Nitrosopumilus maritimus genome reveals unique mechanisms for nitrification and autotrophy in globally distributed marine crenarchaea.

Ammonia-oxidizing archaea are ubiquitous in marine and terrestrial environments and now thought to be significant contributors to carbon and nitrogen cycling. The isolation of Candidatus "Nitrosopumilus maritimus" strain SCM1 provided the opportunity for linking its chemolithotrophic physiology with a genomic inventory of the globally distributed archaea. Here we report the 1,645,259-bp closed genome of strain SCM1, revealing highly copper-dependent systems for ammonia oxidation and electron transport that are distinctly different from known ammonia-oxidizing bacteria. Consistent with in situ isotopic studies of marine archaea, the genome sequence indicates N. maritimus grows autotrophically using a variant of the 3-hydroxypropionate/4-hydroxybutryrate pathway for carbon assimilation, while maintaining limited capacity for assimilation of organic carbon. This unique instance of archaeal biosynthesis of the osmoprotectant ectoine and an unprecedented enrichment of multicopper oxidases, thioredoxin-like proteins, and transcriptional regulators points to an organism responsive to environmental cues and adapted to handling reactive copper and nitrogen species that likely derive from its distinctive biochemistry. The conservation of N. maritimus gene content and organization within marine metagenomes indicates that the unique physiology of these specialized oligophiles may play a significant role in the biogeochemical cycles of carbon and nitrogen.

Improvement in survival and cardiac metabolism after gene transfer of sarcoplasmic reticulum Ca(2+)-ATPase in a rat model of heart failure.

BACKGROUND: In heart failure, sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA2a) activity is decreased, resulting in abnormal calcium handling and contractile dysfunction. We have previously shown that increasing SERCA2a expression by gene transfer improves ventricular function in a rat model of heart failure created by ascending aortic constriction. METHODS AND RESULTS: In this study, we tested the effects of gene transfer of SERCA2a on survival, left ventricular (LV) volumes, and metabolism. By 26 to 27 weeks after aortic banding, all animals developed heart failure (as documented by >25% decrease in fractional shortening) and were randomized to receive either an adenovirus carrying the SERCA2a gene (Ad.SERCA2a) or control virus (Ad.betagal-GFP) by use of a catheter-based technique. Sham-operated rats, uninfected or infected with either Ad.betagal-GFP or Ad.SERCA2a, served as controls. Four weeks after gene transfer, survival in rats with heart failure treated with Ad.betagal-GFP was 9%, compared with 63% in rats receiving Ad.SERCA2a. LV volumes were significantly increased in heart failure (0.64+/-0.05 versus 0.35+/-0.03 mL, P<0.02). Overexpression of SERCA2a normalized LV volumes (0.46+/-0.07 mL) in the failing hearts. (31)P NMR analysis showed a reduced ratio of phosphocreatine to ATP content in failing+Ad.betagal-GFP compared with sham+Ad.betagal-GFP (0.82+/-0.13 versus 1.38+/-0.14, P<0.01). Overexpression of SERCA2a in failing hearts improved the phosphocreatine/ATP ratio (1.23+/-0.28). CONCLUSIONS: In this study, we show that unlike inotropic agents that improve contractile function at the expense of increased mortality and worsening metabolism, gene transfer of SERCA2a improves survival and the energy potential in failing hearts.

Heterodimeric structure of superoxide dismutase in complex with its metallochaperone.

The copper chaperone for superoxide dismutase (CCS) activates the eukaryotic antioxidant enzyme copper, zinc superoxide dismutase (SOD1). The 2.9 A resolution structure of yeast SOD1 complexed with yeast CCS (yCCS) reveals that SOD1 interacts with its metallochaperone to form a complex comprising one monomer of each protein. The heterodimer interface is remarkably similar to the SOD1 and yCCS homodimer interfaces. Striking conformational rearrangements are observed in both the chaperone and target enzyme upon complex formation, and the functionally essential C-terminal domain of yCCS is well positioned to play a key role in the metal ion transfer mechanism. This domain is linked to SOD1 by an intermolecular disulfide bond that may facilitate or regulate copper delivery.

Structure of the yeast ribonucleotide reductase Y2Y4 heterodimer.

The R2 subunits of class I ribonucleotide reductases (RNRs) house a diferric-tyrosyl radical (Y*) cofactor essential for DNA synthesis. In yeast, there are two R2 proteins, Y2 and Y4. Although both Y2 and Y4 are homologous to R2s from other organisms, Y4 lacks three conserved iron-binding residues, and its exact function is unclear. Y4 is required for assembly of the diferric-Y* cofactor in Y2, and the two proteins can form both homodimeric and heterodimeric complexes. The Y2Y4 heterodimer was crystallized from a mixture of the two proteins, and its structure was determined to 2.8 A resolution. Both Y2 and Y4 are completely alpha helical and resemble the mouse and Escherichia coli R2s in overall fold. Three alpha helices not observed in the mouse R2 structure are present at the Y2 N terminus, and one extra N-terminal helix is observed in Y4. In addition, one of the eight principal helices in both Y2 and Y4, alphaD, is shifted significantly from its position in mouse R2. The heterodimer interface is similar to the mouse R2 homodimer interface in size and interacting residues, but loop regions at the interface edges differ. A single metal ion, assigned as Zn(II), occupies the Fe2 position in the Y2 active site. Treatment of the crystals with Fe(II) results in difference electron density consistent with formation of a diiron center. No metal-binding site is observed in Y4. Instead, the residues in the active site region form a hydrogen-bonding network involving an arginine, two glutamic acids, and a water molecule.

Hmg-CoA reductase inhibitor modulates monocyte-endothelial cell interaction under physiological flow conditions in vitro: involvement of Rho GTPase-dependent mechanism.

3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, or statins, have been reported to exert actions independent of their lipid-lowering effects. To critically assess the effects of statins on monocyte-endothelial cell interactions, we used an in vitro model that mimicked physiological flow conditions. Monocytic U937 cells were incubated in the presence of cerivastatin for 48 hours. Adhesive interactions of statin-treated U937 cells were then analyzed by use of activated (interleukin-1beta 10 U/mL, 4 hours) human umbilical vein endothelial cells in an in vitro flow apparatus. Flow cytometric analysis of adhesion molecules and measurement of F-actin content in U937 cells were performed before and after statin treatment. Preincubation with cerivastatin significantly decreased U937 firm adhesion to activated human umbilical vein endothelial cells, whereas U937 rolling was not decreased. Fluorescence-activated cell sorter analysis revealed downregulation of U937 surface expression of CD11a, CD18, and VLA4 after statin treatment. Cerivastatin significantly reduced F-actin content in U937 cells and inhibited RhoA translocation, whereas preincubation with C3 exoenzyme reduced U937 adhesion under flow. Cerivastatin reduces monocyte adhesion to vascular endothelium under physiological flow conditions via downregulation of integrin adhesion molecules and inhibition of actin polymerization via RhoA inactivation. Our findings have important implications for the lipid-independent effects of statins.

Rational reprogramming of the R2 subunit of Escherichia coli ribonucleotide reductase into a self-hydroxylating monooxygenase.

The outcome of O2 activation at the diiron(II) cluster in the R2 subunit of Escherichia coli (class I) ribonucleotide reductase has been rationally altered from the normal tyrosyl radical (Y122*) production to self-hydroxylation of a phenylalanine side-chain by two amino acid substitutions that leave intact the (histidine)2-(carboxylate)4 ligand set characteristic of the diiron-carboxylate family. Iron ligand Asp (D) 84 was replaced with Glu (E), the amino acid found in the cognate position of the structurally similar diiron-carboxylate protein, methane monooxygenase hydroxylase (MMOH). We previously showed that this substitution allows accumulation of a mu-1,2-peroxodiiron(III) intermediate, which does not accumulate in the wild-type (wt) protein and is probably a structural homologue of intermediate P (H(peroxo)) in O2 activation by MMOH. In addition, the near-surface residue Trp (W) 48 was replaced with Phe (F), blocking transfer of the "extra" electron that occurs in wt R2 during formation of the formally Fe(III)Fe(IV) cluster X. Decay of the mu-1,2-peroxodiiron(III) complex in R2-W48F/D84E gives an initial brown product, which contains very little Y122* and which converts very slowly (t1/2 approximately 7 h) upon incubation at 0 degrees C to an intensely purple final product. X-ray crystallographic analysis of the purple product indicates that F208 has undergone epsilon-hydroxylation and the resulting phenol has shifted significantly to become a ligand to Fe2 of the diiron cluster. Resonance Raman (RR) spectra of the purple product generated with 16O2 or 18O2 show appropriate isotopic sensitivity in bands assigned to O-phenyl and Fe-O-phenyl vibrational modes, confirming that the oxygen of the Fe(III)-phenolate species is derived from O2. Chemical analysis, experiments involving interception of the hydroxylating intermediate with exogenous reductant, and Mössbauer and EXAFS characterization of the brown and purple species establish that F208 hydroxylation occurs during decay of the peroxo complex and formation of the initial brown product. The slow transition to the purple Fe(III)-phenolate species is ascribed to a ligand rearrangement in which mu-O2- is lost and the F208-derived phenolate coordinates. The reprogramming to F208 monooxygenase requires both amino acid substitutions, as very little epsilon-hydroxyphenylalanine is formed and pathways leading to Y122* formation predominate in both R2-D84E and R2-W48F.

Structure of beta-lactam synthetase reveals how to synthesize antibiotics instead of asparagine.

The enzyme beta-lactam synthetase (beta-LS) catalyzes the formation of the beta-lactam ring in clavulanic acid, a clinically important beta-lactamase inhibitor. Whereas the penicillin beta-lactam ring is generated by isopenicillin N synthase (IPNS) in the presence of ferrous ion and dioxygen, beta-LS uses ATP and Mg2+ as cofactors. According to sequence alignments, beta-LS is homologous to class B asparagine synthetases (AS-Bs), ATP/Mg2+-dependent enzymes that convert aspartic acid to asparagine. Here we report the first crystal structure of a beta-LS. The 1.95 A resolution structure of Streptomyces clavuligerus beta-LS provides a fully resolved view of the active site in which substrate, closely related ATP analog alpha,beta-methyleneadenosine 5'-triphosphate (AMP-CPP) and a single Mg2+ ion are present. A high degree of substrate preorganization is observed. Comparison to Escherichia coli AS-B reveals the evolutionary changes that have taken place in beta-LS that impede interdomain reaction, which is essential in AS-B, and that accommodate beta-lactam formation. The structural data provide the opportunity to alter the synthetic potential of beta-LS, perhaps leading to the creation of new beta-lactamase inhibitors and beta-lactam antibiotics.

Akt activation preserves cardiac function and prevents injury after transient cardiac ischemia in vivo.

BACKGROUND: The serine-threonine kinase Akt is activated by several ligand-receptor systems previously shown to be cardioprotective. Akt activation reduces cardiomyocyte apoptosis in models of transient ischemia. Its role in cardiac dysfunction or infarction, however, remains unclear. METHODS AND RESULTS: We examined the effects of a constitutively active Akt mutant (myr-Akt) in a rat model of cardiac ischemia-reperfusion injury. In vivo gene transfer of myr-Akt reduced infarct size by 64% and the number of apoptotic cells by 84% (P<0.005 for each). Ischemia-reperfusion injury decreased regional cardiac wall thickening as well as the maximal rate of left ventricular pressure rise and fall (+dP/dt and -dP/dt). Akt activation restored regional wall thickening and +dP/dt and -dP/dt to levels seen in sham-operated rats. To better understand this benefit, we examined the effects of myr-Akt on hypoxic cardiomyocyte dysfunction in vitro. myr-Akt prevented hypoxia-induced abnormalities in cardiomyocyte calcium transients and shortening. Akt activation also enhanced sarcolemmal expression of Glut-4 in vivo and increased glucose uptake in vitro to the level seen with insulin treatment. CONCLUSIONS: Akt activation exerts a powerful cardioprotective effect after transient ischemia that probably reflects its ability to both inhibit cardiomyocyte death and improve function of surviving cardiomyocytes. Akt may represent an important nodal target for therapy in ischemic and other heart disease.

Crystal structure of a novel red copper protein from Nitrosomonas europaea.

Nitrosocyanin (NC) is a mononuclear red copper protein isolated from the ammonia oxidizing bacterium Nitrosomonas europaea. Although NC exhibits some sequence homology to classic blue copper proteins, its spectroscopic and electrochemical properties are drastically different. The 1.65 A resolution crystal structure of oxidized NC reveals an unprecedented trimer of single domain cupredoxins. Each copper center is partially covered by an unusual extended beta-hairpin structure from an adjacent monomer. The copper ion is coordinated by His 98, His 103, Cys 95, a single side chain oxygen of Glu 60, and a solvent molecule. In the 2.3 A resolution structure of reduced NC, His 98 shifts away from the copper ion, and the solvent molecule is not observed. The arrangement of these ligands renders the coordination geometry of the NC red copper center distinct from that of blue copper centers. In particular, the red copper center has a higher coordination number and lacks the long Cu-S(Met) and short Cu-S(Cys) bond distances characteristic of blue copper. Moreover, the red copper center is square pyramidal whereas blue copper is typically distorted tetrahedral. Analysis of the NC structure provides insight into possible functions of this new type of biological copper center.

Xenon and halogenated alkanes track putative substrate binding cavities in the soluble methane monooxygenase hydroxylase.

To investigate the role of protein cavities in facilitating movement of the substrates, methane and dioxygen, in the soluble methane monooxygenase hydroxylase (MMOH), we determined the X-ray structures of MMOH from Methylococcus capsulatus (Bath) cocrystallized with dibromomethane or iodoethane, or by using crystals pressurized with xenon gas. The halogenated alkanes bind in two cavities within the alpha-subunit that extend from one surface of the protein to the buried dinuclear iron active site. Two additional binding sites were located in the beta-subunit. Pressurization of two crystal forms of MMOH with xenon resulted in the identification of six binding sites located exclusively in the alpha-subunit. These results indicate that hydrophobic species bind preferentially in preexisting cavities in MMOH and support the hypothesis that such cavities may play a functional role in sequestering and enhancing the availability of the physiological substrates for reaction at the active site.

Role of phosphoinositide 3-kinase in monocyte recruitment under flow conditions.

Chemokines such as the monocyte chemol attractant protein-1 (MCP-1) convert monocyte rolling to firm arrest under physiological flow conditions via integrin activation and simultaneously activate phosphoinositide 3-kinase (PI3K). Here we used adenoviral gene transfer and biochemical inhibitors to manipulate PI3K-dependent pathways in human monocytes. In in vitro lipid kinase assays from purified human monocytes, we showed that MCP-1 activates the "classical" PI3Kalpha pathway and not PI3Kgamma, a PI3K isoform thought to be activated only by the betagamma complex of heterotrimeric G proteins. The activity of PI3Kalpha in purified human monocytes was evident within 30 s. MCP-1-induced monocyte arrest was significantly inhibited both by wortmannin (n = 4; p < 0.01) and LY294002 (n = 4; p < 0.01) with restoration of the rolling phenotype (p < 0.05 for both inhibitors, compared with rolling of control monocytes after MCP-1 treatment). To test the hypothesis that activation of PI3K is sufficient to induce monocyte adhesion, we transduced the monocytic THP-1 cell line with a recombinant adenovirus (Ad) carrying a constitutively active mutant of PI3K (Ad.BD110). We examined the ability of these cells to adhere to human vascular endothelium (HUVEC) transduced with adenoviruses carrying E-selectin, intercellular adhesion molecule-1 (ICAM-1), and VCAM-1. Under flow conditions, ICAM-1- and VCAM-1-dependent firm adhesion of Ad.BD110-transduced THP-1 cells was enhanced compared with THP-1 cells infected with control Ad (n = 4; p < 0.01 for both). Adhesion augmented by constitutive PI3K activation was entirely abrogated by pretreatment with wortmannin (n = 3; p < 0.01). In contrast, a constitutively active Akt construct had no effect on THP-1 adhesion (n = 3; p = NS). We conclude that PI3K activation is necessary and sufficient to enhance monocytic adhesion under physiological flow conditions. BD110-expressing THP-1 cells should provide a useful tool for identifying the signaling pathways downstream of PI3K that are necessary for monocyte recruitment relevant to a variety of human vascular pathologies.

Copper delivery by metallochaperone proteins.

Copper is an essential element in all living organisms, serving as a cofactor for many important proteins and enzymes. Metallochaperone proteins deliver copper ions to specific physiological partners by direct protein-protein interactions. The Atx1-like chaperones transfer copper to intracellular copper transporters, and the CCS chaperones shuttle copper to copper,zinc superoxide dismutase. Crystallographic studies of these two copper chaperone families have provided insights into metal binding and target recognition by metallochaperones and have led to detailed molecular models for the copper transfer mechanism.

Akt participation in the Wnt signaling pathway through Dishevelled.

Inactivation of glycogen synthase kinase 3beta (GSK3beta) and the resulting stabilization of free beta-catenin are critical steps in the activation of Wnt target genes. While Akt regulates GSK3alpha/beta in the phosphatidylinositide 3-OH kinase signaling pathway, its role in Wnt signaling is unknown. Here we report that expression of Wnt or Dishevelled (Dvl) increased Akt activity. Activated Akt bound to the Axin-GSK3beta complex in the presence of Dvl, phosphorylated GSK3beta and increased free beta-catenin levels. Furthermore, in Wnt-overexpressing PC12 cells, dominant-negative Akt decreased free beta-catenin and derepressed nerve growth factor-induced differentiation. Therefore, Akt acts in association with Dvl as an important regulator of the Wnt signaling pathway.

Structure of the bacteriophage lambda Ser/Thr protein phosphatase with sulfate ion bound in two coordination modes.

The protein phosphatase encoded by bacteriophage lambda (lambda PP) belongs to a family of Ser/Thr phosphatases (Ser/Thr PPases) that includes the eukaryotic protein phosphatases 1 (PP1), 2A (PP2A), and 2B (calcineurin). These Ser/Thr PPases and the related purple acid phosphatases (PAPs) contain a conserved phosphoesterase sequence motif that binds a dinuclear metal center. The mechanisms of phosphoester hydrolysis by these enzymes are beginning to be unraveled. To utilize lambda PP more effectively as a model for probing the catalytic mechanism of the Ser/Thr PPases, we have determined its crystal structure to 2.15 A resolution. The overall fold resembles that of PP1 and calcineurin, including a conserved beta alpha beta alpha beta structure that comprises the phosphoesterase motif. Substrates and inhibitors probably bind in a narrow surface groove that houses the active site dinuclear Mn(II) center. The arrangement of metal ligands is similar to that in PP1, calcineurin, and PAP, and a bound sulfate ion is present in two novel coordination modes. In two of the three molecules in the crystallographic asymmetric unit, sulfate is coordinated to Mn2 in a monodentate, terminal fashion, and the two Mn(II) ions are bridged by a solvent molecule. Two additional solvent molecules are coordinated to Mn1. In the third molecule, the sulfate ion is triply coordinated to the metal center with one oxygen coordinated to both Mn(II) ions, one oxygen coordinated to Mn1, and one oxygen coordinated to Mn2. The sulfate in this coordination mode displaces the bridging ligand and one of the terminal solvent ligands. In both sulfate coordination modes, the sulfate ion is stabilized by hydrogen bonding interactions with conserved arginine residues, Arg 53 and Arg 162. The two different active site structures provide models for intermediates in phosphoester hydrolysis and suggest specific mechanistic roles for conserved residues.

Heterodimer formation between superoxide dismutase and its copper chaperone.

Copper, zinc superoxide dismutase (SOD1) is activated in vivo by the copper chaperone for superoxide dismutase (CCS). The molecular mechanisms by which CCS recognizes and docks with SOD1 for metal ion insertion are not well understood. Two models for the oligomerization state during copper transfer have been proposed: a heterodimer comprising one monomer of CCS and one monomer of SOD1 and a dimer of dimers involving interactions between the two homodimers. We have investigated protein-protein complex formation between copper-loaded and apo yeast CCS (yCCS) and yeast SOD1 for both wild-type SOD1 (wtSOD1) and a mutant SOD1 in which copper ligand His 48 has been replaced with phenylalanine (H48F-SOD1). According to gel filtration chromatography, dynamic light scattering, analytical ultracentrifugation, and chemical cross-linking experiments, yCCS and this mutant SOD1 form a complex with the correct molecular mass for a heterodimer. No higher order oligomers were detected. Heterodimer formation is facilitated by the presence of zinc but does not depend on copper loading of yCCS. The complex formed with H48F-SOD1 is more stable than that formed with wtSOD1, suggesting that the latter is a more transient species. Notably, heterodimer formation between copper-loaded yCCS and wtSOD1 is accompanied by SOD1 activation only in the presence of zinc. These findings, taken together with structural, biochemical, and genetic studies, strongly suggest that in vivo copper loading of yeast SOD1 occurs via a heterodimeric intermediate.

Adenoviral transduction of human E-selectin into isolated, perfused, rat aortic segments: an ex vivo model for studying leukocyte-endothelial interactions.

E-selectin, a member of the selectin family of adhesion molecules, is thought to play an important role in leukocyte-endothelial (EC) interactions during inflammation and atherosclerosis. To critically examine the role of E-selectin in leukocyte-EC interactions in the vascular system, we created a recombinant adenoviral vector containing a human E-selectin cDNA (AdRSVE-sel) and examined the effect of AdRSVE-sel in an ex vivo vascular model of a rat aortic segment. A segment of abdominal aorta was isolated from a male Sprague-Dawley rat transduced with AdRSVE-sel ex vivo. After 72 h, surface expression of transduced E-selectin in the segment was confirmed by Western blotting and immunohistochemistry using anti-E-selectin mAb. Aortic segments were connected to a perfusion system and the adhesion of human polymorphonuclear neutrophils (PMN), and a human monocytic cell line (THP-1) to the EC surface was studied in the presence of a physiological level of flow (0.85 ml/min, approximate luminal surface shear stress=1.76 dyn/cm2). Adhesion of PMN was assessed by scanning electron microscopy and quantified using fluorescently labeled PMN. AdRSVE-sel transduced aortic segments mediated significantly more PMN and THP-1 adhesion than control segments transduced with AdRSVLacZ. Pretreatment of AdRSVE-sel transduced aortic segments with anti-E-selectin mAb inhibited PMN adhesion significantly, as well as THP-1. These data indicate that human E-selectin expressed in rat aortic segments can support the adhesion of human PMN as well as THP-1 under physiological flow conditions. This genetically modified, excised, vascular-segment model provides a useful tool for the study of leukocyte recruitment in the vascular system.

Cytosolic phospholipase A(2) regulates golgi structure and modulates intracellular trafficking of membrane proteins.

The Golgi complex and the trans-Golgi network are critical cellular organelles involved in the endocytic and biosynthetic pathways of protein trafficking. Lipids have been implicated in the regulation of membrane-protein trafficking, vesicular fusion, and targeting. We have explored the role of cytosolic group IV phospholipase A(2) (cPLA(2)) in membrane-protein trafficking in kidney epithelial cells. Adenoviral expression of cPLA(2) in LLC-PK(1) kidney epithelial cells prevents constitutive trafficking to the plasma membrane of an aquaporin 2-green fluorescent protein chimera, with retention of the protein in the rough endoplasmic reticulum. Plasma membrane Na(+)-K(+)-ATPase alpha-subunit localization is markedly reduced in cells expressing cPLA(2), whereas the trafficking of a Cl(-)/HCO(3)(-) anion exchanger to the plasma membrane is not altered in these cells. Expression of cPLA(2) results in dispersion of giantin and beta-COP from their normal, condensed Golgi localization, and in marked disruption of the Golgi cisternae. cPLA(2) is present in Golgi fractions from noninfected LLC-PK(1) cells and rat kidney cortex. The distribution of tubulin and actin was not altered by cPLA(2), indicating that the microtubule and actin cytoskeleton remain intact. Total cellular protein synthesis is unaffected by the increase in cPLA(2) activity. Thus cPLA(2) plays an important role in determining Golgi architecture and selective control of constitutive membrane-protein trafficking in renal epithelial cells.

Glycogen synthase kinase-3beta is a negative regulator of cardiomyocyte hypertrophy.

Hypertrophy is a basic cellular response to a variety of stressors and growth factors, and has been best characterized in myocytes. Pathologic hypertrophy of cardiac myocytes leads to heart failure, a major cause of death and disability in the developed world. Several cytosolic signaling pathways have been identified that transduce prohypertrophic signals, but to date, little work has focused on signaling pathways that might negatively regulate hypertrophy. Herein, we report that glycogen synthase kinase-3beta (GSK-3beta), a protein kinase previously implicated in processes as diverse as development and tumorigenesis, is inactivated by hypertrophic stimuli via a phosphoinositide 3-kinase-dependent protein kinase that phosphorylates GSK-3beta on ser 9. Using adenovirus-mediated gene transfer of GSK-3beta containing a ser 9 to alanine mutation, which prevents inactivation by hypertrophic stimuli, we demonstrate that inactivation of GSK-3beta is required for cardiomyocytes to undergo hypertrophy. Furthermore, our data suggest that GSK-3beta regulates the hypertrophic response, at least in part, by modulating the nuclear/cytoplasmic partitioning of a member of the nuclear factor of activated T cells family of transcription factors. The identification of GSK-3beta as a transducer of antihypertrophic signals suggests that novel therapeutic strategies to treat hypertrophic diseases of the heart could be designed that target components of the GSK-3 pathway.

Akt-mediated survival of oligodendrocytes induced by neuregulins.

Neuregulins have been implicated in a number of events in cells in the oligodendrocyte lineage, including enhanced survival, mitosis, migration, and differentiation. At least two signaling pathways have been shown to be involved in neuregulin signaling: the phosphatidylinositol (PI)-3 kinase and the mitogen-activated protein kinase pathways. In the present studies, we examined the signaling pathway involved in the survival function of heregulin, focusing on heregulin-induced changes in Akt activity in cultured glial cells, and the consequences of Akt activation in cells in the oligodendrocyte lineage. Heregulin binds erbB receptors, and in our studies, primary cultures of both oligodendrocyte progenitor cells and differentiating oligodendrocytes expressed erbB2, erbB3, and erbB4 receptors. In C6 glioma cells and primary cultures of oligodendrocytes, heregulin induced time- and dose-dependent Akt phosphorylation at Ser(473) in a wortmannin-sensitive manner. To investigate further the signaling pathway for heregulin in glial cells, BAD was overexpressed in C6 glioma cells. In these cells, heregulin induced phosphorylation of BAD at Ser(136). Apoptosis of oligodendrocyte progenitor cells induced by growth factor deprivation was effectively blocked by heregulin in a wortmannin-sensitive manner. Overexpression of dominant negative Akt but not of wild-type Akt by adenoviral gene transfer in primary cultures of both oligodendrocytes and their progenitors induced significant apoptosis through activation of the caspase cascade. The present data suggest that the survival function of heregulin is mediated through the PI-3 kinase/Akt pathway in cells in the oligodendrocyte lineage and that the Akt pathway may be quite important for survival of cells in this lineage.