Phosphorylated Form (phosphorylated + form)

Distribution by Scientific Domains
Distribution within Life Sciences

Selected Abstracts

Activation of ADF/cofilin mediates attractive growth cone turning toward nerve growth factor and netrin-1

Bonnie M. Marsick
Abstract Proper neural circuitry requires that growth cones, motile tips of extending axons, respond to molecular guidance cues expressed in the developing organism. However, it is unclear how guidance cues modify the cytoskeleton to guide growth cone pathfinding. Here, we show acute treatment with two attractive guidance cues, nerve growth factor (NGF) and netrin-1, for embryonic dorsal root ganglion and temporal retinal neurons, respectively, results in increased growth cone membrane protrusion, actin polymerization, and filamentous actin (F-actin). ADF/cofilin (AC) family proteins facilitate F-actin dynamics, and we found the inactive phosphorylated form of AC is decreased in NGF- or netrin-1-treated growth cones. Directly increasing AC activity mimics addition of NGF or netrin-1 to increase growth cone protrusion and F-actin levels. Extracellular gradients of NGF, netrin-1, and a cell-permeable AC elicit attractive growth cone turning and increased F-actin barbed ends, F-actin accumulation, and active AC in growth cone regions proximal to the gradient source. Reducing AC activity blunts turning responses to NGF and netrin. Our results suggest that gradients of NGF and netrin-1 locally activate AC to promote actin polymerization and subsequent growth cone turning toward the side containing higher AC activity. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 70: 565,588, 2010 [source]

Deamidation of labile asparagine residues in the autoregulatory sequence of human phenylalanine hydroxylase

FEBS JOURNAL, Issue 5 2003
Structural, functional implications
Two dimensional electrophoresis has revealed a microheterogeneity in the recombinant human phenylalanine hydroxylase (hPAH) protomer, that is the result of spontaneous nonenzymatic deamidations of labile asparagine (Asn) residues [Solstad, T. and Flatmark, T. (2000) Eur. J. Biochem.267, 6302,6310]. Using of a computer algorithm, the relative deamidation rates of all Asn residues in hPAH have been predicted, and we here verify that Asn32, followed by a glycine residue, as well as Asn28 and Asn30 in a loop region of the N-terminal autoregulatory sequence (residues 19,33) of wt-hPAH, are among the susceptible residues. First, on MALDI-TOF mass spectrometry of the 24 h expressed enzyme, the E. coli 28-residue peptide, L15,K42 (containing three Asn residues), was recovered with four monoisotopic mass numbers (i.e., m/z of 3106.455, 3107.470, 3108.474 and 3109.476, of decreasing intensity) that differed by 1 Da. Secondly, by reverse-phase chromatography, isoaspartyl (isoAsp) was demonstrated in this 28-residue peptide by its methylation by protein- l -isoaspartic acid O -methyltransferase (PIMT; EC Thirdly, on incubation at pH 7.0 and 37 °C of the phosphorylated form (at Ser16) of this 28-residue peptide, a time-dependent mobility shift from tR,,34 min to ,,31 min (i.e., to a more hydrophilic position) was observed on reverse-phase chromatography, and the recovery of the tR,,34 min species decreased with a biphasic time-course with t0.5 -values of 1.9 and 6.2 days. The fastest rate is compatible with the rate determined for the sequence-controlled deamidation of Asn32 (in a pentapeptide without 3D structural interference), i.e., a deamidation half-time of ,,1.5 days in 150 mm Tris/HCl, pH 7.0 at 37 °C. Asn32 is located in a cluster of three Asn residues (Asn28, Asn30 and Asn32) of a loop structure stabilized by a hydrogen-bond network. Deamidation of Asn32 introduces a negative charge and a partial ,-isomerization (isoAsp), which is predicted to result in a change in the backbone conformation of the loop structure and a repositioning of the autoregulatory sequence and thus affect its regulatory properties. The functional implications of this deamidation was further studied by site-directed mutagenesis, and the mutant form (Asn32,Asp) revealed a 1.7-fold increase in the catalytic efficiency, an increased affinity and positive cooperativity of L-Phe binding as well as substrate inhibition. [source]

The phosphatidylinositol-3 kinase (PI3K)-Akt pathway suppresses neurite branch formation in NGF-treated PC12 cells

GENES TO CELLS, Issue 8 2003
Maiko Higuchi
Background:, Previous studies have shown that phosphatidylinositol-3 kinase (PI3K) plays an important role in NGF (nerve growth factor)-induced neurite elongation. However, the roles of the PI3K pathway in neurite branch formation were not fully understood. Also, it was not clear where the PI3K pathway is activated during branch formation. Results:, We found that the treatment of PC12 cells with the PI3K inhibitor LY294002 resulted in a marked increase in the number of neurite branch points, suggesting a suppressive role of PI3K in neurite branch formation. Expression of a constitutively active form of Akt, a downstream effector of PI3K, decreased the number of branch points, whereas that of a dominant-negative form of Akt increased it. In contrast, inhibition of neither Rac, mTOR nor GSK3, other effectors of PI3K, promoted branch formation. Importantly, the phosphorylated form of endogenous Akt was localized at the tips of growth cones, but devoid of small branches in NGF-treated PC12 cells. A GFP-fusion protein of the plekstrin-homology (PH) domain of Akt was also localized at the tips of growth cones. Conclusions:, The PI3K-Akt pathway thus plays a key role in suppression of neurite branch formation in NGF-treated PC12 cells. Summary figure, Figure Summary figure,. working model for the regulation of neuritogenesis in PC12 cells. PI3K may mediate NGF regulation of neuritogenesis via two pathways. Rac induces neurite elongation and branch formation. Akt induces neurite elongation, but prevents branch formation. [source]

Critical roles of LGN/GPSM2 phosphorylation by PBK/TOPK in cell division of breast cancer cells

Chikako Fukukawa
To investigate the molecular mechanism of mammary carcinogenesis and identify novel molecular targets for breast cancer therapy, we analyzed genome-wide gene expression profiles of 81 clinical breast cancer samples. Here, we report the critical role of LGN/GPSM2 (Leu-Gly-Asn repeat-enriched protein/G-protein signaling modulator 2) in the growth of breast cancer cells. Semiquantitative RT-PCR and Northern blot analyses confirmed upregulation of LGN/GPSM2 in a large proportion of breast cancers. Immunocytochemical staining identified LGN/GPSM2 at the spindle in cells at metaphase, and at midzone and midbody in cytokinetic cells. Western blot analysis indicated the highest expression and the phosphorylated form of LGN/GPSM2 protein in G2/M phase. Treatment with small-interfering RNAs (siRNAs) targeting LGN/GPSM2 caused incompletion of cell division and resulted in significant growth suppression of breast cancer cells. We found that the 450th threonine (Thr450) of LGN/GPSM2 was phosphorylated by the serine/threonine kinase PBK/TOPK during mitosis. Overexpression of LGN/GPSM2-T450A in which Thr450 was substituted with alanine induced growth suppression and aberrant chromosomal segregation. These findings imply an important role of LGN/GPSM2 in cell division of breast cancer cells and suggest that the PBK/TOPK-LGN/GPSM2 pathway might be a promising molecular target for treatment of breast cancer. © 2010 Wiley-Liss, Inc. [source]

Effect of methylglyoxal modification and phosphorylation on the chaperone and anti-apoptotic properties of heat shock protein 27

Tomoko Oya-Ito
Abstract Heat shock protein 27 (Hsp27) is a stress-inducible protein in cells that functions as a molecular chaperone and also as an anti-apoptotic protein. Methylglyoxal (MGO) is a reactive dicarbonyl compound produced from cellular glycolytic intermediates that reacts non-enzymatically with proteins to form products such as argpyrimidine. We found considerable amount of Hsp27 in phosphorylated form (pHsp27) in human cataractous lenses. pHsp27 was the major argpyrimidine-modified protein in brunescent cataractous lenses. Modification by MGO enhanced the chaperone function of both pHsp27 and native Hsp27, but the effect on Hsp27 was at least three-times greater than on pHsp27. Phosphorylation of Hsp27 abolished its chaperone function. Transfer of Hsp27 using a cationic lipid inhibited staurosporine (SP)-induced apoptotic cell death by 53% in a human lens epithelial cell line (HLE B-3). MGO-modified Hsp27 had an even greater effect (62% inhibition). SP-induced reactive oxygen species in HLE-B3 cells was significantly lower in cells transferred with MGO-modified Hsp27 when compared to native Hsp27. In vitro incubation experiments showed that MGO-modified Hsp27 reduced the activity of caspase-9, and MGO-modified pHsp27 reduced activities of both caspase-9 and caspase-3. Based on these results, we propose that Hsp27 becomes a better anti-apoptotic protein after modification by MGO, which may be due to multiple mechanisms that include enhancement of chaperone function, reduction in oxidative stress, and inhibition of activity of caspases. Our results suggest that MGO modification and phosphorylation of Hsp27 may have important consequences for lens transparency and cataract development. J. Cell. Biochem. © 2006 Wiley-Liss, Inc. [source]

Expression of a releasable form of annexin II by human keratinocytes

Feridoun Karimi-Busheri
Abstract Annexin II is a multifunctional calcium-dependent phospholipid binding protein whose presence in epidermis has previously been reported. However, like other members of annexin family, annexin II has been regarded as either an intracellular protein or associated with the cellular membrane. Here, we report the presence of a releasable annexin II and p11, two monomers of annexin II tetramer, in keratinocyte-conditioned medium (KCM). Proteins present in KCM were fractionated on a gel filtration column and following further evaluation, a releasable protein with apparent MW of 36 kDa was identified. Further characterization identified this protein as the p36 monomer of annexin II tetramer. The phospho-tyrosine antibody did not visualize this protein as the phosphorylated form of p36. Several experiments were conducted to examine whether this protein is soluble or associated with keratinocyte cell membranes in the conditioned medium. A centrifugation of conditioned medium was not able to bring this protein down into the pellet. Surprisingly, the results of Western analysis identified p36 and p11, two monomers of the annexin II tetramer, in conditioned medium derived from either keratinocytes cultured alone or keratinocytes co-cultured with fibroblasts. In contrast to the keratinocyte-conditioned medium in which annexin II was easily detectable, both monomers were barely detectable in conditioned medium collected from dermal fibroblasts. This finding was in contrast to the cell lysates in which p36 was detectable in both keratinocytes and fibroblasts. However, the amount of this protein was markedly higher in keratinocyte lysate relative to that of dermal fibroblasts. Conditioned medium derived from keratinocyte established from adult showed a higher level of annexin II compared to that of keratinocytes established from newborn babies. The expression of p11 seems to increase with differentiation of keratinocytes derived from either adult or newborn skin samples. When the site of annexin synthesis in human skin was examined by immunohistochemical staining, the antibody for p36 localized the annexin to the keratinocyte cell members in the basal and suprabasal keratinocytes. In conclusion, Western blot detection of both p36 and p11 in conditioned medium from skin cells revealed that human keratinocytes, but not fibroblasts, express a releasable monomer form of annexin II which is regulated by differentiation status of keratinocytes. This finding is consistent with the localization of annexin II detected by immunohistochemical staining. J. Cell. Biochem. 86: 737,747, 2002. © 2002 Wiley-Liss, Inc. [source]

Constitutive Phosphorylation of the Vesicular Inhibitory Amino Acid Transporter in Rat Central Nervous System

Cécile Bedet
Abstract:,-Aminobutyric acid (GABA) and glycine are stored into synaptic vesicles by a recently identified vesicular inhibitory amino acid transporter [VIAAT, also called vesicular GABA transporter (VGAT)]. Immunoblotting analysis revealed that rat brain VIAAT migrated as a doublet during sodium dodecyl sulfate,polyacrylamide gel electrophoresis, with a predominant slower band in all areas examined except olfactory bulb and retina. The slower band corresponded to a phosphorylated form of VIAAT as it was converted to the faster one by treating brain homogenates with alkaline phosphatase or with an endogenous phosphatase identified as type 2A protein,serine/threonine phosphatase using okadaic acid. In contrast, the recombinant protein expressed in COS-7 or PC12 cells co-migrated with the faster band of the brain doublet and was insensitive to alkaline phosphatase. To investigate the influence of VIAAT phosphorylation on vesicular neurotransmitter loading, purified synaptic vesicles were treated with alkaline phosphatase and assayed for amino acid uptake. However, neither GABA nor glycine uptake was affected by VIAAT phosphorylation. These results indicate that VIAAT is constitutively phosphorylated on cytosolic serine or threonine residues in most, but not all, regions of the rat brain. This phosphorylation does not regulate the vesicular loading of GABA or glycine, suggesting that it is involved at other stages of the synaptic vesicle life cycle. [source]

Ethanol Uses cAMP-Independent Signal Transduction Mechanisms to Activate Proenkephalin Promoter Activity in Rat C6 Glioma Cells

ALCOHOLISM, Issue 7 2000
Xiaoju Yang
Background: Previous in vivo studies show that acute ethanol exposure sequentially increases protein kinase A (PKA) activity, the phosphorylation of the adenosine 3,:5,-cyclic monophosphate (cAMP) dependent transcription factor, CREB, and finally proenkephalin gene expression. The present study was conducted to determine if ethanol could activate directly the adenylyl cyclase pathway and thus enhance proenkephalin promoter activity. Methods: Cultured rat C6 glioma cells stably transfected with a segment of the five prime flanking region of rat proenkephalin promoter (nucleotide -2700 + 53) ligated to the chloramphenicol acetyltransferase (CAT) reporter gene were employed to study the effects of ethanol on proenkephalin promoter activity. This region of proenkephalin promoter contains two cAMP response elements (CRE-1 and CRE-2) and one AP2 site located in the region upstream of the TATA box. Cultures were exposed to ethanol, isoproterenol, and phorbol-12, myristate 13-acetate (PMA) alone and in combination, in the presence and absence of PKA and protein kinase C (PKC) inhibitors. Results: Ethanol and isoproterenol increased proenkephalin promoter activity in a dose-dependent manner. Ethanol had an additive effect on maximal isoproterenol-stimulated proenkephalin promoter activity, which suggested that ethanol used a cAMP-independent signai transduction pathway to increase proenkephalin promoter activation. In contrast with isoproterenol, ethanol exposure did not increase cAMP accumulation, PKA activity, or the phosphorylated form of CREB. However, ethanol exposure modestly increased PKC activity. The PKA-specific inhibitor, Rp-cAMP, dampened isoproterenol-induced activation of CAT activity but did not alter ethanol's ability to increase CAT activity. However, the PKC inhibitors, chelerthyrine and G07874, abrogated ethanol's effect of CAT activity but did not alter isoproterenol's effects. Conclusions: Ethanol enhanced proenkephalin promoter activity and potentiated isoproterenol stimulated promoter activity through a cAMP-independent pathway. [source]

,-Tocopheryl phosphate , An active lipid mediator?

Jean-Marc Zingg
Abstract The vitamin E (,-tocopherol, ,T) derivative, ,-tocopheryl phosphate (,TP), is detectable in small amounts in plasma, tissues, and cultured cells. Studies done in vitro and in vivo suggest that ,T can become phosphorylated and ,TP dephosphorylated, suggesting the existence of enzyme(s) with ,T kinase or ,TP phosphatase activity, respectively. As a supplement in animal studies, ,TP can reach plasma concentrations similar to ,T and only a part is dephosphorylated; thus, ,TP may act both as pro-vitamin E, but also as phosphorylated form of vitamin E with possibly novel regulatory activities. Many effects of ,TP have been described: in the test tube ,TP modulates the activity of several enzymes; in cell culture ,TP affects proliferation, apoptosis, signal transduction, and gene expression; in animal studies ,TP prevents atherosclerosis, ischemia/reperfusion injury, and induces hippocampal long-term potentiation. At the molecular level, ,TP may act as a cofactor for enzymes, as an active lipid mediator similar to other phosphorylated lipids, or indirectly by altering membrane characteristics such as lipid rafts, fluidity, and curvature. In this review, the molecular and cellular activities of ,TP are examined and the possible functions of ,TP as a natural compound, cofactor and active lipid mediator involved in signal transduction and gene expression discussed. [source]

Extracellular signal-regulated protein kinase is activated in cervical intraepithelial neoplasms but inactivated in invasive cervical carcinoma

Keiko Matsuura
The extracellular signal-regulated protein kinase (ERK) signaling pathway has been reported to play important roles in cell growth in various neoplasms. The purpose of the present study was to immunohistochemically analyze the phosphorylation status (activity) of ERK in 24 cases of cervical carcinoma using an antiphosphorylated ERK antibody (,p-ERK Ab) that specifically recognizes the phosphorylated form of ERK (p-ERK). In normal cervical epithelium, p-ERK was found to be confined to basal cells that were negative for Ki-67, suggesting that ERK was not activated in proliferating normal cervical epithelium. In cervical intraepithelial neoplasms (CIN), increased abnormal parabasal cells were positive for both p-ERK and Ki-67, suggesting that ERK activation in CIN may be involved in tumor cell proliferation. In contrast, it was found that, in invasive cervical carcinomas, almost all the carcinoma cells were positive for Ki-67 but negative for p-ERK, suggesting that, in contrast to many other types of cancers, the ERK signaling pathway is downregulated in invasive cervical carcinoma. These findings suggest that the phosphorylation status of ERK differs between CIN and invasive carcinomas, and that downregulation of the ERK signaling pathway may contribute to transformation of CIN to invasive cervical carcinomas. [source]

Role of p38 MAPKs in Hypericin Photodynamic Therapy-induced Apoptosis of Nasopharyngeal Carcinoma Cells

Pui S. Chan
The present study aims to determine the role of mitogen-activated protein kinases (MAPKs) in hypericin-mediated photodynamic therapy (HY-PDT)-induced apoptosis of the HK-1 nasopharyngeal carcinoma (NPC) cells. HY-PDT was found to induce proteolytic cleavage of procaspase-9 and -3 in HK-1 cells. Apoptotic nuclei were observed at 6 h after PDT whereas B-cell leukemia/lymphoma-2-associated-X-protein (Bax) translocation and formation of Bax channel is responsible for the cell death. Increase in phosphorylation of p38 MAPKs and c-Jun N-terminal kinase 1/2 (JNK1/2) was detected at 15,30 min after HY-PDT. The appearance of phosphorylated form of p38 MAPKs and JNK1/2 was inhibited by the singlet oxygen scavenger l -histidine. HY-PDT-induced cell death was enhanced by the chemical inhibitors for p38 MAPKs (SB202190 and SB203580), but not by the JNKs inhibitor SP600125. Knockdown of the p38, and p38, MAPK isoforms by small interfering RNA (siRNA) are more effective than the p38, in enhancing PDT-induced cell death. Augmentation of apoptosis by p38, or p38, knockdown is also correlated with the increased proteolytic cleavage of procaspase-9 after HY-PDT treatment. Our results suggested that HY-PDT activated p38 MAPKs through the production of singlet oxygen. Inhibition of p38 MAPKs with chemical inhibitors or siRNA enhances HY-PDT-induced apoptosis of the HK-1 NPC cells. [source]

SELDI-TOF MS analysis of the Cardiac Troponin,I forms present in plasma from patients with myocardial infarction

Estelle Peronnet
Abstract The troponin,(Tn) complex is composed of troponin,T, troponin,C and troponin,I. The cardiac isoform of TnI (cTnI) is modified and released in blood of patients with cardiovascular diseases as a heterogeneous mixture of free, complexed and posttranslationally modified forms. With the aim to determine later, whether specific forms of cTnI could be associated with the different pathologies leading to cTnI release, the cTnI forms present in the plasma from 64,patients with acute myocardial infarction,(AMI) have been analysed by SELDI-TOF MS using anti-TnI mAbs coupled to PS20 ProteinChips® arrays. Upfront immunoaffinity enrichment using anti-cTnI,19C7 mAb allowed us to detect cTnI and bis -phosphorylated cTnI in 11/12 and 9/12 analyses respectively, as well as truncated cTnI in plasma with concentration of cTnI as low as 8,ng/mL. Cardiac troponin,C (cTnC) and covalent TnIC complex were also found in pools of plasma with higher concentrations of cTnI. MAb 19C7-affinity SELDI-TOF MS analysis performed after immunopurification of one pool of AMI plasma with anti-free cTnI, anti-cTnC, and anti-phosphorylated cTnI mAbs indicated that intact and bis -phosphorylated cTnI were mostly under the free form. Besides, a 18,718,m/z peak could correspond to a truncated phosphorylated form initially complexed with cTnC. [source]

EGFR Regulates the Side Population in Head and Neck Squamous Cell Carcinoma

Jocelyn S. Chen BS
Abstract Objective: To identify the presence of side population (SP) cells in established head and neck squamous carcinoma cell (HNSCC) lines and to determine the role of EGFR in the regulation of the side population of these cells. Methods: SP cells were identified using flow cytometry analysis by the ability of these cells to extrude the Hoechst 33342 dye via the drug transporter BCRP1/ABCG2. Effect of EGFR on the side population was determined also by difference in Hoechst extrusion and by immunofluorescence. Immunohistochemical staining was performed to show the presence of the BCRP1/ABCG2 transporter and the phosphorylated form of EGFR in HNSCC tissue. Results: SP cells are present in HNSCC cell lines. With the Hoechst 33342 extrusion assay, SP cells were found to comprise an average of 0.69% of the UMSCC10B cells and 0.91% of HN12 cells. Addition of the EGF ligand increased the SP population while inactivation of the EGFR kinase by Iressa significantly decreased SP. Conclusion: In established head and neck squamous cell carcinoma cell lines, SP cells were found using methods that determine expression and function of the drug transporter BCRP1/ABCG2. Activation of EGFR, a gene implicated in tumorigenesis in HNSCC leads to increased SP, and conversely, inhibition of EGFR leads to decrease in SP. This finding could help explain the role of EGFR in regulating cancer stem cells and thus tumorigenesis in HNSCC. [source]

Extracellular signal-regulated kinase (ERK) activation in chicken heterophils stimulated with phorbol 12-myristate 13-acetate (PMA), Formyl-methionylleucyl-phenylalanine (fMLP) and lipopolysaccharide (LPS)

ABSTRACT The signaling pathways leading to the activation of extracellular signal-regulated kinase (ERK) by phorbol 12-myristate 13-acetate (PMA), formyl-methionylleucyl-phenylalanine (fMLP) and lipopolysaccharide (LPS) in chicken heterophils were examined. To determine the mechanism of ERK's activation and its relation with the influx of calcium ions, heterophils were stimulated by PMA, fMLP and LPS. ERK was not activated by fMLP. LPS- and PMA-stimulated activation of ERK, based on Western blotting with antibodies against the phosphorylated form of ERK, was attenuated by the pretreatment of cells with the intracellular calcium chelator BAPTA/AM (1,2-bis (o-aminophenoxy) ethane-N,N,N,,N,-tetraacetic acid) but not with the extracellular calcium chelator EGTA (glycol-bis(2-aminoethylether)-N,N,N,,N,-tetraacetic acid). Exposure of cells to the protein kinase C (PKC) inhibitor GF109203X inhibited the LPS- and PMA-stimulated phosphorylation of ERK in a concentration-dependent manner. The LPS-stimulated phosphorylation was inhibited by pretreatment with the phospholipase C (PLC) inhibitor U73122 but not the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. These results indicate that the LPS-induced phosphorylation of ERK in the chicken heterophils is mediated by PLC, PKC and intracellular calcium, and the PMA-stimulated phosphorylation is dependent on intracellular calcium ion and PKC. [source]

Halofuginone inhibition of COL1A2 promoter activity via a c-Jun,dependent mechanism

Tracy L. McGaha
Objective The naturally occurring compound halofuginone has been shown to antagonize collagen synthesis by fibroblasts both in vitro and in vivo. We previously demonstrated that this inhibitory property was related to the ability of halofuginone to disrupt transforming growth factor , signal transduction. The present study further analyzed the ability of halofuginone to affect transcription factors that can regulate type I collagen gene expression by examining its effect on c-Jun, the negative regulator of collagen gene transcription. Methods The phosphorylation state of c-Jun in the presence of halofuginone was examined via direct Western blotting, and the transcriptional activity of the activator protein 1 (AP-1) binding element via electrophoretic mobility shift assay and luciferase reporter assay. We determined whether the effect of halofuginone on collagen synthesis was dependent on the presence of c-Jun by ectopic expression of a wild-type or dominant-negative c-Jun construct in the presence of halofuginone and assaying ,2(I) collagen promoter strength via luciferase reporter assay. The effect of halofuginone on ,2(I) collagen message levels in fibroblasts when wild-type or dominant-negative c-Jun was overexpressed was determined. We also determined whether halofuginone had an effect on the phosphorylation state of c-Jun in the skin of TSK/+ mice via immunohistochemistry. Results Treatment of fibroblasts with 10,8M halofuginone enhanced basal and mitogen-mediated phosphorylation of c-Jun in culture. This elevated phosphorylation of c-Jun correlated with enhanced DNA binding and transcriptional activation of an AP-1 complex consisting of c-Jun and Fos but lacking the c-Jun antagonist JunB. Overexpression of c-Jun enhanced in a dose-dependent manner the ability of halofuginone to inhibit the activity of a luciferase reporter construct under control of the ,3200-bp to +54-bp COL1A2 promoter, whereas the expression of a dominant-negative c-Jun construct abolished this effect. Northern blotting showed that overexpression of c-Jun enhanced the ability of halofuginone to reduce collagen ,2(I) messenger RNA levels in fibroblasts, whereas expression of the dominant-negative c-Jun abolished this effect. Topical administration of a halofuginone-containing cream for 20 days to TSK mice, which spontaneously develop dermal fibrosis, greatly increased the phosphorylated form of c-Jun in the skin; this was followed by a decrease in skin thickness and type I collagen messenger RNA expression. Conclusion Our findings illustrate the powerful down-regulatory property of c-Jun toward type I collagen and establish that halofuginone exerts its effect on collagen synthesis in a c-Jun,dependent manner. [source]

Modulation of gap junctions by nitric oxide contributes to the anti-arrhythmic effect of sodium nitroprusside?

Márton Gönczi
Background and purpose:, Nitric oxide (NO) donors provide a preconditioning-like anti-arrhythmic protection in the anaesthetized dog. As NO may modulate gap junction (GJ) function, the present study investigated whether this anti-arrhythmic effect is due to a modification of GJs by NO, derived from the NO donor sodium nitroprusside (SNP). Experimental approach:, In chloralose-urethane-anaesthetized, open-chest dogs, either saline (controls; n= 11) or SNP (0.2 µg·kg,1·min,1; n= 10) was infused at a rate of 0.5 mL·min,1 by the intracoronary route. The infusions were started 20 min prior to and maintained throughout the entire 60 min occlusion period of the left anterior descending coronary artery. The severity of ischaemia and of arrhythmias, tissue electrical impedance and permeability, as well as the phosphorylation of connexin43, were assessed. Key results:, Compared with the controls, SNP infusion markedly suppressed the total number of ventricular premature beats (666 ± 202 vs. 49 ± 18; P < 0.05), and the number of ventricular tachycardiac episodes (8.1 ± 2.3 vs. 0.2 ± 0.1; P < 0.05) without significantly modifying the incidence of ventricular tachycardia or ventricular fibrillation. The severity of ischaemia (epicardial ST-segment changes, inhomogeneity of electrical activation) and tissue electrical impedance changes were significantly less in the SNP-treated dogs. SNP improved GJ permeability and preserved the phosphorylated form of connexin43. Conclusion and implications:, The anti-arrhythmic protection resulting from SNP infusion in the anaesthethized dog may, in part, be associated with the modulation of gap junctional function by NO. [source]

Pitavastatin inhibits azoxymethane-induced colonic preneoplastic lesions in C57BL/KsJ- db/db obese mice

CANCER SCIENCE, Issue 7 2010
Yoichi Yasuda
Obesity and related metabolic abnormalities are risk factors for colorectal cancer. A state of chronic inflammation and adipocytokine imbalance may play a role in colorectal carcinogenesis. Statins, which are commonly used for the treatment of hyperlipidemia, are known to possess anti-inflammatory effects. Statins also exert chemopreventive properties against various cancers. The present study examined the effects of pitavastatin, a recently developed lipophilic statin, on the development of azoxymethane (AOM)-initiated colonic premalignant lesions in C57BL/KsJ- db/db (db/db) obese mice. Male db/db mice were administrated weekly subcutaneous injections of AOM (15 mg/kg body weight) for 4 weeks and then were subsequently fed a diet containing 1 ppm or 10 ppm pitavastatin for 8 weeks. Feeding with either dose of pitavastatin significantly reduced the number of colonic premalignant lesions, ,-catenin accumulated crypts, by inhibiting proliferation and the surrounding inflammation. Pitavastatin increased the serum levels of adiponectin while conversely decreasing the serum levels of total cholesterol, tumor necrosis factor-, (TNF-,), interleukin (IL)-6, IL-18, and leptin. Pitavastatin also caused a significant increase in the expression of phosphorylated form of the AMP-activated kinase (AMPK) protein on the colonic mucosa of AOM-treated mice. In addition, the expression levels of TNF-,, IL-6, IL-18, and COX-2 mRNAs on the colonic mucosa of AOM-treated mice were decreased by treatment with this agent. These findings suggest that pitavastatin attenuates chronic inflammation and improves the imbalance of adipocytokines, both of which are caused by the presence of excess adipose tissues, thereby preventing the development of colonic premalignancies in an obesity-related colon cancer model. Therefore, some types of statins, including pitavastatin, may be a useful chemoprevention modality for colon cancer in obese individuals. (Cancer Sci 2010) [source]

Post-translational modifications of the major linear epitope 169,190aa of Ro60 kDa autoantigen alter the autoantibody binding

A. G. Terzoglou
Summary Ro60 kDa is a member of the Ro/LaRNP ribonucleoprotein complex and its major linear B cell epitope, corresponding to the region 169,190aa, has been found to be the initial target of the autoimmune response in patients with systemic lupus erythematosus. This sequence contains one serine and two arginine amino acid residues, which can potentially be modified post-translationally by phosphorylation or citrullination, respectively. The aim of this study was to develop an immunoassay for anti-Ro60 kDa epitope antibody detection and to investigate the changes in the antigenicity of the Ro60 kDa epitope when it is post-translationally modified, by either citrullination or phosphorylation. Peptide analogues corresponding to the unmodified form of the epitope, its phosphorylated form, and a form with both arginine residues citrullinated were synthesized. The peptide coating conditions were investigated and it was found that the use of highly hydrophilic surfaces increase the efficiency of the coating, as well as the sensitivity of the method for anti-peptide antibody detection. All peptides were tested by the optimized enzyme-linked immunosorbent assay (ELISA) against 119 sera from patients with primary Sjögren's syndrome, systemic lupus erythematosus and rheumatoid arthritis with anti-Ro/SSA reactivity, 20 sera from patients with systemic diseases without anti-Ro/SSA immune reactivity, as well as against 65 sera from normal individuals. A large proportion of the tested sera reacted against all three peptide analogues, although with a preference for the unmodified form of the epitope. In conclusion, post-translational modifications of the major Ro60 kDa B cell epitope can alter the autoantibody binding. [source]

Preferential recognition of the phosphorylated major linear B-cell epitope of La/SSB 349,368aa by anti-La/SSB autoantibodies from patients with systemic autoimmune diseases

A. G. Terzoglou
Summary Sera from patients with primary Sjögren Syndrome (pSS) or Systemic Lupus Erythematosus (SLE) often contain autoantibodies directed against La/SSB. The sequence 349,368aa represents the major B-cell epitope of La/SSB, also it contains, at position 366, a serine aminoacid residue which constitutes the main phosphorylation site of the protein. In this study we investigated the differential recognition of the 349,368aa epitope and its phosphorylated form by antibodies found in sera from patients with systemic autoimmune diseases. Peptides corresponding to the sequence of the unphosphorylated (pep349,368aa) and the phosphorylated form (pep349,368aaPh) of the La/SSB epitope 349,368aa, as well as to a truncated form spanning the sequence 349,364aa and lacking the phosphorylation site (pep349,364aa), were synthesized. Sera from 53 patients with pSS and SLE with anti-La/SSB specificity, 30 patients with pSS and SLE without anti-La/SSB antibodies, 25 patients with rheumatoid arthritis and 32 healthy individuals were investigated by ELISA experiments. Autoantibodies to pep349,368aaPh were detected in sera of anti-La/SSB positive patients with a higher prevalence compared to the pep349,368aa (66%versus 45%). Pep349,368aaPh inhibited the antibody binding almost completely (92%), while pep349,368aa inhibited the binding only partially (45%). Anti-La/SSB antibodies presented a higher relative avidity for the phosphorylated than the unphosphorylated peptide. Immunoadsorbent experiments using the truncated peptide pep349,364aa indicated that the flowthrough showed a selective specificity for pep349,368aaPh, while the eluted antibodies reacted with both peptide analogues of the La/SSB epitope. These data suggest that sera from pSS and SLE patients with anti-La/SSB reactivity possess autoantibodies that bind more frequently and with a higher avidity to the phosphorylated major B-cell epitope of the molecule. [source]

Protein kinase C , phosphorylates keratin 8 at Ser8 and Ser23 in GH4C1 cells stimulated by thyrotropin-releasing hormone

FEBS JOURNAL, Issue 13 2007
Yoshiko Akita
Protein kinase C , (PKC,) is activated by thyrotropin-releasing hormone (TRH), a regulator of pituitary function in rat pituitary GH4C1 cells. We analyzed the downstream mechanism after PKC, activation. Exposure of GH4C1 cells to TRH or a phorbol ester increased the phosphorylation of three p52 proteins (p52a, p52b and p52c) and decreased the phosphorylation of destrin and cofilin. GF109203X, an inhibitor of protein kinases including PKC, inhibited phosphorylation of the p52 proteins by TRH stimulation. Peptide mapping, amino-acid sequencing, and immunochemical studies indicated that p52a, p52b, and p52c are the differentially phosphorylated isoforms of keratin 8 (K8), an intermediate filament protein. The unphosphorylated K8 (p52n) localized exclusively to the cytoskeleton, whereas the phosphorylated forms (especially p52c), which are increased in TRH-stimulated cells, localized mainly to the cytosol. K8 phosphorylation was enhanced in PKC,-overexpressing clones, and purified recombinant PKC, directly phosphorylated K8 with a profile similar to that observed in TRH-stimulated cells. PKC, and K8 colocalized near the nucleus under basal conditions and were concentrated in the cell periphery and cell,cell contact area after TRH stimulation. MS analyses of phospho-K8 and K8-synthesized peptide (amino acids 1,53) showed that PKC, phosphorylates Ser8 and Ser23 of K8. Phosphorylation of these sites is enhanced in TRH-stimulated cells and PKC,-overexpressing cells, as assessed by immunoblotting using antibodies to phospho-K8. These results suggest that K8 is a physiological substrate for PKC,, and the phosphorylation at Ser8 and Ser23 transduces, at least in part, TRH,PKC, signaling in pituitary cells. [source]

Phosphorylation and oligomerization states of native pig brain HSP90 studied by mass spectrometry

FEBS JOURNAL, Issue 8 2001
Cyrille Garnier
HSP90 is one of the most abundant proteins in the cytosol of eukaryotic cells. HSP90 forms transient or stable complexes with several key proteins involved in signal transduction including protooncogenic protein kinases and nuclear receptors, it interacts with cellular structural elements such as actin-microfilament, tubulin-microtubule and intermediate filaments, and also exhibits conventional chaperone functions. This protein exists in two isoforms ,-HSP90 and ,-HSP90, and it forms dimers which are crucial species for its biological activity. PAGE, ESI-MS and MALDI-MS were used to study HSP90 purified from pig brain. The two protein isoforms were clearly distinguished by ESI-MS, the , isoform being ,,six times more abundant than the , isoform. ESI-MS in combination with ,,phosphatase treatment provided direct evidence of the existence of four phosphorylated forms of native pig brain ,-HSP90, with the diphosphorylated form being the most abundant. For the , isoform, the di-phosphorylated was also the most abundant. MALDI mass spectra of HSP90 samples after chemical cross-linking showed a high percentage of ,,, homodimers. In addition, evidence for the existence of higher HSP90 oligomers was obtained. [source]

Molecular cloning and expression of Tenebrio molitor ultraspiracle during metamorphosis and in vivo induction of its phosphorylation by 20-hydroxyecdysone

M. Nicolaď
Abstract Using a RT-PCR approach, the Tenebrio molitor homologue of Drosophila Ultraspiracle (TmUSP) was characterized. Its DNA binding domain shows a degree of identity with those of the other insect USPs. However, the ligand binding domain is closer to those of retinoid X receptors. Using an antibody raised against DmUSP, Western blot analysis of proteins from epidermis and other tissues revealed five immunoreactive bands, corresponding to different phosphorylated forms of a unique polypeptide, as shown by ,-phosphatase treatment. The nuclear form of TmUSP seems unphosphorylated. An in vivo 20-hydroxyecdysone treatment increases considerably and rapidly the phosphorylated forms of TmUSP. This post-translational modification may play a role in the 20-hydroxyecdysone response. [source]

Do axonal defects in tau and amyloid precursor protein transgenic animals model axonopathy in Alzheimer's disease?

Jürgen Götz
Abstract The subcellular localization of organelles, mRNAs and proteins is particularly challenging in neurons. Owing to their extended morphology, with axons in humans exceeding a meter in length, in addition to which they are not renewed but persist for the entire lifespan, it is no surprise that neurons are highly vulnerable to any perturbation of their sophisticated transport machinery. There is emerging evidence that impaired transport is not only causative for a range of motor disorders, but possibly also for Alzheimer's disease (AD) and related neurodegenerative disorders. Support for this hypothesis comes from transgenic animal models. Overexpression of human tau and amyloid precursor protein (APP) in mice and flies models the key hallmark histopathological characteristics of AD, such as somatodendritic accumulation of phosphorylated forms of tau and ,-amyloid (A,) peptide-containing amyloid plaques, as well as axonopathy. The latter has also been demonstrated in mutant mice with altered levels of Alzheimer-associated genes, such as presenilin (PS). In A,-producing APP transgenic mice, axonopathy was observed before the onset of plaque formation and tau hyperphosphorylation. In human AD brain, an axonopathy was revealed for early but not late Braak stages. The overall picture is that key players in AD, such as tau, APP and PS, perturb axonal transport early on in AD, causing impaired synaptic plasticity and reducing survival rates. It will be challenging to determine the molecular mechanisms of these different axonopathies, as this might assist in the development of new therapeutic strategies. [source]

The mitogen-activated protein kinases p38 and ERK1/2 are increased in lesional psoriatic skin

C. Johansen
Summary Background, Alterations in specific signal transduction pathways may explain the hyperproliferation and abnormal differentiation of the keratinocytes as well as the increased expression of inflammatory cytokines seen in psoriasis. Major signalling pathways used by eukaryotic cells to transduce extracellular signals into cellular responses impinge on the mitogen-activated protein kinases (MAPKs). Objectives, To investigate the expression of the MAPK p38, extracellular signal-regulated kinase (ERK) and c-Jun NH2 -terminal kinase (JNK) in psoriatic skin. Methods, Keratome biopsies were taken from patients with plaque-type psoriasis. Western blot analysis was used to determine p38, ERK and JNK activity and protein levels, whereas kinase assays were used to examine the kinase activity of p38. Results, We demonstrated increased levels of the phosphorylated forms of p38 and ERK1/2 in lesional psoriatic skin compared with nonlesional psoriatic skin. No abnormality was found in the activation and expression of JNK1/2. Ex vivo kinase assays confirmed the increased activation of p38, and furthermore demonstrated increased kinase activity of the p38 isoforms p38,, p38, and p38, in lesional compared with nonlesional psoriatic skin. p38, was not detected in the psoriatic skin. Clearance of the psoriatic lesions, induced by climatotherapy at the Dead Sea for 4 weeks, led to a normalization in the activity of both p38 and ERK1/2. Conclusions, Taken together, our results demonstrate that the activity of the MAPKs p38,, p38, and p38, and ERK1/2 are increased in lesional psoriatic skin compared with nonlesional psoriatic skin, and that clearance of psoriasis normalizes the p38 and ERK1/2 activity. Thus, p38 and ERK1/2 might be potential targets in the treatment of psoriasis. [source]