Tyrosine phosphorylation of A17 during vaccinia virus infection: involvement of the H1 phosphatase and the F10 kinase.
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Vaccinia virus encodes two protein kinases (B1 and F10) and a dual-specificity phosphatase (VH1), suggesting that phosphorylation and dephosphorylation of substrates on serine/threonine and tyrosine residues are important in regulating diverse aspects of the viral life cycle. Using a recombinant in which expression of the H1 phosphatase can be regulated experimentally (vindH1), we have previously demonstrated that repression of H1 leads to the maturation of noninfectious virions that contain several hyperphosphorylated substrates (K. Liu et al., J. Virol. 69:7823-7834). In this report, we demonstrate that among these is a 25-kDa protein that is phosphorylated on tyrosine residues in H1-deficient virions and can be dephosphorylated by recombinant H1. We demonstrate that the 25-kDa phosphoprotein represents the product of the A17 gene and that A17 is phosphorylated on serine, threonine, and tyrosine residues during infection. Detection of phosphotyrosine within A17 is abrogated when Tyr(203) (but not Tyr(3), Tyr(6), or Tyr(7)) is mutated to phenylalanine, suggesting strongly that this amino acid is the site of tyrosine phosphorylation. Phosphorylation of A17 fails to occur during nonpermissive infections performed with temperature-sensitive mutants defective in the F10 kinase. Our data suggest that this enzyme, which was initially characterized as a serine/threonine kinase, might in fact have dual specificity. This hypothesis is strengthened by the observation that Escherichia coli induced to express F10 contain multiple proteins which are recognized by antiphosphotyrosine antiserum. This study presents the first evidence for phosphotyrosine signaling during vaccinia virus infection and implicates the F10 kinase and the H1 phosphatase as the dual-specificity enzymes that direct this cycle of reversible phosphorylation. (+info)
The B cell antigen receptor activates the Akt (protein kinase B)/glycogen synthase kinase-3 signaling pathway via phosphatidylinositol 3-kinase.
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We have previously shown that the B cell Ag receptor (BCR) activates phosphatidylinositol (PI) 3-kinase. We now show that a serine/threonine kinase called Akt or protein kinase B is a downstream target of PI 3-kinase in B cells. Akt has been shown to promote cell survival as well as the transcription and translation of proteins involved in cell cycle progression. Using an Ab that specifically recognizes the activated form of Akt that is phosphorylated on serine 473, we show that BCR engagement activates Akt in a PI 3-kinase-dependent manner. These results were confirmed using in vitro kinase assays. Moreover, BCR ligation also induced phosphorylation of Akt of threonine 308, another modification that is required for activation of Akt. In the DT40 chicken B cell line, phosphorylation of Akt on serine 473 was completely dependent on the Lyn tyrosine kinase, while the Syk tyrosine kinase was required for sustained phosphorylation of Akt. Complementary experiments in BCR-expressing AtT20 endocrine cells confirmed that Src kinases are sufficient for BCR-induced Akt phosphorylation, but that Syk is required for sustained phosphorylation of Akt on both serine 473 and threonine 308. In insulin-responsive cells, Akt phosphorylates and inactivates the serine/threonine kinase glycogen synthase kinase-3 (GSK-3). Inactivation of GSK-3 may promote nuclear accumulation of several transcription factors, including NF-ATc. We found that BCR engagement induced GSK-3 phosphorylation and decreased GSK-3 enzyme activity. Thus, BCR ligation initiates a PI 3-kinase/Akt/GSK-3 signaling pathway. (+info)
Role of Escherichia coli RpoS, LexA and H-NS global regulators in metabolism and survival under aerobic, phosphate-starvation conditions.
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It has been suggested that Escherichia coli can resist aerobic, glucose-starvation conditions by switching rapidly from an aerobic to a fermentative metabolism, thereby preventing the production by the respiratory chain of reactive oxygen species (ROS) that can damage cellular constituents. In contrast, it has been reported that E. coli cannot resist aerobic, phosphate (Pi)-starvation conditions, probably because of the maintenance of an aerobic metabolism and the continuous production of ROS. This paper presents evidence that E. coli cells starved for Pi under aerobic conditions indeed maintain an active aerobic metabolism for about 3 d, which allows the complete degradation of exogenous nutrients such as arginine (metabolized probably to putrescine via the SpeA-initiated pathway) and glucose (metabolized notably to acetate), but cell viability is not significantly affected because of the protection afforded against ROS through the expression of the RpoS and LexA regulons. The involvement of the LexA-controlled RuvAB and RecA proteins with the RecG and RecBCD proteins in metabolism and cell viability implies that DNA double-strand breaks (DSB), and thus hydroxyl radicals that normally generate this type of damage, are produced in Pi-starved cells. It is shown that induction of the LexA regulon, which helps protect Pi-starved cells, is totally prevented by introduction of a recB mutation, which indicates that DSB are actually the main DNA lesion generated in Pi-starved cells. The requirement of RpoS for survival of cells starved for Pi may thus be explained by the role played by various RpoS-controlled gene products such as KatE, KatG and Dps in the protection of DNA against ROS. In the same light, the degradation of arginine and threonine may be accounted for by the synthesis of polyamines (putrescine and spermidine) that protect nucleic acids from ROS. Besides LexA and RpoS, a third global regulator, the nucleoid-associated protein H-NS, is also shown to play a key role in Pi-starved cells. Through a modulation of the metabolism during Pi starvation, H-NS may perform two complementary tasks: it helps maintain a rapid metabolism of glucose and arginine, probably by favouring the activity of aerobic enzymes such as the NAD-dependent pyruvate dehydrogenase complex, and it may enhance the cellular defences against ROS which are then produced by increasing RpoS activity via the synthesis of acetate and presumably homoserine lactone. (+info)
A missense mutation in canine C1C-1 causes recessive myotonia congenita in the dog.
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Myotonia congenita is an inherited disorder of sarcolemmal excitation leading to delayed relaxation of skeletal muscle following contractions. Mutations in a skeletal muscle voltage-dependent chloride channel, CIC-1, have been identified as the molecular genetic basis for the syndrome in humans, and in two well characterized animal models of the disease: the myotonic goat, and the arrested development of righting (adr) mouse. We now report the molecular genetic and electrophysiological characterization of a canine CIC-1 mutation that causes autosomal recessive myotonia congenita in miniature Schnauzers. The mutation results in replacement of a threonine residue in the D5 transmembrane segment with methionine. Functional characterization of the mutation introduced into a recombinant CIC-1 and heterologously expressed in a cultured mammalian cell line demonstrates a profound effect on the voltage-dependence of activation such that mutant channels have a greatly reduced open probability at voltages near the resting membrane potential of skeletal muscle. The degree of this dysfunction is greatly diminished when heterodimeric channels containing a wild-type and mutant subunit are expressed together as a covalent concatemer strongly supporting the observed recessive inheritance in affected dog pedigrees. Genetic and electrophysiological characterization of the myotonic dog provides a new and potentially valuable animal model of an inherited skeletal muscle disease that has advantages over existing models of myotonia congenita. (+info)
Essential amino acids affect interstitial dopamine metabolites in the anterior piriform cortex of rats.
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The anterior piriform cortex (APC) is essential for the anorectic reactions to an amino acid-imbalanced diet, and it also responds to repletion of the limiting amino acid. In the present study, we examine the dynamic changes of the interstitial dopamine metabolites in the APC following feeding of either an amino acid-corrected or -imbalanced diet. Microdialysates, collected from the APC, were analyzed using HPLC with electrochemical detection. The concentrations were 19.7 +/- 4.8 microg/L for 3, 4-dyhydroxyphenylacetic acid and 25.1 +/- 4.4 microg/L for homovanillic acid, respectively, in the baseline dialysates. After diet treatments, no significant changes occurred in 3, 4-dyhydroxyphenylacetic acid in the corrected (n = 7) or imbalanced (n = 9) groups vs. the basal group (n = 7). However, after feeding the threonine-corrected diet, the concentration of homovanillic acid was significantly less (P < 0.01) than after the basal and imbalanced diets. The homovanillic acid level in the corrected group was already significantly lower than in the basal group by 20 min (P < 0.05), and reached its lowest level at 70 min (P < 0.05). The concentrations of homovanillic acid in the corrected group remained at this low level until the end of the experiment. The present results introduce the idea that the dopaminergic system is involved in the feeding responses to essential amino acid repletion. (+info)
Cold shock response of Bacillus subtilis: isoleucine-dependent switch in the fatty acid branching pattern for membrane adaptation to low temperatures.
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Bacillus subtilis has developed sophisticated mechanisms to withstand fluctuations in temperature. Membrane fatty acids are the major determinants for a sufficiently fluid membrane state to ensure the membrane's function at all temperatures. The fatty acid profile of B. subtilis is characterized by a high content of branched fatty acids irrespective of the growth medium. Here, we report on the importance of isoleucine for B. subtilis to survive cold shock from 37 to 15 degrees C. Cold shock experiments with strain JH642 revealed a cold-protective function for all intermediates of anteiso-branched fatty acid biosynthesis. Metabolites related to iso-branched or straight-chain fatty acid biosynthesis were not protective. Fatty acid profiles of different B. subtilis wild-type strains proved the altered branching pattern by an increase in the anteiso-branched fatty acid content and a concomitant decrease of iso-branched species during cold shock. There were no significant changes in the fatty acid saturation or acyl chain length. The cold-sensitive phenotype of isoleucine-deficient strains in the absence of isoleucine correlated with their inability to synthesize more anteiso-branched fatty acids, as shown by the fatty acid profile. The switch to a fatty acid profile dominated by anteiso-C(15:0) and C(17:0) at low temperatures and the cold-sensitive phenotype of isoleucine-deficient strains in the absence of isoleucine focused our attention on the critical role of anteiso-branched fatty acids in the growth of B. subtilis in the cold. (+info)
Asymmetric contributions to RNA binding by the Thr(45) residues of the MS2 coat protein dimer.
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A prominent feature of the interaction of MS2 coat protein with RNA is the quasi-symmetric insertion of a bulged adenine (A-10) and a loop adenine (A-4) into conserved pockets on each subunit of the coat protein dimer. Because of its presence in both of these adenine-binding pockets, Thr(45) is thought to play an important role in interaction with RNA on both subunits of the dimer. To test the significance of Thr(45), we introduced all 19 amino acid substitutions. However, we were initially unable to determine the effects of the mutations on RNA binding because every substitution compromised the ability of coat protein to fold correctly. Genetic fusion of coat protein subunits reverted these protein structural defects, allowing us to show that the RNA binding activity of coat protein tolerates substitution of Thr(45), but only on one or the other subunit of the dimer. Single-chain heterodimer complementation experiments suggest that the primary site of Thr(45) interaction with RNA is with A-4 in the translational operator. Either contact of Thr(45) with A-10 makes little contribution to stability of the RNA-protein complex, or the effects of Thr(45) substitution are offset by conformational adjustments that introduce new, favorable contacts at nearby sites. (+info)
Mutational analysis of the regulatory mechanism of PKN: the regulatory region of PKN contains an arachidonic acid-sensitive autoinhibitory domain.
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PKN is a fatty acid- and Rho GTPase-activated protein kinase whose catalytic domain in the carboxyl terminus is homologous to those of protein kinase C (PKC) family members. The amino terminal region of PKN is suggested to function as a regulatory domain, since tryptic cleavage or the binding of Rho GTPase to this region results in protein kinase activation of PKN. The structural basis for the regulation of PKN was investigated by analyzing the activity of a series of deletion/site-directed mutants expressed in insect cells. The amino-terminally truncated form of PKN (residue 455-942) showed low basal activity similar to that of the wild-type enzyme, and was arachidonic acid-dependent. However, further deletion (residue 511-942) resulted in a marked increase in the basal activity and a decrease in the arachidonic acid dependency. A (His)(6)-tagged protein comprising residues 455-511 of PKN (designated His-Ialpha) inhibited the kinase activity of the catalytic fragment of PKN in a concentration-dependent manner in competition with substrate (K(i) = 0.6+/-0.2 microM). His-Ialpha also inhibited the activity of the catalytic fragment of PRK2, an isoform of PKN, but had no inhibitory effect on protein kinase A or protein kinase Cdelta. The IC(50) value obtained in the presence of 40 microM arachidonic acid was two orders of magnitude greater than that in the absence of the modifier. These results indicate that this protein fragment functions as a specific inhibitor of PKN and PRK2, and that arachidonic acid relieves the catalytic activity of wild-type PKN from autoinhibition by residues 455-511 of PKN. Autophosphorylation of wild-type PKN increased the protein kinase activity, however, substitution of Thr64, Ser374, or Thr531 in the regulatory region of PKN with alanine, abolished this effect. Substitution of Thr774 in the activation loop of the catalytic domain of PKN with alanine completely abolished the protein kinase activity. These results suggest that these phosphorylation sites are also important in the regulation of the PKN kinase activity. Potential differences in the mechanism of activation between the catalytic regions of PKN and PRK2 are also discussed. (+info)