Transduction cascades initiated by fibroblast growth factor 1 on Xenopus oocytes expressing MDA-MB-231 mRNAs. Role of Grb2, phosphatidylinositol 3-kinase, Src tyrosine kinase, and phospholipase Cgamma

Xenopus oocytes expressing fibroblast growth factor receptors (FGFRs) from the hormone-independent breast cancer cells, MDA-MB-231, are used as a biological system to analyze the signalling cascades initiated by FGF1. FGF1 induces ERK2 phosphorylation and G2/M transition. These events are dependent on the Shc/Grb2/Ras pathway, on Src and PI3Kinase (PI3K), as shown by the use of SH2 domains or dominant negative proteins, and on PLC gamma and calcium as demonstrated by a PLC gamma inhibitory peptide and BAPTA-AM. FGF1 mobilizes Ins(1,4,5)P3-sensitive calcium stores, as recorded through the inhibition by caffeine of a chloride calcium-dependent current in expressing oocytes. This study shows that the transduction cascades induced by FGF1 on FGFRs from MDA-MB-231 cells represent the sum of Ras, Src, PI3K, and PLC gamma pathways. It emphasizes the mitogenic effect of the PLC gamma-calcium cascade.     

Fibroblast growth factors 1 and 2 differently activate MAP kinase in Xenopus oocytes expressing fibroblast growth factor receptors 1 and 4

The mitogen-activated protein kinase (MAP kinase) signalling cascade activated by fibroblast growth factors (FGF1 and FGF2) was analysed in a model system, Xenopus oocytes, expressing fibroblast growth factor receptors (FGFR1 and FGFR4). Stimulation of FGFR1 by FGF1 or FGF2 and FGFR4 by FGF1 induced a sustained phosphorylation of extracellular signal-regulated protein kinase 2 (ERK2) and meiosis reinitiation. In contrast, FGFR4 stimulation by FGF2 induced an early transient activation of ERK2 and no meiosis reinitiation. FGFR4 transduction cascades were differently activated by FGF1 and FGF2. Early phosphorylation of ERK2 was blocked by the dominant negative form of growth factor-bound protein 2 (Grb2) and Ras, for FGF1-FGFR4 and FGF2-FGFR4. The phosphatidylinositol 3-kinase (PI3 kinase) inhibitors wortmannin and LY294002 only prevented the early ERK2 phosphorylation triggered by FGF2-FGFR4 but not by FGF1-FGFR4. ERK2 phosphorylation triggered by FGFR4 depended on the Grb2/Ras pathway and also involved PI3 kinase in a time-dependent manner.     

ERK2 is required for FGF1-induced JNK1 phosphorylation in Xenopus oocyte expressing FGF receptor 1

A possible connection between the ERK2 and JNK1 MAP kinases transduction cascades was investigated in Xenopus oocytes expressing FGFR1 stimulated by FGF1. Injection of various inhibitors for the Shc/Grb2/Ras/Mos/MEK/ERK2 cascade blocked FGF1-induced germinal vesicle breakdown (GVBD), as well as ERK2 and JNK1 phosphorylation. JNK1 was found to be activated downstream of ERK2, since injection of an active ERK2 triggered JNK1 phosphorylation and inhibition of ERK2 either by a MEK inhibitor or the MKP3 phosphatase blocked JNK1 phosphorylation. These results demonstrated that in FGFR1 signalling JNK1 phosphorylation depends on ERK2.     

Glu-256 is a main structural determinant for oligomerisation of human arginase I

The role of RasGAP was investigated in the model system of Xenopus oocytes expressing fibroblast growth factor receptor 1 (FGFR1) stimulated by fibroblast growth factor 1 (FGF1). The injection of the SH2-SH3-SH2 domains of RasGAP suppressed Ras activity, extracellular signal-regulated protein kinase 2 (ERK2) phosphorylation and Mos synthesis. The SH2 domain of Src, and PP2, an inhibitor of Src, also abolished Ras activity, ERK2 phosphorylation and Mos synthesis. In addition, Src activity was blocked by the SH2-SH3-SH2 domains of RasGAP. Immunoprecipitation of a chimera composed of the extracellular domain of the platelet-derived growth factor (PDGF) receptor and the intracellular domain of FGFR1 stimulated by PDGF-BB demonstrates the recruitment of phosphorylated RasGAP. This study shows that the transduction cascade induced by the FGFR1-FGF1 interaction in Xenopus oocytes involves RasGAP as a co-activator of Src to stimulate the Ras/mitogen-activated protein kinase cascade and Mos synthesis. It emphasises a new positive regulatory role for RasGAP in FGFR transduction.     

Association of fibroblast growth factor receptor 1 with the adaptor protein Grb14. Characterization of a new receptor binding partner

Using the cytoplasmic domain of fibroblast growth factor receptor 1 (FGFR1) as bait in a yeast two-hybrid screen, Grb14 was identified as a FGFR1 binding partner. A kinase-inactive mutant of FGFR1 failed to interact with Grb14, indicating that activation of FGFR1 is necessary for binding. Deletion of the C-tail or mutation of both C-tail tyrosine residues of FGFR1 to phenylalanine abolished binding, and deletion of the juxtamembrane domain of the receptor reduced binding, suggesting that Grb14 binds to FGFR1 at multiple sites. Co-immunoprecipitation and in vitro binding assays demonstrated that binding of Grb14 to FGFR1 in mammalian cells was dependent on receptor activation by fibroblast growth factor-2 (FGF-2). Deletion of the Src homology 2 (SH2) domain of Grb14 reduced but did not block binding to FGFR1 and eliminated dependence on receptor activation. The SH2 domain alone bound both FGFR1 and platelet-derived growth factor receptor, whereas full-length Grb14 bound only FGFR1, suggesting that regions upstream of the SH2 domain confer specificity for FGFR1. Grb14 was phosphorylated on serine and threonine residues in unstimulated cells, and treatment with FGF-2 enhanced this phosphorylation. Expression of exogenous Grb14 inhibited FGF-2-induced cell proliferation, whereas a point-mutated form of Grb14 incapable of binding to FGFR1 enhanced FGF-2-induced mitogenesis. These data demonstrate an interaction between activated FGFR1 and Grb14 and suggest a role for Grb14 in FGF signaling.     

Tyrosine phosphorylation of Grb2 by Bcr/Abl and epidermal growth factor receptor: a novel regulatory mechanism for tyrosine kinase signaling.

Growth factor receptor-binding protein-2 (Grb2) plays a key role in signal transduction initiated by Bcr/Abl oncoproteins and growth factors, functioning as an adaptor protein through its Src homology 2 and 3 (SH2 and SH3) domains. We found that Grb2 was tyrosine-phosphorylated in cells expressing BCR/ABL and in A431 cells stimulated with epidermal growth factor (EGF). Phosphorylation of Grb2 by Bcr/Abl or EGF receptor reduced its SH3-dependent binding to Sos in vivo, but not its SH2-dependent binding to Bcr/Abl. Tyr209 within the C-terminal SH3 domain of Grb2 was identified as one of the tyrosine phosphorylation sites, and phosphorylation of Tyr209 abolished the binding of the SH3 domain to a proline-rich Sos peptide in vitro. In vivo expression of a Grb2 mutant where Tyr209 was changed to phenylalanine enhanced BCR/ABL-induced ERK activation and fibroblast transformation, and potentiated and prolonged Grb2-mediated activation of Ras, mitogen-activated protein kinase and c-Jun N-terminal kinase in response to EGF stimulation. These results suggest that tyrosine phosphorylation of Grb2 is a novel mechanism of down-regulation of tyrosine kinase signaling.     

A Src homology 3-binding sequence on the C terminus of Sprouty2 is necessary for inhibition of the Ras/ERK pathway downstream of fibroblast growth factor receptor stimulation

Because the Sprouty (Spry) proteins were shown to be inhibitors of the mainstream Ras/ERK pathway, there has been considerable interest in ascertaining their mechanism of action especially since a possible role as tumor suppressors for these inhibitory proteins has been suggested. We compared the ability of the mammalian Spry isoforms to inhibit the Ras/ERK pathway in the context of fibroblast growth factor receptor (FGFR) signaling. Spry2 is considerably more inhibitory than Spry1 or Spry4, and this correlates with the binding to Grb2 via a C-terminal proline-rich sequence that is found exclusively on Spry2. This PXXPXR motif binds directly to the N-terminal Src homology domain 3 of Grb2, and when added onto the C terminus of Spry4 the resultant chimera inhibits the Ras/ERK pathway. The ability to inhibit neurite outgrowth in PC-12 cells correlates with the propensity of Spry isoforms and engineered constructs to inhibit the phosphorylation of ERK1/2. The PXXPXR motif is cryptic in unstimulated cells, and it is postulated that Spry2 undergoes a conformational change following FGFR stimulation, enabling the subsequent interaction with Grb2. We present evidence that Spry2 can compete with the RasGEF (guanine nucleotide exchange factor) SOS1 for binding to Grb2, resulting in the inhibition of phosphorylation of ERK1/2.     

A truncated form of fibroblast growth factor receptor 1 inhibits signal transduction by multiple types of fibroblast growth factor receptor.

A truncated form of the type 1 fibroblast growth factor receptor (FGFR1) lacking most of its cytoplasmic domain was tested for its ability to inhibit signal transduction by each of three different wild-type FGFRs (FGFR1, 2, and 3). When the truncated FGFR1 was expressed in Xenopus oocytes in excess of each wild-type FGFR, mobilization of intracellular calcium mediated by the wild-type FGFRs was completely blocked. The truncated FGFR did not inhibit signal transduction by the co-expressed platelet-derived growth factor beta-receptor. A form of truncated FGFR1 which lacked the first immunoglobulin-like domain also inhibited signal transduction by wild-type FGFRs. Truncated FGFR formed complexes with wild-type FGFR in the presence of basic FGF in intact cells. These observations were consistent with the hypothesis that the truncated FGFR interacted with wild-type FGFRs to form nonfunctional heterodimers, thus eliminating the signaling by the wild-type FGFRs. The observation that signaling by multiple types of FGFR can be blocked by a single type of truncated FGFR suggests that the different types of FGFR can interact with each other in ligand-mediated complexes. These findings provide a molecular basis for inhibiting the actions of FGFs in vivo.     

Caveolin-1 orchestrates fibroblast growth factor 2 signaling control of angiogenesis in placental artery endothelial cell caveolae

Fibroblast growth factor (FGF) receptor 1 (FGFR1) protein was expressed as the long and short as well as some truncated forms in ovine fetoplacental artery ex vivo and in vitro. Upon FGF2 stimulation, both the long and short FGFR1s were tyrosine phosphorylated and the PI3K/AKT1 and ERK1/2 pathways were activated in a concentration- and time- dependent manner in ovine fetoplacental artery endothelial (oFPAE) cells. Blockade of the PI3K/AKT1 pathway attenuated FGF2-stimulated cell proliferation and migration as well as tube formation; blockade of the ERK1/2 pathway abolished FGF2-stimulated tube formation and partially inhibited cell proliferation and did not alter cell migration. Both AKT1 and ERK1/2 were co-fractionated with caveolin-1 and activated by FGF2 in the caveolae. Disruption of caveolae by methyl-β-cyclodextrin inhibited FGF2 activation of AKT1 and ERK1/2. FGFR1 was found in the caveolae where it physically binds to caveolin-1. FGF2 stimulated dissociation of FGFR1 from caveolin-1. Downregulation of caveolin-1 significantly attenuated the FGF2-induced activation of AKT1 and ERK1/2 and inhibited FGF2-induced cell proliferation, migration and tube formation in oFPAE cells. Pretreatment with a caveolin-1 scaffolding domain peptide to mimic caveolin-1 overexpression also inhibited these FGF2-induced angiogenic responses. These data demonstrate that caveolae function as a platform for regulating FGF2-induced angiogenesis through spatiotemporally compartmentalizing FGFR1 and the AKT1 and ERK1/2 signaling modules; the major caveolar structural protein caveolin-1 interacts with FGFR1 and paradoxically regulates FGF2-induced activation of PI3K/AKT1 and ERK1/2 pathways that coordinately regulate placental angiogenesis.     

Inhibition of FGF receptor signalling in Xenopus oocytes: differential effect of Grb7, Grb10 and Grb14

The role of Grb7 adapters, Grb7, Grb10, and Grb14, was investigated in Xenopus oocytes expressing fibroblast growth factor receptors (FGFR). FGF-induced maturation of FGFR-expressing oocytes was blocked by previous injection of Grb7 or Grb14, but not Grb10. This effect correlated with Grb7/14 binding to the receptor, and inhibition of the Ras-dependent pathway. Interestingly, the phosphorylated insulin receptor interacting region (PIR) and Src 2 homology domains (SH2) of Grb7 and Grb14 were differently implicated in the inhibition of FGFR signalling. This study provided further evidence for specificity of the biological action of the Grb7 adapters on receptor tyrosine kinase signalling.     

Sef inhibits fibroblast growth factor signaling by inhibiting FGFR1 tyrosine phosphorylation and subsequent ERK activation

Signaling through fibroblast growth factor receptors (FGFRs) is essential for many cellular processes including proliferation and migration as well as differentiation events such as angiogenesis, osteogenesis, and chondrogenesis. Recently, genetic screens in Drosophila and gene expression screens in zebrafish have resulted in the identification of several feedback inhibitors of FGF signaling. One of these, Sef (similar expression to fgf genes), encodes a transmembrane protein that belongs to the FGF synexpression group. Here we show that like zebrafish Sef (zSef), mouse Sef (mSef) interacts with FGFR1 and that the cytoplasmic domain of mSef mediates this interaction. Overexpression of mSef in NIH3T3 cells results in a decrease in FGF-induced cell proliferation associated with a decrease in Tyr phosphorylation of FGFR1 and FRS2. As a consequence, there is a reduction in the phosphorylation of Raf-1 at Ser(338), MEK1/2 at Ser(217) and Ser(221), and ERK1/2 at Thr(202) and Tyr(204). Furthermore, mSef inhibits ERK activation mediated by a constitutively activated FGFR1 but not by a constitutively active Ras and decreases FGF but not PDGF-mediated activation of Akt. These results indicate that Sef exerts its inhibitory effects at the level of FGFR and upstream of Ras providing an additional level of negative regulation of FGF signaling.     

Interaction of fibroblast growth factor receptor 3 and the adapter protein SH2-B. A role in STAT5 activation

Fibroblast growth factor receptor 3 (FGFR3) influences a diverse array of biological processes, including cell growth, differentiation, and migration. Activating mutations in FGFR3 are associated with multiple myeloma, cervical carcinoma, and bladder cancer. To identify proteins that interact with FGFR3 and which may mediate FGFR3-dependent signaling, a yeast two-hybrid screen was employed using the cytoplasmic kinase domain of FGFR3 as bait. We identified the adapter protein SH2-B as an FGFR3-interacting protein. Coimmunoprecipitation experiments demonstrate binding of the SH2-B beta isoform to FGFR3 in 293T cells. Tyrosine phosphorylation of SH2-B beta was observed when coexpressed with activated FGFR3 mutants such as the weakly activated mutant N540K or the strongly activated mutant K650E, both associated with human developmental syndromes. The extent of tyrosine phosphorylation of SH2-B beta correlates with receptor activation, suggesting that FGFR3 activation mediates tyrosine phosphorylation of SH2-B beta. Furthermore, two tyrosine phosphorylation sites of FGFR3, Tyr-724 and Tyr-760, are required for optimal binding of the Src homology-2 (SH2) domain of SH2-B beta. We also demonstrate the phosphorylation and nuclear translocation of Stat5 by activated FGFR3, which increases in response to overexpression of SH2-B beta. Taken together, our results identify SH2-B beta as a novel FGFR3 binding partner that mediates signal transduction.     

Fibroblast Growth Factor 2 Causes G2/M Cell Cycle Arrest in Ras-Driven Tumor Cells through a Src-Dependent Pathway

We recently reported that paracrine Fibroblast Growth Factor 2 (FGF2) triggers senescence in Ras-driven Y1 and 3T3^Ras mouse malignant cell lines. Here, we show that although FGF2 activates mitogenic pathways in these Ras-dependent malignant cells, it can block cell proliferation and cause a G2/M arrest. These cytostatic effects of FGF2 are inhibited by PD173074, an FGF receptor (FGFR) inhibitor. To determine which downstream pathways are induced by FGF2, we tested specific inhibitors targeting mitogen-activated protein kinase (MEK), phosphatidylinositol 3 kinase (PI3K) and protein kinase C (PKC). We show that these classical mitogenic pathways do not mediate the cytostatic activity of FGF2. On the other hand, the inhibition of Src family kinases rescued Ras-dependent malignant cells from the G2/M irreversible arrest induced by FGF2. Taken together, these data indicate a growth factor-sensitive point in G2/M that likely involves FGFR/Ras/Src pathway activation in a MEK, PI3K and PKC independent manner.     

FRS2 proteins recruit intracellular signaling pathways by binding to diverse targets on fibroblast growth factor and nerve growth factor receptors

The docking protein FRS2 was implicated in the transmission of extracellular signals from the fibroblast growth factor (FGF) or nerve growth factor (NGF) receptors to the Ras/mitogen-activated protein kinase signaling cascade. The two members of the FRS2 family, FRS2alpha and FRS2beta, are structurally very similar. Each is composed of an N-terminal myristylation signal, a phosphotyrosine-binding (PTB) domain, and a C-terminal tail containing multiple binding sites for the SH2 domains of the adapter protein Grb2 and the protein tyrosine phosphatase Shp2. Here we show that the PTB domains of both the alpha and beta isoforms of FRS2 bind directly to the FGF or NGF receptors. The PTB domains of the FRS2 proteins bind to a highly conserved sequence in the juxtamembrane region of FGFR1. While FGFR1 interacts with FRS2 constitutively, independent of ligand stimulation and tyrosine phosphorylation, NGF receptor (TrkA) binding to FRS2 is strongly dependent on receptor activation. Complex formation with TrkA is dependent on phosphorylation of Y490, a canonical PTB domain binding site that also functions as a binding site for Shc (NPXpY). Using deletion and alanine scanning mutagenesis as well as peptide competition assays, we demonstrate that the PTB domains of the FRS2 proteins specifically recognize two different primary structures in two different receptors in a phosphorylation-dependent or -independent manner. In addition, NGF-induced tyrosine phosphorylation of FRS2alpha is diminished in cells that overexpress a kinase-inactive mutant of FGFR1. This experiment suggests that FGFR1 may regulate signaling via NGF receptors by sequestering a common key element which both receptors utilize for transmitting their signals. The multiple interactions mediated by FRS2 appear to play an important role in target selection and in defining the specificity of several families of receptor tyrosine kinases.     

RasGAP Shields Akt from Deactivating Phosphatases in Fibroblast Growth Factor Signaling but Loses This Ability Once Cleaved by Caspase-3

Fibroblast growth factor receptors (FGFRs) are involved in proliferative and differentiation physiological responses. Deregulation of FGFR-mediated signaling involving the Ras/PI3K/Akt and the Ras/Raf/ERK MAPK pathways is causally involved in the development of several cancers. The caspase-3/p120 RasGAP module is a stress sensor switch. Under mild stress conditions, RasGAP is cleaved by caspase-3 at position 455. The resulting N-terminal fragment, called fragment N, stimulates anti-death signaling. When caspase-3 activity further increases, fragment N is cleaved at position 157. This generates a fragment, called N2, that no longer protects cells. Here, we investigated in Xenopus oocytes the impact of RasGAP and its fragments on FGF1-mediated signaling during G₂/M cell cycle transition. RasGAP used its N-terminal Src homology 2 domain to bind FGFR once stimulated by FGF1, and this was necessary for the recruitment of Akt to the FGFR complex. Fragment N, which did not associate with the FGFR complex, favored FGF1-induced ERK stimulation, leading to accelerated G₂/M transition. In contrast, fragment N2 bound the FGFR, and this inhibited mTORC2-dependent Akt Ser-473 phosphorylation and ERK2 phosphorylation but not phosphorylation of Akt on Thr-308. This also blocked cell cycle progression. Inhibition of Akt Ser-473 phosphorylation and entry into G₂/M was relieved by PHLPP phosphatase inhibition. Hence, full-length RasGAP favors Akt activity by shielding it from deactivating phosphatases. This shielding was abrogated by fragment N2. These results highlight the role played by RasGAP in FGFR signaling and how graded stress intensities, by generating different RasGAP fragments, can positively or negatively impact this signaling.     

ERK negatively regulates the epidermal growth factor-mediated interaction of Gab1 and the phosphatidylinositol 3-kinase

We have examined the ability of epidermal growth factor (EGF)-stimulated ERK activation to regulate Grb2-associated binder-1 (Gab1)/phosphatidylinositol 3-kinase (PI3K) interactions. Inhibiting ERK activation with the MEK inhibitor U0126 increased the EGF-stimulated association of Gab1 with either full-length glutathione S-transferase-p85 or the p85 C-terminal Src homology 2 (SH2) domain, a result reproduced by co-immunoprecipitation of the native proteins from intact cells. This increased association of Gab1 and the PI3K correlates with an increase in PI3K activity and greater phosphorylation of Akt. This result is in direct contrast to what we have previously reported following HGF stimulation where MEK inhibition decreased the HGF-stimulated association of Gab1 and p85. In support of this divergent effect of ERK on Gab1/PI3K association following HGF and EGF stimulation, U0126 decreased the HGF-stimulated association of p85 and the Gab1 c-Met binding domain but did not alter the EGF-stimulated association of p85 and the c-Met binding domain. An examination of the mechanism of this effect revealed that the treatment of cells with EGF + U0126 increased the tyrosine phosphorylation of Gab1 as well as its association with another SH2-containing protein, SHP2. Furthermore, overexpression of a catalytically inactive form of SHP2 or pretreatment with pervanadate markedly increased EGF-stimulated Gab1 tyrosine phosphorylation. These experiments demonstrate that EGF and HGF-mediated ERK activation result in divergent effects on Gab1/PI3K signaling. HGF-stimulated ERK activation increases the Gab1/PI3K association, whereas EGF-stimulated ERK activation results in a decrease in the tyrosine phosphorylation of Gab1 and a decreased association with the PI3K. SHP2 is shown to associate with and dephosphorylate Gab1, suggesting that EGF-stimulated ERK might act through the regulation of SHP2.     

Differential tyrosine phosphorylation of fibroblast growth factor (FGF) receptor-1 and receptor proximal signal transduction in response to FGF-2 and heparin

The sulfated regions in heparan sulfate and heparin are known to affect fibroblast growth factor (FGF) function. We have studied the mechanism whereby heparin directs FGF-2-induced FGF receptor-1 (FGFR-1) signal transduction. FGF-2 alone stimulated maximal phosphorylation of Src homology domain 2 tyrosine phosphatase (SHP-2) and the adaptor molecule Crk, in heparan sulfate-deficient Chinese hamster ovary (CHO) 677 cells expressing FGFR-1. In contrast, for phospholipase Cgamma(1) (PLCgamma(1)) and the adaptor molecule Shb to be maximally tyrosine-phosphorylated, cells had to be stimulated with both FGF-2 and heparin (100 ng/ml). Tyrosine residues 463 in the juxtamembrane domain and 766 in the C-terminal tail in FGFR-1 are known to bind Crk and PLCgamma(1), respectively. Analysis of tryptic phosphopeptide maps of FGFR-1 from cells stimulated with FGF-2 alone and FGF-2 together with heparin showed that FGF-2 alone stimulated a several-fold increase in tyrosine 463 in the juxtamembrane domain. In contrast, heparin had to be included in order for tyrosine 766 to be phosphorylated to the same fold level. Our data imply that tyrosine 463 is phosphorylated and able to transduce signals in response to FGF-2 treatment alone; furthermore, we suggest that FGFR-1 dimerization/kinase activation is stabilized by heparin.     

Regulation of ephexin1, a guanine nucleotide exchange factor of Rho family GTPases, by fibroblast growth factor receptor-mediated tyrosine phosphorylation

Fibroblast growth factor (FGF) signal is implicated in not only cell proliferation, but cell migration and morphological changes. Several different Rho family GTPases downstream of the Ras/ERK pathway are postulated to mediate the latter functions. However, none have been recognized to be directly coupled to FGF receptors (FGFRs). We have previously reported that EphA4 and FGFRs hetero-oligomerize through their cytoplasmic domains, trans-activate each other, and transduce a signal for cell proliferation through a docking protein, FRS2alpha (Yokote, H., Fujita, K., Jing, X., Sawada, T., Liang, S., Yao, L., Yan, X., Zhang, Y., Schlessinger, J., and Sakaguchi, K. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 18866-18871). Here, we have found that ephexin1, a guanine nucleotide exchange factor for Rho family GTPases, constitutes another downstream component of the receptor complex. Ephexin1 directly binds to the kinase domain of FGFR mainly through its DH and PH domains. The binding appears to become weaker and limited to the DH domain when FGFRs become activated. FGFR-mediated phosphorylation of ephexin1 enhances the guanine nucleotide exchange activity toward RhoA without affecting the activity to Rac1 or Cdc42. The FGFR-mediated tyrosine phosphorylation includes, but is not limited to, the residue (Tyr-87) phosphorylated by Src family kinase, which is known to be activated following EphA4 activation. The Tyr-to-Asp mutations that mimic the tyrosine phosphorylation in some of the putative FGFR-mediated phosphorylation sites increase the nucleotide exchange activity for RhoA without changing the activity for Rac1 or Cdc42. From these results, we conclude that ephexin1 is located immediately downstream of the EphA4-FGFR complex and the function is altered by the FGFR-mediated tyrosine phosphorylation at multiple sites.     

The scaffolding adapter Gab1 mediates vascular endothelial growth factor signaling and is required for endothelial cell migration and capillary formation

Vascular endothelial growth factor (VEGF) is involved in the promotion of endothelial cell proliferation, migration, and capillary formation. These activities are mainly mediated by the VEGFR2 receptor tyrosine kinase that upon stimulation, promotes the activation of numerous proteins including phospholipase Cgamma (PLCgamma), phosphatidylinositol 3-kinase (PI3K), Akt, Src, and ERK1/2. However, the VEGFR2-proximal signaling events leading to the activation of these targets remain ill defined. We have identified the Gab1 adapter as a novel tyrosine-phosphorylated protein in VEGF-stimulated cells. In bovine aortic endothelial cells, Gab1 associates with VEGFR2, Grb2, PI3K, SHP2, Shc, and PLCgamma, and its overexpression enhances VEGF-dependent cell migration. Importantly, silencing of Gab1 using small interfering RNAs leads to the impaired activation of PLCgamma, ERK1/2, Src, and Akt; blocks VEGF-induced endothelial cell migration; and perturbs actin reorganization and capillary formation. In addition, co-expression of VEGFR2 with Gab1 mutants unable to bind SHP2 or PI3K in human embryonic kidney 293 cells and bovine aortic endothelial cells mimics the defects observed in Gab1-depleted cells. Our work thus identifies Gab1 as a novel critical regulatory component of endothelial cell migration and capillary formation and reveals its key role in the activation of VEGF-evoked signaling pathways required for angiogenesis.     

Angiogenic oligosaccharides of hyaluronan induce multiple signaling pathways affecting vascular endothelial cell mitogenic and wound healing responses

Hyaluronan (HA) is a large nonsulfated glycosaminoglycan and an important regulator of angiogenesis, in particular, the growth and migration of vascular endothelial cells. We have identified some of the key intermediates responsible for induction of mitogenesis and wound recovery. Treatment of bovine aortic endothelial cells with oligosaccharides of hyaluronan (o-HA) resulted in rapid tyrosine phosphorylation and plasma membrane translocation of phospholipase Cgamma1 (PLCgamma1). Cytoplasmic loading with inhibitory antibodies to PLCgamma1, Gbeta, and Galpha(i/o/t/z) inhibited activation of extracellular-regulated kinase 1/2 (ERK1/2). Treatment with the Galpha(i/o) inhibitor, pertussis toxin, reduced o-HA-induced PLCgamma1 tyrosine phosphorylation, protein kinase C (PKC) alpha and beta1/2 membrane translocation, ERK1/2 activation, mitogenesis, and wound recovery, suggesting a mechanism for o-HA-induced angiogenesis through G-proteins, PLCgamma1, and PKC. In particular, we demonstrated a possible role for PKCalpha in mitogenesis and PKCbeta1/2 in wound recovery. Using antisense oligonucleotides and the Ras farnesylation inhibitor FTI-277, we showed that o-HA-induced bovine aortic endothelial cell proliferation, wound recovery, and ERK1/2 activation were also partially dependent on Ras activation, and that o-HA-stimulated tyrosine phosphorylation of the adapter protein Shc, as well as its association with Sos1. Binding of Src to Shc was required for its activation and for Ras-dependent activation of ERK1/2, cell proliferation, and wound recovery. Neither Src nor Ras activation was inhibited by pertussis toxin, suggesting that their activation was independent of heterotrimeric G-proteins. However, the specific Src kinase inhibitor PP2 inhibited Gbeta subunit co-precipitation with PLCgamma1, suggesting a possible role for Src in activation of PLCgamma1 and interaction between two distinct o-HA-induced signaling pathways.