Src-induced Tyrosine Phosphorylation of VE-cadherin Is Not Sufficient to Decrease Barrier Function of Endothelial Monolayers

Activation of Src family kinases (SFK) and the subsequent phosphorylation of VE-cadherin have been proposed as major regulatory steps leading to increases in vascular permeability in response to inflammatory mediators and growth factors. To investigate Src signaling in the absence of parallel signaling pathways initiated by growth factors or inflammatory mediators, we activated Src and SFKs by expression of dominant negative Csk, expression of constitutively active Src, or knockdown of Csk. Activation of SFK by overexpression of dominant negative Csk induced VE-cadherin phosphorylation at tyrosines 658, 685, and 731. However, dominant negative Csk expression was unable to induce changes in the monolayer permeability. In contrast, expression of constitutively active Src decreased barrier function and promoted VE-cadherin phosphorylation on tyrosines 658 and 731, although the increase in VE-cadherin phosphorylation preceded the increase in permeability by 4–6 h. Csk knockdown induced VE-cadherin phosphorylation at sites 658 and 731 but did not induce a loss in barrier function. Co-immunoprecipitation and immunofluorescence studies suggest that phosphorylation of those sites did not impair VE-cadherin ability to bind p120 and β-catenin or the ability of these proteins to localize at the plasma membrane. Taken together, our data show that Src-induced tyrosine phosphorylation of VE-cadherin is not sufficient to promote an increase in endothelial cell monolayer permeability and suggest that signaling leading to changes in vascular permeability in response to inflammatory mediators or growth factors may require VE-cadherin tyrosine phosphorylation concurrently with other signaling pathways to promote loss of barrier function.     

Phosphorylation of VE-cadherin controls endothelial phenotypes via p120-catenin coupling and Rac1 activation

To establish the role of vascular endothelial (VE)-cadherin in the regulation of endothelial cell functions, we investigated the effect of phosphorylation of a VE-cadherin site sought to be involved in p120-catenin binding on vascular permeability and endothelial cell migration. To this end, we introduced either wild-type VE-cadherin or Y658 phosphomimetic (Y658E) or dephosphomimetic (Y658F) VE-cadherin mutant constructs into an endothelial cell line (rat fat pad endothelial cells) lacking endogenous VE-cadherin. Remarkably, neither wild-type- nor Y658E VE-cadherin was retained at cell-cell contacts because of p120-catenin preferential binding to N-cadherin, resulting in the targeting of N-cadherin to cell-cell junctions and the exclusion of VE-cadherin. However, Y658F VE-cadherin was able to bind p120-catenin and to localize at adherence junctions displacing N-cadherin. This resulted in an enhanced barrier function and a complete abrogation of Rac1 activation and lamellipodia formation, thereby inhibiting cell migration. These findings demonstrate that VE-cadherin, through the regulation of Y658 phosphorylation, competes for junctional localization with N-cadherin and controls vascular permeability and endothelial cell migration.     

ICAM-1-mediated, Src- and Pyk2-dependent vascular endothelial cadherin tyrosine phosphorylation is required for leukocyte transendothelial migration

Leukocyte transendothelial migration (TEM) has been modeled as a multistep process beginning with rolling adhesion, followed by firm adhesion, and ending with either transcellular or paracellular passage of the leukocyte across the endothelial monolayer. In the case of paracellular TEM, endothelial cell (EC) junctions are transiently disassembled to allow passage of leukocytes. Numerous lines of evidence demonstrate that tyrosine phosphorylation of adherens junction proteins, such as vascular endothelial cadherin (VE-cadherin) and beta-catenin, correlates with the disassembly of junctions. However, the role of tyrosine phosphorylation in the regulation of junctions during leukocyte TEM is not completely understood. Using human leukocytes and EC, we show that ICAM-1 engagement leads to activation of two tyrosine kinases, Src and Pyk2. Using phospho-specific Abs, we show that engagement of ICAM-1 induces phosphorylation of VE-cadherin on tyrosines 658 and 731, which correspond to the p120-catenin and beta-catenin binding sites, respectively. These phosphorylation events require the activity of both Src and Pyk2. We find that inhibition of endothelial Src with PP2 or SU6656 blocks neutrophil transmigration (71.1 +/- 3.8% and 48.6 +/- 3.8% reduction, respectively), whereas inhibition of endothelial Pyk2 also results in decreased neutrophil transmigration (25.5 +/- 6.0% reduction). Moreover, overexpression of the nonphosphorylatable Y658F or Y731F mutants of VE-cadherin impairs transmigration of neutrophils compared with overexpression of wild-type VE-cadherin (32.7 +/- 7.1% and 38.8 +/- 6.5% reduction, respectively). Our results demonstrate that engagement of ICAM-1 by leukocytes results in tyrosine phosphorylation of VE-cadherin, which is required for efficient neutrophil TEM.     

CD44 variant,but not standard CD44 isoforms,mediate disassembly of endothelial VE-cadherin junction on metastatic melanoma cells

Loss of endothelial adherens junctions is involved in tumor metastasis. Here, we demonstrate that, in the metastatic Lu1205 melanoma cells, expression of the CD44 variant CD44v8-v10 induced junction disassembly and vascular endothelial (VE)-cadherin phosphorylation at Y658 and Y731. Short interfering RNA (siRNA)-mediated CD44 knockdown or sialic acid cleavage reversed these effects. Moreover, microspheres coated with recombinant CD44v8-v10 promoted endothelial junction disruption. Overexpression of CD44v8-v10 but not of standard CD44 (CD44s) promoted gap formation in the non-metastatic WM35 melanoma cells, whereas CD44 knockdown or neuraminidase treatment dramatically diminished melanoma transendothelial migration. Endothelial cells transfected with the phosphomimetic VE-cadherin mutant Y658E supported transmigration of CD44-silenced Lu1205 cells. Our findings imply that CD44 variant isoform (CD44v) but not CD44s regulates endothelial junction loss, promoting melanoma extravasation.     

Mutant B-Raf(V600E) Promotes Melanoma Paracellular Transmigration by Inducing Thrombin-mediated Endothelial Junction Breakdown

Tumor invasiveness depends on the ability of tumor cells to breach endothelial barriers. In this study, we investigated the mechanism by which the adhesion of melanoma cells to endothelium regulates adherens junction integrity and modulates tumor transendothelial migration (TEM) by initiating thrombin generation. We found that the B-Raf(V600E) mutation in metastatic melanoma cells up-regulated tissue factor (TF) expression on cell membranes and promoted thrombin production. Co-culture of endothelial monolayers with metastatic melanoma cells mediated the opening of inter-endothelial spaces near melanoma cell contact sites in the presence of platelet-free plasma (PFP). By using small interfering RNA (siRNA), we demonstrated that B-Raf(V600E) and TF silencing attenuated the focal disassembly of adherens junction induced by tumor contact. Vascular endothelial-cadherin (VE-cadherin) disassembly was dependent on phosphorylation of p120-catenin on Ser-879 and VE-cadherin on Tyr-658, Tyr-685, and Tyr-731, which can be prevented by treatment with the thrombin inhibitor, hirudin, or by silencing the thrombin receptor, protease-activated receptor-1, in endothelial cells. We also provided strong evidence that tumor-derived thrombin enhanced melanoma TEM by inducing ubiquitination-coupled VE-cadherin internalization, focal adhesion formation, and actin assembly in endothelium. Confocal microscopic analysis of tumor TEM revealed that junctions transiently opened and resealed as tumor cells accomplished TEM. In addition, in the presence of PFP, tumor cells preferentially transmigrated via paracellular routes. PFP supported melanoma transmigration under shear conditions via a B-Raf(V600E)-thrombin-dependent mechanism. We concluded that the activation of thrombin generation by cancer cells in plasma is an important process regulating melanoma extravasation by disrupting endothelial junction integrity.     

VEGF-induced vascular permeability is mediated by FAK

Endothelial cells (ECs) form cell-cell adhesive junctional structures maintaining vascular integrity. This barrier is dynamically regulated by vascular endothelial growth factor (VEGF) receptor signaling. We created an inducible knockin mouse model to study the contribution of the integrin-associated focal adhesion tyrosine kinase (FAK) signaling on vascular function. Here we show that genetic or pharmacological FAK inhibition in ECs prevents VEGF-stimulated permeability downstream of VEGF receptor or Src tyrosine kinase activation in vivo. VEGF promotes tension-independent FAK activation, rapid FAK localization to cell-cell junctions, binding of the FAK FERM domain to the vascular endothelial cadherin (VE-cadherin) cytoplasmic tail, and direct FAK phosphorylation of β-catenin at tyrosine-142 (Y142) facilitating VE-cadherin-β-catenin dissociation and EC junctional breakdown. Kinase inhibited FAK is in a closed conformation that prevents VE-cadherin association and limits VEGF-stimulated β-catenin Y142 phosphorylation. Our studies establish a role for FAK as an essential signaling switch within ECs regulating adherens junction dynamics.     

VE-PTP and VE-cadherin ectodomains interact to facilitate regulation of phosphorylation and cell contacts

VE-cadherin is the essential adhesion molecule in endothelial adherens junctions, and the regulation of protein tyrosine phosphorylation is thought to be important for the control of adherens junction integrity. We show here that VE-PTP (vascular endothelial protein tyrosine phosphatase), an endothelial receptor-type phosphatase, co-precipitates with VE-cadherin, but not with beta-catenin, from cell lysates of transfected COS-7 cells and of endothelial cells. Co-precipitation of VE-cadherin and VE-PTP required the most membrane-proximal extracellular domains of each protein. Expression of VE-PTP in triple-transfected COS-7 cells and in CHO cells reversed the tyrosine phosphorylation of VE-cadherin elicited by vascular endothelial growth factor receptor 2 (VEGFR-2). Expression of VE-PTP under an inducible promotor in CHO cells transfected with VE-cadherin and VEGFR-2 increased the VE-cadherin-mediated barrier integrity of a cellular monolayer. Surprisingly, a catalytically inactive mutant form of VE-PTP had the same effect on VE-cadherin phosphorylation and cell layer permeability. Thus, VE-PTP is a transmembrane binding partner of VE-cadherin that associates through an extracellular domain and reduces the tyrosine phosphorylation of VE-cadherin and cell layer permeability independently of its enzymatic activity.     

Tyr-94 Phosphorylation Inhibits Pyruvate Dehydrogenase Phosphatase 1 and Promotes Tumor Growth

Many cancer cells rely more on aerobic glycolysis (the Warburg effect) than mitochondrial oxidative phosphorylation and catabolize glucose at a high rate. Such a metabolic switch is suggested to be due in part to functional attenuation of mitochondria in cancer cells. However, how oncogenic signals attenuate mitochondrial function and promote the switch to glycolysis remains unclear. We previously reported that tyrosine phosphorylation activates and inhibits mitochondrial pyruvate dehydrogenase kinase (PDK) and phosphatase (PDP), respectively, leading to enhanced inhibitory serine phosphorylation of pyruvate dehydrogenase (PDH) and consequently inhibition of pyruvate dehydrogenase complex (PDC) in cancer cells. In particular, Tyr-381 phosphorylation of PDP1 dissociates deacetylase SIRT3 and recruits acetyltransferase ACAT1 to PDC, resulting in increased inhibitory lysine acetylation of PDHA1 and PDP1. Here we report that phosphorylation at another tyrosine residue, Tyr-94, inhibits PDP1 by reducing the binding ability of PDP1 to lipoic acid, which is covalently attached to the L2 domain of dihydrolipoyl acetyltransferase (E2) to recruit PDP1 to PDC. We found that multiple oncogenic tyrosine kinases directly phosphorylated PDP1 at Tyr-94, and Tyr-94 phosphorylation of PDP1 was common in diverse human cancer cells and primary leukemia cells from patients. Moreover, expression of a phosphorylation-deficient PDP1 Y94F mutant in cancer cells resulted in increased oxidative phosphorylation, decreased cell proliferation under hypoxia, and reduced tumor growth in mice. Together, our findings suggest that phosphorylation at different tyrosine residues inhibits PDP1 through independent mechanisms, which act in concert to regulate PDC activity and promote the Warburg effect.     

The N Terminus of Cbl-c Regulates Ubiquitin Ligase Activity by Modulating Affinity for the Ubiquitin-conjugating Enzyme

Cbl proteins are ubiquitin ligases (E3s) that play a significant role in regulating tyrosine kinase signaling. There are three mammalian family members: Cbl, Cbl-b, and Cbl-c. All have a highly conserved N-terminal tyrosine kinase binding domain, a catalytic RING finger domain, and a C-terminal proline-rich domain that mediates interactions with Src homology 3 (SH3) containing proteins. Although both Cbl and Cbl-b have been studied widely, little is known about Cbl-c. Published reports have demonstrated that the N terminus of Cbl and Cbl-b have an inhibitory effect on their respective E3 activity. However, the mechanism for this inhibition is still unknown. In this study we demonstrate that the N terminus of Cbl-c, like that of Cbl and Cbl-b, inhibits the E3 activity of Cbl-c. Furthermore, we map the region responsible for the inhibition to the EF-hand and SH2 domains. Phosphorylation of a critical tyrosine (Tyr-341) in the linker region of Cbl-c by Src or a phosphomimetic mutation of this tyrosine (Y341E) is sufficient to increase the E3 activity of Cbl-c. We also demonstrate for the first time that phosphorylation of Tyr-341 or the Y341E mutation leads to a decrease in affinity for the ubiquitin-conjugating enzyme (E2), UbcH5b. The decreased affinity of the Y341E mutant Cbl-c for UbcH5b results in a more rapid turnover of bound UbcH5b coincident with the increased E3 activity. These data suggest that the N terminus of Cbl-c contributes to the binding to the E2 and that phosphorylation of Tyr-341 leads to a decrease in affinity and an increase in the E3 activity of Cbl-c.     

Site-specific incorporation of a phosphotyrosine mimetic reveals a role for tyrosine phosphorylation of SHP-2 in cell signaling.

The regulation of protein tyrosine phosphatase (PTPase) SHP-2 is proposed to involve tyrosine phosphorylation on two tail tyrosine residues. Using "expressed protein ligation", nonhydrolyzable phosphotyrosine analogs were introduced at known phosphorylation sites in SHP-2. Biochemical analysis suggests that a phosphonate at Tyr542 interacts intramolecularly with the N-terminal SH2 domain to relieve basal inhibition of the PTPase, whereas a phosphonate at Tyr-580 stimulates the PTPase activity by interaction with the C-terminal SH2 domain. Microinjection experiments indicate that a single phosphorylation of Tyr-542 of SHP-2 is sufficient to activate the MAP kinase pathway in living cells. These studies support a novel mechanism explaining how tyrosine phosphorylation of a PTPase is important in signal transduction.     

Inhibition of endothelial FAK activity prevents tumor metastasis by enhancing barrier function

Pharmacological focal adhesion kinase (FAK) inhibition prevents tumor growth and metastasis, via actions on both tumor and stromal cells. In this paper, we show that vascular endothelial cadherin (VEC) tyrosine (Y) 658 is a target of FAK in tumor-associated endothelial cells (ECs). Conditional kinase-dead FAK knockin within ECs inhibited recombinant vascular endothelial growth factor (VEGF-A) and tumor-induced VEC-Y658 phosphorylation in vivo. Adherence of VEGF-expressing tumor cells to ECs triggered FAK-dependent VEC-Y658 phosphorylation. Both FAK inhibition and VEC-Y658F mutation within ECs prevented VEGF-initiated paracellular permeability and tumor cell transmigration across EC barriers. In mice, EC FAK inhibition prevented VEGF-dependent tumor cell extravasation and melanoma dermal to lung metastasis without affecting primary tumor growth. As pharmacological c-Src or FAK inhibition prevents VEGF-stimulated c-Src and FAK translocation to EC adherens junctions, but FAK inhibition does not alter c-Src activation, our experiments identify EC FAK as a key intermediate between c-Src and the regulation of EC barrier function controlling tumor metastasis.     

Phosphorylation of DEP-1/PTPRJ on threonine 1318 regulates Src activation and endothelial cell permeability induced by vascular endothelial growth factor

The protein tyrosine phosphatase DEP-1/PTPRJ positively regulates Src family kinases and critical biological functions in endothelial and hematopoietic cells. Phosphorylation of DEP-1 on Y1311/Y1320 mediates the association and activation of Src, and promotes Src-dependent angiogenic responses including endothelial cell permeability. We have identified T1318 as a phosphorylated residue proximal to Y1320. The aim of this study was to determine if T1318 phosphorylation exerts a regulatory role over the function of DEP-1. We show that phosphorylation of DEP-1 on Y1320 was reduced when T1318 was mutated. This led to the decreased association of DEP-1 T1318A with Src, and defective Src activation in both HEK 293T and VEGF-stimulated endothelial cells. Consistent with these findings, VEGF-induced tyrosine phosphorylation of VE-cadherin, its association to β-arrestin1/2, and cell permeability were impaired in cells expressing DEP-1 T1318A. Conversely, expression of the phosphomimetic mutant DEP-1 T1318E constitutively enhanced the phosphorylation of Y1320 and VE-cadherin over that induced by WT DEP-1, and resulted in increased VEGF-dependent permeability. DEP-1 T1318 is part of a CK2 consensus phosphorylation site and was identified as a CK2 substrate. Modulation of CK2 expression or activity in endothelial cells regulated T1318 phosphorylation, and correlated with the status of Y1320 phosphorylation, Src activation, and cell permeability. CK2-dependent phosphorylation of DEP-1 T1318 promotes Y1320 phosphorylation and Src activation upon VEGF stimulation. Phosphorylation of T1318 is thus part of a regulatory mechanism that channels the activity of DEP-1 towards Src to allow its optimal activation and the promotion of endothelial cell permeability.     

Src kinase activity is essential for osteoclast function

Deletion of the c-src gene impairs osteoclast bone resorbing activity, causing osteopetrosis. Although it has been concluded that restoring only the Src adaptor function at least partly rescues the cell attachment and skeletal phenotypes, the contribution of Src kinase activity remains controversial. Src forms a complex with Pyk2 and Cbl after adhesion-induced stimulation of alpha(V)beta(3) integrin. To demonstrate the importance of the Pyk2-Src association in osteoclasts and to distinguish the contributions of the Src adaptor and kinase activities in cytoskeletal organization and osteoclast function, we expressed mutants of Src and Pyk2 in osteoclasts using adenovirus vectors. Eliminating the Src-binding site on Pyk2 (Pyk2(Y402F)) markedly inhibited bone resorption by osteoclast-like cells, whereas kinase-dead Pyk2 had little effect. Kinase-dead Src, unlike kinase-dead Pyk2, markedly inhibited the bone-resorbing activity of wild type osteoclasts and failed to significantly restore bone-resorbing activity to Src(-/-) osteoclast-like cells. Activation of Src kinase by overexpressing kinase-dead Csk failed to reverse the inhibitory effect of Pyk2(Y402F), suggesting that osteoclastic bone resorption requires both c-Src kinase activity and the targeting of Src kinase by Pyk2. Src-catalyzed phosphorylation of Cbl on Tyr-731 is reported to induce the activation and recruitment of phosphatidylinositol 3-kinase to the cell membrane in a signaling pathway that is critical for osteoclast function. Expressing the Cbl(Y731F) mutant in osteoclasts markedly reduced their bone resorbing activity, suggesting that phosphorylation of Cbl(Y731) and the subsequent recruitment and activation of phosphatidylinositol 3-kinase may be critical signaling events downstream of Src in osteoclasts.     

Tyrosine phosphorylation of VE-cadherin and claudin-5 is associated with TGF-β1-induced permeability of centrally derived vascular endothelium

Breakdown of the inner blood-retinal barrier and the blood-brain barrier is associated with changes in tight and adherens junction-associated proteins that link vascular endothelial cells. This study aimed to test the hypothesis that transforming growth factor (TGF)-β1 increases the paracellular permeability of vascular endothelial monolayers through tyrosine phosphorylation of VE-cadherin and claudin-5. Bovine retinal and human brain capillary endothelial cells were grown as monolayers on coated polycarbonate membranes. Paracellular permeability was studied by measuring the equilibration of (14)C-inulin or fluorescence-labelled dextran. Changes in VE-cadherin and claudin-5 expression were studied by immunocytochemistry (ICC) and quantified by cell-based enzyme linked immunosorbent assays (ELISA). Tyrosine phosphorylation of VE-cadherin and claudin-5 was studied by ICC, immunoprecipitation and Western blotting. We found that exposure of endothelial cells to TGF-β1 caused a dose-dependent increase in paracellular permeability as reflected by increases in the equilibration of (14)C-inulin. This effect was enhanced by the tyrosine phosphatase inhibitor orthovanadate and attenuated by the tyrosine kinase inhibitor lavendustin A. ICC and cell-based ELISA revealed that TGF-β1 induced both dose- and time-dependent decreases in VE-cadherin and claudin-5 expression. Assessment of cell viability indicated that changes in these junction-associated proteins were not due to endothelial death or injury. ICC revealed that tyrosine phosphorylation of endothelial monolayers was greatly enhanced by TGF-β1 treatment, and immunoprecipitation of cell lysates showed increased tyrosine phosphorylation of VE-cadherin and claudin-5. Our results suggest that tyrosine phosphorylation of VE-cadherin and claudin-5 is involved in the increased paracellular permeability of central nervous system-derived vascular endothelium induced by TGF-β1.     

Tyrosyl phosphorylation of Shp2 is required for normal ERK activation in response to some,but not all,growth factors

The protein-tyrosine phosphatase Shp2 is required for normal activation of the ERK mitogen-activated protein kinase in multiple receptor tyrosine kinase signaling pathways. In fibroblasts, Shp2 undergoes phosphorylation at two C-terminal tyrosyl residues in response to some (fibroblast growth factor and platelet-derived growth factor (PDGF)) but not all (epidermal growth factor and insulin-like growth factor) growth factors. Whereas the catalytic activity of Shp2 is required for all Shp2 actions, the effect of tyrosyl phosphorylation on Shp2 function has been controversial. To clarify the role of Shp2 tyrosyl phosphorylation, we infected Shp2-mutant fibroblasts with retroviruses expressing wild type Shp2 or mutants of either (Y542F or Y580F) or both (Y542F,Y580F) C-terminal tyrosines. Compared with wild type cells, ERK activation was decreased in Y542F- or Y580F-infected cells in response to fibroblast growth factor and PDGF but not the epidermal growth factor. Mutation of both phosphorylation sites resulted in a further decrease in growth factor-evoked ERK activation, although not to the level of the vector control. Immunoblot analyses confirm that Tyr-542 and Tyr-580 are the major sites of Shp2 tyrosyl phosphorylation and that Tyr-542 is the major Grb2 binding site. However, studies with antibodies specific for individual Shp2 phosphorylation sites reveal unexpected complexity in the mechanism of Shp2 tyrosyl phosphorylation by different receptor tyrosine kinases. Moreover, because Y580F mutants retain nearly wild type Grb2-binding ability, yet exhibit defective PDGF-evoked ERK activation, our results show that the association of Grb2 with Shp2 is not sufficient for promoting full ERK activation in response to these growth factors, thereby arguing strongly against the "Grb2-adapter" model of Shp2 action.     

Novel mechanism regulating endothelial permeability via T-cadherin-dependent VE-cadherin phosphorylation and clathrin-mediated endocytosis

T-cadherin is a unique member of the cadherin superfamily of adhesion molecules. In contrast to “classical” cadherins, T-cadherin lacks transmembrane and cytoplasmic domains and is anchored to the cell membrane via a glycosilphosphoinositol moiety. T-cadherin is predominantly expressed in cardiovascular system. Clinical and biochemical studies evidence that expression of T-cadherin increases in post-angioplasty restenosis and atherosclerotic lesions—conditions associated with endothelial dysfunction and pathological expression of adhesion molecules. Here, we provide data suggesting a new signaling mechanism by which T-cadherin regulates endothelial permeability. T-cadherin overexpression leads to VE-cadherin phosphorylation on Y731 (β-catenin-binding site), VE-cadherin clathrin-dependent endocytosis and its degradation in lysosomes. Moreover, T-cadherin overexpression results in activation of Rho GTPases signaling and actin stress fiber formation. Thus, T-cadherin up-regulation is involved in degradation of a key endothelial adhesion molecule, VE-cadherin, resulting in the disruption of endothelial barrier function. Our results point to the role of T-cadherin in regulation of endothelial permeability and its possible engagement in endothelial dysfunction.     

Mechanism of tyrosine phosphorylation and activation of phospholipase C-gamma 1. Tyrosine 783 phosphorylation is not sufficient for lipase activation

Phospholipase C-gamma 1 (PLC-gamma 1) is phosphorylated on three tyrosine residues: Tyr-771, Tyr-783, and Tyr-1253. With the use of antibodies specific for each of these phosphorylation sites, we have now determined the kinetics and magnitude of phosphorylation at each site. Phosphorylation of Tyr-783, which is essential for lipase activation, was observed in all stimulated cell types examined. The extent of phosphorylation of Tyr-1253 was approximately 50 to 70% of that of Tyr-783 in cells stimulated with platelet-derived growth factor (PDGF) or epidermal growth factor (EGF), but Tyr-1253 phosphorylation was not detected in B or T cell lines stimulated through B- and T-cell antigen receptors, respectively. Tyr-771 was phosphorylated only at a low level in all cells studied. In cells stimulated with PDGF, phosphorylation and dephosphorylation of Tyr-783 and of Tyr-1253 occurred with similar kinetics; the receptor kinase appeared to phosphorylate both sites, albeit with Tyr-783 favored over Tyr-1253, before the bound PLC-gamma 1 was released, and phosphorylation at the two sites occurred independently. PDGF and EGF induced similar levels of phosphorylation of Tyr-783 and of Tyr-1253 in a cell line that expressed receptors for both growth factors. However, only PDGF, not EGF, elicited substantial PLC activity, suggesting that Tyr-783 phosphorylation was not sufficient for enzyme activation. Finally, concurrent production of phosphatidylinositol 3,4,5-trisphosphate was found to contribute to the activation of phosphorylated PLC-gamma 1.     

Differential regulation of Pyk2 phosphorylation at Tyr-402 and Tyr-580 in intestinal epithelial cells: roles of calcium, Src, Rho kinase, and the cytoskeleton

The calcium-dependent proline-rich tyrosine kinase Pyk2 is activated by tyrosine phosphorylation, associates with focal adhesion proteins, and has been linked to proliferative and migratory responses in a variety of mesenchymal and epithelial cell types. Full Pyk2 activation requires phosphorylation at functionally distinct sites, including autophosphorylation site Tyr-402 and catalytic domain site Tyr-580, though the mechanisms involved are unclear. The pathways mediating Pyk2 phosphorylation at Tyr-402 and Tyr-580 were therefore investigated. Both sites were rapidly and transiently phosphorylated following cell stimulation by Ang II or LPA. However, only Tyr-580 phosphorylation was rapidly enhanced by intracellular Ca(2+) release, or inhibited by Ca(2+) depletion. Conversely, Tyr-402 phosphorylation was highly sensitive to inhibition of actin stress fibers, or of Rho kinase (ROK), an upstream regulator of stress fiber assembly. Ang II also induced a delayed (30-60 min) secondary phosphorylation peak occurring at Tyr-402 alone. Unlike the homologous focal adhesion kinase (FAK), Pyk2 phosphorylation was sensitive neither to the Src inhibitor PP2, nor to truncation of its N-terminal region, which contains a putative autoinhibitory FERM domain. These results better define the mechanisms involved in Pyk2 activation, demonstrating that autophosphorylation is ROK- and stress fiber-dependent, while transphosphorylation within the kinase domain is Ca(2+)-dependent and Src-independent in intestinal epithelial cells. This contrasts with the tight sequential coupling of phosphorylation seen in FAK activation, and further underlines the differences between these closely related kinases.     

EphB1-mediated cell migration requires the phosphorylation of paxillin at Tyr-31/Tyr-118

Interactions between Eph receptors and their membrane-bound ligands (ephrins) are of critical importance for key developmental processes such as boundary formation or vascular development. Their downstream signaling pathways are intricate and heterogeneous at several levels, the combined effect being a highly complex and flexible system. Here we demonstrate that activated EphB1 induces tyrosine phosphorylation of the focal adhesion protein paxillin at Tyr-31 and Tyr-118 and is recruited to paxillin-focal adhesion kinase (FAK) complexes. Pretreatment with the specific Src inhibitor PP2, or expression of dominant-negative, kinase-dead c-Src abrogates EphB1-induced tyrosine phosphorylation of paxillin. Cells transfected with the paxillin mutant Y31F/Y118F displayed a reduced migration in response to ephrin B2 stimulation. Furthermore, expression of an LD4 deletion mutant (paxillin DeltaLD4) significantly reduces EphB1-paxillin association, paxillin tyrosine phosphorylation, as well as EphB1-dependent cell migration. Finally, mutation of the Nck-binding site of EphB1 (Y594F) interrupts the interaction between Nck, paxillin, and EphB1. These data suggest a model in which ligand-activated EphB1 forms a signaling complex with Nck, paxillin, and focal adhesion kinase and induces tyrosine phosphorylation of paxillin in a c-Src-dependent manner to promote cell migration.