Hyperosmotic stress induces rapid focal adhesion kinase phosphorylation at tyrosines 397 and 577. Role of Src family kinases and Rho family GTPases

Hyperosmotic stress induced by treatment of Swiss 3T3 cells with the non-permeant solutes sucrose or sorbitol, rapidly and robustly stimulated endogenous focal adhesion kinase (FAK) phosphorylation at Tyr-397, the major autophosphorylation site, and at Tyr-577, within the kinase activation loop. Hyperosmotic stress-stimulated FAK phosphorylation at Tyr-397 occurred via a Src-independent pathway, whereas Tyr-577 phosphorylation was completely blocked by exposure to the Src family kinase inhibitor PP-2. Inhibition of p38 MAP kinase or phosphatidylinositol 3-kinases did not prevent FAK phosphorylation stimulated by hyperosmotic stress. Overexpression of N17 RhoA did not reduce hyperosmotic stress-mediated localization of phosphorylated FAK to focal contacts and treatment with the Rho-associated kinase inhibitor Y-27632 did not prevent FAK translocation and tyrosine phosphorylation in response to hyperosmotic stress. Overexpression of N17 Rac only slightly altered the hyperosmotic stress-mediated localization of phosphorylated FAK to focal contacts. In contrast, overexpression of the N17 mutant of Cdc42 disrupted hyperosmotic stress-stimulated FAK Tyr-397 localization to focal contacts. Additionally, treatment of cells with Clostridium difficile toxin B potently inhibited hyperosmotic stress-induced FAK tyrosine phosphorylation. Furthermore, FAK null fibroblasts compared with their FAK containing controls show markedly increased sensitivity, manifest by subsequent apoptosis, to sustained hyperosmotic stress. Our results indicate that FAK plays a fundamental role in protecting cells from hyperosmotic stress, and that the pathway(s) that mediates FAK autophosphorylation at Tyr-397 in response to osmotic stress can be distinguished from the pathways utilized by many other stimuli, including neuropeptides and bioactive lipids (Rho- and Rho-associated kinase-dependent), tyrosine kinase receptor agonists (phosphatidylinositol 3-kinase-dependent), and integrins (Src-dependent).     

Dysregulation of vascular endothelial growth factor receptor 2 phosphorylation is associated with disruption of the blood-brain barrier and brain endothelial cell apoptosis induced by plasma from women with preeclampsia

Disruption of the blood-brain barrier (BBB) is central in the pathophysiology of acute cerebral complications in women who have preeclampsia. Underling mechanisms are unclear.Using female human brain endothelial cells as an in vitro model of BBB, we show that plasma of women with preeclampsia increases cell apoptosis and permeability via activation of the vascular endothelial growth factor receptor 2 (VEGFR2).Since plasma of women with preeclampsia also enhanced VEGFR2 phosphorylation in the tyrosine 951 but decreased phosphorylation at the tyrosine 1175, we propose the former would be the more likely active form of VEGFR2 responsible for BBB alterations.     

Different phosphorylation mechanisms are involved in the activation of sucrose non-fermenting 1 related protein kinases 2 by osmotic stresses and abscisic acid

In Arabidopsis cell suspension, hyperosmotic stresses (mannitol and NaCl) were previously shown to activate nine sucrose non-fermenting 1 related protein kinases 2 (SnRK2s) whereas only five of them were also activated by abscisic acid (ABA) treatment. Here, the possible activation by phosphorylation/dephosphorylation of each kinase was investigated by studying their phosphorylation state after osmotic stress, using the Pro-Q Diamond, a specific dye for phosphoproteins. All the activated kinases were phosphorylated after osmotic stress but the induced phosphorylation changes were clearly different depending on the kinase. In addition, the increase of the global phosphorylation level induced by ABA application was lower, suggesting that different mechanisms may be involved in SnRK2 activation by hyperosmolarity and ABA. On the other hand, SnRK2 kinases remain activated by hyperosmotic stress in ABA-deficient and ABA-insensitive mutants, indicating that SnRK2 osmotic activation is independent of ABA. Moreover, using a mutant form of SnRK2s, a specific serine in the activation loop was shown to be phosphorylated after stress treatments and essential for activity and/or activation. Finally, SnRK2 activity was sensitive to staurosporine, whereas SnRK2 activation by hyperosmolarity or ABA was not, indicating that SnRK2 activation by phosphorylation is mediated by an upstream staurosporine-insensitive kinase, in both signalling pathways. All together, these results indicate that different phosphorylation mechanisms and at least three signalling pathways are involved in the activation of SnRK2 proteins in response to osmotic stress and ABA.     

Vascular endothelial growth factor activates the Tie family of receptor tyrosine kinases

The ability of cells to respond appropriately to changes in their environment requires integration and cross-talk between relevant signalling pathways. The vascular endothelial growth factor (VEGF) and angiopoietin families of ligands are key regulators of blood vessel formation. VEGF binds to receptor tyrosine kinases of the VEGF-receptor family to activate signalling pathways leading to endothelial migration, proliferation and survival whereas the angiopoietins interact with the Tie receptor tyrosine kinases to control vessel stability, survival and maturation. Here we show that VEGF can also activate the angiopoietin receptor Tie2. Activation of human endothelial cells with VEGF caused a four-fold stimulation of tyrosine phosphorylation of Tie2. This stimulation was not due to VEGF-induction of Tie2 ligands as soluble ligand binding domain of Tie2 failed to inhibit VEGF activation of the receptor. Immunoprecipitation analysis demonstrated no physical interaction between VEGF receptors and Tie2. However Tie2 does interact with the related receptor tyrosine kinase Tie1 and this receptor was found to be essential for VEGF activation of Tie2. VEGF stimulated proteolytic cleavage of Tie1 generating a truncated Tie1 intracellular domain. Similarly, phorbol ester also both stimulated Tie1 truncation and activated Tie2 phosphorylation. Inhibition of Tie1 cleavage with the metalloprotease inhibitor TAPI-2 suppressed VEGF- and phorbol ester-induced phosphorylation of Tie2. Truncated Tie1 formed in response to VEGF was also found to be tyrosine phosphorylated and this was independent of Tie2, though Tie2 could enhance Tie1 intracellular domain phosphorylation. Together these data demonstrate that VEGF activates Tie2 via a mechanism involving proteolytic cleavage of the associated tyrosine kinase Tie1 leading to trans-phosphorylation of Tie2. This novel mechanism of receptor tyrosine kinase activation is likely to be important in integrating signalling between two of the key receptor groups regulating angiogenesis.     

Axl is essential for VEGF-A-dependent activation of PI3K/Akt

Herein, we report that vascular endothelial growth factor A (VEGF-A) engages the PI3K/Akt pathway by a previously unknown mechanism that involves three tyrosine kinases. Upon VEGF-A-dependent activation of VEGF receptor-2 (VEGFR-2), and subsequent TSAd-mediated activation of Src family kinases (SFKs), SFKs engage the receptor tyrosine kinase Axl via its juxtamembrane domain to trigger ligand-independent autophosphorylation at a pair of YXXM motifs that promotes association with PI3K and activation of Akt. Other VEGF-A-mediated signalling pathways are independent of Axl. Interfering with Axl expression or function impairs VEGF-A- but not bFGF-dependent migration of endothelial cells. Similarly, Axl null mice respond poorly to VEGF-A-induced vascular permeability or angiogenesis, whereas other agonists induce a normal response. These results elucidate the mechanism by which VEGF-A activates PI3K/Akt, and identify previously unappreciated potential therapeutic targets of VEGF-A-driven processes.     

Hydrogen peroxide activates the Gas6-Axl pathway in vascular smooth muscle cells

Axl, a receptor tyrosine kinase, is involved in cell survival, proliferation, and migration. We have shown that Axl expression increases in the neointima of balloon-injured rat carotids. Because oxidative stress is known to play a major role in remodeling of injured vessels, we hypothesized that H(2)O(2) might activate Axl by promoting autophosphorylation. H(2)O(2) rapidly stimulated Axl tyrosine phosphorylation in rat vascular smooth muscle cells within 1 min that was maximal at 5 min (6-fold). The response to H(2)O(2) was concentration-dependent with EC(50) of approximately 500 microm. Axl phosphorylation was partly dependent on production of its endogenous ligand, growth arrest gene 6 (Gas6), because Axl-Fc, a fragment of Axl extracellular domain that neutralizes Gas6, inhibited H(2)O(2)-induced Axl phosphorylation by 50%. Axl phosphorylation by H(2)O(2) was also attenuated by warfarin, which inhibits Gas6 activity by preventing post-translational modification. In intact vessels Axl was phosphorylated by H(2)O(2), and Axl phosphorylation was inhibited by warfarin treatment in balloon-injured carotids. Akt, a downstream target of Axl, was phosphorylated by H(2)O(2)in Axl(+/+) mouse aorta but significantly inhibited in Axl(-/-) aorta. Intimal proliferation was decreased significantly in a cuff injury model in Axl(-/-) mice compared with Axl(+/+) mice. In summary, Axl is an important signaling mediator for oxidative stress in cultured vascular smooth muscle cells and intact vessels and may represent an important therapeutic target for vascular remodeling and response to injury.     

Intracellular signaling pathways involved in Gas6-Axl-mediated survival of endothelial cells

Gas6 is a gamma-carboxylated ligand for the receptor tyrosine kinase Axl. Gas6-Axl interactions can rescue endothelial cells from apoptosis, and this study examined the intracellular signaling mechanisms responsible for this phenomenon. Using flow cytometry, we first confirmed that Gas6 can abrogate apoptosis induced by serum starvation of primary cultures of human umbilical vein endothelial cells (HUVECs). This effect is mediated through phosphorylation of the serine-threonine kinase Akt, with maximal phosphorylation observed after 4 h of treatment with 100 ng/ml Gas6. Inhibition of Akt phosphorylation and abrogation of gas6-mediated survival of HUVECs by wortmannin implicated phosphatidylinositol 3-kinase as the mediator of Akt phosphorylation. Dominant negative Akt constructs largely abrogated the protective effect of Gas6 on HUVECs, underscoring the importance of Akt activation in Gas6-mediated survival. Several downstream regulators of this survival pathway were identified in HUVECs, namely, NF-kappaB as well as the antiapoptotic and proapoptotic proteins Bcl-2 and caspase 3, respectively. We showed that NF-kappaB is phosphorylated early after Gas6 treatment as evidenced by doublet formation on Western blotting. As well, the level of Bcl-2 protein increased, supporting the notion that the Bcl-2 antiapoptotic pathway is stimulated. The levels of expression of the caspase 3 activation products p12 and p20 decreased with Gas6 treatment, consistent with a reduction in proapoptotic caspase 3 activation. Taken together, these experiments provide new information about the mechanism underlying Gas6 protection from apoptosis in primary endothelial cell cultures.     

Evidence for a role of platelet endothelial cell adhesion molecule-1 in endothelial cell mechanosignal transduction: is it a mechanoresponsive molecule?

Fluid shear stress (FSS) induces many forms of responses, including phosphorylation of extracellular signal-regulated kinase (ERK) in endothelial cells (ECs). We have earlier reported rapid tyrosine phosphorylation of platelet endothelial cell adhesion molecule-1 (PECAM-1) in ECs exposed to FSS. Osmotic changes also induced similar PECAM-1 and ERK phosphorylation with nearly identical kinetics. Because both FSS and osmotic changes should mechanically perturb the cell membrane, they might activate the same mechanosignaling cascade. When PECAM-1 is tyrosine phosphorylated by FSS or osmotic changes, SHP-2 binds to it. Here we show that ERK phosphorylation by FSS or osmotic changes depends on PECAM-1 tyrosine phosphorylation, SHP-2 binding to phospho-PECAM-1, and SHP-2 phosphatase activity. In ECs under flow, detectable amounts of SHP-2 and Gab1 translocated from the cytoplasm to the EC junction. When magnetic beads coated with antibodies against the extracellular domain of PECAM-1 were attached to ECs and tugged by magnetic force for 10 min, PECAM-1 associated with the beads was tyrosine phosphorylated. ERK was also phosphorylated in these cells. Binding of the beads by itself or pulling on the cell surface using poly-l-coated beads did not induce phosphorylation of PECAM-1 and ERK. These results suggest that PECAM-1 is a mechanotransduction molecule.     

Cell shrinkage regulates Src kinases and induces tyrosine phosphorylation of cortactin, independent of the osmotic regulation of Na+/H+ exchangers

The signaling pathways by which cell volume regulates ion transporters, e.g. Na+/H+ exchangers (NHEs), and affects cytoskeletal organization are poorly understood. We have previously shown that shrinkage induces tyrosine phosphorylation in CHO cells, predominantly in an 85-kDa band. To identify volume-sensitive kinases and their substrates, we investigated the effect of hypertonicity on members of the Src kinase family. Hyperosmolarity stimulated Fyn and inhibited Src. Fyn activation was also observed in nystatin-permeabilized cells, where shrinkage cannot induce intracellular alkalinization. In contrast, osmotic inhibition of Src was prevented by permeabilization or by inhibiting NHE-1. PP1, a selective Src family inhibitor, strongly reduced the hypertonicity-induced tyrosine phosphorylation. We identified one of the major targets of the osmotic stress-elicited phosphorylation as cortactin, an 85-kDa actin-binding protein and well known Src family substrate. Cortactin phosphorylation was triggered by shrinkage and not by changes in osmolarity or pHi and was abrogated by PP1. Hyperosmotic cortactin phosphorylation was reduced in Fyn-deficient fibroblasts but remained intact in Src-deficient fibroblasts. To address the potential role of the Src family in the osmotic regulation of NHEs, we used PP1. The drug affected neither the hyperosmotic stimulation of NHE-1 nor the inhibition of NHE-3. Thus, members of the Src family are volume-sensitive enzymes that may participate in the shrinkage-related reorganization of the cytoskeleton but are probably not responsible for the osmotic regulation of NHE.     

Epidermal growth factor (EGF) promotes phosphorylation at threonine-654 of the EGF receptor: possible role of protein kinase C in homologous regulation of the EGF receptor

Treatment of cells with tumor-promoting phorbol diesters, which causes activation of protein kinase C, leads to phosphorylation of the epidermal growth factor (EGF) receptor at threonine-654. Addition of phorbol diesters to intact cells causes inhibition of the EGF-induced tyrosine-protein kinase activity of the EGF receptor and it has been suggested that this effect of phorbol diesters is mediated by the phosphorylation of the receptor by protein kinase C. We measured the activity of protein kinase C in A431 cells by determining the incorporation of [32P]phosphate into peptides containing threonine-654 obtained by trypsin digestion of EGF receptors. After 3 h of exposure to serum-free medium, A431 cells had no detectable protein kinase C activity. Addition of EGF to these cells resulted in [32P] incorporation into threonine-654 as well as into tyrosine residues. This indicates that EGF promotes the activation of protein kinase C in A431 cells. The phosphorylation of threonine-654 induced by EGF was maximal after only 5 min of EGF addition and the [32P] incorporation into threonine-654 reached 50% of the [32P] in a tyrosine-containing peptide. This indicates that a significant percentage of the total EGF receptors are phosphorylated by protein kinase C. A variety of external stimuli activate Na+/H+ exchange, including EGF, phorbol diesters, and hypertonicity. To ascertain whether activation of protein kinase C is an intracellular common effector of all of these systems, we measured the activity of protein kinase C after exposure of A431 cells to hyperosmotic conditions and observed no effect on phosphorylation of threonine-654, therefore, activation of Na+/H+ exchange by hypertonic medium is independent of protein kinase C activity. Since stimulation of protein kinase C by phorbol diesters results in a decrease in EGF receptor activity, the stimulation of protein kinase C activity by addition of EGF to A431 cells contributes to a feedback mechanism which results in the attenuation of EGF receptor function.     

Osmotic stress stimulates generation of superoxide anion by spermatozoa in horses

The objective of this study was to examine the interplay between osmotic and oxidative stress as well as to determine mechanisms by which osmotic stress increases superoxide generation in spermatozoa of horses. Superoxide production, as measured by dihydroethidium (DHE), increased when spermatozoa of horses were incubated under either hyperosmotic or hyposmotic conditions. This increase in superoxide production was inhibited by the MAP kinase p38 inhibitor, SB203580, and by the superoxide scavenger, tiron. Incubation of spermatozoa under hyperosmotic conditions increased overall protein tyrosine phosphorylation as measured by western blotting techniques; however, a similar increase was not detected when spermatozoa were incubated under hyposmotic conditions. The general protein kinase C (PKC) and protein tyrosine kinase (PTK) inhibitor staurosporine inhibited (P < 0.05) tyrosine phosphorylation in samples from cells under hyperosmotic conditions. In addition, the NADPH oxidase inhibitor diphenyleneiodonium (DPI) also inhibited (P < 0.05) protein tyrosine phosphorylation in cells under hyperosmotic conditions. In summary, these data indicate that incubation of equine spermatozoa under both hyposmotic and hyperosmotic conditions can increase superoxide anion generation. Under hyperosmotic conditions, this increased generation of superoxide anion was accompanied by increased protein tyrosine phosphorylation.     

Hyperosmotic stress induces phosphorylation of cytosolic phospholipase A(2) in HaCaT cells by an epidermal growth factor receptor-mediated process

Cytosolic phospholipase A(2) (cPLA(2)) is an enzyme involved in the formation of proinflammatory mediators by catalyzing the release of arachidonic acid, thereby mediating eicosanoid biosynthesis. Using HaCaT keratinocytes as a model system, we present experimental evidence that in these cells, cPLA(2) is constitutively phosphorylated and that the degree of phosphorylation dramatically increases in cells under hyperosmotic stress induced by sorbitol. In parallel, a rapid release of arachidonic acid followed by prostaglandin E(2) formation was detected. Elucidating the mechanism of cPLA(2) upregulation, we observed that it is mediated via epidermal growth factor receptor (EGFR) activation, since tyrphostin AG1478, a selective inhibitor of EGFR tyrosine kinase, completely inhibited cPLA(2) phosphorylation. Furthermore, addition of PD98059, which is an inhibitor of MEK1 activation, but not of SB203580, which is an inhibitor of p38 stress kinase, inhibited cPLA(2) phosphorylation, indicating that the ras-raf-MEK cascade is the major signalling pathway involved in cPLA(2) phosphorylation. In addition, depletion of the cells from intracellular calcium does not prevent sorbitol-elicited cPLA(2) phosphorylation, suggesting that this process is independent of the presence of calcium. Together, our results demonstrate that hyperosmotic stress phosphorylates cPLA(2) in human keratinocytes by an EGFR-mediated process.     

Vitamin K-dependent Gas6 activates ERK kinase and stimulates growth of cardiac fibroblasts

The protein product of growth arrest specific gene 6 (Gas6), is the biological ligand for the Axl subfamily of receptor tyrosine kinases. We investigated the effects of exogenous Gas6 on growth of cardiac fibroblasts isolated from genetically Gas6-deficient mice. Recombinant Gas6, containing N terminal gamma-carboxyglutamic acid residues formed from a vitamin K-dependent reaction, stimulated both DNA synthesis and proliferation of cardiac fibroblasts under serum-free conditions. Gas6 also markedly enhanced survival of cells during prolonged serum starvation. Gas6 stimulated tyrosine phosphorylation of Axl as well as phosphorylation of ERK kinase. The mitogenic effects of Gas6 were inhibited by neutralising anti-Gas6 antibodies and by a soluble Axl ectodomain fusion protein. In contrast, recombinant Gas6 from cells treated with warfarin, which prevents the gamma-carboxylation reaction, neither stimulated fibroblast proliferation nor activated Axl tyrosine phosphorylation. Gas6-induced cell proliferation was additive to the effects of epidermal growth factor, suggesting activation of discrete signalling pathways. In conclusion, Gas6 appears to be a unique growth factor for fibroblasts and post-translational gamma-carboxylation is necessary for its biological activity. These findings implicate vitamin K-dependent biochemical reactions in growth processes in development and in disease.     

Hypertonicity primes malignant melanoma cells for apoptosis

The tumor environment critically influences responsiveness of cancer cells to chemotherapies, most of which activate the mitochondria-regulated (intrinsic) apoptotic cascade to kill malignant cells. Especially skin tumors encounter an environment with remarkable biophysical properties. Cutaneous accumulation of Na⁺ locally establishes osmotic pressure gradients in vivo (hypertonicity or hyperosmotic stress), but whether cutaneous hypertonicity is a factor that modulates the responsiveness of skin cancers to therapeutic apoptosis-induction has thus far not been investigated. Here, we show that hyperosmotic stress lowers the threshold for apoptosis induction in malignant melanoma, the deadliest form of skin cancer. Hypertonic conditions enforce addiction to BCL-2-like proteins to prevent initiation of the mitochondria-regulated (intrinsic) apoptotic pathway. Essentially, hyperosmotic stress primes mitochondria for death. Our work identifies osmotic pressure in the tumor microenvironment as a cell extrinsic factor that modulates responsiveness of malignant melanoma cells to therapy.     

TGF-beta activates Erk MAP kinase signalling through direct phosphorylation of ShcA

Erk1/Erk2 MAP kinases are key regulators of cell behaviour and their activation is generally associated with tyrosine kinase signalling. However, TGF-beta stimulation also activates Erk MAP kinases through an undefined mechanism, albeit to a much lower level than receptor tyrosine kinase stimulation. We report that upon TGF-beta stimulation, the activated TGF-beta type I receptor (TbetaRI) recruits and directly phosphorylates ShcA proteins on tyrosine and serine. This dual phosphorylation results from an intrinsic TbetaRI tyrosine kinase activity that complements its well-defined serine-threonine kinase function. TGF-beta-induced ShcA phosphorylation induces ShcA association with Grb2 and Sos, thereby initiating the well-characterised pathway linking receptor tyrosine kinases with Erk MAP kinases. We also found that TbetaRI is tyrosine phosphorylated in response to TGF-beta. Thus, TbetaRI, like the TGF-beta type II receptor, is a dual-specificity kinase. Recruitment of tyrosine kinase signalling pathways may account for aspects of TGF-beta biology that are independent of Smad signalling.     

Identification of extracellularly phosphorylated membrane proteins

Ecto‐protein kinases phosphorylate extracellular membrane proteins and exhibit similarities to casein kinases and protein kinases A and C. However, the identification of their protein substrates still remains a challenge because a clear separation from intracellular phosphoproteins is difficult. Here, we describe a straightforward method for the identification of extracellularly phosphorylated membrane proteins in human umbilical vein endothelial cells (HUVECs) and K562 cells which used the protease bromelain to selectively remove ectoproteins from intact cells and combined this with the subsequent analysis using IMAC and LC‐MS/MS. A “false‐positive” strategy in which cells without protease treatment served as controls was applied. Using this approach we identified novel phosphorylation sites on five ectophosphoproteins (NOTCH1, otopetrin 1, regulator of G‐protein signalling 13 (RGS13), protein tyrosine phosphatase receptor type D isoform 3 (PTPRD), usherin isoform B (USH2A)). Use of bromelain appears to be a reliable technique for the further identification of phosphorylated surface‐exposed peptides when extracellular adenosine‐5'‐triphosphate is elevated during purinergic signalling.     

Cell volume-dependent phosphorylation of proteins of the cortical cytoskeleton and cell-cell contact sites. The role of Fyn and FER kinases

Cell volume affects diverse functions including cytoskeletal organization, but the underlying signaling pathways remained undefined. We have shown previously that shrinkage induces Fyn-dependent tyrosine phosphorylation of the cortical actin-binding protein, cortactin. Because FER kinase was implicated in the direct phosphorylation of cortactin, we investigated the osmotic responsiveness of FER and its relationship to Fyn and cortactin. Shrinkage increased FER activity and tyrosine phosphorylation. These effects were abolished by the Src family inhibitor PP2 and strongly mitigated in Fyn-deficient but not in Src-deficient cells. FER overexpression caused cortactin phosphorylation that was further enhanced by hypertonicity. Exchange of tyrosine residues 421, 466, and 482 for phenylalanine prevented cortactin phosphorylation by hypertonicity and strongly decreased it upon FER overexpression, suggesting that FER targets primarily the same osmo-sensitive tyrosines. Because constituents of the cell-cell contacts are substrates of Fyn and FER, we investigated the effect of shrinkage on the adherens junctions. Hypertonicity provoked Fyn-dependent tyrosine phosphorylation in beta-catenin, alpha-catenin, and p120(Cas) and caused the dissociation of beta-catenin from the contacts. This process was delayed in Fyn-deficient or PP2-treated cells. Thus, FER is a volume-sensitive kinase downstream from Fyn, and the Fyn/FER pathway may contribute to the cell size-dependent reorganization of the cytoskeleton and the cell-cell contacts.