Structural Basis of RasGRP Binding to High-Affinity PKC Ligands

Rong, Suo Bao; Enyedy, Istvan; Qiao, Lixin; Zhao, Long; Ma, Dawei; Pearce, Larry L.; Lorenzo, Patricia S.; Stone, James C.; Blumberg, Peter M.; Wang, Shaomeng; Kozikowski, Alan P.

doi:10.1021/jm010422z

Cited by 22 publications

(17 citation statements)

References 48 publications

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“…Although PKC isozymes and RasGRP possess similar C1 domains, there are differences in their ligand recognition properties (Lorenzo et al, 2000;Shao et al, 2001;Reuther et al, 2002;Rong et al, 2002;Madani et al, 2004;Pu et al, 2005). These differences may be responsible for the distinct dose-response profiles observed for PMA-induced Erk activation in sensitive versus resistant EL4 cells (Ku and Meier, 2000), as well as for the differential effects of various PKC activators on other signaling events in these cells (Sansbury et al, 1997).…”

Section: Discussionmentioning

confidence: 99%

RasGRP1 Confers the Phorbol Ester-Sensitive Phenotype to EL4 Lymphoma Cells

Han¹,

Knoepp²,

Hallman³

et al. 2007

Molecular Pharmacology

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The murine EL4 lymphoma cell line exists in variants that are either sensitive or resistant to the tumor promoter phorbol 12-myristate 13-acetate (PMA). In sensitive EL4 cells, PMA causes robust Erk mitogen-activated protein kinase activation that results in growth arrest. In resistant cells, PMA induces minimal Erk activation, without growth arrest. PMA stimulates IL-2 production in sensitive, but not resistant, cells. The role of RasGRP1, a PMA-activated guanine nucleotide exchange factor for Ras, in EL4 phenotype was examined. Endogenous RasGRP1 protein is expressed at much higher levels in sensitive than in resistant cells. PMA-induced Ras activation is observed in sensitive cells but not in resistant cells lacking Ras-GRP1. PMA induces down-regulation of RasGRP1 protein in sensitive cells but increases RasGRP1 in resistant cells. Transfection of RasGRP1 into resistant cells enhances PMA-induced Erk activation. In the reverse experiment, introduction of small interfering RNA (siRNA) for RasGRP1 suppresses PMA-induced Ras and Erk activations in sensitive cells. Sensitive cells incubated with siRNA for RasGRP1 exhibit the PMA-resistant phenotype, in that they are able to proliferate in the presence of PMA and do not secrete IL-2 when stimulated with PMA. These studies indicate that the PMA-sensitive phenotype, as previously defined for the EL4 cell line, is conferred by endogenous expression of RasGRP1 protein.EL4, a cell line originated from a carcinogen-induced murine thymoma, provides a unique model system for the study of phorbol ester response and resistance. Responses of sensitive "wild-type" (WT) EL4 cells to phorbol 12-myristate 13-acetate (PMA) include protein kinase C (PKC) activation

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Section: Discussionmentioning

confidence: 99%

RasGRP1 Confers the Phorbol Ester-Sensitive Phenotype to EL4 Lymphoma Cells

Han¹,

Knoepp²,

Hallman³

et al. 2007

Molecular Pharmacology

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“…C1RasGRP1 has been modeled using a docking approach (Rong et al, 2002); the resulting structure shows that its binding pocket is shallower than that of C1bPKC␦. This may explain its better recognition of saturated DAG, which is more compact and has less headgroup spacing than polyunsaturated DAG.…”

Section: Discussionmentioning

confidence: 99%

Diacylglycerol-dependent Binding Recruits PKCθ and RasGRP1 C1 Domains to Specific Subcellular Localizations in Living T Lymphocytes

Carrasco

Mérida

2004

MBoC

130

132

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Diacylglycerol (DAG) signaling relies on the presence of conserved domain 1 (C1) in its target proteins. Phospholipase C-dependent generation of DAG after T cell receptor (TCR) triggering is essential for the correct immune response onset. Accordingly, two C1-containing proteins expressed in T lymphocytes, Ras guanyl nucleotide-releasing protein1 (Ras-GRP1) and protein kinase C (PKC), were shown to be fundamental for T-cell activation and proliferation. Although containing the same regulatory domain, they are proposed to relocate to distinct subcellular locations in response to TCR triggering. Here we studied intracellular localization of RasGRP1 and PKC C1 domains in living Jurkat T cells. The results demonstrate that, in the absence of significant primary sequence differences, the C1 domains of these proteins show specific localization within the cell and distinct responses to pharmacological stimulation and TCR triggering. These differences help explain the divergent localization and distinct functional roles of the full-length proteins, which contains them. The properties of these DAG-binding modules allow their characterization as functional markers that discriminate between DAG pools. Finally, we show that by binding to different diacylglycerol forms, overexpression of distinct C1 modules can attenuate DAG-dependent signals originating from the plasma or internal membranes. This is shown by analyzing the contribution of these two lipid pools to PLC-dependent Ras activation in response to TCR triggering.

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“…Finally, and perhaps more intriguingly from a regulatory point of view, RasGRPs show a different susceptibility toward upstream signals. The activation of RasGRP1, -3, and -4 is mediated via the phospholipase C-␥-dependent generation of diacylglycerol (DAG) (9,(21)(22)(23). This second messenger binds to a C-terminal ZF region present in those GEFs (see Fig.…”

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confidence: 99%

“…This second messenger binds to a C-terminal ZF region present in those GEFs (see Fig. 5), making it possible the translocation of RasGRPs to membranes and their subsequent association with the target GTPases (5,21). In contrast, RasGRP2 shows a very poor response to DAG and, as a consequence, it does not undergo the characteristic rapid translocation of other RasGRPs to the plasma membrane and endomembranes when cells are treated with DAG agonists (13,24).…”

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confidence: 99%

F-actin-dependent Translocation of the Rap1 GDP/GTP Exchange Factor RasGRP2

Caloca

Zugaza

Vicente‐Manzanares

et al. 2004

Journal of Biological Chemistry

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RasGRPs constitute a new group of diacylglycerol-dependent GDP/GTP exchange factors that activate Ras subfamily GTPases. Despite a common structure, RasGRPs diverge in their GTPase specificity, subcellular distribution, and downstream biological effects. The more divergent family member is RasGRP2, a Rap1-specific exchange factor with low affinity toward diacylglycerol. The regulation of RasGRP2 during signal transduction has remained elusive up to now. In this report, we show that the subcellular localization of Ras-GRP2 is highly dependent on actin dynamics. Thus, the induction of F-actin by cytoskeletal regulators such as Vav, Vav2, Dbl, and Rac1 leads to the shift of RasGRP2 from the cytosol to membrane ruffles and its co-localization with F-actin. Treatment of cells with cytoskeletal disrupting drugs abolishes this effect, leading to an abnormal localization of RasGRP2 in cytoplasmic clusters of actin. The use of Rac1 effector mutants indicates that the RasGRP2 translocation is linked exclusively to actin polymerization and is independent of other pathways such as p21-activated kinase JNK, or superoxide production. Biochemical experiments demonstrate that the translocation of RasGRP2 to membrane ruffles is mediated by the direct association of this protein with F-actin, a property contained within its 150 first amino acids. Finally, we show that the RasGRP2/F-actin interaction promotes the regionalized activation of Rap1 in juxtamembrane areas of the cell. These results reveal a novel function of the actin cytoskeleton in mediating the spatial activation of Ras subfamily GTPases through the selective recruitment of GDP/GTP exchange factors.

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Structural Basis of RasGRP Binding to High-Affinity PKC Ligands

Cited by 22 publications

References 48 publications

RasGRP1 Confers the Phorbol Ester-Sensitive Phenotype to EL4 Lymphoma Cells

RasGRP1 Confers the Phorbol Ester-Sensitive Phenotype to EL4 Lymphoma Cells

Diacylglycerol-dependent Binding Recruits PKCθ and RasGRP1 C1 Domains to Specific Subcellular Localizations in Living T Lymphocytes

F-actin-dependent Translocation of the Rap1 GDP/GTP Exchange Factor RasGRP2

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