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Iron chelation can improve endothelial function. However, effect on endothelial function of deferiprone has not been reported. We hypothesized deferiprone could promote nitric oxide (NO) production in endothelial cells. We studied effects of deferiprone on blood nitrite and blood pressure after single oral dose (25 mg/kg) in healthy subjects and hemoglobin E/[beta]-thalassemia patients. Further, effects of deferiprone on NO production and endothelial NO synthase (eNOS) phosphorylation in primary human pulmonary artery endothelial cells (HPAEC) were investigated in vitro. Blood nitrite levels were higher in patients with deferiprone therapy than those without deferiprone (P = 0.023, n = 16 each). Deferiprone increased nitrite in plasma and whole blood of healthy subjects (P = 0.002 and 0.044) and thalassemia patients (P = 0.003 and 0.046) at time 180 min (n = 20 each). Asymptomatic reduction in diastolic blood pressure (P = 0.005) and increase in heart rate (P = 0.009) were observed in healthy subjects, but not in thalassemia patients. In HPAEC, deferiprone increased cellular nitrite and phospho-eNOS (Ser1177) (P = 0.012 and 0.035, n = 6) without alteration in total eNOS protein and mRNA. We conclude that deferiprone can induce NO production by enhancing eNOS phosphorylation in endothelial cells. Key words: deferiprone, endothelial nitric oxide synthase, nitric oxide, nitrite, thalassemia. La chelation du fer peut permettre d'ameliorer la fonction endotheliale. Cependant, l'effet de la deferiprone sur la fonction endotheliale n'a pas ete rapporte. Nous avons formule une hypothese selon laquelle la deferiprone pourrait favoriser la production d'oxyde nitrique (NO) dans les cellules endotheliales. Nous avons etudie les effets de la deferiprone sur les nitrites sanguins et la tension arterielle apres l'administration d'une dose orale unique (25 mg/kg) chez des sujets en bonne sante et chez des patients atteints de thalassemie [beta]--hemoglobine E. De plus, nous avons etudie les effets in vitro de la deferiprone sur la production de NO et la phosphorylation de la NO synthase endotheliale (eNOS) dans des cellules endotheliales primaires d'arteres pulmonaires humaines. Les taux de nitrites sanguins etaient plus eleves chez les patients ayant recu de la deferiprone que chez ceux qui n'en avaient pas recu(P = 0,023; n = 16 chacun). Apres 180 minutes, la deferiprone entrainait une augmentation des taux de nitrites dans le plasma et le sang complet des sujets en bonne sante (P = 0,002 et 0,044) ainsi que chez les patients atteints de thalassemie (P = 0,003 et 0,046; n = 20 chacun). Nous avons observe une diminution de la tension arterielle diastolique (P = 0,005) avec une augmentation de la frequence cardiaque (P = 0,009) chez les sujets en bonne sante, mais pas chez les patients atteints de thalassemie. Dans les cellules endotheliales d'arteres pulmonaires humaines, la deferiprone entrainait une augmentation des taux de nitrites cellulaires et de phospho-eNOS (Ser1177) (P = 0,012 et 0,035; n = 6) sans modification des taux d'eNOS proteique totaux et d'ARNm. Nous en arrivons a la conclusion que la deferiprone peut provoquer la production de NO en faisant augmenter la phosphorylation de l'eNOS dans les cellules endotheliales. [Traduit par la Redaction] Mots-cles : deferiprone, oxyde nitrique synthase endotheliale, oxyde nitrique, nitrites, thalassemie., Introduction Iron chelator is indicated for iron overloading, which is a complication of regular blood transfusion in thalassemia. Iron chelation can reduce serum ferritin, nontransferrin-bound iron, and iron deposition in [...]

Protein turnover is a tightly controlled process that is crucial for the removal of aberrant polypeptides and for cellular signalling. Whereas ubiquitin marks eukaryotic proteins for proteasomal degradation, a general tagging system for the equivalent bacterial Clp proteases is not known. Here we describe the targeting mechanism of the ClpCClpP proteolytic complex from Bacillus subtilis. Quantitative affinity proteomics using a ClpP-trapping mutant show that proteins phosphorylated on arginine residues are selectively targeted to ClpCClpP. In vitro reconstitution experiments demonstrate that arginine phosphorylation by the McsB kinase is required and sufficient for the degradation of substrate proteins. The docking site for phosphoarginine is located in the amino-terminal domain of the ClpC ATPase, as resolved at high resolution in a co-crystal structure. Together, our data demonstrate that phosphoarginine functions as a bona fide degradation tag for the ClpCClpP protease. This system, which is widely distributed across Gram-positive bacteria, is functionally analogous to the eukaryotic ubiquitinproteasome system., Author(s): Dbora Broch Trentini [1]; Marcin Jzef Suskiewicz [1]; Alexander Heuck [1]; Robert Kurzbauer [1]; Luiza Deszcz [1]; Karl Mechtler [1, 2]; Tim Clausen (corresponding author) [1] Proteins destined for [...]

ADP-glucose pyrophosphorylase (AGP), which consists of two large subunits (AGP-L) and two small subunits (AGP-S), controls the rate-limiting step in the starch biosynthetic pathway. In this study, a full-length open [...]

PKA phosphorylates multiple molecules involved in calcium ([Ca.sup.2+]) handling in cardiac myocytes and is considered to be the predominant regulator of β-adrenergic receptor-mediated enhancement of cardiac contractility; however, recent identification of exchange protein activated by cAMP (EPAC), which is independently activated by cAMP, has challenged this paradigm. Mice lacking Epac1 (Epac1 KO) exhibited decreased cardiac contractility with reduced phospholamban (PLN) phosphorylation at serine-16, the major PKA-mediated phosphorylation site. In Epac1 KO mice, intracellular [Ca.sup.2+] storage and the magnitude of [Ca.sup.2+] movement were decreased; however, PKA expression remained unchanged, and activation of PKA with isoproterenol improved cardiac contractility. In contrast, direct activation of EPAC in cardiomyocytes led to increased PLN phosphorylation at serine-16, which was dependent on PLC and PKCe. Importantly, Epac1 deletion protected the heart from various stresses, while Epac2 deletion was not protective. Compared with WT mice, aortic banding induced a similar degree of cardiac hypertrophy in Epac1 KO; however, lack of Epac1 prevented subsequent cardiac dysfunction as a result of decreased cardiac myocyte apoptosis and fibrosis. Similarly, Epac1 KO animals showed resistance to isoproterenol- and aging-induced cardiomyopathy and attenuation of arrhythmogenic activity. These data support Epac1 as an important regulator of PKA-independent PLN phosphorylation and indicate that Epac1 regulates cardiac responsiveness to various stresses., Introduction β-Adrenergic receptor (β-AR) signaling is well established as a primary defense mechanism against acute stress or changes in hemodynamic load; however, its role in cardiac pathogenesis, although studied extensively, [...]

Entry into mitosis in eukaryotes requires the activity of cyclin-dependent kinase 1 (Cdk1). Cdk1 is opposed by protein phosphatases in two ways: They inhibit activation of Cdk1 by dephosphorylating the protein kinases Wee1 and Myt1 and the protein phosphatase Cdc25 (key regulators of Cdk1), and they also antagonize Cdk1's own phosphorylation of downstream targets. A particular form of protein phosphatase 2A (PP2A) containing a B558 subunit (PP2A-B55[delta]) is the major protein phosphatase that acts on model CDK substrates in Xenopus egg extracts and has antimitotic activity. The activity of PPPA-B55[delta] is high in interphase and low in mitosis, exactly opposite that of Cdk1. We report that inhibition of PP2A-B55[delta] results from a small protein, known as [alpha]-endosulfine (Ensa), that is phosphorylated in mitosis by the protein kinase Greatwall (Gwl). This converts Ensa into a potent and specific inhibitor of PP2A-B55[delta]. This pathway represents a previousLy unknown element in the control of mitosis. 10.1126/science.1195689

Pressure overload-induced cardiac hypertrophy results in a pathological type of hypertrophy with activation of signaling cascades like the extracellular signal-regulated kinase (ERK) pathway, which promotes negative cardiac remodeling and decreased contractile function. In contrast, thyroid hormone mediates a physiological type of hypertrophy resulting in enhanced contractile function. In addition, thyroid hormone action is diminished in pressure overload-induced cardiac hypertrophy. We hypothesized that thyroid hormone status modulates ERK activity and that administration of thyroid hormone could alter the activity of this kinase in cardiac hypertrophy induced by pressure overload. ERK is activated by phosphorylation; accordingly, we investigated phosphorylation of ERK in hearts of control, hypothyroid, and hyperthyroid mice. In addition, the effect of T3 treatment on ERK phosphorylation in hypertrophied hearts from transverse aortic-constricted (TAC) mice was investigated. Results showed that phosphorylated ERK (p-ERK) was decreased by 25% in hyperthyroid mice. In contrast, hypothyroid mice presented increased p-ERK by 80%. TAC mice presented a greater than fourfold increase of p-ERK compared with control mice. Interestingly, T3 administration dramatically canceled TAC-induced ERK phosphorylation (36% lower compared with control). Raf-1 is upstream of the ERK pathway. TAC mice presented a 45% increase in phospho-Raf-1 (Ser338). T3 treatment inhibited this effect of pressure overload and further decreased p-Raf-1 (Ser338) by 37%, compared with control. Overexpression of thyroid hormone receptor-[alpha] in cultured cardiomyocytes potentiated the inhibitory effect of T3 on ERK phosphorylation. We concluded that thyroid hormone has an inhibitory effect on the Raf-1/ERK pathway. Furthermore, treatment of TAC mice with T3 inhibited Raf-1/ERK pathway by a thyroid hormone receptor-dependent mechanism. MAPK; thyroid hormone receptors doi: 10.1152/ajpcell.00168.2010.

During the classic 'fight-or-flight' stress response, sympathetic nervous system activation leads to catechol-amine release, which increases heart rate and contractility, resulting in enhanced cardiac output. Catecholamines bind to β-adrenergic receptors, causing cAMP generation and activation of PKA, which phosphorylates multiple targets in cardiac muscle, including the cardiac ryanodine receptor/calcium release channel (RyR2) required for muscle contraction. PKA phosphorylation of RyR2 enhances channel activity by sensitizing the channel to cytosolic calcium ([Ca.sup.2+]). Here, we found that mice harboring RyR2 channels that cannot be PKA phosphorylated (referred to herein as RyR2-[S2808A.sup.+/+] mice) exhibited blunted heart rate and cardiac contractile responses to catecholamines (isoproterenol). The isoproterenol-induced enhancement of ventricular myocyte [Ca.sup.2+] transients and fractional shortening (contraction) and the spontaneous beating rate of sinoatrial nodal cells were all blunted in RyR2-[S2808A.sup.+/+] mice. The blunted cardiac response to catecholamines in RyR2-[S2808A.sup.+/+] mice resulted in impaired exercise capacity. RyR2-[S2808A.sup.+/+] mice were protected against chronic catecholaminergic-induced cardiac dysfunction. These studies identify what we believe to be new roles for PKA phosphorylation of RyR2 in both the heart rate and contractile responses to acute catecholaminergic stimulation., Introduction During exercise, heart rate (chronotropy) and cardiac contractility (inotropy) increase to meet the metabolic demands of the organs. Stress-induced activation of the sympathetic nervous system (SNS) results in catecholamine [...]

Obesity and type 2 diabetes are associated with increased lipogenesis in the liver. This results in fat accumulation in hepatocytes, a condition known as hepatic steatosis, which is a form of nonalcoholic fatty liver disease (NAFLD), the most common cause of liver dysfunction in the United States. Carbohydrate-responsive element- binding protein (ChREBP), a transcriptional activator of glycolytic and lipogenic genes, has emerged as a major player in the development of hepatic steatosis in mice. However, the molecular mechanisms enhancing its transcriptional activity remain largely unknown. In this study, we have identified the histone acetyltransferase (HAT) coactivator p300 and serine/threonine kinase salt-inducible kinase 2 (SIK2) as key upstream regulators of ChREBP activity. In cultured mouse hepatocytes, we showed that glucose-activated p300 acetylated ChREBP on Lys672 and increased its transcriptional activity by enhancing its recruitment to its target gene promoters. SIK2 inhibited p300 HAT activity by direct phosphorylation on Ser89, which in turn decreased ChREBP-mediated lipogenesis in hepatocytes and mice overexpressing SIK2. Moreover, both liver-specific SIK2 knockdown and p300 overexpression resulted in hepatic steatosis, insulin resistance, and inflammation, phenotypes reversed by SIK2/p300 co-overexpression. Finally, in mouse models of type 2 diabetes and obesity, low SIK2 activity was associated with increased p300 HAT activity, ChREBP hyperacetylation, and hepatic steatosis. Our findings suggest that inhibition of hepatic p300 activity may be beneficial for treating hepatic steatosis in obesity and type 2 diabetes and identify SIK2 activators and specific p300 inhibitors as potential targets for pharmaceutical intervention., Introduction The metabolic syndrome, which represents a collection of abnormalities including obesity, type 2 diabetes, dyslipidemia, fatty liver, and a proinflammatory state (1), affects more than 27% of adults in [...]

In situations of stress the heart beats faster and stronger. According to Marks and colleagues, this response is, to a large extent, the consequence of facilitated [Ca.sup.2+] release from intracellular [Ca.sup.2+] stores via ryanodine receptor 2 (RyR2), thought to be due to catecholamine-induced increases in RyR2 phosphorylation at serine 2808 (S2808). If catecholamine stimulation is sustained (for example, as occurs in heart failure), RyR2 becomes hyperphosphorylated and 'leaky,' leading to arrhythmias and other pathology. This 'leaky RyR2 hypothesis' is highly controversial. In this issue of the JCI, Marks and colleagues report on two new mouse lines with mutations in S2808 that provide strong evidence supporting their theory. Moreover, the experiments revealed an influence of redox modifications of RyR2 that may account for some discrepancies in the field., The heart has a remarkable capacity to react to altered demand by changing the rate at which it beats and the force with which it contracts, thereby changing its output. [...]

Intracellular cAMP is compartmentalized to near membrane domains in endothelium, where it strengthens endothelial cell barrier function. Phosphodiesterase 4D4 (PDE4D4) interacts with the spectrin membrane skeleton and prevents cAMP from accessing microtubules. Expression of a dominant-negative PDE4D4 peptide enables cAMP to access microtubules, where it results in phosphorylation of the nonneuronal microtubule-associated protein tau at serine 214. Presently, we sought to determine whether PKA is responsible for tau-Ser214 phosphorylation and furthermore whether PKA phosphorylation of tau-Ser214 is sufficient to reorganize microtubules and induce endothelial cell gaps. In cells expressing the dominant-negative PDE4D4 peptide, forskolin activated transmembrane adenylyl cyclases, increased cAMP, and induced tau-Ser214 phosphorylation that was accompanied by microtubule reorganization. PKA catalytic and regulatory I subunits, but not the regulatory II subunit, coassociated with reorganized microtubules. To determine the functional consequence of tau-Ser214 phosphorylation, wild-type human tau40 and tau40 engineered to possess an alanine point mutation (S214A) were stably expressed in endothelium. In cells expressing the dominant-negative PDE4D4 peptide and tauS214A, PKA-dependent phosphorylation of both the endogenous and heterologously expressed tau were abolished. Expression of tau-S214A prevented forskolin from depolymerizing microtubules, inducing intercellular gaps, and increasing macromolecular permeability. These findings therefore identify nonneuronal tau as a critical cAMP-responsive microtubule-associated protein that controls microtubule architecture and endothelial cell barrier function. adenylyl cyclase; cytoskeleton; phosphodiesterase doi: 10.1152/ajplung.00431.2009.

The swelling-activated [K.sup.+] currents ([I.sub.K,vol]) in Ehrlich ascites tumor cells (EATC) has been reported to be through the two-pore domain ([K.sub.2p]), TWIK-related acid-sensitive [K.sup.+] channel 2 (TASK-2). The regulatory volume decrease (RVD), following hypotonic exposure in EATC, is rate limited by [I.sub.K,vol] indicating that inhibition of RVD reflects inhibition of TASK-2. We find that in EATC the tyrosine kinase inhibitor genistein inhibits RVD by 90%, and that the tyrosine phosphatase inhibitor monoperoxo(picolinato)-oxo-vanadate(V) [mpV(pic)] shifted the volume set point for inactivation of the channel to a lower cell volume. Swelling-activated [K.sup.+] efflux was impaired by genistein and the Src kinase family inhibitor 4-amino-5-(4-chloro-phenyl)-7-(t-butyl) pyrazolo[3,4-d]pyrimidine (PP2) and enhanced by the tyrosine phosphatase inhibitor mpV(pic). With the use of the TASK-2 inhibitor clofilium, it is demonstrated that mpV(pic) increased the volume-sensitive part of the [K.sup.+] efflux 1.3 times. To exclude [K.sup.+] efflux via a KCl cotransporter, cellular [Cl.sup.-] was substituted with N[O.sup.-.sub.3]. Also under these conditions [K.sup.+] efflux was completely blocked by genistein. Thus tyrosine kinases seem to be involved in the activation of the volume-sensitive [K.sup.+] channel, whereas tyrosine phosphatases appears to be involved in inactivation of the channel. Overexpressing TASK-2 in human embryonic kidney (HEK)-293 cells increased the RVD rate and reduced the volume set point. TASK-2 has tyrosine sites, and precipitation of TASK-2 together with Western blotting and antibodies against phosphotyrosines revealed a cell swelling-induced, time-dependent tyrosine phosphorylation of the channel. Even though we found an inhibiting effect of PP2 on RVD, neither Src nor the focal adhesion kinase (FAK) seem to be involved. Inhibitors of the epidermal growth factor receptor tyrosine kinases had no effect on RVD, whereas the Janus kinase (JAK) inhibitor cucurbitacin inhibited the RVD by 40%. It is suggested that the cytokine receptor-coupled JAK/STAT pathway is upstream of the swelling-induced phosphorylation and activation of TASK-2 in EATC. cell volume regulation; Janus kinase; volume-sensitive channels doi: 10.1152/ajpcell.00024.2010.

Mutations in PARK2/Parkin, which encodes a ubiquitin E3 ligase, cause autosomal recessive Parkinson disease (PD). Here we show that the nonreceptor tyrosine kinase c-Abl phosphorylates tyrosine 143 of parkin, inhibiting parkin's ubiquitin E3 ligase activity and protective function, coAbl is activated by dopaminergic stress and by dopaminergic neurotoxins, 1-methyl-4-phenylpyridinium ([Mpp.sup.+]) in vitro and in vivo by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), leading to parkin inactivation, accumulation of the parkin substrates aminoacyl-tRNA synthetase-interacting multifunctional protein type 2 (AIMP2) (p38/JTV-1) and fuse-binding protein 1 (FBP1), and cell death. STI-571, a c-Abl-family kinase inhibitor, prevents the phosphorylation of parkin, maintaining parkin in a catalytically active and protective state. STI-571's protective effects require parkin, as shRNA knockdown of parkin prevents STI-571 protection. Conditional knockout of c-Abl in the nervous system also prevents the phosphorylation of parkin, the accumulation of its substrates, and subsequent neurotoxicity in response to MPTP intoxication. In human postmortem PD brain, c-Abl is active, parkin is tyrosine-phosphorylated, and AIMP2 and FBP1 accumulate in the substantia nigra and striatum. Thus, tyrosine phosphorylation of parkin by c-Abl is a major posttranslational modification that inhibits parkin function, possibly contributing to pathogenesis of sporadic PD. Moreover, inhibition of c-Abl may be a neuroprotective approach in the treatment of PD. AIMP2 | Parkinson disease | STI-571 | ubiquitin doi/ 10.1073/pnas.1006083107

The phosphodie sterase type-5 inhibitor sildenafil has powerful cardioprotective effects against ischemia-reperfusion injury. PKG-mediated signaling has been implicated in this protection, although the mechanism and the downstream targets of this kinase remain to be fully elucidated. In this study we assessed the role of phospholemman (PLM) phosphorylation, which activates the [Na.sup.+]/[K.sup.+]-ATPase, in cardioprotection afforded by sildenafil administered during reperfusion. Isolated perfused mouse hearts were optimally protected against infarction (indexed by tetrazolium staining) by 0.1 [micro]M sildenafil treatment during the first 10 min of reperfusion. Extended sildenafil treatment (30, 60, or 120 min at reperfusion) did not alter the degree of protection provided. This protection was PKG dependent, since it was blocked by KT-5823. Western blot analysis using phosphospecific antibodies to PLM showed that sildenafil at reperfusion did not modulate PLM Ser63 or Set68 phosphorylation but significantly increased Ser69 phosphorylation. The treatment of isolated rat ventricular myocytes with sildenafil or 8-bromo-cGMP (PKG agonist) enhanced PLM Ser69 phosphorylation, which was bisindolylmaleimide (PKC inhibitor) sensitive. Patch-clamp studies showed that sildenafil treatment also activated the [Na.sup.+]/[K.sup.-]-ATPase, which is anticipated in light of PLM Ser69 phosphorylation. [Na.sup.+]/[K.sup.-]-ATPase activation during reperfusion would attenuate [Na.sup.+] overload at this time, providing a molecular explanation of how sildenafil guards against injury at this time. Indeed, using flame photometry and rubidium uptake into isolated mouse hearts, we found that sildenafil enhanced [Na.sup.+]/[K.sup.-]-ATPase activity during reperfusion. In this study we provide a molecular explanation of how sildenafil guards against myocardial injury during postischemic reperfusion. protein kinase G; sodium/potassium-adenosine 5'-triphosphatase; ischemia; heart doi: 10.1152/ajpheart.00129.2010.

ROS, such as [H.sub.2][O.sub.2], are a component of pathological conditions in many organ systems and have been reported to be elevated in cardiac pathophysiology. The experiments presented here test the hypothesis that [H.sub.2][O.sub.2] induces alterations in cardiac myofilament function by the posttranslational modification of sarcomeric proteins indirectly through PKC signaling. In vitro assessment of actomyosin [Mg.sup.2+]-ATPase activity of myofibrillar fractions showed blunted relative ATP consumption in the relaxed state (pCa 8.0) in response to treatment with 0.5 mM [H.sub.2][O.sub.2] before myofilament isolation. The effect was attributable to downstream 'redox signaling,' inasmuch as the direct application of [H.sub.2][O.sub.2] to isolated myofibrils did not alter [Mg.sup.2+]-ATPase activity. [Ca.sup.2+]-ATPase activity, which was used as a measure of myofibrillar myosin function, was unaffected by [H.sub.2][O.sub.2]. Functional experiments using rat cardiac trabeculae treated with 0.5 or 5 mM H202 followed by detergent extraction of membranes demonstrated increased [Ca.sup.2+] sensitivity of force production, a faster rate of force redevelopment, and (for 5 mM) decreased maximum tension. Biochemical analysis of myocardial samples treated with 0.5 mM [H.sub.2][O.sub.2] demonstrated increased phosphorylation of two sarcomeric proteins: cardiac troponin I and myosin-binding protein-C. These changes were eliminated by a general PKC inhibitor. However, [H.sub.2][O.sub.2] and the general PKC activator PMA induced different phosphorylation patterns in cardiomyocytes in which PKC-[delta] was elevated by viral infection. These data provide evidence that PKC-dependent redox signaling affects the function of cardiac myofilaments and indicate modification of specific proteins through this signaling mechanism. force-pCa relationship; ATPase activity; phosphorylation; cardiac troponin I; myosin-binding protein C doi: 10.1152/ajpheart.00050.2010.

Nuclear envelope breakdown (NEBD) is an essential step during the G2/M transition in higher eukaryotic cells. Increasing evidence supports the notion that both microtubules and microtubule-associated motor proteins are critical regulators of NEBD. Although it has been described that [p150.sup.Glued], the major component of the dynein/dynactin complex, localizes in the nuclear envelope during prophase, the exact role of [p150.sup.Glued] and its regulation during NEBD are largely elusive. Polo-like kinase 1 (Plk1), the best characterized Ser/Thr kinase, is involved in mitotic entry in several systems; however, the targets of Plk1 during NEBD are unknown. Herein, we show that in mammalian cells both Plk1 and [p150.sup.Glued] regulate NEBD and that Plk1 interacts with and phosphoryates [p150.sup.Glued] during NEBD at prophase. Using various approaches, we showed that Plk1 phosphorylates [p150.sup.Glued] at Ser-179 and that the pS179 epitope is generated at the nuclear envelope of prophase cells. Significantly, Plk1-mediated phosphorylation of [p150.sup.Glued] at Ser-179 positively regulates its accumulation at the nuclear envelope during prophase. Finally, we found that cells expressing the Plk1-unphosphorylatable mutant ([p150.sup.Glued]-s179A) arrest at G2, as indicated by reduced NEBD, increased levels of cyclin B and phospho-H3, but a decreased level of Cdc2 kinase activity. Taking these data together, we conclude that Plk1 phosphorylation of [p150.sup.Glued] might be one major pathway of NEBD regulation. mitosis | microtubule dynamics | dynactin doi/ 10.1073/pnas.1006615107

We have developed a phosphatase-based phosphopeptide quantitation (PPQ) method for determining phosphorylation stoichiometry in complex biological samples. This PPQ method is based on enzymatic dephosphorylation, combined with specific and accurate peptide identification and quantification by multiple reaction monitoring (MRM) with stable-isotope-labeled standard peptides. In contrast with classical MRM methods for the quantitation of phosphorylation stoichiometry, the PPQ--MRM method needs only one nonphosphorylated SIS (stable isotope-coded standard) and two analyses (one for the untreated sample and one for the phosphatase-treated sample), from which the expression and modification levels can accurately be determined. From these analyses, the percent phosphorylation can be determined. In this manuscript, we compare the PPQ--MRM method with an MRM method without phosphatase and demonstrate the application of these methods to the detection and quantitation of phosphorylation of the classic phosphorylated breast cancer biomarkers (ER[alpha] and HER2), and for phosphorylated RAF and ERK1, which also contain phosphorylation sites of biological importance. Using synthetic peptides spiked into a complex protein digest, we were able to use our PPQ--MRM method to accurately determine the total phosphorylation stoichiometry on specific peptides as well as the absolute amount of the peptide and phosphopeptide present. Analyses of samples containing ER[alpha] protein revealed that the PPQ--MRM method is capable of determining phosphorylation stoichiometry in proteins from cell lines, and is in good agreement with determinations obtained using the direct MRM approach in terms of phosphorylation and total protein amount. 10.1021/ac1005553

Recent investigations demonstrate increased Na/H exchanger-1 (NHE-1) activity and plasma levels of ouabain-like factor in spontaneously hypertensive rats. At nanomolar concentrations, ouabain increases Na-K-ATPase activity, induces cell proliferation, and activates complex signaling cascades. We hypothesize that the activity of NHE-1 and Na-K-ATPase are interdependent. To test whether treatment with picomolar ouabain regulates Na-K-ATPase through an NHE-1-dependent mechanism, we examined the role of NHE-1 in ouabain-mediated stimulation of Na-K-ATPase in kidney proximal tubule cell lines [opossum kidney (OK), HK-2, HKC-5, and HKC-11] and rat kidney basolateral membranes. Ouabain stimulated Na-K-ATPase activity and tyrosine phosphorylation in cells that express NHE-1 (OK, HKC-5, and HKC-11) but not in HK-2 cells that express very low levels of NHE-1. Inhibition of NHE-1 with 5 [micro]M EIPA, a NHE-1specific inhibitor, prevented ouabain-mediated stimulation of S6Rb uptake and Na-K-ATPase phosphorylation in OK, HKC-5, and HKC- 11 cells. Expression of wild-type NHE- 1 in HK2 cells restored regulation of Na-K-ATPase by picomolar ouahain. Treatment with picomolar ouabain increased membrane expression of Na-K-ATPase and enhanced NHE-1-Na-K-ATPase [alpha]-subunit association. Treatment with ouabain (1 [micro]g x kg body [wt.sup.-1] x [day.sup.-1]) increased Na-K-ATPase activity, expression, phosphorylation, and association with NHE-1 increased in rat kidney cortical basolateral membranes. Eight days' treatment with ouabain (1 [micro]g x kg body [wt.sup.-1] x [day.sup.-1]) resulted in increased blood pressure in these rats. These results suggest that the association of NHE-1 with Na-K-ATPase is critical for ouabain-mediated regulation of Na-K-ATPase and that these effects may play a role in cardioglycoside-stimulated hypertension. cardioglycosides; phosphorylation doi: 10.1152/ajprenal.00581.2009.

Norepinephrine (NE) released from the sympathetic nerves innervating white adipose tissue (WAT) is the principal initiator of lipolysis in mammals. Central WAT sympathetic outflow neurons express melanocortin 4-receptor (MC4-R) mRNA. Single central injection of melanotan II (MTII; MC3/4-R agonist) nonuniformly increases WAT NE turnover (NETO), increases interscapular brown adipose tissue (IBAT) NETO, and increases the circulating lipolytic products glycerol and free fatty acid. The WAT pads that contributed to this lipolysis were inferred from the increases in NETO. Because phosphorylation of perilipin A (p-perilipin A) and hormone-sensitive lipase are necessary for NE-triggered lipolysis, we tested whether MTII would increase these intracellular markers of lipolysis. Male Siberian hamsters received a single 3rd ventricular injection of MTII or saline. Trunk blood was collected at 0.5, 1.0, and 2.0 h postinjection from excised inguinal, retroperitoneal, and epididymal WAT (IWAT, RWAT, and EWAT, respectively) and IBAT pads. MTII increased circulating glycerol concentrations at 0.5 and 1.0 h, whereas free fatty acid concentrations were increased at 1.0 and 2.0 h. Western blot analysis showed that MTII specifically increased p-perilipin A and hormone-sensitive lipase only in fat pads that previously had MTII-induced increases in NETO. Phosphorylation increased in IWAT at all time points and IBAT at 0.5 h, but not RWAT or EWAT at any time point. These results show for the first time in rodents that p-perilipin A can serve as an in vivo, fat pad-specific indictor of lipolysis and extend our previous findings showing that central melanocortin stimulation increases WAT lipolysis. sympathetic nervous system; method; Western blot analysis doi: 10.1152/ajpregu.00535.2009.

Transcription networks composed of various transcriptional factors specifically expressed in undifferentiated embryonic stem (ES) cells have been implicated in the regulation of pluripotency in ES cells. However, the molecular mechanisms responsible for self-renewal, maintenance of pluripotency, and lineage specification during differentiation of ES cells are still unclear. The results of this study demonstrate that a phosphorylation-dependent chromatin relaxation factor, transcriptional intermediary factor-1[beta] (TIF1[beta]), is a unique regulator of the pluripotency of ES cells and regulates Oct3/4dependent transcription in a phosphorylation-dependent manner. TIF1[beta] is specifically phosphorylated in pluripotent mouse ES cells at the C-terminal serine 824, which has been previously shown to induce chromatin relaxation. Phosphorylated TIF1[beta] is partially colocalized at the activated chromatin markers, and forms a complex with the pluripotency-specific transcription factor Oct3/4 and subunits of the switching defective/sucrose nonfermenting, ATP-dependent chromatin remodeling complex, Smarcad1, Brg-1, and BAF155, all of which are components of an ES-specific chromatin remodeling complex, esBAF. Phosphorylated TIF1[beta] specifically induces ES cell-specific genes and enables prolonged maintenance of an undifferentiated state in mouse ES cells. Moreover, TIF1[beta] regulates the reprogramming process of somatic cells in a phosphorylation-dependent manner. Our results suggest that TIF1[beta] provides a phosphorylation-dependent, bidirectional platform for specific transcriptional factors and chromatin remodeling enzymes that regulate the cell differentiation process and the pluripotency of stem cells. chromatin remodeling | differentiation | iPS cells | oct3/4 | esBAF doi/ 10.1073/pnas.0907601107

Translation and messenger RNA (mRNA) degradation are important sites of gene regulation, particularly during stress where translation and mRNA degradation are reprogrammed to stabilize bulk mRNAs and to preferentially translate mRNAs required for the stress response. During stress, untranslating mRNAs accumulate both in processing bodies (P-bodies), which contain some translation repressors and the mRNA degradation machinery, and in stress granules, which contain mRNAs stalled in translation initiation. How signal transduction pathways impinge on proteins modulating P-body and stress granule formation and function is unknown. We show that during stress in Saccharomyces cerevisiae, Dcp2 is phosphorylated on serine 137 by the Ste20 kinase. Phosphorylation of Dcp2 affects the decay of some mRNAs and is required for Dcp2 accumulation in P-bodies and specific protein interactions of Dcp2 and for efficient formation of stress granules. These results demonstrate that Ste20 has an unexpected role in the modulation of mRNA decay and translation and that phosphorylation of Dcp2 is an important control point for mRNA decapping. doi/ 10.1083/jcb.200912019

Aurora A is a spindle pole--associated protein kinase required for mitotic spindle assembly and chromosome segregation. In this study, we show that Drosophila melanogaster aurora A phosphorylates the dynactin subunit [p150.sup.glued] on sites required for its association with the mitotic spindle. Dynactin strongly accumulates on microtubules during prophase but disappears as soon as the nuclear envelope breaks down, suggesting that its spindle localization is tightly regulated. If aurora A's function is compromised, dynactin and dynein become enriched on mitotic spindle microtubules. Phosphorylation sites are localized within the conserved microtubule-binding domain (MBD) of the [p150.sup.glued]. Although wild-type [p150.sup.glued] binds weakly to spindle microtubules, a variant that can no longer be phosphorylated by aurora A remains associated with spindle microtubules and fails to rescue depletion of endogenous [p150.sup.glued]. Our results suggest that aurora A kinase participates in vivo to the phosphoregulation of the [p150.sup.glued] MBD to limit the microtubule binding of the dynein--dynactin complex and thus regulates spindle assembly. doi/ 10.1083/jcb.201001144

[Na.sup.+]/[H.sup.+] exchanger 3 (NHE3) is phosphorylated and regulated by multiple kinases, including PKA, SGK1, and CK2; however, the role of phosphatases in the dephosphorylation and regulation of NHE3 remains unknown. The purpose of this study was to determine whether serine/threonine phosphatases alter NHE3 activity and phosphorylation and, if so, at which sites. To this end, we first examined the effects of calyculin A [a combined protein phosphatase 1 (PP1) and PP2A inhibitor] and okadaic acid (a PP2A inhibitor) on general and site-specific NHE3 phosphorylation. Calyculin A induced a phosphorylation-dependent NHE3 gel mobility shift and increased NHE3 phosphorylation at serines 552 and 605. No change in NHE3 phosphorylation was detected after okadaic acid treatment. An NHE3 gel mobility shift was also evident in calyculin A-treated COS-7 cells transfected with either wild-type or mutant (S552A, S605G, S661A, S716A) rat NHE3. Since the NHE3 gel mobility shift occurred despite mutation of known phosphorylation sites, novel sites of phosphorylation must also exist. Next, we assayed NHE3 activity in response to calyculin A and okadaic acid and found that calyculin A induced a 24% inhibition of NHE3 activity, whereas okadaic acid had no effect. When all known NHE3 phosphorylation sites were mutated, calyculin A induced a stimulation of NHE3 activity, demonstrating a functional significance for the novel phosphorylation sites. Finally, we established that the PP1 catalytic subunit can directly dephosphorylate immunopurified NHE3 in vitro. In conclusion, our data demonstrate that a calyculin A-sensitive phosphatase, most likely PP1, is involved in the regulation and dephosphorylation of NHE3 at known and novel sites. [Na.sup.+]/[H.sup.+] exchanger; protein phosphatase 1; protein phosphatase 2A doi:10.1152/ajprenal.00182.2009

MicroRNAs (miRs) can regulate many cellular functions, but their roles in regulating responses of vascular endothelial cells (ECs) to mechanical stimuli remain unexplored. We hypothesize that the physiological responses of ECs are regulated by not only mRNA and protein signaling networks, but also expression of the corresponding miRs. EC growth arrest induced by pulsatile shear (PS) flow is an important feature for flow regulation of ECs. miR profiling showed that 21 miRs are differentially expressed (8 up- and 13 downregulated) in response to 24-h PS as compared to static condition (ST). The mRNA expression profile indicates EC growth arrest under 24-h PS. Analysis of differentially expressed miRs yielded 68 predicted mRNA targets that overlapped with results of microarray mRNA profiling. Functional analysis of miR profile indicates that the cell cycle network is highly regulated. The upregulation of miR-23b and miR-27b was found to correlate with the PS-induced EC growth arrest. Inhibition of miR-23b using antagomir-23b oligonucleotide (AM23b) reversed the PS-induced E2F1 reduction and retinoblastoma (Rb) hypophosphorylation and attenuated the PS-induced G1/G0 arrest. Antagomir AM27b regulated E2F1 expression, but did not affect Rb and growth arrest. Our findings indicate that PS suppresses EC proliferation through the regulation of miR-23b and provide insights into the role of miRs in mechanotransduction. cell cycle | shear | bioinformatics | gene regulation | mechanotransduction www.pnas.org/cgi/doi/10.1073/pnas.0914825107

Two-component signal transduction systems are widespread in prokaryotes and control numerous cellular processes. Extensive investigation of sensor kinase and response regulator proteins from many two-component systems has established conserved sequence, structural, and mechanistic features within each family. In contrast, the phosphatases which catalyze hydrolysis of the response regulator phosphoryl group to terminate signal transduction are poorly understood. Here we present structural and functional characterization of a representative of the CheC/CheX/ FliY phosphatase family. The X-ray crystal structure of Borrelia burgdorferi CheX complexed with its CheY3 substrate and the phosphoryl analogue [BeF.sup.-.sub.3] reveals a binding orientation between a response regulator and an auxiliary protein different from that shared by every previously characterized example. The surface of CheY3 containing the phosphoryl group interacts directly with a long helix of CheX which bears the conserved (E - [X.sub.2] - N) motif. Conserved CheX residues Glu96 and Asn99, separated by a single helical turn, insert into the CheY3 active site. Structural and functional data indicate that CheX Asn99 and CheY3 Thr81 orient a water molecule for hydrolytic attack. The catalytic residues of the CheX x CheY3 complex are virtually superimposable on those of the Escherichia coli CheZ phosphatase complexed with CheY, even though the active site helices of CheX and CheZ are oriented nearly perpendicular to one other. Thus, evolution has found two structural solutions to achieve the same catalytic mechanism through different helical spacing and side chain lengths of the conserved acid/amide residues in CheX and CheZ. CheX | CheY | two-component systems | dephosphorylation | convergent evolution doi/10.1073/pnas.0911185107

Cyclin-dependent kinase 5 (Cdk5) and its activator p35 are critical for radial migration and lamination of cortical neurons. However, how this kinase is regulated by extracellular and intracellular signals during cortical morphogenesis remains unclear. Here, we show that PKC[delta], a member of novel PKC expressing in cortical neurons, could stabilize p35 by direct phosphorylation. PKC[delta] attenuated the degradation of p35 but not its mutant derivative, which could not be phosphorylated by PKC[delta]. Down-regulation of PKC[delta] by in utero electroporation of specific small interference RNA (siRNA) severely impaired the radial migration of cortical neurons. This migration defect was similar to that caused by down-regulation of p35 and could be prevented by cotransfection with the wild-type but not the mutant p35. Furthermore, PKC[delta] could be activated by the promigratory factor brain-derived neurotrophic factor (BDNF) and was required for the activation of Cdk5 by BDNF. Both PKC[delta] and p35 were required for the promigratory effect of BDNF on cultured newborn neurons. Thus, PKC[delta] may promote cortical radial migration through maintaining the proper level of p35 in newborn neurons. BDNF | neuronal migration | phosphorylation | protein stability | ubiquitin doi/ 10.1073/pnas.0812872106

High-energy inositol pyrophosphates, such as [IP.sup.7] (diphosphoinositol pentakisphosphate), can directly donate a [beta]-phosphate to a prephosphorylated serine residue generating pyrophosphorylated proteins. Here, we show that the [beta] subunit of AP-3, a clathrin-associated protein complex required for HIV-1 release, is a target of [IP.sub.7]-mediated pyrophosphoryiation. We have identified Kif3A, a motor protein of the kinesin superfamily, as an AP3B1-binding partner and demonstrate that Kif3A, like the AP-3 complex, is involved in an intracellular process required for HIV-1 Gag release. Importantly, [IP.sub.7]-mediated pyrophosphorylation of AP3B1 modulates the interaction with Kif3A and, as a consequence, affects the release of HIV-1 virus-like particles. This study identifies a cellular process that is regulated by [IP.sub.7]mediated pyrophosphorylation. phosphorylation | trafficking | kinesin doi/10.1073/pnas.0909176106

Perturbation of lipid second messenger networks is associated with the impairment of synaptic function in Alzheimer disease. Underlying molecular mechanisms are unclear. Here, we used an unbiased lipidomic approach to profile alkylacylglycerophosphocholine second messengers in diseased tissue. We found that specific isoforms defined by a palmitic acid (16:0) at the sn-1 position, namely 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (C16:0 PAF) and 1-O-hexadecyl-sn-glycero-3-phosphocholine (C16:0 lysoPAF), were elevated in the temporal cortex of Alzheimer disease patients, transgenic mice expressing human familial disease-mutant amyloid precursor protein, and human neurons directly exposed to amyloid-[[beta].sub.42] oligomers. Acute intraneuronal accumulation of C16:0 PAF but not C16:0 lyso-PAF initiated cyclin-dependent kinase 5-mediated hyperphosphorylation of tau on Alzheimer disease-specific epitopes. Chronic elevation caused a caspase 2 and 3/7-dependent cascade resulting in neuronal death. Pharmacological inhibition of C16:0 PAF signaling, or molecular strategies increasing hydrolysis of C16:0 PAF to C16:0 lyso-PAF, protected human neurons from amyloid-[[beta].sub.42] toxicity. Together, these data provide mechanistic insight into how disruptions in lipid metabolism can determine neuronal response to accumulating oligomeric amyloid-[[beta].sub.42]. Alzheimer disease | glycerophosphocholine | lipidomics | doi/10.1073/pnas.0905654106

Fast synaptic inhibition in the brain is largely mediated by [gamma]aminobutyric acid receptors (GABAAR). While the pharmacological manipulation of GABAAR function by therapeutic agents, such as benzodiazepines can have profound effects on neuronal excitation and behavior, the endogenous mechanisms neurons use to regulate the efficacy of synaptic inhibition and their impact on behavior remains poorly understood. To address this issue, we created a knock-in mouse in which tyrosine phosphorylation of the GABAARs [gamma]2 subunit, a posttranslational modification that is critical for their functional modulation, has been ablated. These animals exhibited enhanced GABAAR accumulation at postsynaptic inhibitory synaptic specializations on pyramidal neurons within the CA3 subdomain of the hippocampus, primarily due to aberrant trafficking within the endocytic pathway. This enhanced inhibition correlated with a specific deficit in spatial object recognition, a behavioral paradigm dependent upon CA3. Thus, phospho-dependent regulation of GABAAR function involving just two tyrosine residues in the [gamma]2 subunit provides an input-specific mechanism that not only regulates the efficacy of synaptic inhibition, but has behavioral consequences. cognition/GABAA receptor/inhibitory synapses doi/10.1073/pnas.0908840106

AMPA receptor (AMPAR) channel properties and function are regulated by its subunit composition and phosphorylation. Certain types of neural activity can recruit [Ca.sup.2+]-permeable (CP) AMPARs, such as GluR1 homomers, to synapses likely via lateral diffusion from extrasynaptic sites. Here we show that GluR1$845 phosphorylation can alter the subunit composition of perisynaptic AMPARs by providing stability to GluR1 homomers. Using mice specifically lacking phosphorylation of the GluR1$845 site (GluR1-S845A mutants), we demonstrate that this site is necessary for maintaining CP-AMPARs. Specifically, in the GluR1-S845A mutants, CP-AMPARs were absent from perisynaptic locations mainly due to lysosomal degradation. This regulation was mimicked by acute desphosphorylation of the GluR1-S845 site in wild-type mice by NMDA application. Furthermore, long-term depression (LTD) was associated with a reduction in perisynaptic CP-AMPAR levels. Our findings suggest that GluR1-S845 is necessary for maintaining CP-AMPARs on the surface, especially at perisynaptic sites, and suggest that the regulation of these receptors is involved in synaptic plasticity. excitatory synaptic transmission/GluR1 homomer doi/10.1073/pnas.0910338106

The analysis of post-translational phosphorylation is a crucial step in understanding the mechanisms of many physiological events. Numerous approaches for the development of analytical methods aimed at the detection and quantification of phosphorylated proteins by mass spectrometry have been reported in the literature. In this paper, we report a new strategy for the identification of phosphorylated serine and threonine residues in phosphoproteins. This method consists of selective derivatization of phosphoproteins coupled with double pseudoneutral loss extraction after nanoLC/ESI-MS/MS analysis. First, we designed and synthesized a new derivatization reagent, N-(4-bromobenzoyl)aminoethanethial, which can selectively react with [alpha],[beta]-unsaturated ketones produced by [beta]-elimination of a phosphoryl group from phosphorylated serine or threonine residues. The mass spectrum of the derivatized peptide shows a product ion with a characteristic isotopic pattern. After derivatization, fragment ions of peptides with phosphoserine or phosphothreonine have twin peaks with an intensity ratio of approximately 1:1 and a difference of two mass units, while product ions from peptides without phosphoserine or phosphothreonine have normal isotopic patterns. Therefore, the neutral loss of the derivatized residue in the product ion mass spectrum includes two difference losses caused by [sup.79]Br and [sup.81]Br. The extraction of the product scan mass spectrum with double pseudoneutral losses is very effective for identification of the derivatized peptides produced from phosphoryiated peptides. The new strategy represents a useful tool for the analysis of phosphorylated serine and threonine residues in phosphorylated proteins. 10.1021/ac9017988

Abscisic acid (ABA) signaling is important for stress responses and developmental processes in plants. A subgroup of protein phosphatase 2C (group A PP2C) or SNF1-related protein kinase 2 (subclass III SnRK2) have been known as major negative or positive regulators of ABA signaling, respectively. Here, we demonstrate the physical and functional linkage between these two major signaling factors. Group A PP2Cs interacted physically with SnRK2s in various combinations, and efficiently inactivated ABA-activated SnRK2s via dephosphorylation of multiple Ser/Thr residues in the activation loop. This step was suppressed by the RCAR/PYR ABA receptors in response to ABA. However the abi1-1 mutated PP2C did not respond to the receptors and constitutively inactivated SnRK2. Our results demonstrate that group A PP2Cs act as 'gatekeepers' of subclass III SnRK2s, unraveling an important regulatory mechanism of ABA signaling. protein phosphorylation | | LC-MS | SnRK2 | PYR1 doi/ 10.1073/pnas.0907095106

Store-operated [Ca.sup.2+] entry (SOCE) is a ubiquitous [Ca.sup.2+] influx pathway activated in response to depletion of intracellular [Ca.sup.2+] stores. SOCE is a primary modulator of intracellular [Ca.sup.2+] dynamics, which specify cellular responses. Interestingly, SOCE inactivates during M phase but the mechanisms involved remain unclear. SOCE is mediated by clustering of the ER [Ca.sup.2+] sensor STIM1 in response to [Ca.sup.2+] store depletion, leading to gating of the plasma membrane SOCE channel Orai1. Here we show that SOCE inactivation in meiosis is the result of internalization of Orai1 into an intracellular vesicular compartment and to the inability of STIM1 to cluster in response to store depletion. At rest, Orai1 continuously recycles between the cell membrane and an endosomal compartment. We further show that STIM1-STIM1 interactions are inhibited during meiosis, which appears to mediate the inability of STIM1 to form puncta following store depletion, in contrast, STIM1-Orai1 interactions remain functional during meiosis. Combined, the removal of Orai1 from the cell membrane and STIM1 clustering inhibition effectively uncouple store depletion from SOCE activation in meiosis. Although STIM1 is phosphorylated during meiosis, phosphomimetic and alanine substitution mutations do not modulate STIM1 clustering, arguing that phosphorylation does not mediate STIM1 clustering inhibition during meiosis. phosphorylation | store-operated calcium entry | trafficking doi/ 10.1073/pnas.0904651106

To explore the mechanisms and evolution of cell-cycle control, we analyzed the position and conservation of large numbers of phosphorylation sites for the cyclin-dependent kinase Cdk1 in the budding yeast Sacchoromyces cerevisiae. We combined specific chemical inhibition of Cdk1 with quantitative mass spectrometry to identify the positions of 547 phosphorylation sites on 308 Cdk1 substrates in vivo. Comparisons of these substrates with orthologs throughout the ascomycete lineage revealed that the position of most phosphorylation sites is not conserved in evolution; instead, dusters of sites shift position in rapidly evolving disordered regions. We propose that the regulation of protein function by phosphorylation often depends on simple nonspecific mechanisms that disrupt or enhance protein-protein interactions. The gain or loss of phosphorylation sites in rapidly evolving regions could facilitate the evolution of kinase-signaling circuits.