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1.
Curr Biol ; 11(12): 981-6, 2001 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-11448776

RESUMO

Ca(2+) is a universal second messenger that is critical for cell growth and is intimately associated with many Ras-dependent cellular processes such as proliferation and differentiation. Ras is a small GTP binding protein that operates as a molecular switch regulating the control of gene expression, cell growth, and differentiation through a pathway from receptors to mitogen-activated protein kinases (MAPKs). A role for intracellular Ca(2+) in the activation of Ras has been previously demonstrated, e.g., via the nonreceptor tyrosine kinase PYK2 and by Ca(2+)/calmodulin-dependent guanine nucleotide exchange factors (GEFs) such as Ras-GRF; however, there is no Ca(2+)-dependent mechanism for direct inactivation. An important advance toward greater understanding of the complex coordination within the Ras-signaling network is the spatio-temporal analysis of signaling events in vivo. Here, we describe the identification of CAPRI (Ca(2+)-promoted Ras inactivator), a Ca(2+)-dependent Ras GTPase-activating protein (GAP) that switches off the Ras-MAPK pathway following a stimulus that elevates intracellular Ca(2+). Analysis of the spatio-temporal dynamics of CAPRI indicates that Ca(2+) regulates the GAP by a fast C2 domain-dependent translocation mechanism.


Assuntos
Cálcio/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Ativadoras de ras GTPase/metabolismo , Proteínas ras/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Meios de Cultura Livres de Soro , Ativadores de GTP Fosfo-Hidrolase/metabolismo , Genes Reporter , Histamina/farmacologia , Humanos , Immunoblotting , Ionomicina/farmacologia , Ionóforos/farmacologia , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência
2.
Curr Biol ; 9(5): 265-8, 1999 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-10074452

RESUMO

GAP1(m) is a member of the GAP1 family of Ras GTPase-activating proteins (GAPs) [1]. In vitro, it has been shown to bind inositol 1, 3,4,5-tetrakisphosphate (IP4), the water-soluble inositol head group of the lipid second messenger phosphatidylinositol 3,4, 5-trisphosphate (PIP3) [2] [3]. This has led to the suggestion that GAP1(m) might function as a PIP3 receptor in vivo [4]. Here, using rat pheochromocytoma PC12 cells transiently transfected with a plasmid expressing a chimera of green fluorescent protein fused to GAP1(m) (GFP-GAP1(m)), we show that epidermal growth factor (EGF) induces a rapid (less than 60 seconds) recruitment of GFP-GAP1(m) from the cytosol to the plasma membrane. This recruitment required a functional GAP1(m) pleckstrin homology (PH) domain, because a specific point mutation (R629C) in the PH domain that inhibits IP4 binding in vitro [5] totally blocked EGF-induced GAP1(m) translocation. Furthermore, the membrane translocation was dependent on PI 3-kinase, and the time course of translocation paralleled the rate by which EGF stimulates the generation of plasma membrane PIP3 [6]. Significantly, the PIP3-induced recruitment of GAP1(m) did not appear to result in any detectable enhancement in its basal Ras GAP activity. From these results, we conclude that GAP1(m) binds PIP3 in vivo, and it is recruited to the plasma membrane, but does not appear to be activated, following agonist stimulation of PI 3-kinase.


Assuntos
Fosfatos de Fosfatidilinositol/metabolismo , Proteínas/metabolismo , Proteínas Ativadoras de ras GTPase , Animais , Células COS , Células PC12 , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas/genética , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismo
4.
Biochem Soc Trans ; 34(Pt 5): 846-50, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17052212

RESUMO

Ras proteins are binary switches that, by cycling between inactive GDP-bound and active GTP-bound conformations, regulate multiple cellular signalling pathways including those that control cell growth, differentiation and survival. Approximately 30% of all human tumours express Ras-containing oncogenic mutations that lock the protein into a constitutively active conformation. The activation status of Ras is regulated by two groups of proteins: GEFs (guanine nucleotide-exchange factors) bind to Ras and enhance the exchange of GDP for GTP, thereby activating it, whereas GAPs (GTPase-activating proteins) inactivate Ras by binding to the GTP-bound form and enhancing the hydrolysis of the bound nucleotide back to GDP. In this review, we focus on a group of key regulators of Ras inactivation, the GAP1 family of Ras-GAPs. The members of this family are GAP1m, GAP1IP4BP, CAPRI (Ca2+-promoted Ras inactivator) and RASAL (Ras-GTPase-activating-like protein) and, as we will discuss, they are emerging as important modulators of Ras and small GTPase signalling that are subject to regulation by a diverse array of events and second messenger signals.


Assuntos
GTP Fosfo-Hidrolases/metabolismo , Proteínas Ativadoras de ras GTPase/metabolismo , Humanos , Interferência de RNA , RNA Interferente Pequeno/genética , Transdução de Sinais/fisiologia , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas Ativadoras de ras GTPase/genética
5.
J Biol Chem ; 275(36): 28261-8, 2000 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-10869341

RESUMO

The group I family of pleckstrin homology (PH) domains are characterized by their inherent ability to specifically bind phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) and its corresponding inositol head-group inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P(4)). In vivo this interaction results in the regulated plasma membrane recruitment of cytosolic group I PH domain-containing proteins following agonist-stimulated PtdIns(3,4,5)P(3) production. Among group I PH domain-containing proteins, the Ras GTPase-activating protein GAP1(IP4BP) is unique in being constitutively associated with the plasma membrane. Here we show that, although the GAP1(IP4BP) PH domain interacts with PtdIns(3,4, 5)P(3), it also binds, with a comparable affinity, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) (K(d) values of 0.5 +/- 0.2 and 0.8 +/- 0.5 microm, respectively). Intriguingly, whereas this binding site overlaps with that for Ins(1,3,4,5)P(4), consistent with the constitutive plasma membrane association of GAP1(IP4BP) resulting from its PH domain-binding PtdIns(4,5)P(2), we show that in vivo depletion of PtdIns(4,5)P(2), but not PtdIns(3,4,5)P(3), results in dissociation of GAP1(IP4BP) from this membrane. Thus, the Ins(1,3,4,5)P(4)-binding PH domain from GAP1(IP4BP) defines a novel class of group I PH domains that constitutively targets the protein to the plasma membrane and may allow GAP1(IP4BP) to be regulated in vivo by Ins(1,3,4,5)P(4) rather than PtdIns(3,4,5)P(3).


Assuntos
Membrana Celular/metabolismo , Fosfatos de Inositol/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/metabolismo , Substituição de Aminoácidos , Animais , Sítios de Ligação , Células COS , Núcleo Celular/metabolismo , Células HeLa , Humanos , Lipossomos , Mutagênese Sítio-Dirigida , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Frações Subcelulares/metabolismo , Sacarose , Transfecção
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