Effects of Combined Phosphorylation at Ser-617 and Ser-1179 in Endothelial Nitric-oxide Synthase on EC50(Ca2+) Values for Calmodulin Binding and Enzyme Activation

We have investigated the possible biochemical basis for enhancements in NO production in endothelial cells that have been correlated with agonist- or shear stress-evoked phosphorylation at Ser-1179. We have found that a phosphomimetic substitution at Ser-1179 doubles maximal synthase activity, partially disinhibits cytochrome c reductase activity, and lowers the EC₅₀(Ca²⁺) values for calmodulin binding and enzyme activation from the control values of 182 ± 2 and 422 ± 22 nm to 116 ± 2 and 300 ± 10 nm. These are similar to the effects of a phosphomimetic substitution at Ser-617 (Tran, Q. K., Leonard, J., Black, D. J., and Persechini, A. (2008) Biochemistry 47, 7557–7566). Although combining substitutions at Ser-617 and Ser-1179 has no additional effect on maximal synthase activity, cooperativity between the two substitutions completely disinhibits reductase activity and further reduces the EC₅₀(Ca²⁺) values for calmodulin binding and enzyme activation to 77 ± 2 and 130 ± 5 nm. We have confirmed that specific Akt-catalyzed phosphorylation of Ser-617 and Ser-1179 and phosphomimetic substitutions at these positions have similar functional effects. Changes in the biochemical properties of eNOS produced by combined phosphorylation at Ser-617 and Ser-1179 are predicted to substantially increase synthase activity in cells at a typical basal free Ca²⁺ concentration of 50–100 nm.The nitric-oxide synthases catalyze formation of NO and l-citrulline from l-arginine and O₂, with NADPH as the electron donor (1). The role of NO generated by endothelial nitricoxide synthase (eNOS)² in the regulation of smooth muscle tone is well established and was the first of several physiological roles for this small molecule that have so far been identified (2). The nitric-oxide synthases are homodimers of 130–160-kDa subunits. Each subunit contains a reductase and oxygenase domain (1). A significant difference between the reductase domains in eNOS and nNOS and the homologous P450 reductases is the presence of inserts in these synthase isoforms that appear to maintain them in their inactive states (3, 4). A calmodulin (CaM)-binding domain is located in the linker that connects the reductase and oxygenase domains, and the endothelial and neuronal synthases both require Ca²⁺ and exogenous CaM for activity (5, 6). When CaM is bound, it somehow counteracts the effects of the autoinhibitory insert(s) in the reductase. The high resolution structure for the complex between (Ca²⁺)₄-CaM and the isolated CaM-binding domain from eNOS indicates that the C-ter and N-ter lobes of CaM, which each contain a pair of Ca²⁺-binding sites, enfold the domain, as has been observed in several other such CaM-peptide complexes (7). Consistent with this structure, investigations of CaM-dependent activation of the neuronal synthase suggest that both CaM lobes must participate (8, 9).Bovine eNOS can be phosphorylated in endothelial cells at Ser-116, Thr-497, Ser-617, Ser-635, and Ser-1179 (10–12). There are equivalent phosphorylation sites in the human enzyme (10–12). Phosphorylation of the bovine enzyme at Thr-497, which is located in the CaM-binding domain, blocks CaM binding and enzyme activation (7, 11, 13, 14). Ser-116 can be basally phosphorylated in cells (10, 11, 13, 15), and dephosphorylation of this site has been correlated with increased NO production (13, 15). However, it has also been reported that a phosphomimetic substitution at this position has no effect on enzyme activity measured in vitro (13). Ser-1179 is phosphorylated in response to a variety of stimuli, and this has been reliably correlated with enhanced NO production in cells (10, 11). Indeed, NO production is elevated in transgenic endothelium expressing an eNOS mutant containing an S1179D substitution, but not in tissue expressing an S1179A mutant (16). Shear stress or insulin treatment is correlated with Akt-catalyzed phosphorylation of Ser-1179 in endothelial cells, and this is correlated with increased NO production in the absence of extracellular Ca²⁺ (17–19). Akt-catalyzed phosphorylation or an S1179D substitution has also been correlated with increased synthase activity in cell extracts at low intracellular free [Ca²⁺] (17). Increased NO production has also been observed in cells expressing an eNOS mutant containing an S617D substitution, and physiological stimuli such as shear-stress, bradykinin, VEGF, and ATP appear to stimulate Akt-catalyzed phosphorylation of Ser-617 and Ser-1179 (12, 13, 20). Although S617D eNOS has been reported to have the same maximum activity in vitro as the wild type enzyme (20), in our hands an S617D substitution increases the maximal CaM-dependent synthase activity of purified mutant enzyme ∼2-fold, partially disinhibits reductase activity, and reduces the EC₅₀(Ca²⁺) values for CaM binding and enzyme activation (21).In this report, we describe the effects of a phosphomimetic Asp substitution at Ser-1179 in eNOS on the Ca²⁺ dependence of CaM binding and CaM-dependent activation of reductase and synthase activities. We also describe the effects on these properties of combining this substitution with one at Ser-617. Finally, we demonstrate that Akt-catalyzed phosphorylation and Asp substitutions at Ser-617 and Ser-1179 have similar functional effects. Our results suggest that phosphorylation of eNOS at Ser-617 and Ser-1179 can substantially increase synthase activity in cells at a typical basal free Ca²⁺ concentration of 50–100 nm, while single phosphorylations at these sites produce smaller activity increases, and can do so only at higher free Ca²⁺ concentrations.     

Glutathione Participates in the Regulation of Mitophagy in Yeast

The antioxidant N-acetyl-l-cysteine prevented the autophagy-dependent delivery of mitochondria to the vacuoles, as examined by fluorescence microscopy of mitochondria-targeted green fluorescent protein, transmission electron microscopy, and Western blot analysis of mitochondrial proteins. The effect of N-acetyl-l-cysteine was specific to mitochondrial autophagy (mitophagy). Indeed, autophagy-dependent activation of alkaline phosphatase and the presence of hallmarks of non-selective microautophagy were not altered by N-acetyl-l-cysteine. The effect of N-acetyl-l-cysteine was not related to its scavenging properties, but rather to its fueling effect of the glutathione pool. As a matter of fact, the decrease of the glutathione pool induced by chemical or genetical manipulation did stimulate mitophagy but not general autophagy. Conversely, the addition of a cell-permeable form of glutathione inhibited mitophagy. Inhibition of glutathione synthesis had no effect in the strain Δuth1, which is deficient in selective mitochondrial degradation. These data show that mitophagy can be regulated independently of general autophagy, and that its implementation may depend on the cellular redox status.Autophagy is a major pathway for the lysosomal/vacuolar delivery of long-lived proteins and organelles, where they are degraded and recycled. Autophagy plays a crucial role in differentiation and cellular response to stress and is conserved in eukaryotic cells from yeast to mammals (1, 2). The main form of autophagy, macroautophagy, involves the non-selective sequestration of large portions of the cytoplasm into double-membrane structures termed autophagosomes, and their delivery to the vacuole/lysosome for degradation. Another process, microautophagy, involves the direct sequestration of parts of the cytoplasm by vacuole/lysosomes. The two processes coexist in yeast cells but their extent may depend on different factors including metabolic state: for example, we have observed that nitrogen-starved lactate-grown yeast cells develop microautophagy, whereas nitrogen-starved glucose-grown cells preferentially develop macroautophagy (3).Both macroautophagy and microautophagy are essentially non-selective, in the way that autophagosomes and vacuole invaginations do not appear to discriminate the sequestered material. However, selective forms of autophagy have been observed (4) that target namely peroxisomes (5, 6), chromatin (7, 8), endoplasmic reticulum (9), ribosomes (10), and mitochondria (3, 11–13). Although non-selective autophagy plays an essential role in survival by nitrogen starvation, by providing amino acids to the cell, selective autophagy is more likely to have a function in the maintenance of cellular structures, both under normal conditions as a “housecleaning” process, and under stress conditions by eliminating altered organelles and macromolecular structures (14–16). Selective autophagy targeting mitochondria, termed mitophagy, may be particularly relevant to stress conditions. The mitochondrial respiratory chain is both the main site and target of ROS⁴ production (17). Consequently, the maintenance of a pool of healthy mitochondria is a crucial challenge for the cells. The progressive accumulation of altered mitochondria (18) caused by the loss of efficiency of the maintenance process (degradation/biogenesis de novo) is often considered as a major cause of cellular aging (19–23). In mammalian cells, autophagic removal of mitochondria has been shown to be triggered following induction/blockade of apoptosis (23), suggesting that autophagy of mitochondria was required for cell survival following mitochondria injury (14). Consistent with this idea, a direct alteration of mitochondrial permeability properties has been shown to induce mitochondrial autophagy (13, 24, 25). Furthermore, inactivation of catalase induced the autophagic elimination of altered mitochondria (26). In the yeast Saccharomyces cerevisiae, the alteration of F₀F₁-ATPase biogenesis in a conditional mutant has been shown to trigger autophagy (27). Alterations of mitochondrial ion homeostasis caused by the inactivation of the K⁺/H⁺ exchanger was shown to cause both autophagy and mitophagy (28). We have reported that treatment of cells with rapamycin induced early ROS production and mitochondrial lipid oxidation that could be inhibited by the hydrophobic antioxidant resveratrol (29). Furthermore, resveratrol treatment impaired autophagic degradation of both cytosolic and mitochondrial proteins and delayed rapamycin-induced cell death, suggesting that mitochondrial oxidation events may play a crucial role in the regulation of autophagy. This existence of regulation of autophagy by ROS has received molecular support in HeLa cells (30): these authors showed that starvation stimulated ROS production, namely H₂O₂, which was essential for autophagy. Furthermore, they identified the cysteine protease hsAtg4 as a direct target for oxidation by H₂O₂. This provided a possible connection between the mitochondrial status and regulation of autophagy.Investigations of mitochondrial autophagy in nitrogen-starved lactate-grown yeast cells have established the existence of two distinct processes: the first one occurring very early, is selective for mitochondria and is dependent on the presence of the mitochondrial protein Uth1p; the second one occurring later, is not selective for mitochondria, is not dependent on Uth1p, and is a form of bulk microautophagy (3). The absence of the selective process in the Δuth1 mutant strongly delays and decreases mitochondrial protein degradation (3, 12). The putative protein phosphatase Aup1p has been also shown to be essential in inducing mitophagy (31). Additionally several Atg proteins were shown to be involved in vacuolar sequestration of mitochondrial GFP (3, 12, 32, 33). Recently, the protein Atg11p, which had been already identified as an essential protein for selective autophagy has also been reported as being essential for mitophagy (33).The question remains as to identify of the signals that trigger selective mitophagy. It is particularly intriguing that selective mitophagy is activated very early after the shift to a nitrogen-deprived medium (3). Furthermore, selective mitophagy is very active on lactate-grown cells (with fully differentiated mitochondria) but is nearly absent in glucose-grown cells (3). In the present paper, we investigated the relationships between the redox status of the cells and selective mitophagy, namely by manipulating glutathione. Our results support the view that redox imbalance is a trigger for the selective elimination of mitochondria.     

Acylated Cholesteryl Galactosides Are Specific Antigens of Borrelia Causing Lyme Disease and Frequently Induce Antibodies in Late Stages of Disease

Borrelia burgdorferi sensu lato is the causative agent of Lyme disease (LD), an infectious disease occurring in North America, Europe, and Asia in different clinical stages. B. burgdorferi sensu lato encompasses at least 12 species, with B. burgdorferi sensu stricto, B. garinii, and B. afzelii being of highest clinical importance. Immunologic testing for LD as well as recent vaccination strategies exclusively refer to proteinaceous antigens. However, B. burgdorferi sensu stricto exhibits glycolipid antigens, including 6-O-acylated cholesteryl β-d-galactopyranoside (ACGal), and first the data indicated that this compound may act as an immunogen. Here we investigated whether B. garinii and B. afzelii also possess this antigen, and whether antibodies directed against these compounds are abundant among patients suffering from different stages of LD. Gas-liquid chromatography/mass spectroscopy and NMR spectroscopy showed that both B. garinii and B. afzelii exhibit ACGal in high quantities. In contrast, B. hermsii causing relapsing fever features 6-O-acylated cholesteryl β-d-glucopyranoside (ACGlc). Sera derived from patients diagnosed for LD contained antibodies against ACGal, with 80% of patients suffering from late stage disease exhibiting this feature. Antibodies reacted with ACGal from all three B. burgdorferi species tested, but not with ACGlc from B. hermsii. These data show that ACGal is present in all clinically important B. burgdorferi species, and that specific antibodies against this compound are frequently found during LD. ACGal may thus be an interesting tool for improving diagnostics as well as for novel vaccination strategies.Lyme disease (LD)² is caused by B. burgdorferi sensu lato (s.l.) and is transmitted by ticks of the genus Ixodes (1, 2). It is the most common tick-borne disease in the U.S. with an incidence of 6 per 100,000, with endemic areas such as Connecticut reaching 111 cases per 100,000 (3). LD is also frequent in Asia and Europe, particularly in Germany, Austria, Slovenia, and Sweden (2, 4). B. burgdorferi s.l. comprises at least 12 species with B. burgdorferi sensu stricto (Bbu), B. garinii (Bga), and B. afzelii (Baf) being of highest clinical importance (2). In the U.S., LD is exclusively caused by Bbu, whereas in Europe all human pathogenic species are found, with Bga and Baf being predominant (2, 5, 6). LD is an infectious disease occurring in different clinical stages: early localized infection is indicated by erythema migrans (EM) in ∼70–90% of the patients (7–9), and early disseminated infection often causes neurological manifestations, such as facial palsy, meningitis, meningoradiculitits, or meningoencephalitis (early neuroborreliosis (NB)) (2,8,9). The cardinal manifestation of late stage LD in the U.S. is Lyme arthritis (LA), with ∼70% of the untreated EM cases developing this syndrome (10, 11). In Europe, next to arthritis, acrodermatitis chronica atrophicans (ACA) is a frequent late manifestation, and has been associated with Baf (11).Currently, diagnosis of LD is generally based on assessment of clinical features in combination with immunologic serum testing, where both ELISA and a confirming immunoblot are required (12, 13). However, because in Europe and Asia at least three species are causing LD, there is a substantial variation of immunodominant antigens, which requires the combination of various homologous antigens for effective serodiagnosis (14–16). Immunologic evaluation in these areas is therefore complicated, and no consensus has been established yet (12). In comparison to diagnostic procedures, vaccination strategies directed against LD so far have also been based on proteinaceous antigens: in the 1990s, recombinant vaccines based on OspA were found to be effective (17), but the production was discontinued, one reason being the high production costs in comparison to early treatment (2). Another concern raised against this approach was a potential triggering of autoimmune diseases by vaccination with Osps due to a similarity between an immunodominant epitope in OspA and human leukocyte function-associated antigen-1 (18).In contrast to proteins, information on membrane glycolipids in Borrelia available today is rather scarce. In 1978, a preliminary compositional analysis of lipid extracts of B. hermsii causing relapsing fever (RF) indicated the presence of monoglucosyldiacylglycerol and acylated as well as non-acylated cholesteryl glucosides (19). Later, studies on Bbu indicated the presence of complex glycolipids as well, but no chemical analysis was performed (20, 21). A more recent study identified mono-α-d-galactosyldiacylglycerol (MGalD) in Bbu, and first data indicated that antibodies present in sera obtained from LD patients detected this antigen (22). We and others were recently able to show that Bbu furthermore exhibits cholesteryl 6-O-acyl-β-d-galactopyranoside (ACGal) as well as its non-acylated counterpart, cholesteryl β-d-galactopyranoside (βCGal) (23, 24). Patient sera reacted with ACGal more frequently as compared with MGalD (23), and antibodies could be raised in mice by intraperitoneal injection (24), indicating that this compound is a strong immunogen.The aim of this study was to elucidate whether ACGal is a common structure present in the most relevant B. burgdorferi species of clinical importance, and whether it is a specific feature of Borrelia causing LD. Furthermore, we aimed at defining the frequency of the occurrence of antibodies against this antigen in patients suffering from LD. To this end, we performed a comparative structural analysis of glycolipid fractions of Bbu as well as the two other B. burgdorferi s.l. species of clinical importance, Baf and Bga, in comparison with B. hermsii (Bhe), the causative agent of relapsing fever. We found ACGal to be present in all B. burgdorferi species tested, whereas Bhe exhibited cholesteryl 6-O-acyl-β-d-glucopyranoside (ACGlc) instead. Antibodies against ACGal could be detected in the majority of patients diagnosed for arthritis or acrodermatitis, and these failed to cross-react with ACGlc. These data demonstrate that ACGal is an abundant, but still highly specific antigen in B. burgdorferi and thus a promising candidate for vaccine development and improvement of serologic methods.     

Amphetamine and Methamphetamine Differentially Affect Dopamine Transporters in Vitro and in Vivo

The psychostimulants d-amphetamine (AMPH) and methamphetamine (METH) release excess dopamine (DA) into the synaptic clefts of dopaminergic neurons. Abnormal DA release is thought to occur by reverse transport through the DA transporter (DAT), and it is believed to underlie the severe behavioral effects of these drugs. Here we compare structurally similar AMPH and METH on DAT function in a heterologous expression system and in an animal model. In the in vitro expression system, DAT-mediated whole-cell currents were greater for METH stimulation than for AMPH. At the same voltage and concentration, METH released five times more DA than AMPH and did so at physiological membrane potentials. At maximally effective concentrations, METH released twice as much [Ca²⁺]_i from internal stores compared with AMPH. [Ca²⁺]_i responses to both drugs were independent of membrane voltage but inhibited by DAT antagonists. Intact phosphorylation sites in the N-terminal domain of DAT were required for the AMPH- and METH-induced increase in [Ca²⁺]_i and for the enhanced effects of METH on [Ca²⁺]_i elevation. Calmodulin-dependent protein kinase II and protein kinase C inhibitors alone or in combination also blocked AMPH- or METH-induced Ca²⁺ responses. Finally, in the rat nucleus accumbens, in vivo voltammetry showed that systemic application of METH inhibited DAT-mediated DA clearance more efficiently than AMPH, resulting in excess external DA. Together these data demonstrate that METH has a stronger effect on DAT-mediated cell physiology than AMPH, which may contribute to the euphoric and addictive properties of METH compared with AMPH.The dopamine transporter (DAT)³ is a main target for psychostimulants, such as d-amphetamine (AMPH), methamphetamine (METH), cocaine (COC), and methylphenidate (Ritalin®). DAT is the major clearance mechanism for synaptic dopamine (DA) (1) and thereby regulates the strength and duration of dopaminergic signaling. AMPH and METH are substrates for DAT and competitively inhibit DA uptake (2, 3) and release DA through reverse transport (4–9). AMPH- and METH-induced elevations in extracellular DA result in complex neurochemical changes and profound psychiatric effects (2, 10–16). Despite their structural and pharmacokinetic similarities, a recent National Institute on Drug Abuse report describes METH as a more potent stimulant than AMPH with longer lasting effects at comparable doses (17). Although the route of METH administration and its availability must contribute to the almost four times higher lifetime nonmedical use of METH compared with AMPH (18), there may also be differences in the mechanisms that underlie the actions of these two drugs on the dopamine transporter.Recent studies by Joyce et al. (19) have shown that compared with d-AMPH alone, the combination of d- and l-AMPH in Adderall® significantly prolonged the time course of extracellular DA in vivo. These experiments demonstrate that subtle structural features of AMPH, such as chirality, can affect its action on dopamine transporters. Here we investigate whether METH, a more lipophilic analog of AMPH, affects DAT differently than AMPH, particularly in regard to stimulated DA efflux.METH and AMPH have been reported as equally effective in increasing extracellular DA levels in rodent dorsal striatum (dSTR), nucleus accumbens (NAc) (10, 14, 20), striatal synaptosomes, and DAT-expressing cells in vitro (3, 6). John and Jones (21), however, have recently shown in mouse striatal and substantia nigra slices, that AMPH is a more potent inhibitor of DA uptake than METH. On the other hand, in synaptosomes METH inhibits DA uptake three times more effectively than AMPH (14), and in DAT-expressing COS-7 cells, METH releases DA more potently than AMPH (EC₅₀ = 0.2 μm for METH versus EC₅₀ = 1.7 μm for AMPH) (5). However, these differences do not hold up under all conditions. For example, in a study utilizing C6 cells, the disparity between AMPH and METH was not found (12).The variations in AMPH and METH data extend to animal models. AMPH- and METH-mediated behavior has been reported as similar (22), lower (20), or higher (23) for AMPH compared with METH. Furthermore, although the maximal locomotor activation response was less for METH than for AMPH at a lower dose (2 mg/kg, intraperitoneal), both drugs decreased locomotor activity at a higher dose (4 mg/kg) (20). In contrast, in the presence of a salient stimuli, METH is more potent in increasing the overall magnitude of locomotor activity in rats yet is equipotent with AMPH in the absence of these stimuli (23).The simultaneous regulation of DA uptake and efflux by DAT substrates such as AMPH and METH, as well as the voltage dependence of DAT (24), may confound the interpretation of existing data describing the action of these drugs. Our biophysical approaches allowed us to significantly decrease the contribution of DA uptake and more accurately determine DAT-mediated DA efflux with millisecond time resolution. We have thus exploited time-resolved, whole-cell voltage clamp in combination with in vitro and in vivo microamperometry and Ca²⁺ imaging to compare the impact of METH and AMPH on DAT function and determine the consequence of these interactions on cell physiology.We find that near the resting potential, METH is more effective than AMPH in stimulating DAT to release DA. In addition, at efficacious concentrations METH generates more current, greater DA efflux, and higher Ca²⁺ release from internal stores than AMPH. Both METH-induced or the lesser AMPH-induced increase in intracellular Ca²⁺ are independent of membrane potential. The additional Ca²⁺ response induced by METH requires intact phosphorylation sites in the N-terminal domain of DAT. Finally, our in vivo voltammetry data indicate that METH inhibits clearance of locally applied DA more effectively than AMPH in the rat nucleus accumbens, which plays an important role in reward and addiction, but not in the dorsal striatum, which is involved in a variety of cognitive functions. Taken together these data imply that AMPH and METH have distinguishable effects on DAT that can be shown both at the molecular level and in vivo, and are likely to be implicated in the relative euphoric and addictive properties of these two psychostimulants.     

Incompatibility with Formin Cdc12p Prevents Human Profilin from Substituting for Fission Yeast Profilin: INSIGHTS FROM CRYSTAL STRUCTURES OF FISSION YEAST PROFILIN*S?

Expression of human profilin-I does not complement the temperature-sensitive cdc3-124 mutation of the single profilin gene in fission yeast Schizosaccharomyces pombe, resulting in death from cytokinesis defects. Human profilin-I and S. pombe profilin have similar affinities for actin monomers, the FH1 domain of fission yeast formin Cdc12p and poly-l-proline (Lu, J., and Pollard, T. D. (2001) Mol. Biol. Cell 12, 1161–1175), but human profilin-I does not stimulate actin filament elongation by formin Cdc12p like S. pombe profilin. Two crystal structures of S. pombe profilin and homology models of S. pombe profilin bound to actin show how the two profilins bind to identical surfaces on animal and yeast actins even though 75% of the residues on the profilin side of the interaction differ in the two profilins. Overexpression of human profilin-I in fission yeast expressing native profilin also causes cytokinesis defects incompatible with viability. Human profilin-I with the R88E mutation has no detectable affinity for actin and does not have this dominant overexpression phenotype. The Y6D mutation reduces the affinity of human profilin-I for poly-l-proline by 1000-fold, but overexpression of Y6D profilin in fission yeast is lethal. The most likely hypotheses to explain the incompatibility of human profilin-I with Cdc12p are differences in interactions with the proline-rich sequences in the FH1 domain of Cdc12p and wider “wings” that interact with actin.The small protein profilin not only helps to maintain a cytoplasmic pool of actin monomers ready to elongate actin filament barbed ends (2), but it also binds to type II poly-l-proline helices (3, 4). The actin (5) and poly-l-proline (6–8) binding sites are on opposite sides of the profilin molecule, so profilin can link actin to proline-rich targets. Viability of fission yeast depends independently on profilin binding to both actin and poly-l-proline, although cells survive >10-fold reductions in affinity for either ligand (1).Fission yeast Schizosaccharomyces pombe depend on formin Cdc12p (9, 10) and profilin (11) to assemble actin filaments for the cytokinetic contractile ring. Formins are multidomain proteins that nucleate and assemble unbranched actin filaments (12). Formin FH2 domains form homodimers that can associate processively with the barbed ends of growing actin filaments (13, 14). FH2 dimers slow the elongation of barbed ends (15). Most formin proteins have an FH1 domain linked to the FH2 domain. Binding profilin-actin to multiple polyproline sites in an FH1 domain concentrates actin near the barbed end of an actin filament associated with a formin FH2 homodimer. Actin transfers very rapidly from the FH1 domains onto the filament end (16) allowing profilin to stimulate elongation of the filament (15, 17).We tested the ability of human (Homo sapiens, Hs)⁷ profilin-I to complement the temperature-sensitive cdc3-124 mutation (11) in the single fission yeast profilin gene with the aim of using yeast to characterize human profilin mutations. The failure of expression of Hs profilin-I to complement the cdc3-124 mutation prompted us to compare human and fission yeast profilins more carefully. We report here a surprising incompatibility of Hs profilin-I with fission yeast formin Cdc12p, a crystal structure of fission yeast profilin, which allowed a detailed comparison with Hs profilin, and mutations that revealed how overexpression of Hs profilin-I compromises the viability of wild-type fission yeast.     

Structural and Mechanistic Insights into Lunatic Fringe from a Kinetic Analysis of Enzyme Mutants

The Notch receptor is critical for proper development where it orchestrates numerous cell fate decisions. The Fringe family of β1,3-N-acetylglucosaminyltransferases are regulators of this pathway. Fringe enzymes add N-acetylglucosamine to O-linked fucose on the epidermal growth factor repeats of Notch. Here we have analyzed the reaction catalyzed by Lunatic Fringe (Lfng) in detail. A mutagenesis strategy for Lfng was guided by a multiple sequence alignment of Fringe proteins and solutions from docking an epidermal growth factor-like O-fucose acceptor substrate onto a homology model of Lfng. We targeted three main areas as follows: residues that could help resolve where the fucose binds, residues in two conserved loops not observed in the published structure of Manic Fringe, and residues predicted to be involved in UDP-N-acetylglucosamine (UDP-GlcNAc) donor specificity. We utilized a kinetic analysis of mutant enzyme activity toward the small molecule acceptor substrate 4-nitrophenyl-α-l-fucopyranoside to judge their effect on Lfng activity. Our results support the positioning of O-fucose in a specific orientation to the catalytic residue. We also found evidence that one loop closes off the active site coincident with, or subsequent to, substrate binding. We propose a mechanism whereby the ordering of this short loop may alter the conformation of the catalytic aspartate. Finally, we identify several residues near the UDP-GlcNAc-binding site, which are specifically permissive toward UDP-GlcNAc utilization.Defects in Notch signaling have been implicated in numerous human diseases, including multiple sclerosis (1), several forms of cancer (2-4), cerebral autosomal dominant arteriopathy with sub-cortical infarcts and leukoencephalopathy (5), and spondylocostal dysostosis (SCD)³ (6-8). The transmembrane Notch signaling receptor is activated by members of the DSL (Delta, Serrate, Lag2) family of ligands (9, 10). In the endoplasmic reticulum, O-linked fucose glycans are added to the epidermal growth factor-like (EGF) repeats of the Notch extracellular domain by protein O-fucosyltransferase 1 (11-13). These O-fucose monosaccharides can be elongated in the Golgi apparatus by three highly conserved β1,3-N-acetylglucosaminyltransferases of the Fringe family (Lunatic (Lfng), Manic (Mfng), and Radical Fringe (Rfng) in mammals) (14-16). The formation of this GlcNAc-β1,3-Fuc-α1, O-serine/threonine disaccharide is necessary and sufficient for subsequent elongation to a tetrasaccharide (15, 19), although elongation past the disaccharide in Drosophila is not yet clear (20, 21). Elongation of O-fucose by Fringe is known to potentiate Notch signaling from Delta ligands and inhibit signaling from Serrate ligands (22). Delta ligands are termed Delta-like (Delta-like1, -2, and -4) in mammals, and the homologs of Serrate are known as Jagged (Jagged1 and -2) in mammals. The effects of Fringe on Drosophila Notch can be recapitulated in Notch ligand in vitro binding assays using purified components, suggesting that the elongation of O-fucose by Fringe alters the binding of Notch to its ligands (21). Although Fringe also appears to alter Notch-ligand interactions in mammals, the effects of elongation of the glycan past the O-fucose monosaccharide is more complicated and appears to be cell type-, receptor-, and ligand-dependent (for a recent review see Ref. 23).The Fringe enzymes catalyze the transfer of GlcNAc from the donor substrate UDP-α-GlcNAc to the acceptor fucose, forming the GlcNAc-β1,3-Fuc disaccharide (14-16). They belong to the GT-A-fold of inverting glycosyltransferases, which includes N-acetylglucosaminyltransferase I and β1,4-galactosyltransferase I (17, 18). The mechanism is presumed to proceed through the abstraction of a proton from the acceptor substrate by a catalytic base (Asp or Glu) in the active site. This creates a nucleophile that attacks the anomeric carbon of the nucleotide-sugar donor, inverting its configuration from α (on the nucleotide sugar) to β (in the product) (24, 25). The enzyme then releases the acceptor substrate modified with a disaccharide and UDP. The Mfng structure (26) leaves little doubt as to the identity of the catalytic residue, which in all likelihood is aspartate 289 in mouse Lfng (we will use numbering for mouse Lunatic Fringe throughout, unless otherwise stated). The structure of Mfng with UDP-GlcNAc soaked into the crystals (26) showed density only for the UDP portion of the nucleotide-sugar donor and no density for two loops flanking either side of the active site. The presence of flexible loops that become ordered upon substrate binding is a common observation with glycosyltransferases in the GT-A fold family (18, 25). Density for the entire donor was observed in the structure of rabbit N-acetylglucosaminyltransferase I (27). In this case, ordering of a previously disordered loop upon UDP-GlcNAc binding may have contributed to increased stability of the donor. In the case of bovine β1,4-galactosyltransferase I, a section of flexible random coil from the apo-structure was observed to change its conformation to α-helical upon donor substrate binding (28). Both loops in Lfng are highly conserved, and we have mutated a number of residues in each to test the hypothesis that they interact with the substrates. The mutagenesis strategy was also guided by docking of an EGF-O-fucose acceptor substrate into the active site of the Lfng model as well as comparison of the Lfng model with a homology model of the β1,3-glucosyltransferase (β3GlcT) that modifies O-fucose on thrombospondin type 1 repeats (29, 30). The β3GlcT is predicted to be a GT-A fold enzyme related to the Fringe family (17, 18, 29).     

Mechanism of Allosteric Inhibition of N-Acetyl-L-glutamate Synthase by L-Arginine

N-Acetylglutamate synthase (NAGS) catalyzes the first committed step in l-arginine biosynthesis in plants and micro-organisms and is subject to feedback inhibition by l-arginine. This study compares the crystal structures of NAGS from Neisseria gonorrhoeae (ngNAGS) in the inactive T-state with l-arginine bound and in the active R-state complexed with CoA and l-glutamate. Under all of the conditions examined, the enzyme consists of two stacked trimers. Each monomer has two domains: an amino acid kinase (AAK) domain with an AAK-like fold but lacking kinase activity and an N-acetyltransferase (NAT) domain homologous to other GCN5-related transferases. Binding of l-arginine to the AAK domain induces a global conformational change that increases the diameter of the hexamer by ∼10 Å and decreases its height by ∼20Å_. AAK dimers move 5Å outward along their 2-fold axes, and their tilt relative to the plane of the hexamer decreases by ∼4°. The NAT domains rotate ∼109° relative to AAK domains enabling new interdomain interactions. Interactions between AAK and NAT domains on different subunits also change. Local motions of several loops at the l-arginine-binding site enable the protein to close around the bound ligand, whereas several loops at the NAT active site become disordered, markedly reducing enzymatic specific activity.l-Arginine biosynthesis in most micro-organisms and plants involves the initial acetylation of l-glutamate by N-acetylglutamate synthase (NAGS, EC 2.3.1.1)² to produce N-acetylglutamate (NAG). NAG is then converted by NAG kinase (NAGK, EC 2.7.2.8) to NAG-phosphate and subsequently to N-acetylornithine (1, 2). Two alternative reactions are used to remove the acetyl group from acetylornithine. The linear pathway uses N-acetylornithine deacetylase (EC 3.5.1.16) to catalyze the metal-dependent hydrolysis of the acetyl group to form l-ornithine and acetate, whereas the acetyl recycling pathway transfers the acetyl group from N-acetylornithine to l-glutamate, producing l-ornithine and NAG. This reaction is catalyzed by ornithine acetyltransferase (EC 2.3.1.35).In the linear pathway, NAGS is the only target of feedback inhibition by l-arginine. In contrast, in the acetyl cycling pathway l-arginine may inhibit NAGS and NAGK or ornithine acetyltransferase (3). Structure determinations of l-arginine-insensitive (4) and l-arginine-sensitive NAGKs (5) provided insights into the structural basis of l-arginine inhibition of NAGK. l-Arginine-insensitive Escherichia coli (ec) NAGK is a homodimer (4), whereas l-arginine-sensitive NAGKs from Thermotoga maritima (tm) and Pseudomonas aeruginosa (pa) are hexamers formed by pair-wise interlacing of the N-terminal helices of three ecNAGK-like dimers, to create a second type of dimer interface. l-Arginine binding to a site close to the C terminus induces global conformational changes that expands the ring by ∼8 Å and decreases the tilt of the ecNAGK-like dimers relative to the plane of the ring by ∼6°. The inhibition mechanism was proposed to involve the enlargement of an active site located close to the l-arginine-binding site.Because of the sequence similarity between NAGK and NAGS, it was speculated that they may have similar l-arginine-binding sites and hexameric ring structures (5). However, our recent structural determination of NAGS from Neisseria gonorrhoeae (ng) revealed the active site to be located in the NAT domain, >25 Å away from the proposed l-arginine-binding site (6). Therefore, the allosteric mechanism of NAGS is likely to be different from that of l-arginine-sensitive NAGKs. Here we compare the structures of ngNAGS in the inactive T-state with l-arginine bound and in the R-state complexed with CoA and l-glutamate and determine the structural basis for the allosteric inhibition of NAGS by l-arginine.     

Full Domain Closure of the Ligand-binding Core of the Ionotropic Glutamate Receptor iGluR5 Induced by the High Affinity Agonist Dysiherbaine and the Functional Antagonist 8,9-Dideoxyneodysiherbaine

The prevailing structural model for ligand activation of ionotropic glutamate receptors posits that agonist efficacy arises from the stability and magnitude of induced domain closure in the ligand-binding core structure. Here we describe an exception to the correlation between ligand efficacy and domain closure. A weakly efficacious partial agonist of very low potency for homomeric iGluR5 kainate receptors, 8,9-dideoxyneodysiherbaine (MSVIII-19), induced a fully closed iGluR5 ligand-binding core. The degree of relative domain closure, ∼30°, was similar to that we resolved with the structurally related high affinity agonist dysiherbaine and to that of l-glutamate. The pharmacological activity of MSVIII-19 was confirmed in patch clamp recordings from transfected HEK293 cells, where MSVIII-19 predominantly inhibits iGluR5-2a, with little activation apparent at a high concentration (1 mm) of MSVIII-19 (<1% of mean glutamate-evoked currents). To determine the efficacy of the ligand quantitatively, we constructed concentration-response relationships for MSVIII-19 following potentiation of steady-state currents with concanavalin A (EC₅₀ = 3.6 μm) and on the nondesensitizing receptor mutant iGluR5-2b(Y506C/L768C) (EC₅₀ = 8.1 μm). MSVIII-19 exhibited a maximum of 16% of full agonist efficacy, as measured in parallel recordings with glutamate. Molecular dynamics simulations and electrophysiological recordings confirm that the specificity of MSVIII-19 for iGluR5 is partly attributable to interdomain hydrogen bond residues Glu⁴⁴¹ and Ser⁷²¹ in the iGluR5-S1S2 structure. The weaker interactions of MSVIII-19 with iGluR5 compared with dysiherbaine, together with altered stability of the interdomain interaction, may be responsible for the apparent uncoupling of domain closure and channel opening in this kainate receptor subunit.Ionotropic glutamate receptors (iGluRs)³ are central to fast excitatory synaptic transmission in the central nervous system and are involved in numerous physiological and pathophysiological processes. The iGluRs consist of three different classes of receptors, N-methyl-d-aspartic acid (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and kainate receptors (1), which are assembled as tetramers in a dimer of dimers configuration (2, 3). These receptors can be considered as multidomain proteins, composed of an extracellular N-terminal domain, a ligand-binding core made of discontinuous S1 and S2 segments that form two lobes (domains D1 and D2), three transmembrane-spanning regions (M1–M3) with a re-entrant loop between M1 and M2, and finally a cytoplasmic region (1).Ligand-binding cores of iGluRs assume tertiary structures in solution that reproduce the pharmacological profiles of full-length receptors. Crystallographic studies of ligand-binding core complexes from representative members of all three iGluR subtypes (4–6) as well as the ligand-binding core of the structurally related δ2 subunit in complex with d-serine (7) have yielded unprecedented insight into structural correlates of iGluR function. Binding of agonists to iGluR ligand-binding cores can be described as a “Venus flytrap” mechanism. In the resting state, the ligand-binding core is present in an open form that is stabilized by antagonists (4, 8, 9). When an agonist binds to the ligand-binding core, a rotational change in conformation occurs, resulting in domain closure of the D1 and D2 lobes around a central hinge region (4, 6). In full-length receptors, this domain closure is thought to result in the opening of the ion channel (receptor activation). The extents of domain closure of ligand-binding cores of AMPA and kainate receptor subunits are correlated with the activation and the desensitization of the receptor (9, 10).However, previous studies have questioned the association between the degree of domain closure of the ligand-binding core and channel opening or agonist efficacy. For example, AMPA was shown to induce a more closed structure of the ligand-binding core of the mutated iGluR2(L650T) than was expected from its partial agonist efficacy (11, 12). Also, no correlation between domain closure and agonist efficacy has been demonstrated for the NR1 subunit of NMDA receptors (13).In this study, we present the first example of a nonmutated kainate receptor that lacks the correlation between domain closure and efficacy. We tested if two structurally related kainate receptor ligands, one an agonist and one described previously as an antagonist (14), conformed to the prevailing structural model of ligand-induced activity. The high affinity agonist dysiherbaine (DH) is a natural excitotoxin originally isolated from a marine sponge (15, 16), whereas 8,9-dideoxyneodysiherbaine (MSVIII-19) is a synthetic analog that inhibits activation of iGluR5 receptors (14). To investigate receptor interactions with the two closely related compounds as well as the degree of domain closure introduced by the compounds, we determined the crystal structures of DH and MSVIII-19 in complex with the ligand-binding core of the kainate receptor subunit iGluR5 (iGluR5-S1S2). These two structures, along with functional studies, provide novel insights into the mechanism of kainate receptor activation, inhibition, and desensitization.     

Sgt1 Dimerization Is Required for Yeast Kinetochore Assembly

The kinetochore, which consists of DNA sequence elements and structural proteins, is essential for high-fidelity chromosome transmission during cell division. In budding yeast, Sgt1 and Hsp90 help assemble the core kinetochore complex CBF3 by activating the CBF3 components Skp1 and Ctf13. In this study, we show that Sgt1 forms homodimers by performing in vitro and in vivo immunoprecipitation and analytical ultracentrifugation analyses. Analyses of the dimerization of Sgt1 deletion proteins showed that the Skp1-binding domain (amino acids 1–211) contains the Sgt1 homodimerization domain. Also, the Sgt1 mutant proteins that were unable to dimerize also did not bind Skp1, suggesting that Sgt1 dimerization is important for Sgt1-Skp1 binding. Restoring dimerization activity of a dimerization-deficient sgt1 mutant (sgt1-L31P) by using the CENP-B (centromere protein-B) dimerization domain suppressed the temperature sensitivity, the benomyl sensitivity, and the chromosome missegregation phenotype of sgt1-L31P. These results strongly suggest that Sgt1 dimerization is required for kinetochore assembly.Spindle microtubules are coupled to the centromeric region of the chromosome by a structural protein complex called the kinetochore (1, 2). The kinetochore is thought to generate a signal that arrests cells during mitosis when it is not properly attached to microtubules, thereby preventing aberrant chromosome transmission to the daughter cells, which can lead to tumorigenesis (3, 4). The kinetochore of the budding yeast Saccharomyces cerevisiae has been characterized thoroughly, genetically and biochemically; thus, its molecular structure is the most well detailed to date. More than 70 different proteins comprise the budding yeast kinetochore, and several of those are conserved in mammals (2).The budding yeast centromere DNA is a 125-bp region that contains three conserved regions, CDEI, CDEII, and CDEIII (5, 6). CDEI is bound by Cbf1 (7–9). CDEIII (25 bp) is essential for centromere function (10) and is the site where CBF3 binds to centromeric DNA. CBF3 contains four proteins: Ndc10, Cep3, Ctf13 (11–18), and Skp1 (17, 18), all of which are essential for viability. Mutations in any of the four CBF3 proteins abolish the ability of CDEIII to bind to CBF3 (19, 20). All of the described kinetochore proteins, except the CDEI-binding Cbf1, localize to kinetochores dependent on the CBF3 complex (2). Therefore, the CBF3 complex is the fundamental structure of the kinetochore, and the mechanism of CBF3 assembly is of major interest.We previously isolated SGT1, the skp1-4 kinetochore-defective mutant dosage suppressor (21). Sgt1 and Skp1 activate Ctf13; thus, they are required for assembly of the CBF3 complex (21). The molecular chaperone Hsp90 is also required for the formation of the Skp1-Ctf13 complex (22). Sgt1 has two highly conserved motifs that are required for protein-protein interaction, the tetratricopeptide repeat (TPR)² (21) and the CS (CHORD protein- and Sgt1-specific) motif. We and others (23–26) have found that both domains are important for the interaction with Hsp90. The Sgt1-Hsp90 interaction is required for the assembly of the core kinetochore complex; this interaction is an initial step in kinetochore assembly (24, 26, 27) that is conserved between yeast and humans (28, 29).In this study, we further characterized the molecular mechanism of this assembly process. We found that Sgt1 forms dimers in vivo, and our results strongly suggest that Sgt1 dimerization is required for kinetochore assembly in budding yeast.     

Characterization of WbpB, WbpE, and WbpD and Reconstitution of a Pathway for the Biosynthesis of UDP-2,3-diacetamido-2,3-dideoxy-d-mannuronic Acid in Pseudomonas aeruginosa

The lipopolysaccharide of Pseudomonas aeruginosa PAO1 contains an unusual sugar, 2,3-diacetamido-2,3-dideoxy-d-mannuronic acid (d-ManNAc3NAcA). wbpB, wbpE, and wbpD are thought to encode oxidase, transaminase, and N-acetyltransferase enzymes. To characterize their functions, recombinant proteins were overexpressed and purified from heterologous hosts. Activities of His₆-WbpB and His₆-WbpE were detected only when both proteins were combined in the same reaction. Using a direct MALDI-TOF mass spectrometry approach, we identified ions that corresponded to the predicted products of WbpB (UDP-3-keto-d-GlcNAcA) and WbpE (UDP-d-GlcNAc3NA) in the coupled enzyme-substrate reaction. Additionally, in reactions involving WbpB, WbpE, and WbpD, an ion consistent with the expected product of WbpD (UDP-d-GlcNAc3NAcA) was identified. Preparative quantities of UDP-d-GlcNAc3NA and UDP-d-GlcNAc3NAcA were enzymatically synthesized. These compounds were purified by high-performance liquid chromatography, and their structures were elucidated by NMR spectroscopy. This is the first report of the functional characterization of these proteins, and the enzymatic synthesis of UDP-d-GlcNAc3NA and UDP-d-GlcNAc3NAcA.Gram-negative organisms such as Pseudomonas aeruginosa produce lipopolysaccharide (LPS)⁴ as an essential component of the outer leaflet of the outer membrane. LPS can be conceptually divided into three parts: lipid A, which anchors LPS into the membrane; core oligosaccharide, which contributes to membrane stability; and the O-antigen, which is a polysaccharide that extends away from the cell surface. In P. aeruginosa, two types of O-antigen are observed: A-band O-antigen, which is common to most strains, and B-band O-antigen, which is variable and therefore used as the basis of the International Antigenic Typing Scheme (1). P. aeruginosa serotypes O2, O5, O16, O18, and O20 collectively belong to serogroup O2, because they all share common backbone sugar structures in their O-antigen repeat units consisting of two di-N-acetylated uronic acids and one 2-acetamido-2,6-dideoxy-d-galactose (N-acetyl-d-fucosamine). The minor structural variations in the O-antigen repeat units that differentiate this serogroup into five serotypes are: the type of glycosidic linkage between O-units (alpha versus beta) that is formed by the O-antigen polymerase (Wzy), isomers present (d-mannuronic or l-guluronic acid), and acetyl group substituents (2–4). The B-band O-antigen of P. aeruginosa PAO1 (serotype O5) contains a repeating trisaccharide of 2-acetamido-3-acetamidino-2,3-dideoxy-d-mannuronic acid (d-ManNAc3NAmA), 2,3-diacetamido-2,3-dideoxy-d-mannuronic acid (d-ManNAc3NAcA), and 2-acetamido-2,6-dideoxy-d-galactose (3).The biosynthesis of the two mannuronic acid derivatives has yet to be fully understood and has been the subject of investigation by our group. To produce UDP-d-ManNAc3NAcA, a five-step pathway has been proposed (Fig. 1) that requires the products of five genes localized to the B-band O-antigen biosynthesis cluster (5). The O-antigen biosynthesis cluster was shown to be identical for all serotypes within serogroup O2, which further underscores the high similarity between these serotypes (5). The five genes, including wbpA, wbpB, wbpE, wbpD, and wbpI, have been shown to be essential for B-band LPS biosynthesis, because knockout mutants of each of these genes are deficient in B-band O-antigen (6–8). Homologs of all five of the proteins required for the UDP-d-ManNAc3NAcA biosynthesis pathway are conserved in other bacterial pathogens, including Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica. Cross-complementation of P. aeruginosa knockout mutants lacking wbpA, wbpB, wbpE, wbpD, or wbpI with the homologues from B. pertussis could fully restore LPS production in the P. aeruginosa LPS mutants, suggesting that the genes from B. pertussis are functional homologs of the wbp genes (7). Homologs of these genes could be identified in diverse bacterial species, demonstrating the importance of UDP-d-ManNAc3NAcA biosynthesis beyond its role in P. aeruginosa (7).Open in a separate windowFIGURE 1.Proposed pathway for the biosynthesis of UDP-d-ManNAc3NAcA in P. aeruginosa PAO1. The full names of the sugars are as follows: GlcNAc, 2-acetamido-2-deoxy-d-glucose; GlcNAcA, 2-acetamido-2-deoxy-d-glucuronic acid; 3-keto-d-GlcNAcA, 2-acetamido-2-deoxy-d-ribo-hex-3-uluronic acid; GlcNAc3NA, 2-acetamido-3-amino-2,3-dideoxy-d-glucuronic acid; GlcNAc3NAcA, 2,3-diacetamido-2,3-dideoxy-d-glucuronic acid; ManNAc3NAcA, 2,3-diacetamido-2,3-dideoxy-d-mannuronic acid. Adapted from Ref. 8.The first enzyme of the UDP-d-ManNAc3NAcA biosynthesis pathway, WbpA, is a 6-dehydrogenase that converts UDP-2-acetamido-2-deoxy-d-glucose (N-acetyl-d-glucosamine; UDP-d-GlcNAc) to UDP-2-acetamido-2-deoxy-d-glucuronic acid (N-acetyl-d-glucosaminuronic acid, UDP-d-GlcNAcA) using NAD⁺ as a coenzyme (9) (Fig. 1). Following this, the second step in UDP-d-ManNAc3NAcA biosynthesis is proposed to be an oxidation reaction catalyzed by WbpB, forming UDP-2-acetamido-2-deoxy-d-ribo-hex-3-uluronic acid (3-keto-d-GlcNAcA), which in turn is used as the substrate for transamination by WbpE, creating UDP-2-acetamido-3-amino-2,3-dideoxy-d-glucuronic acid (d-GlcNAc3NA).This residue is thought to be the substrate for WbpD, a putative N-acetyltransferase of the hexapeptide acyltransferase superfamily (10) that requires acetyl-CoA as a co-substrate (8). WbpD has been proposed to synthesize UDP-2,3-diacetamido-2,3-dideoxy-d-glucuronic acid (UDP-d-GlcNAc-3NAcA), which is utilized in the B-band O-antigen of P. aeruginosa serotype O1. In P. aeruginosa serogroup O2, the UDP-d-GlcNAc3NAcA is then epimerized by WbpI to create the UDP-d-ManNAc3NAcA required for incorporation into B-band LPS (11). A derivative of UDP-d-ManNAc3NAcA is also used in the synthesis of B-band O-antigen of P. aeruginosa serogroup O2. UDP-d-ManNAc3NAmA is thought to be produced through additional modification of UDP-d-ManNAc3NAcA via the action of WbpG, an amidotransferase, which has also been demonstrated to be essential for the production of B-band O-antigen (12, 13).In the current study, our aim was to define the function of WbpB, WbpE, and WbpD, because only genetic evidence has previously been given for the involvement of wbpB and wbpE (7), and the reaction catalyzed by WbpD could not be demonstrated due to the unavailability of its presumed substrate, UDP-d-GlcNAc3NA (8). The functional characterization of these proteins is also important for understanding LPS biosynthesis in B. pertussis, because the genes in the LPS locus of this species, wlbA, wlbC, and wlbB, could cross-complement knockouts of wbpB, wbpE, and wbpD, respectively, when expressed in P. aeruginosa PAO1 (7). Furthermore, these three proteins form a cassette for the generation of C-3 N-acetylated hexoses and may be important for the biosynthesis of a variety of other sugars. Capillary electrophoresis and MALDI-TOF mass spectrometry were used to analyze reaction mixtures of WbpB and WbpE and showed that the expected products were produced only when both enzymes were present together. Achieving the enzymatic synthesis of the product of both enzymes, which was demonstrated to be UDP-d-GlcNAc3NA by ¹H NMR spectroscopy, was a key breakthrough, because this rare sugar has never before been produced by any means. UDP-d-GlcNAc3NA was also essential for use as the substrate of WbpD, which not only allowed us to determine the enzymatic activity of this protein but also allowed the enzymatic synthesis of UDP-d-GlcNAc3NAcA to be achieved as well. Although this sugar had previously been produced through a 17-step chemical synthesis (11, 14), the 4-step concurrent enzymatic reaction demonstrates the advantage of linking chemistry with biology and represents a significant saving of both time and reagents as compared with chemical synthesis. Finally, our data also showed the success in reconstituting in vitro the 5-step pathway for the biosynthesis of UDP-d-ManNAc3NAcA in P. aeruginosa.     

The RIG-I-like Receptor LGP2 Recognizes the Termini of Double-stranded RNA

The RIG-I-like receptors (RLRs), RIG-I and MDA5, recognize single-stranded RNA with 5′ triphosphates and double-stranded RNA (dsRNA) to initiate innate antiviral immune responses. LGP2, a homolog of RIG-I and MDA5 that lacks signaling capability, regulates the signaling of the RLRs. To establish the structural basis of dsRNA recognition by the RLRs, we have determined the 2.0-Å resolution crystal structure of human LGP2 C-terminal domain bound to an 8-bp dsRNA. Two LGP2 C-terminal domain molecules bind to the termini of dsRNA with minimal contacts between the protein molecules. Gel filtration chromatography and analytical ultracentrifugation demonstrated that LGP2 binds blunt-ended dsRNA of different lengths, forming complexes with 2:1 stoichiometry. dsRNA with protruding termini bind LGP2 and RIG-I weakly and do not stimulate the activation of RIG-I efficiently in cells. Surprisingly, full-length LGP2 containing mutations that abolish dsRNA binding retained the ability to inhibit RIG-I signaling.The innate immune response is the first line of defense against invading pathogens; it is the ubiquitous system of defense against microbial infections (1). Toll-like receptors (TLRs)³ and RIG-I (retinoic acid-inducible gene 1)-like receptors (RLRs) play key roles in innate immune response toward viral infection (2-5). Toll-like receptors TLR3, TLR7, and TLR8 sense viral RNA released in the endosome following phagocytosis of the pathogens (6). RIG-I-like receptors RIG-I and MDA5 detect viral RNA from replicating viruses in infected cells (3, 7, 8). Stimulation of these receptors leads to the induction of type I interferons (IFNs) and other proinflammatory cytokines, conferring antiviral activity to the host cells and activating the acquired immune responses (4, 9).RIG-I discriminates between viral and host RNA through specific recognition of the uncapped 5′-triphosphate of single-stranded RNA (5′ ppp ssRNA) generated by viral RNA polymerases (10, 11). In addition, RIG-I also recognizes double-stranded RNA generated during RNA virus replication (7, 12). Transfection of cells with synthetic double-stranded RNA stimulates the activation of RIG-I (13, 14). Synthetic dsRNA mimics, such as polyinosinic-polycytidylic acid (poly(I·C)), can activate MDA5 when introduced into the cytoplasm of cells. Digestion of poly(I·C) with RNase III transforms poly(I·C) from a ligand for MDA5 into a ligand for RIG-I, suggesting that MDA5 recognizes long dsRNA, whereas RIG-I recognizes short dsRNA (15). Studies of RIG-I and MDA5 knock-out mice confirmed the essential roles of these receptors in antiviral immune responses and demonstrated that they sense different sets of RNA viruses (12, 16).RIG-I and MDA5 contain two caspase recruiting domains (CARDs) at their N termini, a DEX(D/H) box RNA helicase domain, and a C-terminal regulatory or repressor domain (CTD). The helicase domain and the CTD are responsible for viral RNA binding, whereas the CARDs are required for signaling (3, 8). The current model of RIG-I activation suggests that under resting conditions RIG-I is in a suppressed conformation, and viral RNA binding triggers a conformation change that leads to the exposure of the CARDs for the recruitment of the downstream protein IPS-1 (also known as MAVS, Cardif, or VISA) (14, 17). Limited proteolysis of the RIG-I·dsRNA complex showed that RIG-I residues 792-925 of the CTD are involved in dsRNA and 5′ ppp ssRNA binding (14). The CTD of RIG-I overlaps with the C terminus of the previously identified repressor domain (18). The structures of RIG-I and LGP2 (laboratory of genetics and physiology 2) CTD in isolation have been determined by x-ray crystallography and NMR spectroscopy (14, 19, 20). A large, positively charged surface on RIG-I recognizes the 5′ triphosphate group of viral ssRNA (14, 19). RNA binding studies by titrating RIG-I CTD with dsRNA and 5′ ppp ssRNA suggested that overlapping sets of residues on this charged surface are involved in RNA binding (14). Mutagenesis of several positively charged residues on this surface either reduces or disrupts RNA binding by RIG-I, and these mutations also affect the induction of IFN-β in vivo (14, 19). However, the exact nature of how the RLRs recognize viral RNA and how RNA binding activates these receptors remains to be established.LGP2 is a homolog of RIG-I and MDA5 that lacks the CARDs and thus has no signaling capability (21, 22). The expression of LGP2 is inducible by dsRNA or IFN treatment as well as virus infection (21). Overexpression of LGP2 inhibits Sendai virus and Newcastle disease virus signaling (21). When coexpressed with RIG-I, LGP2 can inhibit RIG-I signaling through the interaction of its CTD with the CARD and the helicase domain of RIG-I (18). LGP2 could suppress RIG-I signaling by three possible ways (23): 1) binding RNA with high affinity, thereby sequestering RNA ligands from RIG-I; 2) interacting directly with RIG-I to block the assembly of the signaling complex; and 3) competing with IKKi (IκB kinase ε) in the NF-κB signaling pathway for a common binding site on IPS-1. To elucidate the structural basis of dsRNA recognition by the RLRs, we have crystallized human LGP2 CTD (residues 541-678) bound to an 8-bp double-stranded RNA and determined the structure of the complex at 2.0 Å resolution. The structure revealed that LGP2 CTD binds to the termini of dsRNA. Mutagenesis and functional studies showed that dsRNA binding is likely not required for the inhibition of RIG-I signaling by LGP2.     

The Substrate Recognition Domains of the N-end Rule Pathway

The N-end rule pathway is a ubiquitin-dependent system where E3 ligases called N-recognins, including UBR1 and UBR2, recognize type-1 (basic) and type-2 (bulky hydrophobic) N-terminal residues as part of N-degrons. We have recently reported an E3 family (termed UBR1 through UBR7) characterized by the 70-residue UBR box, among which UBR1, UBR2, UBR4, and UBR5 were captured during affinity-based proteomics with synthetic degrons. Here we characterized substrate binding specificity and recognition domains of UBR proteins. Pull-down assays with recombinant UBR proteins suggest that 570-kDa UBR4 and 300-kDa UBR5 bind N-degron, whereas UBR3, UBR6, and UBR7 do not. Binding assays with 24 UBR1 deletion mutants and 31 site-directed UBR1 mutations narrow down the degron-binding activity to a 72-residue UBR box-only fragment that recognizes type-1 but not type-2 residues. A surface plasmon resonance assay shows that the UBR box binds to the type-1 substrate Arg-peptide with K_d of ∼3.4 μm. Downstream from the UBR box, we identify a second substrate recognition domain, termed the N-domain, required for type-2 substrate recognition. The ∼80-residue N-domain shows structural and functional similarity to 106-residue Escherichia coli ClpS, a bacterial N-recognin. We propose a model where the 70-residue UBR box functions as a common structural element essential for binding to all known destabilizing N-terminal residues, whereas specific residues localized in the UBR box (for type 1) or the N-domain (for type 2) provide substrate selectivity through interaction with the side group of an N-terminal amino acid. Our work provides new insights into substrate recognition in the N-end rule pathway.The N-end rule pathway is a ubiquitin (Ub)²-dependent proteolytic system in which N-terminal residues of short-lived proteins function as an essential component of degradation signals (degrons) called N-degrons (Fig. 1A) (1-15). An N-degron can be created from a pre-N-degron through specific N-terminal modifications (12). Specifically, in mammals, N-terminal Asn and Gln are tertiary destabilizing residues that function through their deamidation by N-terminal amidohydrolases into the secondary destabilizing N-terminal residues Asp and Glu, respectively (6, 16) (Fig. 1A). N-terminal Asp and Glu are secondary destabilizing residues that function through their arginylation by ATE1 R-transferase, which creates the primary destabilizing residue Arg at the N terminus (4, 8) (Fig. 1A). N-terminal Cys can also function as a tertiary destabilizing residue through its oxidation in a manner depending on nitric oxide and oxygen (O₂); the oxidized Cys residue is subsequently arginylated by ATE1 (8, 13, 17).Open in a separate windowFIGURE 1.A, the mammalian N-end rule pathway. N-terminal residues are indicated by single-letter abbreviations for amino acids. Yellow ovals denote the rest of a protein substrate. C^* denotes oxidized N-terminal Cys, either Cys-sulfinic acid [CysO₂(H)] or Cys-sulfonic acid [CysO₃(H)]. The Cys oxidation requires nitric oxide and oxygen (O₂) or its derivatives. The oxidized Cys is arginylated by ATE1 Arg-tRNA-protein transferase (R-transferase). N-recognins also recognize internal (non-N-terminal) degrons in other substrates of the N-end rule pathway. B, the X-peptide pull-down assay. Left, a 12-mer peptide bearing N-terminal Arg (type 1), Phe (type 2), Trp (type 2), or Gly (stabilizing control) residue was cross-linked through its C-terminal Cys residue to Ultralink Iodoacetyl beads. Right, the otherwise identical 12-mer peptide, bearing C-terminal biotinylated Lys instead of Cys, was conjugated, via biotin, to the streptavidin-Sepharose beads. C, the X-peptide pull-down assay of endogenous UBR proteins using testes extracts. Extracts from mouse testes were mixed with bead-conjugated X-peptides bearing N-terminal Phe (F), Gly (G), or Arg (R). After centrifugation, captured proteins were separated and subjected to anti-UBR immunoblotting. Mo, a pull-down reaction with mock beads. D, the X-peptide pull-down assays using rat testis extracts were performed in the presence of varying concentrations of NaCl. After incubation and washing, bound proteins were eluted by 10 mm Tyr-Ala for Phe-peptide, 10 mm Arg-Ala for Arg-peptide, and 5 mm Tyr-Ala and 5 mm Arg-Ala for Val-peptide. Eluted proteins were subjected to immunoblotting for UBR1 and UBR5. E, cytoplasmic fractions of wild-type (+/+), Ubr1^-/-, Ubr2^-/-, Ubr1^-/-Ubr2^-/-, and Ubr1^-/-Ubr2^-/-Ubr4^RNAi MEFs were subjected to X-peptide pull-down assay. Precipitated proteins were separated and analyzed by immunoblotting for UBR1 and UBR4.N-terminal Arg together with other primary destabilizing N-terminal residues are directly bound by specific E3 Ub ligases called N-recognins (3, 7, 9). Destabilizing N-terminal residues can be created through the removal of N-terminal Met or the endoproteolytic cleavage of a protein, which exposes a new amino acid at the N terminus (12, 13). N-terminal degradation signals can be divided into type-1 (basic; Arg, Lys, and His) and type-2 (bulky hydrophobic; Phe, Leu, Trp, Tyr, and Ile) destabilizing residues (2, 12). In addition to a destabilizing N-terminal residue, a functional N-degron requires at least one internal Lys residue (the site of a poly-Ub chain formation) and a conformational feature required for optimal ubiquitylation (1, 2, 18). UBR1 and UBR2 are functionally overlapping N-recognins (3, 7, 9). Our proteomic approach using synthetic peptides bearing destabilizing N-terminal residues captured a set of proteins (200-kDa UBR1, 200-kDa UBR2, 570-kDa UBR4, and 300-kDa UBR5/EDD) characterized by a 70-residue zinc finger-like domain termed the UBR box (10-12). UBR5 is a HECT E3 ligase known as EDD (E3 identified by differential display) (19) and a homolog of Drosophila hyperplastic discs (20). The mammalian genome encodes at least seven UBR box-containing proteins, termed UBR1 through UBR7 (10). UBR box proteins are generally heterogeneous in size and sequence but contain, with the exception of UBR4, specific signatures unique to E3s or a substrate recognition subunit of the E3 complex: the RING domain in UBR1, UBR2, and UBR3; the HECT domain in UBR5; the F-box in UBR6 and the plant homeodomain domain in UBR7 (Fig. 2B). The biochemical properties of more recently identified UBR box proteins, such as UBR3 through UBR7, are largely unknown.Open in a separate windowFIGURE 2.The binding properties of the UBR box family members to type-1 and type-2 destabilizing N-terminal residues. A, the X-peptide pull-down assay with overexpressed, full-length UBR proteins: UBR2, UBR3 (in S. cerevisiae cells), UBR4, UBR5 (in COS7 cells), and UBR6 and UBR7 (in the wheat germ lysates). Precipitates were analyzed by immunoblotting (for UBR2, UBR3, UBR4, and UBR5) with tag-specific antibodies as indicated in B or autoradiography (for UBR6 and UBR7). B, the structures of UBR box proteins. Shown are locations of the UBR box, the N-domain, and other E3-related domains. UBR, UBR box; RING, RING finger; UAIN, UBR-specific autoinhibitory domain; CRD, cysteine-rich domain; PHD, plant homeodomain; HECT, HECT domain.Studies using knock-out mice implicated the N-end rule pathway in cardiac development and signaling, angiogenesis (8, 15), meiosis (9), DNA repair (21), neurogenesis (15), pancreatic functions (22), learning and memory (23, 24), female development (9), muscle atrophy (25), and olfaction (11). Mutations in human UBR1 is a cause of Johanson-Blizzard syndrome (22), an autosomal recessive disorder with multiple developmental abnormalities (26). Other functions of the pathway include: (i) a nitric oxide and oxygen (O₂) sensor controlling the proteolysis of RGS4, RGS5, and RGS16 (8, 13, 17), (ii) a heme sensor through hemin-dependent inhibition of ATE1 function (27), (iii) the regulation of short peptide import through the peptide-modulated degradation of the repressor of the import (28, 29), (iv) the control of chromosome segregation through the degradation of a separate produced cohesin fragment (30), (v) the regulation of apoptosis through the degradation of a caspase-processed inhibitor of apoptosis (31, 32), (vi) the control of the human immunodeficiency virus replication cycle through the degradation of human immunodeficiency virus integrase (10, 33), and (vii) the regulation of leaf senescence in plants (34).In the present study we characterized substrate binding specificities and recognition domains of UBR proteins. In our binding assays, UBR1, UBR2, UBR4, and UBR5 were captured by N-terminal degradation determinants, whereas UBR3, UBR6, and UBR7 were not. We also report that in contrast to other E3 systems that usually recognize substrates through protein-protein interface, UBR1 and UBR2 have a general substrate recognition domain termed the UBR box. Remarkably, a 72-residue UBR box-only fragment fully retains its structural integrity and thereby the ability to recognize type-1 N-end rule substrates. We also report that the N-domain, structurally and functionally related with bacterial N-recognins, is required for recognizing type-2 N-end rule substrates. We discuss the evolutionary relationship between eukaryotic and prokaryotic N-recognins.     

Secretory Carrier Membrane Protein 2 Regulates Cell-surface Targeting of Brain-enriched Na+/H+ Exchanger NHE5

NHE5 is a brain-enriched Na⁺/H⁺ exchanger that dynamically shuttles between the plasma membrane and recycling endosomes, serving as a mechanism that acutely controls the local pH environment. In the current study we show that secretory carrier membrane proteins (SCAMPs), a group of tetraspanning integral membrane proteins that reside in multiple secretory and endocytic organelles, bind to NHE5 and co-localize predominantly in the recycling endosomes. In vitro protein-protein interaction assays revealed that NHE5 directly binds to the N- and C-terminal cytosolic extensions of SCAMP2. Heterologous expression of SCAMP2 but not SCAMP5 increased cell-surface abundance as well as transporter activity of NHE5 across the plasma membrane. Expression of a deletion mutant lacking the SCAMP2-specific N-terminal cytosolic domain, and a mini-gene encoding the N-terminal extension, reduced the transporter activity. Although both Arf6 and Rab11 positively regulate NHE5 cell-surface targeting and NHE5 activity across the plasma membrane, SCAMP2-mediated surface targeting of NHE5 was reversed by dominant-negative Arf6 but not by dominant-negative Rab11. Together, these results suggest that SCAMP2 regulates NHE5 transit through recycling endosomes and promotes its surface targeting in an Arf6-dependent manner.Neurons and glial cells in the central and peripheral nervous systems are especially sensitive to perturbations of pH (1). Many voltage- and ligand-gated ion channels that control membrane excitability are sensitive to changes in cellular pH (1-3). Neurotransmitter release and uptake are also influenced by cellular and organellar pH (4, 5). Moreover, the intra- and extracellular pH of both neurons and glia are modulated in a highly transient and localized manner by neuronal activity (6, 7). Thus, neurons and glia require sophisticated mechanisms to finely tune ion and pH homeostasis to maintain their normal functions.Na⁺/H⁺ exchangers (NHEs)³ were originally identified as a class of plasma membrane-bound ion transporters that exchange extracellular Na⁺ for intracellular H⁺, and thereby regulate cellular pH and volume. Since the discovery of NHE1 as the first mammalian NHE (8), eight additional isoforms (NHE2-9) that share 25-70% amino acid identity have been isolated in mammals (9, 10). NHE1-5 commonly exhibit transporter activity across the plasma membrane, whereas NHE6-9 are mostly found in organelle membranes and are believed to regulate organellar pH in most cell types at steady state (11). More recently, NHE10 was identified in human and mouse osteoclasts (12, 13). However, the cDNA encoding NHE10 shares only a low degree of sequence similarity with other known members of the NHE gene family, raising the possibility that this sodium-proton exchanger may belong to a separate gene family distantly related to NHE1-9 (see Ref. 9).NHE gene family members contain 12 putative transmembrane domains at the N terminus followed by a C-terminal cytosolic extension that plays a role in regulation of the transporter activity by protein-protein interactions and phosphorylation. NHEs have been shown to regulate the pH environment of synaptic nerve terminals and to regulate the release of neurotransmitters from multiple neuronal populations (14-16). The importance of NHEs in brain function is further exemplified by the findings that spontaneous or directed mutations of the ubiquitously expressed NHE1 gene lead to the progression of epileptic seizures, ataxia, and increased mortality in mice (17, 18). The progression of the disease phenotype is associated with loss of specific neuron populations and increased neuronal excitability. However, NHE1-null mice appear to develop normally until 2 weeks after birth when symptoms begin to appear. Therefore, other mechanisms may compensate for the loss of NHE1 during early development and play a protective role in the surviving neurons after the onset of the disease phenotype.NHE5 was identified as a unique member of the NHE gene family whose mRNA is expressed almost exclusively in the brain (19, 20), although more recent studies have suggested that NHE5 might be functional in other cell types such as sperm (21, 22) and osteosarcoma cells (23). Curiously, mutations found in several forms of congenital neurological disorders such as spinocerebellar ataxia type 4 (24-26) and autosomal dominant cerebellar ataxia (27-29) have been mapped to chromosome 16q22.1, a region containing NHE5. However, much remains unknown as to the molecular regulation of NHE5 and its role in brain function.Very few if any proteins work in isolation. Therefore identification and characterization of binding proteins often reveal novel functions and regulation mechanisms of the protein of interest. To begin to elucidate the biological role of NHE5, we have started to explore NHE5-binding proteins. Previously, β-arrestins, multifunctional scaffold proteins that play a key role in desensitization of G-protein-coupled receptors, were shown to directly bind to NHE5 and promote its endocytosis (30). This study demonstrated that NHE5 trafficking between endosomes and the plasma membrane is regulated by protein-protein interactions with scaffold proteins. More recently, we demonstrated that receptor for activated C-kinase 1 (RACK1), a scaffold protein that links signaling molecules such as activated protein kinase C, integrins, and Src kinase (31), directly interacts with and activates NHE5 via integrin-dependent and independent pathways (32). These results further indicate that NHE5 is partly associated with focal adhesions and that its targeting to the specialized microdomain of the plasma membrane may be regulated by various signaling pathways.Secretory carrier membrane proteins (SCAMPs) are a family of evolutionarily conserved tetra-spanning integral membrane proteins. SCAMPs are found in multiple organelles such as the Golgi apparatus, trans-Golgi network, recycling endosomes, synaptic vesicles, and the plasma membrane (33, 34) and have been shown to play a role in exocytosis (35-38) and endocytosis (39). Currently, five isoforms of SCAMP have been identified in mammals. The extended N terminus of SCAMP1-3 contain multiple Asn-Pro-Phe (NPF) repeats, which may allow these isoforms to participate in clathrin coat assembly and vesicle budding by binding to Eps15 homology (EH)-domain proteins (40, 41). Further, SCAMP2 was shown recently to bind to the small GTPase Arf6 (38), which is believed to participate in traffic between the recycling endosomes and the cell surface (42, 43). More recent studies have suggested that SCAMPs bind to organellar membrane type NHE7 (44) and the serotonin transporter SERT (45) and facilitate targeting of these integral membrane proteins to specific intracellular compartments. We show in the current study that SCAMP2 binds to NHE5, facilitates the cell-surface targeting of NHE5, and elevates Na⁺/H⁺ exchange activity at the plasma membrane, whereas expression of a SCAMP2 deletion mutant lacking the N-terminal domain containing the NPF repeats suppresses the effect. Further we show that this activity of SCAMP2 requires an active form of a small GTPase Arf6, but not Rab11. We propose a model in which SCAMPs bind to NHE5 in the endosomal compartment and control its cell-surface abundance via an Arf6-dependent pathway.     

Mechanism of Sustained Activation of Ribosomal S6 Kinase (RSK) and ERK by Kaposi Sarcoma-associated Herpesvirus ORF45: MULTIPROTEIN COMPLEXES RETAIN ACTIVE PHOSPHORYLATED ERK AND RSK AND PROTECT THEM FROM DEPHOSPHORYLATION*

As obligate intracellular parasites, viruses exploit diverse cellular signaling machineries, including the mitogen-activated protein-kinase pathway, during their infections. We have demonstrated previously that the open reading frame 45 (ORF45) of Kaposi sarcoma-associated herpesvirus interacts with p90 ribosomal S6 kinases (RSKs) and strongly stimulates their kinase activities (Kuang, E., Tang, Q., Maul, G. G., and Zhu, F. (2008) J. Virol. 82 ,1838 -1850). Here, we define the mechanism by which ORF45 activates RSKs. We demonstrated that binding of ORF45 to RSK increases the association of extracellular signal-regulated kinase (ERK) with RSK, such that ORF45, RSK, and ERK formed high molecular mass protein complexes. We further demonstrated that the complexes shielded active pERK and pRSK from dephosphorylation. As a result, the complex-associated RSK and ERK were activated and sustained at high levels. Finally, we provide evidence that this mechanism contributes to the sustained activation of ERK and RSK in Kaposi sarcoma-associated herpesvirus lytic replication.The extracellular signal-regulated kinase (ERK)² mitogen-activated protein kinase (MAPK) signaling pathway has been implicated in diverse cellular physiological processes including proliferation, survival, growth, differentiation, and motility (1-4) and is also exploited by a variety of viruses such as Kaposi sarcoma-associated herpesvirus (KSHV), human cytomegalovirus, human immunodeficiency virus, respiratory syncytial virus, hepatitis B virus, coxsackie, vaccinia, coronavirus, and influenza virus (5-17). The MAPK kinases relay the extracellular signaling through sequential phosphorylation to an array of cytoplasmic and nuclear substrates to elicit specific responses (1, 2, 18). Phosphorylation of MAPK is reversible. The kinetics of deactivation or duration of signaling dictates diverse biological outcomes (19, 20). For example, sustained but not transient activation of ERK signaling induces the differentiation of PC12 cells into sympathetic-like neurons and transformation of NIH3T3 cells (20-22). During viral infection, a unique biphasic ERK activation has been observed for some viruses (an early transient activation triggered by viral binding or entry and a late sustained activation correlated with viral gene expression), but the responsible viral factors and underlying mechanism for the sustained ERK activation remain largely unknown (5, 8, 13, 23).The p90 ribosomal S6 kinases (RSKs) are a family of serine/threonine kinases that lie at the terminus of the ERK pathway (1, 24-26). In mammals, four isoforms are known, RSK1 to RSK4. Each one has two catalytically functional kinase domains, the N-terminal kinase domain (NTKD) and C-terminal kinase domain (CTKD) as well as a linker region between the two. The NTKD is responsible for phosphorylation of exogenous substrates, and the CTKD and linker region regulate RSK activation (1, 24, 25). In quiescent cells ERK binds to the docking site in the C terminus of RSK (27-29). Upon mitogen stimulation, ERK is activated by its upstream MAPK/ERK kinase (MEK). The active ERK phosphorylates Thr-359/Ser-363 of RSK in the linker region (amino acid numbers refer to human RSK1) and Thr-573 in the CTKD activation loop. The activated CTKD then phosphorylates Ser-380 in the linker region, creating a docking site for 3-phosphoinositide-dependent protein kinase-1. The 3-phosphoinositide-dependent protein kinase-1 phosphorylates Ser-221 of RSK in the activation loop and activates the NTKD. The activated NTKD autophosphorylates the serine residue near the ERK docking site, causing a transient dissociation of active ERK from RSK (25, 26, 28). The stimulation of quiescent cells by a mitogen such as epidermal growth factor or a phorbol ester such as 12-O-tetradecanoylphorbol-13-acetate (TPA) usually results in a transient RSK activation that lasts less than 30 min. RSKs have been implicated in regulating cell survival, growth, and proliferation. Mutation or aberrant expression of RSK has been implicated in several human diseases including Coffin-Lowry syndrome and prostate and breast cancers (1, 24, 25, 30-32).KSHV is a human DNA tumor virus etiologically linked to Kaposi sarcoma, primary effusion lymphoma, and a subset of multicentric Castleman disease (33, 34). Infection and reactivation of KSHV activate multiple MAPK pathways (6, 12, 35). Noticeably, the ERK/RSK activation is sustained late during KSHV primary infection and reactivation from latency (5, 6, 12, 23), but the mechanism of the sustained ERK/RSK activation is unclear. Recently, we demonstrated that ORF45, an immediate early and also virion tegument protein of KSHV, interacts with RSK1 and RSK2 and strongly stimulates their kinase activities (23). We also demonstrated that the activation of RSK plays an essential role in KSHV lytic replication (23). In the present study we determined the mechanism of ORF45-induced sustained ERK/RSK activation. We found that ORF45 increases the association of RSK with ERK and protects them from dephosphorylation, causing sustained activation of both ERK and RSK.