Ral proteins constitute a family of small GTPases that can be activated by Ras in cells. In the GTP-bound state, Ral proteins bind to RalBP1, a GTPase-activating protein for CDC42 and Rac GTPases. We have used the two-hybrid system in yeast to clone a cDNA for a novel ϳ85-kDa protein that can bind to an additional site on RalBP1. This newly identified protein contains an Eps homology (EH) domain, which was first detected in the epidermal growth factor (EGF) receptor substrate Eps15. Recently, the EH domain of Eps15 has been shown to bind to proteins containing an asparagineproline-phenylalanine motif. Moreover, EH domains have been found in proteins involved in endocytosis and/or actin cytoskeleton regulation. The RalBP1 associated Eps-homology domain protein, Reps1, is tyrosinephosphorylated in response to EGF stimulation of cells. In addition, Reps1 has the capacity to form a complex with the SH3 domains of the adapter proteins Crk and Grb2, which may link Reps1 to an EGF-responsive tyrosine kinase. Thus, Reps1 may coordinate the cellular actions of activated EGF receptors and Ral-GTPases.The Ral-GTPases, RalA and RalB, form a family of Rasrelated GTP-binding proteins (1). Like all GTPases, Ral proteins cycle between the active GTP bound and inactive GDP bound states. Activation of Ral proteins occurs upon interaction with a Ral-specific guanine nucleotide exchange factor (for review, see Ref. 2). This promotes the release of GDP from Ral, allowing activating GTP to take its place. Deactivation takes place when bound GTP is hydrolyzed back to GDP upon interaction of Ral with a specific GTPase-activating protein, Ral-GAP (3).It is likely that Ral proteins act as molecular switches that become active in response to specific upstream signals and then alter the activities of specific downstream target proteins. A series of recent findings support the idea that Ras proteins are important upstream activators of Ral proteins. In particular, it has been shown that active GTP-bound Ras binds to (4 -6) and activates (7) Ral-specific guanine nucleotide exchange factors, which then activate Ral proteins in cells. Thus, activation of Ral-GTPases appears to constitute a distinct downstream signaling pathway from Ras (for review, see Ref. 2).Studies are beginning to reveal how Ral proteins contribute to Ras signaling. Overexpression of Ral-GDS (8) or constitutively active Ral (7) can enhance cellular transformation induced by Ras, and expression of a dominant negative form of Ral can suppress Ras-induced transformation (7).

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transcription factors such as Stat3, and components of the Larry A.Feig 2 endocytic machinery such as clathrin (Wilde et al., 1999)

These results indicated that Heparanase expression may be an important role in invasion and hematogenous metastasis, and may be a biologic marker of prognostic significance in colorectal cancer patients.

Ras proteins can activate at least three classes of downstream target proteins: Raf kinases, phosphatidylinositol-3 phosphate (PI3) kinase, and Ral-specific guanine nucleotide exchange factors (Ral-GEFs). In NIH 3T3 cells, activated Ral-GEFs contribute to Ras-induced cell proliferation and oncogenic transformation by complementing the activities of Raf and PI3 kinases. In PC12 cells, activated Raf and PI3 kinases mediate Ras-induced cell cycle arrest and differentiation into a neuronal phenotype. Here, we show that in PC12 cells, Ral-GEF activity acts opposite to other Ras effectors. Elevation of Ral-GEF activity induced by transfection of a mutant Ras protein that preferentially activates Ral-GEFs, or by transfection of the catalytic domain of the Ral-GEF Rgr, suppressed cell cycle arrest and neurite outgrowth induced by nerve growth factor (NGF) treatment. In addition, Rgr reduced neurite outgrowth induced by a mutant Ras protein that preferentially activates Raf kinases. Furthermore, inhibition of Ral-GEF activity by expression of a dominant negative Ral mutant accelerated cell cycle arrest and enhanced neurite outgrowth in response to NGF treatment. Ral-GEF activity may function, at least in part, through inhibition of the Rho family GTPases, CDC42 and Rac. In contrast to Ras, which was activated for hours by NGF treatment, Ral was activated for only ϳ20 min. These findings suggest that one function of Ral-GEF signaling induced by NGF is to delay the onset of cell cycle arrest and neurite outgrowth induced by other Ras effectors. They also demonstrate that Ras has the potential to promote both antidifferentiation and prodifferentiation signaling pathways through activation of distinct effector proteins. Thus, in some cell types the ratio of activities among Ras effectors and their temporal regulation may be important determinants for cell fate decisions between proliferation and differentiation.

The results suggest that CD44v8-10 plays a role in metastasis of colorectal cancer, and tha CD44v8-10 expression may be a biologic marker of prognostic significance.

The Ral proteins are members of the Ras superfamily of GTPases. Because they reside in synaptic vesicles, we used transgenic mice expressing a dominant inhibitory form of Ral to investigate the role of Ral in neurosecretion. Using a synaptosomal secretion assay, we found that while K ؉ -evoked secretion of glutamate was normal, protein kinase C-mediated enhancement of glutamate secretion was suppressed in the mutant mice. Since protein kinase C effects on secretion have been shown to be due to enhancement of the size of the readily releasable pool of synaptic vesicles docked at the plasma membrane, we directly measured the refilling of this readily releasable pool of synaptic vesicles after Ca 2؉ -triggered exocytosis. Refilling of the readily releasable pool was suppressed in synaptosomes from mice expressing dominant inhibitory Ral. Moreover, we found that protein kinase C and calcium-induced phosphorylation of proteins thought to influence synaptic vesicle function, such as MARCKS, synapsin, and SNAP-25, were all reduced in synaptosomes from these transgenic mice. Concomitant with these studies, we searched for new functions of Ral by detecting proteins that specifically bind to it in cells. Consistent with the phenotype of the transgenic mice described above, we found that active but not inactive RalA binds to the Sec6/8 (exocyst) complex, whose yeast counterpart is essential for targeting exocytic vesicles to specific docking sites on the plasma membrane. These findings demonstrate a role for Ral-GTPase signaling in the modulation of the readily releasable pool of synaptic vesicles and suggest the possible involvement of Ral-Sec6/8 (exocyst) binding in modulation of synaptic strength.

BackgroundIn addition to regulating platelet function, the G protein-coupled sub-family member Proteinase-activated receptor-1 (PAR1) has a proposed role in the development of various cancers, but its exact role and mechanism of action in the invasion, metastasis, and proliferation process in gastric cancer have yet to be completely elucidated. Here, we analyzed the relationship between PAR1 activation, proliferation, invasion, and the signaling pathways downstream of PAR1 activation in gastric cancer.MethodsWe established a PAR1 stably transfected MKN45 human gastric cancer cell line (MKN45/PAR1) and performed cell proliferation and invasion assays employing this cell line and MKN28 cell line exposed to PAR1 agonists (α-thrombin and TFLLR-NH2). We also quantified NF-κB activation by electrophoretic mobility shift assay (EMSA) and the level of Tenascin-C (TN-C) expression in conditioned medium by ELISA of MKN45/PAR1 following administration of α-thrombin. A high molecular weight concentrate was derived from the resultant conditioned medium and subsequent cultures of MKN45/PAR1 and MKN28 were exposed to the resultant concentrate either in the presence or absence of TN-C-neutralizing antibody. Lysates of these subsequent cells were probed to quantify levels of phospholyrated Epidermal Growth Factor Receptor (EGFR).ResultPAR1 in both PAR1/MKN45 and MKN28 was activated by PAR1 agonists, resulting in cell proliferation and matrigel invasion. We have shown that activation of NF-κB and EGFR phosphorylation initially were triggered by the activation of PAR1 with α-thrombin. Quantitative PCR and Western blot assay revealed up-regulation of mRNA and protein expression of NF-κB target genes, especially TN-C, a potential EGFR activator. The suppressed level of phosphorylated EGFR, observed in cells exposed to concentrate of conditioned medium in the presence of TN-C-neutralizing antibody, identifies TN-C as a putative autocrine stimulatory factor of EGFR possibly involved in the sustained PAR1 activation responses observed.ConclusionOur data indicate that in gastric carcinoma cells, PAR1 activation can trigger an array of responses that would promote tumor cell growth and invasion. Over expression of NF-κB, EGFR, and TN-C, are among the effects of PAR1 activation and TN-C induces EGFR activation in an autocrine manner. Thus, PAR1 is a potentially important therapeutic target for the treatment of gastric cancer.