several organs including lung, intestine, and brain are infected by SARS-CoV-2. It has been reported that SARS-CoV-2 receptor angiotensin-converting enzyme-2 (ACE2) is expressed in human testis. However, whether testis is also affected by SARS-CoV-2 is still unclear. In this study, we generate a human ACE2 (hACE2) transgenic mouse model in which the expression of hACE2 gene is regulated by hACE2 promoter. Sertoli and Leydig cells from hACE2 transgenic mice can be infected by SARS-CoV-2 pseudovirus in vitro, and severe pathological changes are observed after injecting the SARS-CoV-2 pseudovirus into the seminiferous tubules. Further studies reveal that Sertoli and Leydig cells from hACE2 transgenic mice are also infected by authentic SARS-CoV-2 virus in vitro. After testis interstitium injection, authentic SARS-CoV-2 viruses are first disseminated to the interstitial cells, and then detected inside the seminiferous tubules which in turn cause germ cell loss and disruption of seminiferous tubules. Our study demonstrates that testis is most likely a target of SARS-CoV-2 virus. Attention should be paid to the reproductive function in SARS-CoV-2 patients.     

Ruthenium red-insensitive Ca2+ uptake and release by mitochondria

Calcium uptake by rat liver mitochondria driven by an artificial pH gradient is ruthenium red insensitive, electrically neutral, and inhibited by the local anesthetic, nupercaine. This pH-driven Ca2+ transport is also inhibited by NH3, Pi, and acetate. Direct measurements of Pi indicate it is not translocated with Ca2+ during pH-driven Ca2+ uptake. Calcium is therefore not transported by a Ca2+-Pi symport mechanism. Ruthenium red-insensitive Ca2+ efflux is similar in its inhibition by nupercaine and its kinetics, and is also electroneutral. This suggests that the Ca2+ uptake described here occurs via reversal of the principal pathway of mitochondrial Ca2+ release. From the available data, pH-driven Ca2+ uptake (and presumably Ca2+ efflux) is hypothesized to occur by Ca2+ symport with unidentified anions. Protons may move counter to Ca2+ or reversibly dissociate from cotransported anions, which therefore couples Ca2+ transport to the pH gradient.     

Polycystin-1 and polycystin-2 regulate the cell cycle through the helix-loop-helix inhibitor Id2

Autosomal-dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disease and is characterized by progressive cyst formation and ultimate loss of renal function. Increased cell proliferation is a key feature of the disease. Here, we show that the ADPKD protein polycystin-2 (PC2) regulates the cell cycle through direct interaction with Id2, a member of the helix-loop-helix (HLH) protein family that is known to regulate cell proliferation and differentiation. Id2 expression suppresses the induction of a cyclin-dependent kinase inhibitor, p21, by either polycystin-1 (PC1) or PC2. The PC2-Id2 interaction is regulated by PC1-dependent phosphorylation of PC2. Enhanced Id2 nuclear localization is seen in human and mouse cystic kidneys. Inhibition of Id2 expression by RNA interference corrects the hyperproliferative phenotype of PC1 mutant cells. We propose that Id2 has a crucial role in cell-cycle regulation that is mediated by PC1 and PC2.     

Peroxisomal Degradation of 2-Oxoisocaproate. Evidence for Free Acid Intermediates

Peroxisomes from mung bean hypocotyl (Vigna radiata L.) degrade 2-oxoisocaproate, the transamination product of leucine, via isobutyryl-CoA and propionyl-CoA to acetyl-CoA. The methyl group at the C-3 position forms a barrier to β-oxidation. This barrier is overcome in the peroxisomes by several enzymatic steps. Senecioate (3-methylcrotonate), 2-hydroxyisovalerate, and 2-oxoisovalerate were detected as free acid intermediates. Senecioate, formed from 3-methylcrotonyl-CoA, is transformed by enzymatic hydrolysis to 2-hydroxyisovalerate. 2-Hydroxyisovalerate is then oxidized to 2-oxoisovalerate in an H₂O₂-producing reaction, exhibiting 1:1 stoichiometry of the products, by a 2-hydroxyacid oxidase which is different from the peroxisomal marker enzyme glycollate oxidase. 2-oxoisovalerate is activated by an NAD-dependent oxidative decarboxylation to isobutyryl-CoA. Accumulation of 2-oxoisovalerate in the presence of arsenite, an inhibitor of oxidative decarboxylations, is a feature of this latter pathway of degradation of isovaleryl-CoA or senecioate. It is concluded that the barrier caused by the methyl group of 2-oxoisocaproate is surmounted in higher plant peroxisomes in a manner different to that in mammalian mitochondria.     

Superoxide-dependent oxidation of extracellular reducing agents by isolated neutrophils

Incubation of stimulated neutrophils with sulfhydryl (RSH) compounds or ascorbic acid (ascorbate) results in rapid superoxide (O2-)-dependent oxidation of these reducing agents. Oxidation of RSH compounds to disulfides (RSSR) is faster than the rate of O2- production by the neutrophil NADPH-oxidase, whereas about one ascorbate is oxidized per O2-. Ascorbate is oxidized to dehydroascorbate, which is also oxidized but at a slower rate. Oxidation is accompanied by a large increase in oxygen (O2) uptake that is blocked by superoxide dismutase. Lactoferrin does not inhibit, indicating that ferric (Fe3+) ions are not required, and Fe3+-lactoferrin does not catalyze RSH or ascorbate oxidation. Two mechanisms contribute to oxidation: 1) O2- oxidizes ascorbate or reduced glutathione and is reduced to hydrogen peroxide (H2O2), which also oxidizes the reductants. O2- reacts directly with ascorbate, but reduced glutathione oxidation is mediated by the reaction of O2- with manganese (Mn2+). The H2O2-dependent portion of oxidation is mediated by myeloperoxidase-catalyzed oxidation of chloride to hypochlorous acid (HOCl) and oxidation of the reductants by HOCl. 2) O2- initiates Mn2+-dependent auto-oxidation reactions in which RSH compounds are oxidized and O2 is reduced. Part of this oxidation is due to the RSH-oxidase activity of myeloperoxidase. This activity is blocked by superoxide dismutase but does not require O2- production by the NADPH-oxidase, indicating that myeloperoxidase produces O2- when incubated with RSH compounds. It is proposed that an important role for O2- in the cytotoxic activities of phagocytic leukocytes is to participate in oxidation of reducing agents in phagolysosomes and the extracellular medium. Elimination of these protective agents allows H2O2 and products of peroxidase/H2O2/halide systems to exert cytotoxic effects.     

Characterization of the initiation factor eIF2B and its regulation in Drosophila melanogaster

Eukaryotic initiation factor (eIF) 2B catalyzes a key regulatory step in the initiation of mRNA translation. eIF2B is well characterized in mammals and in yeast, although little is known about it in other eukaryotes. eIF2B is a hetropentamer which mediates the exchange of GDP for GTP on eIF2. In mammals and yeast, its activity is regulated by phosphorylation of eIF2alpha. Here we have cloned Drosophila melanogaster cDNAs encoding polypeptides showing substantial similarity to eIF2B subunits from yeast and mammals. They also exhibit the other conserved features of these proteins. D. melanogaster eIF2Balpha confers regulation of eIF2B function in yeast, while eIF2Bepsilon shows guanine nucleotide exchange activity. In common with mammalian eIF2Bepsilon, D. melanogaster eIF2Bepsilon is phosphorylated by glycogen synthase kinase-3 and casein kinase II. Phosphorylation of partially purified D. melanogaster eIF2B by glycogen synthase kinase-3 inhibits its activity. Extracts of D. melanogaster S2 Schneider cells display eIF2B activity, which is inhibited by phosphorylation of eIF2alpha, showing the insect factor is regulated similarly to eIF2B from other species. In S2 cells, serum starvation increases eIF2alpha phosphorylation, which correlates with inhibition of eIF2B, and both effects are reversed by serum treatment. This shows that eIF2alpha phosphorylation and eIF2B activity are under dynamic regulation by serum. eIF2alpha phosphorylation is also increased by endoplasmic reticulum stress in S2 cells. These are the first data concerning the structure, function or control of eIF2B from D. melanogaster.     

Glycosylation is a novel TGFβ1-independent post-translational modification of Smad2

Smad2 is a crucial component of intracellular signaling by transforming growth factor-β (TGFβ). Here we describe that Smad2 is glycosylated, which is a novel for Smad2 post-translational modification. We showed that the Smad2 glycosylation was inhibited upon treatment of cells with 17β-estradiol, and was enhanced in cells in a dense culture as compared to cells in a sparse culture. The Smad2 glycosylation was not dependent on the C-terminal phosphorylation of Smad2, and was not affected by TGFβ1 treatment of the cells. Smad2 was glycosylated at multiple sites, and one of the predicted sites is Serine110. Thus, Smad2 is glycosylated, and this post-translational modification was modulated by 17β-estradiol but not by TGFβ1.