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001-es BibID:	BIBFORM047427
Első szerző:	Adyshev, Djanybek M.
Cím:	Ezrin/radixin/moesin proteins differentially regulate endothelial hyperpermeability after thrombin / Djanybek M. Adyshev, Steven M. Dudek, Nurgul Moldobaeva, Kyung-mi Kim, Shwu-Fan Ma, Anita Kasa, Joe G. N. Garcia, Alexander D. Verin
Dátum:	2013
ISSN:	1040-0605
Megjegyzések:	Endothelial cell (EC) barrier disruption induced by inflammatory agonists such as thrombin leads to potentially lethal physiological dysfunction such as alveolar flooding, hypoxemia and pulmonary edema. Thrombin stimulates paracellular gap and F-actin stress fiber formation, triggers actomyosin contraction and alters EC permeability through multiple mechanisms that include protein kinase C (PKC) activation. We previously have shown that the ezrin, radixin, and moesin (ERM) actin-binding proteins differentially participate in S1P-induced EC barrier enhancement. Phosphorylation of a conserved threonine residue in the C terminus of ERM proteins causes conformational changes in ERM to unmask binding sites and is considered a hallmark of ERM activation. In the present study we test the hypothesis that ERM proteins are phosphorylated on this critical threonine residue by thrombin-induced signaling events and explore the role of the ERM family in modulating thrombin-induced cytoskeletal rearrangement and EC barrier function. Thrombin promotes ERM phosphorylation at this threonine residue (Ezrin-567, Radixin-564, Moesin-558) in a PKC-dependent fashion and induces translocation of phosphorylated ERM to the EC periphery. Thrombin-induced ERM threonine phosphorylation is likely synergistically mediated by protease-activated receptors PAR1 and PAR2. Using the siRNA approach, depletion of either moesin alone, or of all three ERM proteins, significantly attenuates thrombin-induced increase in EC barrier permeability (TER), cytoskeletal rearrangements, paracellular gap formation and accumulation of di-phospho-MLC. In contrast, radixin depletion exerts opposing effects on these indices. These data suggest that ERM proteins play important differential roles in the thrombin-induced modulation of EC permeability, with moesin promoting barrier dysfunction and radixin opposing it
Tárgyszavak:	Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban thrombin ERM PKC phosphorylation
Megjelenés:	American Journal of Physiology-Lung Cellular and Molecular Physiology 305 : 3 (2013), p. L240-L255. -
További szerzők:	Dudek, Steven Moldobaeva, Nurgul Kim, Kyung-mi Ma, Shwu-Fan Kovács-Kása Anita (1983-) Garcia, Joe G. N. Verin, Alexander
Internet cím:	Szerző által megadott URL DOI Intézményi repozitóriumban (DEA) tárolt változat
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001-es BibID:	BIBFORM074172
Első szerző:	Csortos Csilla (biokémikus)
Cím:	TIMAP is a positive regulator of pulmonary endothelial barrier function / Csilla Csortos, Istvan Czikora, Natalia V. Bogatcheva, Djanybek M. Adyshev, Christophe Poirier, Gabor Olah, Alexander D. Verin
Dátum:	2008
ISSN:	1040-0605 1522-1504
Megjegyzések:	TGF-?-inhibited membrane-associated protein, TIMAP, is expressed at high levels in endothelial cells (EC). It is regarded as a member of the MYPT (myosin phosphatase target subunit) family of protein phosphatase 1 (PP1) regulatory subunits; however, its function in EC is not clear. In our pull-down experiments, recombinant TIMAP binds preferentially the ?-isoform of the catalytic subunit of PP1 (PP1c?) from pulmonary artery EC. As PP1c?, but not PP1c?, binds with MYPT1 into functional complex, these results suggest that TIMAP is a novel regulatory subunit of myosin phosphatase in EC. TIMAP depletion by small interfering RNA (siRNA) technique attenuates increases in transendothelial electrical resistance induced by EC barrier-protective agents (sphingosine-1-phosphate, ATP) and enhances the effect of barrier-compromising agents (thrombin, nocodazole) demonstrating a barrier-protective role of TIMAP in EC. Immunofluorescent staining revealed colocalization of TIMAP with membrane/cytoskeletal protein, moesin. Moreover, TIMAP coimmunoprecipitates with moesin suggesting the involvement of TIMAP/moesin interaction in TIMAP-mediated EC barrier enhancement. Activation of cAMP/PKA cascade by forskolin, which has a barrier-protective effect against thrombin-induced EC permeability, attenuates thrombin-induced phosphorylation of moesin at the cell periphery of control siRNA-treated EC. On the contrary, in TIMAP-depleted EC, forskolin failed to affect the level of moesin phosphorylation at the cell edges. These results suggest the involvement of TIMAP in PKA-mediated moesin dephosphorylation and the importance of this dephosphorylation in TIMAP-mediated EC barrier protection.protein phosphorylation and dephosphorylation are known to be the key signaling events affecting the status of vascular endothelial barrier (11). Cytoskeletal and intercellular junctional proteins are regulated via reversible phosphorylation of serine (Ser), threonine (Thr), or tyrosine (Tyr) side chains. Based on many recent data, it is apparent that several types of protein phosphatases are intimately involved in the regulation of endothelial barrier function (10, 17, 27?29). However, their regulation is not yet completely understood.Protein phosphatase 1 (PP1) is a multimeric phosphoserine/phosphothreonine-specific phosphatase. One of the four different isoforms, ?, ?, ?1, or ?2, of the catalytic subunit (PP1c) binds to one (or two) protein from a pool of regulatory subunits (R). The holoenzyme forms possess diverse cellular functions. A common structural element of R proteins is a short, conserved PP1c binding motif, (R/K)VXF (3, 9, 10). Different R subunits may direct PP1 holoenzymes to distinct subcellular locations and increase or suppress the activity toward specific substrates (3, 9). Myosin light chain phosphatase (or myosin phosphatase, MP), for example, is composed of PP1c? and two regulatory subunits, namely, a larger targeting/regulatory subunit (myosin phosphatase target subunit, MYPT) and a small regulatory subunit (M20) (2, 10, 14). The activity of MP holoenzyme is increased toward phosphorylated myosin compared with the activity of the PP1c monomer (15).It was recently shown that MP function is not limited to myosin dephosphorylation. The MP regulatory subunit MYPT1 can directly bind to F-actin binding proteins including ERM proteins (ezrin-radixin-moesin family). These proteins could be phosphorylated by either protein kinase C? or Rho kinase (12, 20); phosphorylation renders unfolded ERM protein, enabling its interaction with actin and membrane proteins (20, 21). ERM dephosphorylation by MP seems to affect ERM conformation and cytoskeletal/membrane binding capacities (12, 20). These data indicate that MP not only dephosphorylates myosin, but it is also involved in the regulation of F-actin cytoskeleton.Recently, other proteins of the MYPT family, namely MYPT3, TIMAP (TGF-?-inhibited membrane-associated protein), and myosin binding subunit 85 (MBS85), were identified and characterized from different sources (8, 25, 26). They share some structural features with MYPT1, e.g., all of these proteins contain the PP1c binding motif followed by ankyrin repeats. On the other hand, MYPT3, TIMAP, and MBS85 have their own special features as well. For example, both TIMAP and MYPT3 have COOH-terminal prenylation motif suggesting possible membrane association. The high level of homology with MYPT1 implies that TIMAP, MYPT3, and MBS85 may be regulatory subunits of PP1; however, their physiological significance is not known.TIMAP is a 64-kDa protein expressed at high levels in endothelial cells (EC). As TIMAP mRNA synthesis is strongly downregulated by TGF-?1 (8), it is likely to assume that TIMAP may be an important component of endothelial response to TGF-?1, including apoptosis, capillary morphogenesis, and barrier dysfunction. It is highly homologous to MYPT3 (?45% amino acid homology) and shares its structural features, i.e., PP1c binding motif, ankyrin repeats, prenylation motif, and possible nuclear localization signals (8). Yeast and bacterial two-hybrid screening revealed several potential protein partners for TIMAP (1, 16). For instance, TIMAP interacts with the 37/67-kDa laminin receptor (LAMR1). It was suggested that TIMAP targets PP1c to LAMR1, and LAMR1 is a TIMAP-dependent PP1c substrate (16). Although protein-protein interaction between TIMAP and PP1c was shown by immunoprecipitation, its role in regulating PP1c activity is not clarified yet. In the present work, we present evidence for specific interaction between TIMAP and PP1c?. Furthermore, we show that TIMAP has a barrier-protective role in human pulmonary artery endothelial cells (HPAEC), and we propose that ERM proteins are among its targets.
Tárgyszavak:	Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban transendothelial electrical resistance small interfering RNA moesin interaction with protein phosphatase 1
Megjelenés:	American Journal Of Physiology-Lung Cellular And Molecular Physiology. - 295 : 3 (2008), p. L440-L450. -
További szerzők:	Czikora István (1979-) (vegyész, biokémikus) Bogatcheva, Natalia V. Adyshev, Djanybek M. Poirier, Christophe Oláh Gábor Verin, Alexander
Internet cím:	Szerző által megadott URL DOI Intézményi repozitóriumban (DEA) tárolt változat
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001-es BibID:	BIBFORM001291
Első szerző:	Csortos Csilla (biokémikus)
Cím:	Regulation of vascular endothelial cell barrier function and cytoskeleton structure by protein phosphatases of the PPP family / Csortos Cs., Kolosova I., Verin A. D.
Dátum:	2007
Tárgyszavak:	Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban endothelial barrier function Ser/Thr protein phosphatases
Megjelenés:	American Journal of Physiology-Lung Cellular and Molecular Phsiology. - 293 (2007), p. L843-L854. -
További szerzők:	Kolosova, Irina Verin, Alexander
Internet cím:	elektronikus változat DOI
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