renal-capsule-xenografting-subcutaneous-pellet-implantation-for

Renal Capsule Xenografting and Subcutaneous Pellet Implantation for the Evaluation of Prostate Carcinogenesis and Benign Prostatic Hyperplasia

New therapies for two common prostate diseases,  prostate cancer (PrCa) and benign prostatic hyperplasia (BPH), depend  critically on experiments ...

University of Wisconsin-Madison

Biliverdin IXalpha reductase (BVR) catalyzes reduction of the HO activity product, biliverdin, to bilirubin. hBVR is a serine/threonine kinase that contains a bZip domain. Presently, regulation of gene expression by hBVR was examined. 293A cells were infected with adenovirus-doxycycline (Ad-Dox)-inducible hBVR cDNA. High level expression of hBVR was determined at mRNA, protein, and activity levels 8 h after induction. Cell signal transduction microarray analysis of cells infected with expression or with the control Ad-inverted (INV)-hBVR vector identified ATF-2 among several up-regulated genes. ATF-2 is a bZip transcription factor for activation of cAMP response element (CRE) and a dimeric partner to c-jun in MAPK pathway that regulates the stress protein, HO-1, expression. Northern and Western blot analyses showed increases of approximately 10-fold in ATF-2 mRNA and protein at 16 and 24 h after Dox addition. Ad-INV-hBVR did not effect ATF-2 expression. In hBVR-infected cells, levels of HO-1 mRNA and protein were increased. In vitro translated hBVR and nuclear extract containing hBVR in gel mobility-shift assay bound to AP-1 sites in the ATF-2 promoter region and to an oligonucleotide containing the CRE site. Both bindings could be competed out by excess unlabeled probe; in the presence of hBVR antibody, they displayed shifted bands. Co-transfection of hBVR with ATF-2 or c-jun promoters caused a severalfold increase in luciferase activity. hBVR modulation of ATF-2 and HO-1 expression suggests it has a potential role in regulation of AP-1 and cAMP-regulated genes and a role in cell signaling. We propose that increased expression of the protein can be used to alter the gene expression profile in the cell.

Biliverdin reductase, a novel regulator for induction of activating transcription factor-2 and heme oxygenase-1.

R&eacute;ductase de IXalpha de biliverdine (BVR) catalyse la r&eacute;duction du produit activit&eacute; HO, biliverdine, en bilirubine. hBVR est une s&eacute;rine/thr&eacute;onine kinase qui contient un domaine bZip. Actuellement, la r&eacute;gulation de l&#39;expression de g&egrave;ne de hBVR a &eacute;t&eacute; examin&eacute;e. 293 A cellules ont &eacute;t&eacute; infect&eacute;es par l&#39;ad&eacute;novirus-doxycycline (Ad-Dox)-cDNA hBVR inductible. Haut niveau d&#39;expression de hBVR a &eacute;t&eacute; d&eacute;termin&eacute; au niveau de l&#39;ARNm, prot&eacute;ines et l&#39;activit&eacute; 8 h apr&egrave;s l&#39;induction. Cellule analyse de microarray de transduction de signal de cellules infect&eacute;es avec expression ou avec la commande Ad-invers&eacute; (INV)-vecteur de hBVR identifi&eacute; ATF-2 parmi plusieurs g&egrave;nes de r&eacute;gulation. ATF-2 est un facteur de transcription bZip pour l&#39;activation de l&#39;&eacute;l&eacute;ment de r&eacute;ponse de l&#39;AMPc (CRE) et un partenaire dim&egrave;re &agrave; c-jun, en voie MAPK qui r&eacute;gule les prot&eacute;ines de stress, HO-1, expression. Nord et ouest blot analyses montr&eacute;s des augmentations d&#39;environ 10 fois dans ATF-2 ARNm et de prot&eacute;ines &agrave; 16 et 24 h apr&egrave;s addition de Dox. Ad-INV-hBVR n&#39;avait pas entra&icirc;n&eacute; expression ATF-2. Dans les cellules infect&eacute;es par le hBVR, les niveaux d&#39;HO-1 ARNm et de prot&eacute;ines ont &eacute;t&eacute; augment&eacute;s. HBVR traduit in vitro et extrait nucl&eacute;aire contenant hBVR dans l&#39;analyse de la mobilit&eacute;-d&eacute;calage de gel li&eacute; aux sites de l&#39;AP-1 dans la r&eacute;gion promotrice de ATF-2 et d&#39;un oligonucl&eacute;otide contenant le site de la CRE. Deux liaisons pourraient en comp&eacute;tition hors de la sonde en exc&egrave;s sans &eacute;tiquette ; en pr&eacute;sence d&#39;anticorps hBVR, ils affichent des bandes d&eacute;cal&eacute;es. Co-transfection de hBVR avec des promoteurs de l&#39;ATF-2 ou c-jun a provoqu&eacute; une augmentation de plusieurs fois dans l&#39;activit&eacute; de la lucif&eacute;rase. hBVR modulation de l&#39;expression ATF-2 et HO-1 sugg&egrave;re elle a un r&ocirc;le potentiel dans le r&egrave;glement d&#39;AP-1 et de g&egrave;nes r&eacute;gul&eacute;s par le cAMP et un r&ocirc;le dans la signalisation cellulaire. Nous proposons que l&#39;augmentation de l&#39;expression de la prot&eacute;ine peut servir &agrave; modifier le profil d&#39;expression de g&egrave;ne dans la cellule.

R&eacute;ductase de biliverdine, un r&eacute;gulateur de roman pour l&#39;induction d&#39;activer la transcription factor-2 et l&#39;h&egrave;me oxyg&eacute;nase-1.

Riduttasi di Biliverdin IXalpha (BVR) catalizza la riduzione del prodotto attivit&agrave; HO, Biliverdina a bilirubina. hBVR &egrave; una serina/treonina chinasi che contiene un dominio bZip. Attualmente, la regolazione dell&#39;espressione genica da hBVR &egrave; stato esaminato. 293A cellule sono state infettate con adenovirus-doxiciclina (annuncio-Dox)-inducible hBVR cDNA. Espressione di alto livello di hBVR era determinato a livelli di mRNA e proteine attivit&agrave; 8 h dopo l&#39;induzione. Cella di analisi microarray trasduzione dei segnali delle cellule infettate con espressione o con il controllo annuncio-invertito (INV)-vettore hBVR identificato ATF-2 tra diversi geni up-regolati. ATF-2 &egrave; un fattore di trascrizione del bZip per l&#39;attivazione dell&#39;elemento risposta cAMP (CRE) e un partner dimerica di c-jun in via MAPK che regola la proteina dello stress, HO-1, espressione. Settentrionale e occidentale blot analisi ha mostrati aumenti di circa 10 volte in ATF-2 mRNA e proteina a 16 e 24 h dopo l&#39;aggiunta di Dox. Annuncio-INV-hBVR non ha senso espressione ATF-2. Nelle cellule infettate da hBVR, sono stati aumentati livelli di HO-1 mRNA e di proteine. HBVR tradotto in vitro ed estratto nucleare contenente hBVR in gel mobilit&agrave;-shift assay legato ai luoghi di AP-1 nella regione del promotore di ATF-2 e un oligonucleotide contenente il sito CRE. Entrambe le associazioni potrebbero essere gareggiate fuori dalla sonda in eccesso senza etichetta; in presenza di anticorpi hBVR, essi visualizzati bande spostati. Co-transfection di hBVR con i promotori di ATF-2 o c-jun ha causato un aumento molteplicemente nell&#39;attivit&agrave; della luciferasi. hBVR modulazione di ATF-2 e HO 1 espressione suggerisce ha un potenziale ruolo nella regolazione di AP-1 e geni regolati cAMP e un ruolo nella cella di segnalazione. Proponiamo che l&#39;aumentata espressione della proteina pu&ograve; essere utilizzato per modificare il profilo di espressione genica nella cellula.

Riduttasi di Biliverdin, un romanzo regolatore per l&#39;induzione dell&#39;attivazione di fattore di trascrizione-2 ed eme ossigenasi-1.

BVR reduces biliverdin, the HO-1 and HO-2 product, to bilirubin. Human biliverdin (BVR) is a serine/threonine kinase activated by free radicals. It is a leucine zipper (bZip) DNA-binding protein and a regulatory factor for 8/7-bp AP-1-regulated genes, including HO-1 and ATF-2/CREB. Presently, small interference (si) RNA constructs were used to investigate the role of human BVR in sodium arsenite (As)-mediated induction of HO-1 and in cytoprotection against apoptosis. Activation of BVR involved increased serine/threonine phosphorylation but not its protein or transcript levels. The peak activity at 1 h (4-5-fold) after treatment of 293A cells with 5 mum As preceded induction of HO-1 expression by 3 h. The following suggests BVR involvement in regulating oxidative stress response of HO-1: siBVR attenuated As-mediated increase in HO-1 expression; siBVR, but not siHO-1, inhibited As-dependent increased c-jun promoter activity; treatment of cells with As increased AP-1 binding of nuclear proteins; BVR was identified in the DNA-protein complex; and AP-1 binding of the in vitro translated BVR was phosphorylation-dependent and was attenuated by biliverdin. Most unexpectedly, cells transfected with siBVR, but not siHO-1, displayed a 4-fold increase in apoptotic cells when treated with 10 mum As as detected by flow cytometry. The presence of BVR small interference RNA augmented the effect of As on levels of cytochrome c, TRAIL, and DR-5 mRNA and cleavage of poly(ADP-ribose) polymerase. The findings describe the function of BVR in HO-1 oxidative response and, demonstrate, for the first time, not only that BVR advances the role of HO-1 in cytoprotection but also affords cytoprotection independent of heme degradation.

Small interference RNA-mediated gene silencing of human biliverdin reductase, but not that of heme oxygenase-1, attenuates arsenite-mediated induction of the oxygenase and increases apoptosis in 293A kidney cells.

BVR r&eacute;duit la biliverdine, le HO-1 et produit de l&#39;HO-2, de la bilirubine. La biliverdine humaine (BVR) est une s&eacute;rine/thr&eacute;onine kinase activ&eacute;e par les radicaux libres. C&#39;est une prot&eacute;ine de liaison &agrave; l&#39;ADN leucine zipper (bZip) et un facteur de normalisation pour les 8/7-bp de g&egrave;nes r&eacute;gul&eacute;s par AP-1, y compris les HO-1 et ATF-2/CREB. Actuellement, des constructions de RNA interf&eacute;rence (si) ont &eacute;t&eacute; utilis&eacute;es pour enqu&ecirc;ter sur le r&ocirc;le des humain BVR en ars&eacute;nite de sodium (As)-m&eacute;di&eacute;e par induction de HO-1 et dans la cytoprotection contre l&#39;apoptose. Activation de BVR impliqu&eacute; s&eacute;rine/thr&eacute;onine accrue des niveaux phosphorylation mais pas sa prot&eacute;ine ou une transcription. Le pic d&#39;activit&eacute; &agrave; 1 h (4 &agrave; 5 fois) apr&egrave;s le traitement de cellules 293 a avec 5 maman comme pr&eacute;c&eacute;d&eacute;e d&#39;induction de l&#39;expression HO-1 h 3. Ce qui suit sugg&egrave;re l&#39;implication de BVR dans la r&eacute;gulation de r&eacute;ponse au stress oxydatif de HO-1: siBVR a att&eacute;nu&eacute; l&#39;augmentation comme m&eacute;diation dans l&#39;expression HO-1 ; siBVR, mais pas siHO-1, inhib&eacute; comme d&eacute;pendant augmentation de l&#39;activit&eacute; de promoteur de c-jun ; traitement des cellules avec comme liant d&#39;AP-1 accrue de prot&eacute;ines nucl&eacute;aires ; BVR a &eacute;t&eacute; identifi&eacute; dans le complexe ADN-prot&eacute;ine ; et AP-1 liaison de la BVR traduit in vitro &eacute;tait d&eacute;pendante de la phosphorylation et a &eacute;t&eacute; att&eacute;nu&eacute;e par la biliverdine. De fa&ccedil;on plus inattendue, les cellules transfect&eacute;es avec siBVR, mais pas siHO-1, affich&eacute;s une augmentation de 4 fois en apoptose cellules avec 10 maman comme comme d&eacute;tect&eacute;s par cytom&eacute;trie en flux. La pr&eacute;sence de l&#39;ARN d&#39;interf&eacute;rence BVR a augment&eacute; l&#39;effet de comme sur les niveaux du cytochrome c, sentier et DR-5 ARNm et clivage de ribose polym&eacute;rase. Les constatations d&eacute;crivent la fonction de BVR en HO-1 r&eacute;ponse oxydante et, d&eacute;montrent, pour la premi&egrave;re fois, non seulement que BVR avance le r&ocirc;le de HO-1 dans la cytoprotection mais offre aussi la cytoprotection ind&eacute;pendante de la d&eacute;gradation de l&#39;h&egrave;me.

D&#39;interf&eacute;rence RNA-mediated gene silencing of r&eacute;ductase humaine biliverdine, mais pas celui de l&#39;h&egrave;me oxyg&eacute;nase-1, att&eacute;nue l&#39;induction par l&#39;ars&eacute;nite de l&#39;oxyg&eacute;nase et augmente l&#39;apoptose des cellules de rein de 293 a.

BVR riduce Biliverdina, la HO-1 e 2 HO prodotto, bilirubina. Umano Biliverdina (BVR) &egrave; una serina/treonina chinasi attivata dai radicali liberi. &Egrave; una DNA-binding protein di leucina zipper (bZip) e un fattore regolatore per 8/7-bp AP-1-regolato geni, tra cui HO-1 e ATF-2/CREB. Attualmente, piccole interferenze (si) RNA costrutti sono stati utilizzati per studiare il ruolo di BVR umana in sodio arsenito (As)-mediata ad induzione di HO-1 e in cytoprotection contro il apoptosis. Attivazione di BVR coinvolti i livelli di fosforilazione ma non la proteina o trascrizione aumentata serina/treonina. Il picco di attivit&agrave; a 1 h (4-5 volte) dopo il trattamento di cellule 293A con 5 mamma come preceduto induzione dell&#39;espressione di HO-1 da 3 h. Il seguente suggerisce il coinvolgimento di BVR nella regolazione della risposta allo stress ossidativo di HO-1: siBVR attenuato come mediata aumento nell&#39;espressione di HO-1; siBVR, ma non gabrielelippi-1, inibiti come dipendente aumentata attivit&agrave; di c-jun promotore; trattamento delle cellule con maggiore AP-1 associazione di proteine nucleari; BVR &egrave; stato identificato nel complesso DNA-proteina; e AP-1 associazione della BVR tradotto in vitro era fosforilazione-dipendente ed &egrave; stato attenuato dalla Biliverdina. Pi&ugrave; inaspettatamente, celle transfected con siBVR, ma non gabrielelippi-1, visualizzate un aumento di 4 volte in apoptotic cellule quando trattati con 10 mamma come come rilevato tramite flusso cytometry. La presenza di interferenze piccola BVR RNA aumentata l&#39;effetto di come sui livelli di citocromo c, sentiero e DR-5 mRNA e scissione della polimerasi di poly(ADP-ribose). I risultati descrivono la funzione di BVR in HO-1 risposta ossidativa e dimostrano, per la prima volta, non solo che BVR avanza il ruolo di HO-1 in cytoprotection, ma offre anche una cytoprotection indipendente di degradazione dell&#39;eme.

Piccola interferenza RNA-mediata del gene silencing della riduttasi di biliverdin umana, ma non quella dell&#39;eme ossigenasi-1, attenua arsenito-mediated induction of le ossigenasi e aumenta l&#39;apoptosi nelle cellule renali 293A.

Human biliverdin reductase (hBVR), a dual specificity kinase (Ser/Thr/Tyr) is, as protein kinase C (PKC) betaII, activated by insulin and free radicals (Miralem, T., Hu, Z., Torno, M. D., Lelli, K. M., and Maines, M. D. (2005) J. Biol. Chem. 280, 17084-17092; Lerner-Marmarosh, N., Shen, J., Torno, M. D., Kravets, A., Hu, Z., and Maines, M. D. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 7109-7114). Here, by using 293A cells co-transfected with pcDNA3-hBVR and PKC betaII plasmids, we report the co-immunoprecipitation of the proteins and co-purification in the glutathione S-transferase (GST) pulldown assay. hBVR and PKC betaII, but not the reductase and PKC zeta, transphosphorylated in assay systems supportive of activity of only one of the kinases. PKC betaII K371R mutant protein ("kinase-dead") was also a substrate for hBVR. The reductase increased the Vmax but not the apparent Km values of PKC betaII for myelin basic protein; activation was independent of phospholipids and extended to the phosphorylation of S2, a PKC-specific substrate. The increase in substrate phosphorylation was blocked by specific inhibitors of conventional PKCs and attenuated by sihBVR. The effect of the latter could be rescued by subsequent overexpression of hBVR. To a large extent, the activation was a function of the hBVR N-terminal chain of valines and intact ATP-binding site and the cysteine-rich C-terminal segment. The cobalt protoporphyrin-activated hBVR phosphorylated a threonine in a peptide corresponding to the Thr500 in the human PKC betaII activation loop. Neither serine nor threonine residues in peptides corresponding to other phosphorylation sites of the PKC betaII nor PKC zeta activation loop-derived peptides were substrates. The phosphorylation of Thr500 was confirmed by immunoblotting of hBVR.PKC betaII immunocomplex. The potential biological relevance of the hBVR activation of PKC betaII was suggested by the finding that in cells transfected with the PKC betaII, hBVR augmented phorbol myristate acetate-mediated c-fos expression, and infection with sihBVR attenuated the response. Also, in cells overexpressing hBVR and PKC betaII, as well as in untransfected cells, upon treatment with phorbol myristate acetate, the PKC translocated to the plasma membrane and co-localized with hBVR. hBVR activation of PKC betaII underscores its potential function in propagation of signals relayed through PKCs.

Human biliverdin reductase, a previously unknown activator of protein kinase C betaII.

R&eacute;ductase de biliverdine humaine (hBVR), une kinase double sp&eacute;cificit&eacute; (Ser/Thr/Tyr) est, comme la prot&eacute;ine kinase C (PKC) betaII, activ&eacute;e par l&#39;insuline et des radicaux libres (Miralem, T., Hu, Z., Torno, D. M., Lelli, K. M. et Maines, M. D. (2005) J. Biol. Chem. 280, 17084-17092 ; Lerner-Marmarosh, N., Shen, J., Torno, D. M., Kravets, A., Hu, Z. et Maines, M. D. (2005) Proc Natl. Acad. Sci. u. s. a. 102, 7109-7114). Nous rapportons ici, &agrave; l&#39;aide de cellules 293 a cotransfect&eacute;es avec pcDNA3-hBVR et plasmides betaII PKC, le co-immunopr&eacute;cipitation des prot&eacute;ines et des copurification dans l&#39;essai de pulldown glutathion S-transf&eacute;rase (GST). transphosphorylated hBVR et PKC betaII, mais pas la r&eacute;ductase et PKC zeta, dans les syst&egrave;mes de dosage favorables de l&#39;activit&eacute; d&#39;un seul des kinases. PKC betaII K371R prot&eacute;ine mutante (&quot;kinase-mort&quot;) est aussi un substrat pour hBVR. La r&eacute;ductase augmente la Vmax mais pas les valeurs de Km apparents de PKC betaII pour la prot&eacute;ine basique de la my&eacute;line ; activation &eacute;tait ind&eacute;pendante des phospholipides et &eacute;tendu &agrave; la phosphorylation de S2, un substrat sp&eacute;cifique PKC. L&#39;augmentation de la phosphorylation de substrat a &eacute;t&eacute; bloqu&eacute;e par des inhibiteurs sp&eacute;cifiques de PKCs conventionnels et att&eacute;nu&eacute;e par sihBVR. L&#39;effet de ce dernier pourrait &ecirc;tre sauv&eacute;e par surexpression ult&eacute;rieure de hBVR. Dans une large mesure, l&#39;activation est fonction de la cha&icirc;ne N-terminale des valines et intact ATP binding site de hBVR et le segment de C-terminal riche en cyst&eacute;ine. Le hBVR de protoporphyrine-activ&eacute; de cobalt phosphoryl&eacute;e une thr&eacute;onine dans un peptide correspondant &agrave; la Thr500 dans la boucle d&#39;activation de betaII PKC humaine. R&eacute;sidus s&eacute;rine ni thr&eacute;onine en peptides correspondant &agrave; d&#39;autres sites de phosphorylation de la PKC betaII ni de peptides de boucle-d&eacute;riv&eacute;s d&#39;activation PKC zeta sont des substrats. La phosphorylation de Thr500 a &eacute;t&eacute; confirm&eacute;e par immunoblotting de poly&eacute; betaII hBVR.PKC. Le potentiel la pertinence biologique des hBVR d&#39;activation de la PKC betaII a &eacute;t&eacute; sugg&eacute;r&eacute;e par la constatation que, dans les cellules transfect&eacute;es avec le betaII PKC, hBVR augment&eacute;e expression de c-fos induite par l&#39;ac&eacute;tate de phorbol myristate, et infection avec sihBVR a att&eacute;nu&eacute; la r&eacute;ponse. En outre, dans les cellules surexprimant hBVR et PKC betaII, ainsi que dans les cellules celllulaire, trait&eacute;s par l&#39;ac&eacute;tate de phorbol myristate, la PKC translocation vers la membrane plasmique et co-localisaient avec hBVR. hBVR l&#39;activation de la PKC betaII met en &eacute;vidence son r&ocirc;le potentiel dans la propagation de signaux transmis par le biais de PKCs.

R&eacute;ductase humaine biliverdine, un activateur jusqu&#39;alors inconnu de la prot&eacute;ine kinase C betaII.

Riduttasi di biliverdin umana (hBVR), una specificit&agrave; dual chinasi (Ser/Thr/Tyr) &egrave;, come proteina chinasi C (PKC) betaII, attivato dall&#39;insulina e i radicali liberi (Miralem, T., Hu, Z., Torno, m. D., Lelli, K. M. e Maines, M. D. (2005) j Biol Chem. 280, 17084-17092; Lerner-Marmarosh, N., Shen, J., Torno, D. M., Kravets, A., Hu, z e Maines, M. D. (2005) Proc Natl. acad. sci. u. s. a. 102, 7109-7114). Qui, utilizzando cellule 293A co-transfected con pcDNA3-hBVR e PKC betaII plasmidi, riportiamo il co-immunoprecipitazione delle proteine e co-purification nel dosaggio pulldown glutatione S-transferasi (GST). transphosphorylated hBVR e betaII PKC, ma non la reduttasi e PKC zeta, nei sistemi di dosaggio sostegno di attivit&agrave; di uno solo la chinasi. PKC betaII K371R proteina mutante (&quot;chinasi-morto&quot;) &egrave; stato anche un substrato per hBVR. La riduttasi aumentato il Vmax ma non i valori di Km apparenti di PKC betaII per proteina basica mielinica; attivazione indipendente di fosfolipidi ed esteso per la fosforilazione di S2, un substrato di PKC-specifici. L&#39;aumento della fosforilazione del substrato &egrave; stata bloccata da specifici inibitori di PKCs convenzionale e attenuato da sihBVR. L&#39;effetto di quest&#39;ultimo potrebbe essere salvato dalla conseguente sovraespressione di hBVR. In larga misura, l&#39;attivazione &egrave; stata una funzione di hBVR catena valines e intatto ATP-binding sito N-terminale e il segmento C-terminale ricco di cisteina. Il cobalto protoporfirina-attivato hBVR fosforilato una treonina in un peptide corrispondente a Thr500 nel ciclo di attivazione betaII PKC umano. Residui di serina n&eacute; treonina in peptidi corrispondenti ad altri siti di fosforilazione della betaII PKC n&eacute; peptidi derivati dal ciclo PKC zeta attivazione erano substrati. La fosforilazione di Thr500 &egrave; stata confermata da immunoblotting di immunocomplessi betaII hBVR.PKC. Il potenziale rilevanza biologica dell&#39;attivazione della PKC betaII hBVR &egrave; stato suggerito dalla constatazione che nelle celle transfected con il betaII PKC, hBVR aumentata espressione di forbolo miristato acetato-mediata c-fos, e infezione con sihBVR attenuato la risposta. Inoltre, in cellule con sovraesprimono relativa hBVR e PKC betaII, cos&igrave; come nelle cellule untransfected, previo trattamento con forbolo miristato acetato, il PKC traslocata a membrana del plasma e co-ha localizzato con hBVR. hBVR attivazione della PKC betaII sottolinea la funzione potenziale nella propagazione dei segnali inoltrati attraverso PKCs.

Reduttasi umana Biliverdina, un precedentemente sconosciuto attivatore della proteina chinasi C betaII.

Human biliverdin reductase (hBVR) is a dual function enzyme: a catalyst for bilirubin formation and a S/T/Y kinase that shares activators with protein kinase C (PKC) -zeta, including cytokines, insulin, and reactive oxygen species (ROS). Presently, we show that hBVR increases PKC-zeta autophosphorylation, stimulation by TNF-alpha, as well as cytokine stimulation of NF-kappaB DNA binding and promoter activity. S149 in hBVR S/T kinase domain and S230 in YLS230F in hBVR's docking site for the SH2 domain of signaling proteins are phosphorylation targets of PKC-zeta. Two hBVR-based peptides, KRNRYLS230F (#1) and KKRILHC281 (#2), but not their S-->A or C-->A derivatives, respectively, blocked PKC-zeta stimulation by TNF-alpha and its membrane translocation. The C-terminal-based peptide KYCCSRK296 (#3), enhanced PKC-zeta stimulation by TNF-alpha; for this, Lys296 was essential. In metabolically 32P-labeled HEK293 cells transfected with hBVR or PKC-zeta, TNF-alpha increased hBVR phosphorylation. TNF-alpha did not stimulate PKC-zeta in cells infected with small interfering RNA for hBVR or transfected with hBVR with a point mutation in the nucleotide-binding loop (G17), S149, or S230; this was similar to the response of "kinase-dead" PKC-zeta(K281R). We suggest peptide #1 blocks PKC-zeta-docking site interaction, peptide #2 disrupts function of the PKC-zeta C1 domain, and peptide #3 alters ATP presentation to the kinase. The findings are of potential significance for development of modulators of PKC-zeta activity and cellular response to cytokines.

Regulation of TNF-alpha-activated PKC-zeta signaling by the human biliverdin reductase: identification of activating and inhibitory domains of the reductase.

R&eacute;ductase humaine de biliverdine (hBVR) est une enzyme de la double fonction : un catalyseur pour la formation de bilirubine et une S/T/Y kinase qui partage les activateurs avec la prot&eacute;ine kinase C (PKC)-zeta, y compris les cytokines, l&#39;insuline et les esp&egrave;ces r&eacute;actives de l&#39;oxyg&egrave;ne (ROS). Actuellement, nous montrons que hBVR augmente PKC-zeta autophosphorylation, stimulation par le TNF-alpha, ainsi que la stimulation de l&#39;activit&eacute; de liaison et promoteur de NF-kappaB ADN cytokine. S149 dans domaine kinase de hBVR S/T et S230 dans YLS230F dans le site d&#39;accueil de hBVR pour le domaine SH2 de prot&eacute;ines de signalisation sont des cibles de la phosphorylation de PKC-zeta. Deux peptides ax&eacute;e sur les hBVR, les KRNRYLS230F (#1) et KKRILHC281 (#2), mais pas leur S--&gt; A ou C--&gt; un d&eacute;riv&eacute;s, bloqu&eacute; respectivement, stimulation de la PKC-zeta de TNF-alpha et la translocation de sa membrane. Le peptide C-terminal-based KYCCSRK296 (#3), am&eacute;lior&eacute; de stimulation de la PKC-zeta de TNF-alpha ; pour ce faire, il fallait Lys296. Dans m&eacute;taboliquement marqu&eacute;s au 32P les cellules HEK293 transfect&eacute;es avec hBVR ou PKC-zeta, TNF-alpha ont augment&eacute; hBVR phosphorylation. TNF-alpha ne stimule pas la PKC-zeta dans les cellules infect&eacute;es par les petits ARN interf&eacute;rents pour hBVR ou transfect&eacute;es avec hBVR avec une mutation ponctuelle dans la boucle de liaison nucl&eacute;otidique (G17), S149 ou S230 ; C&#39;&eacute;tait semblable &agrave; la r&eacute;ponse de &laquo; kinase-morts &raquo; PKC-zeta(K281R). Nous vous sugg&eacute;rons d&#39;interaction site de peptide #1 blocs PKC-zeta-docking, peptide #2 perturbe la fonction du domaine PKC-zeta C1 et peptide #3 modifie la pr&eacute;sentation d&#39;ATP &agrave; la kinase. Les conclusions sont d&#39;une importance potentielle pour le d&eacute;veloppement des modulateurs de l&#39;activit&eacute; de la PKC-zeta et la r&eacute;ponse cellulaire aux cytokines.

R&egrave;glement du TNF-alpha-activ&eacute; PKC-zeta signalisation par la r&eacute;ductase humaine biliverdine : identification d&#39;activation et domaines inhibitrices de la r&eacute;ductase.

Umano Biliverdina reduttasi (hBVR) &egrave; enzima un duplice funzione: un catalizzatore per la formazione di bilirubina e una chinasi S/T/Y che condivide gli attivatori con protein chinasi C (PKC)-zeta, citochine, insulina e specie reattive dell&#39;ossigeno (ROS). Attualmente, ci mostra che hBVR aumenta PKC zeta autophosphorylation, stimolazione di TNF-alfa, cos&igrave; come citochina stimolazione dell&#39;attivit&agrave; di associazione e promotore di NF-kappaB DNA. S149 nel dominio della chinasi di hBVR S/T e S230 in YLS230F nel sito di ancoraggio di hBVR per il dominio SH2 di proteine di segnalazione sono gli obiettivi di fosforilazione della PKC zeta. Due peptidi hBVR-based, KRNRYLS230F (#1) e KKRILHC281 (#2), ma non loro S--&gt; A o C--&gt; un derivati, rispettivamente, bloccata la stimolazione PKC-zeta di TNF-alfa e la traslocazione di membrana. Il peptide C-terminale basato su KYCCSRK296 (#3), enhanced stimolazione PKC-zeta di TNF-alfa; per questo, Lys296 &egrave; stato essenziale. Metabolicamente marcato con 32P HEK293 celle transfected con hBVR o PKC zeta, TNF-alfa aumentata fosforilazione di hBVR. TNF-alfa ha fatto non stimolare PKC-zeta in cellule infettate con piccolo RNA interferente per hBVR o transfected con hBVR con una mutazione puntiforme nel nucleotide-binding loop (G17), S149 o S230; Questo era simile alla risposta di PKC-zeta(K281R) &quot;chinasi-morti&quot;. Consigliamo di interazione di sito #1 blocchi PKC-zeta-aggancio del peptide, peptide #2 sconvolge la funzione del dominio C1 PKC zeta e peptide #3 altera la presentazione di ATP per la chinasi. I risultati sono di importanza potenziale per lo sviluppo di modulatori dell&#39;attivit&agrave; PKC zeta e risposta cellulare di citochine.

Regolamento del TNF-alfa-attivato PKC-zeta segnalazione dalla riduttasi di biliverdin umano: identificazione di attivazione e domini inibitori della reduttasi.

Activation of the MEK/ERK/Elk-signaling cascade is a mechanism for relaying mitogenic and stress stimuli for gene activation. MEK1 is the proximate kinase for activation of ERK1/2, and nuclear targeting of ERK1/2 is obligatory for Elk1 transcriptional activity. Human biliverdin reductase (hBVR) is a recently described Ser/Thr/Tyr kinase in the MAPK insulin/insulin-like growth factor 1 (IGF1)-signaling cascade. Using 293A cells and in vitro experiments, we detail the formation of a ternary complex of MEK/ERK/hBVR, activation of MEK1 and ERK1/2 kinase activities by hBVR, and phosphorylation of hBVR by ERK1/2. hBVR is nearly as effective as IGF1 in activating ERK; intact hBVR ATP-binding domain is necessary for Elk1 activation, whereas protein-protein interaction is the basis for hBVR activation of MEK1 and ERK. The two MAPK docking consensus sequences present in hBVR, F(162)GFP and K(275)KRILHCLGL (C- and D-box, respectively), are ERK interactive sites; interaction at each site is critical for ERK/Elk1 activation. Transfection with mutant hBVR-P(165) or peptides corresponding to the C- or D-box blocked activation of ERK by IGF1. Transfection with D-box mutant hBVR prevented the activation of ERK by wild-type protein and dramatically decreased Elk1 transcriptional activity. hBVR is a nuclear transporter of ERK; experiments with hBVR nuclear export signal (NES) and nuclear localization signal (NLS) mutants demonstrated its critical role in the nuclear localization of IGF-stimulated ERK for Elk1 activation. These findings, together with observations that si-hBVR blocked activation of ERK and Elk1 by IGF1 and prevented formation of ternary complex between MEK/ERK/hBVR, define the critical role of hBVR in ERK signaling and nuclear functions of the kinase.

Human biliverdin reductase is an ERK activator; hBVR is an ERK nuclear transporter and is required for MAPK signaling.

Activation de la cascade de signalisation MEK/ERK/Elk est un m&eacute;canisme pour le reparcage mitog&egrave;ne et stimuli de stress pour l&#39;activation du g&egrave;ne. MEK1 est la kinase imm&eacute;diate pour l&#39;activation de ERK1/2, et un ciblage nucl&eacute;aire de ERK1/2 est obligatoire pour les activit&eacute; transcriptionnelle Elk1. R&eacute;ductase humaine de biliverdine (hBVR) est une kinase Ser/Thr/Tyr r&eacute;cemment d&eacute;crite dans la MAPK insuline/insulin-like growth factor 1 (IGF1)-cascade de signalisation. &Agrave; l&#39;aide de cellules 293 a et des exp&eacute;riences in vitro, nous d&eacute;taillons la formation d&#39;un complexe ternaire de MEK/ERK/hBVR, activation de MEK1 et ERK1/2 activit&eacute;s de kinase de hBVR et la phosphorylation de hBVR de ERK1/2. hBVR est presque aussi efficace que l&#39;IGF1 dans l&#39;activation de ERK ; hBVR intact ATP binding domaine est n&eacute;cessaire pour l&#39;activation Elk1, consid&eacute;rant que l&#39;interaction prot&eacute;ine-prot&eacute;ine est &agrave; la base pour l&#39;activation de la hBVR de MEK1 et ERK. Les deux s&eacute;quences de consensus d&#39;amarrage de MAPK pr&eacute;sent dans hBVR, F (162) GFP et K (275) KRILHCLGL (C - et D-box, respectivement), sont des sites interactifs ERK ; interaction sur chaque site est critique pour l&#39;activation de ERK/Elk1. Transfection avec hBVR-P(165) mutant ou peptides correspondant &agrave; la zone C - ou D-bloqu&eacute; activation de ERK par IGF1. Transfection avec D-box hBVR mutant emp&ecirc;che l&#39;activation de ERK par la prot&eacute;ine sauvage et Elk1 spectaculaire diminution de l&#39;activit&eacute; transcriptionnelle. hBVR est un transporteur nucl&eacute;aire de ERK ; exp&eacute;riences avec signal d&#39;exportations nucl&eacute;aires hBVR (NES) et mutants (NLS) de signal de localisation nucl&eacute;aire a d&eacute;montr&eacute; son r&ocirc;le crucial dans la localisation nucl&eacute;aire de ERK stimul&eacute;e par l&#39;IGF pour activation Elk1. Ces r&eacute;sultats, ainsi que les observations que TR-hBVR bloqu&eacute; activation de ERK et Elk1 par IGF1 et a emp&ecirc;ch&eacute; la formation d&#39;un complexe ternaire entre MEK/ERK/hBVR, d&eacute;finissent le r&ocirc;le essentiel des hBVR dans les fonctions de signalisation et nucl&eacute;aires de ERK la kinase.

R&eacute;ductase humaine biliverdine est un activateur ERK ; hBVR est un transporteur nucl&eacute;aire de ERK et est requis pour la signalisation MAPK.

Attivazione della cascata del MEK/ERK/Elk-segnalazione &egrave; un meccanismo per l&#39;inoltro mitogeni e stimoli di stress per l&#39;attivazione del gene. MEK1 &egrave; la chinasi proximata per l&#39;attivazione di ERK1/2 e targeting nucleare di ERK1/2 &egrave; obbligatorio per l&#39;attivit&agrave; trascrizionale Elk1. Riduttasi di biliverdin umana (hBVR) sono una chinasi Ser/Thr/Tyr recentemente descritta in MAPK insulina/insulin-like growth factor 1 (IGF1)-cascata di segnalazione. Utilizzando cellule 293A ed esperimenti in vitro, abbiamo dettagliato la formazione di un complesso ternario di MEK/ERK/hBVR, l&#39;attivazione di attivit&agrave; della chinasi MEK1 ed ERK1/2 da hBVR e fosforilazione della hBVR di ERK1/2. hBVR &egrave; quasi altrettanto efficace come IGF1 nell&#39;attivazione di ERK; hBVR intatto ATP-binding dominio &egrave; necessario per l&#39;attivazione di Elk1, considerando interazione proteina-proteina &egrave; la base per l&#39;attivazione di hBVR di MEK1 ed ERK. Le due sequenze consenso docking MAPK presentano in hBVR, F (162) GFP e K (275) KRILHCLGL (C - e D-box, rispettivamente), sono siti interattivi ERK; interazione in ciascun sito &egrave; fondamentale per l&#39;attivazione di ERK/Elk1. Transfezione con hBVR-P(165) mutante o peptidi corrispondenti a C - o D-box bloccato attivazione di ERK di IGF1. Transfezione con D-box hBVR mutante ha impedito l&#39;attivazione di ERK proteina wild-type e drammaticamente diminuita attivit&agrave; transcriptional Elk1. hBVR &egrave; un trasportatore nucleare di ERK; esperimenti con hBVR segnale di esportazione nucleare (NES) e mutanti (NLS) segnale nucleare di localizzazione ha dimostrato suo ruolo critico nella localizzazione nucleare di ERK IGF-stimolato per attivazione Elk1. Questi risultati, insieme con le osservazioni che si-hBVR bloccato sull&#39;attivazione di ERK ed Elk1 di IGF1 e ha impedito la formazione del complesso ternario fra MEK/ERK/hBVR, definiscono il ruolo critico di hBVR nelle funzioni di segnalazione e nucleare ERK della chinasi di.

Umano Biliverdina reduttasi &egrave; un attivatore ERK; hBVR &egrave; un trasportatore ERK nucleare ed &egrave; necessaria per la segnalazione MAPK.

L&#39;activit&eacute; de la kinase Ser/Thr/Tyr de r&eacute;ductase humaine de biliverdine (hBVR) et l&#39;expression de la prot&eacute;ine se liant &agrave; l&#39;antig&egrave;ne Goodpasture (GPBP), une kinase Ser/Thr non conventionnelle pour le collag&egrave;ne de type IV de la membrane basale, sont r&eacute;gies par le facteur de n&eacute;crose tumorale (TNF-alpha). Les cytokines pro-inflammatoires stimule l&#39;activit&eacute; de la kinase de hBVR et active de NF-kappaB, un r&eacute;gulateur transcriptionnel des ARNm GPBP. Augmentation de l&#39;activit&eacute; GPBP est associ&eacute;e &agrave; plusieurs maladies auto-immunes, y compris le syndrome de Goodpasture. Ici, nous montrons que dans les cellules HEK293A hBVR lie &agrave; GPBP et bas-r&eacute;gule son activit&eacute; kinase stimul&eacute;e par le TNF-alpha ; ce n&#39;&eacute;tait pas due &agrave; une diminution de l&#39;expression de GPBP. R&eacute;sultats avec les petits ARN interf&eacute;rents &agrave; hBVR et &agrave; la sous-unit&eacute; p65 de NF-kappaB montrent le r&ocirc;le de hBVR dans la stimulation initiale d&#39;expression GPBP de TNF-alpha-activ&eacute; NF-kappaB ; hBVR n&#39;&eacute;tait pas un facteur dans la m&eacute;diation de stabilit&eacute; de l&#39;ARNm de le GPBP. Le domaine d&#39;interaction a &eacute;t&eacute; mapp&eacute; sur le motif C CX (10) (281) dans le C-terminal 24 r&eacute;sidus de hBVR. Un 7-peptide, KKRILHC(281), correspondant &agrave; la base du consensus D (delta)-bo&icirc;te de motif dans le domaine d&#39;interaction, &eacute;tait aussi efficace que le polypeptide intact 296-r&eacute;sidus hBVR dans l&#39;inhibition de l&#39;activit&eacute; kinase GPBP. Expression hBVR GPBP ni r&eacute;glement&eacute; ni expression NF-kappaB d&eacute;pendante de TNF-alpha. Collectivement, nos donn&eacute;es r&eacute;v&egrave;lent que hBVR est un r&eacute;gulateur de la cascade de signalisation de TNF-alpha-GPBP-collag&egrave;ne type IV et de d&eacute;couvrir une nouvelle interaction biologique qui peut-&ecirc;tre &ecirc;tre pertinents dans la pathog&eacute;nie auto-immune.

R&eacute;ductase humaine biliverdine supprime l&#39;activit&eacute; kinase de Goodpasture-antig&egrave;ne prot&eacute;ique (GPBP): la r&eacute;ductase r&eacute;gule l&#39;expression de GPBP de facteur de n&eacute;crose tumorale-alpha-NF-kappaB-d&eacute;pendante.

L&#39;attivit&agrave; della chinasi Ser/Thr/Tyr della riduttasi di biliverdin umana (hBVR) e l&#39;espressione di Goodpasture antigen-binding protein (GPBP), una chinasi Ser/Thr non convenzionali per il collagene di tipo IV della membrana basale, sono regolati dal fattore di necrosi tumorale (TNF-alfa). Le citochine pro-infiammatorie stimola l&#39;attivit&agrave; della chinasi di hBVR e l&#39;attivazione di NF-kappaB, un regolatore di transcriptional del mRNA GPBP. Aumento dell&#39;attivit&agrave; GPBP &egrave; associata a diverse condizioni autoimmuni, tra cui la sindrome di Goodpasture. Qui vi mostriamo che in HEK293A cellule hBVR associato a GPBP e gi&ugrave;-regola la sua attivit&agrave; chinasica di TNF-alfa-stimolata; Questo non era a causa di una diminuzione nell&#39;espressione di GPBP. Risultati con piccolo RNA interferente per hBVR e per la subunit&agrave; normativo p65 di NF-kappaB mostrano il ruolo di hBVR nella stimolazione iniziale dell&#39;espressione GPBP di TNF-alfa-attivato NF-kappaB; hBVR non era un fattore nella mediazione della stabilit&agrave; del mRNA GPBP. Il dominio di interazione &egrave; stato mappato il motivo C CX (10) (281) nel C-terminale 24 residui di hBVR. Un peptide 7-residui, KKRILHC(281), corrispondente al nucleo del consenso D (delta)-casella motivo nel dominio dell&#39;interazione, &egrave; stato efficace come il polipeptide hBVR 296-residui intatti inibendo l&#39;attivit&agrave; della chinasi GPBP. GPBP regolato n&eacute; hBVR espressione n&eacute; espressione di NF-kappaB dipendente dal TNF-alfa. Collettivamente, i nostri dati rivelano che hBVR &egrave; un regolatore del TNF-alfa-GPBP-collagene tipo IV cascata di segnalazione e scoprire un romanzo interazione biologica che pu&ograve; essere rilevante nella patogenesi autoimmune.

Riduttasi di biliverdin umano sopprime l&#39;attivit&agrave; della chinasi Goodpasture antigen-binding protein (GPBP): la reduttasi regola l&#39;espressione di fattore di necrosi tumorale-alfa-NF-kappaB-dipendente GPBP.

The Ser/Thr/Tyr kinase activity of human biliverdin reductase (hBVR) and the expression of Goodpasture antigen-binding protein (GPBP), a nonconventional Ser/Thr kinase for the type IV collagen of basement membrane, are regulated by tumor necrosis factor (TNF-alpha). The pro-inflammatory cytokine stimulates kinase activity of hBVR and activates NF-kappaB, a transcriptional regulator of GPBP mRNA. Increased GPBP activity is associated with several autoimmune conditions, including Goodpasture syndrome. Here we show that in HEK293A cells hBVR binds to GPBP and down-regulates its TNF-alpha-stimulated kinase activity; this was not due to a decrease in GPBP expression. Findings with small interfering RNA to hBVR and to the p65 regulatory subunit of NF-kappaB show the hBVR role in the initial stimulation of GPBP expression by TNF-alpha-activated NF-kappaB; hBVR was not a factor in mediating GPBP mRNA stability. The interacting domain was mapped to the (281)CX(10)C motif in the C-terminal 24 residues of hBVR. A 7-residue peptide, KKRILHC(281), corresponding to the core of the consensus D(delta)-Box motif in the interacting domain, was as effective as the intact 296-residue hBVR polypeptide in inhibiting GPBP kinase activity. GPBP neither regulated hBVR expression nor TNF-alpha dependent NF-kappaB expression. Collectively, our data reveal that hBVR is a regulator of the TNF-alpha-GPBP-collagen type IV signaling cascade and uncover a novel biological interaction that may be of relevance in autoimmune pathogenesis.

Human biliverdin reductase suppresses Goodpasture antigen-binding protein (GPBP) kinase activity: the reductase regulates tumor necrosis factor-alpha-NF-kappaB-dependent GPBP expression.

hBVR is a Ser/Thr/Tyr kinase/scaffold protein/transcription factor/intracellular transporter of regulators. hBVR is an upstream activator of the insulin/IGF-1/MAPK/PI3K signaling pathway, and of NF-kappaB. As a reductase, it converts biliverdin to the antioxidant, bilirubin. hBVR gene has 8 exons; exon 1 is not translated. We report the characterization of hBVR promoter and its negative and positive regulation, respectively, by TNF-alpha and hypoxia. The 5' end of exon 1 was defined by primer extension analyses; deletion of an inhibitor sequence 350-425 bp upstream of this exon enhanced the promoter activity. One of two NF-kappaB binding sites in the 836-bp promoter was functional; the P65 subunit of NF-kappaB and TNF-alpha acted as inhibitors. On the basis of EMSA and ChIP assays, TNF-alpha treatment increases binding of NF-kappaB to its regulatory element. Overexpression of IkappaB increased hBVR mRNA. Biliverdin, but not bilirubin, was as effective as TNF-alpha in inhibiting hBVR promoter activity. Only one of 4 hypoxia responsive elements (HREs) bound to HIF-1alpha and ARNT expressed in HEK293A cells. An abasic site was introduced at the 3' G of the HRE. This element bound HIF-1 in the gel shift and in in-cell luciferase assays. hBVR was detected in the nucleus at 1, 2, and 4 h after hypoxia (1% O(2)), at which times its kinase and reductase activities were increased. Because hypoxia positively influences hBVR promoter and phosphorylation and TNF-alpha activated NF-kappaB inhibits the promoter, while biliverdin inhibits both NF-kappaB activity and hBVR promoter, we propose a regulatory mechanism for NF-kappaB by hypoxia and TNF-alpha centered on hBVR/biliverdin.

Characterization of the human biliverdin reductase gene structure and regulatory elements: promoter activity is enhanced by hypoxia and suppressed by TNF-alpha-activated NF-kappaB.

hBVR est un transporteur intracellulaire/facteur de Ser/Thr/Tyr kinase/&eacute;chafaudage prot&eacute;ine/transcription des r&eacute;gulateurs. hBVR est un activateur en amont de l&#39;insuline/IGF-1/MAPK/PI3K voie de signalisation et de NF-kappaB. Comme une r&eacute;ductase, il convertit la biliverdine l&#39;antioxydant, bilirubine. g&egrave;ne hBVR a 8 exons ; exon 1 n&#39;est pas traduit. Nous rapportons la caract&eacute;risation des hBVR promoteur et de son r&egrave;glement positif et n&eacute;gatif, respectivement, de TNF-alpha et de l&#39;hypoxie. L&#39;extr&eacute;mit&eacute; 5&#39; de l&#39;exon 1 a &eacute;t&eacute; d&eacute;finie par analyses d&#39;extension amorce ; suppression d&#39;un inhibiteur de la s&eacute;quence 350-425 bp en amont de cette exon a augment&eacute; l&#39;activit&eacute; de promoteur. L&#39;un des deux sites de liaison de NF-kappaB dans le promoteur 836-bp &eacute;tait fonctionnel ; la sous-unit&eacute; P65 de NF-kappaB et TNF-alpha ont agi en tant qu&#39;inhibiteurs. Sur la base d&#39;essais de EMSA et puce, traitement de TNF-alpha augmente la liaison de NF-kappaB &agrave; son &eacute;l&eacute;ment r&eacute;glementaire. La surexpression de la IkappaB a augment&eacute; hBVR mRNA. La biliverdine, mais pas de bilirubine, &eacute;tait aussi efficace que le TNF-alpha, en inhibant l&#39;activit&eacute; de promoteur hBVR. Seul des 4 &eacute;l&eacute;ments sensibles hypoxie (HREs) li&eacute;s &agrave; l&#39;HIF-1alpha et ARNT exprim&eacute;s dans les cellules HEK293A. Un site ABASIQUE a &eacute;t&eacute; introduit au 3&#39; G de l&#39;EDH. Cet &eacute;l&eacute;ment li&eacute; HIF-1 dans le d&eacute;calage de gel et dans les essais de la lucif&eacute;rase dans les cellules. hBVR a &eacute;t&eacute; d&eacute;tect&eacute;e dans le noyau &agrave; 1, 2 et 4 h apr&egrave;s l&#39;hypoxie (1 % O(2)), &agrave; quel moment ses activit&eacute;s r&eacute;ductase kinase ont &eacute;t&eacute; augment&eacute;es. Parce que l&#39;hypoxie influe positivement sur la phosphorylation et promoteur de la hBVR et de TNF-alpha activ&eacute; NF-kappaB inhibe le promoteur, tandis que la biliverdine inhibe l&#39;activit&eacute; de NF-kappaB tant hBVR promoteur, nous proposons un m&eacute;canisme r&eacute;glementaire pour NF-kappaB par hypoxie et TNF-alpha centr&eacute; sur hBVR/biliverdine.

Caract&eacute;risation de la structure des g&egrave;nes humains biliverdine r&eacute;ductase et des &eacute;l&eacute;ments r&eacute;glementaires : activit&eacute; du promoteur est renforc&eacute;e par l&#39;hypoxie et supprim&eacute;e par le TNF-alpha-activ&eacute; NF-kappaB.

hBVR &egrave; un trasportatore fattore/intracellulare Tyr/Ser/Thr chinasi/impalcatura proteica/trascrizione dei regolatori. hBVR &egrave; un attivatore a monte dell&#39;insulina/IGF-1/MAPK/PI3K pathway di segnalazione e di NF-kappaB. Come una reduttasi, converte Biliverdina antiossidante, bilirubina. hBVR gene ha 8 esoni; esone 1 non &egrave; tradotto. Riportiamo la caratterizzazione del promotore hBVR e relativo regolamento positivo e negativo, rispettivamente, di TNF-alfa e ipossia. L&#39;estremit&agrave; 5&#39; dell&#39;esone 1 &egrave; stata definita da analisi di estensione dell&#39;iniettore; eliminazione di un inibitore della sequenza 350-425 bp a monte di questo esone rafforzata l&#39;attivit&agrave; di promotore. Uno dei due siti di legame di NF-kappaB in promotore 836-bp &egrave; stato funzionale; la subunit&agrave; P65 di NF-kappaB e TNF-alfa ha agito come inibitori. Sulla base di saggi EMSA e ChIP, trattamento di TNF-alfa aumenta il legame di NF-kappaB al suo elemento normativo. Sovraespressione della IkappaB aumentato hBVR mRNA. Biliverdina, ma non la bilirubina, era altrettanto efficace come TNF-alfa nell&#39;inibire l&#39;attivit&agrave; di promotore hBVR. Solo uno dei 4 ipossia reattivo elementi (HREs) associati a HIF-1alpha e ARNT espresso nelle cellule HEK293A. Un sito abasic &egrave; stato introdotto al 3&#39; G del Sacro Romano Impero. Questo elemento associato HIF-1 nel turno gel e in saggi di luciferasi in cella. hBVR &egrave; stato rilevato nel nucleo a 1, 2 e 4 ore dopo l&#39;ipossia (1% O(2)), in quale momento l&#39;attivit&agrave; della chinasi e reduttasi sono stati aumentati. Perch&eacute; l&#39;ipossia influenza positivamente la fosforilazione e hBVR promotore e TNF-alfa attivato NF-kappaB inibisce il promotore, mentre Biliverdina inibisce l&#39;attivit&agrave; di NF-kappaB sia hBVR promotore, proponiamo un meccanismo di regolamentazione per NF-kappaB da ipossia e TNF-alfa centrata su hBVR/Biliverdina.

Caratterizzazione della struttura di gene umano Biliverdina reduttasi ed elementi normativi: attivit&agrave; di promotore &egrave; rafforzata da ipossia e soppressa dal TNF-alfa-attivato NF-kappaB.

Growth factors, insulin, oxidative stress, and cytokines activate ERK1/2 by PKCÎ´ and MEK1/2. Human biliverdin reductase (hBVR), a Ser/Thr/Tyr kinase and intracellular scaffold/bridge/anchor, is a nuclear transporter of MEK1/2-stimulated ERK1/2 (Lerner-Marmarosh, N., Miralem, T., Gibbs, P. E., and Maines, M. D. (2008) Proc. Natl. Acad. Sci. U.S.A. 105, 6870-6875). hBVR, PKCÎ´, and MEK1/2 overlap in their tissue expression profile and type of activators. Presently, we report on formation of an hBVR-PKCÎ´-ERK2 ternary complex that is essential for ERK2 signal transduction and activation of genes linked to cell proliferation and cancer. MEK1/2 and the protein phosphatase PP2A were also present in the complex. When cells were stimulated with insulin-like growth factor-1 (IGF-1), an increased interaction between hBVR and PKCÎ´ was detected by FRET-fluorescence lifetime imaging microscopy. hBVR and ERK2 were phosphorylated by PKCÎ´; however, the PKC was not a substrate for either ERK2 or hBVR. IGF-1 and phorbol ester increased hBVR/PKCÎ´ binding; hBVR was required for the activation of PKCÎ´ and its interaction with ERK2. The C-terminal phenylalanine residues of PKCÎ´ (Phe(660), Phe(663), and Phe(665)) were necessary for binding to ERK2 but not for hBVR binding. Formation of the hBVR-PKCÎ´-ERK2 complex required the hBVR docking site for ERK, FXFP (DEF, C-box) and D(Î´)-box (ILXXLXL) motifs. The hBVR-based peptide KKRILHCLGLA inhibited PKC activation and PKCÎ´/ERK2 interaction. Phorbol ester- and TNF-Î±-dependent activation of the ERK-regulated transcription factors Elk1 and NF-ÎºB and expression of the iNOS gene were suppressed by hBVR siRNA; those activities were rescued by hBVR. The findings reveal the direct input of hBVR in PKCÎ´/ERK signaling and identify hBVR-based peptide regulators of ERK-mediated gene activation.

Formation of ternary complex of human biliverdin reductase-protein kinase CÎ´-ERK2 protein is essential for ERK2-mediated activation of Elk1 protein, nuclear factor-ÎºB, and inducible nitric-oxidase synthase (iNOS).

Facteurs de croissance, l&#39;insuline, le stress oxydatif et cytokines activent ERK1/2 par PKC&delta; et MEK1/2. R&eacute;ductase humaine de biliverdine (hBVR), une kinase Ser/Thr/Tyr et intracellulaire &eacute;chafaudage/pont/ancre, est un transporteur nucl&eacute;aire de ERK1/2 MEK1/2-stimul&eacute;e (Lerner-Marmarosh, N., Miralem, T., Gibbs, P. E. et Maines, M. D. (2008) Proc Natl. Acad. Sci. U.S.A. 105, 6870-6875). hBVR, PKC&delta; et MEK1/2 se chevauchent dans leur profil d&#39;expression des tissus et le type d&#39;activateurs. Actuellement, nous rapportons sur la formation d&#39;un complexe ternaire hBVR-PKC&delta;-ERK2 qui est essentiel pour la transduction du signal ERK2 et l&#39;activation des g&egrave;nes li&eacute;s &agrave; la cellule la prolif&eacute;ration et le cancer. MEK1/2 et la prot&eacute;ine phosphatase PP2A &eacute;taient &eacute;galement pr&eacute;sents dans le complexe. Lorsque les cellules sont stimul&eacute;es avec insulin-like growth factor-1 (IGF-1), une interaction accrue entre hBVR et PKC&delta; a &eacute;t&eacute; d&eacute;tect&eacute;e par la frette-fluorescence lifetime imaging microscopy. hBVR et ERK2 sont phosphoryl&eacute;s par PKC&delta; ; Toutefois, la PKC n&#39;&eacute;tait pas un substrat pour ERK2 ou hBVR. Ester de phorbol et de l&#39;IGF-1 a augment&eacute; de liaison hBVR/PKC&delta; ; hBVR &eacute;tait requise pour l&#39;activation de PKC&delta; et son interaction avec ERK2. Les r&eacute;sidus de ph&eacute;nylalanine C-terminal de PKC&delta; (Phe(660), Phe(663) et Phe(665)) ont &eacute;t&eacute; n&eacute;cessaires pour la liaison &agrave; ERK2, mais pas pour la liaison de la hBVR. Le site d&#39;accueil de hBVR de formation du complexe hBVR-PKC&delta;-ERK2 requispour ERK, FXFP (DEF, bo&icirc;te de transfert) et D (&delta;)-bo&icirc;te de motifs (ILXXLXL). Le peptide ax&eacute;e sur les hBVR KKRILHCLGLA inhibe l&#39;activation de la PKC et PKC&delta;/ERK2 interaction. Phorbol fonction ester et TNF-&alpha; activation des facteurs de transcription r&eacute;glement&eacute;s ERK Elk1 et NF-&kappa;B et l&#39;expression du g&egrave;ne iNOS ont &eacute;t&eacute; supprim&eacute;es par les siARN hBVR ; ces activit&eacute;s ont &eacute;t&eacute; secourues par hBVR. Les r&eacute;sultats r&eacute;v&egrave;lent l&#39;apport direct de hBVR dans la PKC&delta;/ERK signalisation et identifient les r&eacute;gulateurs de peptide ax&eacute;e sur les hBVR de l&#39;activation de g&egrave;nes ERK.

Formation du complexe ternaire de biliverdine humaine r&eacute;ductase-prot&eacute;ine kinase C&delta;-ERK2 prot&eacute;ine est essentielle pour l&#39;activation par ERK2 Elk1 prot&eacute;ine facteur nucl&eacute;aire-&kappa;B et inductible oxydase-nitrique synthase (iNOS).

Fattori di crescita, insulina, stress ossidativo e citochine attivano ERK1/2 da PKC&delta; e MEK1/2. Riduttasi di biliverdin umana (hBVR), una Ser/Thr/Tyr chinasi e intracellulare impalcatura/ponte/ancoraggio, &egrave; un trasportatore nucleare di MEK1/2-stimolato ERK1/2 (Lerner-Marmarosh, N., Miralem, T., Maines, M. D. (2008) proc Acad Sci. U.S.A. 105, Natl., P. E. e Gibbs 6870-6875). hBVR, PKC&delta; e MEK1/2 si sovrappongono nel loro profilo di espressione del tessuto e il tipo di attivatori. Attualmente, abbiamo relazione sulla formazione di un complesso ternario hBVR-PKC&delta;-ERK2 che &egrave; essenziale per la trasduzione del segnale di ERK2 e attivazione dei geni collegati alla cella proliferazione e cancro. MEK1/2 e la fosfatasi della proteina PP2A erano anche presenti nel complesso. Quando le cellule sono state stimolate con il fattore di crescita insulino-simile 1 (IGF-1), una maggiore interazione tra hBVR e PKC&delta; &egrave; stata rilevata da lifetime FRET-fluorescenza imaging microscopia. hBVR ed ERK2 erano fosforilato da PKC&delta;; Tuttavia, il PKC non era un substrato per ERK2 o hBVR. Estere di IGF-1 e forbolo aumentata associazione hBVR/PKC&delta;; hBVR &egrave; stato richiesto per l&#39;attivazione del PKC&delta; e della sua interazione con ERK2. I residui di fenilalanina C-terminale di PKC&delta; (Phe(660), Phe(663) e Phe(665)) erano necessarie per l&#39;associazione di ERK2, ma non per l&#39;associazione hBVR. Formazione del complesso hBVR-PKC&delta;-ERK2 richiesto il sito docking hBVR per ERK, FXFP (DEF, C-box) e D (&delta;)-casella motivi (ILXXLXL). Il peptide hBVR-based KKRILHCLGLA inibite attivazione PKC e PKC&delta;/ERK2 interazione. Forbolo estere - e TNF-&alpha;-dipendente attivazione dei fattori di trascrizione regolata ERK Elk1 e NF-&kappa;B ed espressione del gene iNOS furono soppresse da hBVR siRNA; tali attivit&agrave; sono state salvate da hBVR. I risultati rivelano l&#39;input diretto dei hBVR in PKC&delta;/ERK segnalazione e identificano i regolatori basati su hBVR del peptide di attivazione di ERK-mediata del gene.

Formazione del complesso ternario della umana Biliverdina reduttasi-chinasi di proteina C&delta;-ERK2 proteina &egrave; essenziale per l&#39;attivazione di ERK2-mediata della proteina Elk1, fattore-&kappa;B nucleare e viscoelastico ossidasi acido nitrico sintasi (iNOS).

PKCÎ´, a Ser/Thr kinase, promotes cell growth, tumorigenesis, and apoptosis. Human biliverdin reductase (hBVR), a Ser/Thr/Tyr kinase, inhibits apoptosis by reducing biliverdin-IX to antioxidant bilirubin. The enzymes are activated by similar stimuli. Reportedly, hBVR is a kinase-independent activator of PKCÎ´ and is transactivated by the PKC (Gibbs, P. E., Miralem, T., Lerner-Marmarosh, N., Tudor, C., and Maines, M. D. (2012) J. Biol. Chem. 287, 1066-1079). Presently, we examined interactions between the two proteins in the context of regulation of their activities and defining targets of hBVR phosphorylation by PKCÎ´. LC-MS/MS analysis of PKCÎ´-activated intact hBVR identified phosphorylated serine positions 21, 33, 230, and 237, corresponding to the hBVR Src homology-2 domain motif (Ser(230) and Ser(237)), flanking the ATP-binding motif (Ser(21)) and in PHPS sequence (Ser(33)) as targets of PKCÎ´. Ser(21) and Ser(230) were also phosphorylated in hBVR-based peptides. The Ser(230)-containing peptide was a high affinity substrate for PKCÎ´ in vitro and in cells; the relative affinity was PKCÎ´ > PKCÎ²II > PKCÎ¶. Two overlapping peptides spanning this substrate, KRNRYLSF and SFHFKSGSL, were effective inhibitors of PKCÎ´ kinase activity and PKCÎ´-supported activation of transcription factors Elk1 and NF-ÎºB. Only SFHFKSGSL, in PKCÎ´-transfected phorbol 12-myristate 13-acetate-stimulated cells, caused membrane blebbing and cell loss. Biliverdin noncovalently inhibited PKCÎ´, whereas PKCÎ´ potentiated hBVR reductase activity and accelerated the rate of bilirubin formation. This study, together with previous findings, reveals an unexpected regulatory interplay between PKCÎ´ and hBVR in modulating cell death/survival in response to various activating stimuli. In addition, this study has identified novel substrates for and inhibitors of PKCÎ´. We suggest that hBVR-based technology may have utility to modulate PKCÎ´-mediated functions in the cell.

The human biliverdin reductase-based peptide fragments and biliverdin regulate protein kinase CÎ´ activity: the peptides are inhibitors or substrate for the protein kinase C.

PKC&delta;, une Ser/Thr kinase, favorise l&#39;apoptose, tumorigen&egrave;se et la croissance cellulaire. R&eacute;ductase humaine de biliverdine (hBVR), une kinase Ser/Thr/Tyr, inhibe l&#39;apoptose en r&eacute;duisant la biliverdine-IX en bilirubine antioxydant. Les enzymes sont activ&eacute;s par des stimuli similaires. Selon les t&eacute;moignages, hBVR est un activateur de kinase-ind&eacute;pendant de PKC&delta; et est transactivated par la PKC (Gibbs, P. E., Miralem, T., Lerner-Marmarosh, N., Tudor, C. et Maines, M. D. (2012) J. Biol. Chem. 287, 1066-1079). Actuellement, nous avons &eacute;tudi&eacute; les interactions entre deux prot&eacute;ines dans le cadre de r&eacute;glementation de leurs activit&eacute;s et objectifs d&eacute;terminants de la phosphorylation de hBVR de PKC&delta;. S&eacute;rine analyse LC-MS/MS de PKC&delta;-activ&eacute; intact hBVR identifi&eacute; phosphoryl&eacute;e positionne 21, 33, 230 et 237, correspondant au motif domaine hBVR Src homologie-2 (Ser(230) et Ser(237)), flanquant le motif de l&#39;ATP binding (Ser(21)) dans l&#39;ordre de php (Ser(33)) comme cible potentielle de PKC&delta;. SER(21) et Ser(230) ont &eacute;t&eacute; &eacute;galement phosphoryl&eacute; en peptides ax&eacute;e sur les hBVR. Le peptide contenant 230 de Ser est un substrat de haute affinit&eacute; pour la PKC&delta; in vitro et dans les cellules ; l&#39;affinit&eacute; relative &eacute;tait PKC&delta; &gt; PKC&beta;II &gt; PKC &zeta;. Deux peptides qui se chevauchent, s&#39;&eacute;tendant sur ce substrat, KRNRYLSF et SFHFKSGSL, sont des inhibiteurs efficaces de l&#39;activit&eacute; de la kinase PKC&delta; et PKC&delta;-prise en charge de l&#39;activation de facteurs de transcription Elk1 et NF-&kappa;B. Seulement SFHFKSGSL, en 12-myristate 13-ac&eacute;tate-stimul&eacute;e cellules transfect&eacute;es PKC&delta; phorbol, par membrane blebbing et cellule baisse. La biliverdine inhib&eacute;e noncovalent PKC&delta;, tandis que PKC&delta; a potentialis&eacute; l&#39;activit&eacute; hBVR r&eacute;ductase et acc&eacute;l&eacute;r&eacute; le rythme de la formation de bilirubine. Cette &eacute;tude, ainsi que les conclusions pr&eacute;c&eacute;dentes, r&eacute;v&egrave;le une interaction r&eacute;glementaire inattendue entre PKC&delta; et hBVR dans la modulation de la mort/la survie des cellules en r&eacute;ponse &agrave; divers stimuli activant. En outre, cette &eacute;tude a identifi&eacute; de nouveaux substrats pour et inhibiteurs de la PKC&delta;. Nous sugg&egrave;rent que la technologie ax&eacute;e sur les hBVR peut-&ecirc;tre avoir utilitaire pour moduler les fonctions de m&eacute;diation PKC&delta; dans la cellule.

Les fragments de peptide ax&eacute;e sur la r&eacute;ductase humaine biliverdine et la biliverdine r&eacute;glementent l&#39;activit&eacute; prot&eacute;ine kinase C&delta; : les peptides sont inhibiteurs ou substrat pour la prot&eacute;ine kinase C.

PKC&delta;, una Ser/Thr chinasi, promuove la crescita cellulare apoptosi e tumorigenesi. Riduttasi di biliverdin umana (hBVR), una chinasi Ser/Thr/Tyr, inibisce l&#39;apoptosi riducendo IX Biliverdina a bilirubina antiossidante. Gli enzimi sono attivati da stimoli simili. Secondo quanto riferito, hBVR &egrave; un attivatore della chinasi-indipendente di PKC&delta; ed &egrave; transactivated dalla PKC (Gibbs, P. E., Miralem, T., Lerner-Marmarosh, N., Tudor, C. e Maines, M. D. (2012) j Biol Chem. 287, 1066-1079). Attualmente, abbiamo esaminato le interazioni tra le due proteine nel contesto del regolamento delle loro attivit&agrave; e definizione degli obiettivi della fosforilazione di hBVR di PKC&delta;. Serina analisi LC-MS/MS di hBVR intatta attivato PKC&delta; identificato fosforilato posizioni 21, 33, 230 e 237, corrispondente al motivo di dominio hBVR Src homology-2 (Ser(230) e Ser(237)), che fiancheggiano il motivo ATP-binding (Ser(21)) e in sequenza PHPS (Ser(33)) come obiettivi di PKC&delta;. Ser(21) e Ser(230) sono stati anche fosforilato in peptidi hBVR-based. Il peptide contenente 230 Ser era un substrato ad alta affinit&agrave; per PKC&delta; in vitro e in cellule; la relativa affinit&agrave; era PKC&delta; &amp;gt; PKC&beta;II &amp;gt; PKC&zeta;. Due peptidi sovrapposte che abbracciano questo substrato, KRNRYLSF e SFHFKSGSL, sono state efficaci inibitori della attivit&agrave; della chinasi PKC&delta; e supportati da PKC&delta; attivazione di fattori di trascrizione Elk1 e NF-&kappa;B. Solo SFHFKSGSL, in PKC&delta;-transfected forbolo miristato-12 13-acetato-stimolato cellule, ha causato perdita di membrana delle cellule e blebbing. Biliverdina inibito noncovalently PKC&delta;, mentre PKC&delta; potenziate hBVR attivit&agrave; reduttasi e accelerato il tasso di formazione di bilirubina. Questo studio, insieme ai risultati precedenti, rivela un&#39;inaspettata interazione normativo tra PKC&delta; e hBVR nel modulare la morte/la sopravvivenza delle cellule in risposta a vari stimoli di attivazione. Inoltre, questo studio ha individuato nuovi substrati per e inibitori della PKC&delta;. Suggeriamo che la tecnologia basata su hBVR pu&ograve; avere utilit&agrave; di modulare le funzioni PKC&delta;-mediata nella cella.

L&#39;umano Biliverdina reduttasi-basato del peptide frammenti e Biliverdina regolare attivit&agrave; della proteina chinasi C&delta;: i peptidi sono inibitori o substrato per la proteina chinasi C.

Article	Year
Biliverdin reductase, a novel regulator for induction of activating transcription factor-2 and heme oxygenase-1. The Journal of biological chemistry\| PubMed ID: 14988408	2004
Small interference RNA-mediated gene silencing of human biliverdin reductase, but not that of heme oxygenase-1, attenuates arsenite-mediated induction of the oxygenase and increases apoptosi... The Journal of biological chemistry\| PubMed ID: 15741166	2005
Human biliverdin reductase, a previously unknown activator of protein kinase C betaII. The Journal of biological chemistry\| PubMed ID: 17227757	2007
Regulation of TNF-alpha-activated PKC-zeta signaling by the human biliverdin reductase: identification of activating and inhibitory domains of the reductase. FASEB journal : official publication of the Federation of American Societies for Experimental Biology\| PubMed ID: 17639074	2007
Human biliverdin reductase is an ERK activator; hBVR is an ERK nuclear transporter and is required for MAPK signaling. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 18463290	2008
Human biliverdin reductase suppresses Goodpasture antigen-binding protein (GPBP) kinase activity: the reductase regulates tumor necrosis factor-alpha-NF-kappaB-dependent GPBP expression. The Journal of biological chemistry\| PubMed ID: 20177069	2010
Characterization of the human biliverdin reductase gene structure and regulatory elements: promoter activity is enhanced by hypoxia and suppressed by TNF-alpha-activated NF-kappaB. FASEB journal : official publication of the Federation of American Societies for Experimental Biology\| PubMed ID: 20410444	2010
Formation of ternary complex of human biliverdin reductase-protein kinase CÎ´-ERK2 protein is essential for ERK2-mediated activation of Elk1 protein, nuclear factor-ÎºB, and inducible nitric... The Journal of biological chemistry\| PubMed ID: 22065579	2012
The human biliverdin reductase-based peptide fragments and biliverdin regulate protein kinase CÎ´ activity: the peptides are inhibitors or substrate for the protein kinase C. The Journal of biological chemistry\| PubMed ID: 22584576	2012

Tihomir Miralem

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