scholarly journals Ras activation by insulin and epidermal growth factor through enhanced exchange of guanine nucleotides on p21ras

1993 ◽  
Vol 13 (1) ◽  
pp. 155-162
Author(s):  
R H Medema ◽  
A M de Vries-Smits ◽  
G C van der Zon ◽  
J A Maassen ◽  
J L Bos
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Guanine Nucleotide Exchange Factor ◽  
Nucleotide Binding ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Epidermal Growth

A number of growth factors, including insulin and epidermal growth factor (EGF), induce accumulation of the GTP-bound form of p21ras. This accumulation could be caused either by an increase in guanine nucleotide exchange on p21ras or by a decrease in the GTPase activity of p21ras. To investigate whether insulin and EGF affect nucleotide exchange on p21ras, we measured binding of [alpha-32P]GTP to p21ras in cells permeabilized with streptolysin O. For this purpose, we used a cell line which expressed elevated levels of p21 H-ras and which was highly responsive to insulin and EGF. Stimulation with insulin or EGF resulted in an increase in the rate of nucleotide binding to p21ras. To determine whether this increased binding rate is due to the activation of a guanine nucleotide exchange factor, we made use of the inhibitory properties of a dominant negative mutant of p21ras, p21ras (Asn-17). Activation of p21ras by insulin and EGF in intact cells was abolished in cells infected with a recombinant vaccinia virus expressing p21ras (Asn-17). In addition, the enhanced nucleotide binding to p21ras in response to insulin and EGF in permeabilized cells was blocked upon expression of p21ras (Asn-17). From these data, we conclude that the activation of a guanine nucleotide exchange factor is involved in insulin- and EGF-induced activation of p21ras.

scholarly journals Ras activation by insulin and epidermal growth factor through enhanced exchange of guanine nucleotides on p21ras.

1993 ◽  
Vol 13 (1) ◽  
pp. 155-162 ◽  
Author(s):  
R H Medema ◽  
A M de Vries-Smits ◽  
G C van der Zon ◽  
J A Maassen ◽  
J L Bos
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Guanine Nucleotide Exchange Factor ◽  
Nucleotide Binding ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Epidermal Growth

A number of growth factors, including insulin and epidermal growth factor (EGF), induce accumulation of the GTP-bound form of p21ras. This accumulation could be caused either by an increase in guanine nucleotide exchange on p21ras or by a decrease in the GTPase activity of p21ras. To investigate whether insulin and EGF affect nucleotide exchange on p21ras, we measured binding of [alpha-32P]GTP to p21ras in cells permeabilized with streptolysin O. For this purpose, we used a cell line which expressed elevated levels of p21 H-ras and which was highly responsive to insulin and EGF. Stimulation with insulin or EGF resulted in an increase in the rate of nucleotide binding to p21ras. To determine whether this increased binding rate is due to the activation of a guanine nucleotide exchange factor, we made use of the inhibitory properties of a dominant negative mutant of p21ras, p21ras (Asn-17). Activation of p21ras by insulin and EGF in intact cells was abolished in cells infected with a recombinant vaccinia virus expressing p21ras (Asn-17). In addition, the enhanced nucleotide binding to p21ras in response to insulin and EGF in permeabilized cells was blocked upon expression of p21ras (Asn-17). From these data, we conclude that the activation of a guanine nucleotide exchange factor is involved in insulin- and EGF-induced activation of p21ras.

scholarly journals The guanine nucleotide exchange factor Ric-8A induces domain separation and Ras domain plasticity in Gαi1

2015 ◽  
Vol 112 (5) ◽  
pp. 1404-1409 ◽  
Author(s):  
Ned Van Eps ◽  
Celestine J. Thomas ◽  
Wayne L. Hubbell ◽  
Stephen R. Sprang
Keyword(s):  
G Protein ◽  
Bound States ◽  
Guanine Nucleotide Exchange Factor ◽  
Nucleotide Binding ◽  
Conformational Ensemble ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

Heterotrimeric G proteins are activated by exchange of GDP for GTP at the G protein alpha subunit (Gα), most notably by G protein-coupled transmembrane receptors. Ric-8A is a soluble cytoplasmic protein essential for embryonic development that acts as both a guanine nucleotide exchange factor (GEF) and a chaperone for Gα subunits of the i, q, and 12/13 classes. Previous studies demonstrated that Ric-8A stabilizes a dynamically disordered state of nucleotide-free Gα as the catalytic intermediate for nucleotide exchange, but no information was obtained on the structures involved or the magnitude of the structural fluctuations. In the present study, site-directed spin labeling (SDSL) together with double electron-electron resonance (DEER) spectroscopy is used to provide global distance constraints that identify discrete members of a conformational ensemble in the Gαi1:Ric-8A complex and the magnitude of structural differences between them. In the complex, the helical and Ras-like nucleotide-binding domains of Gαi1 pivot apart to occupy multiple resolved states with displacements as large as 25 Å. The domain displacement appears to be distinct from that observed in Gαs upon binding of Gs to the β2 adrenergic receptor. Moreover, the Ras-like domain exhibits structural plasticity within and around the nucleotide-binding cavity, and the switch I and switch II regions, which are known to adopt different conformations in the GDP- and GTP-bound states of Gα, undergo structural rearrangements. Collectively, the data show that Ric-8A induces a conformationally heterogeneous state of Gαi and provide insight into the mechanism of action of a nonreceptor Gα GEF.

scholarly journals Integration of Transforming Growth Factor β and RAS Signaling Silences a RAB5 Guanine Nucleotide Exchange Factor and Enhances Growth Factor-Directed Cell Migration

2007 ◽  
Vol 28 (5) ◽  
pp. 1573-1583 ◽  
Author(s):  
Hailiang Hu ◽  
Marc Milstein ◽  
Joanne M. Bliss ◽  
Minh Thai ◽  
Gautam Malhotra ◽  
...  
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor ◽  
Guanine Nucleotide Exchange Factor ◽  
Transforming Growth Factor Β ◽  
Ras Signaling ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

ABSTRACT Transforming growth factor β (TGF-β) receptor (TβR) signaling contributes to normal development as well as tumorigenesis. Here we report that RIN1, a RAB5 guanine nucleotide exchange factor (GEF) and down regulator of receptor tyrosine kinases (RTKs), promotes TβR signaling through enhanced endocytosis. TβR activation induces SNAI1 (Snail), a transcription repressor that reduces RIN1 expression, providing a negative feedback mechanism to control TβR trafficking and downstream signaling. Persistent RAS signaling disrupts this equilibrium by stabilizing SNAI1 protein, resulting in strong silencing of RIN1 and stabilization of RTKs. TGF-β-induced RIN1 silencing in breast cancer cells prolonged sensitivity to hepatocyte growth factor, a ligand for the MET-type RTK, and enhanced growth factor-directed cell motility. We conclude that in some tumor cells TβR and RAS signals are integrated through the silencing of RIN1, leading to a reduction in RAB5-mediated endocytosis. These findings shed new light on the basis for distinct interpretations of TGF-β signaling by normal versus transformed cells.

scholarly journals Coupling between p210bcr-abl and Shc and Grb2 adaptor proteins in hematopoietic cells permits growth factor receptor-independent link to ras activation pathway.

1994 ◽  
Vol 179 (1) ◽  
pp. 167-175 ◽  
Author(s):  
T Tauchi ◽  
H S Boswell ◽  
D Leibowitz ◽  
H E Broxmeyer
Keyword(s):  
Growth Factor ◽  
Cell Lines ◽  
Hematopoietic Cells ◽  
Guanine Nucleotide Exchange Factor ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Activation Pathway ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

Enforced expression of p210bcr-abl transforms interleukin 3 (IL-3)-dependent hematopoietic cell lines to growth factor-independent proliferation. It has been demonstrated that nonreceptor tyrosine kinase oncogenes may couple to the p21ras pathway to exert their transforming effect. In particular, p210bcr-abl was recently found to effect p21ras activation in hematopoietic cells. In this context, experiments were performed to evaluate a protein signaling pathway by which p210bcr-abl might regulate p21ras. It was asked whether Shc p46/p52, a protein containing a src-homology region 2 (SH2) domain, and known to function upstream from p21ras, might form specific complexes with p210bcr-abl and thus, possibly alter p21ras activity by coupling to the guanine nucleotide exchange factor (Sos/CDC25) through the Grb2 protein-Sos complex. This latter complex has been previously demonstrated to occur ubiquitously. We found that p210bcr-abl formed a specific complex with Shc and with Grb2 in three different murine cell lines transfected with a p210bcr-abl expression vector. There appeared to be a higher order complex containing Shc, Grb2, and bcr-abl proteins. In contrast to p210bcr-abl transformed cells, in which there was constitutive tight association between Grb2 and Shc, binding between Grb2 and Shc was Steel factor (SLF)-dependent in a SLF-responsive, nontransformed parental cell line. The SLF-dependent association between Grb2 and Shc in nontransformed cells involved formation of a complex of Grb2 with c-kit receptor after SLF treatment. Thus, p210bcr-abl appears to function in a hematopoietic p21ras activation pathway to allow growth factor-independent coupling between Grb2, which exists in a complex with the guanine nucleotide exchange factor (Sos), and p21ras. Shc may not be required for Grb2-c-kit interaction, because it fails to bind strongly to c-kit.

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