Endothelial nitric oxide synthase (eNOS) is the nitric oxide synthase isoform responsible for maintaining systemic blood pressure, vascular remodelling and angiogenesis. eNOS is phosphorylated in response to various forms of cellular stimulation, but the role of phosphorylation in the regulation of nitric oxide (NO) production and the kinase(s) responsible are not known. Here we show that the serine/threonine protein kinase Akt (protein kinase B) can directly phosphorylate eNOS on serine 1179 and activate the enzyme, leading to NO production, whereas mutant eNOS (S1179A) is resistant to phosphorylation and activation by Akt. Moreover, using adenovirus-mediated gene transfer, activated Akt increases basal NO release from endothelial cells, and activation-deficient Akt attenuates NO production stimulated by vascular endothelial growth factor. Thus, eNOS is a newly described Akt substrate linking signal transduction by Akt to the release of the gaseous second messenger NO.

The balance of nitric oxide (⅐NO) and superoxide anion (O 2 . ) plays an important role in vascular biology. The association of heat shock protein 90 (Hsp90) with endothelial nitric-oxide synthase (eNOS) is a critical step in the mechanisms by which eNOS generates ⅐NO. As eNOS is capable of generating both ⅐NO and O 2 . , we hypothesized that Hsp90 might also mediate eNOS-dependent O 2 . production. To test this hypothesis, bovine coronary endothelial cells (BCEC) were pretreated with geldanamycin (GA, 10 g/ml; 17.8 M) and then stimulated with the calcium ionophore, A23187 (5 M). GA significantly decreased A23187-stimulated eNOS-dependent nitrite production (p < 0.001, n ‫؍‬ 4) and significantly increased A23187-stimulated eNOS-dependent O 2 . production (p < 0.001, n ‫؍‬ 8). A23187 increased phospho-eNOS(Ser-1179) levels by >1.6-fold over vehicle (V)-treated levels. Pretreatment with GA by itself or with A23187 increased phospho-eNOS levels. In unstimulated V-treated BCEC cultures low amounts of Hsp90 were found to associate with eNOS. Pretreatment with GA and/or A23187 increased the association of Hsp90 with eNOS. These data show that Hsp90 is essential for eNOS-dependent ⅐NO production and that inhibition of ATP-dependent conformational changes in Hsp90 uncouples eNOS activity and increases eNOS-dependent O 2 . production.Nitric oxide (⅐NO) and superoxide anion (O 2 . ) play opposing roles in vascular biology. Nitric oxide generation is increased greatly when Hsp90 associates with eNOS 1 in endothelial cell cultures (1, 2). A decrease in the amount of Hsp90 co-precipitating with eNOS is associated with a decrease in ⅐NO production by pulmonary artery endothelial cells exposed to prolonged periods of hypoxia (3). Geldanamycin (GA) is an ansamycin antibiotic that binds to the ATP binding site of Hsp90, thereby inhibiting the ATP/ADP cycle required for the interaction with client proteins such as eNOS (2-4). GA has been used to demonstrate that ⅐NO production in mesentary arteries and rat aortas depends on Hsp90 activity, implying that factors adversely affecting this interaction between Hsp90 and eNOS may be one of the mechanisms for portal hypertension and increased vascular tone (2, 4). Taken together, these reports indicate that Hsp90 is critical for eNOS generation of ⅐NO.Emerging evidence suggests that under pathological conditions eNOS may also generate O 2. (5-

Abstract-Protein-protein interactions with the molecular chaperone hsp90 and phosphorylation on serine 1179 by the protein kinase Akt leads to activation of endothelial nitric oxide synthase. However, the interplay between these protein-protein interactions remains to be established. In the present study, we show that vascular endothelial growth factor stimulates the coordinated association of hsp90, Akt, and resultant phosphorylation of eNOS. Characterization of the domains of hsp90 required to bind eNOS, using yeast 2-hybrid, cell-based coprecipitation experiments, and GST-fusion proteins, revealed that the M region of hsp90 interacts with the amino terminus of eNOS and Akt. The addition of purified hsp90 to in vitro kinase assays facilitates Akt-driven phosphorylation of recombinant eNOS protein, but not a short peptide encoding the Akt phosphorylation site, suggesting that hsp90 may function as a scaffold for eNOS and Akt. In vivo, coexpression of adenoviral or the cDNA for hsp90 with eNOS promotes nitric oxide release; an effect eliminated using a catalytically functional phosphorylation mutant of eNOS. These results demonstrate that stimulation of endothelial cells with vascular endothelial growth factor recruits eNOS and Akt to an adjacent region on the same domain of hsp90, thereby facilitating eNOS phosphorylation and enzyme activation. Key Words: nitric oxide Ⅲ signaling Ⅲ scaffold Ⅲ hsp90 Ⅲ Akt E ndothelial nitric oxide synthase (eNOS) continually produces low levels of nitric oxide (NO) to regulate several aspects of cardiovascular homeostasis. In endothelial cells and cells transfected with the eNOS cDNA, eNOS behaves as a peripheral membrane protein that is regulated by the allosteric activator, calmodulin (CaM). In vitro, the addition of CaM to recombinant eNOS markedly accelerates NOS catalytic function and NO synthesis. 1 However, in vivo, additional regulatory mechanisms other than CaM participate in eNOS activation/inactivation. This concept is supported by studies demonstrating that mislocalization of eNOS secondary to mutations that block its membrane association do not influence its catalytic function or calcium dependency in vitro; however, agonist-stimulated NO release from cells is markedly diminished. 2-4 These studies imply that spatial and temporal regulation of membrane associated eNOS function must involve other protein-protein or protein-lipid interactions that impact on its activation state.In the past several years, many protein partners that interact with eNOS have been described, including caveolins-1 and -3, 5,6 heat shock protein 90 (hsp90), 7 dynamin-2, 8 G protein-coupled receptors, 9 and certain kinases including Akt and mitogen-activated protein kinase family members. 10,11 All these proteins have been show to interact with eNOS in conventional in vitro assays, including coprecipitations, affinity chromatography, and yeast 2-hybrid analysis. In vivo, there is compelling evidence supporting the importance of caveolin-1, hsp90, and Akt in regulating NO release because overe...

Pathways controlling cell proliferation and cell survival require flexible adaptation to environmental stresses. These mechanisms are frequently exploited in cancer, allowing tumor cells to thrive in unfavorable milieus. Here, we show that Hsp90, a molecular chaperone that is central to the cellular stress response, associates with survivin, an apoptosis inhibitor and essential regulator of mitosis. This interaction involves the ATPase domain of Hsp90 and the survivin baculovirus inhibitor of apoptosis repeat. Global suppression of the Hsp90 chaperone function or targeted Abmediated disruption of the survivin-Hsp90 complex results in proteasomal degradation of survivin, mitochondrial-dependent apoptosis, and cell cycle arrest with mitotic defects. These data link the cellular stress response to an antiapoptotic and mitotic checkpoint maintained by survivin. Targeting the survivin-Hsp90 complex may provide a rational approach for cancer therapy.

Previous efforts to control cellular behaviour have largely relied upon various forms of genetic engineering. Once the genetic content of a living cell is modified, the behaviour of that cell typically changes as well. However, other methods of cellular control are possible. All cells sense and respond to their environment. Therefore, artificial, non-living cellular mimics could be engineered to activate or repress already existing natural sensory pathways of living cells through chemical communication. Here we describe the construction of such a system. The artificial cells expand the senses of Escherichia coli by translating a chemical message that E. coli cannot sense on its own to a molecule that activates a natural cellular response. This methodology could open new opportunities in engineering cellular behaviour without exploiting genetically modified organisms.

Methods to regulate gene expression programs in bacterial cells are limited by the absence of effective gene activators. To address this challenge, we have developed synthetic bacterial transcriptional activators in E. coli by linking activation domains to programmable CRISPR-Cas DNA binding domains. Effective gene activation requires target sites situated in a narrow region just upstream of the transcription start site, in sharp contrast to the relatively flexible target site requirements for gene activation in eukaryotic cells. Together with existing tools for CRISPRi gene repression, these bacterial activators enable programmable control over multiple genes with simultaneous activation and repression. Further, the entire gene expression program can be switched on by inducing expression of the CRISPR-Cas system. This work will provide a foundation for engineering synthetic bacterial cellular devices with applications including diagnostics, therapeutics, and industrial biosynthesis.

Artificial cells capable of both sensing and sending chemical messages to bacteria have yet to be built. Here we show that artificial cells that are able to sense and synthesize quorum signaling molecules can chemically communicate with V. fischeri, V. harveyi, E. coli, and P. aeruginosa. Activity was assessed by fluorescence, luminescence, RT-qPCR, and RNA-seq. Two potential applications for this technology were demonstrated. First, the extent to which artificial cells could imitate natural cells was quantified by a type of cellular Turing test. Artificial cells capable of sensing and in response synthesizing and releasing N-3-(oxohexanoyl)homoserine lactone showed a high degree of likeness to natural V. fischeri under specific test conditions. Second, artificial cells that sensed V. fischeri and in response degraded a quorum signaling molecule of P. aeruginosa (N-(3-oxododecanoyl)homoserine lactone) were constructed, laying the foundation for future technologies that control complex networks of natural cells.

The subcellular localization of endothelial nitric-oxide synthase (eNOS) is critical for optimal coupling of extracellular stimulation to nitric oxide production. Because eNOS is activated by Akt-dependent phosphorylation to produce nitric oxide (NO), we determined the subcellular distribution of eNOS phosphorylated on serine 1179 using a variety of methodologies. Based on sucrose gradient fractionation, phosphorylated-eNOS (P-eNOS) was found in both caveolin-1-enriched membranes and intracellular domains. Co-transfection of eNOS with Akt and stimulation of endothelial cells with vascular endothelial growth factor (VEGF) increased the ratio of P-eNOS to total eNOS but did not change the relative intracellular distribution between these domains. The proper localization of eNOS to intracellular membranes was required for agonist-dependent phosphorylation on serine 1179, since VEGF did not increase eNOS phosphorylation in cells transfected with a nonacylated, mistargeted form of eNOS. Confocal imaging of P-eNOS and total eNOS pools demonstrated co-localization in the Golgi region and plasmalemma of transfected cells and native endothelial cells. Finally, VEGF stimulated a large increase in NO localized in both the perinuclear region and the plasma membrane of endothelial cells. Thus, activated, phosphorylated eNOS resides in two cellular compartments and both pools are VEGFregulated to produce NO. Endothelial nitric-oxide synthase (eNOS)1 is the NOS isoform responsible for cardiovascular homeostasis including regulation of blood pressure, vessel remodeling, and angiogenesis. In addition to the profound physiological role of eNOS-derived NO, eNOS is unique among NOS family members since it is a peripheral membrane protein that is modified by co-translational N-myristoylation and post-translational cysteine palmitoylation (1, 2). Both N-myristoylation and cysteine palmitoylation are necessary for the subcellular targeting of eNOS onto peripheral aspects of the Golgi complex and to cholesterol-rich microdomains of the plasma membrane including caveolae/ lipid rafts (3, 4). Moreover, mislocalization of the enzyme to either domain impairs agonist-stimulated NO release from cells, implying that the proper subcellular localization of eNOS is critical for stimulus-dependent coupling to the enzyme (5, 6). Recently many investigators have shown that protein phosphorylation of eNOS by several serine/threonine kinases is a critical control step for NO production by endothelial cells. Phosphorylation by AMP kinase (7), Akt (or protein kinase B)