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Phospholipase C(epsilon): a novel Ras effector.

ABSTRACT: Three classes of mammalian phosphoinositide-specific phospholipase C (PLC) have been characterized, PLCbeta, PLCgamma and PLCdelta, that are differentially regulated by heterotrimeric G-proteins, tyrosine kinases and calcium. Here we describe a fourth class, PLCepsilon, that in addition to conserved PLC domains, contains a GTP exchange factor (GRF CDC25) domain and two C-terminal Ras-binding (RA) domains, RA1 and RA2. The RA2 domain binds H-Ras in a GTP-dependent manner, comparable with the Ras-binding domain of Raf-1; however, the RA1 domain binds H-Ras with a low affinity in a GTP-independent manner. While G(alpha)q, Gbetagamma or, surprisingly, H-Ras do not activate recombinant purified protein in vitro, constitutively active Q61L H-Ras stimulates PLC(epsilon) co-expressed in COS-7 cells in parallel with Ras binding. Deletion of either the RA1 or RA2 domain inhibits this activation. Site-directed mutagenesis of the RA2 domain or Ras demonstrates a conserved Ras-effector interaction and a unique profile of activation by Ras effector domain mutants. These studies identify a novel fourth class of mammalian PLC that is directly regulated by Ras and links two critical signaling pathways.

SUBMITTER: Kelley GG

PROVIDER: S-EPMC145421 | biostudies-literature | 2001 Feb

REPOSITORIES: biostudies-literature

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Phospholipase C(epsilon): a novel Ras effector.

Kelley G G GG Reks S E SE Ondrako J M JM Smrcka A V AV

The EMBO journal 20010201 4

Three classes of mammalian phosphoinositide-specific phospholipase C (PLC) have been characterized, PLCbeta, PLCgamma and PLCdelta, that are differentially regulated by heterotrimeric G-proteins, tyrosine kinases and calcium. Here we describe a fourth class, PLCepsilon, that in addition to conserved PLC domains, contains a GTP exchange factor (GRF CDC25) domain and two C-terminal Ras-binding (RA) domains, RA1 and RA2. The RA2 domain binds H-Ras in a GTP-dependent manner, comparable with the Ras- ...[more]

PMID: 11179219

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Project description:BackgroundPhospholipase C? (PLC?) is an effector of Ras and Rap small GTPases and expressed in non-immune cells. It is well established that PLC? plays an important role in skin inflammation, such as that elicited by phorbol ester painting or ultraviolet irradiation and contact dermatitis that is mediated by T helper (Th) 1 cells, through upregulating inflammatory cytokine production by keratinocytes and dermal fibroblasts. However, little is known about whether PLC? is involved in regulation of inflammation in the respiratory system, such as Th2-cells-mediated allergic asthma.MethodsWe prepared a mouse model of allergic asthma using PLC?+/+ mice and PLC??X/?X mutant mice in which PLC? was catalytically-inactive. Mice with different PLC? genotypes were immunized with ovalbumin (OVA) followed by the challenge with an OVA-containing aerosol to induce asthmatic response, which was assessed by analyzing airway hyper-responsiveness, bronchoalveolar lavage fluids, inflammatory cytokine levels, and OVA-specific immunoglobulin (Ig) levels. Effects of PLC? genotype on cytokine production were also examined with primary-cultured bronchial epithelial cells.ResultsAfter OVA challenge, the OVA-immunized PLC??X/?X mice exhibited substantially attenuated airway hyper-responsiveness and broncial inflammation, which were accompanied by reduced Th2 cytokine content in the bronchoalveolar lavage fluids. In contrast, the serum levels of OVA-specific IgGs and IgE were not affected by the PLC? genotype, suggesting that sensitization was PLC?-independent. In the challenged mice, PLC? deficiency reduced proinflammatory cytokine production in the bronchial epithelial cells. Primary-cultured bronchial epithelial cells prepared from PLC??X/?X mice showed attenuated pro-inflammatory cytokine production when stimulated with tumor necrosis factor-?, suggesting that reduced cytokine production in PLC??X/?X mice was due to cell-autonomous effect of PLC? deficiency.ConclusionsPLC? plays an important role in the pathogenesis of bronchial asthma through upregulating inflammatory cytokine production by the bronchial epithelial cells.

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Phospholipase C(epsilon): a novel Ras effector.

Publications

Phospholipase C(epsilon): a novel Ras effector.

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