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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
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2.

001-es BibID:BIBFORM016824
Első szerző:Czikora István (vegyész, biokémikus)
Cím:Characterization of the effect of TIMAP phosphorylation on its interaction with protein phosphatase 1 / Czikora István, Kim Kyung-mi, Kása Anita, Bécsi Bálint, Verin Alexander D., Gergely Pál, Erdődi Ferenc, Csortos Csilla
Dátum:2011
ISSN:0300-9084
Megjegyzések:TIMAP, TGF-beta inhibited, membrane-associated protein, is highly abundant in endothelial cells (EC). We have shown earlier the involvement of TIMAP in PKA-mediated ERM (ezrin-radixin-moesin) dephosphorylation as part of EC barrier protection by TIMAP (Csortos et al., 2008). Emerging data demonstrate the regulatory role of TIMAP on protein phosphatase 1 (PP1) activity. We provide here evidence for specific interaction (Ka = 1.80 x 106 M-1) between non-phosphorylated TIMAP and the catalytic subunit of PP1 (PP1c) by surface plasmon resonance based binding studies. Thiophosphorylation of TIMAP by PKA, or sequential thiophosphorylation by PKA and GSK3β slightly modifies the association constant for the interaction of TIMAP with PP1c and decreases the rate of dissociation. However, dephosphorylation of phospho-moesin substrate by PP1cbeta is inhibited to different extent in the presence of non- (not, vert, similar60% inhibition), mono- (not, vert, similar50% inhibition) or double-thiophosphorylated (<10% inhibition) form of TIMAP. Our data suggest that double-thiophosphorylation of TIMAP has minor effect on its binding ability to PP1c, but considerably attenuates its inhibitory effect on the activity of PP1c. PKA activation by forskolin treatment of EC prevented thrombin evoked barrier dysfunction and ERM phosphorylation at the cell membrane (Csortos et al., 2008). With the employment of specific GSK3beta inhibitor it is shown here that PKA activation is followed by GSK3beta activation in bovine pulmonary EC and both of these activations are required for the rescuing effect of forskolin in thrombin treated EC. Our results suggest that the forskolin induced PKA/GSK3beta activation protects the EC barrier via TIMAP-mediated decreasing of the ERM phosphorylation level.
Tárgyszavak:Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban
TIMAP
Protein Phosphatase 1
Moesin
Surface Plasmon Resonance
Molekuláris Medicina
Megjelenés:Biochimie. - 93 : 7 (2011), p. 1139-1145. -
További szerzők:Kim, Kyung-mi Kovács-Kása Anita (1983-) Bécsi Bálint (1981-) (vegyészmérnök) Verin, Alexander Gergely Pál (1947-) (biokémikus) Erdődi Ferenc (1953-) (biokémikus) Csortos Csilla (1956-) (biokémikus)
Pályázati támogatás:TÁMOP-4.2.1/B-09/1/KONV-2010-0007
TÁMOP
I. Protein foszfatázok szerepe az in vitro porcdifferenciációban és a mechano-transzdukcióban, II. Hypoglykaemiás szerek tervezése a glikogén foszforilázra (foszforilációval és defoszforilációval szabályozott kulcsenzim) ható molekulákkal
TÁMOP-4.2.1/B-09/1/KONV-2010-0007
TÁMOP
Biomolekuláris interakciók jellemzőinek kvantitatív meghatározása
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Intézményi repozitóriumban (DEA) tárolt változat
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3.

001-es BibID:BIBFORM029024
Első szerző:Kim, Kyung-mi
Cím:Molecular characterization of myosin phosphatase in endothelium / Kyung-mi Kim, Csilla Csortos, Istvan Czikora, David Fulton, Nagavedi S. Umapathy, Gabor Olah, Alexander D. Verin
Dátum:2012
ISSN:0021-9541
Megjegyzések:The phosphorylation status of myosin light chain (MLC) is regulated by both MLC kinases and type 1 Ser/Thr phosphatase (PPase 1), MLC phosphatase (MLCP) activities. The activity of the catalytic subunit of MLCP (CS1β) towards myosin depends on its associated regulatory subunit, namely myosin PPase targeting subunit 1 (MYPT1). Our previously published data strongly suggested the involvement of MLCP in endothelial cell (EC) barrier regulation. In this study, our new data demonstrate that inhibition of MLCP by either CS1β or MYPT1 siRNA-based depletion results in significant attenuation of purine nucleotide (ATP and adenosine)-induced EC barrier enhancement. Consistent with the data, thrombin-induced EC F-actin stress fiber formation and permeability increase were attenuated by the ectopic expression of constitutively active (C/A) MYPT1. The data demonstrated for the first time direct involvement of MLCP in EC barrier enhancement/protection. Cloning of MYPT1 in human pulmonary artery EC (HPAEC) revealed the presence of two MYPT1 isoforms, long and variant 2 (V2) lacking 56 amino acids from 553 to 609 of human MYPT1 long, which were previously identified in HeLa and HEK 293 cells. Our data demonstrated that in Cos-7 cells ectopically expressed EC MYPT1 isoforms co-immunoprecipitated with intact CS1β suggesting the importance of PPase 1 activity for the formation of functional complex of MYPT1/CS1β. Interestingly, MYPT1 V2 shows decreased binding affinity compared to MYPT1 long for radixin (novel MLCP substrate and a member of ERM family proteins). These results suggest functional difference between EC MYPT1 isoforms in the regulation of MLCP activity and cytoskeleton. J. Cell. Physiol. 227: 1701-1708, 2012. © 2011 Wiley Periodicals, Inc.
Tárgyszavak:Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban
egyetemen (Magyarországon) készült közlemény
Megjelenés:Journal of Cellular Physiology. - 227 : 4 (2012), p. 1701-1708. -
További szerzők:Fulton, David Umapathy, Nagavedi S. Oláh Gábor Verin, Alexander Csortos Csilla (1956-) (biokémikus) Czikora István (1979-) (vegyész, biokémikus)
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Intézményi repozitóriumban (DEA) tárolt változat
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4.

001-es BibID:BIBFORM047041
035-os BibID:PMID:23721711
Első szerző:Kovács-Kása Anita
Cím:Protein phosphatase 2A activity is required for functional adherent junctions in endothelial cells / Anita Kása, István Czikora, Alexander D. Verin, Pál Gergely, Csilla Csortos
Dátum:2013
ISSN:0026-2862
Megjegyzések:Reversible Ser/Thr phosphorylation of cytoskeletal and adherent junction (AJ) proteins has a critical role in the regulation of endothelial cell (EC) barrier function. We have demonstrated earlier that protein phosphatase 2A (PP2A) activity is important in EC barrier integrity. In the present work, macro- and microvascular EC were examined and we provided further evidence on the significance of PP2A in the maintenance of EC cytoskeleton and barrier function with special focus on the Bα (regulatory) subunit of PP2A. Immunofluorescent staining revealed that the inhibition of PP2A results in changes in the organization of EC cytoskeleton as microtubule dissolution and actin re-arrangement were detected. Depletion of Bα regulatory subunit of PP2A had similar effect on the cytoskeleton structure of the cells. Furthermore, transendothelial electric resistance measurements demonstrated significantly slower barrier recovery of Bα depleted EC after thrombin treatment. AJ proteins, VE-cadherin and β-catenin, were detected along with Bα in pull-down assay. Also, the inhibition of PP2A (by okadaic acid or fostriecin) or depletion of Bα caused β-catenin translocation from the membrane to the cytoplasm in parallel with its phosphorylation on Ser552. In conclusion, our data suggest that the A/Bα/C holoenzyme form of PP2A is essential in EC barrier integrity both in micro- and macrovascular EC.
Tárgyszavak:Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban
Megjelenés:Microvascular Research. - 89 (2013), p. 86-94. -
További szerzők:Czikora István (1979-) (vegyész, biokémikus) Verin, Alexander Gergely Pál (1947-) (biokémikus) Csortos Csilla (1956-) (biokémikus)
Pályázati támogatás:TÁMOP-4.2.2.A-11/1/KONV-2012-0025
TÁMOP
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Intézményi repozitóriumban (DEA) tárolt változat
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5.

001-es BibID:BIBFORM063528
Első szerző:Tar Krisztina (biokémikus, molekuláris biológus)
Cím:The role of protein phosphatase 2A in the regulation of endothelial cell cytoskeleton structure / Tar K., Csortos C., Czikora I., Oláh G., Ma S. F., Wadgaonkar R., Gergely P., Garcia J., Verin A. D.
Dátum:2006
ISSN:1081-5589
Tárgyszavak:Orvostudományok Elméleti orvostudományok idézhető absztrakt
Megjelenés:Journal Of Investigative Medicine. - 54 : 2 (2006), p. 350. -
További szerzők:Csortos Csilla (1956-) (biokémikus) Czikora István (1979-) (vegyész, biokémikus) Oláh Gábor Ma, S. F. Wadgaonkar, Raj Gergely Pál (1947-) (biokémikus) Garcia, Javier Verin, Alexander
Internet cím:Intézményi repozitóriumban (DEA) tárolt változat
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6.

001-es BibID:BIBFORM003558
Első szerző:Tar Krisztina (biokémikus, molekuláris biológus)
Cím:Role of protein phosphatase 2A in the regulation of endothelial cell cytoskeleton structure / Krisztina Tar, Csilla Csortos, Istvan Czikora, Gabor Olah, Shwu-Fan Ma, Raj Wadgaonkar, Pal Gergely, Joe G. N. Garcia, Alexander D. Verin
Dátum:2006
Tárgyszavak:Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban
endothelium
phosphatase 2A
permeability
microtubules
microfilaments
tau
HSP27
Megjelenés:Journal of Cellular Biochemistry. - 98 : 4 (2006), p. 931-953. -
További szerzők:Csortos Csilla (1956-) (biokémikus) Czikora István (1979-) (vegyész, biokémikus) Oláh Gábor Ma, Shwu-Fan Wadgaonkar, Raj Gergely Pál (1947-) (biokémikus) Garcia, Joe G. N. Verin, Alexander
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