Ras-GTP imaging studies using the Ras-binding domain (RBD) of the Ras effector c-Raf as a reporter for overexpressed Ras have produced discrepant results about the possible activation of Ras at the Golgi apparatus. We report that RBD oligomerization provides probes for visualization of endogenous Ras-GTP, obviating Ras overexpression and the side effects derived thereof. RBD oligomerization results in tenacious binding to Ras-GTP and interruption of Ras signalling. Trimeric RBD probes fused to green fluorescent protein report agonist-induced endogenous Ras activation at the plasma membrane (PM) of COS-7, PC12 and Jurkat cells, but do not accumulate at the Golgi. PM illumination is exacerbated by Ras overexpression and its sensitivity to dominant-negative RasS17N and pharmacological manipulations matches Ras-GTP formation assessed biochemically. Our data illustrate that endogenous Golgi-located Ras is not under the control of growth factors and argue for the PM as the predominant site of agonist-induced Ras activation.

Material Supplementary 4.DC1http://www.jimmunol.org/content/suppl/2010/08/16/jimmunol.100033

We studied the expression of arachidonate 5-lipoxygenase (5-LO) in a cell line of human keratinocytes (HaCaT) and in normal human skin keratinocytes in tissue culture. In undifferentiated keratinocytes 5-LO gene expression was low or undetectable as determined by 5-LO mRNA, protein, cell-free enzyme activity, and leukotriene production in intact cells. However, after shift to culture conditions that promote conversion of prokeratinocytes into a more differentiated phenotype, 5-LO gene expression was markedly induced in HaCaT cells and, to a lesser extent, in normal keratinocytes. These results show that 5-LO gene expression is an intrinsic property of human skin keratinocytes.After the discovery of arachidonate 5-lipoxygenase (5-LO; arachidonate:oxygen 5-oxidoreductase, EC 1.13.11.34) in polymorphonuclear neutrophils the enzyme was identified in other leukocyte lineages (1, 2). Products of the 5-LO pathway, including leukotriene (LT) B4 and the cysteinyl LTs, in addition to their roles as inflammatory agonists, may act as physiological autocrine or paracrine signaling molecules. Until recently 5-LO expression was thought to be restricted to the hematopoietic system. However, attempts to identify the enzyme in other tissues yielded results that support the hypothesis that it is expressed in distinctive extrahematopoietic epithelia. Thus, Natsui et al. (3) Expression of the 5-LO pathway in skin epithelium likewise remains debatable. Several authors using primary skin keratinocytes reported the production of 5-hydroxyeicosatetraenoic acid (5-HETE) and LTB4, but subsequent work challenged some of the earlier interpretations (refs. 6-11; for review, see ref. 12).We have used a nontransformed human keratinocyte cell line, HaCaT, and normal human skin keratinocytes (NHKs) as in vitro models to study expression of the 5-LO pathway in extrahematopoietic epithelia. HaCaT cells are derived from a normal skin biopsy and maintain a substantial differentiation potential in culture (13,14). In this report we show that the 5-LO gene is expressed in both HaCaT cells and NHKs under conditions that favor their differentiation. MATERIALS AND METHODS

The correct functioning of Ras proteins requires post-translational modification of the GTP hydrolases (GTPases). These modifications provide hydrophobic moieties that lead to the attachment of Ras to the inner side of the plasma membrane. In this study we investigated the role of Ras processing in the interaction with various putative Ras-effector proteins. We describe a specific, GTP-independent interaction between post-translationally modified Ha-and Ki-Ras4B and the G-protein responsive phosphoinositide 3-kinase p110g. Our data demonstrate that post-translational processing increases markedly the binding of Ras to p110g in vitro and in Sf9 cells, whereas the interaction with p110a is unaffected under the same conditions. Using in vitro farnesylated Ras, we show that farnesylation of Ras is sufficient to produce this effect. The complex of p110g and farnesylated RasGTP exhibits a reduced dissociation rate leading to the efficient shielding of the GTPase from GTPase activating protein (GAP) action. Moreover, Ras processing affects the dissociation rate of the RasGTP complex with the Ras binding domain (RBD) of Raf-1, indicating that processing induces alterations in the conformation of RasGTP. The results suggest a direct interaction between a moiety present only on fully processed or farnesylated Ras and the putative target protein p110g.Keywords: Ras; post-translational modifications; phosphoinositide 3-kinase; farnesylation.The Ras GTP hydrolases (GTPases) are a family of related proteins involved in the regulation of numerous cellular processes such as mitogen-induced G0/G1 phase transition, apoptosis, cytoskeletal rearrangements and regular cell-cycle progression. Ras proteins cycle between a GDP-bound and a GTP-bound state, the latter being the active, signal transducing version of the GTPase.The correct subcellular localization of Ras is critical for the activation of its downstream targets. Immunocytochemical studies have shown, that the majority of Ras is localized to the inner side of the plasma membrane. This association with the plasma membrane has been causally connected to a series of post-translational modifications of Ras. Inhibition of the posttranslational modification reactions abolishes the plasma membrane localization and leads to the loss of the biological activity of Ras [1,2]. The transforming potential of oncogenic Ras variants is severely affected by interfering with their post-translational modifications [2±4]. The Raf kinase isoform Raf-1 binds to, and is activated by, Ras in insect Sf9 cells [5] and vertebrate cell lines [6,7] and this activation, although not the association, is lost if post-translational processing of Ras is prevented [7]. A farnesylation-deficient point mutant of oncogenic Ha-Ras has been identified that can no longer activate the phosphoinositide 3-kinase (PI3K) isoform p110a in COS7 cells [8]. These and other findings have been interpreted in such a way that the post-translational processing of Ras functions to localize both Ras and, eventually, its target pro...

The topology of the signalling pathway linking the G-protein-coupled receptor agonist lysophosphatidic acid (LPA) to extracellular-signal-regulated kinase activation remains undeciphered. In the present study, we report that analysis of LPA signals at the level of Ras-GTP formation and Ras nucleotide exchange discriminates true mediatory signals from permissive activities that do not participate in signal relay. Hence, whereas pertussis toxin (PTX) treatment impairs stimulation of nucleotide exchange, epidermal growth factor receptor (EGFR) inhibition does not compromise LPA-induced acceleration of nucleotide exchange, but instead attenuates basal nucleotide turnover on Ras. Our data indicate that LPA activation of Ras proceeds via PTX-sensitive G(i/o)-proteins and requires a permissive input from basal EGFR activity.