scholarly journals The Small GTP-binding Protein R-Ras Can Influence Integrin Activation by Antagonizing a Ras/Raf-initiated Integrin Suppression Pathway

1999 ◽  
Vol 10 (6) ◽  
pp. 1799-1809 ◽  
Author(s):  
Tariq Sethi ◽  
Mark H. Ginsberg ◽  
Julian Downward ◽  
Paul E. Hughes
Keyword(s):  
Binding Protein ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Integrin Activation ◽  
Gtp Binding Protein ◽  
Ovary Cells ◽  
Downstream Effector ◽  
Gtp Binding ◽  
Downstream Effectors ◽  
Rapid Modulation

The rapid modulation of ligand-binding affinity (“activation”) is a central property of the integrin family of cell adhesion receptors. The small GTP-binding protein Ras and its downstream effector kinase Raf-1 suppress integrin activation. In this study we explored the relationship between Ras and the closely related small GTP-binding protein R-Ras in modulating the integrin affinity state. We found that R-Ras does not seem to be a direct activator of integrins in Chinese hamster ovary cells. However, we observed that GTP-bound R-Ras strongly antagonizes the Ras/Raf-initiated integrin suppression pathway. Furthermore, this reversal of the Ras/Raf suppressor pathway does not seem to be via a competition between Ras and R-Ras for common downstream effectors or via an inhibition of Ras/Raf-induced MAP kinase activation. Thus, R-Ras and Ras may act in concert to regulate integrin affinity via the activation of distinct downstream effectors.

scholarly journals Specific role for p85/p110β in GTP-binding-protein-mediated activation of Akt

2005 ◽  
Vol 392 (3) ◽  
pp. 607-614 ◽  
Author(s):  
Hiroshi Kubo ◽  
Kaoru Hazeki ◽  
Shunsuke Takasuga ◽  
Osamu Hazeki
Keyword(s):  
Binding Protein ◽  
Chinese Hamster Ovary Cells ◽  
Point Mutations ◽  
Chinese Hamster ◽  
Middle Part ◽  
Dependent Manner ◽  
Gtp Binding Protein ◽  
Intact Cells ◽  
Ovary Cells ◽  
Gtp Binding

We prepared CHO (Chinese hamster ovary) cells expressing both IR (insulin receptor) and A1R (A1 adenosine receptor). Treatment of the cells with insulin or PIA [N6-(2-phenylisopropyl)adenosine], a specific A1R agonist increased Akt activity in the cells in a PI3K- (phosphoinositide 3-kinase) dependent manner. Transfection of p110β into the cells augmented the action of PIA with little effect on insulin. Introduction of a pH1 vector producing shRNA (short hairpin RNA) that targets p110β abolished PIA-induced Akt activation. By contrast, an shRNA probe targeting p110α did not impair the effects of PIA. The effect of PIA in p110α-deficient cells was attenuated effectively by both Δp85 and βARK-CT (β-adrenergic receptor kinase-C-terminal peptide). A Δp85-derived protein possessing point mutations in its two SH2 domains did not impair PIA action. These results suggest that tyrosine-phosphorylated proteins and Gβγ (βγ subunits of GTP-binding protein) are necessary for the specific function of p110β in intact cells. The p110β-middle (middle part of p110β) may play an important role in signal reception from GPCRs (GTP-binding-protein-coupled receptor), because transfection of the middle part impaired PIA sensitivity.

scholarly journals Polyamine-mediated turnover of ornithine decarboxylase in Chinese-hamster ovary cells

1986 ◽  
Vol 236 (2) ◽  
pp. 351-357 ◽  
Author(s):  
J R Glass ◽  
E W Gerner
Keyword(s):  
Ornithine Decarboxylase ◽  
Chinese Hamster Ovary ◽  
Specific Activity ◽  
Binding Protein ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Defined Medium ◽  
Ovary Cells ◽  
Exogenous Substrate ◽  
Polyamine Content

We have used Chinese-hamster ovary (CHO) cells maintained in a chemically defined medium to study the regulation of ornithine decarboxylase (ODC) by polyamines. Cells maintained in the defined medium had no detectable putrescine, and approx. 1-3 units of ODC activity/10(6) cells, where 1 unit corresponds to 1 nmol of substrate decarboxylated in 30 min. The defined medium is ornithine-deficient, thus limiting the exogenous substrate for ODC, and subsequently decreasing intracellular polyamine accumulation. Restoration of intracellular putrescine and increased formation of spermidine by addition of exogenous ornithine or putrescine led to a marked decrease in ODC activity, which was paralleled by a decrease in a alpha-DL-difluoromethyl[3,4-3H]ornithine (DFMO)-binding protein of Mr approx. 53,000, which is precipitable with anti-ODC antibody. Calculation of DFMO binding per unit of activity showed no change in the specific activity of the enzyme. We identified [35S]methionine-labelled peptides corresponding to ODC by immunoprecipitation of radiolabeled whole cell proteins. Only one protein was precipitated, of Mr approx. 53 000, which co-migrated with the DFMO-binding protein. Immunoprecipitation of radiolabelled proteins from cells incubated in the presence of exogenous ornithine indicated that the observed activity decrease was not due to an inhibition of ODC protein synthesis. Analysis of immunoprecipitable ODC protein from cells that had been pulse-labelled with [35S]methionine, and then treated for 5 h with 100 microM-ornithine, -putrescine or -spermidine, revealed a distinct disappearance of labelled ODC protein after restoration of intracellular polyamine pools. No detectable turnover of ODC was observed in the absence of exogenous polyamine treatment. These data support the hypothesis that ODC protein, and subsequent activity, is regulated by intracellular polyamine content through mechanisms that influence turnover of the enzyme.

scholarly journals rho, a Small GTP-Binding Protein, Is Essential for Shigella Invasion of Epithelial Cells

1997 ◽  
Vol 185 (2) ◽  
pp. 281-292 ◽  
Author(s):  
Masahisa Watarai ◽  
Yoichi Kamata ◽  
Shunji Kozaki ◽  
Chihiro Sasakawa
Keyword(s):  
Epithelial Cells ◽  
Cho Cells ◽  
Mammalian Cells ◽  
Binding Protein ◽  
Shigella Flexneri ◽  
Chinese Hamster ◽  
Gtp Binding Protein ◽  
Gtp Binding ◽  
Causative Agents ◽  
Invasive Capacity

Shigella, the causative agents of bacillary dysentery, are capable of invading mammalian cells that are not normally phagocytic. Uptake of bacteria by the mammalian cells is directed by bacterial factors named IpaB, IpaC, and IpaD invasins, in which Ipa invasins secreted into the bacterial environment can interact with α5β1 integrin. We report here that Shigella invasion of epithelial cells requires rho activity, a ras-related GTP-binding protein. The invasive capacity of Shigella flexneri for Chinese hamster ovary (CHO) cells and other epithelial cells were greatly reduced when treated with Clostridium botulinum exoenzyme C3 transferase. Conversely, uptake of bacteria by CHO cells was promoted upon microinjection of an activated rho variant, Val14RhoA. Attachment of S. flexneri to CHO cells can elicit tyrosine phosphorylation of pp125FAK and paxillin, localized accumulation of F-actin, vinculin, and talin, and activation of protein kinase C, which were all blocked by the treatment with C3 transferase. Our results indicate that cellular signal transduction regulated by rho is essential for Shigella invasion of epithelial cells.

scholarly journals Regulation of Na+ transport by aldosterone: signaling convergence and cross talk between the PI3-K and MAPK1/2 cascades

2004 ◽  
Vol 286 (6) ◽  
pp. F1232-F1238 ◽  
Author(s):  
Qiusheng Tong ◽  
Rachell E. Booth ◽  
Roger T. Worrell ◽  
James D. Stockand
Keyword(s):  
Cross Talk ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Signaling Cascades ◽  
Dependent Manner ◽  
Phosphatidylinositol 3 Kinase ◽  
Ovary Cells ◽  
Steroid Dependent ◽  
Downstream Effectors ◽  
Positive Modulator

Cross talk between the phosphatidylinositol 3-kinase (PI3-K) and mitogen-activating protein kinase (MAPK)1/2 signaling cascades in response to aldosterone-induced K-RasA was investigated in renal A6 epithelial cells. In addition, the contribution of these signaling pathways to aldosterone-stimulated Na+ transport was investigated. Aldosterone increased active K-RasA levels in A6 cells resulting in activation of downstream effectors in both the MAPK1/2 and PI3-K cascades with K-RasA directly interacting with the catalytic p110 subunit of PI3-K in a steroid-dependent manner. Aldosterone-stimulated PI3-K signaling impinged on the MAPK1/2 cascade at the level of Akt-mediated phosphorylation of c-Raf at an established negative regulatory site. Aldosterone also increased Sgk levels as well as stimulated phosphorylation of this kinase in a PI3-K- and K-RasA-dependent manner. Blockade of MAPK1/2 signaling had little effect on Na+ transport. Conversely, inhibition of PI3-K markedly suppressed transport. Likewise, suppression of K-RasA induction decreased transport. However, Na+ transport was subsequently stimulated under these conditions with the PLA2 inhibitor aristolochic acid, an established positive modulator of Na+ transport, suggesting that K-RasA signaling through PI3-K does not directly affect epithelial sodium channel (ENaC) levels but the activity of this channel. Consistent with this possibility, activity of ENaC reconstituted in Chinese hamster ovary cells was increased by coexpression of constitutively active PI3-K. The current study demonstrates that aldosterone increases Na+ transport, in part, by stimulating PI3-K signaling and that during aldosterone actions, there is both signaling convergence between the two aldosterone-induced proteins, K-RasA and Sgk, as well as cross talk between the PI3-K and MAPK1/2 cascades with the prior but not latter cascade enhancing ENaC activity.

scholarly journals Interaction of Nck-associated protein 1 with activated GTP-binding protein Rac

1997 ◽  
Vol 322 (3) ◽  
pp. 873-878 ◽  
Author(s):  
Yukari KITAMURA ◽  
Tadahiro KITAMURA ◽  
Hiroshi SAKAUE ◽  
Tetsuo MAEDA ◽  
Hikaru UENO ◽  
...  
Keyword(s):  
Binding Protein ◽  
Biological Effects ◽  
Chinese Hamster Ovary Cells ◽  
Active Form ◽  
Chinese Hamster ◽  
Bovine Brain ◽  
Brain Extract ◽  
Potential Candidate ◽  
Ovary Cells

Bacterially expressed glutathione S-transferase fusion proteins containing Rac1 were used to identify binding proteins of this Rho family GTPase present in a bovine brain extract. Five proteins of 85, 110, 125, 140 and 170 kDa were detected, all of which were associated exclusively with guanosine 5´-[γ-thio]triphosphate-bound Rac1, not with GDP-bound Rac1. The 85 and 110 kDa proteins were identified as the regulatory and catalytic subunits respectively of phosphatidylinositol 3-kinase. Several lines of evidence suggested that the 125 kDa protein is identical with Nck-associated protein 1 (Nap1). The mobilities of the 125 kDa protein and Nap1 on SDS/PAGE were indistinguishable, and the 125 kDa protein was depleted from brain extract by preincubation with the Src homology 3 domain of Nck to which Nap1 binds. Furthermore, antibodies to Nap1 reacted with the 125 kDa protein. Nap1 was co-immunoprecipitated with a constitutively active form of Rac expressed in Chinese hamster ovary cells. The observation that complex formation between activated Rac and PAK, but not that between Rac and Nap1, could be reproduced in vitro with recombinant proteins indicates that the interaction of Nap1 with Rac is indirect. The 140 kDa Rac-binding protein is a potential candidate for a link that connects Nap1 to Rac. The multimolecular complex comprising Rac, Nap1 and probably the 140 kDa protein might mediate some of the biological effects transmitted by the multipotent GTPase.

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