Role of Cbl-associated protein/ponsin in receptor tyrosine kinase signaling and cell adhesion

The Cbl-associated protein/ponsin (CAP) is an adaptor protein that contains a so-called Sorbin homology (SoHo) domain and three Src homology 3 (SH3) domains which are engaged in diverse protein-protein interactions. CAP has been shown to function in the regulation of the actin cytoskeleton and cell adhesion and to be involved in the differentiation of muscle cells and adipocytes. In addition, it participates in signaling pathways through several receptor tyrosine kinases such as insulin and neurotrophin receptors. In the last couple of years, several studies have shed light on the details of these processes and identified novel interaction partners of CAP. In this review, we summarize these recent findings and provide an overview on the function of CAP especially in cell adhesion and membrane receptor signaling

Cbl-associated protein is tyrosine phosphorylated by c-Abl and c-Src kinases

Background: The c-Cbl-associated protein (CAP), also known as ponsin, localizes to focal adhesions and stress fibers and is involved in signaling events. Phosphorylation has been described for the other two members of the sorbin homology family, vinexin and ArgBP2, but no data exist about the putative phosphorylation of CAP. According to previous findings, CAP binds to tyrosine kinase c-Abl. However, it is not known if CAP is a substrate of c-Abl or other tyrosine kinases or if phosphorylation regulates its localization. Results: We here show that CAP is Tyr phosphorylated by and interacts with both c-Abl and c-Src. One major phosphorylation site, Tyr360, and two minor contributors Tyr326 and Tyr632 were identified as Abl phosphorylation sites, whereas Src preferentially phosphorylates Tyr326 and Tyr360. Phosphorylation of CAP was not necessary for its localization to focal adhesions and stress fibers, but Tyr326Phe substitution alters the function of CAP during cell spreading. Conclusion: This is the first demonstration of phosphorylation of CAP by any kinase. Our findings suggest that coordinated action of Src and Abl might regulate the function of CAP and reveal a functional role especially for the Src-mediated Tyr phosphorylation of CAP in cell spreading

Ltbp4 regulates Pdgfr beta expression via TGF beta-dependent modulation of Nrf2 transcription factor function

Latent transforming growth factor beta binding protein 4 (LTBP4) belongs to the fibrillin/LTBP family of proteins and plays an important role as a structural component of extracellular matrix (ECM) and local regulator of TGF beta signaling. We have previously reported that Ltbp4S knock out mice (Ltbp4S-/-) develop centrilobular emphysema reminiscent of late stage COPD, which could be partially rescued by inactivating the antioxidant protein Sestrin 2 (Sesn2). More recent studies showed that Sesn2 knock out mice upregulate Pdgfr beta-controlled alveolar maintenance programs that protect against cigarette smoke induced pulmonary emphysema. Based on this, we hypothesized that the emphysema of Ltbp4S-/- mice is primarily caused by defective Pdgfr beta signaling. Here we show that LTBP4 induces Pdgfr beta signaling by inhibiting the antioxidant Nr12/Keap1 pathway in a TGF beta-dependent manner. Overall, our data identified Ltbp4 as a major player in lung remodeling and injury repair. (C) 2016 The Authors. Published by Elsevier B.V.Peer reviewe

Quantum Kinetic Theory of BEC Lattice Gas:Boltzmann Equations from 2PI-CTP Effective Action

We continue our earlier work [Ana Maria Rey, B. L. Hu, Esteban Calzetta, Albert Roura and Charles W. Clark, Phys. Rev. A 69, 033610 (2004)] on the nonequilibrium dynamics of a Bose Einstein condensate (BEC) selectively loaded into every third site of a one-dimensional optical lattice. From the two-particle irreducible (2PI) closed-time-path (CTP) effective action for the Bose- Hubbard Hamiltonian, we show how to obtain the Kadanoff-Baym equations of quantum kinetic theory. Using the quasiparticle approximation, we show that the local equilibrium solutions of these equations reproduce the second- order corrections to the self-energy originally derived by Beliaev. This work paves the way for the use of effective action methods in the derivation of quantum kinetic theory of many atom systems.Comment: 21 pages, 0 figures, minor editorial changes were mad

Molecular Networks in FGF Signaling: Flotillin-1 and Cbl-Associated Protein Compete for the Binding to Fibroblast Growth Factor Receptor Substrate 2

Fibroblast growth factor receptor substrate 2 (FRS2α) is a signaling adaptor protein that regulates downstream signaling of many receptor tyrosine kinases. During signal transduction, FRS2 can be both tyrosine and threonine phosphorylated and forms signaling complexes with other adaptor proteins and tyrosine phosphatases. We have here identified flotillin-1 and the cbl-associated protein/ponsin (CAP) as novel interaction partners of FRS2. Flotillin-1 binds to the phosphotyrosine binding domain (PTB) of FRS2 and competes for the binding with the fibroblast growth factor receptor. Flotillin-1 knockdown results in increased Tyr phosphorylation of FRS2, in line with the inhibition of ERK activity in the absence of flotillin-1. CAP directly interacts with FRS2 by means of its sorbin homology (SoHo) domain, which has previously been shown to interact with flotillin-1. In addition, the third SH3 domain in CAP binds to FRS2. Due to the overlapping binding domains, CAP and flotillin-1 appear to compete for the binding to FRS2. Thus, our results reveal a novel signaling network containing FRS2, CAP and flotillin-1, whose successive interactions are most likely required to regulate receptor tyrosine kinase signaling, especially the mitogen activated protein kinase pathway

Audio-description reloaded : an analysis of visual scenes in 2012 and Hero

This article explores whether the so-called new "cinema of attractions", with its supposed focus on visual effects to the detriment of storytelling, requires a specific approach to audio-description (AD). After some thoughts on film narrative in this type of cinema and the way in which it incorporates special effects, selected scenes with AD from two feature films, 2012 (directed by Emmerich) and Hero (directed by Zhang Yimou), are analysed. 2012 is a disaster movie aiming to thrill the audience with action. Hero is an equally visual movie but its imagery has an aesthetic purpose. The analysis investigates how space, time and action are treated in the films and the ADs, and how the information is presented in terms of focalization, timing and phrasing. The results suggest that effect-driven narratives require carefully timed and phrased ADs that devote much attention to the prosody of the AD script, its interaction with sounds and the use of metapho

Molecular networks in signaling pathways: Flotillin-1, Cbl-associated protein and Fibroblast Growth Factor Receptor Substrate 2

The long sought molecular function of membrane raft-associated flotillin proteins is slowly becoming resolved, partially owing to the increasing knowledge about their interaction partners. Being ubiquitously expressed and evolutionarily highly conserved, flotillins carry out important cellular functions, one of which is the regulation of signal transduction pathways. This study shows that the signaling adaptor protein fibroblast growth factor receptor substrate 2 (FRS2) directly interacts both in vivo and in vitro with flotillin-1 (flot-1). FRS2 is an important docking protein of many receptor tyrosine kinases. It regulates downstream signaling by forming molecular complexes with other adaptor proteins and tyrosine phosphatases, and seems to be a critical mediator of sustained extracellular signal regulated kinase (ERK) activity. Flot-1 has also been implicated in the regulation of ERK activity upon EGF and FGF stimuli. Furthermore, flot-1 forms signalosomes with EGFR and the downstream components of the MAP kinase pathway. The newly discovered interaction between FRS2 and flot-1 was shown to be mediated by the phosphotyrosine binding (PTB) domain and, to a lesser extent, the C-terminus (CT) of FRS2 and by the C-terminus of flot-1. Flot-1 coprecipitated together with FRS2 from murine tissues and cell lysates, demonstrating that this interaction also takes place in vivo. Interestingly, flot-2, which shows a high homology to flot-1 and forms stable oligomeric complexes with it, does not appear to directly interact with FRS2. Novel insights into the functional role of the interaction between flot-1 and FRS2 were provided by the results showing that depletion of flot-1 affects the cellular localization of FRS2. In hepatocytes stably depleted of flot-1, FRS2 appeared to be more soluble. Furthermore, upon pervanadate stimulation of the cells, a small fraction of FRS2 was recruited into detergent resistant membranes, but the recruitment did not take place in the absence of flot-1. Triggered by the same stimulus, a fraction of FRS2 was translocated to the nucleus independently of flot-1. Overexpression of FRS2 has previously been shown to result in increased ERK activation. However, in cells depleted of flot-1, FRS2 was not able to compensate for the compromised ERK activation after EGF or FGF stimulation. This might imply that FRS2 and flot-1 are functionally interconnected and that FRS2 resides upstream of flot-1. Taken together, the results presented here indicate that this complex may be involved in the control of signaling downstream of receptor tyrosine kinases and is important for ensuring a proper signaling response. In the absence of flot-1, increased Tyr phosphorylation of FRS2 was observed. It is known that Tyr and Thr phosphorylation of FRS2 are reciprocally regulated. Since ERK is a known executor of the FRS2 Thr phosphorylation, and ERK activity was shown to be severely diminished upon flot-1 depletion, the increased Tyr phosphorylation of FRS2 was in agreement with this and might be a direct consequence of a decreased ERK activity upon flot-1 depletion. FRS2 owes its name to the major and the first described function of this protein as a substrate for FGFR. PTB domain of FRS2 was published to constitutively bind the juxtamembrane domain of FGFR. In this study, the PTB domain was mapped to be involved in the constitutive interaction with flot-1 and the competition was shown to exist between flot-1 and FGFR1 for binding to FRS2. Another novel interaction partner of FRS2 was discovered in the present study. Cbl-associated protein (CAP) is an adaptor protein with three SH3 domains and it plays a role during insulin signaling by recruiting the signaling complex to lipid rafts. CAP was previously shown to interact with flot-1 via the SoHo domain, and this interaction was found to be crucial for the lipid raft recruitment of other signaling components. Both the PTB domain and CT of FRS2 were found to mediate the interaction with CAP, whereas in CAP, the SoHo domain, together with the third SH3 domain, seems to bind to FRS2. SH3 domains mediate the assembly of specific protein complexes by binding to proline rich sequences, several of which are present in FRS2. Due to overlapping interaction domains, FRS2 and flot-1 competed for the binding to CAP. However, the interaction with neither CAP nor flot-1 was necessary for the observed nuclear translocation of FRS2. Since CAP is expressed as several tissue- and developmental stage-specific isoforms, a further aim of this study was to analyze the expression of its isoforms in mouse embryonic fibroblasts (MEFs). Many new isoforms were discovered here which have not been described in the literature so far. They all contain the SoHo domain and three SH3 domains, but differ among themselves by the presence and length of a proline-rich region that preceeds the SoHo domain and by a novel 20-amino acid (AA) stretch between the second and the third SH3 domain. The length of the proline-rich region turned out to be an important factor determining the strength of the interaction with FRS2. The interaction was found to be weakened by the increasing length of this region. The new isoforms possessing the 20-AA stretch are specifically expressed in murine muscular tissues, with the highest level in the heart. During adipogenesis, we observed a shift in the abundance of the isoforms, in that only the isoforms without the insertion were shown to be upregulated on mRNA level. However, during myogenesis, preferentially expressed isoforms were those with the insertion. The collected data implicate that isoforms with the 20-AA insertion might be more ubiquitous in nondifferentiated/embryonic cells and that the observed "isoform-switch" might be dependent on the cell fate and differentiation state.FRS2 (engl. fibroblast growth factor receptor substrate 2) ist ein Membran-gebundenes Adapterprotein, welches Signalkaskaden ausgehend von verschiedenen Rezeptor-Tyrosinkinasen reguliert. Die molekulare Struktur von FRS2 ist identisch mit dem homologen Protein FRS3. FRS2 beinhaltet eine Myristoylierungssequenz in der Nähe des N-Terminus, eine Phosphotyrosin-Bindedomäne (PTB, engl. phosphotyrosine binding domain) und multiple Tyrosin-Phosphorylierungsstellen im Bereich des CTerminus. Namensgebend für die Proteine der FRS-Familie ist ihre essentielle Funktion bei der Signaltransduktion ausgehend vom FGF-Rezeptor, da dieser selbst keine Adapterproteine rekrutieren kann, die das Signal an MAP-Kinasen weiterleiten. Die Phosphorylierung von FRS2, welche nach Stimulation mit NGF, BDNF, FGF und GDNF erfolgt, ermöglicht die Bildung von Signalkomplexen mit weiteren Adapterproteinen (z.B. Grb2 und SOS) und der Tyrosinphosphatase Shp2. Die Rekrutierung dieser Komponenten resultiert in der Aktivierung der MAP-Kinase- und der PI3K/Akt-Signalkaskade. Auf Grund seiner regulatorischen Funktion nach Stimulierung mit FGF oder NGF, spielt FRS2 eine wichtige Rolle bei der Differenzierung von PC12 Zellen. ... In dieser Studie wurden Flotillin-1 und CAP als neue Interaktionspartner von FRS2 identifiziert. Flotillin-1 bindet an die PTB Domäne von FRS2, wodurch es mit dem FGFR um dessen Bindung konkurriert. Darüber hinaus führt die Deletion von Flotillin-1 zu einer verstärkten Tyrosinphosphorylierung von FRS2. Dieses Ergebnis stimmt mit der beobachteten Inhibierung der ERK Aktivität in Abwesenheit von Flotillin- 1 überein. Das Protein CAP bindet ebenfalls direkt an FRS2, wobei diese Interaktion von der dritten SH3- Domäne und der SoHo-Domäne von CAP, sowie der PTB-Domäne und dem C-Terminus von FRS2 vermittelt wird. Sie SoHo-Domäne ist zusätzlich verantwortlich für die Interaktion von CAP mit Flotillin-1, was impliziert das auch CAP mit Flotillin-1 um die Bindung von FRS2 konkurriert. Dieses neu entdeckte Proteinnetzwerk könnte eine wichtige Rolle bei der Regulation der Signaltransduktion ausgehend von Rezeptor-Tyrosinkinasen spielen. Desweiteren wurde in dieser Studie nachgewiesen, dass verschiedene CAP Isoformen in MEFs exprimiert werden. All diese Isoformen beinhalten eine SoHo-Domäne und SH3-Domänen, unterscheiden sich jedoch im Vorkommen und der Länge einer Prolin-reichen Sequenz und einer neu entdeckten 20 Aminosäure-langen Insertion. Isoformen, welche diese Insertion besitzen, wurden hauptsächlich in murinen Muskelgewebe nachgewiesen - mit höchster Expression im Herzen. Die Isoformen ohne Insertion werden auf mRNA-Level während des Vorgangs der Adipogenese hochreguliert. Für Isoformen ohne Insertion konnte auf mRNA-Level keine Veränderung detektiert werden. Diese Ergebnisse lassen vermuten, dass die neuen murinen CAP Isoformen mit Insertion eine Rolle während der Entwicklung des Herzens spielen

Mitogen-Activated Protein (MAP) Kinase Scaffolding Proteins: A Recount

The mitogen-activated protein kinase (MAPK) pathway is the canonical signaling pathway for many receptor tyrosine kinases, such as the Epidermal Growth Factor Receptor. Downstream of the receptors, this pathway involves the activation of a kinase cascade that culminates in a transcriptional response and affects processes, such as cell migration and adhesion. In addition, the strength and duration of the upstream signal also influence the mode of the cellular response that is switched on. Thus, the same components can in principle coordinate opposite responses, such as proliferation and differentiation. In recent years, it has become evident that MAPK signaling is regulated and fine-tuned by proteins that can bind to several MAPK signaling proteins simultaneously and, thereby, affect their function. These so-called MAPK scaffolding proteins are, thus, important coordinators of the signaling response in cells. In this review, we summarize the recent advances in the research on MAPK/extracellular signal-regulated kinase (ERK) pathway scaffolders. We will not only review the well-known members of the family, such as kinase suppressor of Ras (KSR), but also put a special focus on the function of the recently identified or less studied scaffolders, such as fibroblast growth factor receptor substrate 2, flotillin-1 and mitogen-activated protein kinase organizer 1