scholarly journals N Terminus of Sos1 Ras Exchange Factor: Critical Roles for the Dbl and Pleckstrin Homology Domains

1998 ◽  
Vol 18 (2) ◽  
pp. 771-778 ◽  
Author(s):  
Xiaolan Qian ◽  
William C. Vass ◽  
Alex G. Papageorge ◽  
Pieter H. Anborgh ◽  
Douglas R. Lowy
Keyword(s):  
Biological Activity ◽  
Growth Factor ◽  
Map Kinase ◽  
Stable Complex ◽  
Egf Receptors ◽  
Pleckstrin Homology ◽  
3T3 Cells ◽  
N Terminus ◽  
Ph Domains ◽  
Nih 3T3

ABSTRACT We have studied the functional importance of the N terminus of mouse Sos1 (mSos1), a ubiquitously expressed Ras-specific guanine nucleotide exchange factor whose C-terminal sequences bind Grb-2. Consistent with previous reports, addition of a myristoylation signal to mSos1 (MyrSos1) rendered it transforming for NIH 3T3 cells and deletion of the mSos C terminus (MyrSos1-ΔC) did not interfere with this activity. However, an N-terminally deleted myristoylated mSos1 protein (MyrSos1-ΔN) was transformation defective, although the protein was stable and localized to the membrane. Site-directed mutagenesis was used to examine the role of the Dbl and pleckstrin homology (PH) domains located in the N terminus. When mutations in the PH domain were introduced into two conserved amino acids either singly or together in MyrSos1 or MyrSos1-ΔC, the transforming activity was severely impaired. An analogous reduction in biological activity was seen when a cluster of point mutations was engineered into the Dbl domain. The mitogen-activation protein (MAP) kinase activities induced by the various Dbl and PH mutants of MyrSos1 correlated with their biological activities. When NIH 3T3 cells were transfected with a myristoylated Sos N terminus, their growth response to epidermal growth factor (EGF), platelet-derived growth factor, lysophosphatidic acid or serum was greatly impaired. The dominant inhibitory biological activity of the N terminus correlated with its ability to impair EGF-dependent activation of GTP-Ras and of MAP kinase, as well with the ability of endogenous Sos to form a stable complex with activated EGF receptors. The N terminus with mutations in the Dbl and PH domains was much less inhibitory in these biological and biochemical assays. In contrast to wild-type Sos1, nonmyristoylated versions of Sos1-ΔN and Sos1-ΔC did not form a stable complex with activated EGF receptors. We conclude that the Dbl and PH domains are critical for Sos function and that stable association of Sos with activated EGF receptors requires both the Sos N and C termini.

scholarly journals Loss of epidermal growth factor receptors and release of transforming growth factors do not correlate with sarcoma virus-transformation in clonally-related NIH/3T3-derived cell lines

1985 ◽  
Vol 229 (1) ◽  
pp. 119-125 ◽  
Author(s):  
K D Brown ◽  
D M Blakeley ◽  
P Roberts ◽  
R J Avery
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Cell Line ◽  
Conditioned Medium ◽  
Cell Lines ◽  
Egf Receptors ◽  
3T3 Cells ◽  
Sarcoma Virus ◽  
Epidermal Growth ◽  
Nih 3T3

Transformation of NIH/3T3 cells by Kirsten murine sarcoma virus (MSV) caused a dramatic reduction in the number of cell-surface receptors for epidermal growth factor (EGF). However, the number of EGF receptors remained at a very low level in a non-tumourigenic revertant cell line isolated from the virus-transformed cells, indicating that an increase in EGF receptors is not a requirement for the phenotypic reversion of Kirsten MSV-transformed 3T3 cells. Serum-free conditioned medium from normal and virus-transformed cell lines contained similar amounts of cell growth-promoting activity as assayed by the ability to stimulate DNA synthesis in quiescent Swiss 3T3 cell cultures. However, the concentrated conditioned medium from these cell lines showed no evidence of beta-transforming growth factor (TGF) activity as assayed by promotion of anchorage-independent growth of untransformed normal rat kidney (NRK) fibroblasts in agarose. The cellular release of alpha-TGF activity was assayed by measuring the ability of concentrated conditioned medium to inhibit the binding of 125I-EGF to Swiss 3T3 cells. Conditioned medium protein from the virus-transformed cell line inhibited 125I-EGF binding but only to the same extent as conditioned medium protein prepared from the untransformed cell line. The alpha-TGF secretion by these cell lines was estimated to be 30-45-fold lower than the level of alpha-TGF released by a well-characterized alpha-TGF-producing cell line (3B11). These results suggest that the induction of TGF release is not a necessary event in the transformation of NIH/3T3 cells by Kirsten MSV.

Effect of Taurine on Platelet Derived Growth Factor (PDGF)-induced c-fos and c-jun Expressions and MAP kinase activation in NIH/3T3 Cells

2000 ◽  
Vol 28 (5) ◽  
pp. A202-A202
Author(s):  
T. Nabika ◽  
M. Terashima ◽  
Y. Hosokawa ◽  
K. Imada ◽  
Y. Nakashima ◽  
...  
Keyword(s):  
Growth Factor ◽  
Map Kinase ◽  
Platelet Derived Growth Factor ◽  
3T3 Cells ◽  
Kinase Activation ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

scholarly journals Rit Contributes to Nerve Growth Factor-Induced Neuronal Differentiation via Activation of B-Raf-Extracellular Signal-Regulated Kinase and p38 Mitogen-Activated Protein Kinase Cascades

2005 ◽  
Vol 25 (2) ◽  
pp. 830-846 ◽  
Author(s):  
Geng-Xian Shi ◽  
Douglas A. Andres
Keyword(s):  
Nerve Growth Factor ◽  
Growth Factor ◽  
Map Kinase ◽  
Neurite Outgrowth ◽  
Neuronal Differentiation ◽  
3T3 Cells ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal ◽  
Mitogen Activated Protein ◽  
Nih 3T3

ABSTRACT Rit is one of the original members of a novel Ras GTPase subfamily that uses distinct effector pathways to transform NIH 3T3 cells and induce pheochromocytoma cell (PC6) differentiation. In this study, we find that stimulation of PC6 cells by growth factors, including nerve growth factor (NGF), results in rapid and prolonged Rit activation. Ectopic expression of active Rit promotes PC6 neurite outgrowth that is morphologically distinct from that promoted by oncogenic Ras (evidenced by increased neurite branching) and stimulates activation of both the extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinase signaling pathways. Furthermore, Rit-induced differentiation is dependent upon both MAP kinase cascades, since MEK inhibition blocked Rit-induced neurite outgrowth, while p38 blockade inhibited neurite elongation and branching but not neurite initiation. Surprisingly, while Rit was unable to stimulate ERK activity in NIH 3T3 cells, it potently activated ERK in PC6 cells. This cell type specificity is explained by the finding that Rit was unable to activate C-Raf, while it bound and stimulated the neuronal Raf isoform, B-Raf. Importantly, selective down-regulation of Rit gene expression in PC6 cells significantly altered NGF-dependent MAP kinase cascade responses, inhibiting both p38 and ERK kinase activation. Moreover, the ability of NGF to promote neuronal differentiation was attenuated by Rit knockdown. Thus, Rit is implicated in a novel pathway of neuronal development and regeneration by coupling specific trophic factor signals to sustained activation of the B-Raf/ERK and p38 MAP kinase cascades.

scholarly journals Transactivation of Platelet-Derived Growth Factor Receptor α by the GTPase-Deficient Activated Mutant of Gα12

2006 ◽  
Vol 26 (1) ◽  
pp. 50-62 ◽  
Author(s):  
Rashmi N. Kumar ◽  
Ji Hee Ha ◽  
Rangasudhagar Radhakrishnan ◽  
Danny N. Dhanasekaran
Keyword(s):  
Growth Factor ◽  
Cell Growth ◽  
Signaling Pathway ◽  
Growth Factor Receptor ◽  
Platelet Derived Growth Factor ◽  
Dominant Negative Mutant ◽  
Dependent Manner ◽  
3T3 Cells ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

ABSTRACT The GTPase-deficient, activated mutant of Gα12 (Gα12Q229L, or Gα12QL) induces neoplastic growth and oncogenic transformation of NIH 3T3 cells. Using microarray analysis, we have previously identified a role for platelet-derived growth factor receptor α (PDGFRα) in Gα12-mediated cell growth (R. N. Kumar et al., Cell Biochem. Biophys. 41:63-73, 2004). In the present study, we report that Gα12QL stimulates the functional expression of PDGFRα and demonstrate that the expression of PDGFRα by Gα12QL is dependent on the small GTPase Rho. Our results indicate that it is cell type independent as the transient expression of Gα12QL or the activation of Gα12-coupled receptors stimulates the expression of PDGFRα in NIH 3T3 as well as in human astrocytoma 1321N1 cells. Furthermore, we demonstrate the presence of an autocrine loop involving PDGF-A and PDGFRα in Gα12QL-transformed cells. Analysis of the functional consequences of the Gα12-PDGFRα signaling axis indicates that Gα12 stimulates the phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathway through PDGFR. In addition, we show that Gα12QL stimulates the phosphorylation of forkhead transcription factor FKHRL1 via AKT in a PDGFRα- and PI3K-dependent manner. Since AKT promotes cell growth by blocking the transcription of antiproliferative genes through the inhibitory phosphorylation of forkhead transcription factors, our results describe for the first time a PDGFRα-dependent signaling pathway involving PI3K-AKT-FKHRL1, regulated by Gα12QL in promoting cell growth. Consistent with this view, we demonstrate that the expression of a dominant negative mutant of PDGFRα attenuated Gα12-mediated neoplastic transformation of NIH 3T3 cells.

The K-fgf/hst oncogene induces transformation through an autocrine mechanism that requires extracellular stimulation of the mitogenic pathway

1991 ◽  
Vol 11 (2) ◽  
pp. 1138-1145
Author(s):  
D Talarico ◽  
C Basilico
Keyword(s):  
Growth Factor ◽  
Receptor Activation ◽  
Soft Agar ◽  
Serum Free Medium ◽  
Free Medium ◽  
3T3 Cells ◽  
Serum Free ◽  
Nih 3T3 ◽  
Autocrine Mechanism ◽  
Nih 3T3 Cells

The K-fgf/hst oncogene encodes a secreted growth factor of the fibroblast growth factor (FGF) family. The ability of K-fgf-transformed cells to grow in soft agar and in serum-free medium is inhibited by anti-K-FGF neutralizing antibodies, consistent with an autocrine mechanism of transformation. The transformed properties of clones that express high levels of K-FGF are, however, only partially affected. To better define the autocrine mechanism of transformation by K-fgf and to determine whether receptor activation could occur intracellularly, we constructed two mutants of the K-fgf cDNA. Deletion of the sequences encoding the signal peptide suppressed K-fgf ability to induce foci in NIH 3T3 cells. A few morphologically transformed colonies were observed in cotransfection experiments, and they were found to express high levels of cytoplasmic K-FGF. However, their ability to grow in serum-free medium and in soft agar was inhibited by anti-K-FGF antibodies. Addition of a sequence encoding the KDEL endoplasmic reticulum and Golgi retention signal to the K-fgf cDNA led to accumulation of the growth factor in intracellular compartments. The ability of the KDEL mutant to induce foci in NIH 3T3 cells was much lower than that of the wild-type cDNA, and also in this case the transformed phenotype was reverted by anti-K-FGF antibodies. These and other findings indicate that the transformed phenotype of cells expressing a nonsecretory K-FGF is due to the extracellular activation of the receptor by the small amounts of growth factor that these cells still release. Thus, transformation by K-fgf appears to be due to an autocrine growth mechanisms that requires activation of the mitogenic pathway at the cell surface.

Processing, secretion, and biological properties of a novel growth factor of the fibroblast growth factor family with oncogenic potential

1988 ◽  
Vol 8 (7) ◽  
pp. 2933-2941
Author(s):  
P Delli-Bovi ◽  
A M Curatola ◽  
K M Newman ◽  
Y Sato ◽  
D Moscatelli ◽  
...  
Keyword(s):  
Biological Activity ◽  
Growth Factor ◽  
Kaposi Sarcoma ◽  
Biological Properties ◽  
Fibroblast Growth ◽  
Serum Free Medium ◽  
Free Medium ◽  
Serum Free ◽  
The Stability ◽  
Nih 3T3

We recently reported that the protein encoded in a novel human oncogene isolated from Kaposi sarcoma DNA was a growth factor with significant homology to basic and acidic fibroblast growth factors (FGFs). To study the properties of this growth factor (referred to as K-FGF) and the mechanism by which the K-fgf oncogene transforms cells, we have studied the production and processing of K-FGF in COS-1 cells transfected with a plasmid encoding the K-fgf cDNA. The results show that, unlike basic and acidic FGFs, the K-FGF protein is cleaved after a signal peptide, glycosylated, and efficiently secreted as a mature protein of 176 or 175 amino acids. Inhibition of glycosylation impaired secretion, and the stability of the secreted K-FGF was greatly enhanced by the presence of heparin in the cultured medium. We have used the conditioned medium from transfected COS-1 cells to test K-FGF biological activity. Similar to basic FGF, the K-FGF protein was mitogenic for fibroblasts and endothelial cells and induced the growth of NIH 3T3 mouse cells in serum-free medium. Accordingly, K-fgf-transformed NIH 3T3 cells grew in serum-free medium, consistent with an autocrine mechanism of growth. We have also expressed the protein encoded in the K-fgf protooncogene in COS-1 cells, and it was indistinguishable in its molecular weight, glycosylation, secretion, and biological activity from K-FGF. Taken together, these results suggest that the mechanism of activation of this oncogene is due to overexpression rather than to mutations in the coding sequences.

Synthesis of membrane-bound colony-stimulating factor 1 (CSF-1) and downmodulation of CSF-1 receptors in NIH 3T3 cells transformed by cotransfection of the human CSF-1 and c-fms (CSF-1 receptor) genes

1987 ◽  
Vol 7 (7) ◽  
pp. 2378-2387 ◽  
Author(s):  
C W Rettenmier ◽  
M F Roussel ◽  
R A Ashmun ◽  
P Ralph ◽  
K Price ◽  
...  
Keyword(s):  
Growth Factor ◽  
Disulfide Bonds ◽  
Mononuclear Phagocyte ◽  
Transformed Cells ◽  
Colony Stimulating Factor ◽  
3T3 Cells ◽  
Membrane Bound ◽  
Nih 3T3 ◽  
Stimulating Factor ◽  
Nih 3T3 Cells

NIH 3T3 cells cotransfected with the human c-fms proto-oncogene together with a 1.6-kilobase cDNA clone encoding a 256-amino-acid precursor of the human mononuclear phagocyte colony-stimulating factor CSF-1 (M-CSF) undergo transformation by an autocrine mechanism. The number of CSF-1 receptors on the surface of transformed cells was regulated by ligand-induced receptor degradation and was inversely proportional to the quantity of CSF-1 produced. A tyrosine-to-phenylalanine mutation at position 969 near the receptor carboxyl terminus potentiated its transforming efficiency in cells cotransfected by the CSF-1 gene but did not affect receptor downmodulation. CSF-1 was synthesized as an integral transmembrane glycoprotein that was rapidly dimerized through disulfide bonds. The homodimer was externalized at the cell surface, where it underwent proteolysis to yield the soluble growth factor. Trypsin treatment of viable cells cleaved the plasma membrane form of CSF-1 to molecules of a size indistinguishable from that of the extracellular growth factor, suggesting that trypsinlike proteases regulate the rate of CSF-1 release from transformed cells. The data raise the possibility that this form of membrane-bound CSF-1 might stimulate receptors on adjacent cells through direct cell-cell interactions.

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