Despite these marked variations in specific activity between the individual isoforms, a comprehensive analysis of phosphorylation of validated AKT substrates indicated only subtle differences in signalling via individual isoforms in vivo. Therefore, we hypothesise, at least in this model system, that relative tissue/cellular abundance, rather than specific activity, plays the dominant role in determining AKT substrate specificity in situ.The AKT protooncogene mediates many cellular processes involved in normal development and disease states such as cancer. The three structurally similar isoforms: AKT1, AKT2, and AKT3 exhibit both functional redundancy and isoform-specific functions; however the basis for their differential signalling remains unclear. Here we show that  The AKT protooncogene comprises a family of three highly homologous serine/threonine kinases AKT1, 2 and 3) , 2 and 3. These pEach AKT isoform consists of a pleckstrin homology domain (>75% identity), a linker region (>17% identity), a kinase domain (>87% identity), and a carboxyl tail with a hydrophobic motif (>66% identity) . Recent −/− mice are not 100% viable, with surviving mice that reach adulthood being 15–20% smaller than their wild type (wt) and heterozygous counterparts ATP and varying concentrations of RPRAATF peptide or HIS-rpS7 at 30°C for 20 minutes. The assay was terminated by spotting onto p81 paper, washed, dried, and [γ-32P] counted using the Tri-Carb 2100TR liquid scintillation analyzer . For whole cell lysates, the reaction was terminated by adding 10 μL of 10% trichloroacetic acid. The sample was then cleared, spotted onto p81 paper, and counted as above. In the case of HIS-rpS7, samples were resolved by SDS-PAGE, the gel stained with Coomassie R-250, dried, the appropriate bands excised and counted as above. Direct kinase assays were performed as described in . Brieflyμg) were adjusted to pH 8.5 with 100 mM sodium hydroxide, labelled in the dark with 400 pmol of CyDye Fluor minimal dye Cy2  for 30 minutes on ice and then quenched with 10 mM lysine. Labelled samples were combined with unlabelled proteins to a total of 250 μg, adjusted to a final volume of 340 μL  CHAPS, 0.004% (w/v) bromophenol blue (BPB), 1% (w/v) DTT, and 1% (v/v) IPG buffer) and passively rehydrated onto an 18 cm, nonlinear pH 3–11 Immobiline DryStrip gel . Proteins were focused using the Ettan IPGphor3  at 20°C with 50 μA per strip using the following protocol: step and hold at 150 V for 3 hours, step and hold at 300 V for 3 hours, gradient to 1000 V for 6 hours, gradient to 10000 V for 1 hour, step and hold at 10000 V for 3 hours. Immobiline DryStrip gels were incubated in equilibration buffer  SDS, 50 mM Tris·HCl pH 8.8, 0.002% BPB, 30% (v/v) glycerol) containing 1% (w/v) DTT for 15 minutes, and then incubated in equilibration buffer containing 2.5% (w/v) iodoacetamide for 15 minutes. Proteins were resolved in a 12.5% gel at 0.5 W/gel for 1 hour, then 17 W/gel for 3 hours using the Ettan DALTsix gel running tank . Gels were either stained with Coomassie G-250 (10% (w/v) ammonium sulphate, 0.1% Coomassie (w/v) G-250, 3% (v/v) orthophosphoric acid, 20% (v/v) ethanol) or transferred onto Hybond-LFP  and immunoblotted with the Phospho-AKT Substrate (PAS) antibody (Supplementary Table  1) and Cy5 conjugated secondary antibody . Signals were detected by scanning with the Typhoon tri9100 . Total protein (Cy2) and PAS (Cy5) signals were overlayed using ImageQuant . Protein spots of interest were manually excised from the Coomassie G-250 stained gel and identified by mass spectrometry.All chemicals were purchased from GE Healthcare or Sigma. Protein samples lysed in RLB were precipitated using the Ettan 2D Clean up Kit , resuspended in labelling buffer  and the protein concentration determined using the Ettan 2D Quant Kit . Protein samples . Mass spectrometry was performed by the Core Facility at Peter MacCallum Cancer Centre, utilising facilities at Bio21 RTF . The data acquired were analysed by correlating the peptide masses obtained to predict peptide masses from proteins in the NCBI nonredundant database using the Mascot search engine (Matrix Science). Previously data from our laboratory using model peptide substrates revealed that AKT3 was ~15-fold more active than AKT1 which was ~10-fold more active than AKT2 . To confin vivo substrate of the AKT signaling network [in vitro AKT substrate . To confirm that this difference in relative enzyme activity extended to the ability of the isoforms to phosphorylate a protein substrate, expressed GST-tagged AKT1 and 3 isoforms were purified from pervanadate-treated HEK293 cells and the kinetics of phosphorylation of the RPRAATF peptide , 31 comp network , and we Vmax values calculated as 315.90 ± 88.95 nmol/min/mg and 6.73 ± 0.69 nmol/min/mg, respectively  AKT1, 2, and 3 were expressed in serum-starved or stimulated HEK293 cells ; survival ; growth (PRAS40)  . α and GSK3β, in similar tissues [α (Ser21) phosphorylation under serum-starved conditions  is a negative regulator of cell proliferation , 32, 33  tissues , and the tissues . Overexp Figures . However Figures , suggestOne mechanism by which AKT modulates cell survival involves phosphorylation and thus inactivation of the FoxO family of transcription factors which regulate the expression of various genes associated with apoptosis, the cell cycle, and DNA repair . AKT dirIn this study, FoxO1 (Thr24) and FoxO3a (Thr32) were robustly phosphorylated in response to serum  modestlyAKT also regulates cell survival by phosphorylating murine double minute 2 (MDM2) at residue Ser166, which results in its translocation into the nucleus and binding to p53, a tumour suppressor that acts as a transcription factor to express proapoptotic genes. The p53/MDM2 complex is then sequestered to the cytoplasm for proteasomal degradation , 41, 42.AKT signals to mTORC1 via two converging pathways to regulate cell growth. One pathway involves the proline rich AKT substrate of 40 kDa (PRAS40) and the other the tuberous sclerosis complex 2 (TSC2) –45. SpecAKT is known to mediate cell growth by signaling through the mTORC1 pathway primarily due to mTORC1 phosphorylation of initiation factor 4E-Binding Protein 1 (4E-BP1) and activation of S6 Kinase 1 (S6K1) that phosphorylates ribosomal protein S6 (rpS6) . mTORC1 The function and activity of 4E-BP1 is modulated by phosphorylation at multiple residues . The 4E-Activation of the mTORC1/S6K1 pathway results in the phosphorylation of rpS6 at Ser235, Ser236, Ser240, and Ser244 , 53. In in vivo phosphorylation patterns of known substrates is largely isoform independent. Some subtle differences were identified including the lack of phosphorylation of GSK3α by myrAKT2 and its reduced phosphorylation of FoxO1/3a (Thr24/32).In conclusion, these experiments reveal that despite the enforced expression of AKT isoforms with very different intrinsic kinase activities towards defined peptide and protein substrate, the resulting To further assess potential differences in the requirement for specific isoforms for substrate phosphorylation we used siRNA approaches to knockdown the expression of each isoform individually and in combination Figures  and 6. Win vivo was also analysed by examining the levels of phosphorylation at the two regulatory residues Ser473 and Thr308 . α and GSK3β); survival ; growth (PRAS40). However, many direct AKT substrates are also phosphorylated by other kinases; for example, GSK3β and MDM2 are also substrates of S6K1, a downstream kinase of mTORC1 [As with the overexpression studies we took a targeted approach focusing on AKT substrates involved in key cellular processes: proliferation  or GSK3β (Ser9) (α (Ser21) and GSK3 (Ser9) phosphorylation [α (Ser21) and GSK3 (Ser9).Specific reduction in AKT isoform expression individually or in combination did not modulate the level of phosphorylated or total GSK3rylation . In summPhosphorylation of FoxO1/3a (Thr24/32) was markedly reduced upon knockdown of each of the three AKT isoforms with AKT1 and 3 being more potent than AKT2 Figures . This isPhosphorylation of MDM2 (Ser166) was not altered upon the individual or compound knockdown of the AKT isoforms (Phosphorylation of PRAS40 (Thr246) was not significantly modulated by knockdown of any individual AKT isoform Figures  althoughConsistent with the modest increase in 4E-BP1 (Thr37/46) phosphorylation induced by all constitutively active AKT isoforms , knockdoin vitro  and FoxO1/3a (Thr24/32) exhibited clear differential regulation by the AKT isoforms. Thus we took a third approach, to identify potential AKT isoform-specific substrates by performing an unbiased screen on cells overexpressing myrAKT1 or myrAKT3 and utilizing 2DGE combined with western blot analysis with the phospho-AKT substrate (PAS) antibody that detects phosphorylation at AKT-specific motifs [These data demonstrate dramatic differences in peptide kinetics for the three AKT isoforms, which were less marked when using a protein substrate  Figures . Howeverc motifs . ConsistAKT consists of a family of three homologous isoforms, AKT1, 2, and 3, whose role in maintaining cellular homeostasis is thought to be pivotal in regulating processes such as cell survival, growth, proliferation, angiogenesis, and metabolism , 63–65. α (Ser21) and MDM2 (Ser166) phosphorylation was shown to be exclusively dependent on AKT2 expression.Recently, potential AKT isoform-specific substrates have come to light, providing one clear mechanism for differential signaling; using loss-of-function studies, Brognard et al.  showed tin vivo. Indeed, this concept is reinforced by the observation that AKT3 had a 5-fold lower affinity than AKT1 towards the rpS7 protein meaning at limiting substrate concentrations, the activity of the isoforms may be very similar. Similar differences in the Km for peptide phosphorylation were observed previously [Here we have addressed the question of whether isoform-specific signaling was due to differences in the intrinsic activity of the isoforms. Previous reports have shown that AKT3 is the most active isoform towards a range of peptide substrates , 24, 25.eviously . in vivo we determined the effect of enforced expression of constitutively active isoforms on the phosphorylation of key validated substrates, GSK3α and GSK3β, FoxO1/3a, MDM2, and PRAS40. These observations were complemented by loss-of-function studies using isoform-specific siRNA reagents. Regulation of the level of phosphorylation of these in vivo substrates, whether by overexpression or specific knockdown of individual AKT isoforms, was extremely subtle, indicating that relative expression levels are likely to underlie the major isoform-specific signaling observed at least in HEK293 cells. Expression of each AKT isoform contributed to the phosphorylation of FoxO1 at residue Thr24, but not at Ser256  and cell survival (via FoxO1/3a). It is important to note that the basis of isoform-specific signaling is likely to vary depending on the type of cell or tissue. Certainly, recent studies suggest that the substrates targeted by different AKT isoforms can vary depending on cellular and tissue context [Taken together, these data indicate that despite exhibiting very different intrinsic enzyme activity,  context . AKT isoforms have been shown to localise to different subcellular compartments in a cell-line-specific manner as described above , 20. It The authors declare that there is no conflict of interests.Supplementary material containing Supplementary Material and Methods, one Supplementary Table of the antibodies used in the study and three Supplementary Figures is available online at dois 10.4061/2011/720985.Click here for additional data file.Click here for additional data file.


The Grb-2 associated binder (Gab) family of scaffolding/adaptor/docking proteins is a group of three molecules with significant roles in cytokine receptor signaling. Gabs possess structural motifs for phosphorylation-dependent receptor recruitment, Grb2 binding, and activation of downstream signaling pathways through p85 and SHP-2. In addition, Gabs participate in hematopoiesis and regulation of immune response which can be aberrantly activated in cancer and inflammation. The multifunctionality of Gab adapters might suggest that they would be too difficult to consider as candidates for “targeted” therapy. However, the one drug/one target approach is giving way to the concept of one drug/multiple target approach since few cancers are addicted to a single signaling molecule for survival and combination drug therapies can be problematic. In this paper, we cover recent findings on Gab multi-functionality, binding partners, and their role in hematological malignancy and examine the concept of Gab-targeted therapy. Gab proteins integrate and amplify signals from a wide variety of sources including growth factor, cytokine, and antigen receptors, as well as cell adhesion molecules. They are subject to complex regulation by feedforward and feedback phosphorylation events as well as protein-protein interactions. Gab proteins range from 50 to 100 kDa in size [ elegans .Gab1 was originally identified as a binding protein for Grb-2 , and GabγRI and is required for fibroblast growth factor-induced tyrosine phosphorylation of this docking protein. The PH domain might play an important role to localize or to concentrate Gab proteins to membrane areas where receptors are activated [The overall sequence homology between Gab family members is about 40–50%. All Gab proteins share a similar modular structure, including a Pleckstrin homology (PH) domain at their N-terminus, proline-rich regions in the central part, and multiple phosphorylated tyrosine residues . The PH ctivated .The phosphotyrosine domains and the proline-rich sequences (PXXP) are potential binding sites for Src homology2 (SH2) and SH3 domains, respectively. The positions of the SH2-binding tyrosine-based motifs and the SH3-binding proline-rich sequence are conserved among the Gabs. These are one of the most prominent motifs in signaling molecules due to their relevance in binding and “docking” phosphorylated tyrosine residues or directing protein-protein interaction , 11.γ [These adapter proteins serve important roles in cytokine receptor signaling by acting as a scaffold and coordinating interactions between signaling intermediates. Multiple protein binding motifs are present in many of the adapter molecules leading to multimeric complexes that may also include proteins such as CrkL , PLC-γ [γ , SHIP [1γ , SHP-2 [γ , STAT3 [γ , and STAγ .As mentioned above, one of the fundamental mechanisms for regulation of Gab-mediated signal transduction is site-specific tyrosine phosphorylation of these proteins. These molecules are involved in the phosphatidylinositol-3 kinase (PI3-K) and mitogen-associated protein kinase (MAPK) pathways and include multiple protein binding sites . To furt−/− mice have decreased Erk activation in response to SCF [Y604F/Y633F mutant fails to bind to Shp2 and blocks IL-3-evoked gene activation [The significance of the phosphorylation sites present on Gabs not only lies in aiding its interaction with crucial binding partners, but also on how it influences activation of downstream cytokine receptors. Interaction between the protein tyrosine phosphatase Shp2 and Gab2 regulates MAPK pathway activation. Notably, mast cells and macrophages from Gab2e to SCF . Gab2-Shtivation , where Gtivation . Anothertivation , 59.It is noteworthy that persistent activation of c-Kit and c-Mpl induces hematological malignancies. Activation of PI-3K by c-Kit is dependent both on the direct PI3K-binding site in c-Kit and on the phosphorylation of Gab2 . The facGiven their integral role in cytokine signaling, it was proposed that Gabs may play important roles in hematopoiesis. However, to date, very little is known about how multiple Gabs regulate hematopoietic cytokine signaling.−/− cells are defective in response to gp130 activation through IL-6 and the soluble IL-6Rα [Gab1 deficiency results in embryonic lethality due to developmental defects in heart, placenta, skin, and a reduced ratio of liver to body weight at E14.5 , 63. Alse IL-6Rα .−/− mice are viable but lack allergic response [in vivo. A recent study has established Gab2 ΔSHP2 (Y603F/Y632F) and Gab2 ΔPI3K (Y441F/Y465F/Y573F) mutant knockin mice [Gab2 is tyrosine phosphorylated at any of 19 sites by several early-acting cytokine receptors such as Flt3, c-Kit, IL-3R, and c-Mpl. Gab2 contains SH2 domain binding sites and 5 PxxP sites (polyProline sites) that bind SH3 domain , 18, 29.response  since Garesponse  in a manresponse . Use of response , 48, 74,kin mice , with Ga−/− mice show reduced colony forming ability in methylcellulose and impaired KLS (c-Kit+ Lin− Sca-1+ cell surface markers denoting mouse hematopoietic stem cell) cell growth in liquid culture [Despite the normal appearance, normal BM cellularity, and normal blood counts of mice lacking Gab2 expression, Gab2 culture . The defIn contrast to Gab1 and Gab2 which have ubiquitous expression in brain, kidney, lung, heart, and ovary , 6, 9, GIt is well established that Gab proteins promote tumor-genesis by functioning as “accomplices” of certain oncoproteins or by amplifying signaling upon their overexpression. This type of “nononcogene addiction” has been described for molecules that become essential in the setting of cancer, but they are not mutated or capable of transformation on their own. In addition to normal cytokine activation, Gab1 and Gab2 can also be activated by oncogenic tyrosine kinases, oncoproteins, and Src family kinases (summarized in in vivo for disease induction. It was shown in recent studies that Gab1 expression increased cell motility and adhesion of myeloid 32D cells in a hepatocyte growth factor (HGF) stimulated setting. In this setting, Gab1 was also seen to up regulate GATA-2, which has been implicated in CML and could be a key player in malignant transformation [The study of Gab1 in Met signaling and cancer has been researched in recent years. Overexpression of Gab1 promotes cell cycle progression when Met is expressed at suboptimal levels. For this response, it is required for Gab1 to possess an intact Met-binding motif, the PH domain, and the binding sites for PI3-K and SHP-2. In this model, Gab1 sufficiently promoted transformation and proliferation of fibroblasts . A role ormation .via SHP2 and PI-3K, which is crucial for enhanced proliferation and survival. Similarly, the oncogenic Bcr-FGFR1 fusion protein, which is also the product of a chromosomal translocation, drives the tyrosine phosphorylation of Gab2 in murine bone marrow cells and their malignant transformation through phospho-Y177 mediated Grb2 association [in vivo. However, the relative contribution of the Gab2/SHP2 interaction for Erk activation versus the reciprocal inactivation of STAT5 is highly complex and difficult to discern [Involvement of Gab2 in leukemogenesis was highlighted when myeloid progenitors from Gab2-deficient mice were found to be resistant to transformation by Bcr-Abl . Phosphoociation . Anotherociation . After tociation , 99. Likociation . This fu discern , 91, 100in vitro study whereby overexpression of Gab2 in human MCF-10A cells using a retroviral vector approach [It should be pointed out that although the focus of this review is on Gabs as interaction partners of oncoproteins involved in the transformation of hematopoietic cells, the most thoroughly described roles have been in solid tumors. Gab2 amplification has been seen in nonhematopoietic cancers such as breast cancer cell lines . Gab2-meapproach  gave simapproach . A partiapproach . A recenapproach , 103.The key to figuring out the role of Gabs in hematological disease is to understand their role in signaling cascades. It is crucial to visualize these as intertwined loops. Firstly, phosphorylation of a particular residue might affect the phosphorylation of a nearby residue in either a positive or antagonistic fashion, due to phosphorylation-induced changes in protein conformation. Secondly, phosphorylation-induced conformational changes may alter the accessibility of key regions, such as the PH domain.S159A mutant displayed transforming properties in fibroblasts and prolonged signaling through the PI-3K/Akt pathway. This might impact downstream STAT5 activity, as it has been shown that constitutively active STAT5 forms a complex with the p85 subunit of the PI3-K and Gab2 in leukemic bone marrow cells, resulting in the activation of Akt [in vitro models and further complement it by in vivo analysis. Negative feedback is deemed critical in shifting Gab2 signaling from Erk and Akt to STAT5. This mechanism may be particularly relevant during conditions of hematopoietic stress such as recovery from myelosuppression or leukemic hematopoiesis. These conditions are more likely to increase Gab2-mediated Erk and Akt signaling, while inhibiting STAT5, whereas blockage or complete deficiency of Gab2 might be expected to have a reverse effect (Negative feedback regulation of Gab2 can be achieved by serine phosphorylation at sites near the PH domain (Akt) or near the SHP-2 binding domain (Erk). Akt can constitutively associate with Gab2, phosphorylate it on a consensus phosphorylation site (Ser159) and inhibit Gab2 tyrosine phosphorylation . Mutation of Akt . Gab2 isn of Akt . Thus, ie effect .Since Gabs bind many common receptors and are much less studied in hematopoiesis, it is pivotal to understand the role of binding to their partners and how it impacts oncogenic tyrosine kinase signaling. Due to the fact that all three family members are potential players in various signaling pathways, it will be interesting to see the shift in approach for targeting the Gab family. As discussed earlier, total knockdown of these proteins has varied physiological and phenotypic impacts. But, it cannot be emphasized enough that the Gab family members are also key regulators/enhancers of other oncogenic proteins and could compensate for the deficit of a family member.α-helix, SAH, is a stapled peptide to p53 that has been shown to prevent p53-MDM4 binding, enabling activation of p53 response and tumor growth suppression both in vitro and in vivo models [Synthetic peptides that specifically bind to directed targets offer an approach for the modulation of receptor signaling and subsequent gene expression. Stabilized o models . Recentlo models . VisualiAn alternative approach to targeting the specific effectors of Gab functional interactions would be to target the entire molecule as a whole. The risk of this approach is that it would be predicted to have widespread effects on Erk and AKT activation, as well as loss of potential negative regulatory functions. The total Gab targeting approach is also complicated by the potential redundancy of Gab1, Gab2, and Gab3 for specific Gab signaling functions. Only through rational dissection of Gab structure-function as related to disease progression will targeted therapy for Gabs become more rationally guided. It is appealing to consider adapter proteins as drug targets from the respect that they have the ability to impact upon multiple key oncogenic signals which may be cooperative. This “killing a flock of birds with one stone” may be optimal over even “killing two birds with one stone,” and only adapter proteins permit widespread impact on receptor-signaling molecule interactions. Therefore, Gabs are of interest in the field of targeted therapeutics and their complete deletion shows therapeutic potential in mouse models; however, drug development for these targets will require moving forward with caution and greater understanding of structure-function relationships.


However, the specific molecular mechanisms underlying RyR2 hyperphosphorylation in HF remain poorly understood. The objective of the current study was to test the hypothesis that the enhanced expression of muscle-specific microRNAs (miRNAs) underlies the HF-related alterations in RyR2 phosphorylation in ventricular myocytes by targeting phosphatase activity localized to the RyR2. We studied hearts isolated from canines with chronic HF exhibiting increased left ventricular (LV) dimensions and decreased LV contractility. qRT-PCR revealed that the levels of miR-1 and miR-133, the most abundant muscle-specific miRNAs, were significantly increased in HF myocytes compared with controls . Western blot analyses demonstrated that expression levels of the protein phosphatase 2A (PP2A) catalytic and regulatory subunits, which are putative targets of miR-133 and miR-1, were decreased in HF cells. PP2A catalytic subunit mRNAs were validated as targets of miR-133 by using luciferase reporter assays. Pharmacological inhibition of phosphatase activity increased the frequency of diastolic Ca2+ waves and afterdepolarizations in control myocytes. The decreased PP2A activity observed in HF was accompanied by enhanced Ca2+/calmodulin-dependent protein kinase (CaMKII)-mediated phosphorylation of RyR2 at sites Ser-2814 and Ser-2030 and increased frequency of diastolic Ca2+ waves and afterdepolarizations in HF myocytes compared with controls. In HF myocytes, CaMKII inhibitory peptide normalized the frequency of pro-arrhythmic spontaneous diastolic Ca2+ waves. These findings suggest that altered levels of major muscle-specific miRNAs contribute to abnormal RyR2 function in HF by depressing phosphatase activity localized to the channel, which in turn, leads to the excessive phosphorylation of RyR2s, abnormal Ca2+ cycling, and increased propensity to arrhythmogenesis.In heart failure (HF), arrhythmogenic spontaneous sarcoplasmic reticulum (SR) Ca Two phosphatases have been shown to scaffold to the RyR2 complex, PP1 and PP2A. While PP1 has been identified as the phosphatase controlling RyR2 phosphorylation at the PKA site Ser-2808 2+ release and increases CaMKII-dependent phosphorylation of RyR2 by targeting the regulatory subunit of PP2A B56α We have previously reported that phosphatase activity in the vicinity of the RyR2 channel can be regulated by the muscle-specific microRNA (miRNA) 1 (miR-1) 2+-dependent afterdepolarizations. To examine the effects of reduced phosphatase activity on Ca2+ cycling and membrane potential, the PP1 and PP2A inhibitor Calyculin A 2+ waves (SCW) and delayed afterdepolarizations (DADs)  . Howeverhttp://www.targetscan.org), showed significant decreases in expression levels in HF vs. control myocytes  in the presence of Iso (100 nM). In stark contrast to control myocytes, HF myocytes exhibited spontaneous Ca2+ waves that caused delayed and early afterdepolarizations  on spontaneous Ca2+ waves induced on inhibition of PP2A in paced control myocytes from normal hearts in the presence of Iso. The inhibition of CaMKII in these experiments reversed the increase in arrhythmogenic propensity caused in myocytes by a combination of Iso and PP2A inhibition.  and Calyculin + KN93 (n = 12), respectively, *statistically significant at p<0.05, Student's t-test). Thus, these results strongly support the central role of CaMKII-dependent phosphorylation in HF-induced Ca2+-dependent arrhythmogenesis.To investigate the possible involvement of CaMKII in arrhythmogenesis in myocytes isolated from failing hearts we examined the effects of the specific CaMKII inhibitory peptide, ACI izations . Notably2+ release and altered expression levels of two major muscle-specific miRNAs, miR-1 and miR-133 in a canine model of chronic HF. Our main finding is that the increased expression of miR-1 and miR-133 observed in HF was associated with reduced levels of PP2A regulatory (B56α and B56δ) and catalytic subunits. The dissociation of PP2A activity from the RyR2 complex in HF resulted in CaMKII-dependent hyperphosphorylation of the RyR2 and profound pro-arrhythmic disturbances in Ca2+ cycling and membrane potential. Our results suggest that, in chronic HF, increased miR-1 and miR-133 expression levels lead to abnormal myocyte Ca2+ handling through disruption of site-specific PP2A phosphatase activity.In the present study, we have investigated the possible link between enhanced predisposition of ventricular myocytes toward pro-arrhythmic spontaneous Ca2+ release during β-adrenergic stimulation; (2) enhanced levels of miR-1 and miR-133 in HF myocytes were associated with (a) decreased expression of B56α and B56α known to be involved in subcellular targeting of PP2A and (b) reduced total expression levels of catalytic subunits of PP2A, reduced total PP2A activity and reduced levels of PP2A catalytic subunits associated with the RyR2; (3) in HF, the miRNA-associated reduction in PP2A levels led to significant increases in RyR2 phosphorylation at the PP2A-dependent sites Ser-2814 and Ser-2030; and (4) the functional consequences of pharmacological inhibition of phosphatases in control myocytes and of the miRNA-related decreases in total and localized PP2A activity in HF myocytes were reversed by CaMKII inhibitors.Our conclusions regarding the role of miR-1 and miR-133 in HF are supported by the following specific experimental findings: (1) pharmacological inhibition of PP2A activity in control myocytes was sufficient to promote proarrhythmic spontaneous CaHF is associated with pathological remodeling of ion channels and intracellular signaling pathways, including altered phosphorylation of RyR2s 2+ handling.Recent studies have documented the importance of miRNAs in a number of pathological processes in the cardiovascular system, including cardiac arrhythmias 2+-release 2+ cycling were mimicked by broad pharmacological inhibition of phosphatases . A total of eight hound type dogs of either sex (2–3 years of age) had a right ventricular (RV) pacemaker lead implanted in the RV apex as previously described 3, 5 creatine, 5 taurine, 10 U/ml penicillin, 10 µg/ml streptomycin, and 10 µg/ml gentamycin (pH 7.3). Cells were cultured at 37°C in a humidified atmosphere with 95% air/5% CO2. After 2 hours, unattached cells were removed and myocytes were infected with adenoviruses at multiplicity of infection (MOI) 100 and cultured for 36–48 hours before analysis. At such conditions culture-dependent changes in myocyte structure and function are minimal miR-1-2 and miR-133a gene, into 293 cells using Lipofectamine . Through homologous recombination, the test genes integrate into the E1-deleted adenoviral genome. The viruses were propagated in 293 cells and purified using CsCl2 banding, followed by dialysis against 20 mM Tris buffered saline with 2% glycerol. Tittering was performed on 293 cells overlaid with DMEM plus 5% equine serum and 0.5% agarose.Dogs were euthanized with intravenous sodium pentobarbital or sodium thiopental, followed by rapid removal of the heart as previously described 2, 0.5 MgCl2, 10 HEPES, and 5.6 glucose (pH 7.4). Patch pipettes were filled with the following solution (mmol/L): 90 K-aspartate, 50 KCl, 5 MgATP, 5 NaCl, 1 MgCl2, 0.1 Tris GTP, 10 HEPES, and 0.06 Fluo-3  (pH 7.2). The APs were evoked by application of 4-ms-long voltage pulses with amplitude 50% above the threshold level. Intracellular Ca2+ imaging was performed using an Olympus Fluoview 1000 and Leica SP5 confocal systems. Fluo-3 was excited by the 488 nm line of an argon-ion laser, and the fluorescence was acquired at wavelengths >510 nm. Rhod-2 was exited by 543 nm laser and the fluorescence was acquired at 590–690 nm wavelengths.Action potentials (APs) were recorded using the whole-cell patch clamp technique. The patch-clamp system was based on Axopatch 200B amplifier and DIGIDATA 1322A interface . The external solution consisted of (mM): 140 NaCl, 5.4 KCl, 2.0 CaCl2. Cell lysate proteins were subjected to 4% to 20% SDS-PAGE and blotted onto nitrocellulose membranes . Primary antibodies used: anti-phospho-RyR2-S2808 and anti-phospho-RyR2-S2030 were raised against CRTRRIS(PO4)QTSQ-CONH2 and (CG)TIRGRLLS(PO4)LVEKVTYLKK-CONH2, respectively . Anti-phospho-RyR2-S2814 was a gift from Dr. X. H. Wehrens . For RyR2 phosphorylation, cells were studied before being lysed and were periodically field stimulated at 1 Hz for 1 min in Tyrode solution. Anti-RyR2, was from ThermoFisherScientific; anti-PP2A B56α, from Millipore ; anti-PP2A B56δ from Abcam (Cambridge MA); anti-PP2AC from Calbiochem ; anti-GAPDH used as a loading control was from Abcam . For coimmunoprecipitation the samples were incubated with protein A Sepharose beads at 4°C for 1 h, after which the beads were washed three times with buffer. Proteins (40–60 µg) were separated on SDS PAGE and blotted onto nitrocellulose membranes. Membranes were incubated for 1–2 hour at room temperature with primary anti-RyR2, anti-B56α or anti-PP2AC antibodies. Protein bands were visualized using the Super Signal West Pico kit .Western blot analyses were performed as previously described 2+ and ATP. The amount of phosphorylated substrate was measured by binding with peroxidase conjugate of antibody that specifically detects phosphorylated substrate. The color was quantified by measuring absorbance at dual wavelength of 450/540 nm using microplate reader .Local CaMKII activity was measured in immunoprecipitated RyR2s samples using Cyclex non-radioisotopic kit according to manufacturer's instructions . To perform the relative semi-quantitative immunoassay, the samples (10–20 µg) were diluted in Kinase Buffer, pipetted into microplate wells precoated with CaMKII substrate Syntide 2 and allowed to phosphorylate the bound substrate in the presence of MgTissue samples from the left-ventricle free wall were homogenized in buffer: 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 10% glycerol containing 1 mM DTT, 1 mM PMSF with complete protease inhibitors  using Tissue Tearor . The homogenate was centrifuged at 15,000×g for 15 min. The clear supernatant was depleted of free phosphate by using Sephadex G-25 resin spin columns . Protein Phosphatse 2A activity was determined using the serine/threonine phosphatase assay system microplate assay kit , which determines the amount of free phosphate generated in a reaction by measuring the absorbance of a molybedate: malachite green: phosphate complex. Briefly, appropriate phosphate standards diluted in phosphate-free water, 5× PPase reaction mix and 1 mM phosphopeptide were added to a flat-bottom 96-well plate . Reaction was initiated by adding protein sample (15–30 µg) following incubation at room temperature for 15–20 min. The reaction was stopped by adding equal volume (50 µl) of Molybedate dye, followed by incubation for 15–30 min at room temperature. Optical density of the molybedate: malachite green: phosphate complex was read using a plate reader with a 630 nm filter. Specific activity is expressed as picomoles of phosphate released/min/µg protein.5′- TGA AAT TTT AAA CTT GTA CAG TAT TG -3′) and antisense (5′- GGT GAA TGT ACA TAA GAC TAA ATC -3′) primers. A 599-bp fragment encompassing the majority of the PP2A catalytic subunit β (PP2ACβ) 3′-UTR was PCR-amplified utilizing the following sense (5′- ATT TCT CCT GGG AAA CCT GCC TTT G -3′) and antisense (5′- GTT TAT TTG CTG AGT ACA CCA AAT AGG -3′) primers. Both were amplified using standard procedures and a proofreading polymerase . Human male genomic DNA (Promega) was used as template. Following the manufacturer's protocol, the PCR product was treated for 10 minutes with Taq polymerase. The PCR product was subsequently subcloned into the pCR™2.1 vector following the manufacturer's protocol (Invitrogen). Plasmid DNA was subsequently isolated from recombinant colonies and sequenced to ensure authenticity. The PP2ACα and PP2ACβ 3′-UTR inserts were removed from the pCR™2.1 plasmid by EcoRI digestion. The fragments were subsequently gel purified, filled in and blunt-end ligated into a filled-in XhoI site that is located downstream of the Renilla luciferase (r-luc) reporter gene . The authenticity and orientation of the inserts relative to the Renilla luciferase gene were confirmed by dideoxy sequencing. The resulting recombinant plasmids were designated, psiCHECK/PP2ACα and psiCHECK/PP2ACβ.A 1312-bp fragment encompassing the entire PP2A catalytic subunit α (PP2ACα) 3′-untranslated region (3′-UTR) was PCR-amplified utilizing the following sense (CCT GGT TTA GAT GTG CCA TAT AAA AAT -3′5′- AAC TTG TTT TCA CAT ) and a complementary reverse miR-133 mutagenic primer (AA ACC AGG ATG TGA AAA CAA GTT-3′5′- ATT TTT ATA TGG CAC ATC T), were synthesized and utilized in a PCR experiment as described by the manufacturer. The mutant reporter construct psiCHECK/PP2ACβ-mut was generated by utilizing the psiCHECK/PP2ACβ plasmid as template and mutating the putative miR-133 recognition site (located at 139–145 bp) harbored in the PP2ACβ 3′-UTR using the QuikChange site-directed mutagenesis kit (Stratagene). Briefly, a forward miR-133 mutagenic primer (CC TGG TTT TGT GCC ATA CTA ATG ATG -3′5′- CAT TAA ACC ACA TCA T) and a complementary reverse miR-133 mutagenic primer (AAA CCA GGA TGA TGT GGT TTA ATG -3′5′- CAT CAT TAG TAT GGC ACA ), were synthesized and utilized in a PCR experiment as described by the manufacturer. Mutated sequence is shown in bold print. The amplification reactions were treated with DpnI restriction enzyme to eliminate the parental template and the remaining DNA was used for transformation. The mutation of the miR-133a-1 seed binding sites was confirmed by dideoxy chain termination sequencing. Finally, transformed bacterial cultures were grown and each reporter construct was purified using PureLink™ Hipure Plasmid Maxiprep Kit (Invitrogen).The mutant reporter construct psiCHECK/PP2ACα-mut was generated by utilizing the psiCHECK/PP2ACα plasmid as template and mutating the putative miR-133 recognition site (located at 122–128 bp) harbored in the PP2ACα 3′-UTR using the QuikChange site-directed mutagenesis kit (Stratagene). Briefly, a forward miR-133 mutagenic primer  and a luminometer. Renilla luciferase expression in the psiCHECK vector is generated via an SV40 promoter. Additionally, the psiCHECK-2 vector possesses a secondary firefly reporter expression cassette which is under the control of the HSV-TK promoter. This firefly reporter cassette has been specifically designed to be an intraplasmid transfection normalization reporter; thus when using the psiCHECK-2 vector, the Renilla luciferase signal is normalized to the firefly luciferase signal.CHO cells were purchased from the American Type Culture Collection  and maintained in Dulbecco's modified Eagle's medium  supplemented with 10% fetal bovine serum , 1× antibiotic-antimycotic, and 0.0175 mg/ml L-proline(Sigma). miR-133a-1 and negative control mimics  were obtained from Dharmacon . Transfection of CHO cells with small RNAs was optimized utilizing Lipofectamine 2000 (Invitrogen) and a fluorescein-labeled double-stranded RNA oligomer designated BLOCKiT™ (Invitrogen). Once conditions were optimized, CHO cells (approaching 100% transfection efficiency) were transfected with the luciferase reporter constructs described above and the appropriate miRNA precursor as indicated. After 24 hrs, CHO cells were washed and lysed with Passive Lysis Buffer (Promega), and firefly and −ΔΔCt relative quantification method t was calculated by subtracting their average Ct value from the average Ct value of the small noncoding RNA, RNU48.Total RNA was isolated from canine control and HF myocytes with Trizol reagent (Invitrogen). The RNA was subsequently treated with RNase-free DNase I, and mature miR-1 and -133 were quantified by utilizing TaqMan microRNA assay kits specific for each miRNA  as previously described 2+ measurements and biochemical analyses were determined by one-way ANOVA and Student's t-test where appropriate and presented as mean ± SE.Echocardiographic data are presented as mean ± SD. Differences in echocardiographic parameters were evaluated by unpaired t-tests. Statistical significance of Ca


Activation and signal transduction in the Nucleotide binding, leucine-rich repeat containing receptor (NLR) family needs to be tightly regulated in order to control the inflammatory response to exogenous and endogenous danger signals. Phosphorylation is a common cellular mechanism of regulation that has recently been shown to be important in signalling in another family of cytoplasmic pattern recognition receptors, the RIG-I like receptors. In addition, single nucleotide polymorphisms can alter receptor activity, potentially leading to dysfunction and/or a predisposition to inflammatory barrier diseases.We have computationally analysed the N-terminus of NOD1 and found seven theoretical phosphorylation sites in, or immediately before, the NOD1 Caspase Activation Domain (CARD). Two of these, serine 7 and tyrosine 49 are also found as rare polymorphisms in the African-American population and European-American populations respectively. Mutating serine 7 to either an aspartic acid or an asparagine to mimic the potential impact of phosphorylation or the polymorphism respectively did not affect the response of NOD1 to ligand-mediated NFκB signalling.The NOD1 polymorphism S7N does not interfere with receptor function in response to ligand stimulation. Helicobacter Pylori was submitted to the NetPhos 2.0 server  for the http://evs.gs.washington.edu/EVS/).Consensus sequence images were generated using WebLogo v3.3 . MoleculpUNO-NOD1 encodes full-length untagged human NOD1 and was a kind gift from Dr Peter Murray. Point mutants S7N, S7D and E56K were generated in the pUNO-NOD1 vector by site-directed mutagenesis. The plasmids pLuc and phrG (Promega) are components of the dual-luciferase assay and encode Firefly and Renilla luciferase respectively.2. Assays were performed in 96-well plates and 0.1 ng/well of the appropriate NOD1 plasmid was transfected using jetPEI (Polyplus Transfection) along with 1 ng/well of pLuc and phrG. Stimulation with up to 1000 ng/ml of ie-DAP (Invivogen), or 100 ng/ml of the control ligand iE-Lys (Invivogen) was performed simultaneously with transfection. 24 h later cells were lysed with 1 × passive lysis buffer (Promega) and luminescence measured with a FLUORstar Luminometer (BMG Labtech). Statistical analysis was performed on the data using an unpaired t-test with Welch’s correction for unequal variances. Protein expression was checked 24 h after transfection of HEK293 cells with 3 μg/DNA per well in a 6 well plate in the absence of stimulation. Proteins were visualised by western blot using the NOD1 monoclonal antibody 2A10 [HEK293 cells were maintained in DMEM (Sigma) supplemented with 10% FCS, 100 μg/ml Penicillin/Streptomycin and 2 mM L-glutamine at 37°C and 5% COody 2A10  and antiThis work did not involve human subjects, human material or human data and therefore does not require ethical approval.Potential serine, threonine and tyrosine phosphorylation sites in and immediately adjacent to the CARD of the NLR family member NOD1 were identified using the prediction software NETPhos 2.0. Within the first 120 residues of human NOD1 (CARD structure 19-110) there were four predicted serine phosphorylation sites ; one predicted threonine phosphorylation site (residue 37); and two predicted tyrosine phosphorylation sites (residues 49 and 97) . In contrast, the mutation E56K removed the ability of NOD1 to respond to ligand stimulation consistent with previous studies (p < 0.0005)  , numerous other residues were also present. These were: glycine (5/36), arginine (4/36), histidine (4/36), cysteine (1/36) and isoleucine (1/36) (Table This work has identified residues that may be potentially phosphorylated in the NOD1 CARD and which consequently have the potential to modulate receptor activity in a manner analogous to that seen with RIG-I and MDA-5. It also shows that mutation of one of these residues, serine 7, to either an aspartic acid to mimic phosphorylation, or to asparagine to reflect the natural polymorphism S7N does not affect receptor function. We can therefore conclude that the SNP S7N has no impact on NOD1 activation and signalling and that serine 7 is not involved in regulation of NOD1 signalling.CARD: Caspase activation domain; MAVS: Mitochondrial antiviral signalling protein; MDA-5: Melanoma differentiation-associated protein 5; PKC: Protein kinase C; PRR: Pattern recognition receptor; NFκB: Nuclear factor kappa B; NLR: Nucleotide binding, leucine-rich repeat containing receptor; NLRC4: Nucleotide binding, leucine-rich repeat containing receptor, with a CARD 4; NLRP3: Nucleotide binding, leucine-rich repeat containing receptor with a pyrin 3; NOD1/2: Nucleotide oligomerisation domain containing 1/2; RIG-I: Retinoic acid inducible gene I; SNP: Single nucleotide polymorphism.The authors declare that they have no competing interests.SM performed the reporter assays and western blots and edited the manuscript. TPM designed the experiments, performed the bioinformatic analyses, and wrote the manuscript. Both authors read and approved the final manuscript.


The spike glycoprotein of IBV is essential for host cell attachment, neutralization, and is involved in the induction of protective immunity. Previously obtained sequence data of the spike gene of IBV QX-like and Massachusetts strains were subjected to bioinformatics analysis.Infectious bronchitis virus (IBV) is a Gammacoronavirus of the family COn analysis of potential phosphorylation sites, the Ser542 and Ser563 sites were not present in Massachusetts strains, while QX-like isolates did not have the Ser534 site. Massachusetts and QX-like strains showed different cleavage site motifs. The N-glycosylation sites ASN-XAA-SER/THR-55, 147, 200 and 545 were additionally present in QX-like strains. The leucine-rich repeat regions in Massachusetts strains consisted of stretches of 63 to 69 amino acids, while in the QX-like strains they contained 59 amino acids in length. An additional palmitoylation site was observed in CK/SWE/082066/2010 a QX-like strain. Primary structure data showed difference in the physical properties and hydrophobic nature of both genotypes. The comparison of secondary structures revealed no new structural domains in the genotypic variants. The phylogenetic analyses based on avian and mammalian coronaviruses showed the analysed IBV as closely related to turkey coronaviruses and distantly related to thrush and munia coronaviruses.The study demonstrated that spike glycoprotein of the Massachusetts and the QX-like variants of IBV are molecularly distinct and that this may reflect in differences in the behavior of these viruses in vivo. Coronaviridae and to genus of Gamma-coronavirus group 3[Infectious bronchitis virus (IBV) causes avian infectious bronchitis, a highly contagious disease that affects poultry and produces severe economic losses worldwide. IBV belongs to the order of Nidovirales, family  group 3. The gen group 3-4.The Massachusetts strain was initially isolated in 1940 in USA and clinically characterized as causing respiratory signs and decrease in egg production. It is widely distributed and predominant in many countries around the world. The MasO) that is later cleaved into the N-terminal S1 and C-terminal S2 glycopolypeptides. The spike gene is highly variable, especially the S1 part, due to insertions, deletions, substitutions and recombination events[The spike glycoprotein of the virus is translated as a pre-cursor protein  and emerging (QX-like) genotypes of IBV to indentify differences in potential functional motifs of the spike glycoprotein, predicted by bioinformatics analysis. In addition an evolution analysis with regards to avian and mammalian coronavirus was performed.The loss or acquisitions of N-glycosylation sites in the spike protein were predicted. Thirty N-glycosylation sites were found in the Massachusetts strains, while 35 N-glycosylation sites were present in the QX-like strains. The N-glycosylation sites ASN-XAA-SER/THR-55, 147, 200 and 545 were additionally present in spike gene of QX-like strains. The results revealed that most of the N-glycosylation sites were conserved within genotype in the analyzed strains, except in strain CK/SWE/082066/10 that had lost ASN-XAA-SER/THR-533.The analyses revealed that there were different in number and location of potential phosphorlated peptides in the spike glycoprotein. In Massachusetts isolates phosphorylation sites at position Ser54, Ser79, Ser312, Ser404, Ser534, Ser618, Ser685, Ser908 and Ser1061 were found to be conserved. While two isolates CK/SWE/242/95 and CK/SWE/748/95 that did not contain phosphorylation sites at position Ser542 and Ser563. In contrast, they contained additional potential phosphorylation sites at Ser177 and Ser878. QX-like strains did not contain a potential phosphorylation site at position Ser534 and acquired a new potential phosphorylation site at position Ser513.The cleavage site motifs of analyzed strains are presented in Table Leucine-rich repeat (LRR) regions were observed in the spike glycoprotein consisting of 59 to 69 amino acids in length. In isolate CK/SWE/748/95 two LRR regions were found, starting at position at 771 (ATQLQARNNAL) and 1147 (LEKLSILKTYI), respectively. The LRR regions in Massachusetts strains consisted of stretches of 63 to 69 amino acids, while in the QX-like strains they contained 59 amino acids in length.The isolates CK/SWE/748/95, CK/SWE/1096/97 and CK/SWE/062545/09 contained 10 palmitoylation sites in the spike glycoprotein of the virus. The isolate CK/SWE/082066/2010 contained additional palmitoylation site 1130 (CGGCFGI) in comparison to the all other analyzed isolates.The data regarding the primary structures are presented in  SequIn silico technologies.A genetic analysis of the complete spike glycoprotein of selected isolates of Massachustes and QX-like strains was carried out in order to predict molecular features by application of molecular and Coronaviridae, lactate dehydrogenase elevating virus (LDEV), the loss or acquisition of N-glycosylation sites in the protein has been reported to determine changes of virulence and cellular tropism[In the isolate CK/SWE/082066/10, amino acid phenylalanine substituted asparagine resulting in the loss of a potential N-glycosylation site at position 533. The five additional N-glycosylation sites found in QX strains may influence cellular tropism. The variation in N-glycosylation sites may affect survival and transnsmission of the virus. The small changes can lead to disturbance in folding and conformation of the molecule. The variations in N-glycosylation sites may affect the interaction with receptors and render a virus more susceptible to host innate immune system and lower recognition by antibody, hence affecting the virus replication and infectivity-42. In a tropism.The presence of serine, threonine and tyrosine residues, with the right context for potential phosphorylation was investigated in the spike glycoprotein. The results have shown that the spike glycoprotein of IBV genotypes contained serine and thereonine peptides with potential for phosphorylation. The substitution of a serine by amino acid alanine at positions 563 caused loss of a potential phosphorylated peptide in strains CK/SWE/242/95 and CK/SWE/748/95. Wilbur et al. (1986) reported that N protein is the only structural protein that contained phosphorylation activity in coronaviruses. In thisThe difference in cleavage site motifs of the spike glycoprotein of both genotypes, seem to indicate a correlation with geographical distribution of strains. It has been reported that serotypically and genotypically different IBV contain distinct cleavage site in the spike protein. However, the cleavage site sequence did not correlate with pathogenicity by comparing cleavage recognition sites in pathogenic and attenuated strains of IBV. The cleThe data regarding leucine-rich repeat regions of isolate CK/SWE/748/95 demonstrated that two LRR regions may result in strong folding capacity in the spike glycoprotein. It has been reported that leucine-rich repeat (LRR) is a structural motif of protein like a alpha/beta horseshoe fold and is responsible for protein-protein interactions,38.An additional palmitoylation site was found only in isolate CK/SWE/082066/10, indicating that it is strain specific. However, experimental studies are required to identify the effect of additional palmitoylation in the virus biology. In influenza virus, palmitoylation of the cytoplasmic domain of the hemagglutinin has been shown to increase its association with lipid mass. The delThe predicted primary structures of the spike glycoprotein of both genotypic variants have shown a variable trend in physical properties such as isoelectric point, molecular weights and grand hydropathicity index. Grand average of hydropathicity index results suggested a hydrophobic nature of the spike glycoprotein in all variants. Cavanagh et al. (1986) reported that S2 sub-unit in N-terminal side of the membrane spanning hydrophobic domain of spike glycoprotein of IBV is susceptible to hydrolysis by many proteases.The secondary structures prediction revealed that structures of the spike glycoprotein were found variable in both genotypic variants. It was also observed that there were no new structural domains and that no evolution has occurred in the structure of both genotypic variants. However, secondary structural variation in beta strands and residual coils have been observed between both genotypic strains.A number of phylogenetic studies performed on the spike gene were mostly based on the partial sequences of S1 and N gene. Phylogenetic analyses based on the complete spike glycoprotein of avian coronaviruses demonstrated that IBV strains showed higher relatedness to turkey coronaviruses in comparison to other avian coronaviruses  infidelity of RNA-dependant RNA polymerase which lead to incorporation of nucleotide errors during replication,57; b) rIn silico predictions confirm previous data on phylogenetic analysis, showing that the spike glycoprotein of the Massachusetts and QX genotypes are molecularly different. Since the spike glycoprotein has important role in cell attachment, receptor recognition, the genetic diversity seen in this protein of IBV, avian, human, bat, and other animal coronaviruses may bear relation with host specificity. The identification of these characteristics will be foundation for the better understanding of epidemics, molecular mechanisms of infections, evolution and basis for development of effective vaccines. Bioinformatics approach is easy to perform, cheap and suitable to obtain predictions about functional motifs in a genome. However, the predicted findings require validation through proper laboratory experimentation.The sequence data set generated as previously described by Abro et al. (2012) was usedhttp://www.cbs.dtu.dk/services/NetNGlyc. The potential phosphorylation sites were determined by using websitehttp://www.cbs.dtu.dk/services/NetPhos. Leucine-rich repeat regions were determined by LRRfinder available athttp://www.lrrfinder.com/result.php. Antigenic determinants were predicted with the maximum score with the high confidence for positive determinant motif in the amino acid sequence with the help of serverhttp://mobyle.pasteur.fr. The primary structures of the spike glycoprotein were predicted byhttp://expasy.org/tools. Palmitoylation sites were predicted with the medium threshold frequency by using services athttp://csspalm.biocuckoo.org/prediction.php. The secondary structures  of the protein were predicted by using bioinformatics tools available on websitehttp://npsa-pbil.ibcp.fr. The method of GOR4http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_gor4.html was used to identify the alpha helices, beta strands and coil residues. At least minimum three or more turns were taken in account for one helix, strand or coil in the structure of spike glycoprotein.N-glycosylation sites were predicted by services available onThe comprehensive phylogenetic study was carried out based on the complete spike gene of avian and mammalian coronaviruses available in GenBank. The sequences included IBV Massachusetts type, M41, H120, Baudette, Arkansas, QX like strains and other sequences of Turkey coronavirus (TCoV), thrush coronavirus (ThCoV), munia coronavirus (MuCoV), human coronavirus (HCoV), SARS (Severe acute respiratory syndrome) coronavirus, bovine coronavirus (BCoV), equine coronavirus (ECoV), mouse hepatitis virus (MHV), murine coronavirus (MurCoV), feline coronavirus (FCoV), porcine epidemic diarrhea virus (PEDV), porcine respiratory coronavirus (PRCV), porcine hamagglutinating encephalalomylitis virus (PHEV), bat coronavirus and beluga whale coronavirus sequences (Additional fileThe authors have declared that they have no competing interests.Study concepts and design: SHA, CB Performance of experiments: SHA, KU Analysis of the data: SHA, CB Writing of the manuscript: SHA, KU, SB, CB. The authors have read and approved the final manuscript.Predicted primary structures of spike glycoprotein.Click here for filePredicted secondary structures of spike glycoprotein.Click here for fileList of selected Swedish IBV strains and reference strains of avian coronaviruses of which sequences were downloaded from GenBank for the phylogenetic analysis in this study. The IBV strains analyzed in this study are indicated by star *.Click here for fileList of reference strains of mammalian coronavirus sequences were downloaded from GenBank for the phylogenetic analysis in this study.Click here for file


The aim of this in vitro study was the evaluation of the mechanical properties the screws for rapid maxillary expansion (RME).post-hoc test di Tukey (P < 0.05).Three commercially available screws for RME were tested: Leone A2620; Dentaurum Hyrax; Forestadent Palatal Split Screw. All expansion screws were 10 mm in size. For the evaluation of mechanical properties, the screws for RME were adjusted using the same maxillary dental model. An Instron 3365 testing machine with a load cell of 5 kN recorded the forces released by the screws at different amounts of activation . Each type of screw was tested 10 times. Comparisons between the forces released by the different types of screws at different amounts of activation were carried out by means of analysis of Kruskal-Wallis test with The results of this study showed that all 3 expansion devices were able to develop forces that could produce a separation of the palatine processes. The Hyrax and A2620 expanders developed force values over 20 kg and the Palatal Split screws about 16 kg. Both the A2620 and Hyrax expanders showed significantly greater amounts of forces at all the different amounts of activations with respect to the Palatal Split screw.All tested devices showed the capability of developing expansion forces (16-20 kg) adequate for RME. The A2620 and Hyrax expanders showed a greater level of rigidity than the Palatal Split screw. Rapid maxillary expansion has achieved a prominent role in modern orthodontics as a safe, predictable, and effective way to correct maxillary deficiency on the transverse plane in a wide range of clinical conditions -6. From Forces produced by this appliance have been reported in the range of 16.6 to 34.8 pounds (7.54 to 15.8 kg) .The clinical management of RME is well established and only few differences characterize the various expansion protocols, such as the number of turns of the midline screw (activation rate of the screw), i.e. rapid or slow expansion, appliance design (banded or bonded acrylic expander), and anchorage on deciduous or permanent teeth .Currently, several protocols have been proposed that allow either to operate a classic rapid expansion or to alternate rapid expansion and rapid constriction to activate the craniofacial sutures -15. The in vitro investigations concerning the forces released by the expansion screw during activation phases. Muchitsch et al. [in vitro study, therefore, was to analyze the compression strain developed at each activation of three types of screw for RME (body with arms) utilizing a rigid support.The literature  is very h et al.  analyzedThe study used an experimental model reproducing the maxillary dental arch with the palate to adapt all the screws and to standardize the position of the screw body at the same distance from the palate.We analyzed the stiffness of 3 screws for RME:•A2620 rapid expander  Figure A;•Hyrax  Figure B;•Palatal Split Screw   or orthogonal  to the screw guides. All tested screws were 10 mm in size and a full activation turn was equivalent to an expansion of 0.8 mm (4 activations × 0.2 mm). All tested screws presented the arms laser-welded at the body. In order to evaluate the rigidity of the device, the bending of the arms of the three screws was standardized to replicate a clinical setting  with a load cell of 5 kN  was calculated. Preliminary analysis of the data revealed that normal distribution (Kolmogorov-Smirnov test) and/or equality of variances (Levene’s test) could not be assessed for all the variables. Therefore, statistical comparisons were made by means of nonparametric tests to compare the loading capacity to the different activations within the same expander (Friedman test with Tukey ‘s post-hoc test) and between the 3 different expanders  .The power of the study for one-way ANOVA was calculated on the basis of the sample size of 10 screws, an alpha level of 0.05, with an effect size 1.5 . The powThe descriptive statistics and statistical comparisons of the forces generated by the 3 types of expander during the tests are shown in the Table A total of twenty activations for each expander were carried out. The limit of 20 activations was chosen because deformation of the expansion key occurred in all the three types of expander after this amount of activation (after 9–10 activations for the A2620 and Hyrax expanders). As for the comparisons within the same type of expander  for RME. The present study showed that the greater is the stiffness of the expander (see the activation screw/force curve) Figure , the greThe results of this study showed that all 3 devices succeeded in developing enough expansion force to cause a separation of the palatine processes, such as over 20 kg force for both the Hyrax and the A2620 expander and about 16 kg for the Palatal Split Screw. Thus, all expanders showed appropriate clinical skills. In addition there was a very low variation of the values obtained in each group.The between-group statistical comparison showed a significantly greater stiffness of both the A2620 and the Hyrax expanders, compared to the Palatal Split Screw. Such devices are those that should transfer better the force of the activation of the screw to the teeth and consequently to the bony structures, thus potentially reducing the risk of dentoalveolar tipping.In all cases the force-activation curve had a similar trend, with an almost linear behavior in the first 10 activations, which decreased and became null at the maximum number of activations. Both the A2620 and the Hyrax expanders showed a trend in the developed force that increased up to about 18–20 activations, after which a “plateau” was reached, while the Palatal Split Screw succeeded in increasing the force up to 24 activations , that cannot be reached in a clinical setting. The A2620 and Hyrax expansion screws showed a greater rigidity compared to the Palatal Split Screw.The authors declare that they have no competing interests.MC have made substantial contributions to conception experimental design and acquisition of data; LF participated in the design of the study, performed the statistical analysis, and edited the text; TD performed the experiments; ED revised it critically for important intellectual content; and all of the authors read and approved the final version of manuscript.MC Research Fellow, Department of Surgery and Translational Medicine, University of Florence, Florence, Italy.LF Assistant Professor, Department of Surgery and Translational Medicine, University of Florence, Florence, Italy; Thomas M. Graber Visiting Scholar, Department of Orthodontics and Pediatric Dentistry, School of Dentistry, The University of Michigan, Ann Arbor.TD Assistant Professor, Department of Orthodontics, University of Siena, Siena, Italy.ED Associate Professor, Department of Surgery and Translational Medicine, University of Florence, Florence, Italy.


D = 0.003 μM) with approximately 2 orders of magnitude greater affinity than SAP (KD = 0.44 μM). RNA interference in cytotoxicity assays in NK92 cells showed dependence of CRACC on SAP in addition to EAT-2, indicating selectivity of SAP and EAT-2 may depend on the relative concentrations of the two adaptors. The concentration of SAP was four fold higher than EAT-2 in NK92 cells. Compared with SAP, the significance of EAT-2 recruitment and its downstream effectors are not well characterised. We identified PLCγ1 and PLCγ2 as principal binding partners for the EAT-2 tail. Both PLCγ1 and PLCγ2 are functionally important for cytotoxicity in NK92 cells through CD244 (SLAMF4), NTB-A (SLAMF6) and CRACC. Comparison of the specificity of SH2 domains from activating and inhibitory signalling mediators revealed a hierarchy of affinities for CD244 (SLAMF4) ITSMs. While binding of phosphatase SH2 domains to individual ITSMs of CD244 was weak compared with SAP or EAT-2, binding of tandem SH2 domains of SHP-2 to longer peptides containing tandem phosphorylated ITSMs in human CD244 increased the affinity ten fold. The concentration of the tyrosine phosphatase, SHP-2 was in the order of a magnitude higher than the adaptors, SAP and EAT-2. These data demonstrate a mechanism for direct recruitment of phosphatases in inhibitory signalling by ITSMs, while explaining competitive dominance of SAP and EAT-2.SLAM family receptors regulate activation and inhibition in immunity through recruitment of activating and inhibitory SH2 domain containing proteins to immunoreceptor tyrosine based switch motifs (ITSMs). Binding of the adaptors, SAP and EAT-2 to ITSMs in the cytoplasmic regions of SLAM family receptors is important for activation. We analysed the fine specificity of SLAM family receptor phosphorylated ITSMs and the conserved tyrosine motif in EAT-2 for SH2 domain containing signalling proteins. Consistent with the literature describing dependence of CRACC (SLAMF7) on EAT-2, CRACC bound EAT-2 (K The balance between activation and inhibition in an immune response is critically dependent on the specificity of phosphorylated tyrosine motifs in the cytoplasmic regions of leukocyte surface receptors and associated adaptors for recruitment of intracellular enzymes. The division between activation and inhibition is commonly delineated by specificity of Immunoreceptor Tyrosine based Activation Motifs (ITAMs) and Immunoreceptor Tyrosine based Inhibitory motifs (ITIMs) for an activating kinase or an inhibitory phosphatase respectively. For example, the T cell receptor complex containing multiple ITAMs is an activating receptor and SIRPα with multiple ITIMs is inhibitory. The balance between activation and inhibition is then dependent on recruitment of the receptors at the cell surface.In vivo and in vitro studies in mouse and human have shown that activation through SLAM family receptors is dependent on recruitment of SAP and EAT-2. In the absence of SAP and EAT-2, there is loss of activating function through SLAM family receptors, most notably in humans with mutations in the SH2D1A (SAP) gene who develop X-linked lymphoproliferative disease (XLP), a condition characterized by the failure to clear persistent viral infections such as EBV, lymphoid hyperplasia, and lymphoma development An alternative strategy to regulating the balance between activation and inhibition through tyrosine phosphorylation is in the specificity of receptor motifs for the intracellular binding partners. The SLAM family receptors within the CD2 family of leukocyte surface receptors contain multiple Immunoreceptor Tyrosine-based “Switch” Motifs (ITSMs) . These m−/− and EAT-2/ERT−/− mice, ERT being an additional EAT-2 homologue in mice NK cells express both SAP and EAT-2 proteins In addition to loss of activating function in the absence of SAP, EAT-2 and ERT, SLAM family receptors exhibited effects which suggested the receptors were actively attenuating signalling We sought to more clearly define the hierarchy of binding partners for ITSMs and the EAT-2 tail. We distinguished between specificity of ITSMs for the adaptors, SAP and EAT-2 and for inhibitory binding partners −1 cm−1) were produced in house and at the Oxford Protein Production facility. Biotinylated EAT-2 was produced by expression of full length human EAT-2 with a C-terminal BirA tag (NSGSLHHILDAQKMVWNHR) in the E coli expression vector pTrcHisA. Constructs encoding the 6xHis-tagged tandem SH2 domains plus the SH3 domains of PLCγ1  and PLCγ2  were generated in pTrcHisA (5′ joining sequence with BglII site underlined: gag atc tcc translates as EIS). The molecular weight (MW) and theoretical extinction coefficients (EC) of recombinant PLCγ1 and PLCγ2 SH2-SH2-SH3 domains were PLCγ1; MW = 42531, EC = 63,830 M−1 cm−1 and PLCγ2; MW = 42415, EC = 67,270 M−1 cm−1.Synthetic biotinylated phosphopeptides were obtained from Peptide Protein Research Ltd. The peptide sequences from human proteins are: LAT Y132 (Biotin-HNPGpYLVVLPD); EAT-2 Y127 (Biotin-NSNSDpYVDVLP); and CD244 ITSM1 (Biotin-EFLTIpYEDVKD); CD244 ITSM1+ITSM2 ; SIRPα ITIM1+ITIM2 (Biotin-TNDITpYADLNLPKGKKPAPQAAEPNNHTEpYASIQT); SHIP-1 Y865 (Biotin-TREKLpYDFVKT); CRACC ITSM1 (Biotin-GENTEpYDTIPH) and CRACC ITSM2 (Biotin-PANTVpYSTVEI) and as published previously 2+ or Ni2+ column by standard protocols. Protein purification of SAP, EAT-2, PLCγ1 and PLCγ2 was optimized by being performed on the day of SPR experiment in order to use protein at the highest activity with minimum aggregation. Where applicable, proteins were stored frozen in 10–25 mM HEPES pH 7.4, 200–300 mM NaCl (pH 7.4).All proteins were purified by size exclusion chromatography immediately prior to SPR analysis.Soluble recombinant proteins were produced in BL21(DE3)pLysS E. coli  at 37°C in 2TY medium and induced at OD 600 = 0.6 with 0.1 mM IPTG for 4 h or in 2XYT auto-induction (Formedium) medium overnight. Bacterial pellets were resuspended in ∼0.1 volume 10 mM HEPES pH7.4, 150 mM NaCl and used immediately or stored at −20°C in 10 ml aliquots. His-tag mediated protein purification was performed on a Co8) were treated with 500 μM pervanadate for 15 minutes, then lysed in PBS, 1% Triton X-100, 1 mM sodium orthovanadate in the presence of mammalian protease inhibitor solution (Sigma). Lysates were pre-cleared by centrifugation, followed by incubation with streptavidin Sepharose. Peptide or EAT-2-coated streptavidin beads were incubated with NK92 lysates for 2 hours at 4°C with rotation. Beads were washed once with 15 ml ice cold PBS/1% (v/v) Triton X-100. Bound protein was eluted using 100 mM glycine-HCl pH 2.5.For pull-down of associated proteins, streptavidin Sepharose  was pre-coated with saturating amounts of peptide or EAT-2. NK92 cells (4×10Protein samples were prepared for mass spectrometry as described previously Protein identification data were exported into ProteinCenter (v3.8.3013 – Thermo Fisher) for filtering and annotation. Results were filtered to remove any proteins with less than 3 unique peptides, less than 10 percent sequence coverage, or median protein probability scores less than 0.5. To establish sample specificity, any protein with at least 10-fold greater sequence coverage in one sample compared with the other was considered to be specific for that sample.Measurements of protein-protein affinity were performed on BIAcore 3000 and T200 systems using CM5 or biotin capture chips . Streptavidin was coupled to CM5 sensor chips using standard NHS/EDC chemical linkage, followed by immobilization of biotinylated peptides. The levels of peptide immobilized were approximately 25 and 50 response units respectively of single and tandem tyrosine motifs to facilitate saturating binding and limitation of “bridging” Mouse P815 cells (ATCC #TIB-64) and the human B cell line, 721.221 5′-GCGCGCTTTGTAGGATTCGTTCAAGAGACGAATCCTACAAAGCGCGCTTTTTT was expressed in pLKO.1. Lentiviral particles were produced through polyethylenimine (PEI-Sigma) co-transfection of 293 T cells with pLKO.1 along with packaging vectors pMD2.G and pSPAX2 . Virus was produced in RPMI 1640/10% heat-inactivated FCS for 48–72 hours. Viral supernatants were collected and NK92 cells were transduced twice overnight in the presence of 1000 U/ml IL-2. Cells with stable shRNA expression were selected using 3 μg/ml puromycin (Invitrogen) for 72 hours prior to use in functional assays.Lentiviral constructs expressing gene-specific shRNAs in vector pLKO.1 were obtained from Open Biosystems. Panels of 5 shRNAs were tested for each protein to select the most effective construct. The specific clones used were as follows: TRCN0000006979 for PLCγ1, TRCN0000002322 for PLCγ2, TRCN0000082710 for SAP, and TRCN0000082657 for EAT-2. As a non-targeting control, the hairpin sequence 5 cells were developed with: anti-EAT-2 antibody , anti-PLCγ1 (2822) from Cell Signalling Technology or anti-human PLCγ2 (Q-20) from Santa Cruz. For quantitation of SAP and EAT-2 protein levels in NK92 cells, the equivalent of 0.5−2×106 cells from lysates prepared at 5×107 cells/ml was analysed alongside known amounts of recombinant protein standards, recombinant full-length human SAP  or EAT-2 , generated as previously described and developed with anti-SAP antibody  or anti-EAT-2. For chemiluminescent detection, HRP conjugated anti-mouse and anti-rabbit IgG antibodies were obtained from Cell Signaling Technology. SHP-2 levels in 105 cell equivalents were analysed in a similar manner with recombinant SHP-2 N+C as a standard  and anti-SHP-2 . Fluorescent detection of protein bands was performed on the LI-COR Odyssey Sa system. IRDye 680LT-conjugated anti-rabbit IgG and IRDye 800 CW anti-mouse IgG were obtained from LI-COR Biosciences. Quantitation of protein expression was performed using LI-COR Odyssey Sa software version 1.0. In initial experiments, estimations of % knockdown in comparison with another protein blotted with a specific antibody or in comparison with a nonspecific “reference” band gave the same result. Subsequently, a “reference” band was routinely used. After RNA interference, SAP expression was also quantified by intracellular staining using anti-human SAP (XLP-1D12) from Cell Signalling Technology and median fluorescence intensity measured by flow cytometry.For quantitation of protein expression, NK92 cell lysates were prepared using buffer  containing 0.5% Triton X-100. Lysates were resolved by SDS-PAGE under reducing conditions using Novex gradient gels and transferred to nitrocellulose membranes for western blotting by standard protocols. Following RNA interference, blots containing protein lysates from 1035S35S Protein Labeling Mix  in cysteine/methionine-free RPMI (Sigma). Target cells were washed 3 times in complete RPMI and dispensed into 96-well round bottom plates at 5000 cells/well. Lentiviral-transduced NK92 cells were harvested and mixed with target cells at the indicated effector:target ratio in duplicate unless otherwise stated in the presence of IL-2. The indicated antibodies were added to the cellular mixture for the duration of the 4 hour cytotoxicity assay. The antibodies used to induce cytotoxicity were as follows: anti-NKp30 (P30.15) and anti-NTB-A (NT-7) were obtained from Biolegend; anti-CRACC (162) and anti-CD58 (MEM-63) were obtained from Abd Serotec; anti-CD2 (X53) and anti-CD244 (C1.7) were purified in our laboratory. The numbers of independent experiments were: SAP – 2, EAT2 – 3, PLCγ1 - 4, and PLCγ2 - 2.Cytotoxicity through individual receptors was measured in a redirected lysis assay. P815 target cells were labeled for 3 hours with EXPRED = 0.003 μM  and NKp30 (which associates with CD3ζ) were tested. The cytotoxic activity of the SLAM family receptors, including CRACC, was reproducibly reduced (n = 2) following SAP knockdown . InteresSince there is competition between EAT-2 and SAP for ITSMs, the concentrations of SAP and EAT-2 are factors in determining dependence of SLAM family receptors on these adaptors. We measured the concentration of SAP and EAT-2 in NK92 cells by western blotting , Table 2RNA interference in the redirected lysis assay in NK92 cells reduced the concentration of EAT-2 by up to ∼50% . In the 865). In addition to identifying SAP and EAT-2, the SH2 domain containing phosphatases SHIP-1, SHIP-2, SHP-1, SHP-2 and CSK, as well as GRB-2 and PLCγ1 were also pulled down by the ITSM peptide . Quantitation of the levels of SHP-2 in NK92 cells, as was done for SAP and EAT-2, indicated there is an order of magnitude higher concentration compared with SAP, and is approximately an order of magnitude higher than the KD, indicating that SHP-2 will be competitive for binding highly phosphorylated receptor.Binding of SHP-2 tandem SH2 domains to CD244 was two-three fold weaker, but comparable with binding to tandem ITIMs from a well characterized inhibitory receptor SIRPα + T cells 127 phosphopeptide pull-down of proteins using NK92 cell lysates. Proteins identified by at least 3 unique peptides associating with EAT-2 Y127 phosphopeptide but not with EAT-2 SH2 domain are listed in D∼1 μM) for the EAT-2 tail In contrast to the characterization of the specificity of ITSMs, the underlying molecular basis of activation and inhibition by EAT-2 has not been clarified. Activating effects of EAT-2 downstream of SLAM family receptors are dependent on phosphorylation of the tyrosine motif in the tail 127 and LAT Y132 by SPR. Direct binding of recombinant PLCγ1 to EAT-2 Y127 was confirmed (127 and LAT Y132 peptides with comparable affinity (We compared direct binding of PLCγ1 and PLCγ2 to EAT-2 Yonfirmed . Recombiaffinity , Table 6affinity , Table 6−/− mice PLCγ1 and PLCγ2 have distinct functions in NK cells D for binding ITSMs which may explain the marginal effects on cytotoxicity of halving the concentration of EAT-2 by RNA interference. Additional factors including phosphorylation of its tail are likely to be limiting in determining activating effects of EAT-2.Analysis of interactions of activating partners for ITSMs in the cytoplasmic regions of SLAM family receptors distinguished specificity of SAP and EAT-2 SH2 domains which correlates with published functional data In our previous measurements of SH2 domain binding to phosphopeptides we observed a difference in specificity between tyrosine motifs which bound activating and inhibitory SH2 domains Analysis of interactions of tandem SH2 domains of tyrosine phosphatases with tandem ITSMs indicated that there will be productive recruitment if concentrations of the adaptors, SAP and EAT-2 are compromised. This is consistent with functional data from SAP-family deficient mice There was a discernible difference between ITSMs and ITIMs in specificity for inhibitory binding partners. Subtle differences in sequence and consequent binding affinities may have a significant impact on signalling In contrast to ITSMs, the specificity of the conserved phosphotyrosine motif in the tail of EAT-2 appears to be more limited. We have characterized interactions with FYN and PLCγ, the latter binding up to two orders of magnitude more strongly In summary, we have identified differential and overlapping specificities of positive and negative regulators of cell signalling for the ITSMs of SLAM family receptors. The affinities measured indicate an intracellular environment where positive and negative regulators will compete for the phosphorylated ITSMs of SLAM family receptors. The relative affinities of the SH2 domains studied indicate that the adapters SAP and EAT-2 will play a dominant role in promoting cellular activation when present, however, signal attenuation by phosphatases is possible through direct recruitment to ITSM-containing receptors, especially in the absence of SAP and EAT-2.


Phosphorylation is the most common and pleiotropic modification in biology, which plays a vital role in regulating and finely tuning a multitude of biological pathways. Transport across the nuclear envelope is also an essential cellular function and is intimately linked to many degeneration processes that lead to disease. It is therefore not surprising that phosphorylation of cargos trafficking between the cytoplasm and nucleus is emerging as an important step to regulate nuclear availability, which directly affects gene expression, cell growth and proliferation. However, the literature on phosphorylation of nucleocytoplasmic trafficking cargos is often confusing. Phosphorylation, and its mirror process dephosphorylation, has been shown to have opposite and often contradictory effects on the ability of cargos to be transported across the nuclear envelope. Without a clear connection between attachment of a phosphate moiety and biological response, it is difficult to fully understand and predict how phosphorylation regulates nucleocytoplasmic trafficking. In this review, we will recapitulate clue findings in the field and provide some general rules on how reversible phosphorylation can affect the nuclear-cytoplasmic localization of substrates. This is only now beginning to emerge as a key regulatory step in biology. The nucleus is the key organelle where most of the cellular genetic information is stored. Transcription is also compartmentalized to the nucleus to keep it separate from translation, which occurs in the cytoplasm. The nucleus is separated from the cytoplasm by a double lipid bilayer known as the nuclear envelope (NE), which is composed of an outer and an inner nuclear membrane (abbreviated as ONM and INM). At distinct points, the ONM and INM are conjoined by large macromolecular assemblies known as nuclear pore complexes (NPCs). Because of this peculiar morphology, all transport across the nuclear envelope is physically restricted to occur through the NPC. The NPC is not simply a channel, but mediates the exchange of macromolecules into and out of the nucleus imposing selectivity like a semi-permeable filter . Accordiβ-karyopherins were originally identified based on their ability to bind the small GTPase Ran through an N-terminal binding domain . Ran, a classical NLS substrates only certain animal isoforms can recognize specific non-classical cargos. For instance, importin α5  impoed STAT1  and infled STAT1 , while ied STAT1 .In addition to the prototypical receptor importin β1, other β-karyopherins are well characterized. For instance, importin β2  mediates nuclear import of cargos containing an M9-signal ; or Crm1It is impossible to appreciate the complexity of nucleocytoplasmic trafficking without acknowledging the morphology and composition of the NPC. In vertebrates, the NPC is a ~125 MDa macromolecular assembly composed of approximately thirty proteins, termed nucleoporins (nups) (reviewed in -26). TheReversible phosphorylation of amino acid side chains is the most widely characterized post-translational modification in biology (reviewed in -37). PhoPhosphorylation can have either an enhancing or an inhibitory effect on nuclear import, and unfortunately the molecular consequences of phosphorylation have been elucidated in very few systems . Underst385 in the EBNA-1 NLS  nuclear antigen 1 (EBNA-1) protein Figure , which iS Figure  up-regulS Figure , which i385 with the phosphomimetic aspartic acid decreases the binding affinity for importin α5, demonstrating that the specificity of the response is dependent on the phosphate moiety. Additionally, biophysical studies using chemically synthesized peptides and isothermal titration calorimetry determined that phosphorylation of Ser385 in EBNA-NLS increases the binding affinity for importin α5 by ~20-fold, compared to the unphosphorylated NLS (Kd ~3 μM versus 60 μM) [385 within the NLS stimulates nuclear import by enhancing the binding affinity of EBNA-1 for the import adaptor importin α5.Interestingly, replacement of Sers 60 μM) . Thus thet al. that the entire HBV core particle  undergoes an elegant and tightly regulated mechanism of entry into the host nucleus to replicate its genome. A mature HBV particle contains an envelope with surface glycoproteins that surrounds an icosohedral capsid (composed of 180 or 240 copies of the 21 kDa core protein) containing the viral dsDNA genome, a viral polymerase and the host proteins Protein Kinase C (PKC) and hsp90 . Progenydiameter ) traversdiameter . The cordiameter    Figure . Upstrea) Figure . It was  50-fold ,58 and pr import . Howeverr import . Interesr import . Based oSTATs  form an important family of transcription factors that play a critical role in cellular viability, immune response, and development. There are seven known human STAT isoforms that share a fundamentally conserved multi-domain architecture composed of an N-terminal dimerization domain , a core 701 [in trans when attached to an exogenous protein, the STAT1 NLS is only functional in cis, within the context of the pSTAT1 tertiary structure. This was elegantly shown by Meyer et al. who studied a peptide spanning residues 376-427 of the STAT1 DNA-binding domain and found that it did not accumulate in the nucleus in a micro-injection nuclear import assay [The first STAT family member to be characterized, STAT1, mediates the innate immune response . Upon st701 . Phospho701 . This di701  of STAT1701 . The STA701 . Second,701 . The finrt assay , and reqrt assay .in cis [The extracellular signal regulated kinase (ERK) cascade has an important role in cellular development and phase transitions in the cell cycle . The cytin cis . In restin cis . The Mapin cis . Upon phin cis . It is uin cis . AP-1 coin cis . When exin cis , cellulain cis , and impin cis . Thus, EIn addition to basic NLSs Figure , severalSimilar to NTS-containing proteins, serine/arginine-rich (SR) proteins rely on phosphorylation-dependent recognition of a small epitope for their nuclear import. SR proteins are fundamental to mRNA metabolism in the nucleus , and cycet al. investigated the effect of phosphatase and kinase inhibitors on the nuclear transport of NLS-bearing cargos in a nuclear transport assay in digitonin-permeabilized cells [et al. showed that the phosphatase inhibitors Okadoic Acid (OA) and microcystin dramatically inhibit nuclear import pathways mediated by transportin and importin β. Treatment with the broad-spectrum kinase inhibitor, staurosporine, was able to partially reverse the import inhibition, thereby suggesting that staurosporine-resistant kinases are also involved in the inhibition. They concluded that the phosphorylation targets were most likely nucleoporins in the NPC central channel because OA did not affect karyopherin recognition of NLS-cargo or the cellular localization of RanGTP. In addition, they suggested that the responsible kinases were both membrane associated and cytoplasmic. Interestingly, a recent investigation of the Picornaviruses EMCV and Theiler's murine encephalomyelitis virus showed the negative effects of the Leader protein (L-protein) on the nuclear transport machinery. The L-protein blocks nuclear transport in two known ways: 1) direct binding of Ran, which leads to misregulation of its activity [While phosphorylation of the nuclear envelope and NPC are necessary steps in cell cycle progression and the onset of mitosis, phosphorylation of the nuclear transport machinery in resting cells has a dramatic inhibitory effect on nuclear import. Kehlenbach ed cells . This teed cells  This allactivity , and 2) activity . It willA simple way to retain cargos in the cytoplasm is by inactivating the NLS. This can be achieved either by direct binding and sequestering of the NLS, like the inhibitory protein of NF-kB (IKB) , or by p2+ levels stimulate Calcineurin, a Ca2+ dependent serine phosphatase, which then binds to NFAT and dephosphorylates the NLS, making it accessible for recognition by the nuclear import machinery [2+ levels or inactivation of Calcineurin by CsA or FK506, results in the phosphorylation of the NFAT NLS [The nuclear factor of activated T-cells (NFAT) is a well-characterized example of cargo retained in the cytoplasm by a phosphorylation in its NLS. First identified as a critical transcription factor for T-cell activity ,110, NFAachinery . A drop NFAT NLS , which iNFAT NLS , a proceNFAT NLS .85 is located in close proximity to two well conserved tryptophan residues of importin β, which are known to be important for nuclear import of classical NLS cargos [The second important example of phosphorylation-mediated down-regulation of nuclear import that has important physiological consequences in humans is the parathyroid hormone related protein (PTHrP). This small secreted protein is best known for its role in chondrocyte maturation, but it is also expressed in a range of tumors as it has fundamental roles in cell cycle control and apoptosis ,119. TheS cargos . A phospSaccharomyces cerevisiae is highly sensitive to stress response elements (STREs) [The transduction of extracellular signals to the nucleus displayed by higher eukaryotes occurs through analogous pathways in lower eukaryotes. Not surprisingly, phosphorylation plays a critical role in this process. The cellular localization of the transcription factor Msn2p in  (STREs) . Activat (STREs) . In part (STREs) . These c (STREs) . Under gSimilar to Msn2p, phosphorylation-mediated down regulation of nuclear import has been described for the S. cerevisiae transcription factor Pho4. Budding yeast has an extensive network of proteins involved in regulation of inorganic phosphate availability . Upon phin vitro solution binding assays, Harreman et al. demonstrated that phosphorylation of serine 160 upstream of Swi6-NLS causes a 4-fold decreased binding affinity between the Swi6 and importin α1 [Another example of down-regulation of nuclear import by phosphorylation was reported for the S. cerevisiae transcription factor Swi6. Swi6p regulates the expression of several genes during the G1 phase of the cell cycle ,135, wheortin α1 . A simil701. One of the first phosphatases identified in the regulation of STAT signaling was the T-cell protein tyrosine phosphatase (TC-PTP). The TC-PTP gene is alternatively spliced, yielding a longer ER-associated form TC48, and a nuclear form TC45 [Because STAT1 is the most well studied example of phosphorylation-dependent nuclear transport, there has been much interest in the identification of phosphatases that counteract STAT1 activation. IFN signaling cascades undergo rapid activation-inactivation cycles that require tight regulation. The IFNγ signaling pathway is negatively regulated in at least three ways: 1) suppressors of Cytokine Signaling inhibition of JAKs and the IFNγ receptor at the cell membrane , 2) nuclorm TC45 ,140. Theorm TC45 . Upon deorm TC45 . In addiThe Src homology domain (SHP) family of dual-specificity phosphatases (SHP-1 and SHP-2) also plays a regulatory role in STAT nuclear localization; SHP's have a unique dephosphorylation mechanism, utilizing two tandem N-terminal SH2-domains (N-SH2 and C-SH2), a tyrosine phosphatase (PTP) domain and two C-terminal Tyr phosphorylation sites. Structural investigations of SHP-2 reveal an auto-inhibitory mechanism of the PTP domain by the N-SH2 in the absence of a pTyr substrate . When thPoxviridae  are known to encode phosphatases. Members of the Poxviridae family of double stranded DNA viruses carry a large, linear, genome of approximately 200-kbp, and assemble entirely in the cytoplasm of host cells [Poxviridae encodes the dual-specificity phosphatase VH1, which is highly conserved among poxviruses and essential for the viability of Vaccinia virus in tissue cultures [in vivo. Second, VH1 was shown to deactivate host IFNγ signaling through dephosphorylation of specific tyrosine and serine residues required for STAT1 nuclear import and transcriptional activity [in vitro [Out of necessity, viruses have developed a number of mechanisms to evade the host innate immune response and neutralize cytokine signaling pathways, specifically the Jak/STAT pathway. Many virus families encode enzymes responsible for the evasion of host defense mechanisms, but few viruses like st cells . Poxviricultures . VH1 is cultures . VH1 hascultures  and A14 cultures , in vivoactivity . It has in vitro . Moreovein vitro . Dephospin vitro  but doesin vitro . Like phin vitro , VH1 disin vitro . Interesin vitro . This raIn the past decade, an increasing number of phosphatases have been implicated in the deactivation of JaK/STAT signaling through the dephosphorylation of STAT1 or its upstream effectors. For example, two Flaviviruses, the mosquito-borne flavivirus JEV and the tick-borne TBE both encode the protein NS5, which was shown to disrupt IFN signaling. While NS5 does not display a structurally conserved phosphatase motif, treatment of infected cells with the PTP inhibitor sodium orthovanadate restored IFNγ and IFNα signaling ,155. Thiin vivo. This is likely to have a very significant impact on our ability to understand how extracellular stimuli  affect, modulate and control gene expression in the cell nucleus.It has been 55 years since the groundbreaking study on the conversion of phosphorylase b to phosphorylase a, by Fisher and Krebs, that first emphasized the importance of reversible phosphorylation in biology . Today, Vaccinia virus H1 gene product; JEV: Japanese Encephalitis Virus; TBE: Tick-Borne Encephalitis Virus.NLS: nuclear localization signal; NPC: nuclear pore complex; NE: nuclear envelope; ONM: outer nuclear membrane; INM: inner nuclear membrane; IBB: importin β binding; NES: nuclear export signal; NUP: nucleoporin; EBV: Epstein-Barr virus; EBNA-1: Epstein-Barr virus nuclear antigen 1; HBV: Hepatitis B virus; PKC: protein kinase C; STATs: signal transducers and activators of transcription; SH2: Src2 Homology domain; dsNLS: dimer-specific NLS; cNLS: classical NLS; ncNLS: non classical NLS; ARM: armadillo; ERF: extracellular signal regulated kinase; TAD: transcriptional activation domain; TEY: Thr-Glu-Tyr; NTS: Nuclear Transport Signal; SR protein: serine/arginine-rich proteins; SPS: Ser-Pro-Ser; OA: okadoic acid; IKB: inhibitory protein of NF-kB; NFAT: nuclear factor of activated T-cells; SRR2: serine-rich region; PTHrP: parathyroid hormone related protein; STREs: stress response elements; cAPK: cAMP-dependent protein kinase; TC-PTP: T-cell protein tyrosine phosphatase; PTP: protein tyrosine phosphatase; SHP: SH2-containing tyrosine phosphatase; pSTAT1: phosphorylated STAT1; IFN: interferon; JaK: Janus Kinase; PIAS: Protein Inhibitor of STAT1; SOCS: Suppressor of Cytokine Signaling; VH1: The authors declare that they have no competing interestsJN wrote the initial draft of the manuscript; KL expanded and revised the manuscript, and generated all figures. GC revised both text and figures critically for important intellectual content; all three authors have given final approval of the version to be published.