K153R polymorphism in myostatin gene increases the rate of promyostatin activation by furin

Recent studies demonstrated an association between the K153R polymorphism in the myostatin gene with extreme longevity, lower muscle strength and obesity but the molecular basis of these associations has not been clarified. Here, we show that the K153R mutation significantly increases the rate of proteolysis of promyostatin by furin, but has no effect on the activity of the latent complex or the cleavage of the latent complex by bone morphogenetic protein 1 (BMP-1). The increased rate of activation of K153R mutant promyostatin may explain why this polymorphism is associated with obesity, lower muscle strength and extension of lifespan

K-Ras prenylation as a potential anticancer target

KRAS is one of the most commonly mutated oncogene and a negative predictive factor for a number of targeted therapies. Therefore, the development of targeting strategies against mutant KRAS is urgently needed. One potential strategy involves disruption of K-Ras membrane localization, which is necessary for its proper function. In this review, we summarize the current data about the importance of membrane-anchorage of K-Ras and provide a critical evaluation of this targeting paradigm focusing mainly on prenylation inhibition. Additionally, we performed a RAS mutation-specific analysis of prenylation-related drug sensitivity data from a publicly available database (https://depmap.org/repurposing/) of three classes of prenylation inhibitors: statins, N-bisphosphonates, and farnesyl-transferase inhibitors. We observed significant differences in sensitivity to N-bisphosphonates and farnesyl-transferase inhibitors depending on KRAS mutational status and tissue of origin. These observations emphasize the importance of factors affecting efficacy of prenylation inhibition, like distinct features of different KRAS mutations, tissue-specific mutational patterns, K-Ras turnover, and changes in regulation of prenylation process. Finally, we enlist the factors that might be responsible for the large discrepancy between the outcomes in preclinical and clinical studies including methodological pitfalls, the incomplete understanding of K-Ras protein turnover, and the variation of KRAS dependency in KRAS mutant tumors

Az aktin citoszkeleton szabályozásához vezető jelpályák vizsgálata = The role of Rho GTPases in the regulation of actin cytoskeleton

OTKA (T46130) pályázatunk munkatervében azt vállaltuk, hogy tanulmányozni fogunk olyan jelátviteli utakat, melyek a sejtek aktin citoszkeletonját szabályozzák. Balla Tamás (NIH, USA) munkacsoportjával együttműködve számos PH (pleckstrin homológ) domént expresszáltunk COS7 sejtekben és vizsgáltuk, hogy ezek közül melyek képesek a sejtek kiterülésének gátolni fibronektinnel borított felszínen. Bár az általunk használt összes PH domén képes a PI 3-kináz lipid termékeit kötni in vitro, kizárólag a GRP1/ARNO családba tartozó fehérjék PH doménjei gátolták az integrin-függő jelátviteli utat. A PH domének mutációs analízisével kimutattuk, hogy az általunk vizsgált GRP1/ARNO PH domének nagy valószínűséggel nemcsak lipideket kötnek, hanem (membrán) fehérjéket is, s ez a kettős kölcsönhatás szükséges a PH domének sejten belüli funkciójához. Munkacsoportunk az aktin citoszkeleton szabályozásában fontos szerepet játszó cortactin fehérje működését is vizsgálta. A cortactin fehérje N-, illetve C-terminális doménjeit külön-külön expresszálva COS7 sejtekben azt találtuk, hogy mind a két konstrukció egyaránt gátolja a sejtek kiterülését. A fenti eredmény megerősítésére siRNS technikával kiütöttük a cortactint a sejtekből, ami szintén a sejtkiterülés erős gátlásához vezetett. Ebből arra következtettünk, hogy a cortactinnak szerepe lehet az integrin-mediálta aktin citoszkeleton szabályozásban. | In the project proposal of our OTKA application (T46130) we planned to investigate such signalling transduction pathways that regulate the actin cytoskeleton. In cooperation with Tamas Balla?s group (NIH, USA) several PH domains were over-expressed in COS7 cells. Although all investigated PH domain had the ability to bind to the lipid products of PI 3- kinase, only the PH domains of the GRP1/ARNO protein family were capable of inhibiting the integrin-dependent signalling pathway. Based on mutational analysis of the PH domains, we found that these PH domains bind not only lipids but membrane proteins, and this double interaction is required for the their proper function. Our group studied the cortactin proteins implicated in the regulation of actin cytoskeleton. N-, and C-terminal domains of cortactin were over-expressed in COS7 cells. Interestingly, both domain constructs were able to strongly inhibit the integrin-dependent cell spreading. This result was confirmed by cortactin silencing with siRNA technique. We concluded that cortectin may play an important role in the integrin-dependent regulation of actin cytoskeleton

EGF regulates tyrosine phosphorylation and membrane-translocation of the scaffold protein Tks5

Background: Tks5/FISH is a scaffold protein comprising of five SH3 domains and one PX domain. Tks5 is a substrate of the tyrosine kinase Src and is required for the organization of podosomes/invadopodia implicated in invasion of tumor cells. Recent data have suggested that a close homologue of Tks5, Tks4, is implicated in the EGF signaling.Results: Here, we report that Tks5 is a component of the EGF signaling pathway. In EGF-treated cells, Tks5 is tyrosine phosphorylated within minutes and the level of phosphorylation is sustained for at least 2 hours. Using specific kinase inhibitors, we demonstrate that tyrosine phosphorylation of Tks5 is catalyzed by Src tyrosine kinase. We show that treatment of cells with EGF results in plasma membrane translocation of Tks5. In addition, treatment of cells with LY294002, an inhibitor of PI 3-kinase, or mutation of the PX domain reduces tyrosine phosphorylation and membrane translocation of Tks5.Conclusions: Our results identify Tks5 as a novel component of the EGF signaling pathway. © 2013 Fekete et al.; licensee BioMed Central Ltd

Complex formation of EphB1/Nck/Caskin1 leads to tyrosine phosphorylation and structural changes of the Caskin1 SH3 domain.

Scaffold proteins have an important role in the regulation of signal propagation. These proteins do not possess any enzymatic activity but can contribute to the formation of multiprotein complexes. Although scaffold proteins are present in all cell types, the nervous system contains them in the largest amount. Caskin proteins are typically present in neuronal cells, particularly, in the synapses. However, the signaling mechanisms by which Caskin proteins are regulated are largely unknown. Here we demonstrate that EphB1 receptor tyrosine kinase can recruit Caskin1 through the adaptor protein Nck. Upon activation of the receptor kinase, the SH2 domain of Nck binds to one of its tyrosine residues, while Nck SH3 domains interact with the proline-rich domain of Caskin1. Complex formation of the receptor, adaptor and scaffold proteins results in the tyrosine phosphorylation of Caskin1 on its SH3 domain. The phosphorylation sites were identified by mass-spectrometry as tyrosines 296 and 336. To reveal the structural consequence of this phosphorylation, CD spectroscopy was performed. This measurement suggests that upon tyrosine phosphorylation the structure of the Caskin1 SH3 domain changes significantly. Taken together, we propose that the scaffold protein Caskin1 can form a complex with the EphB1 tyrosine kinase via the Nck protein as a linker. Complex formation results in tyrosine phosphorylation of the Caskin1 SH3 domain. Although we were not able to identify any physiological partner of the SH3 domain so far, we could demonstrate that phosphorylation on conserved tyrosine residues results in marked changes in the structure of the SH3 domain

T cell specific adaptor protein (TSAd) promotes interaction of Nck with Lck and SLP-76 in T cells

Background: The Lck and Src binding adaptor protein TSAd (T cell specific adaptor) regulates actin polymerization in T cells and endothelial cells. The molecular details as to how TSAd regulates this process remain to be elucidated. Results: To identify novel interaction partners for TSAd, we used a scoring matrix-assisted ligand algorithm (SMALI), and found that the Src homology 2 (SH2) domain of the actin regulator Non-catalytic region of tyrosine kinase adaptor protein (Nck) potentially binds to TSAd phosphorylated on Tyr280 (pTyr280) and pTyr305. These predictions were confirmed by peptide array analysis, showing direct binding of recombinant Nck SH2 to both pTyr280 and pTyr305 on TSAd. In addition, the SH3 domains of Nck interacted with the proline rich region (PRR) of TSAd. Pull-down and immunoprecipitation experiments further confirmed the Nck-TSAd interactions through Nck SH2 and SH3 domains. In line with this Nck and TSAd co-localized in Jurkat cells as assessed by confocal microscopy and imaging flow cytometry. Co-immunoprecipitation experiments in Jurkat TAg cells lacking TSAd revealed that TSAd promotes interaction of Nck with Lck and SLP-76, but not Vav1. TSAd expressing Jurkat cells contained more polymerized actin, an effect dependent on TSAd exon 7, which includes interactions sites for both Nck and Lck. Conclusions: TSAd binds to and co-localizes with Nck. Expression of TSAd increases both Nck-Lck and Nck-SLP-76 interaction in T cells. Recruitment of Lck and SLP-76 to Nck by TSAd could be one mechanism by which TSAd promotes actin polymerization in activated T cells. © 2015 Hem et al

A Caskin fehérje szerepének vizsgálata = Investigation of the role of Caskin protein

Pályázatunk munkatervében vállaltuk, hogy tanulmányozzuk a Caskin fehérjecsalád szerepét. Élesztő két-hibrid eljárással számos fehérje interakcióját mutattuk ki a Caskin1-gyel, melyek közül több esetben független módszerekkel is igazoltuk a kölcsönhatást. Így bizonyítottuk, hogy az Abi2 kapcsoló fehérje SH3/prolinban-gazdag domén interakcióval kötődik a Caskin1-hez. Tompa Péter munkacsoportjával együttműködésben kimutattuk, hogy a Caskin1 teljes C-terminális fele rendezetlen fehérje-szerkezetű. Azonosítottunk a Caskin1 C-terminálisán olyan új foszforilációs helyeket, melyeket a protein kináz A és C képes foszforiláni. Foszfospecifikus ellenanyagok segítségével bizonyítottunk, hogy ezek a helyek in vivo is foszforilálódnak fiziológiás stimulusok hatására. Végezetül kimutattuk, hogy a Caskin1 képes az Nck kapcsoló fehérjén keresztül asszociálódni az EphB1 receptor tirozin kinázzal, mely képes a Caskin1-et foszforilálni. | In the project proposal of our grant we suggested to investigate the role of Caskin protein family. By the means of yeast two-hybrid technique we identified several binding partners of Caskin1. In some cases, the protein interactions were confirmed by independent techniques, such as co-immunoprecipitation. Studying the interaction of Caskin1 by Abi2, SH3/proline-rich domain-dependent association was revealed. In cooperation with Peter Tompa's laboratory, we demonstrated that the C-terminal part of Caskins1 is fully disordered. Moreover, novel phosphorylation sites were identified in the Caskin1 C-terminal which were phosphorylated by protein kinase A and C. Using phosphospecific antibodies, in vivo phosphorylations of these sites were proved in response to physiological stimuli. Finally, interaction of Caskin1 with the EphB1 receptor tyrosine kinase was shown through the aadaptor protein Nck. We proved that Caskin1 is a substrate of the tyrosine kinase