Biphasic activation of p21ras by endothelin-1 sequentially activates the ERK cascade and phosphatidylinositol 3-kinase.

Endothelin-1 (ET-1) induces cell proliferation and differentiation through multiple G-protein-linked signaling systems, including p21ras activation. Whereas p21ras activation and desensitization by receptor tyrosine kinases have been extensively investigated, the kinetics of p21ras activation induced by engagement of G-protein-coupled receptors remains to be fully elucidated. In the present study we show that ET-1 induces a biphasic activation of p21ras in rat glomerular mesangial cells. The first peak of activation of p21ras, at 2-5 min, is mediated by immediate association of phosphorylated Shc with the guanosine exchange factor Sos1 via the adaptor protein Grb2. This initial activation of p21ras results in activation of the extracellular signal-regulated kinase (ERK) cascade. We demonstrate that ET-1 signaling elicits a negative feedback mechanism, modulating p21ras activity through ERK-dependent Sos1 phosphorylation, findings which were confirmed using an adenovirus MEK construct. Subsequent to p21ras and ERK deactivation, Sos1 reverts to the non-phosphorylated condition, enabling it to bind again to the Grb2/Shc complex, which is stabilized by persistent Shc phosphorylation. However, the resulting secondary activation of p21ras at 30 min does not lead to ERK activation, correlating with intensive, ET-1-induced expression of MAP kinase phosphatase-1, but does result in increased p21ras-associated phosphatidylinositol 3-kinase activity. Our data provide evidence that ET-1-induced biphasic p21ras activation causes sequential stimulation of divergent downstream signaling pathways.     

Thrombin-stimulated Pyk2 phosphorylation in human endothelium is dependent on intracellular calcium and independent of protein kinase C and Src kinases

G-protein-coupled receptor agonists (GPCAs) cause functional responses in endothelial cells including secretion, proliferation, and altering monolayer permeability. These events are mediated in part by activation of the p42/44 mitogen-activated protein kinase (MAPK) cascade. The cytosolic tyrosine kinase Pyk2 is postulated to link GPCA-induced changes in intracellular calcium to activation of the MAP kinase cascade. We have investigated the regulation of Pyk2 in human umbilical vein endothelial cells in response to GPCAs and show that (1) thrombin, a PAR-1 peptide, and histamine cause rapid concentration- and time-dependent phosphorylation on tyrosines 402 (Src kinase binding site), 881 (Grb2 binding site), and 580 (an autophosphorylation site), (2) thrombin-stimulated phosphorylation is dependent on intracellular calcium and independent of PKC and PI-3 kinase, and (3) inhibition of Src kinases has no significant effect on thrombin-stimulated phosphorylation, implying that tyrosine phosphorylation of Pyk2 is independent of Src binding.     

(alpha)v(beta)3 integrins and Pyk2 mediate insulin-like growth factor I activation of Src and mitogen-activated protein kinase in 3T3-L1 cells

IGF-I stimulates cell growth through interaction of the IGF receptor with multiprotein signaling complexes. However, the mechanisms of IGF-I receptor-mediated signaling are not completely understood. We have previously shown that IGF-I-stimulated 3T3-L1 cell proliferation is dependent on Src activation of the ERK-1/2 MAPK pathway. We hypothesized that IGF-I activation of the MAPK pathway is mediated through integrin activation of Src-containing signaling complexes. The disintegrin echistatin decreased IGF-I phosphorylation of Src and MAPK, and blocking antibodies to (alpha)v and beta3 integrin subunits inhibited IGF-I activation of MAPK, suggesting that (alpha)v(beta)3 integrins mediate IGF-I mitogenic signaling. IGF-I increased ligand binding to (alpha)v(beta)3 as detected by immunofluorescent staining of ligand-induced binding site antibody and stimulated phosphorylation of the beta3 subunit, consistent with inside-out activation of (alpha)v(beta)3 integrins. IGF-I increased tyrosine phosphorylation of the focal adhesion kinase (FAK) Pyk2 (calcium-dependent proline-rich tyrosine kinase-2) to a much greater extent than FAK, and increased association of Src with Pyk2 but not FAK. The intracellular calcium chelator BAPTA prevented IGF-I phosphorylation of Pyk2, Src, and MAPK, suggesting that IGF-I activation of Pyk2 is calcium dependent. Transient transfection with a dominant-negative Pyk2, which lacks the autophosphorylation and Src binding site, decreased IGF-I activation of MAPK, but no inhibition was seen with transfected wild-type Pyk2. These results indicate that IGF-I signaling to MAPK is dependent on inside-out activation of (alpha)v(beta)3 integrins and integrin-facilitated multiprotein complex formation involving Pyk2 activation and association with Src.     

Adaptor proteins Grb2 and Crk couple Pyk2 with activation of specific mitogen-activated protein kinase cascades.

The protein tyrosine kinase Pyk2 acts as an upstream regulator of mitogen-activated protein (MAP) kinase cascades in response to numerous extracellular signals. The precise molecular mechanisms by which Pyk2 activates distinct MAP kinase pathways are not yet fully understood. In this report, we provide evidence that the protein tyrosine kinase Src and adaptor proteins Grb2, Crk, and p130Cas act as downstream mediators of Pyk2 leading to the activation of extracellular signal-regulated kinase (ERK) and c-Jun amino-terminal kinase (JNK). Pyk2-induced activation of Src is necessary for phosphorylation of Shc and p130Cas and their association with Grb2 and Crk, respectively, and for the activation of ERK and JNK cascades. Expression of a Grb2 mutant with a deletion of the amino-terminal Src homology 3 domain or the carboxyl-terminal tail of Sos strongly reduced Pyk2-induced ERK activation, with no apparent effect on JNK activity. Grb2 with a deleted carboxyl-terminal Src homology 3 domain partially blocked Pyk2-induced ERK and JNK pathways, whereas expression of dominant interfering mutants of p130Cas or Crk specifically inhibited JNK but not ERK activation by Pyk2. Taken together, our data reveal specific pathways that couple Pyk2 with MAP kinases: the Grb2/Sos complex connects Pyk2 to the activation of ERK, whereas adaptor proteins p130Cas and Crk link Pyk2 with the JNK pathway.     

Novel Role of Src in Priming Pyk2 Phosphorylation

Proline-rich tyrosine kinase 2 (Pyk2) is a member of the focal adhesion kinase (FAK) family of non-receptor tyrosine kinases and plays an important role in diverse cellular events downstream of the integrin-family of receptors, including cell migration, proliferation and survival. Here, we have identified a novel role for Src kinase in priming Pyk2 phosphorylation and subsequent activation upon cell attachment on the integrin-ligand fibronectin. By using complementary methods, we show that Src activity is indispensable for the initial Pyk2 phosphorylation on the Y402 site observed in response to cell attachment. In contrast, the initial fibronectin-induced autophosphorylation of FAK in the homologous Y397 site occurs in a Src-independent manner. We demonstrate that the SH2-domain of Src is required for Src binding to Pyk2 and for Pyk2 phosphorylation at sites Y402 and Y579. Moreover, Y402 phosphorylation is a prerequisite for the subsequent Y579 phosphorylation. While this initial phosphorylation of Pyk2 by Src is independent of Pyk2 kinase activity, subsequent autophosphorylation of Pyk2 in trans is required for full Pyk2 phosphorylation and activation. Collectively, our studies reveal a novel function of Src in priming Pyk2 (but not FAK) phosphorylation and subsequent activation downstream of integrins, and shed light on the signaling events that regulate the function of Pyk2.     

CrkII associates with BCAR3 in response to endothelin-1 in human glomerular mesangial cells

Endothelin-1 (ET-1) effects in human glomerular mesangial cells (GMC) include proliferation, contraction, and extracellular matrix synthesis. Calcium-regulated nonreceptor, proline-rich tyrosine kinase 2 (Pyk2) is a critical mediator of ET-1 signaling in human glomerulae. Working in concert with Pyk2, adaptor protein CrkII and a recently discovered guanidine exchange factor for certain small GTPases BCAR3 can be involved in ET-1 signaling in the kidney. Signaling through CrkII and BCAR3 might be critical in some proliferative kidney pathologies. The current study was designed to determine the possibility of CrkII and BCAR3 interaction in response to ET-1 in human GMC and the role of Pyk2 in the association of these proteins. Using adenovirus-mediated transfer of genes encoding either green fluorescent protein (control) or dominant interfering Pyk2 construct, we demonstrated that CrkII and BCAR3 can be coprecipitated from unstimulated and ET-1 stimulated GMC; ET-1 treatment time-dependently increased CrkII/BCAR3 complex formation; and inhibition of endogenous Pyk2 autophosphorylation led to a significant decrease in CrkII/BCAR3 association both basal and stimulated.     

Alterations in subcellular localization of p38 MAPK potentiates endothelin-stimulated COX-2 expression in glomerular mesangial cells

Endothelin-1 (ET-1) is a potent vasoconstrictor peptide with mitogenic actions linked to activation of tyrosine kinase signaling pathways. ET-1 induces cyclooxygenase-2 (COX-2), an enzyme that converts arachidonic acid to pro-inflammatory eicosanoids. Activation of each of the three major mitogen-activated protein kinase (MAPK) pathways, ERK1/2, JNK/SAPK, and p38 MAPK (p38), have been shown to enhance the expression of COX-2. Negative regulation of MAPK may occur via a family of dual specificity phosphatases referred to as mitogen-activated protein kinase phosphatases (MKP). The goal of this work was to test the hypothesis that wild type MKP-1 regulates the expression of ET-1-induced COX-2 expression by inhibiting the activation of p38 in cultured glomerular mesangial cells (GMC). An adenovirus expressing both wild type and a catalytically inactive mutant of MKP-1 (MKP-1/CS) were constructed to study ET-1-regulated MAPK signaling and COX-2 expression in cultured GMC. ET-1 stimulated the phosphorylation of ERK and p38 alpha MAPK and induced the expression of COX-2. Expression of COX-2 was partially blocked by U0126, a MEK inhibitor, and SB 203580, a p38 MAPK inhibitor. Adenoviral expression of MKP-1/CS augmented basal and ET-1-induced phosphorylation of p38 alpha MAPK with less pronounced effects on ERK1/2 phosphorylation. Ectopic expression of wild type MKP-1 blocked the phosphorylation of p38 alpha MAPK by ET-1 but increased the phosphorylation of p38 gamma MAPK. Co-precipitation studies demonstrated association of MKP-1 with p38 alpha MAPK and ERK1/2. Immunofluorescent image analysis demonstrated trapping of phospho-p38 MAPK in the cytoplasm by MKP-1/CS/green fluorescent protein. ET-1-stimulated expression of COX-2 was increased in MKP-1/CS versus LacZ or green fluorescent protein-infected control cells. These results indicate that MKP-1 demonstrates a relative selectivity for p38 alpha MAPK versus p38 gamma MAPK in GMC and is likely to indirectly regulate the expression of COX-2.     

The proline-rich tyrosine kinase Pyk2 modulates integrin-mediated neutrophil adhesion and reactive oxygen species generation

Neutrophils are first responders in infection and inflammation. They are able to roll, adhere and transmigrate through the endothelium to reach the site of infection, where they fight pathogens through secretion of granule contents, production of reactive oxygen species, extrusion of neutrophil extracellular traps, and phagocytosis. In this study we explored the role of the non-receptor focal adhesion kinase Pyk2 in neutrophil adhesion and activation. Using a specific Pyk2 pharmacological inhibitor, PF-4594755, as well as Pyk2-deficient murine neutrophils, we found that Pyk2 is activated upon integrin αMβ2-mediated neutrophil adhesion to fibrinogen. This process is triggered by Src family kinases-mediated phosphorylation and supported by Pyk2 autophosphorylation on Y402. In neutrophil adherent to fibrinogen, Pyk2 activates PI3K-dependent pathways promoting the phosphorylation of Akt and of its downstream effector GSK3. Pyk2 also dynamically regulates MAP kinases in fibrinogen-adherent neutrophils, as it stimulates p38MAPK but negatively regulates ERK1/2. Pharmacological inhibition of Pyk2 significantly prevented adhesion of human neutrophils to fibrinogen, and neutrophils from Pyk2-knockout mice showed a reduced ability to adhere compared to wildtype cells. Accordingly, neutrophil adhesion to fibrinogen was reduced upon inhibition of p38MAPK but potentiated by ERK1/2 inhibition. Neutrophil adherent to fibrinogen, but not to polylysine, were able to produce ROS upon lipopolysaccharide challenge and ROS production was completely suppressed upon inhibition of Pyk2. By contrast PMA-induced ROS production by neutrophil adherent to either fibrinogen or polylysine was independent from Pyk2. Altogether these results demonstrate that Pyk2 is an important effector in the coordinated puzzle regulating neutrophil adhesion and activation.     

Endothelin-1 induces serine phosphorylation of the adaptor protein p66Shc and its association with 14-3-3 protein in glomerular mesangial cells

Endothelin-1 (ET-1) is a vasoconstrictor peptide known to be a potent mitogen for glomerular mesangial cells (GMC). In the current study, it is demonstrated that ET-1 treatment of GMC results in serine phosphorylation of the 66-kDa isoform of the adapter protein Shc (p66(Shc)). ET-1-induced serine phosphorylation of p66(Shc) requires activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling module and is efficiently inhibited by both a MAPK/ERK kinase (MEK)-selective inhibitor and adenovirus-mediated transfer of a dominant interfering MEK1 mutant. Furthermore, adenovirus-mediated transfer of a constitutively active MEK1 mutant was found to markedly increase p66(Shc) serine phosphorylation. Adenoviruses encoding constitutively active mutants of MAPK kinases 3 and 6 (upstream kinases of p38(MAPK)) and 7 (upstream kinase of c-Jun NH(2)-terminal kinase) failed to induce serine phosphorylation of this adaptor protein. Serine phosphorylation of p66(Shc) resulted in its association with the serine binding motif-containing protein 14-3-3. ET-1-induced phosphorylation of a serine encompassed in the 14-3-3 binding motif of p66(Shc) was confirmed in experiments employing anti-phospho-14-3-3 binding motif antibodies. These studies are the first to demonstrate that G protein-coupled receptors stimulate serine phosphorylation of p66(Shc) and the first to report the formation of a signaling complex between p66(Shc) and 14-3-3.     

Regulation of calcium-sensitive tyrosine kinase Pyk2 by angiotensin II in endothelial cells. Roles of Yes tyrosine kinase and tyrosine phosphatase SHP-2

Calcium-sensitive tyrosine kinase Pyk2 has been implicated in the regulation of ion channels, cellular adhesion, and mitogenic and hypertrophic reactions. In this study, we have investigated the regulation of Pyk2 by angiotensin II (Ang II) in pulmonary vein endothelial cells. We found that the Ang II-induced tyrosine phosphorylation of Pyk2, which requires the activity of Src family kinase, was specifically regulated by the Src family kinase member, Yes kinase. Moreover, we identified for the first time the constitutive association of Pyk2 with an Src homology 2 (SH2) domain-containing tyrosine phosphatase SHP-2. SHP-2 interacts with Pyk2 through a region other than its SH2 domains. Pyk2 can be dephosphorylated in vitro in SHP-2 immunoprecipitates and in intact cells expressing an NH(2) terminus-truncated form of SHP-2, which lacks the two SH2 domains but has an enhanced phosphatase activity. Ang II activates the endogenous SHP-2. Finally, the SHP-2-mediated dephosphorylation of Pyk2 correlates with the negative effect of SHP-2 on the Ang II-induced activation of extracellular signal-regulated kinase and c-Jun NH(2)-terminal kinase. Thus, the balance of Pyk2 tyrosine phosphorylation in response to Ang II is controlled by Yes kinase and by a tyrosine phosphatase SHP-2 in endothelial cells.     

Transforming growth factor-beta1 stimulates vascular endothelial growth factor 164 via mitogen-activated protein kinase kinase 3-p38alpha and p38delta mitogen-activated protein kinase-dependent pathway in murine mesangial cells

Transforming growth factor-beta1 (TGF-beta1) is a potent inducer of extracellular matrix synthesis leading to progressive glomerular fibrosis. The intracellular signaling mechanisms involved in this process remain incompletely understood. The p38 mitogen-activated protein kinase (MAPK) is a major stress signal transducing pathway that is rapidly activated by TGF-beta1 in mesangial cells. We have previously demonstrated MKK3 as the immediate upstream MAPK kinase required for selective activation of p38 MAPK isoforms, p38alpha and p38delta, and stimulation of pro-alpha1(I) collagen by TGF-beta1 in murine mesangial cells. In this study, we further sought to determine MAPK kinase 3 (MKK3)-dependent TGF-beta1 responses by gene expression profiling analysis utilizing mesangial cells isolated from Mkk3-/- mice compared with Mkk3+/+ controls. Interestingly, vascular endothelial growth factor (VEGF) was identified as a TGF-beta1-induced gene affected by deletion of Mkk3. VEGF is a well known endothelial mitogen, whose actions in nonendothelial cell types are still not well understood. We confirmed that TGF-beta1 increased VEGF mRNA and protein synthesis of VEGF164 and VEGF188 isoforms in wild-type mesangial cells. However, in the Mkk3-/- mesangial cells, both TGF-beta1-induced VEGF mRNA and VEGF164 protein expression were inhibited, whereas TGF-beta1-induced VEGF188 protein expression was unaffected. Furthermore, transfection of dominant negative mutants of p38alpha and p38delta resulted in marked inhibition of TGF-beta1-induced VEGF164 expression but not VEGF188, and treatment with recombinant mouse VEGF164 increased collagen and fibronectin mRNA expression in mesangial cells. Taken together, our findings suggest a critical role for the MKK3-p38alpha and p38delta MAPK pathway in mediating VEGF164 isoform-specific stimulation by TGF-beta1 in mesangial cells. Further, VEGF164 stimulates collagen and fibronectin expression in mesangial cells and thus in turn enhances TGF-beta1-induced extracellular matrix and may play an important role in progressive glomerular fibrosis.     

Interactions between two cytoskeleton-associated tyrosine kinases: calcium-dependent tyrosine kinase and focal adhesion tyrosine kinase

The calcium-dependent tyrosine kinase (CADTK), also known as Pyk2/RAFTK/CAKbeta/FAK2, is a cytoskeleton-associated tyrosine kinase. We compared CADTK regulation with that of the highly homologous focal adhesion tyrosine kinase (FAK). First, we generated site-specific CADTK mutants. Mutation of Tyr402 eliminated autophosphorylation and significantly decreased kinase activity. Mutation of Tyr881, a putative Src kinase phosphorylation site predicted to bind Grb2, had little effect on CADTK regulation. Src family tyrosine kinases resulted in CADTK tyrosine phosphorylation even when co-expressed with the Tyr402/Tyr881 double mutant, suggesting that Src/Fyn etc. phosphorylate additional tyrosine residues. Interestingly, CADTK tyrosine-phosphorylated FAK when both were transiently expressed, but FAK did not phosphorylate CADTK. Biochemical experiments confirmed direct CADTK phosphorylation of FAK. This phosphorylation utilized tyrosine residues other than Tyr397, Tyr925, or Tyr576/Tyr577, suggesting that new SH2-binding sites might be created by CADTK-dependent FAK phosphorylation. Last, expression of the CADTK carboxyl terminus (CRNK) abolished CADTK but not FAK autophosphorylation. In contrast, FAK carboxyl terminus overexpression inhibited both FAK and CADTK autophosphorylation, suggesting that a FAK-dependent cytoskeletal function may be necessary for CADTK activation. Thus, CADTK and FAK, which both bind to some, but not necessarily the same, cytoskeletal elements, may be involved in coordinate regulation of cytoskeletal structure and signaling.     

Activation of integrin β1-focal adhesion kinase-RasGTP pathway plays a critical role in TGF beta1-induced podocyte injury

The depletion of glomerular podocytes is the key mechanism of glomerulosclerosis and progressive renal failure. Transforming growth factor-β (TGFβ) is a central mediator of signaling networks that control a diverse set of cellular processes, such as cell proliferation, differentiation, and apoptosis. Though many key events in TGFβ1 signaling have been documented at cellular and molecular level in podocytes, the complete effects of TGFβ1 on podocyte integrity are still elusive. In this study, the function of adhesion protein integrin β1, focal adhesion kinase (FAK), and a small GTPase Ras was explored in TGFβ1-induced podocyte injury. In cultured mouse podocyte, caspase 3-positive cells were counted by flow cytometry to evaluate podocyte damage at different time points after TGFβ1 treatment. Immunoblotting assay showed that integrin β1, FAK, Src kinase, and an adaptor protein Grb2 were activated rapidly after TGFβ1 stimulation. Active Ras Pull-Down assay revealed that the active Ras (GTP-bound Ras) level was upregulated in TGFβ1-treated cell. Immunoprecipitation results displayed that TGFβ1 enhanced the complex formation of integrin β1, FAK and Src kinase, as well as FAK, Grb2 and Ras. The FAK inhibitor TAE226 and the specific knockdown of Grb2 remarkably alleviated TGFβ1-induced podocyte apoptosis. The activation of p38MAPK and Erk1/2, and the nuclear translocation of NFκB(p65) were increased evidently in TGFβ1-treated cell, which could be dramatically prohibited by the application of the p38MAPK inhibitor SB202190 and the Ras inhibitor FPT Inhibitor III. The Src kinase inhibitor PP2 obviously prevented the activation of FAK and Ras, as well as the translocation of NFκB(p65) from cytoplasm to nuclei. The PP2, FPT Inhibitor III, and SB202190 significantly decreased TGFβ1-induced podocyte apoptosis. Taken together, these data demonstrated that the activation of integrin β1/Src/FAK and Grb2/RasGTP should be responsible for TGFβ1-induced podocyte damage through the p38MAPK and Erk1/2-mediated nuclear translocation of NFκB(p65).     

The presence of a single tyrosine residue at the carboxyl domain of vascular endothelial growth factor receptor-2/FLK-1 regulates its autophosphorylation and activation of signaling molecules

Vascular endothelial growth factor receptor (VEGFR)-2 plays a critical role in vasculogenesis during embryonic development and pathological angiogenesis, but little is known about the molecular mechanisms governing its functions. Here we investigated the role of tyrosine 1212 on mouse VEGFR-2 autophosphorylation and its signal transduction relay in endothelial cells. Mutation of tyrosine 1212 on VEGFR-2 to phenylalanine severely impaired the ligand-dependent autophosphorylation of VEGFR-2 and its ability to associate with and activate Src. This mutation also reduced the VEGFR-2 ability to phosphorylate phospholipase Cgamma1 and mitogen-activated protein kinase (MAPK). Unlike mutation of tyrosine 1212 to phenylalanine, replacement of tyrosine 1212 with glutamic acid preserved the ligand-dependent activation of VEGFR-2 and activation of VEGFR-2-associated signaling proteins including Src, phospholipase Cgamma1, and MAPK. Further analysis showed that Src activation is not required for activation of VEGFR-2, since cells co-expressing wild type receptor with kinase dead Src or wild type Src displayed no apparent effect in the ligand-dependent autophosphorylation of VEGFR-2. Similarly, expression of wild type VEGFR-2 in fibroblast (SYF) cells obtained from the triple knockout Src family kinases showed normal ligand-dependent autophosphorylation. Collectively, these results suggest that phosphorylation of tyrosine 1212 of VEGFR-2 plays a crucial role in the activation of VEGFR-2 and subsequently VEGFR-2-mediated angiogenesis.     

Mechanisms of endothelin-1-induced MAP kinase activation in adrenal glomerulosa cells

G protein-coupled receptors (GPCRs) such as angiotensin II, bradykinin and endothelin-1 (ET-1) are critically involved in the regulation of adrenal function, including aldosterone production from zona glomerulosa cells. Whereas, substantial data are available on the signaling mechanisms of ET-1 in cardiovascular tissues, such information in adrenal glomerulosa cells is lacking. Bovine adrenal glomerulosa (BAG) cells express receptors for endothelin-1 (ET-1) and their stimulation caused phosphorylation of Src (at Tyr416), proline-rich tyrosine kinase (Pyk2 at Tyr402), extracellularly regulated signal kinases (ERK1/2), and their dependent proteins, p90 ribosomal S6 kinase (RSK-1) and CREB. ET-1 elicited these responses predominantly through activation of a G_i-linked cascade with a minor contribution from the G_q/PKC pathway. Whereas, selective inhibition of EGF-R kinase with AG1478 caused complete inhibition of EGF-induced ERK/RSK-1/CREB activation, it caused only partial reduction (30–40%) of such ET-1-induced responses. Consistent with this, inhibition of matrix metalloproteinases (MMPs) with GM6001 reduced ERK1/2 activation by ET-1, consistent with partial involvement of the MMP-dependent EGF-R activation in this cascade. Activation of ERK/RSK-1/CREB by both ET-1 and EGF was abolished by inhibition of Src, indicating its central role in ET-1 signaling in BAG cells. Moreover, the signaling characteristics of ET-1 in cultured BAG cells closely resembled those observed in clonal adrenocortical H295R cells. The ET-1-induced proliferation of BAG and H295 R cells was much smaller than that induced by Ang II or FGF. These data demonstrate that ET-1 causes ERK/RSK-1/CREB phosphorylation predominantly through activation of G_i and Src, with a minor contribution from MMP-dependent EGF-R transactivation.     

Implication of phospholipase D2 in oxidant-induced phosphoinositide 3-kinase signaling via Pyk2 activation in PC12 cells

The role of phospholipase D (PLD) activation in hydrogen peroxide (H(2)O(2))-induced signal transduction and cellular responses is not completely understood. Here we present evidence that Ca(2+)-dependent tyrosine kinase, Pyk2, requires PLD activation to mediate survival pathways in rat pheochromocytoma PC12 cells under oxidative stress. The H(2)O(2)-induced phosphorylation of two Pyk2 sites (Tyr(580), and Tyr(881)) was suppressed by 1-butanol, an inhibitor of transphosphatidylation by PLD, and also by transfection of catalytically negative mouse PLD2K758R (PLD2KR). Furthermore, we found that PLD2 was associated with Pyk2 and Src, and that activation of PLD2 was required for H(2)O(2)-enhanced association of Src with Pyk2 leading to full activation of Pyk2. H(2)O(2)-induced phosphorylation of Akt and p70S6K was dependent on phosphatidylinositol 3-kinase (PI3K) activity and was abolished by 1-butanol but not t-butanol. Furthermore, the PI3K/Akt activation in response to H(2)O(2) was reduced by transfection of either PLD2KR or the dominant negative Pyk2DN. This study is the first demonstration that PLD2 activation is implicated in Src-dependent phosphorylation of Pyk2 (Tyr(580) and Tyr(881)) by promoting the complex formation between Pyk2 and activated Src in PC12 cells exposed to H(2)O(2), thereby resulting in activation of the survival signaling pathway PI3K/Akt/p70S6K.     

Reactive oxygen species mediate Endothelin-1-induced activation of ERK1/2, PKB, and Pyk2 signaling, as well as protein synthesis, in vascular smooth muscle cells

Reactive oxygen species (ROS) have been shown to mediate the effects of several growth factors and vasoactive peptides, such as epidermal growth factor, platelet-derived growth factor, and angiotensin II (AII). Endothelin-1 (ET-1) is a vasoactive peptide which also exhibits mitogenic activity in vascular smooth muscle cells (VSMCs), and is believed to contribute to the pathogenesis of vascular abnormalities such as atherosclerosis, hypertension, and restenosis after angioplasty. However, a possible role for ROS generation in mediating the ET-1 response on extracellular signal-regulated kinases 1 and 2 (ERK1/2), protein kinase B (PKB), and protein tyrosine kinase 2 (Pyk2), key components of the growth-promoting and proliferative signaling pathways, has not been examined in detail. Our aim was to investigate the involvement of ROS in ET-1-mediated activation of ERK1/2, PKB, and Pyk2 in A-10 VSMCs. ET-1 stimulated ERK1/2, PKB, and Pyk2 phosphorylation in a dose- and time-dependent manner. Pretreatment of A-10 VSMCs with diphenyleneiodonium (DPI), an inhibitor of reduced nicotinamide adenine dinucleotide phosphate oxidase, attenuated ET-1-enhanced ERK1/2, PKB, and Pyk2 phosphorylation. In addition, in parallel with an inhibitory effect on the above signaling components, DPI also blocked ET-1-induced protein synthesis. ET-1 was also found to increase ROS production, which was suppressed by DPI treatment. N-Acetylcysteine, a ROS scavenger, exhibited a response similar to that of DPI and inhibited ET-1-stimulated ERK1/2, PKB, and Pyk2 phosphorylation. These results demonstrate that ROS are critical mediators of ET-1-induced signaling events linked to growth-promoting proliferative and hypertrophic pathways in VSMCs.     

The role of mitogen-activated protein kinase activation within focal adhesions in chemotaxis toward FGF-2 by murine brain capillary endothelial cells

Fibroblast growth factors (FGFs) regulate a number of angiogenic cellular responses such as migration of endothelial cells. To examine the role of mitogen-activated protein kinase (MAPK) in endothelial cell migration, chemotaxis toward FGF-2 was determined in murine brain capillary endothelial cells, denoted IBE cells. PD98059, a specific inhibitor for MAPK/Erk kinase, inhibited FGF-2-induced chemotaxis of IBE cells. It has been reported that c-Src tyrosine kinase phosphorylates focal adhesion kinase at tyrosine 925 within focal adhesions, which in turn creates the binding site for Grb2, leading to MAPK activation. The Src family tyrosine kinase inhibitor, PP1, as well as overexpression of kinase-inactive c-Src, attenuated chemotaxis toward FGF-2. To investigate the signaling events involved in FGF-2-induced chemotaxis, MAPK activation was monitored in IBE cells by indirect immunofluorescence staining. Activated MAPK was initially observed in the cytoplasm and gradually moved into nuclei. A fraction of MAPK was activated by FGF-2 within focal adhesions, where FGF receptor-1 and Src family kinases were also colocalized. MAPK activation within focal adhesions was remarkably decreased in kinase-inactive c-Src-expressing IBE cells. Our data suggest that activation of MAPK by FGF-2 within focal adhesions may depend on c-Src activity and is crucial for FGF-2-induced migration of IBE cells.