Potent transforming activity of the small GTP‐binding protein Rit in NIH 3T3 cells: evidence for a role of a p38γ‐dependent signaling pathway

Sakabe, Kaoru; Teramoto, Hidemi; Zohar, Muriel; Behbahani, Babak; Miyazaki, Hiroshi; Chikumi, Hiroki; Gutkind, J. Silvio

doi:10.1016/s0014-5793(01)03264-1

Cited by 32 publications

(33 citation statements)

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“…The SRE activity was determined as described previously (Sakabe et al, 2002). Briefly, HEK-293T cells plated in a sixwell plate together with 0.1 mg of pSRE Luc reporter plasmid.…”

Section: Reporter Gene Assaymentioning

confidence: 99%

Homo- and hetero-oligomerization of PDZ-RhoGEF, LARG and p115RhoGEF by their C-terminal region regulates their in vivo Rho GEF activity and transforming potential

et al. 2004

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PDZ-RhoGEF, LARG, and p115RhoGEF are members of a newly identified family of Rho-guanine nucleotide exchange factors (GEFs) exhibiting a unique structural feature consisting of the presence of an area of similarity to regulators of G protein signaling (RGS). This RGS-like (RGL) domain provides a functional motif by which Ga 12 and Ga 13 can bind and regulate the activity of these RhoGEFs, thus providing a direct link from these heterotrimeric G proteins to Rho. PDZ-RhoGEF and LARG can also be phosphorylated by tyrosine kinases, including FAK, and associate with Plexin B, a semaphorin receptor, which controls axon guidance during development, through their PDZ domain, thereby stimulating Rho. Interestingly, while characterizing a PDZ-RhoGEF antiserum, we found that a transfected PDZ-RhoGEF construct associated with the endogenous PDZ-RhoGEF. Indeed, we observed that PDZ-RhoGEF and LARG can form homo-and hetero-oligomers, whereas p115RhoGEF can only homo-oligomerize, and that this intermolecular interaction was mediated by their unique C-terminal regions. Deletion of the C-terminal tail of PDZ-RhoGEF had no significant effect on the GEF catalytic activity towards Rho in vitro, but resulted in a drastic increase in the ability to stimulate a serum response element reporter and the accumulation of the GTP-bound Rho in vivo. Furthermore, removal of the C-termini of each of the three RGL-containing GEFs unleashed their full transforming potential. Together, these findings suggest the existence of a novel mechanism controlling the activity of PDZRhoGEF, LARG, and p115RhoGEF, which involves homo-and hetero-oligomerization through their inhibitory C-terminal region.

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“…The SRE activity was determined as described previously (Sakabe et al, 2002). Briefly, HEK-293T cells plated in a sixwell plate together with 0.1 mg of pSRE Luc reporter plasmid.…”

Section: Reporter Gene Assaymentioning

confidence: 99%

Homo- and hetero-oligomerization of PDZ-RhoGEF, LARG and p115RhoGEF by their C-terminal region regulates their in vivo Rho GEF activity and transforming potential

et al. 2004

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“…NIH3T3 cells were maintained in Dulbecco's modified Eagle's medium (Life Technologies) supplemented with 10% calf serum, and transfected by the calcium-phosphate precipitation technique adjusting the total amount of DNA to 1 mg/plate with vector alone (Sakbe et al, 2002). Mass populations of cells expressing the transfected gene were selected for their ability to grow in the presence of Geneticin (G418) (GIBCO BRL, NY, USA).…”

Section: Cell Lines and Transfectionmentioning

confidence: 99%

“…Of interest, we and others have recently (Rusyn et al, 2000;Sakbe et al, 2002) reported that the newest member of the Ras superfamily, Rit (Ras-like protein in tissues), induces Ras-like foci when its activated mutant, Rit 79L (analogous to the oncogenic mutant H-Ras 61L), is expressed in NIH3T3 cells. Rit was cloned as a homolog of Ric (calmodulin-binding Ras-like GTPase) in Drosophila (Wes et al, 1996).…”

Section: Introductionmentioning

confidence: 99%

“…This GTPase is highly expressed in most embryonic and adult tissues and lacks a C-terminal prenylation motif. Although Rit is 50% identical to Ras, Rit does not bind Raf nor stimulate MAPK potently (Rusyn et al, 2000;Sakbe et al, 2002). Furthermore, the biochemical routes that are stimulated by Rit are still poorly understood (Rusyn et al, 2000;Sakbe et al, 2002).…”

Section: Introductionmentioning

confidence: 99%

“…Although Rit is 50% identical to Ras, Rit does not bind Raf nor stimulate MAPK potently (Rusyn et al, 2000;Sakbe et al, 2002). Furthermore, the biochemical routes that are stimulated by Rit are still poorly understood (Rusyn et al, 2000;Sakbe et al, 2002). Nonetheless, the ability of Rit to induce transformed foci, which are morphologically identical to those of Ras, albeit with a reduced transforming potency, provided an opportunity to explore the nature of small GTPase-regulated genes whose expression may correlate with the acquisition of the transformed phenotype.…”

Section: Introductionmentioning

confidence: 99%

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Autocrine activation of an osteopontin-CD44-Rac pathway enhances invasion and transformation by H-RasV12

Teramoto

Castellone

Malek³

et al. 2004

Oncogene

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Activated forms of Ras family members are prevalent in many cancers where Ras mutants transduce signals essential for transformation, angiogenesis, invasion and metastasis. As a cancer progression model, we used NIH3T3 cells to explore the mechanism of Ras-induced tumorigenesis. Ras family mutants H-RasV12 and Rit79L strongly induced foci formation, while Rho family mutants RhoA-QL, Rac1-QL and Cdc42-QL were less effective. A comparison of downstream transcriptional targets of Ras and Rho family members using a 26 383 element cDNA microarray revealed that the osteopontin (OPN) gene exhibited the best correlation between magnitude of gene expression change and level of foci formation (r ¼ 0.96, Po0.001). In association with H-RasV12-and Rit79L-mediated transformation, foci secreted OPN protein and upregulated the OPN receptor CD44, suggesting the novel initiation of an aberrant OPN-CD44-Rac autocrine pathway. In support of this were the following observations. First, RGD-deficient OPN protein-binding activity was present in H-RasV12-transformed cells but not in control cells, and binding activity was inhibited by the CD44 blocking antibody. Second, foci formation, cell invasion and Rac activity were induced by H-RasV12 and inhibited by the CD44 blocking antibody. Third, foci formation by H-RasV12 was substantially reduced by a short interfering RNA (siRNA) specifically targeting OPN expression for knockdown. Fourth, H-RasV12-mediated transformation was not blocked by the GRGDS peptide, suggesting that OPN effects were not mediated by the integrins. Lastly, OPN knockdown affected the downstream expression of 160 '2nd tier' genes, and at least a subset of these genes appears to be involved in transformation. Indeed, four genes were selected for knockdown, each resulting in a disruption of foci formation and/or invasion. These results underscore the role of aberrant autocrine signaling and transcriptional networking during tumorigenesis.

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Anti‐Inflammatory/Analgesics: An In‐Depth Look atp38MAPKinases

Brown

2011

Pharmaceutical Sciences Encyclopedia

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Inflammation is a complex process controlled by numerous positive and negative regulators. The p38 MAP kinase pathway is integral to the production of mediators that drive inflammation and pain and the cellular responses to these mediators. If not properly controlled, it can cause dire effects in the inflammatory, immune, and nervous systems that manifest in the development of chronic pain and inflammatory disease. As this chapter will discuss, newer versions of these molecular inhibitors must be designed in a manner that is more mindful of the complexity of p38 and the inflammatory response to reach their potential as an effective anti‐inflammatory and analgesic therapy.

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Potent transforming activity of the small GTP‐binding protein Rit in NIH 3T3 cells: evidence for a role of a p38γ‐dependent signaling pathway

Cited by 32 publications

References 15 publications

Homo- and hetero-oligomerization of PDZ-RhoGEF, LARG and p115RhoGEF by their C-terminal region regulates their in vivo Rho GEF activity and transforming potential

Homo- and hetero-oligomerization of PDZ-RhoGEF, LARG and p115RhoGEF by their C-terminal region regulates their in vivo Rho GEF activity and transforming potential

Autocrine activation of an osteopontin-CD44-Rac pathway enhances invasion and transformation by H-RasV12

Anti‐Inflammatory/Analgesics: An In‐Depth Look atp38MAPKinases

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