scholarly journals The E5 gene product of rhesus papillomavirus is an activator of endogenous Ras and phosphatidylinositol-3'-kinase in NIH 3T3 cells

1996 ◽  
Vol 93 (23) ◽  
pp. 12879-12884 ◽  
Author(s):  
J. Ghai ◽  
R. S. Ostrow ◽  
J. Tolar ◽  
R. C. McGlennen ◽  
T. D. Lemke ◽  
...  
Keyword(s):  
Gene Product ◽  
Phosphatidylinositol 3 Kinase ◽  
3T3 Cells ◽  
Nih 3T3 ◽  
Nih 3T3 Cells ◽  
Phosphatidylinositol 3

scholarly journals Transformation of Mouse Fibroblasts by Jaagsiekte Sheep Retrovirus Envelope Does Not Require Phosphatidylinositol 3-Kinase

2003 ◽  
Vol 77 (18) ◽  
pp. 9951-9959 ◽  
Author(s):  
Naoyoshi Maeda ◽  
Yasuo Inoshima ◽  
David A. Fruman ◽  
Saskia M. Brachmann ◽  
Hung Fan
Keyword(s):  
Regulatory Subunit ◽  
Transformed Cells ◽  
Binding Motif ◽  
Phosphatidylinositol 3 Kinase ◽  
3T3 Cells ◽  
Jaagsiekte Sheep Retrovirus ◽  
Downstream Target ◽  
Nih 3T3 ◽  
Nih 3T3 Cells ◽  
Phosphatidylinositol 3

ABSTRACT Jaagsiekte sheep retrovirus (JSRV) is the causative agent of ovine pulmonary adenocarcinoma, a transmissible lung cancer of sheep. The envelope of JSRV may have oncogenic properties, since it can morphologically transform mouse NIH 3T3 cells and other fibroblast lines. Recently, we found that the cytoplasmic tail of the envelope transmembrane (TM) protein is necessary for transformation, and in particular a consensus binding motif (YXXM) for phosphatidylinositol 3-kinase (PI3K) is important. Moreover, JSRV-transformed cells show phosphorylation (activation) of Akt/protein kinase B, a downstream target of PI3K. In these studies, we directly tested for the involvement of PI3K in transformation by JSRV. Contrary to expectations, four different experiments indicated that PI3K is not necessary for JSRV-induced transformation: (i) cotransfection with a dominant negative truncated form of the PI3K regulatory subunit (Δp85) did not affect transformation frequency, (ii) cells stably expressing Δp85 showed the same frequencies of transformation as parental NIH 3T3 cells, (iii) fibroblasts established from double-knockout mice lacking PI3K p85α and p85β could be transformed with JSRV envelope, and (iv) incubation of cells with the PI3K inhibitor LY294002 did not specifically inhibit transformation, nor did the drug reverse transformation of JSRV-transformed cells. One alternate explanation for the lack of transformation by YXXM mutants could be that they were defective in intracellular trafficking. However, confocal microscopy of epitope-tagged envelope proteins of both wild-type and nontransforming YXXM mutants showed a cell surface or plasma membrane localization. While PI3K is not required for JSRV-induced transformation of NIH 3T3 cells, the downstream target Akt kinase was found to be activated (phosphorylated) in JSRV-transformed PI3K-negative cells. Therefore, JSRV envelope can induce PI3K-independent phosphorylation of Akt.

scholarly journals Requirement of phosphatidylinositol 3-kinase-dependent pathway and Src for Gas6-Axl mitogenic and survival activities in NIH 3T3 fibroblasts.

1997 ◽  
Vol 17 (8) ◽  
pp. 4442-4453 ◽  
Author(s):  
S Goruppi ◽  
E Ruaro ◽  
B Varnum ◽  
C Schneider
Keyword(s):  
Tyrosine Kinase ◽  
Specific Inhibitor ◽  
S Phase ◽  
Phosphatidylinositol 3 Kinase ◽  
3T3 Cells ◽  
Src Tyrosine Kinase ◽  
Survival Effect ◽  
Nih 3T3 ◽  
Nih 3T3 Cells ◽  
Phosphatidylinositol 3

Gas6 is a secreted protein previously identified as the ligand of the Axl receptor tyrosine kinase. We have shown that Gas6 is able to induce cell cycle reentry of serum-starved NIH 3T3 cells and to efficiently prevent apoptosis after complete growth factor removal, a survival effect uncoupled from Gas6-induced mitogenesis. Here we report that the mitogenic effect of Gas6 requires phosphatidylinositol 3-kinase (PI3K) activity since it is abrogated both by the specific inhibitor wortmannin and by overexpression of the dominant negative P13K p85 subunit. Consistently, Gas6 activates the P13K downstream targets S6K and Akt, whose activation is abrogated by addition of wortmannin. Moreover, rapamycin treatment blocks Gas6-induced entry into the S phase of serum-starved NIH 3T3 cells. We also demonstrate the requirement of Src tyrosine kinase for Gas6 signalling since stable or transient expression of a catalytically inactive form of Src significantly inhibited Gas6-stimulated entry into the S phase. Accordingly, Gas6 addition to serum-starved NIH 3T3 cells causes activation of the intrinsic Src kinase activity. When specifically analyzed in a survival assay, these elements were found to be required for the survival effect of Gas6. Taken together, the evidence presented here identifies elements involved in the Gas6 transduction pathway that are responsible for its antiapoptotic effect and suggests that Src is involved in the events regulating cell survival.

scholarly journals A Phosphatidylinositol 3-Kinase Docking Site in the Cytoplasmic Tail of the Jaagsiekte Sheep Retrovirus Transmembrane Protein Is Essential for Envelope-Induced Transformation of NIH 3T3 Cells

2001 ◽  
Vol 75 (22) ◽  
pp. 11002-11009 ◽  
Author(s):  
Massimo Palmarini ◽  
Naoyoshi Maeda ◽  
Claudio Murgia ◽  
Claudio De-Fraja ◽  
Andrew Hofacre ◽  
...  
Keyword(s):  
Cell Signaling ◽  
Cytoplasmic Tail ◽  
Phosphatidylinositol 3 Kinase ◽  
3T3 Cells ◽  
Jaagsiekte Sheep Retrovirus ◽  
Phosphorylated Akt ◽  
A Cell ◽  
Nih 3T3 ◽  
Nih 3T3 Cells ◽  
Phosphatidylinositol 3

ABSTRACT Jaagsiekte sheep retrovirus (JSRV) is the causative agent of a transmissible lung cancer of sheep known as ovine pulmonary carcinoma. Recently, we have found that the expression of the JSRV envelope (Env) is sufficient to transform mouse NIH 3T3 cells in classical transformation assays. To further investigate the mechanisms of JSRV oncogenesis, we generated a series of envelope chimeras between JSRV and the JSRV-related endogenous retroviruses of sheep (enJSRVs) and assessed them in transformation assays. Chimeras containing the exogenous JSRV SU region and the enJSRV TM region were unable to transform NIH 3T3 cells. Additional chimeras containing only the carboxy-terminal portion of TM (a region that we previously identified as VR3) of the endogenous envelope with SU and the remaining portion of TM from the exogenous JSRV were also unable to transform NIH 3T3 cells. The VR3 region includes the putative membrane-spanning region and cytoplasmic tail of the JSRV TM glycoprotein; this suggested that the cytoplasmic tail of the JSRV Env mediates transformation, possibly via a cell signaling mechanism. Mutations Y590 and M593 in the cytoplasmic tail of the JSRV envelope were sufficient to inhibit the transforming abilities of these constructs. Y590 and M593 are part of a Y-X-X-M motif that is recognized by the phosphatidylinositol 3-kinase (PI-3K). PI-3K initiates a cell signaling pathway that inhibits apoptosis and is required for a number of mitogens during the G1-to-S-phase transition of the cell cycle. PI-3K activates Akt by phosphorylation of threonine 308 and serine 473. We detected by Western blot analysis phosphorylated Akt in serum-starved MP1 cells (NIH 3T3 cells transformed by JSRV) but not in the parental NIH 3T3 cells. These data indicate that the cytoplasmic tail of the JSRV TM is necessary for cell transformation and suggest a new mechanism of retroviral transformation. In addition, the ability to dissociate the function of the JSRV envelope to mediate viral entry from its transforming capacity has direct relevance for the design of JSRV-based vectors that target the differentiated epithelial cells of the lungs.

scholarly journals The Saccharomyces cerevisiae SDC25 C-domain gene product overcomes the dominant inhibitory activity of Ha-Ras Asn-17.

1993 ◽  
Vol 13 (1) ◽  
pp. 39-43 ◽  
Author(s):  
F Schweighoffer ◽  
H Cai ◽  
M C Chevallier-Multon ◽  
I Fath ◽  
G Cooper ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Product ◽  
Ras Proteins ◽  
3T3 Cells ◽  
Kinase Gene ◽  
Inhibition Of Proliferation ◽  
Negative Effect ◽  
Carboxy Terminal ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

The carboxy-terminal part of the Saccharomyces cerevisiae SDC25 gene product (SDC25 C domain) can elicit activation of mammalian Ras proteins. Specifically, SDC25 C domain functions as an exchange factor for cellular Ras proteins in CHO cells. In this study, we used the dominant inhibitory Ha-Ras Asn-17 mutant and SDC25 C domain to further investigate the interaction between cellular Ras proteins and their putative endogenous guanine nucleotide-releasing factors. Transcription from the polyomavirus thymidine kinase gene (Py tk) promoter is strongly inhibited by the expression of Ha-Ras Asn-17 in NIH 3T3 cells. Coexpression of SDC25 C domain overcomes the negative effect of the Ras mutant on the Py tk promoter. On the other hand, transactivation of the Ras-responsive element of the Py tk promoter induced by SDC25 C domain is lost upon coexpression of increasing amounts of Ha-Ras Asn-17. In addition, coexpression of SDC25 C domain overcomes the inhibition of proliferation of NIH 3T3 cells caused by Ha-Ras Asn-17. These results are consistent with the idea that the Ha-Ras Asn-17 mutant functions by titrating an upstream activator of cellular Ras proteins.

scholarly journals Phosphatidylinositol 3-kinase binds to α-actinin through the p85 subunit

1994 ◽  
Vol 302 (2) ◽  
pp. 551-557 ◽  
Author(s):  
F Shibasaki ◽  
K Fukami ◽  
Y Fukui ◽  
T Takenawa
Keyword(s):  
Signal Transduction ◽  
Kinase Activity ◽  
Cytoskeletal Reorganization ◽  
Phosphatidylinositol 3 Kinase ◽  
3T3 Cells ◽  
Rich Region ◽  
Phosphatidylinositol 4 ◽  
Nih 3T3 ◽  
Proline Rich Region ◽  
Phosphatidylinositol 3

Phosphatidylinositol 3-kinase (PI 3-kinase) has been shown to play an important role in the signal transduction of cell growth. It is also suggested that it is involved in cytoskeletal reorganization. We have found that alpha-actinin copurifies with PI 3-kinase from bovine thymus. The antibody against PI 3-kinase 85 kDa subunit (p85) also co-immunoprecipitates alpha-actinin from lysates of NIH/3T3 cells. In addition, anti-alpha-actinin antibody coprecipitates PI 3-kinase activity. This coprecipitation was observed even after depolymerization of actin fibres, suggesting that PI 3-kinase binds directly to alpha-actinin. As alpha-actinin is a phosphatidylinositol 4,5-bisphosphate (PI4,5P2)-binding protein, binding experiments using various constructs of truncated p85 were carried out in the presence or absence of PI4,5P2. In the absence of PI4,5P2, chicken gizzard alpha-actinin binds only to the whole p85 construct, but it binds to the proline-rich region of p85 fragments in the presence of PI4,5P2. This binding is enhanced with increased concentrations of Pi4,5P2 up to 10 microM, whereas phosphatidylinositol and phosphatidylinositol 4-phosphate were not good activators of alpha-actinin binding. These results suggest that PI 3-kinase binds to alpha-actinin and regulates cytoskeletal reorganization.

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