Direct stimulation of Vav guanine nucleotide exchange activity for Ras by phorbol esters and diglycerides.

We recently identified Vav as a Ras-activating guanine nucleotide exchange factor (GEF) stimulated by a T-cell antigen receptor-coupled protein tyrosine kinase (PTK). Here, we describe a novel, protein kinase-independent alternative pathway of Vav activation. Phorbol ester, 1,2-diacylglycerol, or ceramide treatment of intact T cells, Vav immunoprecipitates, or partially purified Vav generated by in vitro translation or COS-1 cell transfection stimulated the Ras exchange activity of Vav in the absence of detectable tyrosine phosphorylation. GEF activity of gel-purified Vav was similarly stimulated by phorbol myristate acetate (PMA). Stimulation was resistant to PTK and protein kinase C inhibitors but was blocked by calphostin, a PMA and diacylglycerol antagonist. In vitro-translated Vav lacking its cysteine-rich domain, or mutated at a single cysteine residue within this domain (C528A), was not stimulated by PMA but was fully activated by p56lck. This correlated with increased binding of radiolabeled phorbol ester to COS-1 cells expressing wild-type, but not C528A-mutated, Vav. Thus, Vav itself is a PMA-binding and -activated Ras GEF. Recombinant interleukin-1 alpha stimulated Vav via this pathway, suggesting that diglyceride-mediated Vav activation may couple PTK-independent receptors which stimulate production of lipid second messengers to Ras in hematopoietic cells.

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In Vitro Guanine Nucleotide Exchange Activity of DHR‐2/DOCKER/CZH2 Domains

Methods in Enzymology - Regulators and Effectors of Small GTPases: Rho Family ◽

10.1016/s0076-6879(06)06004-6 ◽

2006 ◽

pp. 41-57 ◽

Cited By ~ 28

Author(s):

Jean‐François Côté ◽

Kristiina Vuori

Keyword(s):

Guanine Nucleotide ◽

Nucleotide Exchange ◽

Guanine Nucleotide Exchange ◽

Exchange Activity

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Activation of Ras in vitro and in intact fibroblasts by the Vav guanine nucleotide exchange protein.

Molecular and Cellular Biology ◽

10.1128/mcb.14.2.906 ◽

1994 ◽

Vol 14 (2) ◽

pp. 906-913 ◽

Cited By ~ 45

Author(s):

E Gulbins ◽

K M Coggeshall ◽

C Langlet ◽

G Baier ◽

N Bonnefoy-Berard ◽

...

Keyword(s):

Map Kinases ◽

Guanine Nucleotide ◽

Nucleotide Exchange ◽

3T3 Cells ◽

Transfected Cells ◽

Guanine Nucleotide Exchange ◽

Nih 3T3 ◽

Exchange Activity ◽

Nih 3T3 Cells

We recently identified Vav, the product of the vav proto-oncogene, as a guanine nucleotide exchange factor (GEF) for Ras. Vav is enzymatically activated by lymphocyte antigen receptor-coupled protein tyrosine kinases or independently by diglycerides. To further evaluate the physiological role of Vav, we assessed its GDP-GTP exchange activity against several Ras-related proteins in vitro and determined whether Vav activation in transfected NIH 3T3 fibroblasts correlates with the activity status of Ras and mitogen-activated protein (MAP) kinases. In vitro translated purified Vav activated by phorbol myristate acetate (PMA) or phosphorylation with recombinant p56lck displayed GEF activity against Ras but not against recombinant RacI, RacII, Ral, or RhoA proteins. Expression of vav or proto-vav in stably transfected NIH 3T3 cells led to a approximately 10-fold increase in basal or PMA-stimulated Ras exchange activity, respectively, in total-cell lysates and Vav immunoprecipitates. Elevated GEF activity was paralleled in each case by a significant increase in the proportion of active, GTP-bound Ras. PMA had a minimal effect on the low Ras. GTP level in untransfected control fibroblasts but increased it from 20 to 37% in proto-vav-transfected cells. vav-transfected cells displayed a constitutively elevated Ras. GTP level (35%), which was not increased further by PMA treatment. MAP kinases, known downstream intermediates in Ras-dependent signaling pathways, similarly exhibited increased basal or PMA-stimulated activity in Vav-expressing cells by comparison with normal NIH 3T3 cells. These results demonstrate a physiologic interaction between Vav and its target, Ras, leading to MAP kinase activation.

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Regulation of Sos Activity by Intramolecular Interactions

Molecular and Cellular Biology ◽

10.1128/mcb.18.2.880 ◽

1998 ◽

Vol 18 (2) ◽

pp. 880-886 ◽

Cited By ~ 69

Author(s):

Senena Corbalan-Garcia ◽

Steluta M. Margarit ◽

Dalia Galron ◽

Shao-song Yang ◽

Dafna Bar-Sagi

Keyword(s):

Catalytic Activity ◽

Tyrosine Kinases ◽

Catalytic Domain ◽

Intramolecular Interactions ◽

Carboxyl Terminus ◽

Guanine Nucleotide ◽

Nucleotide Exchange ◽

Guanine Nucleotide Exchange ◽

Exchange Activity

ABSTRACT The guanine nucleotide exchange factor Sos mediates the coupling of receptor tyrosine kinases to Ras activation. To investigate the mechanisms that control Sos activity, we have analyzed the contribution of various domains to its catalytic activity. Using human Sos1 (hSos1) truncation mutants, we show that Sos proteins lacking either the amino or the carboxyl terminus domain, or both, display a guanine nucleotide exchange activity that is significantly higher compared with that of the full-length protein. These results demonstrate that both the amino and the carboxyl terminus domains of Sos are involved in the negative regulation of its catalytic activity. Furthermore, in vitro Ras binding experiments suggest that the amino and carboxyl terminus domains exert negative allosteric control on the interaction of the Sos catalytic domain with Ras. The guanine nucleotide exchange activity of hSos1 was not augmented by growth factor stimulation, indicating that Sos activity is constitutively maintained in a downregulated state. Deletion of both the amino and the carboxyl terminus domains was sufficient to activate the transforming potential of Sos. These findings suggest a novel negative regulatory role for the amino terminus domain of Sos and indicate a cooperation between the amino and the carboxyl terminus domains in the regulation of Sos activity.

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Activation of Ras in vitro and in intact fibroblasts by the Vav guanine nucleotide exchange protein

Molecular and Cellular Biology ◽

10.1128/mcb.14.2.906-913.1994 ◽

1994 ◽

Vol 14 (2) ◽

pp. 906-913

Author(s):

E Gulbins ◽

K M Coggeshall ◽

C Langlet ◽

G Baier ◽

N Bonnefoy-Berard ◽

...

Keyword(s):

Map Kinases ◽

Guanine Nucleotide ◽

Nucleotide Exchange ◽

3T3 Cells ◽

Transfected Cells ◽

Guanine Nucleotide Exchange ◽

Nih 3T3 ◽

Exchange Activity ◽

Nih 3T3 Cells

We recently identified Vav, the product of the vav proto-oncogene, as a guanine nucleotide exchange factor (GEF) for Ras. Vav is enzymatically activated by lymphocyte antigen receptor-coupled protein tyrosine kinases or independently by diglycerides. To further evaluate the physiological role of Vav, we assessed its GDP-GTP exchange activity against several Ras-related proteins in vitro and determined whether Vav activation in transfected NIH 3T3 fibroblasts correlates with the activity status of Ras and mitogen-activated protein (MAP) kinases. In vitro translated purified Vav activated by phorbol myristate acetate (PMA) or phosphorylation with recombinant p56lck displayed GEF activity against Ras but not against recombinant RacI, RacII, Ral, or RhoA proteins. Expression of vav or proto-vav in stably transfected NIH 3T3 cells led to a approximately 10-fold increase in basal or PMA-stimulated Ras exchange activity, respectively, in total-cell lysates and Vav immunoprecipitates. Elevated GEF activity was paralleled in each case by a significant increase in the proportion of active, GTP-bound Ras. PMA had a minimal effect on the low Ras. GTP level in untransfected control fibroblasts but increased it from 20 to 37% in proto-vav-transfected cells. vav-transfected cells displayed a constitutively elevated Ras. GTP level (35%), which was not increased further by PMA treatment. MAP kinases, known downstream intermediates in Ras-dependent signaling pathways, similarly exhibited increased basal or PMA-stimulated activity in Vav-expressing cells by comparison with normal NIH 3T3 cells. These results demonstrate a physiologic interaction between Vav and its target, Ras, leading to MAP kinase activation.

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TcR and TcR-CD28 Engagement of Protein Kinase B (PKB/AKT) and Glycogen Synthase Kinase-3 (GSK-3) Operates Independently of Guanine Nucleotide Exchange Factor VAV-1

Journal of Biological Chemistry ◽

10.1074/jbc.m604878200 ◽

2006 ◽

Vol 281 (43) ◽

pp. 32385-32394 ◽

Cited By ~ 28

Author(s):

Joanne E. Wood ◽

Helga Schneider ◽

Christopher E. Rudd

Keyword(s):

T Cells ◽

Protein Kinase ◽

Glycogen Synthase ◽

Protein Kinase B ◽

Guanine Nucleotide Exchange Factor ◽

Glycogen Synthase Kinase 3 ◽

Guanine Nucleotide ◽

Nucleotide Exchange ◽

Guanine Nucleotide Exchange ◽

Nucleotide Exchange Factor

TcRζ/CD3 and TcRζ/CD3-CD28 signaling requires the guanine nucleotide exchange factor (GEF) Vav-1 as well as the activation of phosphatidylinositol 3-kinase, protein kinase B (PKB/AKT), and its inactivation of glycogen synthase kinase-3 (GSK-3). Whether these two pathways are connected or operate independently of each other in T-cells has been unclear. Here, we report that anti-CD3 and anti-CD3/CD28 can induce PKB and GSK-3α phosphorylation in the Vav-1–/– Jurkat cell line J. Vav.1 and in primary CD4-positive Vav-1–/– T-cells. Reduced GSK-3α phosphorylation was observed in Vav-1,2,3–/– T-cells together with a complete loss of FOXO1 phosphorylation. Furthermore, PKB and GSK-3 phosphorylation was unperturbed in the presence of GEF-inactive Vav-1 that inhibited interleukin-2 gene activation and a form of Src homology 2 domain-containing lymphocytic protein of 76-kDa (SLP-76) that is defective in binding to Vav-1. The pathway also was intact under conditions of c-Jun N-terminal kinase (JNK) inhibition and disruption of the actin cytoskeleton by cytochalasin D. Both events are down-stream targets of Vav-1. Overall, our findings indicate that the TcR and TcR-CD28 driven PKB-GSK-3 pathway can operate independently of Vav-1 in T-cells.

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Essential Role of Vav Family Guanine Nucleotide Exchange Factors in EphA Receptor-Mediated Angiogenesis

Molecular and Cellular Biology ◽

10.1128/mcb.02215-05 ◽

2006 ◽

Vol 26 (13) ◽

pp. 4830-4842 ◽

Cited By ~ 84

Author(s):

Sonja G. Hunter ◽

Guanglei Zhuang ◽

Dana Brantley-Sieders ◽

Wojciech Swat ◽

Christopher W. Cowan ◽

...

Keyword(s):

Guanine Nucleotide Exchange Factors ◽

Guanine Nucleotide ◽

Nucleotide Exchange ◽

Guanine Nucleotide Exchange ◽

Nucleotide Exchange Factors ◽

Rac1 Gtpase ◽

Rho Family ◽

Epha Receptor

ABSTRACT Angiogenesis, the process by which new blood vessels are formed from preexisting vasculature, is critical for vascular remodeling during development and contributes to the pathogenesis of diseases such as cancer. Prior studies from our laboratory demonstrate that the EphA2 receptor tyrosine kinase is a key regulator of angiogenesis in vivo. The EphA receptor-mediated angiogenic response is dependent on activation of Rho family GTPase Rac1 and is regulated by phosphatidylinositol 3-kinase. Here we report the identification of Vav2 and Vav3 as guanine nucleotide exchange factors (GEFs) that link the EphA2 receptor to Rho family GTPase activation and angiogenesis. Ephrin-A1 stimulation recruits the binding of Vav proteins to the activated EphA2 receptor. The induced association of EphA receptor and Vav proteins modulates the activity of Vav GEFs, leading to activation of Rac1 GTPase. Overexpression of either Vav2 or Vav3 in primary microvascular endothelial cells promotes Rac1 activation, cell migration, and assembly in response to ephrin-A1 stimulation. Conversely, loss of Vav2 and Vav3 GEFs inhibits Rac1 activation and ephrin-A1-induced angiogenic responses both in vitro and in vivo. In addition, embryonic fibroblasts derived from Vav2−/− Vav3−/− mice fail to spread on an ephrin-A1-coated surface and exhibit a significant decrease in the formation of ephrin-A1-induced lamellipodia and filopodia. These findings suggest that Vav GEFs serve as a molecular link between EphA2 receptors and the actin cytoskeleton and provide an important mechanism for EphA2-mediated angiogenesis.

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Expression of mutant eukaryotic initiation factor 2 alpha subunit (eIF-2 alpha) reduces inhibition of guanine nucleotide exchange activity of eIF-2B mediated by eIF-2 alpha phosphorylation

Molecular and Cellular Biology ◽

10.1128/mcb.14.7.4546-4553.1994 ◽

1994 ◽

Vol 14 (7) ◽

pp. 4546-4553

Author(s):

K V Ramaiah ◽

M V Davies ◽

J J Chen ◽

R J Kaufman

Keyword(s):

Protein Synthesis ◽

Initiation Factor ◽

Guanine Nucleotide ◽

Eukaryotic Initiation Factor ◽

Nucleotide Exchange ◽

Wild Type ◽

Guanine Nucleotide Exchange ◽

Initiation Factor 2 ◽

Exchange Activity

The inhibition of protein synthesis that occurs upon phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2 alpha) at serine 51 correlates with reduced guanine nucleotide exchange activity of eIF-2B in vivo and inhibition of eIF-2B activity in vitro, although it is not known if phosphorylation is the cause of the reduced eIF-2B activity in vivo. To characterize the importance of eIF-2 alpha phosphorylation in the regulation of eIF-2B activity, we studied the overexpression of mutant eIF-2 alpha subunits in which serine 48 or 51 was replaced by an alanine (48A or 51A mutant). Previous studies demonstrated that the 51A mutant was resistant to phosphorylation, whereas the 48A mutant was a substrate for phosphorylation. Additionally, expression of either mutant partially protected Chinese hamster ovary (CHO) cells from the inhibition of protein synthesis in response to heat shock treatment (P. Murtha-Riel, M. V. Davies, J. B. Scherer, S. Y. Choi, J. W. B. Hershey, and R. J. Kaufman, J. Biol. Chem. 268:12946-12951, 1993). In this study, we show that eIF-2B activity was inhibited in parental CHO cell extracts upon addition of purified reticulocyte heme-regulated inhibitor (HRI), an eIF-2 alpha kinase that phosphorylates Ser-51. Preincubation with purified HRI also reduced the eIF-2B activity in extracts from cells overexpressing wild-type eIF-2 alpha. In contrast, the eIF-2B activity was not readily inhibited in extracts from cells overexpressing either the eIF-2 alpha 48A or 51A mutant. In addition, eIF-2B activity was decreased in extracts prepared from heat-shocked cells overexpressing wild-type eIF-2 alpha, whereas the decrease in eIF-2B activity was less in heat-shocked cells overexpressing either mutant 48A or mutant 51A. While the phosphorylation at serine 51 in eIF-2 alpha impairs the eIF-2B activity, we propose that serine 48 acts to maintain a high affinity between phosphorylated eIF-2 alpha and eIF-2B, thereby inactivating eIF-2B activity. These findings support the hypothesis that phosphorylation of eIF-2 alpha inhibits protein synthesis directly through reducing eIF-2B activity and emphasize the importance of both serine 48 and serine 51 in the interaction with eIF-2B and regulation of eIF-2B activity.

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A conserved C-terminal domain of EFA6-family ARF6-guanine nucleotide exchange factors induces lengthening of microvilli-like membrane protrusions

Journal of Cell Science ◽

10.1242/jcs.115.14.2867 ◽

2002 ◽

Vol 115 (14) ◽

pp. 2867-2879 ◽

Cited By ~ 4

Author(s):

Valérie Derrien ◽

Carole Couillault ◽

Michel Franco ◽

Stéphanie Martineau ◽

Philippe Montcourrier ◽

...

Keyword(s):

Amino Acid ◽

Coiled Coil ◽

Terminal Region ◽

Guanine Nucleotide ◽

Nucleotide Exchange ◽

Ph Domain ◽

Exchange Factor ◽

Guanine Nucleotide Exchange ◽

Sec7 Domain

We recently reported the identification of EFA6 (exchange factor for ARF6), a brain-specific Sec7-domain-containing guanine nucleotide exchange factor that works specifically on ARF6. Here, we have characterized the product of a broadly expressed gene encoding a novel 1056 amino-acid protein that we have named EFA6B. We show that EFA6B, which contains a Sec7 domain that is highly homologous to EFA6, works as an ARF6-specific guanine exchange factor in vitro. Like EFA6, which will be referred to as EFA6A from now on, EFA6B is involved in membrane recycling and colocalizes with ARF6 in actin-rich membrane ruffles and microvilli-like protrusions on the dorsal cell surface in transfected baby hamster kidney cells. Strikingly, homology between EFA6A and EFA6B is not limited to the Sec7 domain but extends to an adjacent pleckstrin homology (PH) domain and a ∼150 amino-acid C-terminal region containing a predicted coiled coil motif. Association of EFA6A with membrane ruffles and microvilli-like structures depends on the PH domain, which probably interacts with phosphatidylinositol 4,5-biphosphate. Moreover, we show that overexpression of the PH domain/C-terminal region of EFA6A or EFA6B in the absence of the Sec7 domain promotes lengthening of dorsal microvillar protrusions. This morphological change requires the integrity of the coiled-coil motif. Lastly, database analysis reveals that the EFA6-family comprises at least four members in humans and is conserved in multicellular organisms throughout evolution. Our results suggest that EFA6 family guanine exchange factors are modular proteins that work through the coordinated action of the catalytic Sec7 domain to promote ARF6 activation, through the PH domain to regulate association with specific subdomains of the plasma membrane and through the C-terminal region to control actin cytoskeletal reorganization.

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