Bimolecular Fluorescence Complementation

Defining the subcellular distribution of signaling complexes is imperative to understanding the output from that complex. Conventional methods such as ...

Previous studies of ion channel regulation by G proteins have focused on the larger, heterotrimeric GTPases, which are activated by heptahelical membrane receptors. In contrast, studies of the Rho family of smaller, monomeric, Ras-related GTPases, which are activated by cytoplasmic guanine nucleotide exchange factors, have focused on their role in cytoskeletal regulation.

Rac and Rho mediate opposing hormonal regulation of the ether-a-go-go-related potassium channel.

Rac und Rho vermitteln gegnerischen hormonelle Regulation des &#xC4;thers-a-go-go-bezogenen Kaliumkanal.

The covalent attachment of ubiquitin to proteins is an evolutionarily conserved signal for rapid protein degradation. However, additional cellular functions for ubiquitination are now emerging, including regulation of protein trafficking and endocytosis. For example, recent genetic studies suggested a role for ubiquitination in regulating epsin, a modular endocytic adaptor protein that functions in the assembly of clathrin-coated vesicles; however, biochemical evidence for this notion has been lacking. Epsin consists of an epsin NH(2)-terminal homology (ENTH) domain that promotes the interaction with phospholipids, several AP2 binding sites, two clathrin binding sequences, and several Eps15 homology (EH) domain binding motifs. Interestingly, epsin also possesses several recently described ubiquitin-interacting motifs (UIMs) that have been postulated to bind ubiquitin. Here, we demonstrate that epsin is predominantly monoubiquitinated and resistant to proteasomal degradation. The UIMs are necessary for epsin ubiquitination but are not the site of ubiquitination. Finally, we demonstrate that the isolated UIMs from both epsin and an unrelated monoubiquitinated protein, Eps15, are sufficient to promote ubiquitination of a chimeric glutathione-S-transferase (GST)-UIM fusion protein. Thus, our data suggest that UIMs may serve as a general signal for ubiquitination.

The ubiquitin-interacting motifs target the endocytic adaptor protein epsin for ubiquitination.

Die Ubiquitin-interagierenden Motive zielen die endozytischen Adaptorprotein Epsin f&#xFC;r Ubiquitinierung.

Intersectin (ITSN) is a molecular scaffold involved in regulating endocytosis and mitogenic signaling. We previously demonstrated that ITSN transformed rodent fibroblasts, accelerated hormone-induced maturation of Xenopus oocytes, and activated the Elk-1 transcription factor through an MEK- and Erk-independent mechanism. We now demonstrate that ITSN complexes with the Ras guanine nucleotide exchange factor Sos1 leading to increased RasGTP levels. Using fluorescence resonant energy transfer analysis, we demonstrate that ITSN complexes with Ras in living cells leading to Ras activation on intracellular vesicles. These vesicles contain epidermal growth factor receptor but are distinct from transferrin-positive vesicles. However, Ras is not required for ITSN stimulation of transcription. Rather, we demonstrate that ITSN signals through JNK to activate Elk-1. Although ITSN activation of Elk-1 was Ras-independent, ITSN cooperates with Ras to synergistically activate JNK. These findings indicate that ITSN activates multiple intracellular signaling pathways and suggest that this adaptor protein may coordinately regulate the activity of these pathways in vivo.

Intersectin activates Ras but stimulates transcription through an independent pathway involving JNK.

Intersectin Ras aktiviert, sondern stimuliert die Transkription durch eine unabh&#xE4;ngige Biosyntheseweg mit JNK.

Neuroblastoma (NB) and the Ewing sarcoma (ES)/peripheral primitive neuroectodermal tumor (PNET) family are pediatric cancers derived from neural crest cells. Although NBs display features of the sympathetic nervous system, ES/PNETs express markers consistent with parasympathetic differentiation. To examine the control of these differentiation markers, we generated NB x ES/PNET somatic cell hybrids. NB-specific markers were suppressed in the hybrids, whereas ES/PNET-specific markers were unaffected. These results suggested that the Ews/Fli-1 fusion gene, resulting from a translocation unique to ES/PNETs, might account for the loss of NB-specific markers. To test this hypothesis, we generated two different NB cell lines that stably expressed the Ews/Fli-1 gene. We observed that heterologous expression of the Ews/Fli-1 protein led to the suppression of NB-specific markers and de novo expression of ES/PNET markers. To determine the extent of changes in differentiation, we used the Affymetrix GeneChip Array system to observe global transcriptional changes of genes. This analysis revealed that the gene expression pattern of the Ews/Fli-1-expressing NB cells resembled that observed in pooled ES/PNET cell lines and differed significantly from the NB parental cells. Therefore, we propose that Ews/Fli-1 contributes to the etiology of ES/PNET by subverting the differentiation program of its neural crest precursor cell to a less differentiated and more proliferative state.

The Ews/Fli-1 fusion gene switches the differentiation program of neuroblastomas to Ewing sarcoma/peripheral primitive neuroectodermal tumors.

Die Ews/Fli-1 Fusionsgen schaltet das Programm Differenzierung von Neuroblastomen zu Ewing-Sarkom / periphere primitiven neuroektodermalen Tumoren.

An emerging concept in signal transduction is the organization of neuronal receptors and channels into microdomains in which signaling proteins are brought together to regulate functional responses. With the multiplicity of potential protein-protein interactions arises the need for the regulation and timing of these interactions. We have identified N-type Ca(2+) channel-signaling molecule complexes formed at different times upon activation of gamma-aminobutyric acid, type B, receptors. The first type of interaction involves pre-association of signaling proteins such as Src kinase with the Ca(2+) channel, because it is rapidly activated by the receptors and regulates the magnitude of the inhibition of the Ca(2+) channel. The second type of interaction involves signaling molecules that are recruited to the channel by receptor activation and control the rate of the channel response. Recruitment of members of the Ras pathway has two effects as follows: 1) modulation of the rate of onset of the gamma-aminobutyric acid-mediated inhibition of Ca(2+) current, and 2) activation of MAP kinase. Our results suggest that the Ca(2+) channel alpha(1) subunit functions as a dynamic scaffold allowing assembly of intracellular signaling components that alter channel activity and route signals to the MAP kinase pathway.

N-type Ca2+ channels as scaffold proteins in the assembly of signaling molecules for GABAB receptor effects.

N-Typ-Ca 2 +-Kan&#xE4;le als Ger&#xFC;stproteine &#x200B;&#x200B;in der Anordnung von Signalmolek&#xFC;len f&#xFC;r GABAB-Rezeptor Effekte.

The ubiquitin-interacting motif (UIM) is a short peptide motif with the dual function of binding ubiquitin and promoting ubiquitylation. This motif is conserved throughout eukaryotes and is present in numerous proteins involved in a wide variety of cellular processes including endocytosis, protein trafficking, and signal transduction. We previously reported that the UIMs of epsin were both necessary and sufficient for its ubiquitylation. In this study, we found that many, but not all, UIM-containing proteins were ubiquitylated. When expressed as chimeric fusion proteins, most UIMs promoted ubiquitylation of the chimera. In contrast to previous studies, we found that UIMs do not exclusively promote monoubiquitylation but rather a mixture of mono-, multi-, and polyubiquitylation. However, UIM-dependent polyubiquitylation does not lead to degradation of the modified protein. UIMs also bind polyubiquitin chains of varying lengths and to different degrees, and this activity is required for UIM-dependent ubiquitylation. Mutational analysis of the UIM revealed specific amino acids that are important for both polyubiquitin binding and ubiquitin conjugation. Finally we provide evidence that UIM-dependent ubiquitylation inhibits the interaction of UIM-containing proteins with other ubiquitylated cellular proteins. Our results suggest a new model for the ubiquitylation of UIM-containing proteins.

Analysis of the role of ubiquitin-interacting motifs in ubiquitin binding and ubiquitylation.

Analyse der Rolle von Ubiquitin-interagierenden Motive in Ubiquitin-bindende und Ubiquitinierung.

Die U-Box-Ligase Carboxyl-Terminus von HSC 70-interagierende Protein ubiquitiny Epsin.

Epsin is an endocytic adaptor protein involved in the regulation of clathrin-dependent endocytosis. We and others have demonstrated that Epsin is ubiquitylated in cells and requires its ubiquitin interacting motifs (UIMs) for this modification. To further elucidate the mechanism of Epsin ubiquitylation, we initiated studies to identify the E3 ligase(s) that modifies Epsin. In this study, we discovered that the U-box ubiquitin ligase carboxyl-terminus of Hsc70 interacting protein (CHIP) ubiquitylated Epsin. Using an in vitro ubiquitylation assay, we demonstrate that CHIP specifically ubiquitylated Epsin in a UIM-dependent manner. Furthermore, overexpression of CHIP in cells increased Epsin ubiquitylation also in a UIM-dependent manner. Together, these data provide evidence that CHIP functions to ubiquitylate the endocytic protein Epsin.

The U-box ligase carboxyl-terminus of Hsc 70-interacting protein ubiquitylates Epsin.

Receptor tyrosine kinases (RTKs) are critical for normal cell growth, differentiation, and development, but they contribute to various pathological conditions when disrupted. Activation of RTKs stimulates a plethora of pathways, including the ubiquitylation and endocytosis of the receptor itself. Although endocytosis terminates RTK signaling, it has emerged as a requisite step in RTK activation of signaling pathways. We have discovered that the endocytic scaffolding protein intersectin (ITSN) cooperated with epidermal growth factor receptor (EGFR) in the regulation of cell growth and signaling. However, a biochemical link between ITSN and EGFR was not defined. In this study, we demonstrate that ITSN is a scaffold for the E3 ubiquitin ligase Cbl. ITSN forms a complex with Cbl in vivo mediated by the Src homology (SH) 3 domains binding to the Pro-rich COOH terminus of Cbl. This interaction stimulates the ubiquitylation and degradation of the activated EGFR. Furthermore, silencing ITSN by RNA interference attenuated EGFR internalization as well as activation of the extracellular signal-regulated kinasemitogen-activated protein kinase pathway, thereby demonstrating the importance of ITSN in EGFR function. Given the cooperativity between ITSN and additional RTKs, these results point to an important evolutionarily conserved, regulatory role for ITSN in RTK function that is necessary for both signaling from receptors as well as the ultimate termination of receptor signaling.

Intersectin regulates epidermal growth factor receptor endocytosis, ubiquitylation, and signaling.

Rezeptor-Tyrosin-Kinasen (RTKs) sind entscheidend f&uuml;r die normale Zelle Wachstum, Differenzierung und Entwicklung, aber sie tragen zu verschiedenen pathologischen Bedingungen wenn gest&ouml;rt. Aktivierung des RTKs stimuliert eine F&uuml;lle von Optionen, einschlie&szlig;lich der Ubiquitylation und der Endozytose des Rezeptors selbst. Obwohl Endozytose RTK Signalisierung k&uuml;ndigt, hat sich als notwendigen Schritt in RTK-Aktivierung der Signalwege herausgestellt. Wir haben entdeckt, dass das endocytic Ger&uuml;st-Protein intersectin (ITSN) mit epidermalen Wachstumsfaktor-Rezeptor (EGFR) an der Regulierung des Zellwachstums und Signalisierung zusammengearbeitet. Eine biochemische Verbindung zwischen ITSN und EGFR wurde jedoch nicht definiert. In dieser Studie zeigen wir, dass ITSN, dass ein Ger&uuml;st ist f&uuml;r die E3 Ubiquitin Ligase Cbl. ITSN einen Komplex mit Cbl in-vivo bildet durch die Src-Homologie (SH) 3 Bereiche binden an die Pro-rich-COOH-Endstation der Cbl vermittelt. Diese Interaktion stimuliert die Ubiquitylation und den Abbau des aktivierten EGFR. Dar&uuml;ber hinaus vermindert Unterdr&uuml;ckender ITSN von RNA-Interferenz EGFR Internalisierung sowie die Aktivierung des extrazellul&auml;re Signal-regulierten Kinasemitogen aktivierte Protein-Kinase Signalweges, demonstrieren und damit die Bedeutung der ITSN im EGFR-Funktion. Angesichts der Kooperativit&auml;t ITSN bis zus&auml;tzliche RTKs, verweisen diese Ergebnisse auf eine evolution&auml;r konservierte, regulatorische Rolle f&uuml;r ITSN in RTK-Funktion, die f&uuml;r beide Signalisierung von Rezeptoren sowie die ultimative K&uuml;ndigung Rezeptor signalisieren notwendig.

Intersectin regelt die epidermalen Wachstumsfaktor-Rezeptor Endozytose und Ubiquitylation signalisieren.

Huntingon's disease is a progressive neurodegenerative disease arising from expansion of a polyglutamine (polyQ) tract in the protein huntingtin (Htt) resulting in aggregation of mutant Htt into nuclear and/or cytosolic inclusions in neurons. Mutant Htt affects multiple processes including protein degradation, transcription, signal transduction, fast axonal transport and endocytosis [reviewed in Ross, C.A. and Poirier, M.A. (2005) Opinion: what is the role of protein aggregation in neurodegeneration? Nat. Rev. Mol. Cell. Biol., 6, 891-898]. Here, we report that the endocytic and signal transduction scaffold intersectin (ITSN) increased aggregate formation by mutant Htt through activation of the c-Jun-NH(2)-terminal kinase (JNK)-MAPK pathway. Conversely, silencing ITSN or inhibiting JNK attenuated aggregate formation. Using a Drosophila model for polyQ repeat disease, we observed that ITSN enhanced polyQ-mediated neurotoxicity. A reciprocal relationship was observed between ITSN and Htt. While ITSN enhanced Htt aggregation and toxicity, Htt, in turn, inhibited the cooperativity between ITSN and the epidermal growth factor receptor signal transduction pathway. Finally, we observed that ITSN overexpression enhanced aggregation of polyQ-expanded androgen receptor (AR) as well as wild-type versions of both Htt and AR suggesting a broader involvement of ITSN in neurodegenerative diseases through destabilization of polyQ-containing proteins.

Intersectin enhances huntingtin aggregation and neurodegeneration through activation of c-Jun-NH2-terminal kinase.

Huntingon Krankheit ist eine progressive Neurodegenerative Erkrankung, die durch Ausbau der eine bei Polyglutamin (PolyQ)-Darm-Trakt in der Protein-Chorea Huntington (Htt) die Aggregation der Mutant Htt in nuklearen und/oder Zytosolische Einschl&uuml;ssen in Neuronen. Mutant Htt betrifft mehrere Prozesse einschlie&szlig;lich Proteinabbau, Transkription, Signaltransduktion, schnell axonale Transport und Endozytose [&uuml;berpr&uuml;ft in Ross, C.A. und Poirier, M.A. (2005)-Stellungnahme: Was ist die Rolle der Proteinanh&auml;ufung in Neurodegeneration? Nat Rev. Mol Cell. Biol., 6, 891-898]. Hier berichten wir, dass die endocytic und Signal-Transduktion Ger&uuml;st intersectin (ITSN) aggregieren Bildung von Mutant Htt durch Aktivierung der c-Jun-NH 2-Terminal kinase (JNK) erh&ouml;ht-MAPK Weg. Umgekehrt vermindert Unterdr&uuml;ckender ITSN oder Hemmung JNK Aggregat Bildung. Mit einem Drosophila-Modell f&uuml;r PolyQ wiederholen Krankheit, beobachteten wir, dass ITSN PolyQ-vermittelte Neurotoxizit&auml;t verbessert. Eine reziproke Beziehung wurde zwischen ITSN und Htt beobachtet. W&auml;hrend ITSN Htt-Aggregation und Toxizit&auml;t gesteigert, hemmte Htt, wiederum die Kooperativit&auml;t ITSN bis den epidermalen Wachstumsfaktor-Rezeptor Signaltransduktion Weg. Schlie&szlig;lich beobachteten wir, dass ITSN &Uuml;berexpression Aggregation von PolyQ erweiterten Androgenrezeptor (AR) sowie Wildtyp Versionen von Htt und AR schl&auml;gt eine breitere Beteiligung der ITSN bei neurodegenerativen Krankheiten durch Destabilisierung des PolyQ-haltige Proteine verst&auml;rkt.

Intersectin verbessert die Chorea Huntington-Aggregation und Neurodegeneration durch Aktivierung der c-Jun-NH2-Terminal Kinase.

While endocytosis attenuates signals from plasma membrane receptors, recent studies suggest that endocytosis also serves as a platform for the compartmentalized activation of cellular signaling pathways. Intersectin (ITSN) is a multidomain scaffolding protein that regulates endocytosis and has the potential to regulate various biochemical pathways through its multiple, modular domains. To address the biological importance of ITSN in regulating cellular signaling pathways versus in endocytosis, we have stably silenced ITSN expression in neuronal cells by using short hairpin RNAs. Decreasing ITSN expression dramatically increased apoptosis in both neuroblastoma cells and primary cortical neurons. Surprisingly, the loss of ITSN did not lead to major defects in the endocytic pathway. Yeast two-hybrid analysis identified class II phosphoinositide 3'-kinase C2beta (PI3K-C2beta) as an ITSN binding protein, suggesting that ITSN may regulate a PI3K-C2beta-AKT survival pathway. ITSN associated with PI3K-C2beta on a subset of endomembrane vesicles and enhanced both basal and growth factor-stimulated PI3K-C2beta activity, resulting in AKT activation. The use of pharmacological inhibitors, dominant negatives, and rescue experiments revealed that PI3K-C2beta and AKT were epistatic to ITSN. This study represents the first demonstration that ITSN, independent of its role in endocytosis, regulates a critical cellular signaling pathway necessary for cell survival.

Regulation of neuron survival through an intersectin-phosphoinositide 3'-kinase C2beta-AKT pathway.

Wenn Endozytose Signale von Plasma Membranrezeptoren d&auml;mpft, deuten neuere Studien, dass die Endozytose dient auch als Plattform f&uuml;r die zergliederten Aktivierung der zellul&auml;ren Signalisierung Wege. Intersectin (ITSN) ist ein mit Ger&uuml;sten-Protein, die Endozytose reguliert und hat das Potenzial zur Regulierung von verschiedenen biochemischen Bahnen durch seine mehrere, modulare Dom&auml;nen. Um die biologische Bedeutung der ITSN bei der Regulierung der zellul&auml;re Signalwege in Endozytose im Vergleich zu beheben, haben wir stabil ITSN Ausdruck in Nervenzellen verstummen, mit kurzen Haarnadel RNAs. ITSN Ausdruck drastisch verringern, erh&ouml;ht Apoptosis in den Neuroblastoma Zellen und prim&auml;ren kortikalen Neuronen. &Uuml;berraschenderweise f&uuml;hrte der Verlust von ITSN nicht zu gro&szlig;e M&auml;ngel in der endocytic Weg. Hefe-zwei-Hybrid-Analyse identifiziert die Klasse II Phosphoinositide 3&#39;-Kinase C2beta (PI3K-C2beta) als ein ITSN verbindliches Protein, darauf hindeutet, dass ITSN PI3K-C2beta-AKT &uuml;berleben Weg Regeln kann. ITSN zugeordnete PI3K-C2beta auf einer Untermenge von Endomembrane Bl&auml;schen und zur Verbesserung der basalen und Wachstumsfaktor stimuliert PI3K-C2beta T&auml;tigkeit, was zu AKT Aktivierung. Die Verwendung von pharmakologische Inhibitoren, dominante negative und Rettung Experimente ergaben, dass PI3K-C2beta und AKT zu ITSN epistatic waren. Diese Studie stellt die erste Demonstration, dass ITSN, unabh&auml;ngig von seiner Rolle bei der Endozytose, eine kritische zellular signalisieren Weg notwendig f&uuml;r das &Uuml;berleben der Zelle reguliert.

Verordnung von Neuron &Uuml;berleben durch eine intersectin-Phosphoinositid-3&#39;-Kinase-C2beta-AKT-Pathway.

S5a/Rpn10 is a ubiquitin (Ub)-binding protein that is a subunit of the 26S proteasome but also exists free in the cytosol. It binds poly-Ub chains through its two Ub-interacting motifs (UIMs). We discovered that, unlike typical substrates of Ub ligases (E3s), S5a can be ubiquitinated by all E3s tested including multimeric and monomeric Ring finger E3s (MuRF1, Siah2, Parkin, APC, and SCF(betaTRCP1)), the U-box E3, CHIP, and HECT domain E3s (E6AP and Nedd4) when assayed with UbcH5 or related Ub-conjugating enzymes. However, the E2s, UbcH1 and UbcH13/Uev1a, which function by distinct mechanisms, do not support S5a ubiquitination. Thus, S5a can be used for assay of probably all E3s with UbcH5. Ubiquitination of S5a results from its binding to Ub chains on the E3 (after self-ubiquitination) or on the substrate, as a mutant lacking the UIM domain was not ubiquitinated. Furthermore, if the S5a UIM domains were fused to GST, the protein was rapidly ubiquitinated by MuRF1 and CHIP. In addition, polyubiquitination (but not monoubiquitination) of MuRF1 allowed S5a to bind to MuRF1 and accelerated S5a ubiquitination. This tendency of S5a to associate with the growing Ub chain can explain how S5a, unlike typical substrates, which are recognized by certain E3s through specific motifs, is ubiquitinated by all E3s tested and is rapidly degraded in vivo.

The ubiquitin-interacting motif protein, S5a, is ubiquitinated by all types of ubiquitin ligases by a mechanism different from typical substrate recognition.

S5a/Rpn10 ist ein Ubiquitin (Ub)-verbindliches Protein, das eine Untereinheit des der 26S Proteasom aber auch kostenlos in das Zytosol existiert. Es bindet Poly-Ub-Ketten durch seine zwei Ub-Interaktion Motive (UIMs). Wir entdeckten, dass im Gegensatz zu typischen Substrate der Ub-Verkn&uuml;pfung (E3s), S5a Ubiquitinated von allen E3s getestet werden und Monomer Ringfinger E3s sein kann (MuRF1, Siah2, Parkin, APC, und SCF(betaTRCP1)), die U-Box E3, CHIP und HECT Dom&auml;ne E3s (E6AP und Nedd4) mit UbcH5 normalisiert oder Ub-konjugierenenzym Enzyme in Verbindung stehen. Allerdings unterst&uuml;tzen die E2s, UbcH1 und UbcH13/Uev1a, die durch verschiedene Mechanismen funktionieren, keine S5a Ubiquitination. So k&ouml;nnen S5a f&uuml;r Probe der wahrscheinlich alle E3s mit UbcH5 verwendet werden. Ubiquitination S5a resultiert aus seiner Bindung an Ub-Ketten auf der E3 (nach selbst-Ubiquitination) oder auf dem Substrat, wie eine Mutante fehlt der UIM-Dom&auml;ne nicht Ubiquitinated war. Au&szlig;erdem wenn die S5a UIM-Dom&auml;nen zu GST verschmolzen waren, war das Protein schnell Ubiquitinated durch MuRF1 und CHIP. Dar&uuml;ber hinaus Polyubiquitination (aber nicht Monoubiquitination) des MuRF1 erlaubt S5a binden an MuRF1 und S5a Ubiquitination beschleunigt. Diese Tendenz der S5a zuordnen zu der wachsenden Ub-Kette kann erkl&auml;re, S5a, im Gegensatz zu typischen Substraten, die von bestimmten E3s durch bestimmte Motive erkannt werden, ist Ubiquitinated von allen E3s getestet und ist schnell in-vivo degradiert.

Das Ubiquitin-Interaktion Motiv Protein, S5a, ist Ubiquitinated durch alle Arten von Ubiquitin-Verkn&uuml;pfung durch einen Mechanismus, der anders als typische Substrat Anerkennung.

The endocytic pathway is involved in activation and inhibition of cellular signaling. Thus, defining the regulatory mechanisms that link endocytosis and cellular signaling is of interest. An emerging link between these processes is a family of proteins called intersectins (ITSNs). These multidomain proteins serve as scaffolds in the assembly of endocytic vesicles and also regulate components of various signaling pathways, including kinases, guanosine triphosphatases, and ubiquitin ligases. This review summarizes research on the role of ITSNs in regulating both endocytic and signal transduction pathways, discusses the link between ITSNs and human disease, and highlights future directions in the study of ITSNs.

Intersecting pathways in cell biology.

Der endocytic Weg ist Aktivierung und Hemmung der zellul&auml;ren Signalisierung beteiligt. Deshalb definieren die Regulierungsmechanismen, die Endozytose und zellularen Signalisierens Verkn&uuml;pfen von Interesse. Eine neue Verbindung zwischen diesen Prozessen ist eine Familie von Proteinen, genannt Intersectins (ITSNs). Diese Dom&auml;nen Proteine dienen als Ger&uuml;ste in der Versammlung des endocytic Bl&auml;schen und auch Komponenten von verschiedenen Signalwege, darunter Kinasen, Guanosin Triphosphatases und Ubiquitin-Verkn&uuml;pfung zu regulieren. Diesen Bericht fasst Forschung &uuml;ber die Rolle der ITSNs bei der Regulierung der beiden endocytic und Signal Transduction Bahnen, beschreibt den Zusammenhang zwischen ITSNs und menschlicher Erkrankungen und zuk&uuml;nftige Richtungen in der Studie des ITSNs unterstreicht.

Sich &uuml;berschneidende Pfade in der Zellbiologie.

In the present study, we assessed the cooperative roles of C-terminal Src kinase (Csk) binding protein (Cbp) and Caveolin-1 (Cav-1) in the mechanism of Src family tyrosine kinase (SFK) inhibition by Csk. SFKs are inactivated by phosphorylation of their C-terminal tyrosine by Csk. Whereas SFKs are membrane-associated, Csk is a cytoplasmic protein and therefore requires membrane adaptors such as Cbp or Cav-1 for recruitment to the plasma membrane to mediate SFK inhibition. To determine the specific role of Cav-1 and Cbp in SFK inhibition, we measured c-Src activity in the absence of each membrane adaptor. It is noteworthy that in lungs and fibroblasts from Cav-1(-/-) mice, we observed increased expression of Cbp compared with wild-type (WT) controls. However, both c-Src activity and Csk localization at the membrane were similar between Cav-1(-/-) fibroblasts and WT cells. Likewise, Cbp depletion by small interfering RNA (siRNA) treatment of WT cells had no effect on basal c-Src activity, but it increased the phosphorylation state of Cav-1. Immunoprecipitation then confirmed increased association of Csk with phosphomimicking Cav-1. Knockdown of Cbp by siRNA in Cav-1(-/-) cells revealed increased basal c-Src activity, and re-expression of WT Cav-1 in the same cells reduced basal c-Src activity. Taken together, these results indicate that Cav-1 and Cbp cooperatively regulate c-Src activity by recruiting Csk to the membrane where it phosphorylates c-Src inhibitory tyrosine 529. Furthermore, when either Cav-1 or Cbp expression is reduced or absent, there is a compensatory increase in the phosphorylation state or expression level of the other membrane-associated Csk adaptor to maintain SFK inhibition.

Cooperative role of caveolin-1 and C-terminal Src kinase binding protein in C-terminal Src kinase-mediated negative regulation of c-Src.

In der vorliegenden Studie wir beurteilen die kooperativen Rollen der C-terminalen Src Kinase (Csk) verbindliches Protein (Cbp) und Caveolin-1 (Cav-1) in den Mechanismus der Src-Familie Tyrosin Kinase (SFK) Hemmung von Csk. SFKs sind durch Phosphorylierung ihre C-terminalen Tyrosin durch Csk. w&auml;hrend SFKs inaktiviert sind Membran-assoziierte, Csk ist ein zellplasmatisches Protein und erfordert daher Membran-Adapter wie Cbp oder Cav-1 f&uuml;r Rekrutierung durch die Plasmamembran SFK Hemmung zu vermitteln. Um die besondere Rolle der Cav-1 und Cbp SFK Hemmung zu bestimmen, messen wir c-Src Aktivit&auml;t ohne jeden Membran-Adapter. Es ist bemerkenswert, dass in Lunge und Fibroblasten aus Cav-1(-/-) M&auml;use, wir erh&ouml;hte Expression von Cbp im Vergleich zu Wildtyp (WT) Steuerelemente beobachtet. Allerdings waren die c-Src-Aktivit&auml;t und Csk Lokalisierung auf der Membran zwischen Cav-1(-/-) Fibroblasten und WT-Zellen &auml;hnlich. Ebenso Cbp Ersch&ouml;pfung durch kleine interferierender RNA (SiRNA) Behandlung von WT-Zellen hatten keine Auswirkungen auf basale c-Src-Aktivit&auml;t, aber es erh&ouml;ht den Phosphorylierung Zustand der Cav-1. Immunopr&auml;zipitation best&auml;tigt dann erh&ouml;hte Assoziation von Csk mit Phosphomimicking Cav-1. Niederschlag des Cbp von SiRNA in Cav-1(-/-) Zellen ergab erh&ouml;hte basale c-Src-Aktivit&auml;t und Re-expression WT-Cav-1 in denselben Zellen geringer basaler c-Src-Aktivit&auml;t. Zusammengenommen zeigen diese Ergebnisse, dass Cav-1 Cbp kooperativ Regulierung und c-Src-Aktivit&auml;t durch die Rekrutierung von Csk an der Membran, wo es c-Src hemmenden Tyrosin 529 phosphorylates. Dar&uuml;ber hinaus Wenn Cav-1 oder Cbp Ausdruck wird verringert oder abwesend, gibt es eine kompensatorische Erh&ouml;hung Phosphorylierung Zustand oder Ausdruck des anderen Membran-assoziierte Csk Adapters SFK Hemmung beibehalten.

Kooperative Rolle von Caveolin-1 und C-terminalen Src Kinase verbindliches Protein in der C-terminalen Src Kinase-vermittelte negative Regulation des c-Src.

Intersectin 1 (ITSN1) ist eine menschliche Chromosom 21 (HSA21)-Genprodukt Codierung ein Ger&uuml;st mit mehreren Domains-Protein, das in der Endozytose funktioniert und Signaltransduktion in Down-Syndrom, Morbus Alzheimer und potentiell andere Neurodegenerative Krankheiten durch Aktivierung der c-Jun N-Terminal Kinase verwickelt ist. Wir Berichten f&uuml;r die erste Zeit, die ITSN1 Proteine bei Patienten mit Down-Syndrom unterschiedlicher Altersgruppen erh&ouml;ht werden. Jedoch sank um ITSN1 Ebenen im Alter von F&auml;llen mit Down-Syndrom mit Alzheimer-Krankheit-wie Neuropathologie. Analyse eines Romans ITSN1 transgenen Maus zeigt, dass ITSN1 eine &Uuml;berexpression Ergebnisse in ein Geschlecht-abh&auml;ngigen im lokomotorischen T&auml;tigkeit zu verringern. Diese Studie zeigt einen Zusammenhang zwischen eine &Uuml;berexpression von spezifischen ITSN1-Isoformen und verhaltensbasierte Ph&auml;notypen und hat Auswirkungen auf die menschliche neurodegenerativer Erkrankungen wie das Down-Syndrom und Alzheimer-Krankheit.

Intersectin 1 tr&auml;gt zur in-vivo Ph&auml;notypen: Implikationen f&uuml;r Down-Syndrom.

Intersectin 1 (ITSN1) is a human chromosome 21 (HSA21) gene product encoding a multidomain scaffold protein that functions in endocytosis, signal transduction, and is implicated in Down's syndrome, Alzheimer's Disease, and potentially other neurodegenerative diseases through activation of c-Jun N-terminal kinase. We report for the first time that ITSN1 proteins are elevated in individuals with Down's syndrome of varying ages. However, ITSN1 levels decreased in aged cases with Down's syndrome with Alzheimer's disease-like neuropathology. Analysis of a novel ITSN1 transgenic mouse reveals that ITSN1 overexpression results in a sex-dependent decrease in locomotor activity. This study reveals a link between overexpression of specific ITSN1 isoforms and behavioral phenotypes and has implications for human neurodegenerative diseases such as Down's syndrome and Alzheimer's disease.

Intersectin 1 contributes to phenotypes in vivo: implications for Down's syndrome.

Ubiquitylation of receptor tyrosine kinases plays a critical role in regulating the trafficking and lysosomal degradation of these important signaling molecules. We identified the multidomain scaffolding protein intersectin 1 (ITSN1) as an important regulator of this process (N. P. Martin et al., Mol. Pharmacol. 70:1463-1653, 2006) ITSN1 stimulates ubiquitylation of the epidermal growth factor receptor (EGFR) through enhancing the activity of the Cbl E3 ubiquitin ligase. However, the precise mechanism through which ITSN1 enhances Cbl activity was unclear. In this study, we found that ITSN1 enhances Cbl activity through disrupting the interaction of Cbl with the Sprouty2 (Spry2) inhibitory protein. We demonstrate that ITSN1 binds Pro-rich regions in both Cbl and Spry2 and that interaction of ITSN1 with Spry2 disrupts Spry2-Cbl interaction, resulting in enhanced ubiquitylation of the EGFR. Disruption of ITSN1 binding to Spry2 through point mutation of the Pro-rich ITSN1 binding site in Spry2 results in enhanced Cbl-Spry2 interaction and inhibition of receptor ubiquitylation. This study demonstrates that ITSN1 enhances Cbl activity by modulating the interaction of Cbl with Spry2. In addition, our results reveal a new level of complexity in the regulation of Cbl through the interaction with ITSN1 and Spry2.

Intersectin 1 Enhances Cbl Ubiquitylation of Epidermal Growth Factor Receptor through Regulation of Sprouty2-Cbl Interaction.

Ubiquitylation von Rezeptor-Tyrosin-Kinasen spielt eine wichtige Rolle bei der Regulierung des Menschenhandel und lysosomalen Abbau dieser wichtigen signalisierende Molek&uuml;le. Wir das mit Ger&uuml;sten-Protein intersectin 1 (ITSN1) als ein wichtiger Regulator dieses Prozesses identifiziert (N. s. Martin Et Al., Mol Pharmacol. 70:1463-1653, 2006) ITSN1 stimuliert die Ubiquitylation von den epidermalen Wachstumsfaktor-Rezeptor (EGFR) durch die St&auml;rkung der Aktivit&auml;t der Cbl E3 Ubiquitin Ligase. Der genaue Mechanismus, durch den ITSN1 Cbl-Aktivit&auml;t erh&ouml;ht, war jedoch unklar. In dieser Studie fanden wir, dass ITSN1 Cbl-Aktivit&auml;t durch St&ouml;rung der Interaktion von Cbl mit den Sprouty2 erh&ouml;ht (Spry2) hemmendes Protein. Wir zeigen, dass ITSN1 reichenfreundlichen Regionen in Cbl und Spry2 bindet und die Interaktion von ITSN1 mit Spry2 Spry2-Cbl-Interaktion, st&ouml;rt, was zu verbesserten Ubiquitylation des EGFR. Unterbrechung der ITSN1 Bindung an Spry2 durch Punktmutation von der Pro-rich ITSN1-Bindungsstelle in Spry2 f&uuml;hrt zu verbesserten Cbl-Spry2-Interaktion und Hemmung der Rezeptor-Ubiquitylation. Diese Studie zeigt, dass ITSN1 Cbl Aktivit&auml;t verbessert durch Modulation der Interaktion von Cbl mit Spry2. Dar&uuml;ber hinaus zeigen unsere Ergebnisse eine neue Ebene der Komplexit&auml;t bei der Regulierung der Cbl durch die Interaktion mit ITSN1 und Spry2.

Intersectin 1 optimiert Cbl Ubiquitylation des epidermalen Wachstumsfaktor-Rezeptors durch Regulierung der Sprouty2-Cbl-Interaktion.

Mice expressing the tetracycline transactivator (tTA) transcription factor driven by the rat Î±-myosin heavy chain promoter (Î±-MHC-tTA) are widely used to dissect the molecular mechanisms involved in cardiac development and disease. However, these Î±-MHC-tTA mice exhibit a gain-of-function phenotype consisting of robust protection against ischemia/reperfusion injury in both in vitro and in vivo models in the absence of associated cardiac hypertrophy or remodeling. Cardiac function, as assessed by echocardiography, did not differ between Î±-MHC-tTA and control animals, and there were no noticeable differences observed between the two groups in HW/TL ratio or LV end-diastolic and end-systolic dimensions. Protection against ischemia/reperfusion injury was assessed using isolated perfused hearts where Î±-MHC-tTA mice had robust protection against ischemia/reperfusion injury which was not blocked by pharmacological inhibition of PI3Ks with LY294002. Furthermore, Î±-MHC-tTA mice subjected to coronary artery ligation exhibited significantly reduced infarct size compared to control animals. Our findings reveal that Î±-MHC-tTA transgenic mice exhibit a gain-of-function phenotype consisting of robust protection against ischemia/reperfusion injury similar to cardiac pre- and post-conditioning effects. However, in contrast to classical pre- and post-conditioning, the Î±-MHC-tTA phenotype is not inhibited by the classic preconditioning inhibitor LY294002 suggesting involvement of a non-PI3K-AKT signaling pathway in this phenotype. Thus, further study of the Î±-MHC-tTA model may reveal novel molecular targets for therapeutic intervention during ischemic injury.

Cardiac-specific expression of the tetracycline transactivator confers increased heart function and survival following ischemia reperfusion injury.

M&auml;use, die Ausdruck der Tetracyclin-Transactivator (tTA) &Uuml;bertragungfaktor, getrieben von der Ratte &alpha;-Myosin heavy Chain Projekttr&auml;ger (&alpha;-MHC-tTA) sind verbreitet, die molekularen Mechanismen, die kardiale Entwicklung und Krankheit beteiligt zu zergliedern. Allerdings zeigen diese &alpha;-MHC-tTA-M&auml;use einen Gain of Function-Ph&auml;notyp, bestehend aus robusten Schutz gegen Isch&auml;mie/Reperfusion Verletzung in in vitro und in-vivo-Modelle in Ermangelung von zugeordneten Herzhypertrophie oder Umbau. Unterschied Herzfunktion, die Echokardiographie, bewerten sich nicht zwischen &alpha;-MHC-tTA und Kontrolle Tiere, und es gab keine merkliche Unterschiede zwischen den beiden Gruppen in HW/TL-Verh&auml;ltnis oder LV End-diastolischen und End-systolische Dimensionen beobachtet. Schutz vor Isch&auml;mie/Reperfusion Verletzungen wurde anhand isolierte PERFUNDIERTE Herzen wo &alpha;-MHC-tTA M&auml;use hatte robusten Schutz gegen Isch&auml;mie/Reperfusion Verletzung, die nicht durch pharmakologische Hemmung der PI3Ks mit LY294002 blockiert wurde. Dar&uuml;ber hinaus stellte &alpha;-MHC-tTA M&auml;use ausgesetzt koronaren Verbindung deutlich reduzierten Infarktgr&ouml;&szlig;e im Vergleich zu Kontrolltieren. Unsere Ergebnisse zeigen, dass &alpha;-MHC-tTA transgene M&auml;use einen Gain of Function-Ph&auml;notyp, bestehend aus robusten Schutz gegen Isch&auml;mie/Reperfusion Verletzung &auml;hnlich kardiologische Pre- und post-conditioning Wirkungen zeigen. Jedoch wird im Gegensatz zu klassischen vor- und post-conditioning, der &alpha;-MHC-tTA-Ph&auml;notyp nicht von der klassischen Vorkonditionierung Inhibitor LY294002 vorschlagen Einbeziehung der eine PI3K-AKT-Signalweges in diesem Ph&auml;notyp gehemmt. So kann weitere Studie des Modells &alpha;-MHC-tTA neuartige Molekulare Ziele f&uuml;r therapeutische Intervention bei isch&auml;mischen Sch&auml;digung offenbaren.

Herz-spezifischen Ausdruck der Tetracyclin-Transactivator verleiht erh&ouml;hte Herz-Funktion und das &Uuml;berleben nach Isch&auml;mie Reperfusion Verletzung.

Article	Year
Rac and Rho mediate opposing hormonal regulation of the ether-a-go-go-related potassium channel. Current biology : CB\| PubMed ID: 11790300	2002
The ubiquitin-interacting motifs target the endocytic adaptor protein epsin for ubiquitination. Current biology : CB\| PubMed ID: 12121618	2002
Intersectin activates Ras but stimulates transcription through an independent pathway involving JNK. The Journal of biological chemistry\| PubMed ID: 12970366	2003
The Ews/Fli-1 fusion gene switches the differentiation program of neuroblastomas to Ewing sarcoma/peripheral primitive neuroectodermal tumors. Cancer research\| PubMed ID: 14973077	2004
N-type Ca2+ channels as scaffold proteins in the assembly of signaling molecules for GABAB receptor effects. The Journal of biological chemistry\| PubMed ID: 15047708	2004
Analysis of the role of ubiquitin-interacting motifs in ubiquitin binding and ubiquitylation. The Journal of biological chemistry\| PubMed ID: 15155768	2004
The U-box ligase carboxyl-terminus of Hsc 70-interacting protein ubiquitylates Epsin. Biochemical and biophysical research communications\| PubMed ID: 15694383	2005
Intersectin regulates epidermal growth factor receptor endocytosis, ubiquitylation, and signaling. Molecular pharmacology\| PubMed ID: 16914641	2006
Intersectin enhances huntingtin aggregation and neurodegeneration through activation of c-Jun-NH2-terminal kinase. Human molecular genetics\| PubMed ID: 17550941	2007
Regulation of neuron survival through an intersectin-phosphoinositide 3'-kinase C2beta-AKT pathway. Molecular and cellular biology\| PubMed ID: 17875942	2007
The ubiquitin-interacting motif protein, S5a, is ubiquitinated by all types of ubiquitin ligases by a mechanism different from typical substrate recognition. The Journal of biological chemistry\| PubMed ID: 19240029	2009
Intersecting pathways in cell biology. Science signaling\| PubMed ID: 21156937	2010
Cooperative role of caveolin-1 and C-terminal Src kinase binding protein in C-terminal Src kinase-mediated negative regulation of c-Src. Molecular pharmacology\| PubMed ID: 21778303	2011
Intersectin 1 contributes to phenotypes in vivo: implications for Down's syndrome. Neuroreport\| PubMed ID: 21876463	2011
Intersectin 1 Enhances Cbl Ubiquitylation of Epidermal Growth Factor Receptor through Regulation of Sprouty2-Cbl Interaction. Molecular and cellular biology\| PubMed ID: 22158968	2012
Cardiac-specific expression of the tetracycline transactivator confers increased heart function and survival following ischemia reperfusion injury. PloS one\| PubMed ID: 22272284	2012

John P. O'Bryan

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