Tyrosine Phosphorylation Of Cadherin Research Articles

Disruption of endothelial adherens junctions by high glucose is mediated by protein kinase C-β-dependent vascular endothelial cadherin tyrosine phosphorylation.

BackgroundHyperglycemia has been recognized as a primary factor in endothelial barrier dysfunction and in the development of micro- and macrovascular diseases associated with diabetes, but the underlying biochemical mechanisms remain elusive. Tyrosine phosphorylation of vascular endothelial cadherin (VE-cad) leads to the disruption of endothelial adherens junctions and increases the transendothelial migration (TEM) of leukocytes.MethodsVE-cad tyrosine phosphorylation, adherens junction integrity and TEM of monocytes in human umbilical vein endothelial cells (HUVECs) treated with high-concentration glucose were evaluated. The role of protein kinase C (PKC) in induction of endothelial cells adherence junction disruption by exposure of HUVECs to high concentration of glucose was explored.ResultsThe treatment of HUVEC with high-concentration glucose increased VE-cad tyrosine phosphorylation, whereas mannitol or 3-O-methyl-D-glucose had no effect. In addition, high-concentration glucose increased the dissociation of the VE-cad–β-catenin complex, activation of the Wnt/β-catenin pathway, and the TEM of monocytes. These alterations were accompanied by the activation of endothelial PKC and increased phosphorylation of ERK and myosin light chain (MLC). High-concentration glucose-induced tyrosine phosphorylation of VE-cad was attenuated by: 1- the inhibition of PKC-β by overexpression of dominant-negative PKC-β 2- inhibition of MLC phosphorylation by overexpression of a nonphosphorylatable dominant-negative form of MLC, 3- the inhibition of actin polymerization by cytochalasin D and 4- the treatment of HUVECs with forskolin (an activator of adenylate cyclase).ConclusionsOur findings show that the high-concentration glucose-induced disruption of endothelial adherens junctions is mediated by tyrosine phosphorylation of VE-cad through PKC-β and MLC phosphorylation.

Dynamic phosphorylation of VE-cadherin Y685 throughout mouse estrous cycle in ovary and uterus.

We previously reported that vascular endothelial growth factor induced vascular endothelial (VE)-cadherin tyrosine phosphorylation at Y685 in a Src-dependent manner in vitro. Here, we studied the occurrence of Y685 phosphorylation in vivo in the female reproductive tract because it is a unique model of physiological vascular remodeling dependent on vascular endothelial growth factor. We first developed and characterized an anti-phospho-specific antibody against the site Y685 of VE-cadherin to monitor VE-cadherin phosphorylation along the four phases of mouse estrous cycle, termed proestrus, estrus, metestrus, and diestrus. A dynamic profile of tyrosine phosphorylated proteins was observed in both uterus and ovary throughout mouse estrous cycle, including kinase Src, which was found highly active at the estrus phase. The extent of tyrosine phosphorylated VE-cadherin was low at proestrus but strongly increased at estrus and returned to baseline at metestrus and diestrus, suggesting a potent hormonal regulation of this specific process. Indeed, C57Bl/6 female mice treatment with pregnant mare serum gonadotropin and human chorionic gonadotropin confirmed a significant increase in phosphoY685-VE-cadherin compared with that in untreated mice. These results demonstrate that VE-cadherin tyrosine phosphorylation at Y685 is a physiological and hormonally regulated process in female reproductive organs. In addition, this process was concomitant with the early steps of vascular remodeling taking place at estrus stage, suggesting that phosphoY685-VE-cadherin is a biomarker of endothelial cell activation in vivo.

Disruption of endothelial adherens junctions by high glucose is mediated by protein kinase C-β–dependent vascular endothelial cadherin tyrosine phosphorylation

BackgroundHyperglycemia has been recognized as a primary factor in endothelial barrier dysfunction and in the development of micro- and macrovascular diseases associated with diabetes, but the underlying biochemical mechanisms remain elusive. Tyrosine phosphorylation of vascular endothelial cadherin (VE-cad) leads to the disruption of endothelial adherens junctions and increases the transendothelial migration (TEM) of leukocytes.MethodsVE-cad tyrosine phosphorylation, adherens junction integrity and TEM of monocytes in human umbilical vein endothelial cells (HUVECs) treated with high-concentration glucose were evaluated. The role of protein kinase C (PKC) in induction of endothelial cells adherence junction disruption by exposure of HUVECs to high concentration of glucose was explored.ResultsThe treatment of HUVEC with high-concentration glucose increased VE-cad tyrosine phosphorylation, whereas mannitol or 3-O-methyl-D-glucose had no effect. In addition, high-concentration glucose increased the dissociation of the VE-cad–β-catenin complex, activation of the Wnt/β-catenin pathway, and the TEM of monocytes. These alterations were accompanied by the activation of endothelial PKC and increased phosphorylation of ERK and myosin light chain (MLC). High-concentration glucose-induced tyrosine phosphorylation of VE-cad was attenuated by: 1- the inhibition of PKC-β by overexpression of dominant-negative PKC-β 2- inhibition of MLC phosphorylation by overexpression of a nonphosphorylatable dominant-negative form of MLC, 3- the inhibition of actin polymerization by cytochalasin D and 4- the treatment of HUVECs with forskolin (an activator of adenylate cyclase).ConclusionsOur findings show that the high-concentration glucose-induced disruption of endothelial adherens junctions is mediated by tyrosine phosphorylation of VE-cad through PKC-β and MLC phosphorylation.

Atorvastatin preserves the integrity of endothelial adherens junctions by inhibiting vascular endothelial cadherin tyrosine phosphorylation

Vascular endothelial cadherin (VE-cad) tyrosine (Tyr) phosphorylation has been implicated in the disruption of adherens junctions (AJs) induced by inflammatory reactions. The impacts of statins on integrity of AJs and VE-cad Tyr phosphorylation have not been explored. The effects of atorvastatin on IL-1β and monocyte-induced VE-cad Tyr phosphorylation in human umbilical vein endothelial cells (ECs) were studied. In ECs treated with interleukin (IL)-1β for 30min, VE-cad Tyr phosphorylation, dissociation of the VE-cad/β-catenin complex and transendothelial migration (TEM) of monocytes were increased. These processes were mediated by activation of HRas and RhoA that leads to phosphorylation of myosin light chain (MLC). Atorvastatin inhibited IL-1β-induced Tyr phosphorylation of VE-cad by inhibiting RhoA and by dephosphorylating MLC. The attenuating effect of atorvastatin on VE-cad Tyr phosphorylation was reversed when RhoA was activated or MLC phosphatase was inhibited. Furthermore, inhibiting farnesyl transferase or geranylgeranyl transferase reproduced the inhibitory effects of atorvastatin on VE-cad Tyr phosphorylation. In addition, atorvastatin inhibited monocyte-induced VE-cad Tyr phosphorylation in ECs and attenuated IL-1β-induced TEM of monocytes. Our study introduces a novel pleiotropic effect of atorvastatin and suggests that statins protect the integrity of AJs in ECs by inhibiting RhoA-mediated Tyr phosphorylation of VE-cad.

Immunoregulatory functions of KLRG1 cadherin interactions are dependent on forward and reverse signaling

AbstractKLRG1 is an inhibitory receptor expressed on a subset of mature T and NK cells. Recently, E-, N-, and R-cadherin have been identified as ligands for KLRG1. Cadherins are a large family of transmembrane or membrane-associated glycoproteins that were thought to only bind specifically to other cadherins to mediate specific cell-to-cell adhesion in a Ca2+-dependent manner. The consequences of cadherin KLRG1 molecular interactions are not well characterized. Here, we report that the first 2 extracellular domains of cadherin are sufficient to initiate a KLRG1-dependent signaling. We also demonstrate that KLRG1 engagement inhibits cadherin-dependent cellular adhesion and influences dendritic cell secretion of inflammatory cytokines, thereby exerting immunosuppressive effects. Consistent with this, engagement of cadherin by KLRG1 molecule induces cadherin tyrosine phosphorylation. Therefore, KLRG1/cadherin interaction leads to the generation of a bidirectional signal in which both KLRG1 and cadherin activate downstream signaling cascades simultaneously. Taken together, our results provide novel insights on how KLRG1 and E-cadherin interactions are integrated to differentially regulate not only KLRG1+ cells, but also E-cadherin–expressing cells, such as dendritic cells.

Myosin Phosphatase Regulatory Pathways

In many ways, the smooth muscle cell (SMC) has been at the forefront in defining cellular signaling pathways. For example, the initial description of agonist-dependent intracellular calcium ([Ca2+]i) transients was made on vertebrate vascular SMC using aqueorin as the indicator.1 Delineation of nitric oxide (NO) and cGMP signaling was first accomplished in smooth muscle.2,3 And more recently, insights into the general mechanisms of gene transcription that dictate phenotypic traits of cells are being defined by smooth muscle specific gene expression.4,5 It should come as no surprise then that the interactions among many signaling pathways in cells should be explained first in the SMC. Perhaps the reason for the central position of the SMC in defining paradigms in cellular biology is that there are well-defined physiologic end points amenable to measurement (ie, contractile force) and even better described and easily quantified biochemical pathways that underlie said physiologic end points (ie, myosin regulatory light chain phosphorylation). From a historical perspective, the role of protein phosphorylation in smooth muscle contraction as a paradigm for cell signaling follows only that for the control of glycogen metabolism (see 6,7 for reviews). Increases in the levels of cytosolic [Ca2+]i initiate smooth muscle contraction by binding to the universal intracellular Ca2+ receptor protein, calmodulin (CaM), which in turn binds to and activates smooth muscle myosin light chain kinase (MLCK). Activated MLCK catalyzes the phosphorylation serine-19 of the regulatory myosin light chain (MLC) thereby increasing cross-bridge cycling and the rate of tension development. Dephosphorylation of MLC is catalyzed by smooth muscle specific myosin light chain phosphatase (MLCP). Decreases in cytosolic [Ca2+]i and MLC dephosphorylation are considered salient events in smooth muscle relaxation. What has become apparent only within the last decade is the fact that …

Inhibition of vascular endothelial growth factor-induced angiogenesis by resveratrol through interruption of Src-dependent vascular endothelial cadherin tyrosine phosphorylation.

Resveratrol, a polyphenolic compound found in grapes and other fruits, has been reported to inhibit angiogenesis with an as yet elusive mechanism. Here, we investigate the detailed mechanism by which resveratrol inhibits vascular endothelial growth factor (VEGF)-induced angiogenic effects in human umbilical endothelial cells (HUVECs). Exposure of HUVECs to 1 to 2.5 muM resveratrol significantly blocked VEGF-mediated migration and tube formation but not cell proliferation. Under the same concentrations, resveratrol failed to affect VEGF-stimulated activation of VEGF receptor, extracellular signal-regulated protein kinase 1/2, p38 mitogen-activated protein kinase, and Akt. Of interest, resveratrol, at the dose of 1 or 2.5 muM, effectively abrogated VEGF-mediated tyrosine phosphorylation of vascular endothelial (VE)-cadherin and its complex partner, beta-catenin. This inhibitory effect of resveratrol reflected on the retention of VE-cadherin at cell-cell contacts as demonstrated by immunofluorescence. Src kinase assay showed that VEGF-induced endogenous Src kinase activation was strongly inhibited by 1 and 2.5 muM resveratrol. Supportively, inhibition of Src activity by overexpression of Csk resulted in attenuation of the tyrosine phosphorylation of VE-cadherin and endothelial cell (EC) tube formation. Again, transfection with v-Src, an active form of Src, could reverse resveratrol inhibition of VE-cadherin tyrosine phosphorylation and EC tube formation. Reactive oxygen species (ROS) has been shown to be involved in VE-cadherin phosphorylation and its related functions. Flow cytometric analysis showed that VEGF stimulated an evident increase of peroxide, which was strongly attenuated by resveratrol. In addition, antioxidant N-acetyl-cysteine was demonstrated to strongly inhibit VEGF-mediated Src activation, VE-cadherin tyrosine phosphorylation, and HUVEC tube formation. Together, our data suggest that resveratrol inhibition of VEGF-induced angiogenesis was mediated by disruption of ROS-dependent Src kinase activation and the subsequent VE-cadherin tyrosine phosphorylation.

Identification of cadherin tyrosine residues that are phosphorylated and mediate Shc association

Previously, we reported association of the adaptor protein Shc through its SH2 domain with the cytoplasmic domain of the adhesion molecule cadherin (Xu et al. [1997] J. Biol. Chem. 272:13463–13466). This association was dependent on tyrosine phosphorylation of cadherin and could be modulated by extracellular Ca2+ and epidermal growth factor in intact cells. There are six tyrosine residues in the cytoplasmic domain of cadherin. To define the tyrosine residue(s) that mediate Shc recognition, site-directed mutagenesis was employed to alter Tyr851 and/or Tyr883 in cadherin, which both conform to a predicted Shc SH2 domain recognition sequence. Mutation of either Tyr851 or Tyr883, but mostly the latter, decreased Src phosphorylation of cadherin and the binding of Shc to cadherin, as determined by Sepharose bead binding and gel overlay assays. Of the two tyrosine residues, Tyr883 is the major Src phosphorylation and Shc binding site. However, the double mutant (Tyr851, 883 Phe) exhibited less Shc association than the single Tyr883 Phe mutant, suggesting a role for Tyr851 also. In addition, the binding of Shc to the cadherin cytoplasmic domain was competitively inhibited by tyrosine phosphorylated peptides containing either Tyr851 or Tyr883, but not by the corresponding non-phosphorylated peptides. Mutation of Tyr851 and/or Tyr883 did not alter the capacity of the cytoplasmic domain of cadherin to bind β-catenin in vitro. However, Shc binding to cadherin did negatively influence β-catenin binding to the same molecule. J. Cell. Biochem. 75:264–271, 1999. © 1999 Wiley-Liss, Inc.

Identification of cadherin tyrosine residues that are phosphorylated and mediate Shc association.

Previously, we reported association of the adaptor protein Shc through its SH2 domain with the cytoplasmic domain of the adhesion molecule cadherin (Xu et al. [1997] J. Biol. Chem. 272:13463-13466). This association was dependent on tyrosine phosphorylation of cadherin and could be modulated by extracellular Ca(2+) and epidermal growth factor in intact cells. There are six tyrosine residues in the cytoplasmic domain of cadherin. To define the tyrosine residue(s) that mediate Shc recognition, site-directed mutagenesis was employed to alter Tyr851 and/or Tyr883 in cadherin, which both conform to a predicted Shc SH2 domain recognition sequence. Mutation of either Tyr851 or Tyr883, but mostly the latter, decreased Src phosphorylation of cadherin and the binding of Shc to cadherin, as determined by Sepharose bead binding and gel overlay assays. Of the two tyrosine residues, Tyr883 is the major Src phosphorylation and Shc binding site. However, the double mutant (Tyr851, 883 Phe) exhibited less Shc association than the single Tyr883 Phe mutant, suggesting a role for Tyr851 also. In addition, the binding of Shc to the cadherin cytoplasmic domain was competitively inhibited by tyrosine phosphorylated peptides containing either Tyr851 or Tyr883, but not by the corresponding non-phosphorylated peptides. Mutation of Tyr851 and/or Tyr883 did not alter the capacity of the cytoplasmic domain of cadherin to bind beta-catenin in vitro. However, Shc binding to cadherin did negatively influence beta-catenin binding to the same molecule.