Saccharomyces cerevisiae. In this study, we further explore the relationship between the subunits Not4p and Bur2p.The evolutionarily conserved Ccr4-Not and Bur1/2 kinase complexes are functionally related in bur1-23 mutant. Finally, phenotypic analyses of the Not4p phosphomutant (not4S/T5A) and bur2Δ strains showed overlapping sensitivities to drugs that abolish cellular stress responses. The double-mutant not4S/T5A and bur2Δ strain even revealed enhanced phenotypes, indicating that phosphorylation of Not4p and BUR2 are active in parallel pathways for drug tolerance.First, we investigated the presence of post-translational modifications on the Ccr4-Not complex. Using mass spectrometry analyses we identified several SP/TP phosphorylation sites on its Not4p, Not1p and Caf1p subunits. Secondly, the influence of Not4p phosphorylation on global H3K4 tri-methylation status was examined by immunoblotting. This histone mark is severely diminished in the absence of Not4p or of Bur2p, but did not require the five identified Not4p phosphorylation sites. Thirdly, we found that Not4p phosphorylation is not affected by the kinase-defective BUR2 to affect stress responses in Saccharomyces cerevisiae.Not4p is a phospho-protein with five identified phosphorylation sites that are likely targets of a cyclin-dependent kinase(s) other than the Bur1/2p complex. Not4p phosphorylation on the five Not4 S/T sites is not required for global H3K4 tri-methylation. In contrast, Not4p phosphorylation is involved in tolerance to cellular stresses and acts in pathways parallel to  Saccharomyces cerevisiae and regulates mRNA biogenesis at multiple levels  Bur1p Synthetic lethal interactions of several in vivo. Mass spectrometry analyses identified several serine/threonine sites as phosphoacceptor-sites for Not4p. Substitution of these sites to alanine (Not4p-S/T5A) indicates that the mechanism by which the Ccr4-Not complex and the Bur1/2p complex regulate H3K4 tri-methylation is independent of Not4p phosphorylation. In addition, we found that a severe kinase-defective allele of BUR1 did not affect Not4p phosphorylation. Further analysis indicates that Not4p phosphorylation is functionally important for tolerance to drugs that induce replication stress and protein translation errors. Yeast strains containing the Not4p penta-phosphomutant in combination with a BUR2 deletion show a more severe phenotype than either single mutant, which argues against a linear pathway relationship between NOT4 and BUR2. Taken together, our data indicate that phosphorylation of Not4p is involved in tolerance of DNA replication stress and protein processing errors, likely in pathways parallel to BUR2.To investigate the functional relationship between the Ccr4-Not and Bur1/2 kinase complexes, we first explore the phosphorylation status of Ccr4-Not components. We confirmed that Not4p is a phospho-protein TPAPVPAGSNPWGVTQSATPVTSINLSK) was identified by mass spectrometry as a singly-phosphorylated peptide (data not shown), but the phospho-acceptor site could not be identified unequivocally. Manual inspection of the MS/MS spectrograms indicates that T334 and/or S342 of this Not4p peptide are the most likely phospho-acceptor sites. The phosphorylation status of several Ccr4-Not subunits was assessed by their electrophoretic mobility upon dephosphorylation . To this end, wild-type NOT4 or the not4S/T5A mutant allele was chromosomally integrated into the NCY1 strain, which carries a TAP-tagged NOT1 allele and a NOT4 deletion  were analysed by immunoblot analysis  Ccr4-Not and Bur1/2p complexes function in parallel molecular pathways to resist DNA replication stress and cellular stress upon misfolded and/or mistranslated proteins.The drug sensitivity assays showed overlapping effects for phosphorylated Not4p and Bur2p . To expl mutants . Taken tin vivo , indicating that this CDK is not the required kinase for Not4p. Interestingly, yeast mutants deleted for PHO85 showed an increased electrophoretic mobility of Not4p (data not shown), suggesting that Pho85p is involved in Not4p phosphorylation. Pho85p is a CDK that interacts with ten different cyclin partners to exert its diverse roles in the regulation of cellular responses to nutrient levels, environmental conditions and progression through the cell cycle The identification of Not4p phospho-acceptor sites on SP/TP positions  suggestsNOT4 deletion  contained the BUR1 allele on a pRS316-URA3 marked plasmid NOT4 gene in this strain was TAP-tagged, followed by a transformation with pRS315-BUR1-HA3 or pRS315-bur1-23-HA3pRS316-BUR1. The not4:KanMX6 and bur2:URA3 strains were generated using a PCR fragment from genomic DNA of strain KMY58 and KMY187, respectively. Genomic NOT4 or not4S/T5A mutants were obtained by integrating the pRS305-NOT4 or pRS305-not4S/T5A into the NOT4 locus in a not4Δ background using the SmaI restriction site in the NOT4 promoter region (nt -226 relative to the ATG). Integrated mutants and gene disruption were verified by PCR and/or phenotypic rescue. For yeast strains and mRNA analysis used in the Supplementary material please see the Yeast strains used in this study are listed in −1 and precursor ions were sequenced in MS/MS mode at a threshold of 150 counts. Additional analyses were performed by nanoLC-LTQ-Orbitrap-MS (Thermo). Data were processed and subjected to database searches using Proteinlynx Global Server version 2.1 (Micromass) or the MASCOT software (Matrixscience) against SWISSPROT and the NCBI nonredundant database, with a 0.25 Da mass tolerance for both precursor ion and fragment ion. The identified phospho-peptides were confirmed by manual interpretation of the spectra.TAP-tag mediated protein purifications were performed essentially as described NOT4 or not4S/T5A strains occurs via Not1-TAP. A fraction of these purified proteins were resolved on a 4–12% SDS-PAGE gradient gel and silver stained. Other fractions were incubated at 37°C for 45 min with or without SAP. All reactions were quenched by addition of 2x sample buffer and incubated at 95°C for 5 min. Proteins were subjected to immunoblot analyses.Proteins of TAP-tagged Notp strains were captured on IgG beads and washed three times with E-buffer . Immunoprecipitated material was (mock-) treated at 37°C for 45 min with shrimp alkaline phosphatase (SAP) or SAP pre-incubated with 4 mM Na-Vanadate and 800 mM NaF. Alternatively, TAP-tagged purification of proteins from TAP-tagged proteins were detected using the antibody against the protein A moiety of the TAP-tag . Rabbit polyclonal antibody against Not1p and Not4p was generously provided by Dr. M.A. Collart. TBP antiserum was a kind gift from Dr. P.A. Weil. For detection of H3K4 methylation status, yeast were grown in YPD and extracts were prepared as described previously Ten-fold serial dilutions of the indicated strains were spotted on YPD plates without or with the indicated concentrations of hydroxyurea, hygromycin B or cycloheximide. The indicated strains were also 10-fold serial diluted and spotted on SC plates or SC-R plates containing the indicated concentration of canavanine. The plates were grown at 30°C (or 37°C for YPD) for 3 days.Figure S1not4Δ and bur2Δ strains were spotted in 10-fold serial dilutions on YPD or YPD containing 25 mM or 50 mM HU. B: HU-induced RNR3 transcription in cells lacking NOT4 or BUR2. Exponentially growing BY4741, not4Δ and bur2Δ strains were treated with 200 mM HU for 2 hours in YPD. RNA was extracted and subjected to quantitative reverse-transcriptase PCR. Standard deviations of four experiments are indicated as error bars.Deletion of NOT4 or BUR2 leads to similar hydroxyurea sensitivity. A: Hydroxyurea (HU) sensitivity of cells lacking NOT4 or BUR2. BY4741, (0.47 MB TIF)Click here for additional data file.Figure S2not3Δ) with an integration of not4S/TxA  at the NOT4 locus. Lane 12 shows yeast strain NYC1 with wild-type NOT4 at the NOT4 locus  containing the indicated concentration of canavanine. Lane 1-11 show the yeast strain NYC1 (NOT1-TAP (2.57 MB TIF)Click here for additional data file.Materials and Methods S1(0.03 MB DOC)Click here for additional data file.Table S1(0.04 MB DOC)Click here for additional data file.


The yeast cell cycle is largely controlled by the cyclin-dependent kinase (CDK) Cdc28. Recent evidence suggests that both CDK complex stability as well as function during mitosis is determined by precise regulation of Swe1, a CDK inhibitory kinase and cyclin binding partner. A model of mitotic progression has been provided by study of filamentous yeast. When facing nutrient-limited conditions, Ras2-mediated PKA and MAPK signaling cascades induce a switch from round to filamentous morphology resulting in delayed mitotic progression.cdc28 mutants exhibiting constitutive filamentation were subjected to epistasis analyses with RAS2 signaling effectors. It was found that Swe1-mediated inhibitory tyrosine phosphorylation of Cdc28 during filamentous growth is in part mediated by Ras2 activation of PKA, but not Kss1-MAPK, signaling. This pathway is further influenced by Cks1, a conserved CDK-binding partner of elusive function with multiple proposed roles in CDK activation, transcriptional regulation and ubiquitin-mediated proteasome degradation.To delineate how the dimorphic switch contributes to cell cycle regulation, temperature sensitive The dynamic balance between Cks1- and Swe1-dependent regulation of Cdc28 and, thereby, the timing of mitosis during yeast dimorphism is regulated in part by Ras2/cAMP-mediated PKA signaling, a key pathway controlling filamentous growth. Yeast cell division is tightly coupled to morphology. Progression through mitosis activates an apical-isotropic switch in which polarized bud growth is redirected to produce round daughter cells . ConversRAS2 diminishes filamentation, while the dominant allele RAS2-VAL19 induces constitutive pseudohyphal differentiation [TEC1 decreases filament formation [FLO8 diminishes filamentation, whereas deletion of SFL1 enhances filamentation [RAS2-V19 phenotypes [The switch from round to filamentous growth is stimulated physiologically by environmental conditions including low nitrogen availability or fusel alcohol exposure . Althougntiation . The MAPntiation . Accordintiation , while dormation . On the ormation ,14. Deleentation . Productenotypes ,16.HSL1 results in increased Cdc28 tyrosine-19 phosphorylation [HSL1 leads to diminished Swe1 protein levels [HSL1 restores filamentation in the absence of FLO8 and to a lesser degree TEC1, but not in flo8 tec1 double mutants [One possible target of Ras2 signaling may be the Wee kinase Swe1. Swe1 inhibits Cdc28-Clb2 activity at G2/M via phosphorylation of Cdc28 Tyr19 and a phosphorylation-independent mechanism . Overexprylation  and enharylation ,18, whern levels . FurtherCDC20 as well as negative regulators of the PKA pathway PDE2 and BCY1 were found to suppress the temperature sensitivity of a cohesin mutant [cdc16-1 mutant defective in APC function [PKA has also been shown to mediate Ras2 and glucose-dependent inhibition of the anaphase-promoting complex (APC) ,25. The n mutant . Furtherfunction . Togethecdc28-Y18V, T19F, a mutant resistant to Swe1 mediated phosphorylation [CDC20. The precise mechanism remains obscure but has been proposed to involve Cks1 recruitment of the proteasome to Cdc28 complexes [cks1 temperature-sensitive alleles confer G2/M delay [Importantly, Cdc28 is also intimately linked to APC activation. Cdc20-dependent APC activity during mitosis is positively regulated via phosphorylation by Cdc28 [omplexes . Cks1 haomplexes . Interes/M delay , Clb cyc/M delay ,32.cdc28 mutants that perform enhanced filamentous growth. A preponderance of the mutations localized to the binding region of Cks1. We examined the genetic interactions of these mutants with cell cycle regulators and Ras2 signaling components. Our results indicate that the PKA-cAMP pathway may be an additional signaling mechanism by which Swe1 inhibition of Cdc28 is affected. However, Swe1 function is downregulated by a mechanism requiring association of Cdc28 with Cks1.To further elucidate the pathway by which extracellular signals impinge on cell cycle, we screened for temperature sensitive cdc28 mutants, which experience growth arrest at elevated temperatures would be valuable tools to identify connections between cell cycle and extracellular signals. Therefore, a cdc28 shuffle strain was created in which homozygous replacement of CDC28 with TRP1 gene was covered by a URA3 marked plasmid harboring CDC28. Transformation of this strain with a library of degenerate-PCR derived cdc28 alleles on a HIS3 marked plasmid, followed by negative selection against URA3 allowed screening colonies for mutants exhibiting enhanced filamentous growth on low nitrogen media. Five hundred colonies were selected [We hypothesized that constitutively filamentous Cyclin-dependent kinase is a bi-lobed protein with an amino terminal domain composed primarily of β-sheets and a larger carboxy-terminal domain containing several α-helices. Between these two domains lies the catalytic core, the opening of which is covered by the T-loop (activation loop) in the inactive monomeric state ,33-35. WCKS1. Indeed, 11 out of 15 alleles exhibited improved growth by CKS1 overexpression  motif, previously implicated in Cdc28 binding to Cks1 [We were struck by the high number of mutations potentially affecting Cks1 binding, as this factor had yet to be directly linked to the apical-isotropic switch or filamentous differentiation. We examined if normal growth could be restored in these mutants by high copy  in Cks1 . Importa to Cks1 . Mutatiocdc28-E217V, we performed genomic replacement of endogenous CDC28 with cdc28-E217V. As expected, transformation of cdc28-E217V with high copy CKS1 similarly relieved mutant phenotypes as previously found in the cdc28-E217V shuffle strain . Thus, cdc28-E217V phenotypes are not affected by alteration of activating phosphorylation of Cdc28. This further substantiates that cdc28-E217V defects are specific to interaction with Cks1.However, we further questioned if the defects of this mutant were specifically attributable to its diminished interaction with Cks1. As a means to examine this possibility we tested overexpression of the CDK-activating kinase  binding ,41. If Ccts Fig. . Dephospcdc28-E217V phenotypes by CLB2 and CLN1. CLB2 overexpression partly restored cdc28-E217V growth at semi-permissive temperature . These results further support that the phenotype of cdc28-E217V is due to delayed mitotic progression.Given the well-established roles of Cln1 and Clb2 cyclins at the G1/S and the G2/M transitions respectively, as well as their opposing effects on the apical-isotropic switch, we examined modulation of ure Fig.  while cduds Fig. . In turnure Fig.  while decdc28-E217V phenotypes were dependent on Ras2 signaling. Deletion of RAS2 restored near wild-type growth and morphology to cdc28-E217V cells  [In filamentous growth signaling, Ras2 activates both the PKA pathway and the Kss1-MAPK cascade . We queslls Fig. . Two tra17V Fig. , whereasect Fig. . Deletioogy Fig. . These rt shown) ,45.GCN4 provided some suppression to cdc28-E217V cells . Together, these results argue against Swe1 and Cks1 binding competitively to Cdc28.One interpretation of these data is that Swe1 functions upstream of Cks1. Nonetheless, arguing against a role for Swe1 binding or Cdc28 Tyr19 phosphorylation in modulating Cks1 binding, Cdc28 was pulled down by p13IH1 Fig. . An altecdc28-E217V cells. Western blot analysis of the cdc28-E217V cells revealed accumulation of Myc epitope-tagged Swe1, which appeared as a range of apparent molecular weights, a pattern previously ascribed to Swe1 phosphorylation [CDC55, a protein phosphatase 2A (PP2A) regulatory subunit that promotes Swe1 degradation. cdc55 mutants contain elevated levels of Swe1 and show morphological defects including slow growth, elongated morphology and increased Cdc28 Tyr19 hyperphosphorylation at 16°C, which are relieved by deletion of SWE1 [CKS1 also strongly suppressed the growth and morphology defects of cdc55 mutants and reduced Cdc28 Tyr19 phosphorylation ; SKY3012 – MATa gcn4::HIS3 ura3 leu22 μ URA3 GAL> (gift of M. Solomon); pSKB4251 – YEplac195 2 μ URA3 GAL>PTC2 (gift of M. Solomon); pSKB4252 – YEplac195 2 μ URA3 GAL>PTC3 (gift of M. Solomon); pSKB4253 – pRS202 2 μ URA3 CLB2; pSKB4254 – pRS 202 2 μ URA3 CAK1; pSKB4255 – pRS202 2 μ URA3 CLN1; pSKB4256 – pRS202 2 μ URA3 CKS1; pSKB4257 – pRS202 2 μ URA3; pSKB4258 – pCT3 CEN URA3 CDC28; pSKB4259 – pGEX-3x-GST-SWE1 [cdc28 library CEN HIS3pSKB4250 – YEplac195 GST-SWE1   media. Five hundred colonies exhibiting enhanced filamentous growth were isolated and from these, 64 showed dramatically reduced growth at 35°C. Plasmids were rescued and re-introduced in SKY750. After 5-FOA treatment, 39 were found to confer a strong phenotype and were sequenced. Genomic integration of the E217V mutation into SKY2256 was as described [A degenerate-PCR derived library of escribed . Tetrad The effect of polymorphism at E217 was studied as described . MoleculYeast cultures were grown to saturation in rich or selective liquid media at 22°C. Saturated cultures were diluted serially at a ratio of 1/5 and spotted onto rich or selective agar medium. We have found the cdc28 mutants, possibly in part of because of the essential nature of many Cdc28 functions, have a tendency to acquire genomic and intragenic suppressor mutations. Because of this, we repeated spot testing at least four times for each experiment and a representative assay is shown. Each of the experimental groups shown within a given experiment were performed together on the same plate at each temperature. Images were acquired with a Pixera Professional camera on a Zeiss inverted microscope using brightfield illumination and contrast-enhanced in Adobe Photoshop. Magnification for detail of growth and morphology was performed in accord with previously published assessments of filamentation ,73.Suc1 pull-down reactions, 1 mg of protein extract was incubated overnight at 4°C with 50 μ l of p13Suc1 Sepharose bead slurry (Upstate). Beads were washed five times with extract buffer, boiled in sample buffer and the supernatants subjected to SDS-PAGE. For binding assays using GST-Swe1 coated glutathione beads, 1 mg of yeast extract was incubated overnight with 100 μ l of 50% glutathione bead slurry and treated as for p13Suc1 Sepharose beads. GST-Swe1 was isolated from E. coli BL21(DE3) plysS harboring pGEX-GST-SWE1 [600 of 0.6, induced for 3 h at 37°C with 0.2 mM IPTG, and briefly sonicated in PBS buffer containing protease inhibitor cocktail, 1 mM PMSF, 1 mM Orthovanadate, 0.5 mM DTT, and 1% Triton X-100. Glutathione beads were incubated with 1–2 mg of protein extract overnight at 4°C. GST-Swe1 coated beads were washed five times with PBS and incubated with extracts.Yeast extracts were prepared from log phase cultures by bead beating in buffer containing 50 mM Tris pH 7.6, 10% glycerol, 0.5% NP-40, 2 mM EDTA, 250 mM NaCl, 1 mM phenylmethylsulphonyl fluoride, and protease inhibitor cocktail (Roche). Protein content was determined by Bradford assay. For Western blot analysis, proteins separated by SDS-PAGE were transferred to nitrocellulose membranes, and blocked in TBS-Tween-20 containing 5% milk, or 5% BSA for anti-phospho-Tyr15 Cdc2 antibody . Membranes were probed with polyclonal anti-Myc  at 1/1000 dilution, anti-PSTAIRE (Santa Cruz) at 1/500, anti-phospho-Tyr15 Cdc2  at 1/1000, or anti-PGK (Molecular Probes) at 1/2500. Membranes were washed 3× with TBS-Tween-20 and probed with HRP-linked anti-rabbit or anti-mouse IgG (Amersham) at 1/2500. For p13GST-SWE1 , a kind (CDK): cyclin-dependent kinase; (SCF): Skp1-cullin-F-box-protein complex; (PKA): protein kinase A; (MAPK): mitogen-activated protein kinase; (PP2A): protein phosphatase 2A; (G1): growth phase 1; (S): synthesis phase; (G2): growth phase 2; (M): mitosis.The authors declare that they have no competing interests.cdc28 alleles. JC provided intellectual contributions, technical support and aided in review and revision of the manuscript. DC conducted bioinformatic analyses of each of the cdc28 alleles. SK conceived the initial study, directed and coordinated all studies, and provided resources and materials for the studies presented.BT generated the hypotheses and conclusions presented and conducted the genetic and biochemical experiments as well as wrote the manuscript. AK made significant contributions to the conception, design, hypotheses and interpretation of data of all of the studies presented, provided substantial technical assistance and optimization of genetic and biochemical assays, developed a large proportion of the specific reagents for testing of genetic and biochemical interactions of cell cycle genes, and extensively reviewed and revised the manuscript. JS and RG participated in repeating and confirming experimental results, as well as compilation and organization of the data. BB performed the primary screen for temperature sensitive


Estimation of the glomerular filtration rate (GFR) is essential for the evaluation of patients with kidney disease, and for treating patients with drugs that are eliminated from the circulation by the kidneys. Cystatin C has been shown to be superior to creatinine for estimating GFR in several studies. However, studies showing that thyroid function has an impact on cystatin C have not addressed the question of whether the changes in cystatin C levels are due to changes in GFR or in cystatin C synthesis.2), while the creatinine concentration was lower (36 μmol/L) and gave a corresponding creatinine-estimated GFR that was higher (145 mL/min/1.73 m2) than the iohexol-estimated GFR (121 mL/min/1.73 m2) during the hyperthyroid period. After thyroidectomy, the creatinine concentration was 36 μmol/L and creatinine-estimated GFR was calculated as 73 mL/min/1.73 m2, while the cystatin C concentration and cystatin C-calculated GFR was 0.78 mg/L and 114 mL/min/1.73 m2, respectively.We report an account of a hyperthyroid patient with a discrepancy between the GFR estimates from cystatin C and creatinine. The cystatin C concentration (1.36 mg/L) was higher and gave an estimated GFR which was lower (51 mL/min/1.73 mIn contrast to creatinine, cystatin C levels rose in the hyperthyroid state as compared to the euthyroid state. The cystatin C-estimated GFR was reduced compared to the iohexol-estimated GFR. This patient case shows that the hyperthyroid-associated changes in cystatin C levels are not due to changes in GFR. Thyroid function should thus be considered when both cystatin C and creatinine are used as markers of kidney function. Glomerular filtration rate (GFR) is generally accepted as the best overall indicator of renal function and is therefore an important marker for renal disease. Reduced GFR influences the clearance of many pharmaceuticals used today. In the last decades, serum or plasma creatinine has become the most commonly used marker for estimating GFR in clinical practice ,2. DespiA Caucasian female (age 25), smoker, was admitted to the emergency unit with symptoms of tachycardia and anxiety. Thyroid tests were ordered on suspicion of hyperthyroidism. The test results showed values characteristic of severe hyperthyroidism: TSH was below the detection limit of the assay (<0.001 mU/L), 40 pmol/L free T4  and 6.4 nmol T3/L . Antibody levels against thyroid peroxidase (anti-TPO) and TSH receptor were increased as follows: 29 kIU anti-TPO/L  and 11 anti-TSH receptor U/L . The patient was diagnosed with Graves's disease showing disseminated thyroid enlargement. Initial treatment included a β-receptor blocker, propranolol and an inhibitor of thyroxin synthesis called timazol. Later, a thyroidectomy was performed.2 according to the bias-corrected MDRD equation [2 as calculated from cystatin C [2.Plasma levels of creatinine and cystatin C were analyzed as markers of kidney function. When the patient was in the hyperthyroid state, plasma creatinine was 36 μmol/L  and cystatin C was 1.36 mg/L. Calculated glomerular filtration rate (GFR) from creatinine was 145 mL/min/1.73 mequation  and 51 m2 compared to 114 mL/min/1.73 m2 using the cystatin C concentration.Six months later (after surgery), the patient was clinically euthyroid with a TSH value of 3.69 mIE/L and 15 pmol free T4/L. Creatinine and cystatin C levels were 65 μmol/L and 0.78 mg/L, respectively. GFR-calculated from the creatinine concentration was 73 mL/min/1.73 mGFR was determined by measuring the plasma clearance of iohexol, an x-ray contrast agent that is a reliable marker for GFR. The patient was given 5 mL iohexol solution  intravenously in an antecubital vein. The GFR was calculated from the iohexol concentration after the injection. Serum iohexol levels were determined by high-pressure liquid chromatography. The total analytical imprecision of the method was 2.5% using a control sample with an assigned value of 33.5 mg/L and 2.4% for a control sample with an assigned value of 65.7 mg/L.Plasma creatinine measurements were performed by means of the modified kinetic Jaffe reaction on an Architect ci8200 analyzer  and reported using S.I. units (μmol/L). The total analytical imprecision of the creatinine method was 3.2% both at 94 and 337 μmol/L.Cystatin C immunoparticles (code 1014), assay buffer (code 1007), calibrator (code 1012) and control set (code 1019) were all obtained from Gentian . The immunoparticles are coated with purified chicken antibodies to cystatin C on uniform polystyrene particles. Plasma cystatin C measurements on the Architect ci8200 was performed using the following instrument settings: primary wavelength 548 nm, secondary wavelength 700 nm, sample blank position 18, and spline calibration method. 220 μL reagent 1 and 3 μL sample were mixed with 45 μL reagent 2. The total analytical imprecision of the cystatin C method was 1.7% at 0.77 mg/L, 1.1% at 1.25 mg/L and 1.4% at 5.45 mg/L.TSH, free T4 and T3 were also analyzed using the Architect ci8200. The measuring range of the TSH method was 0.001–100 mU/L. The total analytical imprecision of the TSH method was 2.4% at 0.52 mU/L and 2.4% at 5.07 mU/L. The total analytical imprecision was 7.7% at 0.81 nmol/L and 2.53% at 4.06 nmol/L for the T3 method, and 5.2% at 11.5 pmol/L and 4.4% at 16.8 pmol/L for the free T4 method.Anti-TPO was analyzed on a Liaison instrument . The total analytical imprecision of the anti-TPO method was 5.8% at 17.7 kU/L and 8.1% at 161.8 kU/L. Anti-TSH receptor antibodies were analyzed with an ELISA kit from RSR Ltd. .2) during the hyperthyroid period was lower than the corresponding iohexol-estimated GFR (121 mL/min/1.73 m2). On the other hand, the cystatin C-estimated GFR postoperatively (114 mL/min/1.73 m2) was in close agreement with the iohexol-estimated GFR during the hyperthyroid period. This indicates that the increase in cystatin C and subsequent decrease in cystatin C-estimated GFR is not due to a change in glomerular clearance, but rather due to an increased synthesis related to the hyperthyroid state. Cystatin C is considered to be produced in all nucleated cells at a constant rate. No iohexol clearance was performed in the euthyroid state. The iohexol clearance performed during the hyperthyroid state was part of the patient's treatment to evaluate whether the patient had a decreased GFR or not. During the euthyroid state, the cystatin C-estimated GFR was 114 mL/min/1.73 m2 and the creatinine-estimated GFR was 73 mL/min/1.73 m2. According to previous reports, the MDRD equation should only be used to report numeric results below 60 mL/min/1.73 m2 [Hypo- and hyperthyroid diseases occur frequently. Hence, it is important to be aware of erroneous results due to these diseases. The cystatin C-estimated GFR . Thus, both cystatin C and creatinine gave erroneous GFRs in comparison to iohexol clearance, which was used as the reference method.The creatinine values (36 μmol/L) in this patient were reduced during the hyperthyroid period with an ensuing catabolic state resulting in an overestimation of the creatinine-calculated GFR  declare that they have no competing interests.MR was responsible for patient care and data collection. MK and AL conceived the research question. MK and AL wrote the first draft. Subsequent revisions were carried out with input from all authors. The final version was approved by all authors.Written consent was obtained from the patient for publication of the study.


Bacteria and Archaea. Despite severe conditions and significant decreases in microbial abundance, no significant changes in richness or community structure were detected in the ice. Communities of Bacteria and Archaea in the ice, as in under-ice seawater, were dominated by SAR11 clade Alphaproteobacteria and Marine Group I Crenarchaeota, neither of which is known from later season sea ice. The bacterial ice library contained clones of Gammaproteobacteria from oligotrophic seawater clades  but no clones from gammaproteobacterial genera commonly detected in later season sea ice by similar methods . The only common sea ice bacterial genus detected in winter ice was Polaribacter. Overall, selection during ice formation and mortality during winter appear to play minor roles in the process of microbial succession that leads to distinctive spring and summer sea ice communities.The structure of bacterial communities in first-year spring and summer sea ice differs from that in source seawaters, suggesting selection during ice formation in autumn or taxon-specific mortality in the ice during winter. We tested these hypotheses by weekly sampling (January–March 2004) of first-year winter sea ice  that experienced temperatures from −9°C to −26°C, generating community fingerprints and clone libraries for  Betaproteobacteria and high G+C Gram-positives (Actinobacteria) have also been obtained in culture from Antarctic sea ice (in situ hybridization (FISH), overlap remarkably well with culture-based results, confirming the prevalence of these bacterial groups in spring and summer sea ice  lifetime of this ‘mild’ ice were linked to exchange processes at the ice–water interface and progression of the ice-algal bloom. These features do not pertain to upper horizons of much colder, thicker and light-limited Arctic winter sea ice, the subject of this study.Possible shifts in community composition and richness in Arctic winter ice over time and increasingly severe conditions are not known. From cultivation studies of Antarctic sea ice, psychrophilic bacteria are thought to outlive psychrotolerant species during winter . The dynAmundsen was immobilized in landfast sea ice of Franklin Bay (Western Arctic) through the winter, we first investigated the spatial heterogeneity and temporal dynamics of particulate matter, including microorganisms and particulate extracellular polymeric substances (pEPS), within the ice (in situ temperatures had ranged from −15°C to −26°C (25 cm) and −12°C to −22°C , when the CCGS  the ice , where iBacteria already known from spring and summer sea ice. To test this hypothesis we performed community fingerprinting of Bacteria  and Archaea  on the entire sample set collected during the CASES overwintering expedition  in community structure similarity  over time in community structure similarity  and Thermoplasma (228 bp) were found primarily in upper ice horizons; OTUs with best matches to Marine Group I.1c Crenarchaeota (499 bp), Methanobacteria (477 bp), RC-V Euryarchaeota (531 bp) and unknown groups  were found primarily in lower ice horizons  or thermophiles , based on a database of predicted terminal restriction fragment lengths of archaeal sequences from GenBank.All of the phylotypes detected in the archaeal clone libraries were also detected as T-RFLP OTUs, although many OTUs persisting through the winter had no representatives in our clone libraries, possibly as a result of the different primers used for cloning and fingerprinting. Several of these OTUs were putatively identified using a database of sequences from a recent study , includiAlphaproteobacteria . Differences were evident in the relative occurrence of Gammaproteobacteria, which appeared primarily in the sea ice library, and of Bacteroidetes, found primarily in the seawater library , Verrucomicrobia and Deltaproteobacteria (sea ice), and the uncultured ‘Marine Group A’ division (seawater). The seawater library had a greater Chao1 index of richness , likely underestimated due to lesser coverage , but the libraries had similar Shannon diversity indices .Overlap between the sea ice and seawater libraries was limited: 6 of 44 phylotypes were shared between ice  and water , but due to the common dominance of SAR11, no statistical difference was detected between the libraries using WebLIBSHUFF , similar richness  and similar indices of Shannon diversity .Archaeal sequences were present in both sea ice and seawater clone libraries; seven phylotypes had high similarities to existing Arctic archaeal clone sequences. The great majority (91%) of sequences from the archaeal libraries belonged to oup I.1a . The remoup I.1a . Two phyamoA  that simultaneously buffer against external ice-crystal damage, osmotic shock and viral attack has been proposed . EPS areAlphaproteobacteria, with only much smaller complements of the Alphaproteobacteria and Flavobacteriales well known from sea ice. While studies of later season sea ice have failed to detect Archaea, all of our winter ice (and seawater) samples contained them, with the dominant archaeon belonging to Marine Group I.1a Crenarchaeota. Clearly, these pelagic Bacteria and Archaea entrained into sea ice upon its formation and persisted through the winter. Our community structure results also indicate that microorganisms from other habitats, including terrestrial soil, riverine waters and marine sediment, entrain into sea ice and persist through the winter. Likely sources of the non-marine microorganisms include freshwater from the nearby Horton River, eroded soils from the Smoking Hills, and terrestrial organic matter from the Mackenzie River, the largest source of suspended particulates to the Beaufort Shelf  and clone library sequencing (91% of sequences in both libraries) in Franklin Bay is consistent with FISH counts in surface waters at the same site, showing a winter high for this group (to 16% of DAPI counts) that decreased to undetectable levels by late summer  on seasonal time scales . Others in the uncultured Roseobacter RCA cluster were detected infrequently in our libraries, consistent with their low abundance in seawater at our overwintering station  may help elucidate their adaptations to sea ice, just as the genome sequence analysis of Polaribacter MED152 has revealed their adaptations to sunlit surface waters  was melted into 0.22 µm filtered artificial brine solution  to concentrate and de-salt the genomic DNA, according to the manufacturer's recommendations. The recovered volume was increased to 600 µl with TE buffer before three rounds of phenol/chloroform extraction, followed by ethanol precipitation and re-suspension in 50 µl of TE buffer. Total DNA concentration was measured in a SpectraMaxM2 plate reader (Molecular Devices) using PicoGreen fluorescence (Invitrogen) according to the manufacturer's recommendations. Recovery of genomic DNA was < 1–33% based on cell counts  from each sampling day were combined in a single tube. In horizon I, due to low DNA yields, six filters – three from each of two sampling days in adjacent weeks – were combined in a single tube, i.e. from the following pairs of calendar sampling days: 10 and 17, 24 and 31, 60 and 67, and 74 and 81. Each tube then received, per filter, 800 µl of STE buffer  and 40 µl of 20% SDS. After incubation at 65°C for 20 min, each tube was vortexed, then centrifuged at 1400 l counts . No atteBacterial DNA fragments were PCR-amplified for ARISA using fluorescently labelled (6-HEX) forward primer Uni1392F, and unlabelled reverse primer R23S-125R (sequences located in 4 RFUs (ARISA) or 8 × 104 RFUs (T-RFLP) were removed from the analysis. The peaks in the remaining samples were binned using in-house software (http://rocaplab.ocean.washington.edu/cgi/dakster/index.html) and distance cut-offs of 1 bp for fragment lengths of 70–700 bp, 2 bp for 700–1200 bp and 4 bp for > 1200 bp. Each bin, representing a 16S-ITS-23S ribosomal DNA fragment (ARISA) or terminal restriction fragment (T-RFLP), was designated an OTU. The cumulative peaks heights of each OTU were used as gross measures of relative abundance to compare fingerprinting with clone libraries. A presence/absence matrix containing OTUs with at least one peak height greater than 1.0% (ARISA) or 0.25% (T-RFLP) of the sample's cumulative peak height was used for calculation of richness and pairwise similarity, and non-parametric multivariate analyses, performed with PRIMER v6 . A low-pass Fourier transform was applied to ARISA electropherograms to reduce noise and increase peak calling efficiency. Heights of saturated peaks in several T-RFLP electropherograms were estimated by fitting a Gaussian function to the non-saturated points, using the open-source statistical package R . For botRIMER v6 . PairwisRIMER v6 .Bacteria, a = Archaea, w = water and i = ice. Bacterial 16S-ITS-23S ribosomal DNA was amplified using primer pair Uni515F/R23S-125R  was used to call bases and construct contigs which were checked and edited manually as necessary. Most of the bacterial sequences included the 16S–23S ITS region, which we excluded from phylogenetic analyses but from which we calculated predicted ARISA fragment lengths and defined subtypes of 16S rRNA gene phylotypes. Seventeen sequences related to Stenotrophomonas maltophila were removed from the bacterial sea ice library as probable contaminants. ARISA fragment lengths predicted from these sequences (777 and 779 bp) overlapped with those predicted from other clone library sequences, but these fragments were observed only rarely in the ARISA data set. Bacterial sequences were deposited in GenBank with Accession No. EU836892–EU837057 and FJ753995–FB754002; archaeal sequences: EU486859–EU486955; and eukaryotic sequences: FJ753982–FJ753994.Four clone libraries were constructed for phylogenetic analysis, using the ribosomal RNA operon, from two samples: under-ice seawater from day 35, used to create libraries FB04bw and FB04aw; and winter sea ice from horizon I (combined days 74 and 81), used to create libraries FB04bi and FB04ai, where FB04 = Franklin Bay 2004, b = 23S-125R . Archaeahttp://greengenes.lbl.gov). All sequences were checked for chimeras with Bellerophon (paup* v4.10beta (paup* using the dotur (Small-subunit ribosomal rRNA gene sequences were aligned using the NAST aligner (4.10beta . ModelTe4.10beta . In ARB,he dotur  to definhe dotur .


Recent cancer therapies include drugs that target both tumor growth and angiogenesis including mammalian target of rapamycin (mTOR) inhibitors. Since mTOR inhibitor therapy is associated with significant side effects, we examined potential agents that can reduce the therapeutic dose.Methylnaltrexone (MNTX), a peripheral mu opioid receptor (MOR) antagonist, in combination with the mTOR inhibitors temsirolimus and/or rapamycin, was evaluated for inhibition of VEGF-induced human pulmonary microvascular endothelial cell (EC) proliferation and migration as well as in vivo angiogenesis (mouse Matrigel plug assay).MNTX inhibited VEGF-induced EC proliferation and migration with an IC50 of ~100 nM. Adding 10 nM MNTX to EC shifted the IC50 of temsirolimus inhibition of VEGF-induced proliferation and migration from ~10 nM to ~1 nM and from ~50 to ~10 nM respectively. We observed similar effects with rapamycin. On a mechanistic level, we observed that MNTX increased EC plasma membrane-associated tyrosine phosphate activity. Inhibition of tyrosine phosphatase activity  blocked the synergy between MNTX and temsirolimus and increased VEGF-induced tyrosine phosphorylation of Src with enhanced PI3 kinase and mTOR Complex 2-dependent phosphorylation of Akt and subsequent activation of mTOR Complex 1 (rapamycin and temsirolimus target), while silencing Src, Akt or mTOR complex 2 components blocked VEGF-induced angiogenic events.Our data indicate that MNTX exerts a synergistic effect with rapamycin and temsirolimus on inhibition of VEGF-induced human EC proliferation and migration and in vivo angiogenesis. Therefore, addition of MNTX could potentially lower the dose of mTOR inhibitors which could improve therapeutic index. Recent therapeutic interventions for the inhibition of cancer progression include drugs that target both tumor growth and angiogenesis. Mammalian target of rapamycin (mTOR) inhibitors, including sirolimus (rapamycin) and temsirolimus, are potential therapeutic agents for hepatocellular cancer and renal cell carcinoma due to their anti-proliferative and anti-angiogenic properties. However, these mTOR inhibitors are often associated with unwanted side effects including rash, asthenia, mucositis, nausea, edema, anemia, hyperglycemia, thrombocytopenia, hyperlipaenia and anorexia -5. ThereIn this study, we demonstrate that methylnaltrexone (MNTX) acts synergistically with the mTOR inhibitors, rapamycin and temsirolimus, on inhibition of VEGF-induced angiogenic events. Specifically, MNTX inhibited EC proliferation with an IC50 of ~100 nM. Adding 10 nM MNTX shifted the IC50 of temsirolimus on EC proliferation from ~10 nM to ~1 nM. Further, adding 10 nM MNTX shifted the IC50 of temsirolimus on inhibition of EC migration from ~50 nM to ~10 nM. The synergistic effects of MNTX and temsirolimus were also demonstrated in an in vivo model of angiogenesis (mouse Matrigel plug assay). There was a shift in the IC50 on inhibition of VEGF-induced EC proliferation and migration with MNTX and rapamycin. The synergistic mechanism involves MNTX activation of tyrosine phosphatase activity with consequent inhibition of VEGF-induced Src activation. MNTX-induced Src inactivation results in inhibition of PI3 kinase and mTOR signaling required for Akt activation (serine/threonine phosphorylation). These results suggest addition of MNTX could potentially lower the therapeutic doses of mTOR inhibitors including rapamycin and temsirolimus.2, 95% air, with passages 6-10 used for experimentation. Unless otherwise specified, reagents were obtained from Sigma . Vascular endothelial growth factor (VEGF) was purchased from R&D Systems . Methylnaltrexone bromide or methylnaltrexone (MNTX) was purchased from Mallinckrodt Specialty Chemicals . Temsirolimus was acquired through Wyeth Pharmaceuticals. Rapamycin was purchased from Sigma . Reagents for SDS-PAGE electrophoresis were purchased from Bio-Rad  and Immobilon-P transfer membrane was purchased from Millipore . Rabbit anti-pSer473Akt, rabbit anti-pThr308Akt, rabbit anti-Akt, rabbit anti-pThr389 p70 S6K and anti-p70 S6K antibodies were purchased from Cell Signaling Technologies . Rabbit anti-mTOR, rabbit anti-Rictor and rabbit anti-FKBP12 antibodies were purchased from Santa Cruz Biotechnology . Mouse anti-pp60src antibody was purchased from Upstate Biotechnologies . LY294002 was purchased from EMD Biosciences . Mouse anti-β-actin antibody, rabbit anti-phospho-tyrosine418 Src antibody and naltrexone, were purchased from Sigma . Secondary horseradish peroxidase (HRP)-labeled antibodies were purchased from Amersham Biosciences .Human pulmonary microvascular EC (HPMVEC) were obtained from Cambrex  and cultured as previously described ,8 in EBM2, 1% Nonidet P-40 (NP-40), 0.4 mM Na3VO4, 40 mM NaF, 50 μM okadaic acid, 0.2 mM phenylmethylsulfonyl fluoride, 1:250 dilution of Calbiochem protease inhibitor mixture 3). The samples were then immunoprecipitated with either anti-Raptor or anti-Rictor IgG followed by SDS-PAGE in 4-15% polyacrylamide gels, transfer onto Immobilon™ membranes, and developed with specific primary and secondary antibodies. Visualization of immunoreactive bands was achieved using enhanced chemiluminescence (Amersham Biosciences).Cellular materials from treated or untreated HPMVEC were incubated with IP buffer (50 mM HEPES (pH 7.5), 150 mM NaCl, 20 mM MgClThe siRNA for human mTOR, Src, Rictor, FKBP12 and Akt were purchased from Santa Cruz Biotechnology  and were utilized according to the manufacturer's specifications. Briefly, human lung EC were transfected with siRNA using siPORTamine™ as the transfection reagent . Cells (~40% confluent) were serum-starved for 1 hour followed by incubated with 250 nM of target siRNA (or scramble siRNA or no siRNA) for 6 hours in serum-free media. The serum-containing media was then added  for 42 h before biochemical experiments and/or functional assays were conducted.in vitro cell migration. HPMVEC (~1 × 104 cells/well) were plated with various treatments  to the upper chamber and VEGF (100 nM) was added to the lower chamber. Cells were allowed to migrate for 18 hours. Cells from the upper and lower chamber were quantitated using the CellTiter96™ MTS assay  and read at 492 nm. % migration was defined as the # of cells in the lower chamber % the number of cells in both the upper and lower chamber. Each assay was set up in triplicate, repeated at least five times and analyzed statistically by Student's t test .Twenty-four transwell units with 8 μM pore size were used for monitoring 3 cells/well pretreated with various agents  were incubated with 0.2 ml of serum-free media containing 100 nM VEGF for 24 h at 37°C in 5%CO2/95% air in 96-well culture plates. The in vitro cell proliferation assay was analyzed by measuring increases in cell number using the CellTiter96™ MTS assay  and read at 492 nm. Each assay was set up in triplicate, repeated at least five times and analyzed statistically by Student's t test .For measuring cell growth, HPMVEC .We focused our studies on temsirolimus and rapamycin based on our previous published data that MNTX regulates VEGF-induced Akt activation  and the The effects of MNTX on inhibition of mTOR described in this manuscript go beyond VEGF receptor activation and extend to downstream signaling pathways. We and others have previously reported that inhibition of Src protects from EC barrier disruption and angiogenesis ,8,10. SrTemsirolimus was approved by the FDA in 2007 for the treatment of advanced renal cell carcinoma, a disease resistant to existing chemotherapies  ,38-40. TOur results indicate that MNTX exerts a synergistic effect with rapamycin and temsirolimus on inhibition of VEGF-induced human EC proliferation and migration and in vivo angiogenesis. These synergistic effects are achieved through inhibition of different components of a common VEGF-induced angiogenic signaling pathway. MNTX inhibits the mu opioid receptor and stimulates tyrosine phosphatase activity which inhibits VEGF-induced Src activation and Src-regulated PI3 kinase and mTOR Complex 2-mediated Akt activation. Temsirolimus and rapamycin inhibit the downstream target of activated Akt, mTOR Complex 1. Inhibition of these events promotes synergistic inhibition of VEGF-induced angiogenesis Figure . TherefoDr. Moss serves as a paid consultant to Progenics Pharmaceuticals, Inc., has a financial interest in methylnaltrexone as a patent holder through the University of Chicago, and receives stock options from Progenics. Drs. Singleton and Garcia are co-inventors on patents with Dr. Moss. The other authors declare they have no competing interests.PAS helped conceive of the study, and participated in its design and coordination, performed the in vitro inhibition and isobologram assays and helped draft the manuscript. NM participated in the study design and conducted immunoprecipitation and immunoblot assays. FEL, BM and JHS participated in the study design and performed the in vivo Matrigel plug assays. LMV coordinated the animal studies by FEL, BM and JHS. RS participated in the design and coordination of the studies. JM helped conceive of the study, and participated in its design and coordination, and helped draft the manuscript. JGNG coordinated the work of LMV, BM and JHS. All authors read and approve the final manuscript.Determination of methylnaltrexone (MNTX) synergistic effects with rapamycin on inhibition of VEGF-induced human endothelial cell (EC) proliferation and migration. This file shows through inhibition curves and isobologram analysis that MNTX acts synergistically with the mTOR inhibitor, rapamycin, on inhibition of VEGF-induced angiogenic events.Click here for file


We show that PR55γ binds c-SRC and modulates the phosphorylation of serine 12 of c-SRC, a residue we demonstrate to be required for JNK activation by c-SRC. We also find that the physical interaction between PR55γ and c-SRC is sensitive to UV irradiation. Our data reveal a novel mechanism of c-SRC regulation whereby in response to stress c-SRC activity is regulated, at least in part, through loss of the interaction with its inhibitor, PR55γ.Protein Phosphatase type 2A (PP2A) represents a family of holoenzyme complexes with diverse biological activities. Specific holoenzyme complexes are thought to be deregulated during oncogenic transformation and oncogene-induced signaling. Since most studies on the role of this phosphatase family have relied on the use of generic PP2A inhibitors, the contribution of individual PP2A holoenzyme complexes in PP2A-controlled signaling pathways is largely unclear. To gain insight into this, we have constructed a set of shRNA vectors targeting the individual PP2A regulatory subunits for suppression by RNA interference. Here, we identify PR55γ and PR55δ as inhibitors of c-Jun NH 2-terminal kinase (JNK) kinase pathway, which, depending on stimulus, can affect either cell survival or cell proliferation. Here we report that the “B” subunit PR55γ acts as a negative regulator of JNK activity and cell death. We show that PR55γ influences JNK activity by inhibiting one of its upstream regulators, the proto-oncogene c-SRC, through dephosphorylation at one of the key residues on c-SRC, a site we show to be critical for c-SRC activation following cell stress. Overall our work describes the novel function of a specific PP2A subunit involved in cell survival and identifies a novel mechanism of c-SRC regulation.Protein Phosphatase type 2A (PP2A) represent a family of holoenzyme complexes involved in wide range of activities such as growth, differentiation, and cell death. The PP2A holoenzyme complex is made up of a catalytic, a structural, and one of various “B” subunits. These “B” subunits are thought to provide the substrate specificity required for PP2A activity. Previous work on PP2A has mostly been derived by inhibiting the catalytic subunit through chemical inhibition, as such inhibiting all of the pathways associated with PP2A. To identify individual “B” subunits involved in specific cellular processes we have generated a “B” subunit gene knockdown library, which allows us to inhibit each of the known “B” subunits individually. One of the many pathways regulated by PP2A is the c-Jun NH PMA at a final concentration of 200 nM was added for 30 min at 37 °C or the cells were treated with UV irradiation (100 J/m2) and incubated for a further 30 min at 37 °C. c-SRC was immunoprecipitated with Flag antibody (Sigma) as described above. The entire sample was loaded onto an SDS-PAGE gel, run, and then dried. The film was then exposed for 3 h at room temperature. The radioactive bands were then isolated, proteins eluted, digested with trypsin, and phosphopeptide mapping was performed as described previously [32 P] orthophosphate was added to the cells and incubated for a further 2 h in the same medium. c-SRC was immunoprecipitated with Flag antibody (Sigma) and an HA antibody  as described above. The entire sample was loaded onto an SDS-PAGE gel, run, and then dried.For tryptic phosphopeptide analysis U2-OS cells were cotransfected with 4 μg Flag-Src or 4μg or Flag-Src (12A) and 20 μg PR55γ or control vector. Cells were phospho-starved for 45 min and 2 mCi of [eviously ,62. For Table S1(114 KB DOC)Click here for additional data file.Table S2(26 KB DOC)Click here for additional data file.


O-linked β-N-acetylglucosamine transferase  and is O-GlcNAc modified; and also undergoes tyrosine phosphorylation in response to insulin. Tyrosine 114 (Tyr114) and tyrosine 259 (Tyr259) in PHB are in the close proximity of potential O-GlcNAc sites serine 121 (Ser121) and threonine 258 (Thr258) respectively. Substitution of Tyr114 and Tyr259 residues in PHB with phenylalanine by site-directed mutagenesis results in reduced tyrosine phosphorylation as well as reduced O-GlcNAc modification of PHB. Surprisingly, this also resulted in enhanced tyrosine phosphorylation and activity of OGT. This is attributed to the presence of similar tyrosine motifs in PHB and OGT. Substitution of Ser121 and Thr258 with alanine and isoleucine respectively resulted in attenuation of O-GlcNAc modification and increased tyrosine phosphorylation of PHB suggesting an association between these two dynamic modifications. Sequence analysis of O-GlcNAc modified proteins having known O-GlcNAc modification site(s) or known tyrosine phosphorylation site(s) revealed a strong potential association between these two posttranslational modifications in various proteins. We speculate that O-GlcNAc modification and tyrosine phosphorylation of PHB play an important role in tyrosine kinase signaling pathways including insulin, growth factors and immune receptors signaling. In addition, we propose that O-GlcNAc modification and tyrosine phosphorylation is a novel previously unidentified binary switch which may provide new mechanistic insights into cell signaling pathways and is open for direct experimental examination.Prohibitin (PHB or PHB1) is an evolutionarily conserved, multifunctional protein which is present in various cellular compartments including the plasma membrane. However, mechanisms involved in various functions of PHB are not fully explored yet. Here we report for the first time that PHB interacts with  Prohibitin  and its homologue PHB2 are evolutionarily conserved proteins and have been shown to be involved in cell cycle progression, cell differentiation, gene transcription and mitochondrial functions O-GlcNAc (O-linked β-N-acetylglucosamine) modification of tyrosine kinase signaling intermediates by O-GlacNAc transferase (OGT) has been shown to play a regulatory role in receptor tyrosine kinase signaling pathways N-acetylglucosamine from UDP-GlcNAc to a hydroxyl oxygen of serine or threonine in a β confirmation (β-O-glycosylation) O-GlcNAc modification under different conditions) exists O-GlcNAc modification and vice versa. Here we report that PHB interact with OGT both in vivo and in vitro and is an O-GlcNAc modified protein. In addition, we provide data which suggests that O-GlcNAc modification and tyrosine phosphorylation in a protein interact with each other. Implication of this study extends beyond protein specific binary switch and offers new mechanistic insights into tyrosine kinase signaling pathways.In addition to tyrosine phosphorylation, in situ co-localization of PHB and OGT using confocal microscopy  in C2C12 and NIH-3T3 cell lines and subsequent tagging with biotin alkyne (O-GlcNAc modification of PHB is not cell specific (O-GlcNAc modified PHB band was observed in comparison to untreated controls suggesting that insulin facilitates O-GlcNAc modification of PHB. A similar result was found with RINm5F cells (data not shown). These data together suggest that PHB interacts with OGT and is an O-GlcNAc modified protein.Because PHB and OGT both interact with insulin receptor and undergo tyrosine phosphorylation croscopy . This prfication . Recombiantibody . O-GlcNAn alkyne . In bothspecific . In insuO-GlcNAc modification and tyrosine phosphorylation, we asked the question whether these modifications occur in neighboring residues and affect each other. Analysis of PHB protein sequence by Human Protein Reference Database, NetPhos and Scansite servers identified Tyr114 and Tyr259 as potential sites of phosphorylation. Interestingly two (i.e. Ser121 and Thr258) out of three potential O-GlcNAc sites  predicted (discussed later) in PHB are either adjacent to or in the close proximity of tyrosine phosphorylation sites (Tyr114 and Tyr259). Therefore, we used two synthetic peptides (Pep I - Biotin-105RIFTSIGEDYDERVLPSIT123-NH2 and Pep II - Biotin-250QLSRSRNITYLPAGQSVLL268-NH2) biotinylated at the N-terminus and amidated at the C-terminus spanning tyrosine phosphorylation and O-GlcNAc modification sites in PHB. In in vitro kinase assays using catalytic domain of insulin receptor  and ATP  and subsequent analysis with immunoblotting we found that both peptides were phosphorylated at tyrosine residue  significantly enhanced the O-GlcNAc modification of both peptides in comparison to non-phosphorylated control peptides  were immobilized on streptavidin-agarose beads and processed for in vitro O-GlcNAc assays using immunoprecipitated OGT and UDP-GlcNAc. After washing, immobilized control peptides (without any modification) and OGT induced O-GlcNAc modified peptides were used for in vitro kinase assays using catalytic domain of insulin receptor and ATP. Interestingly tyrosine phosphorylation of these peptides was attenuated in the presence of O-GlcNAc modification in comparison to non-glycosylated control peptides . Surprisingly a significant up regulation of O-GlcNAc modification of some proteins in the molecular weight range of ∼50–100 kDa was also observed in response to insulin in cells transfected with Tyr114Phe and Tyr259Phe PHB mutants  and β-1,4-N-acetyl-galactosaminyl transferase 2 (Accession no. XP_548187.2) were identified with significant homology with PHB  is due to increased OGT activity, in vitro O-GlcNAc assays were performed using immunoprecipitated OGT from insulin treated cells co-transfected with OGT and wild type PHB or tyrosine (Tyr114 and Tyr259) mutant-PHB. An increase in OGT enzyme activity was found consistent with increased tyrosine phosphorylation of OGT in comparison with controls  is the most favored residue for O-GlcNAc modification in a protein  having potential tyrosine phosphorylation site(s) in the close proximity of a known O-GlcNAc site(s) with proline residue(s) at −/+1 and/or −/+3 position of O-GlcNAc site(s) or (ii) having potential O-GlcNAc site(s) with proline residue(s) at −/+1 and/or −/+3 position in the close proximity of a known tyrosine phosphorylation site (in this category only those proteins were considered which are known to undergo O-GlcNAc modification however exact sites of modification are not known yet). A number of proteins were identified using each selection criteria  in C2C12 and NIH-3T3 cell lines  and second (Tyr114) is flanked by two other potential O-GlcNAc modification sites i.e. Ser101 (101SQLP104) and Ser121 (120PSITT124)  in PHB is also conserved in PHB2 and corresponding residue in PHB2 is Tyr128. Tyr128 in PHB2 has been identified as phosphorylation site during phospho-tyrosine profiling in cancer cells O-GlcNAc modification in PHB2 occurs at Ser135. It is possible that O-GlcNAc modification and tyrosine phosphorylation in PHB2 interact in the same way as in PHB.Prediction of potential proteins  identifion sites . InteresSITT124) . O-GlcNAs by OGT  suggeste254RSRNIT258 (R-x-R-x-x-S/T) for Akt. Interestingly Tyr259 in PHB is present at tandem position from Akt motif and Thr258 O-GlcNAc site is part of Akt consensus motif (R-x-R-x-x-S/T). This would imply that Thr258 may be subjected to O-GlcNAc modification or phosphorylation under different conditions and phosphorylation status of adjacent tyrosine residue (Tyr259) may play a regulatory role in this process.In another study, PHB has been identified as a substrate for Akt however exact phosphorylation site remains to be determined O-GlcNAc modification and tyrosine phosphorylation and these modification sites are either adjacent or in the close proximity to each other we hypothesize that O-GlcNAc modification and tyrosine phosphorylation may be a previously unidentified novel binary switch. This would imply that these modifications may interact and regulate each other under different conditions, provide read-out for different interacting partners, attribute multiple functions to the same protein and may play an important role in cell signaling cascades . However, both OGT and PHB contain similar phospho tyrosine motifs therefore recruitment and subsequent modulation by other phospho tyrosine binding (PTB) domain containing proteins cannot be ruled out and warrant further investigation.Because PHB  undergoes both cascades . Using bch other . This isagenesis . In addi insulin . BecauseO-GlcNAc sites or vice versa (O-GlcNAc site (59–93%), as well as O-GlcNAc and tyrosine phosphorylation sites (71–87%)  a number of proteins were identified with potential tyrosine phosphorylation sites in the proximity of known ce versa  and S2. (71–87%) . Given t5′ CAGCATCGGAGAGGACTTTGATGAGCGTGTGC 3′ and reverse - 5′ GCACACGCTCATCAAAGTCCTCTCCGATGCTG 3′; Y259F: Forward -5′ TCTCGGAACATCACCTTCCTGCCAGCGG 3′ and reverse - 5′ CCGCTGGCAGGAAGGTGATGTTCCGAGA 3′; S121A Forward - 5′ AGCGTGTGCTGCCGGCCATCACAACTGAG 3′ and reverse - 5′ CTCAGTTGTGATGGCCGGCAGCACACGCT 3′; T258I Forward - 5′ CTCTCGGAACATCATCTACCTGCCAGCGG 3′ and reverse - 5′ CCGCTGGCAGGTAGATGATGTTCCGAGAG 3′. Authenticity of all constructs was confirmed by DNA sequencing at Cancer Care Manitoba. Transfections were performed using lipofectamine , according to manufacturer's protocol. After 48 hrs of transfection cells were serum starved and treated with insulin (100 nM) for 20 minutes. Cell lysates were prepared in lysis buffer  containing protease and phosphatase inhibitors For all cell based experiments cells were grown in 100 mm culture dish in RPMI-1640  complete growth media containing 10% FBS, 1% antibiotic-antimycotic at 37°C. OGT construct was obtained from Prof. Gerald Hart, University of John Hopkins. The pCMV6-XL5 vector containing human PHB clone (Reference sequence XM_056688) was obtained from Origene technologies, USA. PHB mutants were made using site-directed mutagenesis kit  following manufacturer's instructions. The following primers were used for generating PHB mutants: Y114F, Forward - Escherichia coli and purified by His-affinity column was obtained from AmProx Inc. . Custom made PHB105–123 and PHB2250–268 peptides (Pep I and Pep II) were chemically synthesized as 19mer peptide amidated at C-terminus and biotinylated at N-terminus. The correct peptides were obtained in greater than 90% yield and were homogeneous after purification as confirmed by mass and analytical HPLC.Recombinant human His-tagged PHB (His-PHB) expressed in Immunoprecipitation of PHB and OGT was performed using Dynabeads Protein A and DynaMag  according to manufacturer's protocol. In brief, to 1 ml of cell lysate , 10 µl of rabbit polyclonal anti-PHB antibody or monoclonal OGT antibody were added and incubated overnight on a rotating device at 4°C. At the end of the incubation, 25 µl of Dynabeads protein A suspension were added to each tube and further incubated for 2 h. Subsequently, pellets were washed (5×) in ice cold PBS, resuspended and used for in vitro assays and Western blot. Immunoprecipitated proteins were separated on 12% SDS-PAGE and transferred to nitrocellulose membrane for Western blotting as described earlier RINm5F cells were cultured in chambered slides, fixed with 4% formaldehyde in PBS and incubated with antibodies to OGT , and PHB  at a concentration of 1 µg/ml in PBS containing 0.1% BSA. Immune complexes were detected with TRITC and FITC conjugated antibodies to mice and rabbit  respectively.O-GlcNAc metabolic labeling was performed using Click-iT GlcNAz metabolic glycoprotein labeling reagent  from Invitrogen, USA. In brief, cultures were seeded at 2.5×105 cells/ml in 100 mm tissue culture dish and allowed to grow for 72 hrs in the presence of 40 µM tetraacetylated azido-sugar. Cell lysates were prepared with or without insulin treatment in lysis buffer  containing protease and phosphatase inhibitors 105RIFTSIGEDYDERVLPSIT123-NH2 and Pep II - Biotin-250QLSRSRNITYLPAGQSVLL268-NH2, amidated at C-terminus and biotinylated at N-terminus) were incubated with 20 ng of recombinant active insulin receptor in kinase buffer  for 20 min at 30°C. At the end of assays, 10 µl of loading buffer was added to each tube and analyzed on 16% SDS-PAGE, transferred to nitrocellulose membranes and processed for Western blot as described above. To study the effect of O-GlcNAc modification on tyrosine phosphorylation peptides were first immobilized on streptavidin-agarose and then processed for in vitro O-glycosylation assay using immunoprecipitated OGT and UDP-GlcNAc. At the end of incubation, tubes were transferred to the ice and subsequently washed (3×) to remove reaction mixture and finally resuspended in 50 mM Tris-Cl buffer pH 7.4 for subsequent use in an in vitro kinase assay using catalytic domain of insulin receptor and ATP. Subsequently peptides were analyzed by gel-electrophoresis and Western blotting as described previously PHB synthetic peptides (2 µg)  containing Ser121 and Thr258 were incubated with immunoprecipitated OGT in assay buffer  in a total reaction volume of 40 µl for 2 h at 30°C Table S1O-GlcNAc site.Protein sequences having potential tyrosine phosphorylation site in the vicinity of known (0.09 MB PDF)Click here for additional data file.Table S2O-GlcNAc site in the vicinity of known tyrosine phosphorylation known.Protein sequences having potential (0.12 MB PDF)Click here for additional data file.Table S3O-GlcNAc and phospho tyrosine binary switch.Summary of potential (0.14 MB PDF)Click here for additional data file.


Neurofibrillary tangles (NFT), a cardinal neuropathological feature of Alzheimer's disease (AD) that is highly correlated with synaptic loss and dementia severity, appear to be partly attributable to increased phosphorylation of the microtubule stabilizing protein tau at certain AD-related residues. Identifying the kinases involved in the pathologic phosphorylation of tau may provide targets at which to aim new AD-modifying treatments.We report results from a screen of 572 kinases in the human genome for effects on tau hyperphosphorylation using a loss of function, high-throughput RNAi approach. We confirm effects of three kinases from this screen, the eukaryotic translation initiation factor 2 α kinase 2 (EIF2AK2), the dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A (DYRK1A), and the A-kinase anchor protein 13 (AKAP13) on tau phosphorylation at the 12E8 epitope (serine 262/serine 356). We provide evidence that EIF2AK2 effects may result from effects on tau protein expression, whereas DYRK1A and AKAP13 are likely more specifically involved in tau phosphorylation pathways.These findings identify novel kinases that phosphorylate tau protein and provide a valuable reference data set describing the kinases involved in phosphorylating tau at an AD-relevant epitope. Alzheimer's disease (AD) is a secondary tauopathy, generally thought to result from the upstream effects of toxic amyloid aggregates. While there are a growing number of amyloid-modifying therapeutics in clinical trials, targeting amyloid alone may not be sufficient to mitigate the cognitive deficits that occur during the full course of AD. Other tIn AD, tau protein becomes hyperphosphorylated and aggregates into paired helical filaments (PHF), the main component of NFTs -7. IndeeIn vitro, numerous Ser/Thr kinases phosphorylate tau protein[in vivo role of these kinases in the etiology of neurofibrillary tangle formation remains unclear.u protein. Kinasesu protein,17, protu protein, microtuu protein, phosphou protein, and glyu protein,22. HoweWe report the use of a high-content siRNA based screening strategy, surveying 572 kinases throughout the human genome, to identify the kinases involved in ser262 tau phosphorylation. After screening validated siRNAs, we identified candidate kinases that either increased or decreased pS262 tau levels. We provide evidence that the eukaryotic translation initiation factor 2 α kinase 2 (EIF2AK2), dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A (DYRK1A), and a-kinase anchor protein 13 (AKAP13) proteins contribute to phosphorylation of tau at the 12E8 epitope (pS262/pS356).MAPT) mRNA , which has previously been shown to phosphorylate tau protein on serine 262[MARK2 siRNA, which reduced MARK2 expression >95% at the mRNA level, led to a 26% reduction in 12E8 phosphorylated tau (p = 0.029 for three replicates) and to an insignificant change in total tau protein expression  protein expression. The assay uses an H4 neuroglioma cell line engineered to overexpress four repeat tau (4R0N). We confirmed that the assay was sensitive to changes in both total tau protein levels and phosphorylated tau protein levels using siRNA directed at the tau  for kinases that affect tau phosphorylation status. Cataloging of kinases from the human genome has identified at least 518 kinases,24. ThisTo identify potential therapeutic targets that might modify the course of tau phosphorylation and dysfunction in AD, we first identified those kinases that significantly affected the ratio of 12E8 tau to total tau. The ratio of 12E8 tau/total tau could be altered in several ways, either by changes to phosphorylated tau, by changes to total tau, or by a combination of changes to both phosphorylated tau and total tau. For this reason we initially identified siRNAs that significantly reduced 12E8 tau levels with no significant effects on total tau levels relative to non-silencing siRNA controls. Of these kinases, we identified those that significantly affected the ratio of 12E8 tau/total tau relative to the non-silencing siRNA control. These kinases are listed in Table AKAP13) and dual specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A), which were selected both for the significance of the effect  and for the known biology of these candidates (see Discussion). The gene encoding DYRK1A is located within the Down Syndrome critical region (DSCR) on chromosome 21. Screening results showed 12E8 tau levels to be reduced to 77 ± 5% of control (p = 0.017), total tau to be 85 ± 10% of control (p = 0.127), which lead to a significant reduction in the ratio of 12E8 tau/total tau to 86 ± 4% of control .Notably, the microtubule affinity regulating kinase 2 (MARK2) protein, which has been shown to phosphorylate serine 262 of tau protein, showed n Figure . ResultsAKAP13 showed that this protein kinase A (PKA) associated protein is also involved in 12E8 tau phosphorylation. Screening results showed 12E8 tau levels to be reduced to 74 ± 7% (p = 0.023) of control non-silencing siRNA samples, total tau levels to be 93 ± 7% (p = 0.223) of control, leading to a reduction in the ratio of 12E8 tau/total tau to 79 ± 9% (p = 0.054) of control  gene significantly reduced 12E8 and total tau levels  may be exposed and phosphorylation pathways that do not normally regulate tau would have an effect in this assay. However, the identification of MARK2 as a significant pS262 tau phosphorylator Table  suggestsEIF2AK2) and the dual specificity tyrosine phosphorylation regulated kinase 1A gene (DYRK1A) have been implicated previously in neurodegeneration in AD. Polymorphisms within the eukaryotic translation initiation factor 2 α kinase 2 (EIF2AK2) gene have been genetically associated with AD[Both the eukaryotic translation initiation factor 2 α kinase 2 gene  was transfected with a 4R0N tau construct in pcDNA3.1. Following positive selection using geneticin (Invitrogen), individual clones were isolated and screened for tau expression using immunocytochemistry with a rabbit anti-tau antibody (Dako). A single stable tau overexpressing cell line was selected for subsequent siRNA studies. Cells were maintained in Dulbecco's Modified Eagle Medium (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen), 1% penicillin-streptomycin, geneticin (0.25 mg/ml), and 2 mM L-Glutamine (Invitrogen). Cells were split 1:10 at 90% confluency, twice a week.Chemically synthesized siRNA (18.6 μg) from Qiagen's validated human kinase siRNA Set 2.0 were printed into 96-well black clear bottom plates (Corning #3904) using a BioMek FX (Beckman Coulter). Printed plates were then foil sealed and stored at -80°C until use.2, for 96 hours.In order to identify genes involved in modulation of tau phosphorylation, we developed a high content siRNA screening assay. Briefly, 4R0N tau overexpressing H4 cells were reverse-transfected with library siRNA and siLentfect (Bio-Rad) using semi-automated transfection as follows: Screening plates were thawed and siRNA was complexed by addition of 50 μl of diluted siLentfect in OptiMEM (Invitrogen) using a μFill (BioTek) followed by incubation for thirty minutes at RT. Cells were trypsinized and resuspended in growth media without penicillin-streptomycin and added at a concentration of 3000 cell/well in 50 μl using a μFill followed by incubation for 5 minutes at room temperature (RT). Assay plates were incubated at 37°C and 5% COPlates were washed twice with 1× Tris-buffered saline (TBS) (Fisher) and fixed for 15 minutes with4% paraformaldehyde (PFA) at RT. Plates were then washed twice with 1× TBS and incubated with blocking buffer  and 0.1% NP-40) for 1 hour at RT. Fixed cells were next incubated with 1 μg/ml of phospho-tau antibody 12E8  at 4°C overnight. Plates were washed three times with 1× TBS-T (TBS with 0.1% Tween-20) followed by incubation with 1:200 dilution of rabbit anti-human tau (Dako) for 1 hr at RT. Plates were then washed twice with 1× TBS-T followed by incubation with a secondary antibody cocktail of 2 μg/ml FITC-Goat anti-rabbit IgG, 2 μg/ml Cy5- Goat anti-mouse IgG (Jackson Immunore search) and 10 μg/ml Hoechst 33342 (Invitrogen) in blocking buffer for 30 minutes at RT. Plates were then washed once with TBS-T followed by twice with TBS leaving the last wash in the plates. Plates were stored at 4°C overnight prior to analysis. Plates were imaged and then analyzed on the INCell 3000 . The analysis module (Object Intensity 01) identified each cell by the blue staining of the nuclei, and measured both red (Cy5) and green (FITC) intensities in a 4 pixel ring around the nuclei.2. Cell lysates were prepared using the Complete Lysis-M, EDTA-free kit (Roche Applied Science) and quantitated using the BCA protein assay (Pierce). Protein from lysates (20 μg) were separated by SDS-PAGE and transferred to nitrocellulose. Membrane was blocked in 5% BSA for one hour at RT. Membranes were probed with primary antibody overnight at 4°C on a rocker. Membranes were subsequently washed with TBS-T and probed with secondary antibody 1:25000 dilution of HRP-GAM (Jackson Immunoresearch) for forty-five minutes. Membranes were further washed and developed with Super Signal West Femto Maximum Sensitivity Substrate Kit (Promega) and imaged. To test multiple primary antibodies, membranes were stripped for 15 minutes at RT using ReBlot Plus Mild Antibody Stripping Solution (Millipore). Membranes were then washed again for 5 minutes at RT then blocked for one hour in 5% BSA. Membrane was reprobed overnight at 4°C with an anti-Tubulin antibody . Antibodies used for detection included anti-tau  primary antibody, 12E8 antibody (1:7500), anti-EIF2AK2 , anti-DYRK1A , and anti-AKAP13 .4R0N tau overexpressing H4 cells were reverse-transfected with siLentfect complexed with target siRNA in 6 well plates. Cells were grown for 72 hours at 37°C, 5% COAll are defined in the text upon first use.All authors have read and approved the final manuscript. DOA, RR ; GRB, CB, GDB, DRH, JAH, KMB, LG, AG, JR (assisted with siRNA assay development); CD(helped generate the H4-tau cell line); DF, BM ; EMR, Hutton MH, DAS, SM ; TD . All authors read and approved the final manuscript.Total tau and 12E8 tau data for all siRNAs in the Qiagen Kinome siRNA library. All data from the kinome siRNA library screen. p Tau refers to 12E8 tau. Cumulative results columns represent the average of the three independent experiments reported.Click here for file


Drosophila mitosis. We found that the Aurora B kinase complex is essential for this phosphorylation at centromeres, while Polo kinase is required to down-regulate H2A phosphorylation on chromosome arms in mitosis. Cyclin B degradation triggers loss of centromeric H2A phosphorylation at anaphase onset. Epistasis analysis indicated that Polo functions upstream of the H2A kinase NHK-1 but parallel to Aurora B. Therefore, multiple mitotic kinases work together to specify the spatial and temporal pattern of H2A T119 phosphorylation.The spatial and temporal control of histone modifications is crucial for precise regulation of chromatin structure and function. Here we report that phosphorylation of H2A at threonine 119 (T119) is enriched at centromere regions in  In eukaryotes, genomic DNA is first packaged into nucleosomes and then organised into higher-order chromatin structures. Chromatin organisation is locally or globally changed in response to external and internal signals. The changes are required for executing critical biological functions, most notably in regulated gene expression and chromosome segregation. Various post-translational modifications take place on histones, mostly in their tail domains, and play critical roles in the regulation of chromatin structure and function, either directly or indirectly through the recruitment of specific chromatin binding proteins Upon entry into mitosis, chromatin undergoes dramatic morphological changes to form mitotic chromosomes. On mitotic chromosomes, centromeres form unique chromosomal domains which are crucial for chromosome segregation in two respects Drosophila H2A Drosophila mitosis. The Aurora B complex is required for this phosphorylation in centromeric regions, while Polo kinase suppresses phosphorylation by NHK-1 on chromosome arms. Inactivation of Cdc2 kinase is required for loss of centromeric phosphorylation at the metaphase-anaphase transition. Therefore, these mitotic kinases together control the temporal and spatial pattern of H2A phosphorylation at centromeres.Recently a novel phosphorylation site was identified at threonine 119 (T119) in the C-terminal tail of Standard immunological, DNA manipulation and protein techniques were followed throughout Culture and RNAi of S2 cells were carried out as described Cultured cells were examined using a Plan-Apochromat objective lense  attached to an Axioplan2 (Zeiss). Images were captured by a CCD camera  using OpenLab2 (Improvision). Larval central nervous systems were taken using a Plan-Apochromat lense  attached to an Axiovert 200 M (Zeiss) with a confocal scan head . Confocal images were presented as a maximum intensity projection of the Z-stacks. All digital images were imported to Photoshop (Adobe) and adjusted for brightness and contrast.p-nitrophenylphosphate) and incubating on ice for 10 min. Lambda phosphatase (NEB) was added to the cell extract without phosphatase inhibitors and both samples incubated for 30 min at 37 °C. 2× SDS sample buffer was then added to the extracts and boiled for 3 min. Samples were then western blotted with anti-dH2A-pT119 to compare phospho-protein levels. In addition, cells immediately resuspended in 1× SDS sample buffer were included for comparison.For western blotting of phosphatase treated cell extract, cell extracts were obtained by resuspending S2 cells in lysis buffer  with or without phosphatase inhibitors .nhk-1 mutant (nhk-1E107) used in this study was previously described Standard techniques for fly manipulation were followed Drosophila S2 cells using an antibody which specifically recognises this phosphorylated form of H2A  recognised by the antibody on immunoblots . FurtherIn syncytial embryos and oocytes, entire mitotic/meiotic chromosomes are stained with the anti-dH2ApT119 antibody NHK-1 greatly reduced phosphorylation at this site in oocytes, but not in follicle or nurse cells NHK-1 .Centromeric distribution and regulation by conserved mitotic kinases may tempt us to speculate possible involvement of H2A T119 phosphorylation in chromosome segregation in mitosis. The phosphorylation might be important for generating or sensing tension between sister chromatids, or modes of microtubule attachment to kinetochores through the formation of centromere-specific chromatin or recruitment of centromere proteins during mitosis. A loss or misregulation of the H2A phosphorylation may be responsible for a subset of the highly pleiotropic phenotypes observed after down-regulation of Aurora B or Polo


In some cases downstream targets of the Gαq/Ca2+ signaling arm are directly inhibited by β-arrestins, while in other cases the two pathways are synergistic; thus β-arrestins act as molecular switches capable of modifying the signal generated by the receptor.Proteinase-activated-receptor-2 (PAR2 can activate adenosine monophosphate-activated protein kinase (AMPK), a key regulator of cellular energy balance, through Ca2+-dependent Kinase Kinase β (CAMKKβ), while inhibiting AMPK through interaction with β-arrestins. The ultimate outcome of PAR2 activation depended on the cell type studied; in cultured fibroblasts with low endogenous β-arrestins, PAR2 activated AMPK; however, in primary fat and liver, PAR2 only activated AMPK in β-arrestin-2-/- mice. β-arrestin-2 could be co-immunoprecipitated with AMPK and CAMKKβ under baseline conditions from both cultured fibroblasts and primary fat, and its association with both proteins was increased by PAR2 activation. Addition of recombinant β-arrestin-2 to in vitro kinase assays directly inhibited phosphorylation of AMPK by CAMKKβ on Thr172.Here we demonstrate that PAR2 signaling may act as a molecular switch turning a positive signal to AMPK into an inhibitory one.Studies have shown that decreased AMPK activity is associated with obesity and Type II Diabetes, while AMPK activity is increased with metabolically favorable conditions and cholesterol lowering drugs. These results suggest a role for β-arrestin in the inhibition of AMPK signaling, raising the possibility that β-arrestin-dependent PAR Blots were imaged with Alexa680-conjugated rabbit (Invitrogen) and IR800-conjugated mouse (Rockland) secondary antibodies  using the LICOR Odyssey imaging system, and LICOR software was used to calculate integrated intensities of bands; phospho-AMPK and ACC levels were normalized total AMPK and actin respectively. Fold increases in phosphorylation were determined by dividing the band density observed with treatment by that observed in the absence of treatment. Images of Western blots were assembled using Adobe Photoshop 6.0 and imported into Adobe Illustrator. Some gels were spliced to eliminate blank lanes or lanes containing samples unrelated to the figure and splicing is indicated by a white space.Lysates from cells and liver or fat tissue (10 μg protein) were subjected to 8% SDS-PAGE gel and transfer to PVDFfl (Millipore) membranes, followed by Western blotting with the following antibodies: Rabbit polyclonal anti-phosphor-AMPK-ThrCleared lysates (500 μg of protein) were incubated with 20  μl of mouse anti-FLAG agarose conjugated antibodies pre-bound to protein A-agarose with mouse anti- AMPKα1 and 2 (1 μg/200 μg protein) coupled to Protein G agarose for 2hrs at 4°C on a rotator. Immune complexes were resolved by 10% SDS-PAGE and western blotting performed as described.32P-ATP (Perkin Elmer) into a synthetic substrate of AMPK, SAMS (HMRSAMSGLHLVKRR) peptide (Upstate Technologies), in the presence of 5 mM MgCl2, 200  μM AMP and 200  μM [γ-32P] ATP (0.05  μCi/μl). Phosphorylated SAMS peptide was captured on phospho-cellulose strips (Pierce) and counted in a Beckman Scintillation counter; levels of AMPK present in each reaction was determined by western blotting of AMPK immune complexes after removal of reaction mixture, by comparing band density to that of a known quantity of purified recombinant AMPKα. Either the fold increase in activity was determined by dividing the normalized cpm incorporated with 2fAP treatment by that observed in the absence of stimulus or the moles ATP incorporated into each reaction was determined and expressed as nmoles ATP/mg enzyme/min.AMPK was immunoprecipitated from cleared lysates with anti-AMPKα1/2 (1 μg/200  μg protein) as described above. Washed immune complexes were then used for AMPK assays. AMPK activity was determined by the incorporation of 32P] ATP) and 5 mM MgCl2 in the presence of increasing concentrations of recombinant GST alone or GST-β-arrestin-2 at 30°C for 15 min. The enzyme concentration chosen represented the IC50 value determine in Figure GST alone and GST-tagged β-arrestin-2  was purified as described previously . Recombit-tests using graphing software Microsoft Excel or GraphPad Prism, with P < 0.05 considered significant.All experiments were repeated a minimum of three times and results are presented as mean  μ S.E.M. Differences between multiple groups were examined by two-way ANOVA and Tukey PW performed all AMPK phosphorylation experiments and AMPK activity assays, in vitro CAMKK assays and co-immunoprecipitations and wrote the 1st draft of the manuscript.YJ and YW performed HPLC and Mass spectrometric analysis of nucleotides and provided the data shown in Figure JS assisted in the initial set up of AMPK assays in the laboratory.KD designed the majority of the experiments, performed data analysis and was the senior investigator on this project.All authors read and approve the final draft.


Although numerous non-radioactive methods are in use to measure the catalytic activity of protein kinases, most require specialized equipment and reagents and are not sufficiently sensitive for the detection of endogenous kinase activity in biological samples. Kinases of the DYRK family have important functions in developmental and pathophysiological processes in eukaryotic organisms including mammals. We aimed to develop a highly sensitive, low-tech assay suitable to determine the activity of DYRK family kinases in tissues or cells from diverse sources.Xenopus laevis oocytes and identified this kinase as a DYRK1 isoform distinct from the Xenopus DYRK1A ortholog.Phosphorylation-site specific antibodies can be used to monitor the accumulation of the phosphorylated product in kinase assays. We present a modified configuration of an enzyme-linked immunosorbent assay (ELISA)-based kinase assay by using the phosphospecific antibody as the capture antibody. This assay format allowed the detection of small amounts of phosphopeptide in mixtures with an excess of the unphosphorylated substrate peptide (10 fmol phosphorylated peptide over a background of 50 pmol unphosphorylated peptide). Consequently, low substrate turnover rates can be determined. We applied this method to the measurement of endogenous DYRK1A activity in mouse heart tissue by immunocomplex kinase assay. Furthermore, we detected DYRK1-like kinase activity in Xenopus laevis oocytes contain an active DYRK1-related protein kinase more similar to mammalian DYRK1B than DYRK1A.We present a non-radioactive and highly sensitive method for the measurement of endogenous activities of DYRKs in biological samples.  Most cellular processes are controlled by protein phosphorylation, and aberrant kinase activity has been implicated in the etiology of a wide spectrum of diseases, including cancer, chronic inflammatory disorders and neurodegeneration. Studies on protein kinases are important not only to elucidate molecular mechanisms of signal transduction, but also for drug development. Therefore, methods for measuring kinase activity and for the identification of kinase inhibitors have become increasingly important in biomedical research ,2.32P or 33P). Use of radioactivity requires special handling, is associated with inherent high costs of waste disposal, and restricts the flexibility because of the short half life of 32P and 33P. Furthermore, these assays are carried out at subphysiological levels of ATP owing to the necessity of keeping ATP levels, and thus the usage of radioisotopes, within reasonable limits.A widely employed type of assay is based on the use of radioactively labelled ATP as phosphate donor and subsequent detection of phosphate incorporation into a protein or peptide substrate that contains the respective kinase recognition motif ,4. This in vitro-kinase reaction takes place in the wells after coating of the substrate to the surface of the microplate wells, and the phosphorylated molecules are detected with a phosphospecific antibody ATP under identical conditions. All other kinase assays were incubated for 30 min at 30°C.To prepare phosphorylated tau207-219 at 30°C for 30 min and stopped by addition of 50 μL 10 mM EDTA. Samples were stored at -20°C until evaluation by ELISA. Radiometric assays were run in the presence of [γ-33P]ATP (~ 2000 cpm/pmol). Incorporation of 33P into the biotinylated tau peptide was determined by spotting aliquots of the reaction mix onto a streptavidin membrane , which was then washed as described by the manufacturer and evaluated by scintillation counting. Radiometric immunocomplex kinase assays with the substrate peptide DYRKtide (at 100 μM) were run with 10 μM ATP as described before [Immunocomplex kinase assays were carried out in a total reaction volume of 50 μl in kinase buffer, 100 μM ATP and 50 μM taud before .2HPO4, 1.8 mM KH2PO4, pH 7.4), the antigen was added in a volume of 100 μL/well and the plates were incubated for 2 h at room temperature. Bound antigen was detected with streptavidin-HRP conjugate  and subsequent incubation with 100 μL/well TMB  substrate solution until colour development was observed. Other detection reagents  were less sensitive, and higher concentration of the streptavidin-HRP conjugate resulted in higher background in empty wells. The reaction was then stopped by adding 50 μl/well 1 M HCl, and plates were read at 450 nm (reference wavelength 620 nm). After each incubation step, plates were washed 4 times with PBS, pH 7.4, 0.1% Tween-20. Antibody and antigen were diluted in PBS.MaxiSorp F96 MicroWell™ Plates  were coated with 100 ng/well TAU(pT212) antibody at 4°C overnight. To keep the antibody consumption low, we used 100 ng/well of capturing antibody for coating in the standard protocol, although 200 ng/well gave a slightly higher readout (1.2-fold). After blocking for 1 hour with 200 μl/well 2% (w/v) bovine serum albumin  in phosphate buffered saline (PBS)  were loaded with the indicated concentrations of the biotinylated peptides (100 μL/well) and incubated at RT for 2 h. After washing, bound phosphopeptide was detected with rabbit anti TAU(pT212) antibody (1:16.000) and HRP-linked goat anti rabbit IgG (Fc) (Pierce) (1:40.000) and TMB substrate as described above. PBS with 2% BSA and 0.1% Tween-20 was used for plate washing and for dilution of antigen and antibodies.Standard curves of phosphorylated tau peptide and the graphical representation of the time course shown in Figure Xenopus DYRK1. WB conceived of and coordinated the study and wrote the manuscript. All authors read and approved the final manuscript.EL designed and carried out most of the experiments and analysed the results. KK carried out the experiments with Figure S1. This PDF file contains a supplementary figure that shows a multiple sequence alignment of human and Xenopus laevis DYRK1 isoforms.Click here for file