Phosphatase Treatment Research Articles

Applying the Brakes to the Immune Response

Activation of the inflammatory response mediated by the transcription factor nuclear factor κB (NF-κB) is a one-way street, because activation involves the phosphorylation (by the kinase complex IKK) and ubiquitin-mediated degradation of the protein (IκB) that sequesters NF-κB in the cytosol to inhibit its activity. In T and B cells, a signaling adaptor complex consisting of CARMA1 [caspase recruitment domain (CARD) 11], Bcl10, and MALT1 connects receptor activation to activation of IKK, IκB degradation, and NF-κB translocation to the nucleus to activate proinflammatory gene expression. Lobry et al . provide evidence from cultured T cell lines that IKK also phosphorylates Bcl10, which leads to its ubiquitination and proteasome-mediated degradation, thereby providing a mechanism by which the extent of NF-κB activation can be limited. Degradation of Bcl10 in response to T cell receptor ligation or phorbol esters (which directly activate protein kinase C, bypassing the receptor) was inhibited in Jurkat cells deficient for CARMA1 or NEMO (which is a regulatory component of the IKK complex), which suggested that activation of the pathway through IKK was required. Pharmacological inhibition of IKK or down-regulation of IKKβ by RNA interference (RNAi) also prevented Bcl10 degradation. (IKKβ is one of two IKK isoforms; RNAi for IKKα was less effective at inhibiting phosphorylation of Bcl10.) Overexpression of tagged Bcl10 with tagged IKKβ resulted in the accumulation of slower migrating forms of Bcl10, and phosphatase treatment suggested that these were phosphorylated forms of Bcl10. In vitro assays confirmed that IKKβ directly phosphorylated Bcl10, and mutational analysis identified several phosphorylation sites including two (Thr 81 and Ser 85 ) that were in a consensus recognition sequence for phosphorylation-mediated binding of the ubiquitin ligase β-transducin repeat-containing protein (β-TrCP). In transfected HEK293 cells, Bcl10 and β-TrCP were coimmunoprecipitated when active IKKβ was also expressed. Two mutant forms of Bcl10, in which Thr 81 was replaced by Ala (T81A) and Ser 85 by Ala (S85A), did not interact with β-TrCP, and this mutant was also resistant to T cell receptor-stimulated degradation. Interleukin 2 production was enhanced in T cell lines (E29.1) expressing the T81A and S85A mutant compared with cells expressing wild-type Bcl10; thus, Bcl10 degradation appears to limit NF-κB signaling. Finally, two different IKK phosphorylation-deficient mutant forms of Bcl10 accumulated in the nucleus after T cell receptor stimulation. This is intriguing because this localization has also been noted in mucosa-associated lymphoid tissue (MALT) lymphoma. The results regarding Bcl10 phosphorylation reported here are not entirely consistent with other studies and it may be that, under different conditions, different regulatory mechanisms involving Bcl10 exist. C. Lobry, T. Lopez, A. Israël, R. Weil, Negative feedback loop in T cell activation through IκB kinase-induced phosphorylation and degradation of Bcl10. Proc. Natl. Acad. Sci. USA 104 , 908-913 (2007). [Abstract] [Full Text]

Chemical validation of molecular mimicry: interaction of cholera toxin with Campylobacter lipooligosaccharides

It is generally believed that molecular mimicry between bacterial lipooligosaccharide (LOS) and nerve glycolipids may play an important pathogenic role in immune-mediated peripheral neuropathy. One of the putative infectious agents is Campylobacter jejuni (C. jejuni). To elucidate the structural basis for the molecular mimicry, we investigated the structure of the lipooligosaccharide (LOS) fraction of C. jejuni, strain HS19, and found that it includes at least two components, characterized as fast-and slow-moving bands (LF and LS) by thin-layer chromatography as revealed by cholera toxin B subunit (Ctxb) overlay. Structural analysis of the oligosaccharide portion of LS established that it had the following structure: Gal-GalNAc-(NeuAc)Gal-Hep-(Glc;PO(3)H)Hep-Kdo. The GM1-like epitope was validated by a terminal tetrasaccharide unit within this structure. On the other hand, analysis of LF revealed an entirely different structure: 1, 4'-bisphosphoryl glucosamine disaccharide N, N'-acylated by 3-(2-hydroxytetracosanoyloxy)octadecanoic acid at 2- and 2'-positions, which is consistent with that of lipid A. No GM1-like epitope was observed in LF. Both LS and LF interacted with Ctxb as demonstrated by TLC-overlay and sucrose density gradient centrifugation. Surprisingly, LF does not have the basic GM1 structure for interacting with Ctxb. Instead, the affinity of LF to Ctxb required that one or both of the phosphate groups be present in the glucosamine disaccharide residue because after alkaline phosphatase treatment the dephosphorylated LF was unable to bind to Ctxb. We conclude that LS is likely the component contributing to GM1-mimicry in autoimmune peripheral neuropathy and that the role of LF is not clear but may be associated with the initial activation of autoreactive T cells.

Cyclin dependent kinase 2 and the regulation of human progesterone receptor activity

The function of the S phase kinase cyclin A/Cdk2 in maintaining and regulating cell cycle kinetics is well established. However an alternative role in the regulation of progesterone receptor (PR) signaling is emerging. PR and its coactivators are phosphoproteins. Cyclin A/Cdk2 phosphorylates several of the PR phosphorylation sites in vitro and there is evidence that it participates in PR phosphorylation in vivo. Cyclin A/Cdk2 also functions as a PR coactivator. Overexpression increases PR transcriptional activity independent of PR phosphorylation. In the presence of hormone, cyclin A/Cdk2 is recruited to PR bound to DNA of target genes. Inhibition of Cdk activity prevents recruitment of the p160 coactivator steroid receptor coactivator-1 (SRC-1), suggesting that Cdk2 phosphorylates SRC-1. Consistent with this finding, phosphatase treatment of SRC-1 reduces its ability to interact with PR in vitro. Moreover, PR transcriptional activity is highest in S phase where cyclin A is expressed. In G1, PR activity is reduced and the capacity to recruit SRC-1 to a progestin responsive promoter is diminished. Future studies will focus on the importance of cyclin A/Cdk2 phosphorylation of other components of the PR transcription complex, such as the p160 coactivator SRC-1, and the specific role of Cdk2 target sites in the regulation of PR activity.

Alkaline phosphatase treatment improves renal function in patients with severe sepsis or septic shock

We previously demonstrated that upregulation of renal inducible nitric oxide synthase (iNOS) during systemic inflammation is associated with proximal tubule injury. In several in vitro and animal studies alkaline phosphatase (AP) was found to be effective in attenuating the inflammatory response by dephosphorylating LPS and may prevent organ damage. The objective of this study was to investigate the effect of AP on renal iNOS expression and kidney damage in patients with severe sepsis or septic shock. Fifteen patients (nine male/six female, age 55 ± 5 years) with Gram-negative bacterial infection, two out of four SIRS criteria (<24 hours) and acute onset of end-organ dysfunction (<12 hours) were included in a randomized, double-blind, placebo-controlled phase IIa study (2:1 ratio). An intravenous bolus injection of 67.5 U/kg bovine intestinal AP was followed by a maintenance dose of 177.5 U/kg for 24 hours. Arterial blood and urine were collected at different time points and analyzed for stable metabolites of NO. iNOS mRNA was determined by quantitative real-time RT-PCR using RNA isolated from renal cells in urine. The urinary excretion of the cytosolic glutathione S-transferase-A1 (GSTA1-1), a marker for proximal tubule damage, was measured using an ELISA. Data are depicted as the median (25–75% range). NO metabolites in blood were not significantly different between AP-treated (n = 10) and placebo-treated (n = 5) patients. However, the urinary excretion of NO metabolites decreased by 80% (75–85) from 227 (166–531) at baseline to 41 (28–84) μmol/10 mmol creatinine (P < 0.05) after 24 hours of AP administration. After placebo treatment, the amount of urinary NO metabolites increased by 70% (45–570) (from 81 (64–419) to 628 (65–1,479) μmol/10 mmol creatinine, P < 0.05). Baseline expression levels of iNOS in renal cells were 42-fold induced at baseline (vs healthy subjects), and AP administration reduced this induction by 80 ± 5% (Figure ​(Figure1).1). Creatinine clearance improved by 45% (30–180) in patients treated with AP and declined by 25% (15–35) in placebo-treated patients. During the first 24 hours the amount of GSTA1-1 in urine of AP-treated patients decreased by 70% (50–80), compared with an increase of 200% (45–525) in placebo-treated patients, which correlated with urinary NO metabolites, indicating NO-induced proximal tubular damage. Figure 1 In conclusion, in septic patients, infusion of AP results in an attenuated upregulation of iNOS and, subsequent, reduced NO production in the kidney, associated with an improvement in renal function.

RPA2 Is a Direct Downstream Target for ATR to Regulate the S-phase Checkpoint

Upon DNA damage, replication is inhibited by the S-phase checkpoint. ATR (ataxia telangiectasia mutated- and Rad3-related) is specifically involved in the inhibition of replicon initiation when cells are treated with DNA damage-inducing agents that stall replication forks, but the mechanism by which it acts to prevent replication is not yet fully understood. We observed that RPA2 is phosphorylated on chromatin in an ATR-dependent manner when replication forks are stalled. Mutation of the ATR-dependent phosphorylation sites in RPA2 leads to a defect in the down-regulation of DNA synthesis following treatment with UV radiation, although ATR activation is not affected. Threonine 21 and serine 33, two residues among several phosphorylation sites in the amino terminus of RPA2, are specifically required for the UV-induced, ATR-mediated inhibition of DNA replication. RPA2 mutant alleles containing phospho-mimetic mutations at ATR-dependent phosphorylation sites have an impaired ability to associate with replication centers, indicating that ATR phosphorylation of RPA2 directly affects the replication function of RPA. Our studies suggest that in response to UV-induced DNA damage, ATR rapidly phosphorylates RPA2, disrupting its association with replication centers in the S-phase and contributing to the inhibition of DNA replication.

EDD Mediates DNA Damage-induced Activation of CHK2

EDD, the human orthologue of Drosophila melanogaster "hyperplastic discs," is overexpressed or mutated in a number of common human cancers. Although EDD has been implicated in DNA damage signaling, a definitive role has yet to be demonstrated. Here we report a novel interaction between EDD and the DNA damage checkpoint kinase CHK2. EDD and CHK2 associate through a phospho-dependent interaction involving the CHK2 Forkhead-associated domain and a region of EDD spanning a number of putative Forkhead-associated domain-binding threonines. Using RNA interference, we demonstrate a critical role for EDD upstream of CHK2 in the DNA damage signaling pathway. EDD is necessary for the efficient activating phosphorylation of CHK2 in response to DNA damage following exposure to ionizing radiation or the radiomimetic, phleomycin. Cells depleted of EDD display impaired CHK2 kinase activity and an inability to respond to DNA damage. These results identify EDD as a novel mediator in DNA damage signal transduction via CHK2 and emphasize the potential importance of EDD in cancer.

Accumulation of Epstein–Barr virus (EBV) BMRF1 protein EA-D during latent EBV activation of Burkitt's lymphoma cell line Raji

As a new model to elucidate molecular mechanisms in Epstein–Barr virus (EBV) activation, we tested the tetracycline-inducible (Tet-On)/BZLF1-oriP plasmid system in Raji cells. Cells transfected with this Tet-On plasmid did not activate EBV by doxycycline and surprisingly EBV latency was disrupted with large amounts of BMRF1 protein (EA-D) being accumulated in the cells. Brilliant EA-D fluorescence was markedly condensed in small sized cells, intra-cellular vesicles, and extra-cellular particles. Scanning electron microscopy demonstrated the extra-cellular particles to be covered with a membrane. EA-D molecules of 58, 50, 48, and 44 kDa were expressed in the cells. The high (58 and 50 kDa) and low (48 and 44 kDa) EA-D molecules appeared in the early and late stages, respectively. Low EA-D molecules were detected mostly in EA-D positive cells separated into the heaviest density layer of a discontinuous Percoll gradient. Such molecules could be created from high EA-D molecules by protein phosphatase treatment. The EA-D molecules that appeared similar were detected in EBV-activated P3HR-1 and Akata cells. Several hypotheses concerning the accumulation of EA-D molecules of various polymorphic forms and their phosphorylation/dephosphorylation in this model system are presented, with possible biological and clinical relevance.

DNA polymerase ε associates with the elongating form of RNA polymerase II and nascent transcripts

DNA polymerase epsilon co-operates with polymerases alpha and delta in the replicative DNA synthesis of eukaryotic cells. We describe here a specific physical interaction between DNA polymerase epsilon and RNA polymerase II, evidenced by reciprocal immunoprecipitation experiments. The interacting RNA polymerase II was the hyperphosphorylated IIO form implicated in transcriptional elongation, as inferred from (a) its reduced electrophoretic mobility that was lost upon phosphatase treatment, (b) correlation of the interaction with phosphorylation of Ser5 of the C-terminal domain heptapeptide repeat, and (c) the ability of C-terminal domain kinase inhibitors to abolish it. Polymerase epsilon was also shown to UV crosslink specifically alpha-amanitin-sensitive transcripts, unlike DNA polymerase alpha that crosslinked only to RNA-primed nascent DNA. Immunofluorescence microscopy revealed partial colocalization of RNA polymerase IIO and DNA polymerase epsilon, and immunoelectron microscopy revealed RNA polymerase IIO and DNA polymerase epsilon in defined nuclear clusters at various cell cycle stages. The RNA polymerase IIO-DNA polymerase epsilon complex did not relocalize to specific sites of DNA damage after focal UV damage. Their interaction was also independent of active DNA synthesis or defined cell cycle stage.

Control of Phospholipid Synthesis by Phosphorylation of the Yeast Lipin Pah1p/Smp2p Mg2+-dependent Phosphatidate Phosphatase

Phosphorylation of the conserved lipin Pah1p/Smp2p in Saccharomyces cerevisiae was previously shown to control transcription of phospholipid biosynthetic genes and nuclear structure by regulating the amount of membrane present at the nuclear envelope (Santos-Rosa, H., Leung, J., Grimsey, N., Peak-Chew, S., and Siniossoglou, S. (2005) EMBO J. 24, 1931-1941). A recent report identified Pah1p as a Mg2+-dependent phosphatidate (PA) phosphatase that regulates de novo lipid synthesis (Han G.-S., Wu, W. I., and Carman, G. M. (2006) J. Biol. Chem. 281, 9210-9218). In this work we use a combination of mass spectrometry and systematic mutagenesis to identify seven Ser/Thr-Pro motifs within Pah1p that are phosphorylated in vivo. We show that phosphorylation on these sites is required for the efficient transcriptional derepression of key enzymes involved in phospholipid biosynthesis. The phosphorylation-deficient Pah1p exhibits higher PA phosphatase-specific activity than the wild-type Pah1p, indicating that phosphorylation of Pah1p controls PA production. Opi1p is a transcriptional repressor of phospholipid biosynthetic genes, responding to PA levels. Genetic analysis suggests that Pah1p regulates transcription of these genes through both Opi1p-dependent and -independent mechanisms. We also provide evidence that derepression of phospholipid biosynthetic genes is not sufficient to induce the nuclear membrane expansion shown in the pah1delta cells.

Identification of Two Forms of Vitellogenin-derived Phosvitin and Elucidation of Their Fate and Roles During Oocyte Maturation in the Barfin Flounder, Verasper moseri

A new method for visualizing small and multiple phosvitins (Pvs) in oocytes from a marine teleost was developed by a combination of gel filtration, alkaline phosphatase treatment, and SDS-PAGE followed by silver staining. Three distinct Pv polypeptides having molecular masses of 15 kDa, 8 kDa, and 7 kDa were visualized in vitellogenic follicle extract of barfin flounder, Verasper moseri. N-terminal amino acid sequencing identified two different N-termini that fell into the PvA (7 kDa) and PvB (15 kDa and 8 kDa) groups, which were derived from two forms of vitellogenin (Vg), VgA and VgB, respectively. Analysis of time-course change in phosphorus-rich peaks of gel chromatography fractions of follicle extracts from different maturational stages demonstrated a rapid degradation of Pvs during mid-phase of oocyte maturation. Quantitative analysis of free amino acids in maturing follicles revealed an increment of serine content but not of phosphoserine, indicating the occurrence of dephosphorylation concomitant with Pv degradation. Measurement of phosphatase activity in follicles and eggs at different maturational stages demonstrated a significant activation of phosphatase especially under acidic conditions. This suggested that Pv degradation and dephosphorylation are regulated by changes in ooplasm pH during oocyte maturation. Our results also suggested that the Pvs in barfin flounder vitellogenic oocytes bind to much lower amounts of calcium and magnesium than those of masu salmon, Oncorhynchus masou. This indicates that the Pvs in the barfin flounder, a marine teleost spawning its eggs in seawater, do not play a role in the transport and deposition of calcium and magnesium into oocytes.

Enzymatic treatment of duck hepatitis B virus: Topology of the surface proteins for virions and noninfectious subviral particles

The large surface antigen L of duck hepatitis B virus exhibits a mixed topology with the preS domains of the protein alternatively exposed to the particles' interior or exterior. After separating virions from subviral particles (SVPs), we compared their L topologies and showed that both particle types exhibit the same amount of L with the following differences: 1—preS of intact virions was enzymatically digested with chymotrypsin, whereas in SVPs only half of preS was accessible, 2—phosphorylation of L at S118 was completely removed by phosphatase treatment only in virions, 3—iodine-125 labeling disclosed a higher ratio of exposed preS to S domains in virions compared to SVPs. These data point towards different surface architectures of virions and SVPs. Because the preS domain acts in binding to a cellular receptor of hepatocytes, our findings implicate the exclusion of SVPs as competitors for the receptor binding and entry of virions.

Metaphase Arrest by Cyclin E-Cdk2 Requires the Spindle-Checkpoint Kinase Mps1

Cytostatic factor (CSF) arrests vertebrate eggs in metaphase of meiosis II through several pathways that inhibit activation of the anaphase-promoting complex/cyclosome (APC/C). In Xenopus, the Mos-MEK1-MAPK-p90(Rsk) cascade utilizes spindle-assembly-checkpoint components to effect metaphase arrest. Another pathway involves cyclin E-Cdk2, and sustained cyclin E-Cdk2 activity in egg extracts causes metaphase arrest in the absence of Mos; this latter finding suggests that an independent pathway contributes to CSF arrest. Here, we demonstrate that metaphase arrest with cyclin E-Cdk2, but not with Mos, requires the spindle-checkpoint kinase monopolar spindles 1 (Mps1), a cyclin E-Cdk2 target that is also implicated in centrosome duplication. xMps1 is synthesized and activated during oocyte maturation and inactivated upon CSF release. In egg extracts, CSF release by calcium was inhibited by constitutively active cyclin E-Cdk2 and delayed by wild-type xMps1. Ablation of cyclin E by antisense oligonucleotides blocked accumulation of xMps1, suggesting that cyclin E-Cdk2 controls Mps1 levels. During meiosis II, activated cyclin E-Cdk2 significantly inhibited the APC/C even in the absence of the Mos-MAPK pathway, but this inhibition was not sufficient to suppress S phase between meiosis I and II. These results uniquely place xMps1 downstream of cyclin E-Cdk2 in mediating a pathway of APC/C inhibition and metaphase arrest.

Contrasting Genome-Wide Distribution of 8-Hydroxyguanine and Acrolein-Modified Adenine during Oxidative Stress-Induced Renal Carcinogenesis

Oxidative stress is a persistent threat to the genome and is associated with major causes of human mortality, including cancer, atherosclerosis, and aging. Here we established a method to generate libraries of genomic DNA fragments containing oxidatively modified bases by using specific monoclonal antibodies to immunoprecipitate enzyme-digested genome DNA. We applied this technique to two different base modifications, 8-hydroxyguanine and 1,N6-propanoadenine (acrotein-Ade), in a ferric nitrilotriacetate-induced murine renal carcinogenesis model. Renal cortical genomic DNA derived from 10- to 12-week-old male C57BL/6 mice, of untreated control or 6 hours after intraperitoneal injection of 3 mg iron/kg ferric nitrilotriacetate, was enzyme digested, immunoprecipitated, cloned, and mapped to each chromosome. The results revealed that distribution of the two modified bases was not random but differed in terms of chromosomes, gene size, and expression, which could be partially explained by chromosomal territory. In the wild-type mice, low GC content areas were more likely to harbor the two modified bases. Knockout of OGG1, a repair enzyme for genomic 8-hydroxyguanine, increased the amounts of acrolein-Ade as determined by quantitative polymerase chain reaction analyses. This versatile technique would introduce a novel research area as a high-throughput screening method for critical genomic loci under oxidative stress.

The Na+:Cl– Cotransporter Is Activated and Phosphorylated at the Amino-terminal Domain upon Intracellular Chloride Depletion

The renal Na(+):Cl(-) cotransporter rNCC is mutated in human disease, is the therapeutic target of thiazide-type diuretics, and is clearly involved in arterial blood pressure regulation. rNCC belongs to an electroneutral cation-coupled chloride cotransporter family (SLC12A) that has two major branches with inverse physiological functions and regulation: sodium-driven cotransporters (NCC and NKCC1/2) that mediate cellular Cl(-) influx are activated by phosphorylation, whereas potassium-driven cotransporters (KCCs) that mediate cellular Cl(-) efflux are activated by dephosphorylation. A cluster of three threonine residues at the amino-terminal domain has been implicated in the regulation of NKCC1/2 by intracellular chloride, cell volume, vasopressin, and WNK/STE-20 kinases. Nothing is known, however, about rNCC regulatory mechanisms. By using rNCC heterologous expression in Xenopus laevis oocytes, here we show that two independent intracellular chloride-depleting strategies increased rNCC activity by 3-fold. The effect of both strategies was synergistic and dose-dependent. Confocal microscopy of enhanced green fluorescent protein-tagged rNCC showed no changes in rNCC cell surface expression, whereas immunoblot analysis, using the R5-anti-NKCC1-phosphoantibody, revealed increased phosphorylation of rNCC amino-terminal domain threonine residues Thr(53) and Thr(58). Elimination of these threonines together with serine residue Ser(71) completely prevented rNCC response to intracellular chloride depletion. We conclude that rNCC is activated by a mechanism that involves amino-terminal domain phosphorylation.

D1 dopamine receptor hyperphosphorylation in renal proximal tubules in hypertension

A defect in the coupling of the D(1) receptor (D(1)R) to its G protein/effector complex in renal proximal tubules plays a role in the pathogenesis of spontaneous hypertension. As there is no mutation of the D(1)R gene in the spontaneously hypertensive rat (SHR), we tested the hypothesis that the coupling defect is associated with constitutive desensitization/phosphorylation of the D(1)R. The following experiments were performed: (1) Cell culture and membrane preparations from rat kidneys and immortalized rat renal proximal tubule cells (RPTCs); (2) immunoprecipitation and immunoblotting; (3) cyclic adenosine 3',5' monophosphate and adenylyl cyclase assays; (4) immunofluorescence and confocal microscopy; (5) biotinylation of cell surface proteins; and (6) in vitro enzyme dephosphorylation. Basal serine-phosphorylated D(1)Rs in renal proximal tubules, brush border membranes, and membranes from immortalized RPTCs were greater in SHRs (21.0+/-1.5 density units, DU) than in normotensive rats (7.4+/-2.9 DU). The increased basal serine phosphorylation of D(1)Rs in SHRs was accompanied by decreased expression of D(1)R at the cell surface, and decreased ability of a D(1)-like receptor agonist (fenoldopam) to stimulate cyclic adenosine 3',5' monophosphate (cAMP) production. Increasing protein phosphatase 2A activity with protamine enhanced the ability of fenoldopam to stimulate cAMP accumulation (17+/-4%) and alter D(1)R cell surface expression in intact cells from SHRs. Alkaline phosphatase treatment of RPTC membranes decreased D(1)R phosphorylation and enhanced fenoldopam stimulation of adenylyl cyclase activity (26+/-6%) in SHRs. Uncoupling of the D(1)R from its G protein/effector complex in renal proximal tubules in SHRs is caused, in part, by increased D(1)R serine phosphorylation.

Functional characterization of DHN-5, a dehydrin showing a differential phosphorylation pattern in two Tunisian durum wheat ( Triticum durum Desf.) varieties with marked differences in salt and drought tolerance

Water-deficit stress caused by drought and soil salinization adversely affects plant growth and crop productivity. Dehydrins are involved in the adaptation to water and osmotic stress. We have identified a wheat dehydrin named DHN-5 that is closely related to the maize RAB17. The full-length cDNA of Dhn- 5 gene encodes a putative protein of 227 amino acids and contains 2 conserved lysine-rich-K-segment (EKKGIMDKIKEKLPG) repeats preceded by a stretch of eight serine residues, characteristic of group 2 LEA family. The Northern blot analyses showed a strong accumulation of Dhn- 5 transcript in mature wheat embryos and to a lesser extent in ABA and salt-treated seedlings. Interestingly, DHN-5 protein accumulated differentially in two Tunisian durum wheat ( Triticum durum Desf.) varieties with marked differences in salt and drought tolerance. By using specific dehydrin antibodies and 2D immunoblot analysis on proteins extracted from mature embryos in these two varieties, a differential phosphorylation pattern of DHN-5 was observed. In the resistant variety (R), beside a basic protein spot, a series of acidic spots were detected whereas in the sensitive variety (S) the acidic spots were weakly detectable. These acidic forms correspond to highly phosphorylated forms of DHN-5, which can be removed by alkaline phosphatase treatment. Accumulation of phosphorylated DHN-5 mainly in the R variety suggests a role of P-DHN-5 in preservation of cell integrity during late embryogenesis and desiccation. Subcellular localization of the DHN-5:GFP fusion protein indicated that DHN-5 would be primarily nuclear, suggesting a nuclear role in wheat osmotic stress response. The observed differential phosphorylation pattern of DHN-5 in the resistant and sensitive wheat varieties could be used as a basis for a molecular screen of tolerance/sensitivity to drought and salt stresses in wheat germplasm.

Peroxisome Proliferator-activated Receptor-γ1 Is Dephosphorylated and Degraded during BAY 11-7085-induced Synovial Fibroblast Apoptosis

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) plays a central role in whole body metabolism by regulating adipocyte differentiation and energy storage. Recently, however, PPAR-gamma has also been demonstrated to affect proliferation, differentiation, and apoptosis of different cell types. As we have previously shown that BAY 11-7085-induced synovial fibroblast apoptosis is prevented by PPAR-gamma agonist 15d-PGJ2; the expression of PPAR-gamma in these cells was studied. Both PPAR-gamma1 and PPAR-gamma2 isoforms were cloned from synovial fibroblast RNA, but only PPAR-gamma1 was detected by Western blot, showing constitutive nuclear expression. Within minutes of BAY 11-7085 treatment, a PPAR-gamma1-specific band was shifted into a form of higher mobility, suggesting dephosphorylation, as confirmed by phosphatase treatment of cell extracts. Of interest, BAY 11-7085-induced PPAR-gamma1 dephosphorylation was followed by PARP and caspase-8 cleavage as well as by PPAR-gamma1 protein degradation. PPAR-gamma1 dephosphorylation was followed by the loss of PPAR-DNA binding activity ubiquitously present in synovial fibroblast nuclear extracts. Unlike the phosphorylated form, dephosphorylated PPAR-gamma1 was found in insoluble membrane cell fraction and was not ubiquitinated before degradation. PPAR-gamma1 dephosphorylation coincided with ERK1/2 phosphorylation that accompanies BAY 11-7085-induced synovial fibroblasts apoptosis. 15d-PGJ2, PGD2, and partially UO126, down-regulated ERK1/2 phosphorylation, protected cells from BAY 11-7085-induced apoptosis, and reversed both PPAR-gamma dephosphorylation and degradation. Furthermore, PPAR-gamma antagonist BADGE induced PPAR-gamma1 degradation, ERK1/2 phosphorylation, and synovial fibroblasts apoptosis. The results presented suggest an anti-apoptotic role for PPAR-gamma1 in synovial fibroblasts. Since apoptotic marker PARP is cleaved after PPAR-gamma1 dephosphorylation but before PPAR-gamma1 degradation, dephosphorylation event might be enough to mediate BAY 11-7085-induced apoptosis in synovial fibroblasts.

Dmp53 Activates the Hippo Pathway to Promote Cell Death in Response to DNA Damage

Developmental and environmental signals control a precise program of growth, proliferation, and cell death. This program ensures that animals reach, but do not exceed, their typical size . Understanding how cells sense the limits of tissue size and respond accordingly by exiting the cell cycle or undergoing apoptosis has important implications for both developmental and cancer biology. The Hippo (Hpo) pathway comprises the kinases Hpo and Warts/Lats (Wts), the adaptors Salvador (Sav) and Mob1 as a tumor suppressor (Mats), the cytoskeletal proteins Expanded and Merlin, and the transcriptional cofactor Yorkie (Yki) . This pathway has been shown to restrict cell division and promote apoptosis. The caspase repressor DIAP1 appears to be a primary target of the Hpo pathway in cell-death control. Firstly, Hpo promotes DIAP1 phosphorylation, likely decreasing its stability. Secondly, Wts phosphorylates and inactivates Yki, decreasing DIAP1 transcription. Although we understand some of the events downstream of the Hpo kinase, its mode of activation remains mysterious. Here, we show that Hpo can be activated by Ionizing Radiations (IR) in a Dmp53 (Drosophila melanogaster p53)-dependent manner and that Hpo is required (though not absolutely) for the cell death response elicited by IR or Dmp53 ectopic expression.

Phosphorylation of Microtubule-associated Protein STOP by Calmodulin Kinase II

STOP proteins are microtubule-associated, calmodulin-regulated proteins responsible for the high degree of stabilization displayed by neuronal microtubules. STOP suppression in mice induces synaptic defects affecting both short and long term synaptic plasticity in hippocampal neurons. Interestingly, STOP has been identified as a component of synaptic structures in neurons, despite the absence of microtubules in nerve terminals, indicating the existence of mechanisms able to induce a translocation of STOP from microtubules to synaptic compartments. Here we have tested STOP phosphorylation as a candidate mechanism for STOP relocalization. We show that, both in vitro and in vivo, STOP is phosphorylated by the multifunctional enzyme calcium/calmodulin-dependent protein kinase II (CaMKII), which is a key enzyme for synaptic plasticity. This phosphorylation occurs on at least two independent sites. Phosphorylated forms of STOP do not bind microtubules in vitro and do not co-localize with microtubules in cultured differentiating neurons. Instead, phosphorylated STOP co-localizes with actin assemblies along neurites or at branching points. Correlatively, we find that STOP binds to actin in vitro. Finally, in differentiated neurons, phosphorylated STOP co-localizes with clusters of synaptic proteins, whereas unphosphorylated STOP does not. Thus, STOP phosphorylation by CaMKII may promote STOP translocation from microtubules to synaptic compartments where it may interact with actin, which could be important for STOP function in synaptic plasticity.

Phosphorylation of Viral RNA-dependent RNA Polymerase and Its Role in Replication of a Plus-strand RNA Virus

Central to the process of plus-strand RNA virus genome amplification is the viral RNA-dependent RNA polymerase (RdRp). Understanding its regulation is of great importance given its essential function in viral replication and the common architecture and catalytic mechanism of polymerases. Here we show that Turnip yellow mosaic virus (TYMV) RdRp is phosphorylated, when expressed both individually and in the context of viral infection. Using a comprehensive biochemical approach, including metabolic labeling and mass spectrometry analyses, phosphorylation sites were mapped within an N-terminal PEST sequence and within the highly conserved palm subdomain of RNA polymerases. Systematic mutational analysis of the corresponding residues in a reverse genetic system demonstrated their importance for TYMV infectivity. Upon mutation of the phosphorylation sites, distinct steps of the viral cycle appeared affected, but in contrast to other plus-strand RNA viruses, the interaction between viral replication proteins was unaltered. Our results also highlighted the role of another TYMV-encoded replication protein as an antagonistic protein that may prevent the inhibitory effect of RdRp phosphorylation on viral infectivity. Based on these data, we propose that phosphorylation-dependent regulatory mechanisms are essential for viral RdRp function and virus replication.