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Phosphorylation Status (phosphorylation + status)
Selected AbstractsPhosphorylation status of pyruvate dehydrogenase distinguishes metabolic phenotypes of cultured rat brain astrocytes and neuronsGLIA, Issue 10 2010Nader D. Halim Abstract Glucose metabolism in nervous tissue has been proposed to occur in a compartmentalized manner with astrocytes contributing largely to glycolysis and neurons being the primary site of glucose oxidation. However, mammalian astrocytes and neurons both contain mitochondria, and it remains unclear why in culture neurons oxidize glucose, lactate, and pyruvate to a much larger extent than astrocytes. The objective of this study was to determine whether pyruvate metabolism is differentially regulated in cultured neurons versus astrocytes. Expression of all components of the pyruvate dehydrogenase complex (PDC), the rate-limiting step for pyruvate entry into the Krebs cycle, was determined in cultured astrocytes and neurons. In addition, regulation of PDC enzymatic activity in the two cell types via protein phosphorylation was examined. We show that all components of the PDC are expressed in both cell types in culture, but that PDC activity is kept strongly inhibited in astrocytes through phosphorylation of the pyruvate dehydrogenase alpha subunit (PDH,). In contrast, neuronal PDC operates close to maximal levels with much lower levels of phosphorlyated PDH,. Dephosphorylation of astrocytic PDH, restores PDC activity and lowers lactate production. Our findings suggest that the glucose metabolism of astrocytes and neurons may be far more flexible than previously believed. © 2010 Wiley-Liss, Inc. [source] The regulation of muscle glycogen: the granule and its proteinsACTA PHYSIOLOGICA, Issue 4 2010T. E. Graham Abstract Despite decades of studying muscle glycogen in many metabolic situations, surprisingly little is known regarding its regulation. Glycogen is a dynamic and vital metabolic fuel that has very limited energetic capacity. Thus its regulation is highly complex and multifaceted. The stores in muscle are not homogeneous and there appear to be various metabolic pools. Each granule is capable of independent regulation and fundamental aspects of the regulation appear to be associated with a complex set of proteins (some are enzymes and others serve scaffolding roles) that associate both with the granule and with each other in a dynamic fashion. The regulation includes altered phosphorylation status and often translocation as well. The understanding of the roles and the regulation of glycogenin, protein phosphatase 1, glycogen targeting proteins, laforin and malin are in their infancy. These various processes appear to be the mechanisms that give the glycogen granule precise, yet dynamic regulation. [source] MAP65 in tubulin/colchicine paracrystals of Vigna sinensis root cells: Possible role in the assembly and stabilization of atypical tubulin polymers,CYTOSKELETON, Issue 3 2010Emmanuel Panteris Abstract Members of the MAP65 family, colocalizing with microtubule arrays, have been identified in Vigna sinensis root cells by Western blotting and immunofluorescence. MAP65 proteins were also found in tubulin/colchicine paracrystals, which were formed during colchicine treatment by both immunofluorescence and immunogold microscopy. During recovery from colchicine, MAP65 signal was depleted from disintegrating paracrystals appearing in the reinstating microtubule arrays. MAP65-free perinuclear tubulin/colchicine aggregates were observed in plasmolyzed colchicine-treated cells. Deplasmolysis of the above cells resulted in the formation of MAP65-decorated paracrystals. As confirmed by appropriate biochemical assays with the Phos-tag reagent, MAP65 proteins underwent phosphorylation during plasmolysis, which was reversible by deplasmolysis. According to the effect of the mitogen-activated protein kinase (MAPK) inhibitor UO126, the phosphorylation status of MAP65, as well as its presence in tubulin/colchicine polymers is probably controlled by MAPK-mediated phosphorylation. According to the above, it seems likely that apart from binding to microtubules, MAP65 proteins may act as "tubulin associated proteins" in a broader manner, promoting the polymerization and/or stabilization of atypical polymers such as tubulin/colchicine paracrystals. © 2010 Wiley-Liss, Inc. [source] Fatty acid incorporation in endothelial cells and effects on endothelial nitric oxide synthaseEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 9 2007S. Couloubaly Abstract Background The nature of fatty acids provided by the diet as well as plasma lipid metabolism can modify the composition and properties of plasma membrane and thus the activity of membrane proteins. In humans, as well as in experimental models, diabetes is associated with both an alteration in serum lipid profile and a documented endothelial dysfunction. This in vitro study investigated on an immortalized human endothelial cell line (EA.hy 926) the specific effects of several free fatty acids (FFAs) on the composition of cellular membranes and the regulation of endothelial nitric oxide synthase (eNOS). Materials and methods 0·1% of lipid deprived serum was added to the incubation medium with 25 mm glucose in order to study the effects of individual fatty acids: myristic acid, palmitic acid, stearic acid, oleic acid or linoleic acid at 100 µm bound with albumin. The effects of the FFAs on the endothelial nitric oxide synthase were investigated on mRNA level by quantitative PCR, on protein level and Ser1177 phosphorylation by Western blot and on enzymatic activity on living cells using radiolabelled arginine. Results Free linoleic acid increased the membrane content in n-6 fatty acids (mainly C18: n-6 and its metabolites) with a decrease in saturated and monounsaturated fatty acids. These conditions decreased the basal eNOS activity and reduced the phosphorylation of eNOS-Ser1177 due to activation by histamine. Free palmitic acid enriched the membranes with 16 : 0 with a slight decrease in monounsaturated fatty acids. These conditions increased eNOS activation without increasing Ser1177 phosphorylation upon histamine activation. The addition of the other FFAs also resulted in modifications of membrane composition, which did not to affect eNOS-Ser1177 phosphorylation. Conclusion Among the fatty acids used, only modification of the membrane composition due to linoleic acid supply disturbed the basal enzymatic activity and Ser1177 phosphorylation of eNOS in a way that limited the role of histamine activation. Linoleic acid might involve the dysfunction of both eNOS basal activity and its phosphorylation status and may then contribute to an impaired vasodilatation in vivo. [source] Cholinergic control of epidermal cohesionEXPERIMENTAL DERMATOLOGY, Issue 4 2006Sergei A. Grando Abstract:, The non-neuronal cholinergic system of human epidermis includes the keratinocyte (KC) acetylcholine (ACh) axis composed of the enzymes mediating ACh synthesis and degradation, and two classes of ACh receptors, the nicotinic and muscarinic ACh receptors, mediating biological effects of the cutaneous cytotransmitter ACh. Regulation of KC cell,cell and cell,matrix adhesion is one of the important biological functions of cutaneous ACh. The downstream targets of ACh effects mediated by distinct ACh receptor subtypes include both the intercellular adhesion molecules, such as classical and desmosomal cadherins, and integrins mediating KC adhesion to a substrate. The signaling pathways include activation or inhibition of kinase cascades resulting in either up- or down-regulation of the expression of cell adhesion molecules or changes in their phosphorylation status, or both. The components of the KC ACh axis are involved in cutaneous blistering in patients with autoimmune pemphigus, junctional and dystrophic forms of epidermolysis bullosa, thermal burns, and mustard-induced vesication. Recent progress with the development of antiacantholytic therapies of patients with pemphigus using cholinomimetics indicates that cholinergic drugs may be a promising approach for other cutaneous blistering disorders. [source] Human inhibitor of growth 1 inhibits hepatoma cell growth and influences p53 stability in a variant-dependent manner,HEPATOLOGY, Issue 2 2009Zhi Zhu Inhibitor of growth 1 (ING1) is a type II tumor suppressor that affects cell function by altering chromatin structure and regulating transcription. Recently, three ING1 splice variants have been cloned, but their roles in apoptosis and p53 regulation in human hepatocellular carcinoma (HCC) have not been fully elucidated. The present study found that ING1, in a variant-dependent manner, inhibited hepatoma cell proliferation and colony formation, induced apoptosis and cell cycle arrest at G0/G1 phase, and postponed tumor formation in nude mice. Expression of p33ING1b and p24ING1c variants, but not p47ING1a, was markedly reduced in HCC samples. Reverse transcription polymerase chain reaction and western blotting analysis revealed that ectopic overexpression of p33ING1b or p24ING1c variant increased the expression of p53 downstream genes such as p21waf1 and bax, and repressed bcl-2 expression (P < 0.01), whereas p47ING1a inactivated p21waf1 promoter (P < 0.01). Furthermore, we found that p33ING1b and p24ING1c repressed Mdm2 expression (P < 0.01) and competed with Mdm2 for binding to p53. Interestingly, p33ING1band p24ING1c did not directly bind to Mdm2 protein but strongly increased p14arf expression (P < 0.01) and interacted with p14arf protein to stimulate p53. Moreover, we found that ectopic overexpression of p33ING1b or p24ING1c significantly induced p53 protein acetylation at Lys-373/Lys-382 residue, but did not alter the phosphorylation status of p53. Conclusion: ING1 variants p33ING1b and p24ING1c may modulate p53 activity and subsequently inhibit hepatoma cell growth by at least two possible mechanisms: interacting with Mdm2 and p14arf to stabilize and activate p53, or increasing p53 acetylation. (HEPATOLOGY 2009.) [source] Her-2/neu and EGFR tyrosine kinase activation predict the efficacy of trastuzumab-based therapy in patients with metastatic breast cancerINTERNATIONAL JOURNAL OF CANCER, Issue 5 2006Gernot Hudelist Abstract Her-2/neu overexpression in human breast cancer leads to an aggressive biological behavior and poor prognosis. Although the anti-Her-2/neu antibody trastuzumab (Herceptin®) has become a valuable therapeutic option for patients with Her-2/neu -overexpressing breast cancer, many patients do not benefit from this therapy. To evaluate the effect of receptor activation on tumor response, we have investigated the phosphorylation status of Her-2/neu and EGFR in 46 Her-2/neu -overexpressing tumor samples from trastuzumab-treated metastatic breast cancer patients by immunohistochemistry. Activated (p)tyr-1248 Her-2/neu was detected in 9 of 46 breast cancers (20%), and activated (p)tyr-845 and (p)tyr-1173 EGFR were both present in 6 tumors (13%) while EGFR was present in 16 cases (35%). ptyr-1248 Her-2/neu showed a trend to correlate with increased response to trastuzumab (p = 0.063), while ptyr-845, ptyr-1173 EGFR and EGFR did not. The presence of ptyr-1248 Her-2/neu and ptyr-845 or ptyr-1173 EGFR, however, was a strong predictor of both response to trastuzumab-based treatment (OR = 8.0, p = 0.021 and OR = 8.0, p = 0.021) and clinical benefit (OR = 5.47, p = 0.041 and OR = 6.22, p = 0.028 multivariate logistic regression analysis). Furthermore, ptyr-845 EGFR and ptyr-1248 Her-2/neu were both independent predictors of progression-free survival (RR = 0.21, p = 0.01 and RR = 0.45, p = 0.026, multivariate analysis). Patients with ptyr-845 EGFR positive tumors also tended toward increased overall survival (RR = 0.17, p = 0.082). Taken together, we have demonstrated that the determination of activated EGFR improves the utility of ptyr-1248 Her-2/neu staining in predicting the clinical outcome of patients undergoing trastuzumab treatment. We hypothesize that the activation state of both Her-2/neu and EGFR are key determinants for trastuzumab efficacy. © 2005 Wiley-Liss, Inc. [source] Tamoxifen resistance in MCF7 cells promotes EMT-like behaviour and involves modulation of ,-catenin phosphorylationINTERNATIONAL JOURNAL OF CANCER, Issue 2 2006Stephen Hiscox Abstract We have previously demonstrated that, following acquisition of endocrine resistance, breast cancer cells display an altered growth rate together with increased aggressive behaviour in vitro. Since dysfunctional cell,cell adhesive interactions can promote an aggressive phenotype, we investigated the integrity of this protein complex in our breast cancer model of tamoxifen resistance. In culture, tamoxifen-resistant MCF7 (TamR) cells grew as loosely packed colonies with loss of cell,cell junctions and demonstrated altered morphology characteristic of cells undergoing epithelial-to-mesenchymal transition (EMT). Neutralising E-cadherin function promoted the invasion and inhibited the aggregation of endocrine-sensitive MCF7 cells, whilst having little effect on the behaviour of TamR cells. Additionally, TamR cells had increased levels of tyrosine-phosphorylated ,-catenin, whilst serine/threonine-phosphorylated ,-catenin was decreased. These cells also displayed loss of association between ,-catenin and E-cadherin, increased cytoplasmic and nuclear ,-catenin and elevated transcription of ,-catenin target genes known to be involved in tumour progression and EMT. Inhibition of EGFR kinase activity in TamR cells reduced ,-catenin tyrosine phosphorylation, increased ,-catenin,E-cadherin association and promoted cell,cell adhesion. In such treated cells, the association of ,-catenin with Lef-1 and the transcription of c-myc, cyclin-D1, CD44 and COX-2 were also reduced. These results suggest that homotypic adhesion in tamoxifen-resistant breast cancer cells is dysfunctional due to EGFR-driven modulation of the phosphorylation status of ,-catenin and may contribute to an enhanced aggressive phenotype and transition towards a mesenchymal phenotype in vitro. © 2005 Wiley-Liss, Inc. [source] Nerve growth factor blocks thapsigargin-induced apoptosis at the level of the mitochondrion viaregulation of BimJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 6a 2008E. Szegezdi Abstract This study examined how the neurotrophin, nerve growth factor (NGF), protects PC12 cells against endoplasmic reticulum (ER) stress-induced apoptosis. ER stress was induced using thapsigargin (TG) that inhibits the sarcoplasmic/ER Ca2+ -ATPase pump (SERCA) and depletes ER Ca2+ stores. NGF pre-treatment inhibited translocation of Bax to the mitochondria, loss of mitochondrial transmembrane potential, cytochrome c release, activation of caspases (,3, ,7 and ,9) and apoptosis induction by TG. Notably, TG also caused a marked induction of Bimel mRNA and protein, and knockdown of Bim with siRNA protected cells against TG-induced apoptosis. NGF delayed the induction and increased the phosphorylation of Bimel. NGF-mediated protection was dependent on phosphatidylinositol-3 kinase (PI3K) signalling since all above apoptotic events, including expression and phosphorylation status of Bimel protein, could be reverted by the PI3K inhibitor LY294002. In contrast, NGF had no effect on the TG-mediated induction of the unfolded protein response (increased expression of Grp78, GADD34, splicing of XBP1 mRNA) or ER stress-associated pro-apoptotic responses (induction of C/EBP homologous protein [CHOP], induction and processing of caspase-12). These data indicate that NGF-mediated protection against ER stress-induced apoptosis occurs at the level of the mitochondria by regulating induction and activation of Bim and mitochondrial translocation of Bax. [source] PI3K/AKT regulates aggrecan gene expression by modulating Sox9 expression and activity in nucleus pulposus cells of the intervertebral discJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2009Chin-Chang Cheng The goal of the investigation was to test the hypothesis that the phosphoinositide-3 kinase (PI3K)/AKT signaling pathway regulates the expression of the major extracellular matrix component of the intervertebral disc, aggrecan, in nucleus pulposus cells. Primary rat nucleus pulposus cells were treated with PI3K inhibitor to measure changes in gene and protein expression. In addition, cells were transfected with various luciferase reporter plasmids to investigate mechanisms of regulation of aggrecan gene expression. We found that treatment of nucleus pulposus cells with a PI3K inhibitor, LY294002 resulted in decreased expression of aggrecan and a reduction in deposition of sulfated glycosaminoglycans. Moreover, pharmacological suppression or co-expression of dominant negative (DN)-PI3K or DN-AKT resulted in downregulation of aggrecan promoter activity. Expression of constitutively active (CA)-PI3K significantly induced aggrecan promoter activity. We observed that PI3K maintained Sox9 gene expression and activity: inhibition of PI3K/AKT resulted in decreased Sox9 expression, lowered promoter activity, and mediated a reduction in Sox9 transcriptional activity. PI3K effects were independent of phosphorylation status of C-terminus transactivation domain (TAD) of Sox9. Finally, we noted that in nucleus pulposus cells, PI3K signaling controlled transactivation of p300 (p300-TAD activity), an important transcriptional co-activator of Sox9. Results of these studies demonstrate for the first time that PI3K/AKT signaling controls aggrecan gene expression, in part by modulating Sox9 expression and activity in cells of the nucleus pulposus. J. Cell. Physiol. 221: 668,676, 2009. © 2009 Wiley-Liss, Inc. [source] Integrin ,3,1 interacts with I1PP2A/lanp and phosphatase PP1JOURNAL OF NEUROSCIENCE RESEARCH, Issue 8 2006Diana Mutz Abstract Integrin ,3,1 is a receptor for the extracellular matrix component laminin 5. To elucidate possible signaling pathways induced by integrin ,3,1, we looked for proteins that interact with the cytoplasmic part of the ,3A integrin subunit. We identified several multifunctional proteins by affinity chromatography and subsequent MALDI-TOF-MS and focused on the inhibitor 1 of serine/threonine phosphatase PP2A (I1PP2A, synonym: lanp) which also plays a role during the development of the mouse cerebellum. I1PP2A/lanp colocalizes with the ,3A integrin subunit in differentiated PC12 cells in the cell body and in neurites as well as in Purkinje cells of mouse cerebellum. Overexpression of GFP-I1PP2A/lanp in PC12 cells leads to markedly reduced neurite length on laminin 5 after induction with nerve growth factor. By affinity chromatography the protein phosphatase PP1 can also be identified as a ,3A/cyto-binding protein. PP1 and integrin ,3,1 can be pulled down by GST-I1PP2A/lanp from cell lysates of differentiated and undifferentiated PC12 cells. The phosphatase binds to the cytoplasmic membrane-proximal conserved GFFKR motif of the , integrin subunit, whereas I1PP2A/lanp requires a longer sequence for binding. PP1 but not PP2A is able to dephosphorylate precipitated integrin ,3,1 in vitro. Furthermore, PP1 releases phosphate from T1046 of phosphopeptides that mimic the phosphorylation consensus sequence in the cytoplasmic part of the ,3A integrin subunit. These data suggest that I1PP2A/lanp forms a complex with PP1 and the ,3A integrin subunit and might possibly regulate the phosphorylation status of integrin ,3,1 and/or integrin downstream targets. © 2006 Wiley-Liss, Inc. [source] Apple polyphenols diminish the phosphorylation of the epidermal growth factor receptor in HT29 colon carcinoma cellsMOLECULAR NUTRITION & FOOD RESEARCH (FORMERLY NAHRUNG/FOOD), Issue 5 2007Diana Fridrich Abstract Previously, we showed that an apple juice extract (AE) potently inhibits the protein tyrosine kinase (PTK) activity of the epidermal growth factor receptor (EGFR). In the present study, an apple pomace extract (APE) was found to exceed the EGFR inhibitory properties of AE in a cell-free system. The impact of the extracts on the phosphorylation status of the EGFR in intact cells (HT29) was sensitive to catalase, added to suppress the accumulation of hydrogen peroxide. In the absence of catalase, the formation of hydrogen peroxide was observed, achieving 1.1 ± 0.1 ,M (AE) and 1.5 ± 0.1 ,M (APE) after 45 min of incubation. In the presence of catalase, suppressing the hydrogen peroxide level to the solvent control, APE effectively suppressed EGFR phosphorylation, even exceeding the effects of AE. Both extracts inhibited the growth of HT29 cells, albeit the enhanced EGFR inhibitory properties of APE compared to AE were not reflected by a higher growth inhibitory potential. The results clearly show that the effect of apple extracts on the EGFR and cell growth are not simply artefacts of hydrogen peroxide formation. However, the formation of hydrogen peroxide has to be considered to modulate and/or mask cellular responses to apple extracts. [source] Tyrphostin-A47 inhibitable tyrosine phosphorylation of flagellar proteins is associated with distinct alteration of motility pattern in hamster spermatozoaMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 2 2006Daniel Mariappa Abstract To acquire fertilizing potential, mammalian spermatozoa must undergo capacitation and acrosome reaction. Our earlier work showed that pentoxifylline (0.45 mM), a sperm motility stimulant, induced an early onset of hamster sperm capacitation associated with tyrosine phosphorylation of 45,80 kDa proteins, localized to the mid-piece of the sperm tail. To assess the role of protein tyrosine phosphorylation in sperm capacitation, we used tyrphostin-A47 (TP-47), a specific protein tyrosine kinase inhibitor. The dose-dependent (0.1,0.5 mM) inhibition of tyrosine phosphorylation by TP-47 was associated with inhibition of hyperactivated motility and 0.5 mM TP-47-treated spermatozoa exhibited a distinct circular motility pattern. This was accompanied by hypo-tyrosine phosphorylation of 45,60 kDa proteins, localized to the principal piece of the intact-sperm and the outer dense fiber-like structures in detergent treated-sperm. Sperm kinematic analysis (by CASA) of spermatozoa, exhibiting circular motility (at 1st hr), showed lower values of straight line velocity, curvilinear velocity and average path velocity, compared to untreated controls. Other TP-47 analogues, tyrphostin-AG1478 and -AG1296, had no effect either on kinematic parameters or sperm protein tyrosine phosphorylation. These studies indicate that TP-47-induced circular motility of spermatozoa is compound-specific and that the tyrosine phosphorylation status of 45,60 kDa flagellum-localized proteins could be key regulators of sperm flagellar bending pattern, associated with the hyperactivation of hamster spermatozoa. Mol. Reprod. Dev. © 2005 Wiley-Liss, Inc. [source] Extracellular signal-regulated protein kinase is activated in cervical intraepithelial neoplasms but inactivated in invasive cervical carcinomaPATHOLOGY INTERNATIONAL, Issue 7 2006Keiko 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] Molecular Analysis of Brassinosteroid ActionPLANT BIOLOGY, Issue 3 2006C. Müssig Abstract: Brassinosteroids (BRs) are steroidal plant hormones with important regulatory roles in various physiological processes, including growth, xylem differentiation, disease resistance, and stress tolerance. Several components of the BR signal transduction pathway have been identified. The extracellular domains of receptor kinases such as BRI1 perceive BRs and transduce the signal via intracellular kinase domains. Within the cell further kinases and phosphatases determine the phosphorylation status of transcription factors such as BES1 and BZR1. These factors mediate major BR effects. Studies of BR-regulated genes shed light on the molecular mode of BR action. Genes encoding cell-wall-modifying enzymes, enzymes of the BR biosynthetic pathway, transcription factors, and proteins involved in primary and secondary metabolism are subject to BR-regulation. Gene expression data also point at interactions with other phytohormones and a role of BR in stress responses. This article gives a survey of the BR-signaling pathway. Two BR-responsive genes, OPR3 and EXO, are described in detail. [source] Identification of serine 205 as a site of phosphorylation on Pax3 in proliferating but not differentiating primary myoblastsPROTEIN SCIENCE, Issue 11 2008Patrick J. Miller Abstract Pax3, a member of the paired class homeodomain family of transcription factors, is essential for early skeletal muscle development. Previously, others and we have shown that the stability of Pax3 is regulated on a post-translational level. Evidence in the literature and from our laboratory suggests that phosphorylation, a common form of regulation, may play a role. However, at present, the sites of Pax3 phosphorylation are not known. We demonstrate here the first evidence that Pax3 exists as a phosphoprotein in proliferating mouse primary myoblasts. Using an in vitro kinase assay, deletion, and point mutant analysis, we conclusively identify Ser205 as a site of phosphorylation. The phosphorylation of Ser205 on endogenously expressed Pax3 was confirmed in vivo using antibodies specific for phosphorylation at Ser205. Finally, we demonstrate for the first time that the phosphorylation status of endogenous Pax3 changes rapidly upon the induction of myogenic differentiation. The presence of phosphorylation in a region of Pax3 important for mediating protein,protein interactions, and the fact that phosphorylation is lost upon induction of differentiation, allow for speculation on the biological relevance of phosphorylation. [source] A novel method for analyzing phosphoproteins using SELDI-TOF MS in combination with a series of recombinant proteinsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 14 2007Tetsuyuki Akashi Abstract A novel SELDI-TOF MS-based method for analyzing phosphoproteins was developed using a series of recombinant wild-type and mutant ribosomal P2 proteins. We demonstrated that the phosphorylation status of the overexpressed proteins in cells was easily and rapidly confirmed using this method. The ribosomal P2 protein contained two phosphorylation sites, which were sequentially phosphorylated in vivo. We also quantitatively detected the phosphoprotein by using SELDI-TOF MS. [source] Mammalian Sperm Energy Resources Management and Survival during Conservation in RefrigerationREPRODUCTION IN DOMESTIC ANIMALS, Issue 2006JE Rodriguez-Gil Contents The present review has as its main aim to present an overview regarding the mechanisms utilized by mammalian sperm to manage its intracellular energy levels. This management will strongly influence the sperm's ability to maintain its overall function during its entire life span. Thus, the precise knowledge of these mechanisms will be of the utmost interest to optimize the systems utilized to conserve mammalian sperm for a medium-to-long time-lapse. Briefly, utilization of hexoses as energy substrates by mammalian sperm is very finely regulated from the very first step of its metabolization. Furthermore, the equilibrium among the separate, monosaccharide metabolization pathways in mammalian sperm depends on many factors. This prevents the possibility to draw a general vision of sperm energy utilization, which explains the results of all mammalian species in all points of the sperm life-cycle. To complicate the matter further, there are separate energy phenotypes among mammalian spermatozoa. The precise knowledge of these phenotypes is of the greatest importance in order to optimize the design of new extenders for sperm conservation in refrigerated conditions. Moreover, sugars can act on sperm not only as passive metabolic substrates, but also as direct function activators through mechanisms like specific changes in the tyrosine phosphorylation status of distinct proteins. Finally, mammalian sperm utilizes non-glucidic substrates like citrate and lactate to obtain energy in a regular form. This utilization is also finely regulated and of importance to maintain overall sperm function. This implies that the exact proportion of glucidic and non-glucidic energy substrates could be very important to optimize the survival ability of these cells in conservation. [source] ORIGINAL RESEARCH: Phosphodiesterase Type 5 Regulation in the Penile Corpora CavernosaTHE JOURNAL OF SEXUAL MEDICINE, Issue S3 2009Ching-Shwun Lin PhD ABSTRACT Introduction., Penile detumescence depends on the hydrolysis of cyclic guanosine monophosphate (cGMP) by phosphodiesterase type 5 (PDE5). It is hoped that a review of publications relevant to the regulation of PDE5 in the penis will be helpful to both scientists and clinicians who are interested in the sciences of erectile function/dysfunction. Aims., The aim of this article is to comprehensively review the mechanisms by which PDE5 activity and expression in the penis are regulated. All published studies relevant to PDE5 regulation in the penis or penile cells will be reviewed. Methods., Entrez (PubMed) was used to search for publications relevant to the topics of this review. Keywords used in the searches included vascular, cavernous, penis, smooth muscle, signaling molecules, erection, priapism, and PDE5. Articles that are dedicated to the study of erectile function/dysfunction were prioritized for citation. Results., Regulation of PDE5 can occur at both protein and gene levels. At protein level, PDE5 is activated by phosphorylation and/or allosteric cGMP binding. Deactivation is carried out by protein phosphatase 1 and thus linked to the Rho-kinase signaling pathway. Cleavage of PDE5 into an inactive form has been shown as carried out by caspase-3. At the gene level, PDE5 expression is regulated at two alternative promoters, PDE5A and PDE5A2, both of which are positively regulated by cyclic adenosine monophosphate and cGMP. Downregulation of PDE5 has been observed in the penis of castrated animals; however, proof of androgen regulation of PDE5 gene requires examination of the smooth muscle content. Hyperoxia and hypoxia, respectively, regulate PDE5 expression positively and negatively. Hypoxic downregulation of PDE5 is a possible mechanism for the development of priapism. Conclusions., PDE5 can be regulated at protein and gene levels. In the penis, changes of PDE5 activity have been linked to its phosphorylation status, and downregulation of PDE5 expression has been associated with hypoxia. Lin CS. PDE5 regulation in the penile corpora nervosa. J Sex Med 2009;6(suppl 3):203,209. [source] Phosphorylation of inositol 1,4,5-triphosphate receptor 1 during in vitro maturation of porcine oocytesANIMAL SCIENCE JOURNAL, Issue 1 2010Junya ITO ABSTRACT During fertilization in mammalian species, a sperm-induced intracellular Ca2+ signal ([Ca2+]i) mediates both exit of meiosis and oocyte activation. Recently, we demonstrated in mouse oocytes that the phosphorylation levels of inositol 1,4,5 trisphosphate receptor type1 (IP3R1), the channel responsible for Ca2+ release and oscillations during fertilization, changed during maturation and fertilization. Therefore, we examined the expression and phosphorylation of IP3R1 during in vitro maturation of pig oocytes. Here, our present study shows that expression of IP3R1 protein did not change during maturation, although the phosphorylation status of the receptor, specifically at an MPM-2 epitope, did. We found that while at the beginning of maturation IP3R1 lacked MPM-2 immunoreactivity, it became MPM-2 reactive by 24 h and reached maximal reactivity by 36 h. Interestingly, the acquisition of MPM-2 reactivity coincided with the activation of p34cdc2 kinase and mitogen-activated protein kinase (MAPK), which are involved in meiotic progression. Following completion of maturation, inactivation of MAPK by U0126 did not affect IP3R1 phosphorylation, although inactivation of p34cdc2 kinase by roscovitine dramatically reduced IP3R1 phosphorylation. Neither inhibitor affected total expression of IP3R1. Altogether, our results show that IP3R1 undergoes dynamic phosphorylation during maturation and this might underlie the generation of oscillations at fertilization. [source] The role of retinoblastoma protein family in the control of germ cell proliferation, differentiation and survivalAPMIS, Issue 1 2003JORMA TOPPARI Retinoblastoma family proteins pRb, p107 and p130 are differentially expressed in the rat testis. They function in specific cell types during testicular development and spermatogenesis, participating in the control of proliferation, differentiation, and survival. Their expression levels and phosphorylation status are modulated during germ cell cycle progression and apoptosis. Hyperphosphorylated states and elevated levels of p107 are correlated with cell cycle progression, whereas hypophosphorylated states and reduced levels are associated with suppression of proliferation and apoptosis in germ cells and Leydig cells. These proteins may also serve as markers of cell cycle status of germ cells during spermatogenesis. [source] Dihydrosphingosine 1-phosphate has a potent antifibrotic effect in scleroderma fibroblasts via normalization of phosphatase and tensin homolog levelsARTHRITIS & RHEUMATISM, Issue 7 2010Shizhong Bu Objective Previous studies have revealed a phosphatase and tensin homolog (PTEN),dependent interaction between the sphingolipid agonist dihydrosphingosine 1-phosphate (dhS1P) and the transforming growth factor ,/Smad3 signaling pathway. This study was undertaken to examine responses of systemic sclerosis (SSc) fibroblasts to sphingosine 1-phosphate (S1P) and dhS1P and to gain further insight into the regulation of the S1P/dhS1P/PTEN pathway in SSc fibrosis. Methods Fibroblast cultures were established from skin biopsy samples obtained from patients with SSc and matched healthy controls. Western blotting and quantitative polymerase chain reaction were used to measure protein and messenger RNA levels, respectively. PTEN protein was examined in skin biopsy samples by immunohistochemistry. Results PTEN protein levels were low in SSc fibroblasts and correlated with elevated levels of collagen and phospho-Smad3 and reduced levels of matrix metalloproteinase 1 (MMP-1). Treatment with dhS1P restored PTEN levels and normalized collagen and MMP-1 expression, as well as Smad3 phosphorylation status in SSc fibroblasts. S1P was strongly profibrotic in SSc and control fibroblasts. Distribution of S1P receptor isoforms was altered in SSc fibroblasts, which had reduced levels of S1P receptor 1 and S1P receptor 2 and elevated levels of S1P receptor 3. Only depletion of S1P receptor 1 abrogated the effects of dhS1P and S1P in control dermal fibroblasts. In contrast, depletion of either S1P receptor 1 or S1P receptor 2 prevented the effects of S1P and dhS1P in SSc fibroblasts. Conclusion Our findings demonstrate that PTEN deficiency is a critical determinant of the profibrotic phenotype of SSc fibroblasts. The antifibrotic effect of dhS1P is mediated through normalization of PTEN expression, suggesting that dhS1P or its derivatives may be effective as therapeutic antifibrotic agents. The distribution and function of S1P receptors differ in SSc and healthy fibroblasts, suggesting that alteration in the sphingolipid signaling pathway may contribute to SSc fibrosis. [source] Hsp90 mediates insulin-like growth factor 1 and interleukin-1, signaling in an age-dependent manner in equine articular chondrocytesARTHRITIS & RHEUMATISM, Issue 7 2007Amber K. Boehm Objective Many metabolic processes in chondrocytes thought to contribute to age-related changes in the extracellular matrix are influenced by known roles of Hsp90. Age-related decreases in the level of Hsp90 have been documented in numerous cell types and could contribute to cartilage degeneration. The aim of this study was to investigate the roles of age and Hsp90 in insulin-like growth factor 1 (IGF-1) and interleukin-1, (IL-1,) signaling in chondrocytes. Methods Levels of Hsp90 messenger RNA (mRNA) and protein, with respect to age, were determined by quantitative real-time polymerase chain reaction (PCR) and Western blot analysis, respectively. The Hsp90 inhibitor geldanamycin (50 nM, 100 nM, or 500 nM) was used to assess age-related responses to Hsp90 with concurrent IGF-1 or IL-1, stimulation of chondrocytes. Quantitative real-time PCR was used to measure COL2A1 and matrix metalloproteinase 13 (MMP13) gene expression; Western blot analysis was performed to determine the phosphorylation status of p42/44 and Akt/protein kinase B. Results The effects of Hsp90 inhibition with geldanamycin were concentration dependent. Inhibition of Hsp90 with 100 nM or 500 nM geldanamycin blocked IGF-1,induced cell proliferation, Akt and p42/44 activation, and COL2A1 expression. Basal and IL-1,,induced up-regulation of MMP13 mRNA was blocked by all concentrations of geldanamycin tested. Gain-of-function assays with Hsp90 resulted in increased expression of MMP13 mRNA. Conclusion These results suggest that Hsp90 is involved in opposing signaling pathways of cartilage homeostasis, and that catabolic responses are more sensitive to Hsp90 inhibition than are anabolic responses. Further studies are needed to determine the role of Hsp90 inhibition in osteoarthritis in order to assess its potential as a therapeutic target. [source] THIS ARTICLE HAS BEEN RETRACTED Epigenetic regulator polycomb group protein complexes control cell fate and cancerCANCER SCIENCE, Issue 6 2008Rieko Kanno The chromatin-associated Polycomb group (PcG) proteins were first identified in genetic screens for homeotic transformations in Drosophila melanogaster. Besides body patterning, members of the PcG are now known to regulate epigenetic cellular memory, stem cell self-renewal, and cancer development. Here, we discuss the multifarious functions of the PcG family, isoforms of protein complexes, and its enzymatic activities, for example histone methylation, links to DNA methylation, its phosphorylation status, H2A mono-ubiquitination, SUMOylation, and links to non-coding RNA. We also discuss the function of cytosolic PcG complexes as a regulator of receptor-induced actin polymerization and proliferation in a methylation-dependent manner. We propose that the functional versatility of PcG protein complexes contributed significantly to the complexity of heritable gene repression mechanisms, signal transduction, and cell proliferation in cancer development. (Cancer Sci 2008; 99: 1077,1084) [source] | ||||||||||||||||