The AMP-activated protein kinase (AMPK) in rat skeletal and cardiac muscle is activated by vigorous exercise and ischaemic stress. Under these conditions AMPK phosphorylates and inhibits acetyl-coenzyme A carboxylase causing increased oxidation of fatty acids. Here we show that AMPK coimmunoprecipitates with cardiac endothelial NO synthase (eNOS) and phosphorylates Ser-1177 in the presence of Ca 2+ -calmodulin (CaM) to activate eNOS both in vitro and during ischaemia in rat hearts. In the absence of Ca 2+ -calmodulin, AMPK also phosphorylates eNOS at Thr-495 in the CaMbinding sequence, resulting in inhibition of eNOS activity but Thr-495 phosphorylation is unchanged during ischaemia. Phosphorylation of eNOS by the AMPK in endothelial cells and myocytes provides a further regulatory link between metabolic stress and cardiovascular function.z 1999 Federation of European Biochemical Societies.

Endothelial nitric oxide synthase (eNOS) is an important modulator of angiogenesis and vascular tone [1]. It is stimulated by treatment of endothelial cells in a phosphatidylinositol 3-kinase (PI 3-kinase)-dependent fashion by insulin-like growth factor-1 (IGF-1) and vascular endothelial growth factor (VEGF) [2] [3] and is activated by phosphorylation at Ser1177 in the sequence RIRTQS(1177)F (in the single-letter amino acid code) [4]. The protein kinase Akt is an important downstream target of PI 3-kinase [5] [6], regulating VEGF-stimulated endothelial cell survival [7]. Akt phosphorylates substrates within a defined motif [8], which is present in the sequence surrounding Ser1177 in eNOS. Both Akt [5] [6] and eNOS [9] are localized to, and activated at, the plasma membrane. We found that purified Akt phosphorylated cardiac eNOS at Ser1177, resulting in activation of eNOS. Phosphorylation at this site was stimulated by treatment of bovine aortic endothelial cells (BAECs) with VEGF or IGF-1, and Akt was activated in parallel. Preincubation with wortmannin, an inhibitor of Akt signalling, reduced VEGF- or IGF-1-induced Akt activity and eNOS phosphorylation. Akt was detected in immunoprecipitates of eNOS from BAECs, and eNOS in immunoprecipitates of Akt, indicating that the two enzymes associate in vivo. It is thus apparent that Akt directly activates eNOS in endothelial cells. These results strongly suggest that Akt has an important role in the regulation of normal angiogenesis and raise the possibility that the enhanced activity of this kinase that occurs in carcinomas may contribute to tumor vascularization and survival.

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Nitric oxide is implicated in a variety of signaling pathways in different systems, notably in endothelial cells. Some of its effects can be exerted through covalent modifications of proteins and, among these modifications, increasing attention is being paid to S-nitrosylation as a signaling mechanism. In this work, we show by a variety of methods (ozone chemiluminescence, biotin switch, and mass spectrometry) that the molecular chaperone Hsp90 is a target of S-nitrosylation and identify a susceptible cysteine residue in the region of the C-terminal domain that interacts with endothelial nitric oxide synthase (eNOS). We also show that the modification occurs in endothelial cells when they are treated with S-nitroso-L-cysteine and when they are exposed to eNOS activators. Hsp90 ATPase activity and its positive effect on eNOS activity are both inhibited by S-nitrosylation. Together, these data suggest that S-nitrosylation may functionally regulate the general activities of Hsp90 and provide a feedback mechanism for limiting eNOS activation.atherosclerosis ͉ nitrosation ͉ vascular wall ͉ chaperone R ecent years have witnessed an increasing interest in the roles of nitric oxide (NO) in signal transduction pathways other than its activation of the cGMP pathway. Many of these roles rely on NO's ability to alter protein function through posttranslational modifications. Among these modifications, S-nitrosylation has emerged as a potential and fundamental regulator of protein function. S-nitrosylation is a covalent modification of thiol groups by formation of a thionitrite (-S-NϭO) group, facilitated by the formation of higher nitrogen oxides (1, 2). To date, several dozens of proteins have been shown to become S-nitrosylated and, in many cases, this modification was accompanied by altered function (see table S1 of ref. 1 for review).Nitric oxide, synthesized in the endothelium by endothelial nitric oxide synthase (eNOS), plays crucial roles in the vascular wall, including the maintenance of vascular tone. The possibility that NO might modify eNOS, or elements of the complex system involved in its activation, is an attractive hypothesis, suggesting a potential autoregulatory feedback mechanism. The eNOS enzyme is regulated by several posttranslational modifications including myristoylation, palmitoylation, and phosphorylation (3). This enzyme is also tightly regulated by specific interactions with inhibitory proteins such as caveolin-1 and by positive modulation by the scaffolding protein Hsp90. These interactions have been described in detail, and a relatively complete picture is beginning to emerge (4).We have previously used a proteomic approach to identify several proteins that were S-nitrosylated after exposure of vascular endothelial cells to the physiological nitrosothiol, Snitroso-L-cysteine (CSNO) (5). Further work led to the identification of Hsp90 as a protein susceptible to S-nitrosylation. This chaperone protein, known for its functions in protein folding, degradation, and scaffolding, has attracted renewed ...

Dynein is a minus-end-directed microtubule-associated motor protein involved in cargo transport in the cytoplasm. African swine fever virus (ASFV), a large DNA virus, hijacks the microtubule motor complex cellular transport machinery during virus infection of the cell through direct binding of virus protein p54 to the light chain of cytoplasmic dynein (LC8). Interaction of p54 and LC8 occurs both in vitro and in cells, and the two proteins colocalize at the microtubular organizing center during viral infection. p50/dynamitin, a dominant-negative inhibitor of dynein-dynactin function, impeded ASFV infection, suggesting an essential role for dynein during virus infection. A 13-amino-acid domain of p54 was sufficient for binding to LC8, an SQT motif within this domain being critical for this binding. Direct binding of a viral structural protein to LC8, a small molecule of the dynein motor complex, could constitute a molecular mechanism for microtubulemediated virus transport.

Dysregulation of intracellular Ca2+ homeostasis may underlie amyloid β peptide (Aβ) toxicity in Alzheimer's Disease (AD) but the mechanism is unknown. In search for this mechanism we found that Aβ1–42 oligomers, the assembly state correlating best with cognitive decline in AD, but not Aβ fibrils, induce a massive entry of Ca2+ in neurons and promote mitochondrial Ca2+ overload as shown by bioluminescence imaging of targeted aequorin in individual neurons. Aβ oligomers induce also mitochondrial permeability transition, cytochrome c release, apoptosis and cell death. Mitochondrial depolarization prevents mitochondrial Ca2+ overload, cytochrome c release and cell death. In addition, we found that a series of non-steroidal anti-inflammatory drugs (NSAIDs) including salicylate, sulindac sulfide, indomethacin, ibuprofen and R-flurbiprofen depolarize mitochondria and inhibit mitochondrial Ca2+ overload, cytochrome c release and cell death induced by Aβ oligomers. Our results indicate that i) mitochondrial Ca2+ overload underlies the neurotoxicity induced by Aβ oligomers and ii) inhibition of mitochondrial Ca2+ overload provides a novel mechanism of neuroprotection by NSAIDs against Aβ oligomers and AD.

Bovine endothelial nitric-oxide synthase (eNOS) expressed in Escherichia coli does not have the post-translational modifications found in the native enzyme and is free of tetrahydrobiopterin (BH4). In the presence of BH4, eNOS has an absorption maximum at 400 nm that shifts to 395 nm when the substrate L-arginine is added. The low-spin component of the spectrum of the BH4-free protein is decreased by the addition of BH4 without a corresponding increase in the high-spin component. Addition of BH4 decreases the low-spin population of eNOS even in the presence of excess L-arginine. These results indicate that BH4 directly modulates the heme environment. BH4-free eNOS is completely inactive, but catalytic activity is recovered when BH4 (EC50 approximately 200 nM) is added. The spectroscopically determined binding constants for L-arginine are approximately 1.9 microM in the presence and approximately 4.0 microM in the absence of BH4. The BH4-supplemented enzyme has an activity of 90-120 nmol of citrulline.min-1.mg-1 and Km values of 3 and 14 microM for L-arginine and N-hydroxy-L-arginine, respectively. Of particular interest is the finding by SDS-polyacrylamide gel electrophoresis that BH4-free eNOS exists in a monomer-dimer equilibrium very similar to that observed with the BH4-reconstituted protein. Addition of BH4, increases the percent of the dimer by only approximately 5%. The results establish that BH4 influences the heme environment and stabilizes the protein with respect to heme loss but is not required for dimer formation.

Serine racemase (SR) is a brain enzyme present in glial cells, where it isomerizes L-serine into D-serine that, in turn, diffuses and coactivates the N-methyl-D-aspartate receptor through the binding to the so-called "glycine site." We have developed a method for the slow expression of SR in a eukaryotic vector that permits the correct insertion of the prosthetic group into the active site, rendering functional SR with a K m toward L-serine of 4.8 mM. Divalent cations such as calcium or manganese were necessary for complete enzyme activity, whereas the presence of chelators such as EDTA completely inhibited the enzyme. Moreover, direct binding of calcium to SR was evidenced using 45 Ca 2؉ . Gel filtration of the recombinant SR revealed the protein to be in a dimer-tetramer equilibrium. The addition of EDTA to a calcium-saturated serine racemase evokes a profound conformational change, as monitored by both fluorescence and circular dichroism techniques. Fluorescence titration allowed us to calculate a binding constant for calcium of 6.2 M. Reagents that react with sulfhydryl groups, such as cystamine, were potent inhibitors of SR, in a clear reflection that one or more cysteine residues are important for enzyme activity. Additionally, 16 serine analogues were tested as a putative SR substrate or inhibitors. Significant inhibition was only observed for L-Ser-O-sulfate, L-cycloserine, and L-cysteine. Finally, activation of brain SR as a result of the changes in calcium concentration was studied in primary astrocytes. Treatment of astrocytes with the calcium ionophore A23187, as well as with compounds that augment the intracellular calcium levels such as glutamate or kainate led to an increase in the amount of D-serine present in the extracellular medium. These results suggest that there might be a glutamatergic-mediated regulation of SR activity by intracellular calcium concentration.D-Amino acids have been known for decades to be present in bacteria, where they are important constituents of peptidoglycan in the cell wall. Interestingly, recent improvement in the detection techniques has allowed the identification of significant levels of both D-serine (1-3) and D-aspartic acid (3, 4) in the nervous system of vertebrates. Snyder and co-workers (5, 6) have elegantly purified and subsequently cloned the cDNA for a novel enzyme responsible for the synthesis of D-serine in the brain and identified it as a 37-kDa pyridoxal phosphate-containing racemase present in astrocytes. These protoplasmic astrocytes typically ensheath synapses, strongly suggesting a role for D-serine in synaptic transmission. Serine racemase is highly enriched in the brain and co-localizes with D-serine according to immunohistochemical analysis (6). Additionally, D-serine is concentrated in regions enriched in N-methyl-Daspartate (NMDA) 1 receptors (i.e. highest in the forebrain), whereas the levels of the previously identified NMDA receptor coactivator, glycine, are lowest in this region (7,8). Remarkably, recent reports have also identified D-ser...