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1.
Cell Signal ; 19(6): 1249-57, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17314030

RESUMO

Arap3 is a phosphoinositide (PI) 3 kinase effector that serves as a GTPase activating protein (GAP) for both Arf and Rho G-proteins. The protein has multiple pleckstrin homology (PH) domains that bind preferentially phosphatidyl-inositol-3,4,5-trisphosphate (PI(3,4,5,)P3) to induce translocation of Arap3 to the plasma membrane upon PI3K activation. Arap3 also contains a Ras association (RA) domain that interacts with the small G-protein Rap1 and a sterile alpha motif (SAM) domain of unknown function. In a yeast two-hybrid screen for new interaction partners of Arap3, we identified the PI 5'-phosphatase SHIP2 as an interaction partner of Arap3. The interaction between Arap3 and SHIP2 was observed with endogenous proteins and shown to be mediated by the SAM domain of Arap3 and SHIP2. In vitro, these two domains show specificity for a heterodimeric interaction. Since it was shown previously that Arap3 has a higher affinity for PI(3,4,5,)P3 than for PI(3,4)P2, we propose that the SAM domain of Arap3 can function to recruit a negative regulator of PI3K signaling into the effector complex.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Ativadoras de GTPase/química , Proteínas Ativadoras de GTPase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/metabolismo , Animais , Dimerização , Células HeLa , Humanos , Camundongos , Complexos Multiproteicos/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases , Ligação Proteica , Estrutura Terciária de Proteína , Especificidade por Substrato , Técnicas do Sistema de Duplo-Híbrido , Proteínas rap1 de Ligação ao GTP/metabolismo
2.
Curr Biol ; 14(15): 1380-4, 2004 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-15296756

RESUMO

Rho and Arf family small GTPases are well-known regulators of cellular actin dynamics. We recently identified ARAP3, a member of the ARAP family of dual GTPase activating proteins (GAPs) for Arf and Rho family GTPases, in a screen for PtdIns(3,4,5)P(3) binding proteins. PtdIns(3,4,5)P(3) is the lipid product of class I phosphoinositide 3OH-kinases (PI3Ks) and is a signaling molecule used by growth factor receptors and integrins in the regulation of cell dynamics. We report here that as a Rho GAP, ARAP3 prefers RhoA as a substrate and that it can be activated in vitro by the direct binding of Rap proteins to a neighbouring Ras binding domain (RBD). This activation by Rap is GTP dependent and specific for Rap versus other Ras family members. We found no evidence for direct regulation of ARAP3's Rho GAP activity by PtdIns(3,4,5)P(3) in vitro, but PI3K activity was required for activation by Rap in a cellular context, suggesting that PtdIns(3,4,5)P(3)-dependent translocation of ARAP3 to the plasma membrane may be required for further activation by Rap. Our results indicate that ARAP3 is a Rap-effector that plays an important role in mediating PI3K-dependent crosstalk between Ras, Rho, and Arf family small GTPases.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas rap de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Western Blotting , Células Cultivadas , Testes de Precipitina , Proteínas Ativadoras de ras GTPase/metabolismo
3.
Methods Enzymol ; 406: 91-103, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16472652

RESUMO

ARAP3 is a dual Arf and Rho GTPase activating protein (GAP) that was identified from pig leukocyte cytosol using a phosphatidylinositol-(3,4,5)-trisphosphate (PtdIns[3,4,5]P3) affinity matrix in a targeted proteomics study. ARAP3's domain structure includes five PH domains, an Arf GAP domain, three ankyrin repeats, a Rho GAP domain, and a Ras association domain. ARAP3 is a PtdIns(3,4,5)P3-dependent GAP for Arf6 both in vitro and in vivo. It acts as a Rap-GTP-activated RhoA GAP in vitro, and this activation depends on a direct interaction between ARAP3 and Rap-GTP; in vivo PtdIns(3,4,5)P3 seems to be required to allow ARAP3's activation as a RhoA GAP by Rap-GTP. Overexpression of ARAP3 in pig aortic endothelial (PAE) cells causes the PI3K-dependent loss of adhesion to the substratum and interferes with lamellipodium formation. This overexpression phenotype depends on ARAP3's intact abilities to bind PtdIns(3,4,5)P3, to interact with Rap-GTP, and to be a catalytically active RhoA and Arf6 GAP.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/isolamento & purificação , Proteínas Ativadoras de GTPase/isolamento & purificação , Proteínas Ativadoras de GTPase/metabolismo , Fator 6 de Ribosilação do ADP , Fatores de Ribosilação do ADP/análise , Fatores de Ribosilação do ADP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Citosol/química , Escherichia coli/enzimologia , Leucócitos/química , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas Recombinantes/isolamento & purificação , Spodoptera , Suínos , Proteínas ras/isolamento & purificação , Proteínas rho de Ligação ao GTP/isolamento & purificação
4.
FEBS Lett ; 572(1-3): 172-6, 2004 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-15304343

RESUMO

We have identified a new guanine-nucleotide exchange factor, P-Rex2, and cloned it from human skeletal muscle and brain libraries. It has widespread tissue distribution but is not expressed in neutrophils. P-Rex2 is a 183 kDa protein that activates the small GTPase Rac and is regulated by phosphatidylinositol (3,4,5)-trisphosphate and the beta gamma subunits of heterotrimeric G proteins in vitro and in vivo. P-Rex2 has structure, activity and regulatory properties similar to P-Rex1 but has divergent tissue distribution, as P-Rex1 is mainly expressed in neutrophils. Together, they form an enzyme family capable of mediating Rac signalling downstream of G protein-coupled receptors and phosphoinositide 3-kinase.


Assuntos
Proteínas rac de Ligação ao GTP/metabolismo , Northern Blotting , Clonagem Molecular , Biblioteca Gênica , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Humanos , Inositol 1,4,5-Trifosfato/metabolismo , Especificidade de Órgãos , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas rac de Ligação ao GTP/genética
5.
J Cell Sci ; 119(Pt 3): 425-32, 2006 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-16418224

RESUMO

Rho and Arf family small GTPases control dynamic actin rearrangements and vesicular trafficking events. ARAP3 is a dual GAP for RhoA and Arf6 that is regulated by phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P(3)], a product of the phosphoinositide 3-kinase (PI3K) signalling pathway. To investigate the physiological function of ARAP3, we used an RNAi-based approach in an endothelial cell model. ARAP3-deficient cells showed increased activities of RhoA and Arf6. Phenotypically, they were more rounded than control counterparts and displayed very fine stress fibres. ARAP3-deficient cells were not capable of producing lamellipodia upon growth factor stimulation, a process known to depend on PI3K and Rac activities. Rac was transiently activated in stimulated ARAP3 RNAi cells although its cellular localisation was altered, a likely consequence of increased Arf6 activity. We conclude that ARAP3 recruitment to sites of elevated PtdIns(3,4,5)P(3) is crucial to allow localised inactivation of RhoA and cycling of Arf6, both of which are necessary to allow growth factor-stimulated formation of lamellipodia.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Células Endoteliais/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Pseudópodes/metabolismo , Transdução de Sinais/fisiologia , Fator 6 de Ribosilação do ADP , Fatores de Ribosilação do ADP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Animais , Transporte Biológico Ativo/fisiologia , Linhagem Celular , Vesículas Citoplasmáticas/metabolismo , Células Endoteliais/citologia , Proteínas Ativadoras de GTPase/deficiência , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo
6.
J Biol Chem ; 280(6): 4166-73, 2005 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-15545267

RESUMO

P-Rex1 is a guanine-nucleotide exchange factor (GEF) for the small GTPase Rac. We have investigated here the mechanisms of stimulation of P-Rex1 Rac-GEF activity by the lipid second messenger phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) and the Gbetagamma subunits of heterotrimeric G proteins. We show that a P-Rex1 mutant lacking the PH domain (DeltaPH) cannot be stimulated by PtdIns(3,4,5)P3, which implies that the PH domain confers PtdIns(3,4,5)P3 regulation of P-Rex1 Rac-GEF activity. Consistent with this, we found that PtdIns(3,4,5)P3 binds to the PH domain of P-Rex1 and that the DH/PH domain tandem is sufficient for PtdIns(3,4,5)P3-stimulated P-Rex1 activity. The Rac-GEF activities of the DeltaPH mutant and the DH/PH domain tandem can both be stimulated by Gbetagamma subunits, which infers that Gbetagamma subunits regulate P-Rex1 activity by binding to the catalytic DH domain. Deletion of the DEP, PDZ, or inositol polyphosphate 4-phosphatase homology domains has no major consequences on the abilities of either PtdIns(3,4,5)P3 or Gbetagamma subunits to stimulate P-Rex1 Rac-GEF activity. However, the presence of any of these domains impacts on the levels of basal and/or stimulated P-Rex1 Rac-GEF activity, suggesting that there are important functional interactions between the DH/PH domain tandem and the DEP, PDZ, and inositol polyphosphate 4-phosphatase homology domains of P-Rex1.


Assuntos
Subunidades beta da Proteína de Ligação ao GTP/química , Subunidades gama da Proteína de Ligação ao GTP/química , Fatores de Troca do Nucleotídeo Guanina/biossíntese , Fatores de Troca do Nucleotídeo Guanina/química , Animais , Linhagem Celular , Epitopos/química , Deleção de Genes , Regulação da Expressão Gênica , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Guanosina Difosfato/química , Humanos , Insetos , Lipídeos/química , Mutagênese , Mutação , Monoéster Fosfórico Hidrolases/química , Mutação Puntual , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas rac de Ligação ao GTP/química , Proteínas rac de Ligação ao GTP/fisiologia
7.
Biochem J ; 362(Pt 3): 725-31, 2002 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-11879201

RESUMO

Type IB phosphoinositide 3OH-kinase (PI3K) is activated by G-protein betagamma subunits (Gbetagammas). The enzyme is soluble and largely cytosolic in vivo. Its substrate, PtdIns(4,5)P(2), and the Gbetagammas are localized at the plasma membrane. We have addressed the mechanism by which Gbetagammas regulate the PI3K using an in vitro approach. We used sedimentation assays and surface plasmon resonance to determine association of type IB PI3K with lipid monolayers and vesicles of varying compositions, some of which had Gbetagammas incorporated. Association and dissociation rate constants were determined. Our results indicated that in an assay situation in vitro the majority of PI3K will be associated with lipid vesicles, irrespective of the presence or absence of Gbetagammas. In line with this, a constitutively active membrane-targeted PI3K construct could still be activated substantially by Gbetagammas in vitro. We conclude that Gbetagammas activate type IB PI3K by a mechanism other than translocation to the plasma membrane.


Assuntos
Subunidades beta da Proteína de Ligação ao GTP , Subunidades gama da Proteína de Ligação ao GTP , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Recombinantes/metabolismo , Animais , Células COS , Linhagem Celular , Chlorocebus aethiops , Dimerização , Cinética , Subunidades Proteicas , Spodoptera , Ressonância de Plasmônio de Superfície , Transfecção
8.
J Biol Chem ; 279(43): 44763-74, 2004 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-15310762

RESUMO

Sphingosine kinase 1 (SK1) phosphorylates sphingosine to generate sphingosine 1-phosphate (S1P). Because both substrate and product of the enzyme are potentially important signaling molecules, the regulation of SK1 is of considerable interest. We report that SK1, which is ordinarily a cytosolic enzyme, translocates in vivo and in vitro to membrane compartments enriched in phosphatidic acid (PA), the lipid product of phospholipase D. This translocation depends on direct interaction of SK1 with PA, because recombinant purified enzyme shows strong affinity for pure PA coupled to Affi-Gel. The SK1-PA interaction maps to the C terminus of SK1 and is independent of catalytic activity or of the diacylglycerol kinase-like domain of the enzyme. Thus SK1 constitutes a novel, physiologically relevant PA effector.


Assuntos
Ácidos Fosfatídicos/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/fisiologia , Animais , Transporte Biológico , Células CHO , Células COS , Domínio Catalítico , Linhagem Celular , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Cricetinae , Citosol/metabolismo , Diacilglicerol Quinase/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Immunoblotting , Metabolismo dos Lipídeos , Lipídeos/química , Lipossomos/metabolismo , Microscopia de Fluorescência , Modelos Biológicos , Fosfolipase D/metabolismo , Plasmídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Ratos , Proteínas Recombinantes/química , Transdução de Sinais
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