Endothelial contraction and monolayer hyperpermeability are regulated by Src kinase

2003 ◽  
Vol 284 (3) ◽  
pp. H994-H1002 ◽  
Author(s):  
David R. Mucha ◽  
Carter L. Myers ◽  
Richard C. Schaeffer
Keyword(s):  
Tyrosine Phosphorylation ◽  
Myosin Light Chain ◽  
Kinase Activity ◽  
Kinase Inhibitor ◽  
Src Kinase ◽  
Endothelial Monolayer ◽  
Herbimycin A ◽  
Barrier Formation ◽  
Monolayer Barrier Function

Endothelial monolayer hyperpermeability is regulated by a myosin light chain phosphorylation (MLCP)-dependent contractile mechanism. In this study, we tested the role of Src-dependent tyrosine phosphorylation to modulate endothelial contraction and monolayer barrier function with the use of the myosin phosphatase inhibitor calyculin A (CalA) to directly elevate MLCP with the Src family tyrosine kinase inhibitor herbimycin A (HA) in bovine pulmonary artery endothelial cells (EC). CalA stimulated an increase in MLCP, Src kinase activity, an increase in the tyrosine phosphorylation of paxillin and focal adhesion (FA) kinase (p125FAK), and monolayer hyperpermeability. Microscopic examination of CalA-treated EC revealed a contractile morphology characterized by peripheral contractile bands of actomyosin filaments and stress fibers linked to phosphotyrosine-containing FAs. These CalA-dependent events were HA sensitive. HA alone stimulated an improvement in monolayer barrier formation by reducing the levels of MLCP and phosphotyrosine-containing proteins and the number of large paracellular holes. These data show that Src kinase plays an important role in regulating monolayer hyperpermeability through adjustments in tyrosine phosphorylation, MLCP, and EC contraction.

Src kinase and PI 3-kinase as a transduction pathway in ceramide-induced contraction of colonic smooth muscle

1998 ◽  
Vol 275 (4) ◽  
pp. G705-G711 ◽  
Author(s):  
Adenike I. Ibitayo ◽  
Yasuhiro Tsunoda ◽  
Fumihiko Nozu ◽  
Chung Owyang ◽  
Khalil N. Bitar
Keyword(s):  
Smooth Muscle ◽  
Map Kinase ◽  
Tyrosine Phosphorylation ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
Src Kinase ◽  
Sustained Contraction ◽  
Herbimycin A ◽  
Phosphoinositide 3 Kinase ◽  
In Cells

Ceramide mediates sustained contraction of smooth muscle cells. C2 ceramide induced a rapid increase in Src kinase activity within 15 s, peaked at 1 min, and was sustained up to 8 min. Contraction and Src kinase activity were inhibited in cells incubated in Ca2+-free medium containing 2 mM EGTA and in cells preincubated with herbimycin A, a Src kinase inhibitor. Immunoblotting using a phosphospecific anti-Src (416Y) antibody showed a ceramide-induced increase in pp60 src tyrosine phosphorylation. Immunoprecipitation using an anti-phosphotyrosine antibody followed by Western immunoblotting using a monoclonal IgG anti-phosphoinositide 3-kinase NH2 terminal-SH2 domain antibody showed a ceramide-induced increase in phosphoinositide 3-kinase (PI 3-kinase) tyrosine phosphorylation at a protein mass corresponding to 85 kDa, the regulatory subunit of PI 3-kinase, which contains the Src kinase binding site. PI 3-kinase phosphorylation was inhibited by herbimycin A and by the PI 3-kinase inhibitors wortmannin and LY-294002. Preincubation of cells with herbimycin A or PI 3-kinase inhibitors also resulted in an inhibition of mitogen-activated protein (MAP) kinase p42 and p44 activities as seen on Western blots. In summary, we found that 1) the maintenance of sustained contraction is dependent on extracellular Ca2+; 2) ceramide activates a nonreceptor tyrosine kinase pathway through activation of pp60 src and PI 3-kinase; and 3) the converging signals are probably through activation of MAP kinase.

H2O2-mediated permeability II: importance of tyrosine phosphatase and kinase activity

2001 ◽  
Vol 281 (6) ◽  
pp. C1940-C1947 ◽  
Author(s):  
Christopher G. Kevil ◽  
Naotsuka Okayama ◽  
J. Steven Alexander
Keyword(s):  
Phosphatase Activity ◽  
Protein Tyrosine Phosphatase ◽  
Kinase Activity ◽  
Kinase Inhibitor ◽  
Biological Effects ◽  
Tyrosine Phosphatase ◽  
Src Kinase ◽  
Cell Junctions ◽  
Endothelial Monolayer ◽  
Solute Permeability

We previously reported that exposure of endothelial cells to H2O2results in a loss of cell-cell apposition and increased endothelial solute permeability. The purpose of this study was to determine how tyrosine phosphorylation and tyrosine phosphatases contribute to oxidant-mediated disorganization of endothelial cell junctions. We found that H2O2caused a rapid decrease in total cellular phosphatase activity that facilitates a compensatory increase in cellular phosphotyrosine residues. H2O2exposure also results in increased endothelial monolayer permeability, which was attenuated by pp60, an inhibitor of src kinase. Inhibition of protein tyrosine phosphatase activity by phenylarsine oxide (PAO) demonstrated a similar permeability profile compared with H2O2, suggesting that tyrosine phosphatase activity is important in maintaining a normal endothelial solute barrier. Immunofluorescence shows that H2O2exposure caused a loss of pan-reactive cadherin and β-catenin from cell junctions that was not blocked by the src kinase inhibitor PP1. H2O2also caused β-catenin to dissociate from the endothelial cytoskeleton, which was not prevented by PP1. Finally, we determined that PP1 did not prevent cadherin internalization. These data suggest that oxidants like H2O2produce biological effects through protein phosphotyrosine modifications by decreasing total cellular phosphatase activity combined with increased src kinase activity, resulting in increased endothelial solute permeability.

scholarly journals Src Catalytic but Not Scaffolding Function Is Needed for Integrin-Regulated Tyrosine Phosphorylation, Cell Migration, and Cell Spreading

2002 ◽  
Vol 22 (8) ◽  
pp. 2427-2440 ◽  
Author(s):  
Leslie A. Cary ◽  
Richard A. Klinghoffer ◽  
Christoph Sachsenmaier ◽  
Jonathan A. Cooper
Keyword(s):  
Cell Migration ◽  
Tyrosine Phosphorylation ◽  
Kinase Activity ◽  
Src Kinase ◽  
Focal Adhesions ◽  
Indirect Evidence ◽  
Integrin Signaling ◽  
Primary Role ◽  
Lower Efficiency

ABSTRACT Src family kinases (SFKs) are crucial for signaling through a variety of cell surface receptors, including integrins. There is evidence that integrin activation induces focal adhesion kinase (FAK) autophosphorylation at Y397 and that Src binds to and is activated by FAK to carry out subsequent phosphorylation events. However, it has also been suggested that Src functions as a scaffolding molecule through its SH2 and SH3 domains and that its kinase activity is not necessary. To examine the role of SFKs in integrin signaling, we have expressed various Src molecules in fibroblasts lacking other SFKs. In cells plated on fibronectin, FAK could indeed autophosphorylate at Y397 independently of Src but with lower efficiency than when Src was present. This step was promoted by kinase-inactive Src, but Src kinase activity was required for full rescue. Src kinase activity was also required for phosphorylation of additional sites on FAK and for other integrin-directed functions, including cell migration and spreading on fibronectin. In contrast, Src mutations in the SH2 or SH3 domain greatly reduced binding to FAK, Cas, and paxillin but had little effect on tyrosine phosphorylation or biological assays. Furthermore, our indirect evidence indicates that Src kinase activity does not need to be regulated to promote cell migration and FAK phosphorylation. Although Src clearly plays important roles in integrin signaling, it was not concentrated in focal adhesions. These results indicate that the primary role of Src in integrin signaling is as a kinase. Indirect models for Src function are proposed.

Activation of c-Src in cells bearing v-Crk and its suppression by Csk

1992 ◽  
Vol 12 (10) ◽  
pp. 4706-4713
Author(s):  
H Sabe ◽  
M Okada ◽  
H Nakagawa ◽  
H Hanafusa
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Protein Tyrosine Kinase ◽  
Kinase Activity ◽  
Src Kinase ◽  
Cellular Protein ◽  
Morphological Transformation ◽  
Protein Tyrosine ◽  
In Cells

The protein product of the CT10 virus, p47gag-crk (v-Crk), which contains Src homology region 2 (SH2) and 3 (SH3) domains but lacks a kinase domain, is believed to cause an increase in cellular protein tyrosine phosphorylation. A candidate tyrosine kinase, Csk (C-terminal Src kinase), has been implicated in c-Src Tyr-527 phosphorylation, which negatively regulates the protein tyrosine kinase of pp60c-src (c-Src). To investigate how c-Src kinase activity is regulated in vivo, we first looked at whether v-Crk can activate c-Src kinase. We found that cooverexpression of v-Crk and c-Src caused elevation of c-Src kinase activity, resulting in an increase of tyrosine phosphorylation of cellular proteins and morphological transformation of rat 3Y1 fibroblasts. v-Crk and c-Src complexes were not detected, although v-Crk bound to a variety of tyrosine-phosphorylated proteins in cells overexpressing v-Crk and c-Src. Overexpression of Csk in these transformed cells caused reversion to normal phenotypes and also reduced the level of c-Src kinase activity. However, Csk did not cause reversion of cells transformed by v-Src or c-Src527F, in which Tyr-527 was changed to Phe. These results strongly suggest that Csk acts on Tyr-527 of c-Src and suppresses c-Src kinase activity in vivo. Because Csk can suppress transformation by cooverexpression of v-Crk and c-Src, we suggest that v-Crk causes activation of c-Src in vivo by altering the phosphorylation state of Tyr-527.

Activation of pp60(src) is critical for stretch-induced orienting response in fibroblasts

1999 ◽  
Vol 112 (9) ◽  
pp. 1365-1373 ◽  
Author(s):  
X. Sai ◽  
K. Naruse ◽  
M. Sokabe
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Time Course ◽  
Kinase Inhibitor ◽  
Orienting Response ◽  
Cyclic Stretch ◽  
Wild Type ◽  
Herbimycin A ◽  
Molecular Masses

When subjected to uni-axial cyclic stretch (120% in length, 1 Hz), fibroblasts (3Y1) aligned perpendicular to the stretch axis in a couple of hours. Concomitantly with this orienting response, protein tyrosine phosphorylation of cellular proteins (molecular masses of approximately 70 kDa and 120–130 kDa) increased and peaked at 30 minutes. Immuno-precipitation experiments revealed that paxillin, pp125(FAK), and pp130(CAS) were included in the 70 kDa, and 120–130 kDa bands, respectively. Treatment of the cells with herbimycin A, a tyrosine kinase inhibitor, suppressed the stretch induced tyrosine phosphorylation and the orienting response suggesting that certain tyrosine kinases are activated by stretch. We focused on pp60(src), the most abundant tyrosine kinase in fibroblasts. The kinase activity of pp60(src) increased and peaked at 20 minutes after the onset of cyclic stretch. Treatment of the cells with an anti-sense S-oligodeoxynucleotide (S-ODN) against pp60(src), but not the sense S-ODN, inhibited the stretch induced tyrosine phosphorylation and the orienting response. To further confirm the involvement of pp60(src), we performed the same sets of experiments using c-src-transformed 3Y1 (c-src-3Y1) fibroblasts. Cyclic stretch induced a similar orienting response in c-src-3Y1 to that in wild-type 3Y1, but with a significantly faster rate. The time course of the stretch-induced tyrosine phosphorylation was also much faster in c-src-3Y1 than in 3Y1 fibroblasts. These results strongly suggest that cyclic stretch induces the activation of pp60(src) and that pp60(src) is indispensable for the tyrosine phosphorylation of pp130(CAS), pp125(FAK) and paxillin followed by the orienting response in 3Y1 fibroblasts.

scholarly journals An Anti-Inflammatory Role of VEGFR2/Src Kinase Inhibitor in Herpes Simplex Virus 1-Induced Immunopathology

2011 ◽  
Vol 85 (12) ◽  
pp. 5995-6007 ◽  
Author(s):  
S. Sharma ◽  
S. Mulik ◽  
N. Kumar ◽  
A. Suryawanshi ◽  
B. T. Rouse
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Kinase Inhibitor ◽  
Src Kinase ◽  
Herpes Simplex Virus 1 ◽  
Anti Inflammatory ◽  
Simplex Virus

scholarly journals Role of JAM-A tyrosine phosphorylation in epithelial barrier dysfunction during intestinal inflammation

2019 ◽  
Vol 30 (5) ◽  
pp. 566-578 ◽  
Author(s):  
Shuling Fan ◽  
Caroline M. Weight ◽  
Anny-Claude Luissint ◽  
Roland S. Hilgarth ◽  
Jennifer C. Brazil ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Barrier Function ◽  
Intestinal Inflammation ◽  
Kinase Inhibitor ◽  
Src Kinase ◽  
Small Gtpase ◽  
Epithelial Barrier ◽  
Barrier Dysfunction ◽  
Junction Protein

Junctional adhesion molecule-A (JAM-A), an epithelial tight junction protein, plays an important role in regulating intestinal permeability through association with a scaffold signaling complex containing ZO-2, Afadin, and the small GTPase Rap2. Under inflammatory conditions, we report that the cytoplasmic tail of JAM-A is tyrosine phosphorylated (p-Y280) in association with loss of barrier function. While barely detectable Y280 phosphorylation was observed in confluent monolayers of human intestinal epithelial cells under basal conditions, exposure to cytokines TNFα, IFNγ, IL-22, or IL-17A, resulted in compromised barrier function in parallel with increased p-Y280. Phosphorylation was Src kinase dependent, and we identified Yes-1 and PTPN13 as a major kinase and phosphatase for p-JAM-A Y280, respectively. Moreover, cytokines IL-22 or IL-17A induced increased activity of Yes-1. Furthermore, the Src kinase inhibitor PP2 rescued cytokine-induced epithelial barrier defects and inhibited phosphorylation of JAM-A Y280 in vitro. Phosphorylation of JAM-A Y280 and increased permeability correlated with reduced JAM-A association with active Rap2. Finally, we observed increased phosphorylation of Y280 in colonic epithelium of individuals with ulcerative colitis and in mice with experimentally induced colitis. These findings support a novel mechanism by which tyrosine phosphorylation of JAM-A Y280 regulates epithelial barrier function during inflammation.

scholarly journals Transformation and pp60v-src autophosphorylation correlate with SHC-GRB2 complex formation in rat and chicken cells expressing host-range and kinase-active, transformation-defective alleles of v-src.

1995 ◽  
Vol 6 (8) ◽  
pp. 953-966 ◽  
Author(s):  
M F Verderame ◽  
J L Guan ◽  
K M Woods Ignatoski
Keyword(s):  
Host Range ◽  
Tyrosine Phosphorylation ◽  
Kinase Activity ◽  
Biochemical Properties ◽  
Morphological Transformation ◽  
Anchorage Independent Growth ◽  
Parental Allele ◽  
Morphological Phenotype ◽  
In Cells

The biochemical properties of several pp60v-src substrates believed to participate in src-mediated transformation were examined in cells expressing a kinase-active, transformation-defective v-src allele (v-src-F172 delta/Y416F) and its parental allele, v-src-F172 delta, a host-range--dependent allele that transforms chicken cells to a fusiform morphology, but does not transform rat cells. Because pp60v-src-F172 delta is dependent on autophosphorylation for transforming ability, these alleles provide a unique opportunity to examine the role of pp60v-src autophosphorylation in regulating substrate interactions. Increased pp125FAK tyrosine phosphorylation and high levels of pp60v-src-associated phosphotidylinositol-3' kinase activity were detected specifically in chicken cells exhibiting round, refractile transformation but not in cells transformed to a fusiform morphology. Increased pp125FAK kinase activity, but not increased pp125FAK tyrosine-phosphorylation correlated with pp60v-src autophosphorylation and increased anchorage-independent growth. Thus, pp125FAK and PI3'K may participate in morphological transformation by v-src. Furthermore, association of phosphorylated SHC with the adapter GRB2 correlated with increased anchorage-independent growth (and autophosphorylation) in both rat and chicken cells independent of the morphological phenotype induced. Therefore, host-range dependence for transformation may be regulated through association of SHC with GRB2, thus implicating SHC as a crucial substrate for src-dependent transformation.

VEGF stimulates tyrosine phosphorylation of β-catenin and small-pore endothelial barrier dysfunction

1999 ◽  
Vol 277 (5) ◽  
pp. H2038-H2049 ◽  
Author(s):  
Alex W. Cohen ◽  
José M. Carbajal ◽  
Richard C. Schaeffer
Keyword(s):  
Tyrosine Phosphorylation ◽  
Kinase Inhibitor ◽  
Endothelial Barrier ◽  
Vegf Receptor ◽  
Barrier Dysfunction ◽  
Transport Pathways ◽  
Endothelial Barrier Dysfunction ◽  
Herbimycin A ◽  
Selective Permeability ◽  
Small Pore

The purpose of this study was to test the hypothesis that tyrosine phosphorylation signaling events and protein kinase C (PKC) activation mediate vascular endothelial growth factor-A165(VEGF)-induced endothelial cell (EC) proliferation and barrier dysfunction in bovine pulmonary artery EC monolayers. A size-selective permeability assay showed that VEGF stimulated a delayed, prolonged (6–45 h), concentration-dependent (50–200 ng/ml, ∼1–4 nM) increase in the number of predominantly small-“pore” transport pathways (<60 Å) across EC monolayers. The tyrosine kinase inhibitor herbimycin A (HA) and the selective PKC inhibitor bisindolylmaleimide (BIM) prevented this phenomenon. After 6–24 h, VEGF-treated monolayers displayed an HA- and BIM-sensitive reorganization of β-catenin adherens junctions with fingerlike projections and the loss of β-catenin at sites of small paracellular hole formation. HA and BIM prevented the VEGF-induced increase in EC growth. HA blocked the VEGF-induced rapid and prolonged (10 min–45 h) increases in the phosphotyrosine (PY) contents of VEGF receptor 2, phospholipase C-γ1, paxillin, and β-catenin as well as ∼140- and 128- to 117-kDa proteins, whereas BIM inhibited only the tyrosine phosphorylation of β-catenin. These data suggest that VEGF initiates increased EC growth and chronic, small-pore endothelial barrier dysfunction by PY signaling through β-catenin that depends on PKC.

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