Activation of RhoA by thrombin in endothelial hyperpermeability: role of Rho kinase and protein tyrosine kinases.

Endothelial cells (ECs) actively regulate the extravasation of blood constituents. On stimulation by vasoactive agents and thrombin, ECs change their cytoskeletal architecture and small gaps are formed between neighboring cells. These changes partly depend on a rise in [Ca(2+)](i) and activation of the Ca(2+)/calmodulin-dependent myosin light chain kinase. In this study, mechanisms that contribute to the thrombin-enhanced endothelial permeability were further investigated. We provide direct evidence that thrombin induces a rapid and transient activation of RhoA in human umbilical vein ECs. Under the same conditions, the activity of the related protein Rac was not affected. This was accompanied by an increase in myosin light chain phosphorylation, the generation of F-actin stress fibers, and a prolonged increase in endothelial permeability. Inhibition of the RhoA target Rho kinase with the specific inhibitor Y-27632 reduced all of these effects markedly. In the presence of Y-27632, the thrombin-enhanced permeability was additionally reduced by chelation of [Ca(2+)](i) by BAPTA. These data indicate that RhoA/Rho kinase and Ca(2+) represent 2 pathways that act on endothelial permeability. In addition, the protein tyrosine kinase inhibitor genistein reduced thrombin-induced endothelial permeability without affecting activation of RhoA by thrombin. Our data support a model of thrombin-induced endothelial permeability that is regulated by 3 cellular signal transduction pathways.

Induction of cell scattering by expression of beta1 integrins in beta1-deficient epithelial cells requires activation of members of the rho family of GTPases and downregulation of cadherin and catenin function.

Adhesion receptors, which connect cells to each other and to the surrounding extracellular matrix (ECM), play a crucial role in the control of tissue structure and of morphogenesis. In this work, we have studied how intercellular adhesion molecules and beta1 integrins influence each other using two different beta1-null cell lines, epithelial GE11 and fibroblast-like GD25 cells. Expression of beta1A or the cytoplasmic splice variant beta1D, induced the disruption of intercellular adherens junctions and cell scattering in both GE11 and GD25 cells. In GE11 cells, the morphological change correlated with the redistribution of zonula occluden (ZO)-1 from tight junctions to adherens junctions at high cell confluency. In addition, the expression of beta1 integrins caused a dramatic reorganization of the actin cytoskeleton and of focal contacts. Interaction of beta1 integrins with their respective ligands was required for a complete morphological transition towards the spindle-shaped fibroblast-like phenotype. The expression of an interleukin-2 receptor (IL2R)-beta1A chimera and its incorporation into focal adhesions also induced the disruption of cadherin-based adhesions and the reorganization of ECM-cell contacts, but failed to promote cell migration on fibronectin, in contrast to full-length beta1A. This indicates that the disruption of cell-cell adhesion is not simply the consequence of the stimulated cell migration. Expression of beta1 integrins in GE11 cells resulted in a decrease in cadherin and alpha-catenin protein levels accompanied by their redistribution from the cytoskeleton-associated fraction to the detergent-soluble fraction. Regulation of alpha-catenin protein levels by beta1 integrins is likely to play a role in the morphological transition, since overexpression of alpha-catenin in GE11 cells before beta1 prevented the disruption of intercellular adhesions and cell scattering. In addition, using biochemical activity assays for Rho-like GTPases, we show that the expression of beta1A, beta1D, or IL2R-beta1A in GE11 or GD25 cells triggers activation of both RhoA and Rac1, but not of Cdc42. Moreover, dominant negative Rac1 (N17Rac1) inhibited the disruption of cell-cell adhesions when expressed before beta1. However, all three GTPases might be involved in the morphological transition, since expression of either N19RhoA, N17Rac1, or N17Cdc42 reversed cell scattering and partially restored cadherin-based adhesions in GE11-beta1A cells. Our results indicate that beta1 integrins regulate the polarity and motility of epithelial cells by the induction of intracellular molecular events involving a downregulation of alpha-catenin function and the activation of the Rho-like G proteins Rac1 and RhoA.

Rac downregulates Rho activity: reciprocal balance between both GTPases determines cellular morphology and migratory behavior.

Using biochemical assays to determine the activation state of Rho-like GTPases, we show that the guanine nucleotide exchange factor Tiam1 functions as a specific activator of Rac but not Cdc42 or Rho in NIH3T3 fibroblasts. Activation of Rac by Tiam1 induces an epithelial-like morphology with functional cadherin-based adhesions and inhibits migration of fibroblasts. This epithelial phenotype is characterized by Rac-mediated effects on Rho activity. Transient PDGF-induced as well as sustained Rac activation by Tiam1 or V12Rac downregulate Rho activity. We found that Cdc42 also downregulates Rho activity. Neither V14Rho or N19Rho affects Rac activity, suggesting unidirectional signaling from Rac towards Rho. Downregulation of Rho activity occurs independently of Rac- induced cytoskeletal changes and cell spreading. Moreover, Rac effector mutants that are defective in mediating cytoskeleton changes or Jun kinase activation both downregulate Rho activity, suggesting that neither of these Rac signaling pathways are involved in the regulation of Rho. Restoration of Rho activity in Tiam1-expressing cells by expression of V14Rho results in reversion of the epithelioid phenotype towards a migratory, fibroblastoid morphology. We conclude that Rac signaling is able to antagonize Rho activity directly at the GTPase level, and that the reciprocal balance between Rac and Rho activity determines cellular morphology and migratory behavior in NIH3T3 fibroblasts.

Rac regulates phosphorylation of the myosin-II heavy chain, actinomyosin disassembly and cell spreading.

GTPases of the Rho family regulate actinomyosin-based contraction in non-muscle cells. Activation of Rho increases contractility, leading to cell rounding and neurite retraction in neuronal cell lines. Activation of Rac promotes cell spreading and interferes with Rho-mediated cell rounding. Here we show that activation of Rac may antagonize Rho by regulating phosphorylation of the myosin-II heavy chain. Stimulation of PC12 cells or N1E-115 neuroblastoma cells with bradykinin induces phosphorylation of threonine residues in the myosin-II heavy chain; this phosphorylation is Ca2+ dependent and regulated by Rac. Both bradykinin-mediated and constitutive activation of Rac promote cell spreading, accompanied by a loss of cortical myosin II. Our results identify the myosin-II heavy chain as a new target of Rac-regulated kinase pathways, and implicate Rac as a Rho antagonist during myosin-II-dependent cell-shape changes.

Regulation of p21rac activation in human neutrophils.

The small guanosine triphosphate (GTPase) p21rac is highly expressed in human neutrophils where it is thought to play a role in cytoskeletal reorganization and superoxide production. Using the p21rac binding domain of PAK (PAK-RBD) as an activation-specific probe, we have investigated agonist-stimulated activation of p21rac. Stimulation of neutrophils with the chemoattractants fMet-Leu-Phe (fMLP) or platelet-activating factor (PAF) induced an extremely rapid and transient p21rac activation, being optimal within 5 seconds. This activation correlates with the rapid changes of intracellular free Ca(2+) ([Ca(2+)](i)) stimulated by fMLP; however, changes in [Ca(2+)](i) were neither sufficient nor required for p21rac activation. Furthermore, fMLP-induced p21rac activation was not inhibited by broad tyrosine kinase inhibitors or specific inhibitors of ERK, p38 mitogen activated protein kinase, Src, or phosphatidylinositol 3-kinases. Surprisingly, the cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor-alpha did not cause p21rac activation or modulate fMLP-induced p21rac activation. AlF(-), a potent activator of heterotrimeric G-protein alpha-subunits, however, was found to activate p21rac. Stimulation of neutrophils with phorbol myristate acetate (PMA) strongly activated the respiratory burst, but did not induce p21rac activation, suggesting that superoxide production per se can occur independently of p21rac activation. These data suggest that in human granulocytes, G-protein coupled receptors, but not cytokine receptors, activate p21rac via a rapid, novel exchange-mechanism independently of changes in [Ca(2+)](i), tyrosine phosphorylation, or PI3K.

Matrix-dependent Tiam1/Rac signaling in epithelial cells promotes either cell-cell adhesion or cell migration and is regulated by phosphatidylinositol 3-kinase.

We previously demonstrated that both Tiam1, an activator of Rac, and constitutively active V12Rac promote E-cadherin-mediated cell-cell adhesion in epithelial Madin Darby canine kidney (MDCK) cells. Moreover, Tiam1 and V12Rac inhibit invasion of Ras-transformed, fibroblastoid MDCK-f3 cells by restoring E-cadherin-mediated cell-cell adhesion. Here we show that the Tiam1/Rac-induced cellular response is dependent on the cell substrate. On fibronectin and laminin 1, Tiam1/Rac signaling inhibits migration of MDCK-f3 cells by restoring E-cadherin-mediated cell- cell adhesion. On different collagens, however, expression of Tiam1 and V12Rac promotes motile behavior, under conditions that prevent formation of E-cadherin adhesions. In nonmotile cells, Tiam1 is present in adherens junctions, whereas Tiam1 localizes to lamellae of migrating cells. The level of Rac activation by Tiam1, as determined by binding to a glutathione-S-transferase- PAK protein, is similar on fibronectin or collagen I, suggesting that rather the localization of the Tiam1/Rac signaling complex determines the substrate-dependent cellular responses. Rac activation by Tiam1 requires PI3-kinase activity. Moreover, Tiam1- but not V12Rac-induced migration as well as E-cadherin-mediated cell- cell adhesion are dependent on PI3-kinase, indicating that PI3-kinase acts upstream of Tiam1 and Rac.

Epidermal growth factor induces ubiquitination of Eps15.

Epidermal growth factor (EGF) receptor pathway substrate clone 15 (Eps15) has been described as a 142-kDa EGF receptor substrate. It has been shown to bind to the EGF receptor, adaptor protein-2, and clathrin and is present at clathrin-coated pits and vesicles. Upon stimulation of cells with EGF or transforming growth factor alpha, Eps15 becomes rapidly and transiently phosphorylated on tyrosine residues. This phosphorylation coincides with an increase of 8 kDa in molecular mass. Here we show that this increase in molecular mass is not due to tyrosine phosphorylation. Instead, we found both by Western blotting and protein sequencing that this EGF-induced increase in molecular mass is the result of monoubiquitination. Eps15 ubiquitination but not tyrosine phosphorylation was inhibited under conditions that blocked EGF-induced internalization of the EGF receptor. Our results establish ubiquitination as a second form of EGF-stimulated covalent modification of Eps15.

Association and colocalization of Eps15 with adaptor protein-2 and clathrin.

Eps15 has been identified as a substrate of the EGF receptor tyrosine kinase. In this report, we show that activation of the EGF receptor by either EGF or TGF-alpha results in phosphorylation of Eps15. Stimulation of cells with PDGF or insulin did not lead to Eps15 phosphorylation, suggesting that phosphorylation of Eps15 is a receptor-specific process. We demonstrate that Eps15 is constitutively associated with both alpha-adaptin and clathrin. Upon EGF stimulation, Eps15 and alpha-adaptin are recruited to the EGF receptor. Using a truncated EGF receptor mutant, we demonstrate that the regulatory domain of the cytoplasmic tail of the EGF receptor is essential for the binding of Eps15. Fractionation studies reveal that Eps15 is present in cell fractions enriched for plasma membrane and endosomal membranes. Immunofluorescence studies show that Eps15 colocalizes with adaptor protein-2 (AP-2) and partially with clathrin. No colocalization of Eps15 was observed with the early endosomal markers rab4 and rab5. These observations indicate that Eps15 is present in coated pits and coated vesicles of the clathrin-mediated endocytic pathway, but not in early endosomes. Neither AP-2 nor clathrin are required for the binding of Eps15 to coated pits or coated vesicles, since in membranes lacking AP-2 and clathrin, Eps15 still shows the same staining pattern. These findings suggest that Eps15 may play a critical role in the recruitment of active EGF receptors into coated pit regions before endocytosis of ligand-occupied EGF receptors.

Epidermal growth factor induces serine phosphorylation of actin.

Stimulation of cells by epidermal growth factor induces a rapid polymerisation of actin in the cortical skeleton. Activation of the EGF-receptor leads to autophosphorylation and to phosphorylation of specific intracellular substrates. Here we show that actin is phosphorylated in vitro and in vivo upon EGF stimulation. Two-dimensional phospho-amino acid analysis shows that phosphorylation occurs on serine, not on tyrosine residues.

c-fos induction by stress can be mediated by the SRE.

The proto-oncogene c-fos is important in cell growth, differentiation and development. Several promoter elements have been identified as mediators for the induction of c-fos by serum, tumor promoting agents, epidermal growth factor and other stimuli. Stressors, including heat shock, arsenite and cadmium can induce c-fos transcription, but a heat shock element in the c-fos promoter has not been described. In this report we show that the induction of c-fos by heat shock, arsenite and cadmium can be mediated by the serum response element. Furthermore we show that casein kinase II, which has been proposed to be involved in serum induction via the serum response element, may also be involved in heat shock, arsenite and cadmium induction of c-fos.