Sphingosine 1-phosphate induces platelet/endothelial cell adhesion molecule-1 phosphorylation in human endothelial cells through cSrc and Fyn

Sphingosine 1-phosphate (S1P) is a multifunctional phospholipid which acts through a specific family of G protein-coupled receptors. Platelet/endothelial cell adhesion molecule-1 (PECAM-1) form trans-homophilic binding at lateral cell border. Upon stimulation, its cytoplasmic tyrosine residues could be phosphorylated and interact with various downstream signaling molecules. In this study, we demonstrated that S1P induced PECAM-1 tyrosine phosphorylation in human umbilical cord vein cells (HUVECs). By pharmacological inhibitors, it was suggested that G(i) and Src family kinases were involved in PECAM-1 phosphorylation. Moreover, cSrc and Fyn siRNA significantly suppressed S1P-induced PECAM-1 phosphorylation. These results suggested that S1P-induced PECAM-1 phosphorylation through G(i) and subsequent cSrc and Fyn. Our findings provide further understanding of S1P and PECAM-1 signaling as well as their functions in endothelial cells.     

Intrinsic tyrosine kinase activity is required for vascular endothelial growth factor receptor 2 ubiquitination, sorting and degradation in endothelial cells

The human endothelial vascular endothelial growth factor receptor 2 (VEGFR2/kinase domain region, KDR/fetal liver kinase-1, Flk-1) tyrosine kinase receptor is essential for VEGF-mediated physiological responses including endothelial cell proliferation, migration and survival. How VEGFR2 kinase activation and trafficking are co-coordinated in response to VEGF-A is not known. Here, we elucidate a mechanism for endothelial VEGFR2 response to VEGF-A dependent on constitutive endocytosis co-ordinated with ligand-activated ubiquitination and proteolysis. The selective VEGFR kinase inhibitor, SU5416, blocked the endosomal sorting required for VEGFR2 trafficking and degradation. Inhibition of VEGFR2 tyrosine kinase activity did not block plasma membrane internalization but led to endosomal accumulation. Lysosomal protease activity was required for ligand-stimulated VEGFR2 degradation. Activated VEGFR2 codistributed with the endosomal hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs)/signal-transducing adaptor molecule (STAM) complex in a ligand and time-dependent manner, implying a role for this factor in sorting of ubiquitinated VEGFR2. Increased tyrosine phosphorylation of the Hrs subunit in response to VEGF-A links VEGFR2 activation and Hrs/STAM function. In contrast, VEGFR2 in quiescent cells was present on both the endothelial plasma membrane and early endosomes, suggesting constitutive recycling between these two compartments. This pathway was clathrin-linked and dependent on the AP2 adaptor complex as the A23 tyrphostin inhibited VEGFR2 trafficking. We propose a mechanism whereby the transition of endothelial VEGFR2 from a constitutive recycling itinerary to a degradative pathway explains ligand-activated receptor degradation in endothelial cells. This study outlines a mechanism to control the VEGF-A-mediated response within the vascular system.     

Sphingosine-1-phosphate signaling regulates lamellipodia localization of cortactin complexes in endothelial cells

Sphingosine-1-phosphate (S1P), a serum-borne lipid mediator, was demonstrated to be a potent chemoattractant of endothelial cells. It was recently shown that the colocalization of cortactin and actin related protein 2/3 (Arp2/3) in the lamellipodia is critical to S1P-induced endothelial chemotaxis. In this report, we describe that S1P-stimulated cortactin translocation to the cell periphery to form lamellipodia is specifically mediated by the endothelial S1P1 G-protein coupled receptor, and is regulated by G_i-mediated Akt-dependent S1P1 receptor phosphorylation and Cdc42/Rac activation pathways. In contrast to Src-dependent fibroblast growth factor-induced cortactin translocation, tyrosine phosphorylation cascades are not required for S1P-mediated lamellipodia formation and chemotaxis. Furthermore, we also demonstrate that S1P signaling, via the G_i/Akt/S1P1 phosphorylation/Rac pathway, regulates the cortactin–Arp2/3 complex formation, which ultimately results in membrane ruffling, formation of the lamellipodia and endothelial migration.J.F. Lee and H. Ozaki contributed equally to this work     

Flow shear stress stimulates Gab1 tyrosine phosphorylation to mediate protein kinase B and endothelial nitric-oxide synthase activation in endothelial cells

Fluid shear stress generated by blood flow modulates endothelial cell function via specific intracellular signaling events. We showed previously that flow activated the phosphatidylinositol 3-kinase (PI3K), Akt, and endothelial nitric-oxide synthase (eNOS) via Src kinase-dependent transactivation of vascular endothelial growth factor receptor 2 (VEGFR2). The scaffold protein Gab1 plays an important role in receptor tyrosine kinase-mediated signal transduction. We found here that laminar flow (shear stress = 12 dynes/cm2) rapidly stimulated Gab1 tyrosine phosphorylation in both bovine aortic endothelial cells and human umbilical vein endothelial cells, which correlated with activation of Akt and eNOS. Gab1 phosphorylation as well as activation of Akt and eNOS by flow was inhibited by the Src kinase inhibitor PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) and VEGFR2 kinase inhibitors SU1498 and VTI, suggesting that flow-mediated Gab1 phosphorylation is Src kinase-dependent and VEGFR2-dependent. Tyrosine phosphorylation of Gab1 by flow was functionally important, because flow stimulated the association of Gab1 with the PI3K subunit p85 in a time-dependent manner. Furthermore, transfection of a Gab1 mutant lacking p85 binding sites inhibited flow-induced activation of Akt and eNOS. Finally, knockdown of endogenous Gab1 by small interference RNA abrogated flow activation of Akt and eNOS. These data demonstrate a critical role of Gab1 in flow-stimulated PI3K/Akt/eNOS signal pathway in endothelial cells.     

Transactivation of vascular endothelial growth factor (VEGF) receptor Flk-1/KDR is involved in sphingosine 1-phosphate-stimulated phosphorylation of Akt and endothelial nitric-oxide synthase (eNOS)

Sphingosine 1-phosphate (S1P) and vascular endothelial growth factor (VEGF) elicit numerous biological responses including cell survival, growth, migration, and differentiation in endothelial cells mediated by the endothelial differentiation gene, a family of G-protein-coupled receptors, and fetal liver kinase-1/kinase-insert domain-containing receptor (Flk-1/KDR), one of VEGF receptors, respectively. Recently, it was reported that S1P or VEGF treatment of endothelial cells leads to phosphorylation at Ser-1179 in bovine endothelial nitric oxide synthase (eNOS), and this phosphorylation is critical for eNOS activation. S1P stimulation of eNOS phosphorylation was shown to involve G(i) protein, phosphoinositide 3-kinase, and Akt. VEGF also activates eNOS through Flk-1/KDR, phosphoinositide 3-kinase, and Akt, which suggested that S1P and VEGF may share upstream signaling mediators. We now report that S1P treatment of bovine aortic endothelial cells acutely increases the tyrosine phosphorylation of Flk-1/KDR, similar to VEGF treatment. S1P-mediated phosphorylation of Flk-1/KDR, Akt, and eNOS were all inhibited by VEGF receptor tyrosine kinase inhibitors and by antisense Flk-1/KDR oligonucleotides. Our study suggests that S1P activation of eNOS involves G(i), calcium, and Src family kinase-dependent transactivation of Flk-1/KDR. These data are the first to establish a critical role of Flk-1/KDR in S1P-stimulated eNOS phosphorylation and activation.     

Rac1 modulates sphingosine 1-phosphate-mediated activation of phosphoinositide 3-kinase/Akt signaling pathways in vascular endothelial cells

Sphingosine 1-phosphate (S1P) is a platelet-derived sphingolipid that activates G protein-coupled S1P receptors and initiates a broad range of responses in vascular endothelial cells. The small GTPase Rac1 is implicated in diverse S1P-modulated cellular responses in endothelial cells, yet the molecular mechanisms involved in S1P-mediated Rac1 activation are incompletely understood. We studied the pathways involved in S1P-mediated Rac1 activation in bovine aortic endothelial cells (BAEC) and found that S1P-induced Rac1 activation is impaired following chelation of G protein betagamma subunits by transfection of betaARKct. Treatment with the Src tyrosine kinase inhibitor PP2 completely attenuated S1P-mediated Rac1 activation; however, pretreatment of BAEC with wortmannin, an inhibitor of phosphoinositide (PI) 3-kinase, had no effect on Rac1 activation while completely blocking S1P-induced Akt phosphorylation. We used Rac1-specific small interfering RNA (siRNA) duplexes to "knock down" endogenous Rac1 expression and found that siRNA-mediated Rac1 knockdown significantly impaired basal as well as S1P-induced phosphorylation of protein kinase Akt, as well as several downstream targets of Akt including endothelial nitric-oxide synthase and glycogen synthase kinase 3beta. By contrast, S1P-induced phosphorylation of the mitogen-activated protein kinases ERK1/2 was unperturbed by siRNA-mediated Rac1 knockdown. We found that overexpression of the Rac1 guanine nucleotide exchange factor (GEF) Tiam1 markedly enhanced Rac1 activity, whereas a dominant negative Tiam1 mutant significantly attenuated S1P-mediated Rac1 activation. Taken together, these studies identify G protein betagamma subunits, Src kinase and the GEF Tiam1 as upstream modulators of S1P-mediated Rac1 activation, and establish a central role for Rac1 in S1P-mediated activation of PI 3-kinase/Akt/endothelial nitric-oxide synthase signaling in vascular endothelial cells.     

New role for the protein tyrosine phosphatase DEP-1 in Akt activation and endothelial cell survival

Functional inactivation of the protein tyrosine phosphatase DEP-1 leads to increased endothelial cell proliferation and failure of vessels to remodel and branch. DEP-1 has also been proposed to contribute to the contact inhibition of endothelial cell growth via dephosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2), a mediator of vascular development. However, how DEP-1 regulates VEGF-dependent signaling and biological responses remains ill-defined. We show here that DEP-1 targets tyrosine residues in the VEGFR2 kinase activation loop. Consequently, depletion of DEP-1 results in the increased phosphorylation of all major VEGFR2 autophosphorylation sites, but surprisingly, not in the overall stimulation of VEGF-dependent signaling. The increased phosphorylation of Src on Y529 under these conditions results in impaired Src and Akt activation. This inhibition is similarly observed upon expression of catalytically inactive DEP-1, and coexpression of an active Src-Y529F mutant rescues Akt activation. Reduced Src activity correlates with decreased phosphorylation of Gab1, an adapter protein involved in VEGF-dependent Akt activation. Hypophosphorylated Gab1 is unable to fully associate with phosphatidylinositol 3-kinase, VEGFR2, and VE-cadherin complexes, leading to suboptimal Akt activation and increased cell death. Overall, our results reveal that despite its negative role on global VEGFR2 phosphorylation, DEP-1 is a positive regulator of VEGF-mediated Src and Akt activation and endothelial cell survival.     

Sphingosine 1-phosphate and isoform-specific activation of phosphoinositide 3-kinase beta. Evidence for divergence and convergence of receptor-regulated endothelial nitric-oxide synthase signaling pathways

Sphingosine 1-phosphate (S1P) is a platelet-derived sphingolipid that elicits diverse biological responses, including angiogenesis, via the activation of G protein-coupled EDG receptors. S1P activates the endothelial isoform of nitric-oxide synthase (eNOS), associated with eNOS phosphorylation at Ser-1179, a site phosphorylated by protein kinase Akt. We explored the proximal signaling pathways that mediate Akt activation and eNOS regulation by S1P/EDG receptors. Akt is regulated by the lipid kinase phosphoinositide 3-kinase (PI3-K). We found that bovine aortic endothelial cells (BAEC) express both alpha and beta isoforms of PI3-K, while lacking the gamma isoform. S1P treatment led to the rapid and isoform-specific activation of PI3-Kbeta in BAEC. PI3-Kbeta can be regulated by G protein betagamma subunits (Gbetagamma). The overexpression of a peptide inhibitor of Gbetagamma attenuated S1P-induced eNOS enzyme activation, as well as S1P-induced phosphorylation of eNOS and Akt. In contrast, bradykinin, a classical eNOS agonist, neither activated any PI3-K isoform nor induced eNOS phosphorylation at Ser-1179, despite activating eNOS in BAEC. Vascular endothelial growth factor activated both PI3-Kalpha and PI3-Kbeta via tyrosine kinase pathways and promoted eNOS phosphorylation that was unaffected by Gbetagamma inhibition. These findings indicate that PI3-Kbeta (regulated by Gbetagamma) may represent a novel molecular locus for eNOS activation by EDG receptors in vascular endothelial cells. These studies also indicate that different eNOS agonists activate distinct signaling pathways that diverge proximally following receptor activation but converge distally to activate eNOS.     

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.     

Phosphotyrosine protein dynamics in cell membrane rafts of sphingosine-1-phosphate-stimulated human endothelium: Role in barrier enhancement

Sphingosine-1-phosphate (S1P), a lipid growth factor, is critical to the maintenance and enhancement of vascular barrier function via processes highly dependent upon cell membrane raft-mediated signaling events. Anti-phosphotyrosine 2 dimensional gel electrophoresis (2-DE) immunoblots confirmed that disruption of membrane raft formation (via methyl-β-cyclodextrin) inhibits S1P-induced protein tyrosine phosphorylation. To explore S1P-induced dynamic changes in membrane rafts, we used 2-D techniques to define proteins within detergent-resistant cell membrane rafts which are differentially expressed in S1P-challenged (1 μM, 5 min) human pulmonary artery endothelial cells (EC), with 57 protein spots exhibiting > 3-fold change. S1P induced the recruitment of over 20 cell membrane raft proteins exhibiting increasing levels of tyrosine phosphorylation including known barrier-regulatory proteins such as focal adhesion kinase (FAK), cortactin, p85α phosphatidylinositol 3-kinase (p85αPI3K), myosin light chain kinase (nmMLCK), filamin A/C, and the non-receptor tyrosine kinase, c-Abl. Reduced expression of either FAK, MLCK, cortactin, filamin A or filamin C by siRNA transfection significantly attenuated S1P-induced EC barrier enhancement. Furthermore, S1P induced cell membrane raft components, p-caveolin-1 and glycosphingolipid (GM1), to the plasma membrane and enhanced co-localization of membrane rafts with p-caveolin-1 and p-nmMLCK. These results suggest that S1P induces both the tyrosine phosphorylation and recruitment of key actin cytoskeletal proteins to membrane rafts, resulting in enhanced human EC barrier function.     

Pulmonary endothelial cell barrier enhancement by sphingosine 1-phosphate: roles for cortactin and myosin light chain kinase

We recently reported the critical importance of Rac GTPase-dependent cortical actin rearrangement in the augmentation of pulmonary endothelial cell (EC) barrier function by sphingosine 1-phosphate (S1P). We now describe functional roles for the actin-binding proteins cortactin and EC myosin light chain kinase (MLCK) in mediating this response. Antisense down-regulation of cortactin protein expression significantly inhibits S1P-induced barrier enhancement in cultured human pulmonary artery EC as measured by transendothelial electrical resistance (TER). Immunofluorescence studies reveal rapid, Rac-dependent translocation of cortactin to the expanded cortical actin band following S1P challenge, where colocalization with EC MLCK occurs within 5 min. Adenoviral overexpression of a Rac dominant negative mutant attenuates TER elevation by S1P. S1P also induces a rapid increase in cortactin tyrosine phosphorylation (within 30 s) critical to subsequent barrier enhancement, since EC transfected with a tyrosine-deficient mutant cortactin exhibit a blunted TER response. Direct binding of EC MLCK to the cortactin Src homology 3 domain appears essential to S1P barrier regulation, since cortactin blocking peptide inhibits both S1P-induced MLC phosphorylation and peak S1P-induced TER values. These data support novel roles for the cytoskeletal proteins cortactin and EC MLCK in mediating lung vascular barrier augmentation evoked by S1P.     

A direct interaction between JNK1 and CrkII is critical for Rac1-induced JNK activation.

CrkII, a cellular homolog of v-crk, belongs to a family of adaptor proteins that play a central role in signal transduction cascades. We demonstrate that CrkII interacts directly with c-Jun N-terminal kinase 1 (JNK1). A proline-rich sequence of JNK1 is critical for the interaction of the kinase with the N-terminal Src homology 3 (SH3) domain of CrkII. JNK1 is localized with CrkII in membrane ruffles of Crk-overexpressing cells in a Rac1-dependent manner. A JNK1 mutant (K340A) that fails to interact with CrkII is defective in Rac/epidermal growth factor-induced activation, but remains responsive to UVC irradiation. Furthermore, CrkII recruits JNK1 to a p130Cas multiprotein complex where it may be activated through a hematopoietic progenitor kinase 1- and mitogen-activated protein kinase kinase 4-dependent pathway. Together, the results presented here argue for a new mechanism of regulation of the JNK pathway through the CrkII-p130Cas adaptor complex.     

Sphingosine 1-phosphate and activation of endothelial nitric-oxide synthase. differential regulation of Akt and MAP kinase pathways by EDG and bradykinin receptors in vascular endothelial cells

Sphingosine 1-phosphate (S1P) is a platelet-derived sphingolipid that elicits numerous biological responses in endothelial cells mediated by a family of G protein-coupled EDG receptors. Stimulation of EDG receptors by S1P has been shown to activate the endothelial isoform of nitric-oxide synthase (eNOS) in heterologous expression systems (Igarashi, J., and Michel, T. (2000) J. Biol. Chem. 275, 32363-32370). However, the signaling pathways that modulate eNOS regulation by S1P/EDG in vascular endothelial cells remain less well understood. We now report that S1P treatment of bovine aortic endothelial cells (BAEC) acutely increases eNOS enzyme activity; the EC(50) for S1P activation of eNOS is approximately 10 nm. The magnitude of eNOS activation by S1P in BAEC is equivalent to that elicited by the agonist bradykinin. S1P treatment activates Akt, a protein kinase implicated in phosphorylation of eNOS. S1P treatment of BAEC leads to eNOS phosphorylation at Ser(1179), a residue phosphorylated by Akt; an eNOS mutant in which this Akt phosphorylation site is inactivated shows attenuated S1P-induced eNOS activation. S1P-induced activation both of Akt and of eNOS is inhibited by pertussis toxin, by the phosphoinositide 3-kinase inhibitor wortmannin, and by the intracellular calcium chelator BAPTA (1,2-bis(aminophenoxy)ethane-N,N,N',N'-tetraacetic acid). By contrast to S1P, activation of G protein-coupled bradykinin B2 receptors neither activates kinase Akt nor promotes Ser(1179) eNOS phosphorylation despite robustly activating eNOS enzyme activity. Understanding the differential regulation of protein kinase pathways by S1P and bradykinin may lead to the identification of new points for eNOS regulation in vascular endothelial cells.     

Vascular endothelial growth factor receptor-3 activity is modulated by its association with caveolin-1 on endothelial membrane

Vascular endothelial growth factor receptor-3 (VEGFR-3) is constitutively expressed in lymphatic vessels and transiently in endothelial cells of blood vessels during angiogenesis. Here we report that VEGFR-3 localizes in the caveolae membrane of endothelial cells and co-immunoprecipitates with caveolin-1. Caveolin-1 silencing or its depletion from the cell membrane by cholesterol increases VEGFR-3 autophosphorylation, suggesting that caveolin acts as a negative regulator of VEGFR-3 activity. Receptor activation induces caveolin-1 phosphorylation on tyrosine residues including tyrosine 14. Cell treatment with Src or Abl inhibitors PP2 or STI571, prior to receptor stimulation, affects caveolin-1 phosphorylation without affecting receptor autophosphorylation, suggesting that both Src and Abl are involved in VEGFR-3-dependent caveolin-1 phosphorylation. Caveolin-1 phosphorylation in Src/Fyn/Yes knockout cells demonstrated that Abl phosphorylates caveolin-1 independently from Src family members. These results suggest a functional interaction between VEGFR-3 and caveolin-1 to modulate endothelial cell activation during angiogenesis.     

Selective Inhibition of Vascular Endothelial Growth Factor Receptor‐2 (VEGFR‐2) Identifies a Central Role for VEGFR‐2 in Human Aortic Endothelial Cell Responses to VEGF

Abstract

Vascular endothelial growth factor receptors (VEGFR) are considered essential for angiogenesis. The VEGFR‐family proteins consist of VEGFR‐1/Flt‐1, VEGFR‐2/KDR/Flk‐1, and VEGFR‐3/Flt‐4. Among these, VEGFR‐2 is thought to be principally responsible for angiogenesis. However, the precise role of VEGFRs1–3 in endothelial cell biology and angiogenesis remains unclear due in part to the lack of VEGFR‐specific inhibitors. We used the newly described, highly selective anilinoquinazoline inhibitor of VEGFR‐2 tyrosine kinase, ZM323881 (5‐[[7‐(benzyloxy) quinazolin‐4‐yl]amino]‐4‐fluoro‐2‐methylphenol), to explore the role of VEGFR‐2 in endothelial cell function. Consistent with its reported effects on VEGFR‐2 [IC(50) < 2 nM], ZM323881 inhibited activation of VEGFR‐2, but not of VEGFR‐1, epidermal growth factor receptor (EGFR), platelet‐derived growth factor receptor (PDGFR), or hepatocyte growth factor (HGF) receptor. We studied the effects of VEGF on human aortic endothelial cells (HAECs), which express VEGFR‐1 and VEGFR‐2, but not VEGFR‐3, in the absence or presence of ZM323881. Inhibition of VEGFR‐2 blocked activation of extracellular regulated‐kinase, p38, Akt, and endothelial nitric oxide synthetase (eNOS) by VEGF, but did not inhibit p38 activation by the VEGFR‐1‐specific ligand, placental growth factor (PlGF). Inhibition of VEGFR‐2 also perturbed VEGF‐induced membrane extension, cell migration, and tube formation by HAECs. Vascular endothelial growth factor receptor‐2 inhibition also reversed VEGF‐stimulated phosphorylation of CrkII and its Src homology 2 (SH2)‐binding protein p130^Cas, which are known to play a pivotal role in regulating endothelial cell migration. Inhibition of VEGFR‐2 thus blocked all VEGF‐induced endothelial cellular responses tested, supporting that the catalytic activity of VEGFR‐2 is critical for VEGF signaling and/or that VEGFR‐2 may function in a heterodimer with VEGFR‐1 in human vascular endothelial cells.     

Neuropilin-1 signaling through p130Cas tyrosine phosphorylation is essential for growth factor-dependent migration of glioma and endothelial cells

Neuropilin-1 (NRP1) is a receptor for vascular endothelial growth factor (VEGF) and plays an important role in mediating cell motility. However, the NRP1 signaling pathways important for cell motility are poorly understood. Here we report that p130(Cas) tyrosine phosphorylation is stimulated by hepatocyte growth factor and platelet-derived growth factor in U87MG glioma cells and VEGF in endothelial cells and is dependent on NRP1 via its intracellular domain. In endothelial cells, NRP1 silencing reduced, but did not prevent, VEGF receptor 2 (VEGFR2) phosphorylation, while expression of a mutant form of NRP1 lacking the intracellular domain (NRP1ΔC) did not affect receptor phosphorylation in U87MG cells or human umbilical vein endothelial cells (HUVECs). In HUVECs, NRP1 was also required for VEGF-induced phosphorylation of proline-rich tyrosine kinase 2, which was necessary for p130(Cas) phosphorylation. Importantly, knockdown of NRP1 or p130(Cas) or expression of either NRP1ΔC or a non-tyrosine-phosphorylatable substrate domain mutant protein (p130(Cas15F)) was sufficient to inhibit growth factor-mediated migration of glioma and endothelial cells. These data demonstrate for the first time the importance of the NRP1 intracellular domain in mediating a specific signaling pathway downstream of several receptor tyrosine kinases and identify a critical role for a novel NRP1-p130(Cas) pathway in the regulation of chemotaxis.     

Identification of Tyr900 in the kinase domain of c-Kit as a Src-dependent phosphorylation site mediating interaction with c-Crk

We have previously demonstrated that ligand-stimulation of c-Kit induces phosphorylation of Tyr568 and Tyr570 in the juxtamembrane region of the receptor, leading to recruitment, phosphorylation and activation of members of the Src family of tyrosine kinases. In this paper, we demonstrate that members of the Src family of tyrosine kinases are able to phosphorylate c-Kit selectively on one particular tyrosine residue, Tyr900, located in the second part of the tyrosine kinase domain. In order to identify potential docking partners of Tyr900, a synthetic phosphopeptide corresponding to the amino acid sequence surrounding Tyr900 was used as an affinity matrix. By use of MALDI-TOF mass spectrometry, CrkII was identified as a protein that specifically bound to Tyr900 in a phosphorylation dependent manner, possibly via the p85 subunit of PI3-kinase. Expression of a mutant receptor where Tyr900 had been replaced with a phenylalanine residue (Y900F) resulted in a receptor with reduced ability to phosphorylate CrkII. Together these data support a model where c-Src phosphorylates the receptor, thereby creating docking sites for SH2 domain containing proteins, leading to recruitment of Crk to the receptor.     

Phosphatidylinositol 3-kinase interacts with the adaptor protein Dab1 in response to Reelin signaling and is required for normal cortical lamination

Reelin is a large secreted signaling protein that binds to two members of the low density lipoprotein receptor family, the apolipoprotein E receptor 2 and the very low density lipoprotein receptor, and regulates neuronal positioning during brain development. Reelin signaling requires activation of Src family kinases as well as tyrosine phosphorylation of the intracellular adaptor protein Disabled-1 (Dab1). This results in activation of phosphatidylinositol 3-kinase (PI3K), the serine/threonine kinase Akt, and the inhibition of glycogen synthase kinase 3beta, a protein that is implicated in the regulation of axonal transport. Here we demonstrate that PI3K activation by Reelin requires Src family kinase activity and depends on the Reelin-triggered interaction of Dab1 with the PI3K regulatory subunit p85alpha. Because the Dab1 phosphotyrosine binding domain can interact simultaneously with membrane lipids and with the intracellular domains of apolipoprotein E receptor 2 and very low density lipoprotein receptor, Dab1 is preferentially recruited to the neuronal plasma membrane, where it is phosphorylated. Efficient Dab1 phosphorylation and activation of the Reelin signaling cascade is impaired by cholesterol depletion of the plasma membrane. Using a neuronal migration assay, we also show that PI3K signaling is required for the formation of a normal cortical plate, a step that is dependent upon Reelin signaling.     

Tyrosine 221 in Crk regulates adhesion-dependent membrane localization of Crk and Rac and activation of Rac signaling

The adaptor protein CrkII plays a central role in signal transduction cascades downstream of a number of different stimuli. We and others have previously shown that CrkII mediates attachment-induced JNK activation, membrane ruffling and cell motility in a Rac-dependent manner. We report here that cell attachment leads to tyrosine phosphorylation of CrkII on Y221, and that CrkII-Y221F mutant demonstrates enhanced association with the Crk-binding partners C3G and paxillin. Despite this enhanced signaling complex formation, CrkII-Y221F fails to induce JNK and PAK activation, membrane ruffling and cell migration, suggesting that it is defective in activating Rac signaling. Wild-type CrkII has no effect on adhesion-induced GTP loading of Rac, but its expression results in enhanced membrane localization of Rac, which is known to be required for Rac signaling. In contrast, CrkII-Y221F is deficient in enhancing membrane localization of Rac. Mutations in Rac and CrkII-Y221F that force membrane targeting of these molecules restore Rac signaling in adherent cells. Together, these results indicate that the Y221 site in CrkII regulates Rac membrane translocation upon cell adhesion, which is necessary for activation of downstream Rac signaling pathways.     

Src family kinases mediate receptor-stimulated, phosphoinositide 3-kinase-dependent, tyrosine phosphorylation of dual adaptor for phosphotyrosine and 3-phosphoinositides-1 in endothelial and B cell lines

DAPP-1 (dual-adaptor for phosphotyrosine and 3-phosphoinositides-1) is a broadly distributed pleckstrin homology (PH) and Src homology 2 domain containing protein that can bind phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) and can be phosphorylated on tyrosine 139 and internalised in response to activation of type I phosphoinositide 3-kinases (PI3K). Tyrosine phosphorylation of DAPP-1 appears important for appropriate intracellular targeting and creates a potential binding site for Src homology 2 domain-containing proteins. In endothelial cells overexpressing wild-type platelet-derived growth factor beta (PDGFbeta) receptors, which express Bmx and Src as their major Btk (Bruton's tyrosine kinase) family and Src family tyrosine kinases, respectively, PDGF can stimulate PI3K-dependent tyrosine phosphorylation of DAPP-1. Transient overexpression of Src most effectively, compared with Bmx and Syk, augments basal and PDGF-stimulated tyrosine phosphorylation of DAPP-1, whereas overexpression of dominant-negative Src, but not dominant-negative Bmx, inhibits PDGF-stimulated phosphorylation of DAPP-1. Cells expressing mutant PDGFbeta (Y579F/Y581F) receptors (which fail to bind and activate Src-type kinases) fail to tyrosine phosphorylate DAPP-1 in response to PDGF. We show that in DT40 chicken B cell lines, antibody stimulation leads to PI3K-dependent tyrosine phosphorylation of DAPP-1 that is lost in Lyn- or Syk-deficient cell lines but not Btk-deficient cell lines. PI3K-dependent activation of PKB is only lost in Syk-deficient lines. Finally, in vitro we find lipid-modified Src to be the most effective DAPP-1 tyrosine kinase (versus Syk, Lyn, Btk, and Bmx); phosphorylation of DAPP-1 but not Src autophosphorylation is stimulated approximately 10-fold by PtdIns(3,4,5)P(3) (IC(50) = 150 nm) and phosphatidylinositol 3,4-bisphosphate but not by their nonbiological diastereoisomers and depends on PH domain mediated binding of DAPP-1 to PtdIns(3,4,5)P(3)-containing membranes. We conclude that Src family kinases are responsible for tyrosine phosphorylation of DAPP-1 in vivo and that PI3K regulation is at the level of PH domain-mediated translocation of DAPP-1 to PI3K products in the membrane.