http://www.biochemj.org Biochemical Journal RSS feed -- BJ Signal Immediate Publications 0264-6021 1470-8728 Biochemical Journal http://www.biochemj.org/images/BJ_Name.gif http://www.biochemj.org http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20120035 cat/K_m values) of optimal peptidyl substrates are in the same order (10³ – 10⁴ M^-1 s^-1) as for a putative target protein CYLD. Thus, MALT1 has many similarities to caspase 8, even cleaving the putative target protein CYLD with comparable efficiencies, but with diametrically opposite primary substrate specificity.]]> J Hachmann, S J Snipas, B J van Raam, E M Cancino, E J Houlihan, M Poreba, P Kasperkiewicz, M Drag, G S. Salvesen 2012-02-06T15:29:47Z doi:10.1042/BJ20120035 Portland Press Limited 2012-02-06 BJ Signal http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111644 4B) activates dioxygen at the heme site by electron transfer. The subsequent events are poorly understood due to the lack of stable intermediates. By analogy with cytochromes P450, a heme-iron oxo species may be formed, or direct reaction between a heme-peroxy intermediate and substrate may occur. The two steps may also occur via different mechanisms. Here we analyse the two reaction steps using the G586S mutant of nNOS, which introduces an additional H-bond in the active site and provides an additional proton source. In the mutant, H₄B activates dioxygen as in the wild-type enzyme, but an interesting intermediate heme species is then observed. This may be a stabilized form of the active oxygenating species. The mutant is able to perform step 2 (reaction with NOHA), but not step 1 (with L-Arg) indicating that the extra H-bond enables it to discriminate between the two monooxygenation steps. This implies that the two steps follow different chemical mechanisms.]]> D Papale, C Bruckmann, B Gazur, C S Miles, C G Mowat, S Daff 2012-02-03T11:49:02Z doi:10.1042/BJ20111644 Portland Press Limited 2012-02-03 BJ ChemBio http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111584 M Kang, S Choi, S Jeong, S Lee, T Kwak, H Kim, O Jung, M Lee, Y Ko, J Ryu, Y Choi, D Jeong, H Lee, S Ye, S Kim, J Lee 2012-01-31T14:13:54Z doi:10.1042/BJ20111584 Portland Press Limited 2012-01-31 BJ Signal http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111839 in vitro kinase reaction. The phosphorylation sites were identified by mass spectrometry and the major phosphorylation site was found to be at the highly conserved S1366 residue. A phospho-specific antibody was generated that can specifically recognize ROCKII S1366 phosphorylation. We found that the extent of S1366 phosphorylation of endogenous ROCKII is correlated with that of myosin light chain phosphorylation in cells in response to RhoA stimulation, showing that S1366 phosphorylation reflects its kinase activity. In addition, ROCKII S1366 phosphorylation could be detected in human breast tumors by immunohistochemical staining. Our study provides a new approach for revealing ROCKII activation status by directly probing ROCKII S1366 phosphorylation in cells or tissues.]]> H Chuang, C Yang, Y Tsay, C Hsu, L Tseng, Z Chang, H Lee 2012-01-24T14:38:48Z doi:10.1042/BJ20111839 Portland Press Limited 2012-01-24 BJ Cell http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111052 2 plays an important role in the modulation of the immune response and the inflammatory process. In this study, we describe a PGE₂ positive feedback for Cyclooxygenase (COX) -2 and microsomal PGE Synthase (mPGES) -1 expression in the macrophage cell line RAW 264.7. Our results show that PGE₂ induces COX-2 and mPGES-1 expression, an effect mimicked by dibutyryl-cAMP (dbcAMP) or Forskolin. Furthermore, cAMP signaling pathway cooperates with LPS in the induction of COX-2 and mPGES-1 transcriptional activation. Analysis of the involvement of EP receptors showed that incubation with EP2 agonists up-regulated both COX-2 and mPGES-1 mRNA levels. Moreover, EP2 receptor over expression enhanced the transcriptional activation of COX-2 and mPGES-1 promoters, being this induction abolished by the PKA inhibitor, H89. Activation of PGE₂/EP2/PKA signaling pathway induced the phosphorylation of the cAMP response element-binding protein (CREB) in macrophages and stimulated the specific binding of this transcription factor to COX-2 and mPGES-1 promoters. Deletion or mutation of potential CRE sites in both promoters diminished their transcriptional activity. In summary, our data demonstrate that activation of PKA/CREB signaling through the EP2 receptor by PGE₂ plays a key role in the expression of COX-2 and mPGES-1 in activated macrophages.]]> M D. Díaz-Muñoz, I C. Osma-García, M Fresno, M A. Iñiguez 2012-01-23T14:40:59Z doi:10.1042/BJ20111052 Portland Press Limited 2012-01-23 BJ Gene http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111351 A Hafner, N Obermajer, J Kos 2012-01-19T11:42:24Z doi:10.1042/BJ20111351 Portland Press Limited 2012-01-19 BJ Signal http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111589 2) resides in raft domains and DHA can alter the size of rafts, we hypothesized that PI(4,5)P₂ and downstream actin remodeling are perturbed by the incorporation of n-3 PUFA into membranes, resulting in suppressed T cell activation. CD4⁺ T cells isolated from Fat-1 transgenic mice (membranes enriched in n-3 PUFA) exhibited a 50% decrease in PI(4,5)P₂. Upon activation by plate bound anti-CD3/anti-CD28 or PMA/ionomycin, Fat-1 CD4⁺ T cells failed to metabolize PI(4,5)P₂. Furthermore, actin remodeling, failed to initiate in Fat-1 CD4⁺ T cells upon stimulation; however, the defect was reversed by incubation with exogenous PI(4,5)P₂. When Fat-1 CD4⁺ T cells were stimulated with anti-CD3/anti-CD28 coated beads, the Wiskott-Aldrich syndrome protein (WASP) failed to translocate to the immunological synapse. The suppressive phenotype, consisting of defects in PI(4,5)P₂ metabolism and actin remodeling, were recapitulated in CD4⁺ T cells isolated from mice fed a 4% DHA triglyceride-enriched diet. Collectively, these data demonstrate that DHA alters PI(4,5)P₂ in CD4⁺ T cells, thereby suppressing the recruitment of WASP to the IS, and impairing actin remodeling in CD4⁺ T cells.]]> T Y Hou, J M Monk, Y Fan, R Barhoumi, Y Q Chen, G M Rivera, D N McMurray, R S Chapkin 2012-01-18T11:24:15Z doi:10.1042/BJ20111589 Portland Press Limited 2012-01-18 BJ Cell http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111792 in vitro. A MBP kinase assay of the immunoprecipitation complex indicated that OsMPK3 and OsMPK6 were activated in response to a moderately low temperature (12°C) but a severely low temperature (4°C) in rice seedlings. A constitutively active form of OsMKK6, OsMKK6DD, showed elevated phosphorylation activity against OsMPK3 and OsMPK6 in vitro. OsMPK3, but not OsMPK6, was constitutively activated in transgenic plants overexpressing OsMKK6DD, indicating that OsMPK3 is an in vivo target of OsMKK6. Enhanced chilling tolerance was observed in the transgenic plants overexpressing OsMKK6DD. Taken together, our data suggest that OsMKK6 and OsMPK3 constitute a moderately low-temperature signaling pathway and regulate cold stress tolerance in rice.]]> G Xie, H Kato, R Imai 2012-01-16T14:27:38Z doi:10.1042/BJ20111792 Portland Press Limited 2012-01-16 BJ Plant http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20112078 3 and Gbg subunits and inhibited by PKA. Here, we show that Protein Phosphatase 1a (PP1a) binds P‑Rex1 through an RVxF-type docking motif. PP1a activates P‑Rex1 directly in vitro, both independently of and additively to PIP₃ and Gbg. PP1a also substantially activates P‑Rex1 in vivo, both in basal and PDGF- or LPA-stimulated cells. The phosphatase activity of PP1a is required for P‑Rex1 activation. PP1b, a close homologue of PP1a, is also able to activate P‑Rex1, but less effectively. PP1a stimulates P-Rex1-mediated, Rac-dependent changes in endothelial cell morphology. Mass spectrometric analysis of wild-type P‑Rex1 and a PP1a-binding deficient mutant revealed that endogenous PP1a dephosphorylates P‑Rex1 on at least three residues, S834, S1001 and S1165. Site-directed mutagenesis of S1165 to alanine caused activation of P-Rex1 to a similar degree as did PP1a, confirming S1165 as a dephosphorylation site important in regulating P‑Rex1 Rac-GEF activity. In summary, we have identified a novel mechanism for direct activation of P‑Rex1 through PP1a-dependent dephosphorylation.]]> M A Barber, A Hendrickx, M Beullens, H Ceulemans, D Oxley, S Thelen, M Thelen, M Bollen, H C E Welch 2012-01-16T12:34:24Z doi:10.1042/BJ20112078 Portland Press Limited 2012-01-16 BJ Signal http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20112026 L Bultot, B Guigas, A Von Wilamowitz-Moellendorff, L Maisin, D Vertommen, N Hussain, M Beullens, J J Guinovart, M Foretz, B Viollet, K Sakamoto, L Hue, M H Rider 2012-01-11T12:10:57Z doi:10.1042/BJ20112026 Portland Press Limited 2012-01-11 BJ Signal http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111700 in vivo. Strikingly, PKD2 loss caused increases in thymic output, lymphadenopathy and splenomegaly in TCR transgenic mice. The precise magnitude and timing of PKD2 activation during T cell development is thus critical to regulate thymic homeostasis. PKD2 null T cells that exit the thymus have a normal transcriptome but show a limited and abnormal transcriptional response to antigen. Transcriptional profiling reveals the full consequences of PKD2 loss and maps in detail the selective but critical function for PKD2 in signaling by a/b mature TCR complexes in peripheral T cells.]]> M N Navarro, L V Sinclair, C Feijoo-Carnero, R Clarke, S A Matthews, D A Cantrell 2012-01-10T16:33:45Z doi:10.1042/BJ20111700 Portland Press Limited 2012-01-10 BJ Signal http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20112000 178-), induction of Ser-/Thr-protein kinase activity and nuclear localisation. We identified an alternative ‘non-canonical’ pathway of MST3 activation (regulated primarily through dephosphorylation) which may also be applicable to other GCKIII (and GCKVI) subgroup members. In the basal state, inactive MST3 co-immunoprecipitated with the Golgi protein, GOLGA2/gm130. Activation of MST3 by calyculin A (a protein Ser-/Thr- phosphatase 1/2A inhibitor) stimulated (auto)phosphorylation of MST3(Thr¹⁷⁸-) in the catalytic domain with essentially simultaneous cis-autophosphorylation of MST3(Thr³²⁸-) in the regulatory domain, an event also requiring the MST3(341-376) sequence which acts as a putative docking domain. MST3(Thr¹⁷⁸-) phosphorylation increased MST3 kinase activity but this activity was independent of MST3(Thr³²⁸-) phosphorylation. Interestingly, MST3(Thr³²⁸-) lies immediately C-terminal to a STRAD pseudokinase-like site recently identified as being involved in binding of GCKIII/GCKVI members to MO25 scaffolding proteins. MST3(Thr¹⁷⁸- /Thr³²⁸-) phosphorylation was concurrent with dissociation of MST3 from GOLGA2/gm130 and association of MST3 with MO25, and MST3(Thr³²⁸-) phosphorylation was necessary for formation of the activated MST3-MO25 holocomplex.]]> S J Fuller, L J McGuffin, A K Marshall, A Giraldo, S Pikkarainen, A Clerk, P H Sugden 2012-01-09T16:14:27Z doi:10.1042/BJ20112000 Portland Press Limited 2012-01-09 BJ Signal http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111817 J Mittag, T Behrends, K Nordström, J Anselmo, B Vennström, L Schomburg 2012-01-06T11:15:00Z doi:10.1042/BJ20111817 Portland Press Limited 2012-01-06 BJ Gene http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20110921 K J. Wilson, C P. Mill, R M. Gallo, E M. Cameron, H VanBrocklin, J Settleman, D J. Riese II 2012-01-05T10:31:15Z doi:10.1042/BJ20110921 Portland Press Limited 2012-01-05 BJ Signal http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111530 2+ and calmodulin. eEF2K has previously been shown to undergo autophosphorylation, the stoichiometry of which suggested the existence of multiple sites. Here we identified several autophosphorylation sites including Thr-348, Thr-353, Ser-366 and Ser-445, all of which are highly conserved among vertebrate eEF2Ks. We also identified a number of other sites, including Ser-78, a known site of phosphorylation, and others, some of which are less well conserved. None of the sites lies in the catalytic domain, but three affect eEF2K activity. Mutation of Ser-78, Thr-348 and Ser-366 to a non-phosphorylatable alanine residue decreased eEF2K activity. Phosphorylation of Thr-348 was detected by immunoblotting after transfecting wild-type eEF2K into HEK 293 cells, but not after transfection with a kinase-inactive construct confirming that it is indeed a site of autophosphorylation. Thr-348 appears to be constitutively autophosphorylated in vitro. Interestingly, other recent data suggest that the corresponding residue in other a-kinases is also autophosphorylated and contributes to the activation of these enzymes (Crawley et al., J. Biol. Chem. 2011, 286, 2607-2616). Ser-366 phosphorylation was also detected in intact cells, but was still observed in the kinase-inactive construct, demonstrating that this site is not only phosphorylated autocatalytically but also in trans by other kinases.]]> S Pyr Dit Ruys, X Wang, E M Smith, G Herinckx, N Hussain, M H Rider, D Vertommen, C Gregory Proud 2012-01-04T15:16:12Z doi:10.1042/BJ20111530 Portland Press Limited 2012-01-04 BJ Signal http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111035 Saccharomyces cerevisiae, was investigated using the phosphatidylcholine analogue [¹²⁵I]TID-PC/16, which was incorporated into mixtures of dimyristoyl-phosphatidylcholine and the nonionic detergent C₁₂E₁₀. We found no differences between the recombinant PMCA4 and PMCA purified from erythrocytes (ePMCA). However, titration of the half-maximal activation by Ca²⁺-calmodulin of PMCA2 showed 30 times higher affinity than PMCA4. PMCA2 exhibited a lower level labeling in the autoinhibited conformation relative to PMCA4 indicating that the lower autoinhibition was correlated with a lower exposure to lipids in the autoinhibited state. Analysis of the lipid:protein stoichiometry show that the lipid annulus of PMCA varies: (i) in accordance to the conformational state of the enzyme and (ii) depending on the different isoforms of PMCA. PMCA2 during Ca²⁺ transport changes its conformation to a lesser extent than PMCA4, an isoform more sensitive to modulation by calmodulin and acidic phospholipids. This is the first demonstration of a dynamic behavior of annular lipids and PMCA.]]> I C Mangialavori, G Corradi, D E Rinaldi, M de la Fuente, H P Adamo, J F.C. Rossi 2012-01-04T12:04:22Z doi:10.1042/BJ20111035 Portland Press Limited 2012-01-04 BJ Structure http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111280 C Arden, S J Tudhope, J L Petrie, Z H Al-Oanzi, K S Cullen, A J. Lange, H C Towle, L Agius 2012-01-03T16:07:36Z doi:10.1042/BJ20111280 Portland Press Limited 2012-01-03 BJ Metabolism http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111867 2+-dependent, disulfide-rich, complement-like repeats (CR). While lysine residues from the ligands have been implicated in binding, there has been no quantitation of the energetic contributions of such interactions and hence of their relative importance in overall affinity, or of the ability of arginine or histidine to bind. We have used four representative CR domains from the principal ligand cluster of LRP to determine the energetics of interaction with well defined small ligands, that include methyl esters of lysine, arginine, histidine, and aspartic acid, as well as N-terminally blocked lysine methyl ester. We found that, not only lysine, but also arginine and histidine bound well, and when present with an additional proximal positive charge, accounted for about half the total binding energy of a protein ligand such as PAI-1. Two such sets of interactions, one to each of two CR domains could thus account for almost all the necessary binding energy of a real ligand such as PAI-1. For the CR domains, a central aspartate in the sequence DxDxD tightens Kd by ~20-fold, while DxDDD is no more effective. Together these findings establish the rules for determining binding specificity for protein ligands to LRP and to other LDL receptor family members.]]> P GW Gettins, K Dolmer 2011-12-20T11:59:53Z doi:10.1042/BJ20111867 Portland Press Limited 2011-12-20 BJ Structure http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111647 D values in the nanomolar range. This peptide was tested in human keratinocyte cultures and reduced the phosphorylation of STAT1 and the expression levels of the Interferon Regulatory Factor-1 (IRF-1).]]> N Doti, P L. Scognamiglio, S Madonna, C Scarponi, M Ruvo, G Perretta, C Albanesi, D Marasco 2011-12-14T12:37:44Z doi:10.1042/BJ20111647 Portland Press Limited 2011-12-14 BJ ChemBio http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20112037 in vitro and in vivo, and this effect is dependent on intact signaling via phosphatidylinositol 3-kinase (PI3K), but not protein kinase B (PKB). To test the hypothesis that insulin activates IKKβ as a means of negative feedback, we employed a variety of experimental approaches. Firstly, pharmacological inhibition of IKKβ via BMS-345541 did not potentiate insulin-induced IRS1 tyrosine phosphorylation, PKB phosphorylation or 2-deoxyglucose uptake in differentiated 3T3-L1 adipocytes. BMS-345541 did not prevent insulin-induced IRS1 serine phosphorylation on known IKKβ target sites. Secondly, adenoviral-mediated over-expression of wild type (WT) IKKβ in differentiated 3T3-L1 adipocytes did not suppress insulin-stimulated 2-deoxyglucose uptake, insulin receptor substrate 1 (IRS1) tyrosine phosphorylation, IRS1 association with the p85 regulatory subunit of PI3K or PKB phosphorylation. Thirdly, insulin signaling was not potentiated in mouse embryo fibroblasts lacking IKKβ (Ikkβ^-/- MEF). Finally, insulin treatment of 3T3-L1 adipocytes did not promote the recruitment of IKKβ to IRS1, supporting our data that IKKβ, while activated by insulin, does not promote direct serine phosphorylation of IRS1 and does not contribute to the feedback inhibition of the insulin-signaling cascade.]]> G I Lancaster, B Skiba, C Yang, H T Nicholls, K G Langley, M Chan, C R Bruce, G W Rewcastle, P R Shepherd, M Karin, M A. Febbraio 2011-12-14T12:16:28Z doi:10.1042/BJ20112037 Portland Press Limited 2011-12-14 BJ Signal http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111341 Gaussia luciferase fusions to investigate dimerization of CXCL12 secreted from mammalian cells. By column chromatography and Gaussia luciferase complementation, we established that CXCL12 was secreted from mammalian cells as both monomers and dimers. Secreted CXCL12 also formed homodimers in the extracellular space. Monomeric CXCL12 preferentially activated CXCR4 signaling through Gαi and AKT, while dimeric CXCL12 more effectively promoted recruitment of β-arrestin 2 to CXCR4 and chemotaxis of CXCR4-expressing breast cancer cells. We also showed that CXCR7 preferentially sequestered monomeric CXCL12 from the extracellular space and had minimal effects on dimeric CXCL12 in cell-based assays and an orthotopic tumor xenograft model of human breast cancer. These studies establish that CXCL12 secreted from mammalian cells forms homodimers under physiologic conditions. Since monomeric and dimeric CXCL12 have distinct effects on cell signaling and function, our results have important implications for ongoing efforts to target CXCL12 pathways for therapy.]]> P Ray, S A Lewin, L Anne Mihalko, S Lesher-Perez, S Takayama, K E Luker, G D Luker 2011-12-06T11:29:39Z doi:10.1042/BJ20111341 Portland Press Limited 2011-12-06 BJ Signal http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111322 Synopsis Aldo-keto reductase1B10 (AKR1B10) is overexpressed in live and lung cancer and plays a critical role in tumor development and progression through promoting lipogenesis and eliminating cytotoxic carbonyls. AKR1B10 is a secretory protein and potential tumor marker. However, little is known about the regulatory mechanism of AKR1B10 expression. This study showed that AKR1B10 is induced by mitogens epidermal growth factor (EGF) and insulin through the activator protein-1 (AP-1) signaling pathway. In human hepatocellular carcinoma cells (HepG2 and Hep3B), EGF (50ng/ml) and insulin (10nM) stimulated endogenous AKR1B10 expression and promoter activity. In the AKR1B10 promoter, a putative AP-1 element was found at -222 to -212bp. Deletion or mutations of this AP-1 element abrogated the basal promoter activity and response to EGF and AP-1 proteins. This AP-1 element bound to nuclear proteins extracted from HepG2 cells, and this binding was stimulated by EGF and insulin in a dose-dependent manner. Chromatin immunoprecipitation showed that AP-1 proteins c-Fos and c-Jun were predominant factors bound to the AP-1 consensus, followed by JunD and then JunB. The same order was followed in the stimulation of endogenous AKR1B10 expression by AP-1 proteins. Furthermore, c-Fos shRNA and AP-1 inhibitors/antagonists (U0126 and Tanshinone IIA) inhibited the endogenous AKR1B10 expression and promoter activity in HepG2 cells cultured in vitro or inoculated subcutaneously in nude mice. U0126 also inhibited AKR1B10 expression induced by EGF. Together these data suggest that AKR1B10 is upregulated by EGF and insulin through the AP-1 mitogenic signaling and may be implicated in hepatocarcinogenesis.]]> Z Liu, R Yan, A Al-salman, Y Shen, Y Bu, J Ma, D Luo, C Huang, Y Jiang, A Wilber, Y Mo, M Huang, Y Zhao, D Cao 2011-12-02T16:18:34Z doi:10.1042/BJ20111322 Portland Press Limited 2011-12-02 BJ Signal http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111466 Saccharomyces cerevisiae Hal3 protein is a moonlighting protein, able to function both as an inhibitory subunit of the Ppz1 protein phosphatase and as a constituent protomer of an unprecedented heterotrimeric phosphopantothenoylcysteine decarboxylase (PPCDC), the third enzyme of the CoA biosynthetic pathway. We initiated here the dissection of the structural elements required for both disparate cellular tasks by using a combination of biochemical and genetic approaches. We show that the conserved Hal3 core (the PPCDC domain) is necessary for both functions, as determined by in vitro and in vivo assays. The Hal3 N-terminal domain is not functional by itself, although in vitro experiments indicate that when this domain is combined with the core it has a relevant function in Hal3’s heteromeric PPCDC activity. Both the N-terminal and the acidic C-terminal domain also appear to be important for Hal3’s Ppz1 regulatory function, although our results indicate that the C-terminal domain fulfils the key role in this regard. Finally, we show that the introduction of two key Asn and Cys residues, essential for monofunctional PPCDC activity but absent in Hal3, is not sufficient to convert it to such a homomeric PPCDC, and that additional modifications of Hal3’s PPCDC domain aimed at increasing its resemblance to known PPCDC also fails to introduce this activity. This suggests that Hal3 has undergone significant evolutionary drift from ancestral PPCDC proteins. Taken together, our work highlights specific structural determinants that could be exploited for full understanding of Hal3’s cellular functions.]]> J Abrie, A González, E Strauss, J Ariño 2011-11-29T15:03:13Z doi:10.1042/BJ20111466 Portland Press Limited 2011-11-29 BJ Signal http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20100993 N Tachi, Y Hashimoto, M Matsuoka 2011-11-24T15:29:46Z doi:10.1042/BJ20100993 Portland Press Limited 2011-11-24 BJ Disease http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111418 2), a key modulator of glycolysis and gluconeogenesis. PFKFB3 gene is extensively involved in cell proliferation due to its key role in carbohydrate metabolism. Here we analyse its mechanism of regulation by progestins in breast cancer cells. We report that exposure of T47D cells to synthetic progestins (ORG2058 or Norgestrel) leads to a rapid increase in Fru-2,6-P₂ concentration. Western blot results are compatible with a short-term activation due to PFKFB3 isoenzyme phosphorylation and a long-term sustained action due to increased PFKFB3 protein levels. Transient transfection of T47D cells with deleted gene promoter constructs allowed us to identify a progesterone regulatory element (PRE) to which progesterone receptor binds and thus transactivates PFKFB3 gene transcription. Progesterone Receptor (PR) expression, in the PR-negative cell line (MDA-MB-231), induces endogen PFKFB3 expression in response to Norgestrel. Direct Binding of PR to the PRE box (-3490 nt) was confirmed by ChIP experiments. A dual mechanism affecting PFKFB3 protein and gene regulation operates in order to assure glycolysis in breast cancer cells. An immediate early response through the ERK-RSK pathway leading to phosphorylation of PFKFB3 on Ser 461 is followed by activation of mRNA transcription via cis-acting sequences on PFKFB3 promoter.]]> L Novellasdemunt, M Obach, L Millán-Ariño, A Manzano, F Ventura, J Rosa, A Jordan, A Navarro-Sabate, R Bartrons 2011-11-24T14:08:00Z doi:10.1042/BJ20111418 Portland Press Limited 2011-11-24 BJ Metabolism http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111005 Porphyrias are diseases caused by partial deficiencies of haem biosynthesis enzymes. Acute porphyrias are characterized by a neuropsychiatric syndrome with intermittent induction of hepatic δ-aminolevulinate synthase 1 (ALAS-1), first and rate-limiting enzyme of haem pathway. Porphyria acute attacks are usually treated with glucose administration, its effect is apparently related to its ability of inhibiting ALAS-1 by modulating insulin plasma levels. It was shown that insulin blunts hepatocytes ALAS-1 induction, by disrupting the interaction of the Forkhead box O1 (FOXO1) and the proliferator-activated receptor γ coactivator 1α (PGC-1α). We evaluated the expression of ALAS-1 in a murine model of diabetes and determined the effects of the insulinomimetic vanadate, on the enzyme regulation to evaluate its potential for the treatment of porphyria acute attacks. Both ALAS-1 mRNA and protein content were induced in diabetic animals, accompanied by decreased Akt phosphorylation and increased nuclear FOXO1, PGC-1α and FOXO1-PGC-1α complex. Vanadate reversed ALAS-1 induction with a concomitant PI3K/Akt pathway activation and subsequent reduction of nuclear FOXO1, PGC-1α and FOXO1-PGC-1α complex levels. These finding support that the FOXO1-PGC-1α complex is involved in the control of ALAS-1 expression and further suggest that a vanadate-based therapy could be beneficial for the treatment of porphyria acute attacks. ]]> L Maria Oliveri, C Davio, A María del Cármen Batlle, E Noemi Gerez 2011-11-10T09:13:02Z doi:10.1042/BJ20111005 Portland Press Limited 2011-11-10 BJ Metabolism http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20110282 in vitro experiments, while in vivo animal experiments also demonstrated that mice treated with MAPK/ERK or JNK-specific inhibitors could decrease intra-tumor MMP-7 expression, delay tumor growth, and extend the survival of the mice. In conclusion, mesothelin enhances ovarian cancer invasion by MMP-7 expression through the MAPK/ERK and JNK signal transduction pathways. Blocking the mesothelin-related pathway can be a potential strategy for inhibiting the growth of ovarian cancer.]]> M Chang, C Chen, P Chen, Y Chiang, Y Chen, T Mao, H Lin, W Lin Chiang, W Cheng 2011-10-14T13:22:35Z doi:10.1042/BJ20110282 Portland Press Limited 2011-10-14 BJ Disease