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.

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title: Angiogenesis in cancer and other diseases
abstract: Pathological angiogenesis is a hallmark of cancer and various ischaemic and inflammatory diseases. Concentrated efforts in this area of research are leading to the discovery of a growing number of pro- and anti-angiogenic molecules, some of which are already in clinical trials. The complex interactions among these molecules and how they affect vascular structure and function in different environments are now beginning to be elucidated. This integrated understanding is leading to the development of a number of exciting and bold approaches to treat cancer and other diseases. But owing to several unanswered questions, caution is needed.

title: Role of Angiogenesis in Cardiovascular Disease: A Critical Appraisal
abstract: The role of angiogenesis in atherosclerosis and other cardiovascular diseases has emerged as a major unresolved issue. Angiogenesis has attracted interest from opposite perspectives. Angiogenic cytokine therapy has been widely regarded as an attractive approach both for treating ischemic heart disease and for enhancing arterioprotective functions of the endothelium; conversely, a variety of studies suggest that neovascularization contributes to the growth of atherosclerotic lesions and is a key factor in plaque destabilization leading to rupture. Here, we critically review the evidence supporting a role for angiogenesis and angiogenic factors in atherosclerosis and neointima formation, emphasizing the problems raised by some of the landmark studies and the suitability of animal models of atherosclerosis and neointimal thickening for investigating the role of angiogenesis. Because many of the relevant studies have focused on the role of vascular endothelial growth factor (VEGF), we consider this work in the wider context of VEGF biology and in light of recent experience from clinical trials of VEGF and other angiogenic cytokines for ischemic heart disease. Also discussed are recent findings suggesting that, although angiogenesis may contribute to neointimal growth, it is not required for the initiation of intimal thickening. Our assessment of the evidence leads us to conclude that, although microvessels are a feature of advanced human atherosclerotic plaques, it remains unclear whether angiogenesis either plays a central role in the development of atherosclerosis or is responsible for plaque instability. Furthermore, current evidence from clinical trials of both proangiogenic and antiangiogenic therapies does not suggest that inhibition of angiogenesis is likely to be a viable therapeutic strategy for cardiovascular disease.

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title: Cloning and characterization of rat density-enhanced phosphatase-1, a protein tyrosine phosphatase expressed by vascular cells.
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title: CD148 is a membrane protein tyrosine phosphatase present in all hematopoietic lineages and is involved in signal transduction on lymphocytes.
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title: Expression of DEP-1, a receptor-like protein-tyrosine-phosphatase, is enhanced with increasing cell density.
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title: The protein tyrosine phosphatase DEP-1 is induced during differentiation and inhibits growth of breast cancer cells.
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title: Rat Protein Tyrosine Phosphatase η Suppresses the Neoplastic Phenotype of Retrovirally Transformed Thyroid Cells through the Stabilization of p27Kip1
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title: A Mutant Receptor Tyrosine Phosphatase, CD148, Causes Defects in Vascular Development
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title: Targeted Deficiency or Cytosolic Truncation of the VE-cadherin Gene in Mice Impairs VEGF-Mediated Endothelial Survival and Angiogenesis
abstract: Vascular endothelial cadherin, VE-cadherin, mediates adhesion between endothelial cells and may affect vascular morphogenesis via intracellular signaling, but the nature of these signals remains unknown. Here, targeted inactivation (VEC-/-) or truncation of the beta-catenin-binding cytosolic domain (VECdeltaC/deltaC) of the VE-cadherin gene was found not to affect assembly of endothelial cells in vascular plexi, but to impair their subsequent remodeling and maturation, causing lethality at 9.5 days of gestation. Deficiency or truncation of VE-cadherin induced endothelial apoptosis and abolished transmission of the endothelial survival signal by VEGF-A to Akt kinase and Bcl2 via reduced complex formation with VEGF receptor-2, beta-catenin, and phosphoinositide 3 (PI3)-kinase. Thus, VE-cadherin/ beta-catenin signaling controls endothelial survival.

title: Contact inhibition of VEGF-induced proliferation requires vascular endothelial cadherin, β-catenin, and the phosphatase DEP-1/CD148
abstract: Confluent endothelial cells respond poorly to the proliferative signals of VEGF. Comparing isogenic endothelial cells differing for vascular endothelial cadherin (VE-cadherin) expression only, we found that the presence of this protein attenuates VEGF-induced VEGF receptor (VEGFR) 2 phosphorylation in tyrosine, p44/p42 MAP kinase phosphorylation, and cell proliferation. VE-cadherin truncated in β-catenin but not p120 binding domain is unable to associate with VEGFR-2 and to induce its inactivation. β-Catenin–null endothelial cells are not contact inhibited by VE-cadherin and are still responsive to VEGF, indicating that this protein is required to restrain growth factor signaling. A dominant-negative mutant of high cell density–enhanced PTP 1 (DEP-1)//CD148 as well as reduction of its expression by RNA interference partially restore VEGFR-2 phosphorylation and MAP kinase activation. Overall the data indicate that VE-cadherin–β-catenin complex participates in contact inhibition of VEGF signaling. Upon stimulation with VEGF, VEGFR-2 associates with the complex and concentrates at cell–cell contacts, where it may be inactivated by junctional phosphatases such as DEP-1. In sparse cells or in VE-cadherin–null cells, this phenomenon cannot occur and the receptor is fully activated by the growth factor.

title: The C. elegans homolog of the mammalian tumor suppressor Dep-1/Scc1 inhibits EGFR signaling to regulate binary cell fate decisions.
abstract: Protein phosphorylation by kinases and the subsequent dephosphorylation by phosphatases are key mechanisms that regulate intracellular signal transduction during development. Here, we report the identification of the receptor protein tyrosine phosphatase DEP-1 as a negative regulator of the Caenorhabditis elegans EGF receptor. DEP-1 amplifies in the developing vulva and the excretory system the small differences in the amount of EGF signal received by equivalent precursor cells to achieve binary cell fate decisions. During vulval development, DEP-1 inhibits EGFR signaling in the secondary cell lineage in parallel with the NOTCH-mediated lateral inhibition, while EGFR signaling simultaneously down-regulates DEP-1 and NOTCH expression in the primary cell lineage. This regulatory network of inhibitors results in the full activation of the EGFR/RAS/MAPK pathway in the primary vulval cells and at the same time keeps the EGFR/RAS/MAPK pathway inactive in the adjacent secondary cells. Mammalian Dep-1/Scc1 functions as a tumor-suppressor gene in the intestinal epithelium. Thus, mutations in human Dep-1 may promote tumor formation through a hyperactivation of the EGF receptor.

title: The transmembrane receptor protein tyrosine phosphatase DEP1 interacts with p120ctn
abstract: The receptor-like protein tyrosine phosphatase DEP1, also known as CD148, is expressed predominantly in epithelial cells, in a variety of tumor cell lines, and in lymphocytes. Expression of DEP1 is enhanced at high cell density, and this observation suggests that DEP1 may function in the regulation of cell adhesion and possibly contact inhibition of cell growth. In order to investigate the function of DEP1, substrate-trapping mutants of the phosphatase were used to identify potential substrates. GST-fusion proteins containing the DEP1 catalytic domain with a substrate-trapping D/A mutation were found to interact with p120ctn, a component of adherens junctions. DEP1 also interacted with other members of the catenin gene family including β-catenin and γ-catenin. The interaction with p120ctn is likely to be direct, as the interaction occurs in K562 cells lacking functional adherens junctions and E-cadherin expression. Catalytic domains of the tyrosine phosphatases PTP-PEST, CD45, and PTPβ did not interact with proteins of the catenin family to detectable levels, suggesting that the interaction of DEP1 with these proteins is specific. DEP1 expression was concentrated at sites of cell–cell contact in A549 cells. p120ctn was found to colocalize with these structures. Together these data suggest an important role for DEP-1 in the function of cell–cell contacts and adherens junctions.

title: The receptor-type protein tyrosine phosphatase J antagonizes the biochemical and biological effects of RET-derived oncoproteins.
abstract: Thyroid cancer is frequently associated with the oncogenic conversion of the RET receptor tyrosine kinase. RET gene rearrangements, which lead to the generation of chimeric RET/papillary thyroid carcinoma (PTC) oncogenes, occur in PTC, whereas RET point mutations occur in familial multiple endocrine neoplasia type 2 (MEN2) and sporadic medullary thyroid carcinomas (MTC). We showed previously that the expression of the receptor-type protein tyrosine phosphatase J (PTPRJ) is suppressed in neoplastically transformed follicular thyroid cells. We now report that PTPRJ coimmunoprecipitates with wild-type RET and with the MEN2A-associated RET(C634R) oncoprotein but not with the RET/PTC1 and RET-MEN2B isoforms. Using mutated forms of PTPRJ and RET-MEN2A, we show that the integrity of the respective catalytic domains is required for the PTPRJ/RET-MEN2A interaction. PTPRJ expression induces dephosphorylation of the RET(C634R) and, probably via an indirect mechanism, RET/PTC1 oncoproteins on two key RET autophosphorylation sites (Tyr1062 and Tyr905). This results in a significant decrease of RET-induced Shc and extracellular signal-regulated kinase 1/2 phosphorylation levels. In line with this finding, adoptive PTPRJ expression reduced the oncogenic activity of RET(C634R) in an in vitro focus formation assay of NIH3T3 cells. As expected from the coimmunoprecipitation results, the RET(M918T) oncoprotein, which is associated to MEN2B and sporadic MTC, was resistant to the dephosphorylating activity of PTPRJ. Taken together, these findings identify RET as a novel substrate of PTPRJ and suggest that PTPRJ expression levels may affect tumor phenotype associated with RET/PTC1 and RET(C634R) mutants. On the other hand, resistance to PTPRJ may be part of the mechanism of RET oncogenic conversion secondary to the M918T mutation.

title: The rat tyrosine phosphatase η increases cell adhesion by activating c-Src through dephosphorylation of its inhibitory phosphotyrosine residue
abstract: The expression of the receptor protein tyrosine phosphatase r-PTPη is drastically reduced in rat and human malignant thyroid cells, whereas its restoration reverts the neoplastic phenotype of retrovirally transformed rat thyroid cells. Moreover, reduced levels and loss of heterozygosity of DEP-1, the human homolog of r-PTPη, have been found in many human neoplasias. Here, we report that the r-PTPη protein binds to c-Src in living cells and dephosphorylates the c-Src inhibitory tyrosine phosphorylation site (Tyr 529), thereby increasing c-Src tyrosine kinase activity in malignant rat thyroid cells stably transfected with r-PTPη. Tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin was enhanced in r-PTPη-expressing cells. This was associated with increased adhesion of malignant r-PTPη-transfected thyroid cells vs both untransfected cells and cells stably transfected with an inactive r-PTPη mutant. Treatment of rat thyroid cells with the c-Src inhibitor PP2 decreased cell adhesion to a higher extent in r-PTPη-transfected cells than in mock-transfected or stably transfected cells with the inactive r-PTPη mutant, indicating that r-PTPη regulates cell–substratum adhesion by activating c-Src. Interestingly, the extent of both c-Src dephosphorylation at Tyr 529, FAK and paxillin phosphorylation, and the increased cell adhesion were associated with the degree of r-PTPη expression.

title: Hepatocyte Growth Factor Receptor Tyrosine Kinase Met Is a Substrate of the Receptor Protein-tyrosine Phosphatase DEP-1*
abstract: The receptor protein-tyrosine phosphatase (PTP) DEP-1 (CD148/PTP-η) has been implicated in the regulation of cell growth, differentiation, and transformation, and most recently has been identified as a potential tumor suppressor gene mutated in colon, lung, and breast cancers. We have generated constructs comprising the cytoplasmic segment of DEP-1 fused to the maltose-binding protein to identify potential substrates and thereby suggest a physiological function for DEP-1. We have shown that the substrate-trapping mutant form of DEP-1 interacted with a small subset of tyrosine-phosphorylated proteins from lysates of the human breast tumor cell lines MDA-MB-231, T-47D, and T-47D/Met and have identified the hepatocyte growth factor/scatter factor receptor Met, the adapter protein Gab1, and the junctional component p120 catenin as potential substrates. Following ligand stimulation, phosphorylation of specific tyrosyl residues in Met induces mitogenic, motogenic, and morphogenic responses. When co-expressed in 293 cells, the full-length substrate-trapping mutant form of DEP-1 formed a stable complex with the chimeric receptor colony stimulating factor 1 (CSF)-Met and wild type DEP-1 dephosphorylated CSF-Met. Furthermore, we observed that DEP-1 preferentially dephosphorylated a Gab1 binding site (Tyr1349) and a COOH-terminal tyrosine implicated in morphogenesis (Tyr1365), whereas tyrosine residues in the activation loop of Met (Tyr1230, Tyr1234, and Tyr1235) were not preferred targets of the PTP. The ability of DEP-1 preferentially to dephosphorylate particular tyrosine residues that are required for Met-induced signaling suggests that DEP-1 may function in controlling the specificity of signals induced by this PTK, rather than as a simple “off-switch” to counteract PTK activity.

title: Src mediates stimulation by vascular endothelial growth factor of the phosphorylation of focal adhesion kinase at tyrosine 861, and migration and anti-apoptosis in endothelial cells.
abstract: Vascular endothelial growth factor (VEGF) stimulates the tyrosine phosphorylation of focal adhesion kinase (FAK), increases focal adhesion formation and is chemotactic for human umbilical-vein endothelial cells (HUVECs). In the present study we identified the major sites of VEGF-induced FAK tyrosine phosphorylation and investigated the mechanism mediating this pathway in the action of VEGF. VEGF increased the focal adhesion localization of FAK phosphorylated at Tyr-397 (Y397) and Y861 but stimulated a marked increase in phosphorylation at Y861 without significantly affecting the total level of phospho-Y397 FAK. Inhibition of Src with the specific inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) completely blocked VEGF-induced Y861 phosphorylation without decreasing the level of phospho-Y397 FAK. We also examined the role of Src in mediating endothelial functions of VEGF in which FAK has been implicated as having a role. PP2 markedly inhibited VEGF-induced chemotaxis and wound-healing cell migration. The Src inhibitor also decreased the anti-apoptotic effect of VEGF determined by surface staining of annexin V but did not increase FAK proteolysis or prevent the VEGF-dependent inhibition of FAK proteolysis. In contrast, the specific PtdIns 3-kinase inhibitor LY294002 induced apoptosis and markedly decreased p125(FAK) expression and increased FAK proteolysis but had little effect on Y861 phosphorylation. These findings identify Src-dependent FAK phosphorylation at Y861 as a novel VEGF-induced signalling pathway in endothelial cells and suggest that this pathway might be involved in the mechanisms mediating VEGF-induced endothelial cell migration and anti-apoptosis.

title: Selective requirement for Src kinases during VEGF-induced angiogenesis and vascular permeability.
abstract: Src kinase activity was found to protect endothelial cells from apoptosis during vascular endothelial growth factor (VEGF)-, but not basic fibroblast growth factor (bFGF)-, mediated angiogenesis in chick embryos and mice. In fact, retroviral targeting of kinase-deleted Src to tumor-associated blood vessels suppressed angiogenesis and the growth of a VEGF-producing tumor. Although mice lacking individual Src family kinases (SFKs) showed normal angiogenesis, mice deficient in pp60c-src or pp62c-yes showed no VEGF-induced vascular permeability (VP), yet fyn-/- mice displayed normal VP. In contrast, inflammation-mediated VP appeared normal in Src-deficient mice. Therefore, VEGF-, but not bFGF-, mediated angiogenesis requires SFK activity in general, whereas the VP activity of VEGF specifically depends on the SFKs, Src, or Yes.

title: Akt Mediates Cytoprotection of Endothelial Cells by Vascular Endothelial Growth Factor in an Anchorage-dependent Manner*
abstract: Regulation of endothelial cell apoptosis is a critical modulator of normal and pathological angiogenesis. In this study, we examined the role of the protein kinase Akt/PKB in endothelial cell survival in response to growth factor and matrix attachment signals. Vascular endothelial growth factor(VEGF)-induced cytoprotection of endothelial cell monolayers correlated with the wortmannin-sensitive induction of Akt activity. Transfection of an adenovirus expressing a dominant-negative Akt mutant decreased endothelial cell viability in the presence of VEGF. Conversely, adenoviral transduction of wild-type Akt facilitated the cell survival effects of VEGF, whereas transduction of constitutively active Akt conferred endothelial cell survival in the absence of VEGF. Constitutively active Akt also conferred survival to endothelial cells in suspension culture, whereas stimulation with VEGF did not. In suspension cultures, VEGF stimulation was unable to activate Akt, and Akt protein levels were repressed in cells undergoing anoikis. These data suggest that cross-talk between growth factor- and anchorage-dependent signaling pathways are essential for Akt activation and endothelial cell survival.

title: Vascular Endothelial Growth Factor Signals Endothelial Cell Production of Nitric Oxide and Prostacyclin through Flk-1/KDR Activation of c-Src*
abstract: Vascular endothelial growth factor (VEGF) is a potent endothelial cell-specific mitogen that promotes angiogenesis, vascular hyperpermeability, and vasodilation by autocrine mechanisms involving nitric oxide (NO) and prostacyclin (PGI2) production. These experiments used immunoprecipitation and immunoassay procedures to characterize the signaling pathways by which VEGF induces NO and PGI2 formation in cultured endothelial cells. The data showed that VEGF stimulates complex formation of the flk-1/kinase-insert domain-containing receptor (KDR) VEGF receptor with c-Src and that Src activation is required for VEGF induction of phospholipase C γ1 activation and inositol 1,4,5-trisphosphate formation. Reporter cell assays showed that VEGF promotes a ∼50-fold increase in NO formation, which peaks at 5–20 min. This effect is mediated by a signaling cascade initiated by flk-1/KDR activation of c-Src, leading to phospholipase C γ1 activation, inositol 1,4,5-trisphosphate formation, release of [Ca2+] i and nitric oxide synthase activation. Immunoassays of VEGF-induced 6-keto prostaglandin F1α formation as an indicator of PGI2 production revealed a 3–4-fold increase that peaked at 45–60 min. The PGI2 signaling pathway follows the NO pathway through release of [Ca2+] i , but diverges prior to NOS activation and also requires activation of mitogen-activated protein kinase. These results suggest that NO and PGI2 function in parallel in mediating the effects of VEGF.

title: c-Src Mediates Mitogenic Signals and Associates with Cytoskeletal Proteins upon Vascular Endothelial Growth Factor Stimulation in Kaposi’s Sarcoma Cells1
abstract: Vascular endothelial growth factor (VEGF) appears to be a critical cytokine modulating the growth and spread of Kaposi’s sarcoma (KS). Furthermore, infection with the KS herpes virus results in up-regulation of VEGF and triggering of VEGF receptor activation. The molecular mechanisms regulating such cytokine-driven proliferation of KS cells are not well characterized. We investigated the role of Src-related tyrosine kinases in VEGF-mediated signaling in model KS 38 tumor cells. VEGF stimulation specifically activated c-Src kinase activity but not that of other related Src kinases such as Lyn, Fyn, or Hck in KS cells. Pyrazolopyrimidine, a selective inhibitor of Src family tyrosine kinases, significantly blocked the VEGF-induced growth of KS cells. Further studies using mutants of c-Src kinase revealed that Src mediates mitogen-activated protein kinase activation induced by VEGF. We also observed that VEGF stimulation resulted in increased tyrosine phosphorylation of the focal adhesion components paxillin and p130cas. Furthermore, VEGF induction enhanced the complex formation between Src kinase and paxillin. Src kinase appears to play an important functional role in VEGF-induced signaling in KS cells and may act to link pathways from the VEGF receptor to mitogen-activated protein kinase and cytoskeletal components, thereby effecting tumor proliferation and migration.

title: Characterization of vascular endothelial growth factor's effect on the activation of protein kinase C, its isoforms, and endothelial cell growth.
abstract: Vascular endothelial growth factor (VEGF) is a potent endothelial cell mitogen which mediates its effects by binding to tyrosine kinase receptors. We have characterized the VEGF-activated intracellular signal transduction pathway in bovine aortic endothelial cells and correlated this to its mitogenic effects. VEGF induced concentration- and time-dependent increases in protein kinase C (PKC) activation with a maximum of 2.2-fold above the basal level at 5 x 10(-10) M within 10 min as measured both by in situ and translocation assays. Immunoblotting analysis of PKC isoforms in cytosolic and membrane fractions indicated that after VEGF stimulation the content of Ca(2+)-sensitive PKC isoforms (alpha and betaII) was increased in the membrane fractions, whereas no changes were observed for PKC isoforms delta and epsilon. The stimulation of PKC activity by VEGF was preceded by the activation of phospholipase Cgamma (PLCgamma). This was demonstrated by parallel increases in PLCgamma tyrosine phosphorylation, [3H]inositol phosphate production, and [3H]arachidonic acid-labeled diacylglycerol formation in bovine aortic endothelial cells. In addition, VEGF increased phosphatidylinositol 3-kinase activity 2.1-fold which was inhibited by wortmannin, a phosphatidylinositol 3-kinase inhibitor, without decreasing the VEGF-induced increase in PKC activity or endothelial cell growth. Interestingly, genistein, a tyrosine kinase inhibitor, and GFX or H-7, PKC inhibitors, abolished both VEGF-induced PKC activation and endothelial cell proliferation. VEGF's mitogenic effect was inhibited by a PKC isoform beta-selective inhibitor, LY333531, in a concentration-dependent manner. In contrast, antisense PKC-alpha oligonucleotides enhanced VEGF-stimulated cell growth with a simultaneous decrease of 70% in PKC-alpha protein content. Thus, VEGF appears to mediate its mitogenic effects partly through the activation of the PLCgamma and PKC pathway, involving predominately PKC-beta isoform activation in endothelial cells.

title: The Scaffolding Adapter Gab1 Mediates Vascular Endothelial Growth Factor Signaling and Is Required for Endothelial Cell Migration and Capillary Formation*
abstract: 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 Cγ (PLCγ), 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 PLCγ, and its overexpression enhances VEGF-dependent cell migration. Importantly, silencing of Gab1 using small interfering RNAs leads to the impaired activation of PLCγ, 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.

title: Cell transformation by the middle T-antigen of polyoma virus
abstract: The polyoma virus region expressed early in the lytic cycle encodes three proteins, or T-antigens, that together cause the infected cell to enter the cell cycle and so provide a suitable cellular environment for replication of the viral genome. Under some circumstances infection does not kill the cell, but the T-antigens are still produced, resulting in the cell becoming transformed and tumorigenic. Most of this transforming action is exerted by the middle T-antigen, which has the ability to convert established cell lines to an oncogenic state. Middle T is a membrane bound polypeptide that interacts with a number of the proteins used by tyrosine kinase associated receptors to stimulate mitogenesis, so MT can be considered as a permanently active analogue of a receptor. Through a defined series of interactions, MT assembles a large multi-protein complex at the cell membrane, consisting of MT, the core dimer of protein phosphatase 2A, an src-family tyrosine kinase, and via phosphotyrosines, ShcA, phosphatidylinositol (3′) kinase, and phospholipase Cγ-1. Tyrosine phosphorylation stimulates PI3K and PLCγ-1 enzymatic activity, and on ShcA creates binding sites for Grb2 with its associated Sos1 and Gab1. This activates p21ras, and hence, the MAP kinase cascade. Consequently, MT can be used as a model for studying cell transformation and growth factor receptor signalling pathways.

title: Mechanism of Inhibition of Protein-tyrosine Phosphatases by Vanadate and Pervanadate*
abstract: Vanadate and pervanadate (the complexes of vanadate with hydrogen peroxide) are two commonly used general protein-tyrosine phosphatase (PTP) inhibitors. These compounds also have insulin-mimetic properties, an observation that has generated a great deal of interest and study. Since a careful kinetic study of the two inhibitors has been lacking, we sought to analyze their mechanisms of inhibition. Our results show that vanadate is a competitive inhibitor for the protein-tyrosine phosphatase PTP1B, with a Ki of 0.38 ± 0.02 μM. EDTA, which is known to chelate vanadate, causes an immediate and complete reversal of the inhibition due to vanadate when added to an enzyme assay. Pervanadate, by contrast, inhibits by irreversibly oxidizing the catalytic cysteine of PTP1B, as determined by mass spectrometry. Reducing agents such as dithiothreitol that are used in PTP assays to keep the catalytic cysteine reduced and active were found to convert pervanadate rapidly to vanadate. Under certain conditions, slow time-dependent inactivation by vanadate was observed; since catalase blocked this inactivation, it was ascribed to in situ generation of hydrogen peroxide and subsequent formation of pervanadate. Implications for the use of these compounds as inhibitors and rationalization for some of their in vivo effects are considered.

title: Structurally distinct phosphatases CD45 and CD148 both regulate B cell and macrophage immunoreceptor signaling.
abstract: The receptor-type protein tyrosine phosphatase (RPTP) CD148 is thought to have an inhibitory function in signaling and proliferation in nonhematopoietic cells. However, its role in the immune system has not been thoroughly studied. Our analysis of CD148 loss-of-function mice showed that CD148 has a positive regulatory function in B cells and macrophages, similar to the role of CD45 as a positive regulator of Src family kinases (SFKs). Analysis of CD148 and CD45 doubly deficient B cells and macrophages revealed hyperphosphorylation of the C-terminal inhibitory tyrosine of SFKs accompanied by substantial alterations in B and myeloid lineage development and defective immunoreceptor signaling. Because these findings suggest the C-terminal tyrosine of SFKs is a common substrate for both CD148 and CD45 phosphatases and imply a level of redundancy not previously appreciated, a reassessment of the function of CD45 in the B and myeloid lineages based on prior data from the CD45-deficient mouse is warranted.

title: Control of adhesion-dependent cell survival by focal adhesion kinase
abstract: The interactions of integrins with extracellular matrix proteins can activate focal adhesion kinase (FAK) and suppress apoptosis in normal epithelial and endothelial cells; this subset of apoptosis has been termed "anoikis." Here, we demonstrate that FAK plays a role in the suppression of anoikis. Constitutively activated forms of FAK rescued two established epithelial cell lines from anoikis. Both the major autophosphorylation site (Y397) and a site critical to the kinase activity (K454) of FAK were required for this effect. Activated FAK also transformed MDCK cells, by the criteria of anchorage-independent growth and tumor formation in nude mice. We provide evidence that this transformation resulted primarily from the cells' resistance to anoikis rather than from the activation of growth factor response pathways. These results indicate that FAK can regulate anoikis and that the conferral of anoikis resistance may suffice to transform certain epithelial cells.

title: Inhibition of pp125FAK in cultured fibroblasts results in apoptosis
abstract: The tyrosine kinase called pp125FAK is believed to play an important role in integrin-mediated signal transduction. pp125FAK is associated both functionally and spatially with integrins, which are the cell surface receptors for extracellular matrix components. Although the precise function of pp125FAK is not known, two possibilities have been proposed: pp125FAK may regulate the assembly of focal adhesions in spreading or migrating cells, or pp125FAK may participate in a signal transduction cascade to inform the nucleus that the cell is anchored. To test these models in living cells, a peptide representing the focal adhesion kinase (FAK)-binding site of the beta 1 tail was coupled to carrier protein and injected into cultured cells to competitively inhibit the binding of pp125FAK to endogenous integrin, thus inhibiting activation of pp125FAK on a cell-by-cell basis. In addition, an antibody directed against an epitope adjacent to the focal adhesion targeting sequence on pp125FAK was microinjected, as an alternative means of inhibiting pp125FAK activation. It was observed that when rounded cells were injected with either the integrin peptide or the anti-FAK antibody, the cells rapidly began to apoptose, within 4 h after injection. These results indicate that pp125FAK may play a critical role in suppressing apoptosis in fibroblasts.

title: Regulation of endothelium-derived nitric oxide production by the protein kinase Akt
abstract: Endothelial nitric oxide synthase (eNOS) is the nitric oxide synthase isoform responsible for maintaining systemic blood pressure, vascular remodelling and angiogenesis. eNOS is phosphorylated in response to various forms of cellular stimulation but the role of phosphorylation in the regulation of nitric oxide (NO) production and the kinase(s) responsible are not known. Here we show that the serine/threonine protein kinase Akt (protein kinase B) can directly phosphorylate eNOS on serine 1179 and activate the enzyme, leading to NO production, whereas mutant eNOS (S1179A) is resistant to phosphorylation and activation by Akt. Moreover, using adenovirus-mediated gene transfer, activated Akt increases basal NO release from endothelial cells, and activation-deficient Akt attenuates NO production stimulated by vascular endothelial growth factor. Thus, eNOS is a newly described Akt substrate linking signal transduction by Akt to the release of the gaseous second messenger NO.

title: AKT/PKB Signaling: Navigating Downstream
abstract: The serine/threonine kinase Akt, also known as protein kinase B (PKB), is a central node in cell signaling downstream of growth factors, cytokines, and other cellular stimuli. Aberrant loss or gain of Akt activation underlies the pathophysiological properties of a variety of complex diseases, including type-2 diabetes and cancer. Here, we review the molecular properties of Akt and the approaches used to characterize its true cellular targets. In addition, we discuss those Akt substrates that are most likely to contribute to the diverse cellular roles of Akt, which include cell survival, growth, proliferation, angiogenesis, metabolism, and migration.

title: The Akt kinase signals directly to endothelial nitric oxide synthase
abstract: Endothelial nitric oxide synthase (eNOS) is an important modulator of angiogenesis and vascular tone . It is stimulated by treatment of endothelial cells in a phosphatidylinositol 3-kinase (PI 3-kinase)-dependent fashion by insulin-like growth factor-1 (IGF-1) and vascular endothelial growth factor (VEGF)   and is activated by phosphorylation at Ser1177 in the sequence RIRTQS(1177)F (in the single-letter amino acid code) . The protein kinase Akt is an important downstream target of PI 3-kinase [5] , regulating VEGF-stimulated endothelial cell survival [7]. Akt phosphorylates substrates within a defined motif , which is present in the sequence surrounding Ser1177 in eNOS. Both Akt   and eNOS [9] are localized to, and activated at, the plasma membrane. We found that purified Akt phosphorylated cardiac eNOS at Ser1177, resulting in activation of eNOS. Phosphorylation at this site was stimulated by treatment of bovine aortic endothelial cells (BAECs) with VEGF or IGF-1, and Akt was activated in parallel. Preincubation with wortmannin, an inhibitor of Akt signalling, reduced VEGF- or IGF-1-induced Akt activity and eNOS phosphorylation. Akt was detected in immunoprecipitates of eNOS from BAECs, and eNOS in immunoprecipitates of Akt, indicating that the two enzymes associate in vivo. It is thus apparent that Akt directly activates eNOS in endothelial cells. These results strongly suggest that Akt has an important role in the regulation of normal angiogenesis and raise the possibility that the enhanced activity of this kinase that occurs in carcinomas may contribute to tumor vascularization and survival.

title: Gab1, SHP2, and Protein Kinase A Are Crucial for the Activation of the Endothelial NO Synthase by Fluid Shear Stress
abstract: Fluid shear stress enhances NO production in endothelial cells by a mechanism involving the activation of the phosphatidylinositol 3-kinase and the phosphorylation of the endothelial NO synthase (eNOS). We investigated the role of the scaffolding protein Gab1 and the tyrosine phosphatase SHP2 in this signal transduction cascade in cultured and native endothelial cells. Fluid shear stress elicited the phosphorylation and activation of Akt and eNOS as well as the tyrosine phosphorylation of Gab1 and its association with the p85 subunit of phosphatidylinositol 3-kinase and SHP2. Overexpression of a Gab1 mutant lacking the pleckstrin homology domain abrogated the shear stress–induced phosphorylation of Akt but failed to affect the phosphorylation or activity of eNOS. The latter response, however, was sensitive to a protein kinase A (PKA) inhibitor. Mutation of Gab1 Tyr627 to phenylalanine (YF-Gab1) to prevent the binding of SHP2 completely prevented the shear stress–induced phosphorylation of eNOS, leaving the Akt response intact. A dominant-negative SHP2 mutant prevented the activation of PKA and phosphorylation of eNOS without affecting that of Akt. Moreover, shear stress elicited the formation of a signalosome complex including eNOS, Gab1, SHP2 and the catalytic subunit of PKA. In isolated murine carotid arteries, flow-induced vasodilatation was prevented by a PKA inhibitor as well as by overexpression of either the YF-Gab1 or the dominant-negative SHP2 mutant. Thus, the shear stress–induced activation of eNOS depends on Gab1 and SHP2, which, in turn, regulate the phosphorylation and activity of eNOS by a PKA-dependent but Akt-independent mechanism.

title: Post-translational control of endothelial nitric oxide synthase: why isn't calcium/calmodulin enough?
abstract: Endothelial nitric oxide synthase (eNOS) is important for cardiovascular homeostasis, vessel remodeling, and angiogenesis. Given the impact of endothelium- derived nitric oxide (NO) in vascular biology, much work in the past several years has focused on the control of NO synthesis by regulatory proteins that influence its function. Indeed calcium-activated calmodulin is important for regulation of NOS activity. Herein we discuss why other proteins, in addition to calmodulin, are necessary for eNOS regulation and summarize the biology of negative and positive regulators of eNOS function in vitro, in cells, and in blood vessels.

title: Mechanism of Endothelial Nitric Oxide Synthase Phosphorylation and Activation by Thrombin
abstract: Thrombin has been shown to activate endothelial NO synthase (eNOS) leading to endothelium-dependent vasorelaxation. In addition to its activation by Ca2+/calmodulin, eNOS has several regulatory sites. Ser1179 phosphorylation of eNOS by the phosphatidylinositol 3-kinase-dependent Akt stimulates its catalytic activity. In this study, we have elucidated the signaling mechanism of thrombin-induced phosphorylation of eNOS in the regulation of NO production. Immunoblot analysis showed that thrombin rapidly phosphorylates eNOS at Ser1179 in cultured bovine aortic endothelial cells. Also, thrombin was unable to stimulate eNOS if the Ser1179 was mutated to Ala. Akt is phosphorylated in response to thrombin at Ser473 at a later time point than eNOS. In this regard, a phosphatidylinositol 3-kinase inhibitor, LY294002, blocked Akt phosphorylation without affecting eNOS phosphorylation and cGMP production by thrombin. The Ca2+ ionophore A23187 stimulated eNOS phosphorylation, as well as cGMP production, and pretreatment with intracellular or extracellular Ca2+ chelators inhibited thrombin-induced eNOS phosphorylation and cGMP production. Moreover, infection of bovine aortic endothelial cell with adenovirus encoding dominant-negative mutants of protein kinase C (PKC)&agr; and PKC&dgr; or pretreatment of bovine aortic endothelial cells with PKC inhibitors revealed that PKC&dgr; is indispensable for thrombin-induced eNOS phosphorylation and activation. From these data, we concluded that thrombin induces the Ser1179 phosphorylation-dependent eNOS activation through a Ca2+-dependent, PKC&dgr;-sensitive, but phosphatidylinositol 3-kinase/Akt-independent pathway.

title: ShcA and Grb2 mediate polyoma middle T antigen‐induced endothelial transformation and Gab1 tyrosine phosphorylation
abstract: Middle T antigen (PymT) is the principal transforming component of polyomavirus, and rapidly induces hemangiomas in neonatal mice. PymT, a membrane‐associated scaffold, recruits and activates Src family tyrosine kinases, and, once tyrosine phosphorylated, binds proteins with PTB and SH2 domains such as ShcA, phosphatidylinositol 3‐kinase (PI3K) and phospholipase Cγ‐1 (PLCγ‐1). To explore the pathways required for endothelial transformation in vivo, we introduced PymT mutant forms into mice. PymT variants unable to bind PI3K and PLCγ‐1 directly induced hemangiomas similarly to wild type, but a mutant unable to bind ShcA was transformation compromised. Requirement for a ShcA PTB domain‐ binding site was suppressed by replacing this motif in PymT with YXN sequences, which bind the Grb2 SH2 domain upon phosphorylation. Surprisingly, PymT recruitment of ShcA and Grb2 correlated with PI3K activation. PymT mimics activated receptor tyrosine kinases by forming a ShcA—Grb2—Gab1 complex, thus inducing Gab1 tyrosine phosphorylation, which itself is associated with PI3K. Therefore, PymT activation of ShcA–Grb2 signaling is critical for endothelial transformation, and PymT can stimulate Grb2 signaling to both the MAP kinase and PI3K pathways.

title: Polyoma middle T antigen activates the Ser/Thr kinase Akt in a PI3-kinase-dependent manner.
abstract: Polyoma middle T antigen (PMT) was originally identified as the tumorigenic component of the polyomavirus genome. To investigate whether the serine/ threonine kinase Akt/PKB, which is the proto-oncogene transduced by the transforming AKT8 retrovirus, is activated by PMT, 3T3-L1 fibroblasts were stably transfected with wild type PMT. PMT expression accelerated glucose transport and increased phosphorylation of p70 S6-kinase and MAPK. PMT expression also stimulated Akt kinase activity 7 fold as compared to untreated, mock infected cells. This stimulation rivaled that obtained following insulin treatment of both mock and PMT infected cells. Akt activation and phosphorylation were eliminated in a PMT mutant incapable of interacting with PI3-kinase, but not one which does not interact with Shc, and correlated closely to the amount of PI3-kinase activity in anti-phosphotyrosine immunoprecipitates. These results indicate that the PI3-kinase pathway is requisite, but the Shc pathway is dispensable, for Akt activation. The studies further suggest that Akt may participate in PMT and PI3-kinase's regulation of cellular transformation and tumorigenesis.

title: Akt1 regulates pathological angiogenesis, vascular maturation and permeability in vivo
abstract: Akt kinases control essential cellular functions, including proliferation, apoptosis, metabolism and transcription, and have been proposed as promising targets for treatment of angiogenesis-dependent pathologies, such as cancer and ischemic injury. But their precise roles in neovascularization remain elusive. Here we show that Akt1 is the predominant isoform in vascular cells and describe the unexpected consequences of Akt1 knockout on vascular integrity and pathological angiogenesis. Angiogenic responses in three distinct in vivo models were enhanced in Akt1−/− mice; these enhanced responses were associated with impairment of blood vessel maturation and increased vascular permeability. Although impaired vascular maturation in Akt1−/− mice may be attributed to reduced activation of endothelial nitric oxide synthase (eNOS), the major phenotypic changes in vascular permeability and angiogenesis were linked to reduced expression of two endogenous vascular regulators, thrombospondins 1 (TSP-1) and 2 (TSP-2). Re-expression of TSP-1 and TSP-2 in mice transplanted with wild-type bone marrow corrected the angiogenic abnormalities in Akt1−/− mice. These findings establish a crucial role of an Akt-thrombospondin axis in angiogenesis.