Adaptor fehérjék vizsgálata a vaszkuláris endotéliumban

Vascular endothelial cell monolayer acts as a semiselective barrier between blood and the interstitium. Phosphorylation level of many cytoskeleton and cytoskeleton-associated proteins playing crucial role in the EC barrier function is critical to tissue and organ function. ERM-binding phosphoprotein 50 (EBP50) is a phosphorylatable PDZ domain-containing adaptor protein that is abundantly expressed in epithelium but was not yet studied in the endothelium. We found unusual nuclear localization of EBP50 in bovine pulmonary artery endothelial cells (BPAEC). Immunofluorescent staining and cellular fractionation demonstrated that EBP50 is present in the nuclear and perinuclear region in interphase cells. In the prophase of mitosis EBP50 redistributes to the cytoplasmic region in a phosphorylation dependent manner and during mitosis EBP50 co-localizes with protein phosphatase 2A. Furthermore, in vitro wound healing of BPAEC expressing phospho-mimic mutant of EBP50 was accelerated indicating that EBP50 is involved in the regulation of the cell division. Cell cycle dependent specific interactions were detected between EBP50 and the subunits of PP2A (A, C, and Bα) with immunoprecipitation and pull-down experiments. The interaction of EBP50 with the Bα containing form of PP2A suggests that this holoenzyme of PP2A can be responsible for the dephosphorylation of EBP50 in cytokinesis. Moreover, our results underline the significance of EBP50 in cell division via reversible phosphorylation of the protein with cyclin dependent kinase and PP2A in normal cells. TIMAP, TGF-β inhibited membrane-associated protein, is most abundant in endothelial cells with a regulatory effect on the endothelial barrier function, yet little is known about its interacting partners. RACK1, receptor for activated protein kinase C, serves as an anchor in multiple signaling pathways. We found that RACK1 binds to the TIMAP-PP1c complex in endothelial cells. WD1-4 repeats of RACK1 were identified as critical regions of the interaction both with TIMAP and farnesyl transferase. Phosphorylation of TIMAP by activation of the cAMP/PKA pathway reduced the amount of TIMAP-RACK1 complex and enhanced translocation of TIMAP to the cell membrane in vascular endothelial cells. However, both membrane localization of TIMAP and transendothelial resistance were attenuated after RACK1 depletion. Farnesyl transferase, the enzyme responsible for prenylation and consequent membrane localization of TIMAP, is present in the RACK1-TIMAP complex in control cells, but it does not co-immunoprecipitate with TIMAP after RACK1 depletion. Our results suggest that transient parallel linkage of TIMAP and farnesyl transferase to RACK1 could ensure prenylation and transport of TIMAP to the plasma membrane where it may attend in maintaining the endothelial barrier as a phosphatase regulator. A vaszkuláris endotél sejtek szemiszelektív barrierként választják el a keringő vért a környező szövetektől. A jól működő endotél barrier funció fenntartásában számos citoszkeletális és citoszkeleton asszociált fehérje foszforilációja fontos szerepet játszik. Az EBP50 (ezrin-radixin-moesin-binding phosphoprotein 50), egy olyan foszforilálható adaptor fehérje, mely epitél sejtekben magas expressziós szinttel rendelkezik azonban endotél sejtekben még nem vizsgálták. Immunfluoreszcens festéssel, valamint sejtfrakcionálással az EBP50 fehérjét az interfázisban lévő endotél sejtek magi régiójában detektáltuk. A mitózis profázisában foszforiláció-függő módon a sejtek citoplazmájába transzlokálódott és kolokalizáció volt megfigyelhető a protein foszfatáz 2A enzimmel. Az EBP50 foszforilációt utánzó mutánsát expresszáló sejtek gyorsabb in vitro sebgyógyálást mutattak, ami az EBP50 sejtosztódásban betöltött szabályozó szerepére utal. Immunprecipitációs és pull down kísérletekkel sikerült igazolnuk az EBP50 és a PP2A (A, C, és Bα) alegységei közötti sejtciklus függő kölcsönhatást. Eredményeink arra utalnak, hogy a PP2A enzim szerepet játszik az EBP50 fehérje defoszforilálásában normál sejtekben a sejtciklus során. A TIMAP (TGFβ inhibited membrane-associated protein) endotél sejtekben rendelkezik a legmagasabb expressziós szinttel, szerepéről és kölcsönható partnereiről azonban csak keveset tudunk. A RACK1 (Receptor for Activated C Kinase 1) adaptor fehérje, számos jelátviteli útvonalban játszik szerepet. Eredményeink alapján endotél sejtekben a RACK1 a TIMAP-PP1c komplexhez kötődik. A kölcsönhatásban a RACK1 WD1-4 doménjei vesznek részt, melyhez kötődik továbbá a farnezil-transzferáz enzim is. A cAMP/PKA útvonal aktiválásával elért TIMAP foszforiláció csökkentette a RACK1-TIMAP komplex mennyiségét, valamint a TIMAP membránban való feldúsulásához vezetett. A RACK1 depléciójával a TIMAP membránlokalizációja valamint a farnezil-transzferáz enzimmel való kölcsönhatása is megszűnt. Eredményeink alapján a RACK1 adaptor fehérje biztosítja a TIMAP prenilációját az ehhez szükséges farnezil-transzferáz enzimmel való kölcsönhatással

Ezrin-Radixin-Moesin Binding Phosphoprotein 50:A Potential Novel Biomarker in Human Papilloma Virus-Associated Head and Neck Squamous Cell Carcinomas

High-risk human papilloma virus (HR-HPV) has increasingly been associated with head and neck squamous cell carcinoma (HNSCC), in particular oropharyngeal cancers. Ezrin-Radixin-Moesin Binding Phosphoprotein 50 (EBP50), a putative tumour suppressor, localises to the plasma membrane in suprabasal epithelium and to the cytoplasm in proliferative basal layers, and is a target for degradation by the HR-HPV E6 oncoprotein. The aim of this study was to investigate EBP50 protein expression patterns in HNSCC in a large Scottish cohort to determine if there was a correlation with HPV status and clinical outcomes. EBP50 expression patterns were assessed in 156 HNSCC including oropharyngeal (37.8%), laryngeal (24%), oral (19%) and other sites (18.5%), which were genotyped for presence of HR-HPV. HNSCC were generally negative for membranous EBP50. EBP50 expression was either cytoplasmic/absent, being 'predominantly cytoplasmic' in 76 (49%), 'weak/negligible cytoplasmic' in 44 (28%), 'strongly cytoplasmic' in 5 (3%), 'heterogeneous' in 26 (17%) and 'other' in 5 (3%) samples. Forty tumours (25%) were positive for HPV DNA, predominantly HR-HPV 16, and 44 (28%) were p16 positive. The majority of tumours (71%) with 'weak/negligible cytoplasmic' EBP50 expression originated in the oropharynx were more likely to have positive neck nodes, overexpression of p16 and positive tumour HR-HPV status (P &lt; 0.001). Differences in EBP50 levels between oropharyngeal and non-oropharyngeal tumours may be linked to degradation of EBP50 by HR-HPV, and loss of EBP50 may therefore be a surrogate biomarker for HR-HPV infection in oropharyngeal tumours.</p

Regulation of merlin by protein phosphatase 1-TIMAP and EBP50 in endothelial cells

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Drug design and synthesis of first in class PDZ1 targeting NHERF1 inhibitors as anticancer agents

Targeted approaches aiming at modulating NHERF1 activity, rather than its overall expression, would be preferred to preserve the normal functions of this versatile protein. We focused our attention on the NHERF1/PDZ1 domain that governs its membrane recruitment/displacement through a transient phosphorylation switch. We herein report the design and synthesis of novel NHERF1 PDZ1 domain inhibitors. These compounds have potential therapeutic value when used in combination with antagonists of β-catenin to augment apoptotic death of colorectal cancer cells refractory to currently available Wnt/β-catenin-targeted agents

NHERF1 regulates the progression of colorectal cancer through the interplay with VEGFR2 pathway

The oncogenic role of ectopic expression of Na+/H+ exchanger regulatory factor 1 (NHERF1) was recently suggested in colorectal cancer, where it was implicated in playing a role in the tumor hypoxia microenvironment. Here we showed that a high level expression of NHERF1 was found in colorectal cancer tissues and that the expression of NHERF1 was positively correlated with VEGFR2 expression. The prognostic value of VEGFR2 expression in colorectal cancer relied on the expression of NHERF1. The up-regulation of NHERF1 induced by the exposure to hypoxia in colon cancer cells depended on the activation of VEGFR2 signaling. NHERF1 in turn inhibited the activation of VEGFR2 signaling which could be regulated by the interaction between NHERF1 and VEGFR2, resulting in the reduction of migration and invasion of colon cancer cells. These results suggest a dynamic interplay between NHERF1 and VEGFR2 signaling in colorectal cancer, which could explain the contribution of NHERF1 to the regulation of tumor cell responses to the hypoxia microenvironment

Isotropic actomyosin dynamics promote organization of the apical cell cortex in epithelial cells

Although cortical actin plays an important role in cellular mechanics and morphogenesis, there is surprisingly little information on cortex organization at the apical surface of cells. In this paper, we characterize organization and dynamics of microvilli (MV) and a previously unappreciated actomyosin network at the apical surface of Madin–Darby canine kidney cells. In contrast to short and static MV in confluent cells, the apical surfaces of nonconfluent epithelial cells (ECs) form highly dynamic protrusions, which are often oriented along the plane of the membrane. These dynamic MV exhibit complex and spatially correlated reorganization, which is dependent on myosin II activity. Surprisingly, myosin II is organized into an extensive network of filaments spanning the entire apical membrane in nonconfluent ECs. Dynamic MV, myosin filaments, and their associated actin filaments form an interconnected, prestressed network. Interestingly, this network regulates lateral mobility of apical membrane probes such as integrins or epidermal growth factor receptors, suggesting that coordinated actomyosin dynamics contributes to apical cell membrane organization

Nanoclustering as a dominant feature of plasma membrane organization

Early studies have revealed that some mammalian plasma membrane proteins exist in small nanoclusters. The advent of super-resolution microscopy has corroborated and extended this picture, and led to the suggestion that many, if not most, membrane proteins are clustered at the plasma membrane at nanoscale lengths. In this Commentary, we present selected examples of glycosylphosphatidyl-anchored proteins, Ras family members and several immune receptors that provide evidence for nanoclustering. We advocate the view that nanoclustering is an important part of the hierarchical organization of proteins in the plasma membrane. According to this emerging picture, nanoclusters can be organized on the mesoscale to form microdomains that are capable of supporting cell adhesion, pathogen binding and immune cell-cell recognition amongst other functions. Yet, a number of outstanding issues concerning nanoclusters remain open, including the details of their molecular composition, biogenesis, size, stability, function and regulation. Notions about these details are put forth and suggestions are made about nanocluster function and why this general feature of protein nanoclustering appears to be so prevalent.Postprint (published version

The cellular distribution of Na+/H+ exchanger regulatory factor 1 is determined by the PDZ-I domain and regulates the malignant progression of breast cancer

The oncogenic role of ectopic expression of Na+/H+ exchanger regulatory factor 1 (NHERF1) was recently suggested. Here, we show that NHERF1 was upregulated in high grades compared with low grades. Increased NHERF1 expression was correlated with poor prognosis and poor survival. NHERF1 expression was higher in the nucleus of cancer cells than in contiguous non- mammary epithelial cells. A novel mutation, namely NHERF1 Y24S, was identified in human breast cancer tissues and shown to correspond to a conserved residue in the PDZ-I domain of NHERF1. Truncation and mutation of the PDZ-I domain of NHERF1 increased the nuclear distribution of the NHERF1 protein, and this redistribution was associated with the malignant phenotype of breast cancer cells, including growth, migration, and adhesion. The present results suggest a role for NHERF1 in the progression of breast cancer mediated by the nuclear distribution of the NHERF1 protein, as determined by the truncation or key site mutation of the PDZ-I domain