PP2A Affects Angiogenesis via Its Interaction with a Novel Phosphorylation Site of TSP1.

Alterations in angiogenic properties play a pivotal role in the manifestation and onset of various pathologies, including vascular diseases and cancer. Thrombospondin-1 (TSP1) protein is one of the master regulators of angiogenesis. This study unveils a novel aspect of TSP1 regulation through reversible phosphorylation. The silencing of the B55α regulatory subunit of protein phosphatase 2A (PP2A) in endothelial cells led to a significant decrease in TSP1 expression. Direct interaction between TSP1 and PP2A-B55α was confirmed via various methods. Truncated TSP1 constructs were employed to identify the phosphorylation site and the responsible kinase, ultimately pinpointing PKC as the enzyme phosphorylating TSP1 on Ser93. The biological effects of B55α-TSP1 interaction were also analyzed. B55α silencing not only counteracted the increase in TSP1 expression during wound closure but also prolonged wound closure time. Although B55α silenced cells initiated tube-like structures earlier than control cells, their spheroid formation was disrupted, leading to disintegration. Cells transfected with phosphomimic TSP1 S93D exhibited smaller spheroids and reduced effectiveness in tube formation, revealing insights into the effects of TSP1 phosphorylation on angiogenic properties. In this paper, we introduce a new regulatory mechanism of angiogenesis by reversible phosphorylation on TSP1 S93 by PKC and PP2A B55α.

TIMAP, a Regulatory Subunit of Protein Phosphatase 1, Inhibits In Vitro Neuronal Differentiation.

TIMAP (TGF-ß-inhibited membrane associated protein) is abundant in endothelial cells, and it has been regarded as a member of the myosin phosphatase targeting protein (MYPT) family. Our workgroup previously identified several interacting protein partners of TIMAP and proved its regulatory subunit role for protein phosphatase 1 catalytic subunit (PP1c). TIMAP is also expressed in neuronal cells, but details of its function have not been studied yet. Therefore, we aimed to explore the role of TIMAP in neuronal cells, especially during differentiation. Expression of TIMAP was proved both at mRNA and protein levels in SH-SY5Y human neuroblastoma cells. Differentiation of SH-SY5Y cells was optimized and proved by the detection of neuronal differentiation markers, such as ß3-tubulin, nestin and inhibitor of differentiation 1 (ID1) using qPCR and Western blot. We found downregulation of TIMAP during differentiation. In accordance with this, overexpression of recombinant TIMAP attenuated the differentiation of neuronal cells. Moreover, the subcellular localization of TIMAP has changed during differentiation as it translocated from the plasma membrane into the nucleus. The nuclear interactome of TIMAP revealed more than 50 proteins, offering the possibility to further investigate the role of TIMAP in several key physiological pathways of neuronal cells.

Ser69 phosphorylation of TIMAP affects endothelial cell migration.

PURPOSE/AIM: TIMAP (TGF-ß-inhibited membrane-associated protein) is a regulatory subunit of protein phosphatase 1 (PP1). The N-terminal region contains a binding motif for the catalytic subunit of PP1 (PP1c) and a nuclear localization signal (NLS). Phosphorylation of TIMAP on Ser331, Ser333 and Ser337 side chains was shown to regulate the activity of the TIMAP-PP1c complex. Several studies, however, reported an additional side chain of TIMAP. Ser69 is located near to the PP1c binding motif and NLS, therefore, we hypothesized that the phosphorylation of this side chain perhaps may regulate the interaction between TIMAP and PP1c, or may affect the nuclear transport of TIMAP. Materials and Methods: To study the significance of Ser69 phosphorylation, GST-tagged or c-myc-tagged wild type, phosphomimic S69D and phosphonull S69A recombinant TIMAP proteins were expressed in bacteria or endothelial cells, respectively. Protein-protein interactions of the wild type or mutant forms of TIMAP were studied by pull-down and Western blot. Localization of TIMAP S69 mutants in pulmonary artery endothelial cells was detected by immunofluorescent staining and expression and localization of the recombinants were investigated by subcellular fractionation and Western blot. Results: Modifications of Ser69 of TIMAP had no effect on binding of PP1c, ERM or RACK1. However, S69D TIMAP showed enhanced membrane localization and an increased number of membrane protrusions were observed in the cells overexpressing this phosphomimic mutant. Furthermore, significantly faster wound healing and migration rate of the S69D mutant overexpressing cells were detected by endothelial barrier resistance measurements (ECIS). Specific interaction was shown between TIMAP and polo-like kinase 4 (PLK4), a potential kinase to phosphorylate Ser69. Conclusions: Altogether, our results indicate that Ser69 phosphorylation by PLK4 may evoke an enrichment of TIMAP in the plasma membrane region and may play an important role in endothelial cell migration without affecting the PP1c binding ability of TIMAP.

Dephosphorylation of annexin A2 by protein phosphatase 1 regulates endothelial cell barrier.

Annexin A2 (ANXA2) is a multifunctional protein expressed in nearly all human tissues and cell types, playing a role in various signaling pathways. It is subjected to phosphorylation, but no specific protein phosphatase has been identified in its posttranslational regulation yet. Using pull-down assay followed by liquid chromatography-mass spectrometry analysis we found that ANXA2 interacts with TIMAP (TGF-beta-inhibited membrane-associated protein) in pulmonary artery endothelial cells. TIMAP is highly expressed in endothelial cells, where it acts as a regulatory and targeting subunit of protein phosphatase 1 (PP1). TIMAP plays an important role in the regulation of the endothelial barrier maintenance through the dephosphorylation of its several substrate proteins. In the present work, phosphorylation of Ser25 side chain in ANXA2 by protein kinase C (PKC) was shown both in vivo and in vitro. Phosphorylation level of ANXA2 at Ser25 increased greatly by inhibition of PP1 and by depletion of its regulatory subunit, TIMAP, implying a role of this PP1 holoenzyme in the dephosphorylation of ANXA2. Immunofluorescence staining and subcellular fractionations revealed a diffuse subcellular localization for the endogenous ANXA2, but phospho-Ser25 ANXA2 was mainly detected in the membrane. ANXA2 depletion lowered the basal endothelial barrier and inhibited cell migration, but had no significant effect on cell proliferation or viability. ANXA2 depleted cells failed to respond to PMA treatment, indicating an intimately involvement of phospho-ANXA2 in PKC signaling. Moreover, phosphorylation of ANXA2 disrupted its interaction with S100A10 suggesting a phosphorylation dependent multiple regulatory role of ANXA2 in endothelial cells. Our results demonstrate the pivotal role of PKC-ANXA2-PP1 pathway in endothelial cell signaling, especially in barrier function and cell migration.

Protein phosphatase 2A-mediated flotillin-1 dephosphorylation up-regulates endothelial cell migration and angiogenesis regulation.

Endothelial cells have key functions in endothelial barrier integrity and in responses to angiogenic signals that promote cell proliferation, cell migration, cytoskeletal reorganization, and formation of new blood vessels. These functions highly depend on protein-protein interactions in cell-cell junction and cell attachment complexes and on interactions with cytoskeletal proteins. Protein phosphatase 2A (PP2A) dephosphorylates several target proteins involved in cytoskeletal dynamics and cell adhesion. Our goal was to find new interacting and substrate proteins of the PP2A-B55α holoenzyme in bovine pulmonary endothelial cells. Using LC-MS/MS analysis, we identified flotillin-1 as a protein that binds recombinant GSH S-transferase-tagged PP2A-B55α. Immunoprecipitation experiments, proximity ligation assays, and immunofluorescent staining confirmed the interaction between these two endogenous proteins in endothelial cells. Originally, flotillins were described as regulatory proteins for axon regeneration, but they appear to function in many cellular processes, such as membrane receptor signaling, endocytosis, and cell adhesion. Ser315 is a known PKC-targeted site in flotillin-1. Utilizing phosphomutants of flotillin-1 and the NanoBiT luciferase assay, we show here that phosphorylation/dephosphorylation of Ser315 in flotillin-1 significantly affects its interaction with PP2A-B55α and that PP2A-B55α dephosphorylates phospho-Ser315 Spreading, attachment, migration, and in vitro tube formation rates of S315A variant-overexpressing cells were faster than those of nontransfected or S315D-transfected cells. These results indicate that the PP2A-flotillin-1 interaction identified here affects major physiological activities of pulmonary endothelial cells.

TIMAP, the versatile protein phosphatase 1 regulator in endothelial cells.

Transforming growth factor (TGF)-ß inhibited membrane associated protein, TIMAP, is the member of the myosin phosphatase targeting protein (MYPT) family of protein phosphatase 1 (PP1) regulatory subunits. The N-terminal part of TIMAP has a typical MYPT family structure with a sequence element called MyPhone (myosin phosphatase N-terminal element), a putative bipartite nuclear localization signal, a PP1 catalytic subunit binding motif, and five ankyrin repeats. The C-terminal half of TIMAP is intrinsically disordered, but ends with a functional CAAX box for lipid modification which allows localization of TIMAP at the plasma membrane. TIMAP is prenylated by farnesyl transferase with the contribution of the anchoring protein, RACK1 in the cytoplasm. The controlling effect of TIMAP on PP1 is moderated by PKA/GSK3ß and PKC mediated phosphorylation of TIMAP, the sites are located in the disordered region of the protein. TIMAP is abundant in endothelial cells. A growing body of evidence attained through characterization of newly identified protein partners calls attention to its critical role in normal and pathological activities of the endothelium via regulation of PP1. TIMAP binds the non-integrin laminin receptor 1 and the endothelin converting enzyme 1, which may connect TIMAP to angiogenesis, tumor invasion and metastasis. Barrier protecting role of TIMAP was shown for pulmonary artery endothelial cells. ERM (ezrin-radixin-moesin) proteins, as potential in vivo PP1-TIMAP substrates, are critical targets in the barrier maintenance. TIMAP affects phosphorylation level and subcellular localization of merlin and eukaryotic elongation factor-1A1. Merlin is a key component of signaling pathways regulating cell proliferation, membrane domain formation and cell-cell junction organization. Noncanonical functions of the elongation factor include a role in organization of cytoskeleton dynamics and in apoptosis. The interacting/binding partners identified so far demonstrate a rather complex role of TIMAP in key functions of the endothelium offering TIMAP as a plausible target in pathological issues. © 2017 IUBMB Life, 69(12):918-928, 2017.

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

Merlin (moesin-ezrin-radixin like protein), the product of neurofibromatosis type 2 gene, was primarily recognized as a tumor suppressor, but it also functions as a membrane-cytoskeletal linker and regulator of multiple signaling pathways. The activity and localization of merlin is regulated by head to tail folding that is controlled by phosphorylation of the Ser518 side chain. Merlin localizes in the nucleus when the Ser518 side chain is not phosphorylated, while the phosphorylated form is present in the cytoplasm and the plasma membrane. In this work interactions and their impact on the subcellular localization and phosphorylation state of the Ser518 side chain of merlin were investigated in endothelial cells. It is shown that merlin (dephospho-Ser518 form) interacts in the nucleus of endothelial cells with the scaffolding protein EBP50, a member of the Na+/H+exchanger regulatory factor family. Upon EBP50 depletion, merlin translocated from the nucleus, suggesting that binding of merlin to EBP50 is critical in the nuclear localization of merlin. Along with the translocation, the phosphorylation level of phospho-Ser518-merlin was increased in EBP50 depleted cells. TIMAP (TGFß-inhibited membrane-associated protein), a type 1 protein phosphatase (PP1) regulatory subunit, was newly recognized as an interacting partner for merlin. Domain mapping using truncated mutant forms in GST pull down revealed that the N-terminal half of TIMAP (aa 1-290) and the FERM domain of merlin are the regions responsible for the interaction.The catalytic subunit of PP1 (PP1c) was present in all merlin-TIMAP pull down or immunoprecipitation samples demonstrating that merlin actually interacts with the PP1c-TIMAP holoenzyme. On the other hand, from TIMAP depleted cells, without its targeting protein, PP1c could not bind to merlin. Also, when the phosphatase activity of PP1c-TIMAP was inhibited either with depletion of TIMAP or by treatment of the cells with specific PP1 inhibitor, there was an increase in the amount of phospho-Ser518 form of merlin in the membrane of the cells. These data strongly suggest that the PP1c-TIMAP- complex dephosphorylates phospho-Ser518-merlin. ECIS measurements indicate that phospho-merlin accelerates in vitro wound healing of the endothelial monolayer. In conclusion, in endothelial cells, EBP50 is required for the nuclear localization of merlin and the PP1c-TIMAP holoenzyme plays an important role in the dephosphorylation of merlin on its Ser518 side chain, which influence cell migration and proliferation.

PKC mediated phosphorylation of TIMAP regulates PP1c activity and endothelial barrier function.

TGF-ß inhibited membrane-associated protein (TIMAP) is greatly expressed in endothelial cell lines and serves as a protein phosphatase 1 (PP1) regulatory subunit. Phosphorylation state of TIMAP, through affecting PP1 activity, has a remarkable effect on endothelial barrier function. Here we present evidence for a previously unidentified PKC phosphorylation site in TIMAP. Protein-protein interaction was detected in pulmonary endothelial cells between endogenous TIMAP and activated PKCα. PKCα phosphorylated the full length recombinant TIMAP in in vitro kinase assay and Ser331 of TIMAP was shown to be phosphorylated by PKC. Phosphorylation of TIMAP upon PKC activation in endothelial cells results in enrichment of TIMAP in the membrane, but no such change can be observed in PKC depleted cells. However, the previously identified PKA/GSK-3ß induced enrichment of TIMAP at the plasma membrane was not affected in the absence of PKC. Interaction between TIMAP and the TIMAP-PP1 substrate phospho-ERM was described earlier, but now we show that binding of PKC phosphorylated TIMAP to ERM is severely reduced. This suggests an inhibitory effect of phospho-Ser331 on TIMAP-PP1 activity toward phospho-ERM. Accordingly, phospho-ERM level in the membrane fraction of the phospho-mimic S331D TIMAP mutant transfected cells was increased, but the S331A mutant overexpressing endothelial cells had a lower phospho-ERM level. Consistent with the phospho-ERM level, electric resistance measurements showed that the S331A mutation of TIMAP resulted in faster recovery from the PMA treatment. Taken together, phosphorylation of TIMAP on Ser331 by PKC represents a new mechanism of endothelial barrier regulation, through the inhibition of phospho-ERM dephosphorylation.

Role of Human Corneal Stroma-Derived Mesenchymal-Like Stem Cells in Corneal Immunity and Wound Healing.

Corneal tissue regeneration is of crucial importance for maintaining normal vision. We aimed to isolate and cultivate human corneal stroma-derived mesenchymal stem-like cells (CSMSCs) from the central part of cadaver corneas and study their phenotype, multipotency, role in immunity and wound healing. The isolated cells grew as monolayers in vitro, expressed mesenchymal- and stemness-related surface markers (CD73, CD90, CD105, CD140b), and were negative for hematopoietic markers as determined by flow cytometry. CSMSCs were able to differentiate in vitro into fat, bone and cartilage. Their gene expression profile was closer to bone marrow-derived MSCs (BMMSCs) than to limbal epithelial stem cells (LESC) as determined by high-throughput screening. The immunosuppressive properties of CSMSCs were confirmed by a mixed lymphocyte reaction (MLR), while they could inhibit proliferation of activated immune cells. Treatment of CSMSCs by pro-inflammatory cytokines and toll-like receptor ligands significantly increased the secreted interleukin-6 (IL-6), interleukin-8 (IL-8) and C-X-C motif chemokine 10 (CXCL-10) levels, as well as the cell surface adhesion molecules. CSMSCs were capable of closing a wound in vitro under different stimuli. These cells thus contribute to corneal tissue homeostasis and play an immunomodulatory and regenerative role with possible implications in future cell therapies for treating sight-threatening corneal diseases.

TIMAP-protein phosphatase 1-complex controls endothelin-1 production via ECE-1 dephosphorylation.

Endothelin induced signaling pathways can affect blood pressure and vascular tone, but the influence of endothelins on tumor cells is also significant. We have detected elevated endothelin-1 secretion from TIMAP (TGF-ß inhibited membrane associated protein) depleted vascular endothelial cells. The autocrine signaling activated by the elevated endothelin-1 level through the ETB receptors evoked an angiogenic-like phenotype, the cells assumed an elongated morphology, and enhanced tube formation and wound healing abilities. The depleted protein, TIMAP, is a highly specific and abundant protein in the endothelial cells, and it is a regulatory/targeting subunit for the catalytic subunit of protein phosphatase 1 (PP1c). Protein-protein interaction between the TIMAP-PP1c complex and the endothelin converting enzyme-1 (ECE-1) was detected, the latter of which is a transmembrane protein that produces the biologically active 21-amino acid form of endothelin-1 from proendothelin. The results indicate that silencing of TIMAP induces a reduction in TIMAP-PP1c activity connected to ECE-1. This leads to an increase in the amount of ECE-1 protein in the plasma membrane and a consequent increase in endothelin-1 secretion. Similarly, activation of PKC, the kinase responsible for ECE-1 phosphorylation increased ECE-1 protein level in the membrane fraction of the endothelial cells. The elevated ECE-1 level was mitigated in time in normal cells, but was clearly preserved in TIMAP-depleted cells. Overall, our results indicate that PKC-phosphorylated ECE-1 is a TIMAP-PP1c substrate and this phosphatase complex has an important role in endothelin-1 production of EC through the regulation of ECE-1 activity.

Elongation factor-1A1 is a novel substrate of the protein phosphatase 1-TIMAP complex.

TIMAP (TGF-ß inhibited membrane associated protein) is a protein phosphatase 1 (PP1) regulatory subunit highly abundant in endothelial cells and it is involved in the maintenance of pulmonary endothelial barrier function. It localizes mainly in the plasma membrane, but it is also present in the nuclei and cytoplasm. Direct interaction of TIMAP with the eukaryotic elongation factor 1 A1 (eEF1A1) is shown by pull-down, LC-MS/MS, Far-Western and immunoprecipitations. In connection with the so called moonlighting functions of the elongation factor, eEF1A is thought to establish protein-protein interactions through a transcription-dependent nuclear export motif, TD-NEM, and to aid nuclear export of TD-NEM containing proteins. We found that a TD-NEM-like motif of TIMAP has a critical role in its specific binding to eEF1A1. However, eEF1A1 is not or not exclusively responsible for the nuclear export of TIMAP. On the contrary, TIMAP seems to regulate membrane localization of eEF1A1 as the elongation factor co-localized with TIMAP in the plasma membrane fraction of control endothelial cells, but it has disappeared from the membrane in TIMAP depleted cells. It is demonstrated that membrane localization of eEF1A1 depends on the phosphorylation state of its Thr residue(s); and ROCK phosphorylated eEF1A1 is a novel substrate for TIMAP-PP1 underlining the complex regulatory role of TIMAP in the endothelium. The elongation factor seems to be involved in the regulation of endothelial cell attachment and spreading as silencing of eEF1A1 positively affected these processes which were monitored by transendothelial resistance measurements.

Cytoskeletal mechanisms regulating vascular endothelial barrier function in response to acute lung injury.

Endothelial cells (EC) form a semi-permeable barrier between the interior space of blood vessels and the underlying tissues. In acute lung injury (ALI) the EC barrier is weakened leading to increased vascular permeability. It is widely accepted that EC barrier integrity is critically dependent upon intact cytoskeletal structure and cell junctions. Edemagenic agonists, like thrombin or endotoxin lipopolysaccharide (LPS), induced cytoskeletal rearrangement, and EC contractile responses leading to disruption of intercellular contacts and EC permeability increase. The highly clinically-relevant cytoskeletal mechanisms of EC barrier dysfunction are currently under intense investigation and will be described and discussed in the current review.

NHERF2 is crucial in ERM phosphorylation in pulmonary endothelial cells.

BACKGROUND: EBP50 and NHERF2 adaptor proteins are incriminated in various signaling pathways of the cell. They can bind ERM proteins and mediate ERM-membrane protein interactions. RESULTS: Binding of ERM to EBP50 and NHERF2 was compared in pulmonary artery endothelial cells by immunoprecipitation. NHERF2 associates with all three ERM, but EBP50 appeared to be a weak binding partner if at all. Furthermore, we detected co-localization of NHERF2 and phospho-ERM at the cell membrane and in the filopodia of dividing cells. Silencing of NHERF2 prevented agonist or angiogenesis induced phosphorylation of ERM, while overexpression of the adaptor elevated the phosphorylation level of ERM, likely catalyzed by Rho kinase 2, which co-immunoprecipitated with NHERF2/ERM in control EC, but did not bind to ERM in NHERF2 depleted cells. Dependence of ERM phosphorylation on NHERF2 was also shown in Matrigel tube formation assay, and NHERF2 was proved to be important in angiogenesis as well. Furthermore, when NHERF2 was depleted or cells were overexpressing a mutant form of NHERF2 unable to bind ERM, we found attenuated cell attachment with ECIS measurements, while it was supported by overexpression of wild type NHERF2. CONCLUSIONS: Pivotal role of NHERF2 in the phosphorylation process of ERM in pulmonary artery endothelial cells is shown. We propose that NHERF2 provides a common anchoring surface for ERM and Rho kinase 2. Our results demonstrate the essential role of NHERF2 in endothelial cell adhesion/migration and angiogenesis.

Protein phosphatase 2A activity is required for functional adherent junctions in endothelial cells.

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 ß-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 ß-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.

Putative protein partners for the human CPI-17 protein revealed by bacterial two-hybrid screening.

We have previously demonstrated that PKC-potentiated inhibitory protein of protein phosphatase-1 (CPI-17) is expressed in lung endothelium. CPI-17, a specific inhibitor of myosin light chain phosphatase (MLCP), is involved in the endothelial cytoskeletal and barrier regulation. In this paper, we report the identification of fourteen putative CPI-17 interacting proteins in the lung using BacterioMatch Two-Hybrid System. Five of them: plectin 1 isoform 1, alpha II spectrin, OK/SW-CL.16, gelsolin isoform a, and junction plakoglobin are involved in actin cytoskeleton organization and cell adhesion, suggesting possible significance of these binding partners in CPI-17-mediated cytoskeletal reorganization of endothelial cells. Furthermore, we confirmed the specific interaction between plakoglobin and CPI-17, which is affected by the phosphorylation status of CPI-17 in human lung microvascular endothelial cells.

RACK1 is involved in endothelial barrier regulation via its two novel interacting partners.

BACKGROUND: RACK1, receptor for activated protein kinase C, serves as an anchor in multiple signaling pathways. TIMAP, TGF-ß inhibited membrane-associated protein, is most abundant in endothelial cells with a regulatory effect on the endothelial barrier function. The interaction of TIMAP with protein phosphatase 1 (PP1cδ) was characterized, yet little is known about its further partners. RESULTS: We identified two novel interacting partners of RACK1, namely, TGF-ß inhibited membrane-associated protein, TIMAP, and farnesyl transferase. TIMAP is most abundant in endothelial cells where it is involved in the regulation of the barrier function. 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. CONCLUSIONS: 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.

Cell cycle dependent association of EBP50 with protein phosphatase 2A in endothelial cells.

Ezrin-radixin-moesin (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 report 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 (PP2A). 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, the results underline the significance of EBP50 in cell division via reversible phosphorylation of the protein with cyclin dependent kinase and PP2A in normal cells.

Molecular characterization of myosin phosphatase in endothelium.

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 (CS1ß) 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 CS1ß 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 CS1ß suggesting the importance of PPase 1 activity for the formation of functional complex of MYPT1/CS1ß. 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.

Characterization of the effect of TIMAP phosphorylation on its interaction with protein phosphatase 1.

TIMAP, TGF-ß 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 (K(a) = 1.80 × 10(6) 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 GSK3ß 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 PP1cß is inhibited to different extent in the presence of non- (~60% inhibition), mono- (~50% 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 GSK3ß inhibitor it is shown here that PKA activation is followed by GSK3ß 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/GSK3ß activation protects the EC barrier via TIMAP-mediated decreasing of the ERM phosphorylation level.

TIMAP is a positive regulator of pulmonary endothelial barrier function.

TGF-beta-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 beta-isoform of the catalytic subunit of PP1 (PP1cbeta) from pulmonary artery EC. As PP1cbeta, but not PP1calpha, 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.