scholarly journals GTP binding to translation factor eIF2B stimulates its guanine nucleotide exchange activity

iScience ◽  
2021 ◽  
pp. 103454
Author(s):  
Christopher J. Kershaw ◽  
Martin D. Jennings ◽  
Francesco Cortopassi ◽  
Margherita Guaita ◽  
Hawra Al-Ghafli ◽  
...  
Keyword(s):  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Translation Factor ◽  
Guanine Nucleotide Exchange ◽  
Gtp Binding ◽  
Exchange Activity

scholarly journals GTP Binding to Translation Factor eIF2B Stimulates Its Guanine Nucleotide Exchange Activity

2021 ◽  
Author(s):  
Christopher J. Kershaw ◽  
Martin D. Jennings ◽  
Francesco Cortopassi ◽  
Margherita Guaita ◽  
Hawra Al-Ghafli ◽  
...  
Keyword(s):  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Translation Factor ◽  
Guanine Nucleotide Exchange ◽  
Gtp Binding ◽  
Exchange Activity

Expression of mutant eukaryotic initiation factor 2 alpha subunit (eIF-2 alpha) reduces inhibition of guanine nucleotide exchange activity of eIF-2B mediated by eIF-2 alpha phosphorylation

1994 ◽  
Vol 14 (7) ◽  
pp. 4546-4553
Author(s):  
K V Ramaiah ◽  
M V Davies ◽  
J J Chen ◽  
R J Kaufman
Keyword(s):  
Protein Synthesis ◽  
Initiation Factor ◽  
Guanine Nucleotide ◽  
Eukaryotic Initiation Factor ◽  
Nucleotide Exchange ◽  
Wild Type ◽  
Guanine Nucleotide Exchange ◽  
Initiation Factor 2 ◽  
Exchange Activity

The inhibition of protein synthesis that occurs upon phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2 alpha) at serine 51 correlates with reduced guanine nucleotide exchange activity of eIF-2B in vivo and inhibition of eIF-2B activity in vitro, although it is not known if phosphorylation is the cause of the reduced eIF-2B activity in vivo. To characterize the importance of eIF-2 alpha phosphorylation in the regulation of eIF-2B activity, we studied the overexpression of mutant eIF-2 alpha subunits in which serine 48 or 51 was replaced by an alanine (48A or 51A mutant). Previous studies demonstrated that the 51A mutant was resistant to phosphorylation, whereas the 48A mutant was a substrate for phosphorylation. Additionally, expression of either mutant partially protected Chinese hamster ovary (CHO) cells from the inhibition of protein synthesis in response to heat shock treatment (P. Murtha-Riel, M. V. Davies, J. B. Scherer, S. Y. Choi, J. W. B. Hershey, and R. J. Kaufman, J. Biol. Chem. 268:12946-12951, 1993). In this study, we show that eIF-2B activity was inhibited in parental CHO cell extracts upon addition of purified reticulocyte heme-regulated inhibitor (HRI), an eIF-2 alpha kinase that phosphorylates Ser-51. Preincubation with purified HRI also reduced the eIF-2B activity in extracts from cells overexpressing wild-type eIF-2 alpha. In contrast, the eIF-2B activity was not readily inhibited in extracts from cells overexpressing either the eIF-2 alpha 48A or 51A mutant. In addition, eIF-2B activity was decreased in extracts prepared from heat-shocked cells overexpressing wild-type eIF-2 alpha, whereas the decrease in eIF-2B activity was less in heat-shocked cells overexpressing either mutant 48A or mutant 51A. While the phosphorylation at serine 51 in eIF-2 alpha impairs the eIF-2B activity, we propose that serine 48 acts to maintain a high affinity between phosphorylated eIF-2 alpha and eIF-2B, thereby inactivating eIF-2B activity. These findings support the hypothesis that phosphorylation of eIF-2 alpha inhibits protein synthesis directly through reducing eIF-2B activity and emphasize the importance of both serine 48 and serine 51 in the interaction with eIF-2B and regulation of eIF-2B activity.

scholarly journals Modulation of a GEF switch: Autoinhibition of the intrinsic guanine nucleotide exchange activity of p115-RhoGEF

2010 ◽  
Vol 20 (1) ◽  
pp. 107-117 ◽  
Author(s):  
Zhe Chen ◽  
Liang Guo ◽  
Stephen R. Sprang ◽  
Paul C. Sternweis
Keyword(s):  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
P115 Rhogef ◽  
Exchange Activity

scholarly journals Structural and Spatial Determinants Regulating TC21 Activation by RasGRF Family Nucleotide Exchange Factors

2009 ◽  
Vol 20 (20) ◽  
pp. 4289-4302 ◽  
Author(s):  
Fernando Calvo ◽  
Piero Crespo
Keyword(s):  
Posttranslational Modifications ◽  
Guanine Nucleotide Exchange Factors ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Guanosine Triphosphate ◽  
Guanosine Diphosphate ◽  
Ras Gtpases ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors ◽  
Exchange Activity

RasGRF family guanine nucleotide exchange factors (GEFs) promote guanosine diphosphate (GDP)/guanosine triphosphate (GTP) exchange on several Ras GTPases, including H-Ras and TC21. Although the mechanisms controlling RasGRF function as an H-Ras exchange factor are relatively well characterized, little is known about how TC21 activation is regulated. Here, we have studied the structural and spatial requirements involved in RasGRF 1/2 exchange activity on TC21. We show that RasGRF GEFs can activate TC21 in all of its sublocalizations except at the Golgi complex. We also demonstrate that TC21 susceptibility to activation by RasGRF GEFs depends on its posttranslational modifications: farnesylated TC21 can be activated by both RasGRF1 and RasGRF2, whereas geranylgeranylated TC21 is unresponsive to RasGRF2. Importantly, we show that RasGRF GEFs ability to catalyze exchange on farnesylated TC21 resides in its pleckstrin homology 1 domain, by a mechanism independent of localization and of its ability to associate to membranes. Finally, our data indicate that Cdc42-GDP can inhibit TC21 activation by RasGRF GEFs, demonstrating that Cdc42 negatively affects the functions of RasGRF GEFs irrespective of the GTPase being targeted.

scholarly journals Activation of Ras in vitro and in intact fibroblasts by the Vav guanine nucleotide exchange protein.

1994 ◽  
Vol 14 (2) ◽  
pp. 906-913 ◽  
Author(s):  
E Gulbins ◽  
K M Coggeshall ◽  
C Langlet ◽  
G Baier ◽  
N Bonnefoy-Berard ◽  
...  
Keyword(s):  
Map Kinases ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
3T3 Cells ◽  
Transfected Cells ◽  
Guanine Nucleotide Exchange ◽  
Nih 3T3 ◽  
Exchange Activity ◽  
Nih 3T3 Cells

We recently identified Vav, the product of the vav proto-oncogene, as a guanine nucleotide exchange factor (GEF) for Ras. Vav is enzymatically activated by lymphocyte antigen receptor-coupled protein tyrosine kinases or independently by diglycerides. To further evaluate the physiological role of Vav, we assessed its GDP-GTP exchange activity against several Ras-related proteins in vitro and determined whether Vav activation in transfected NIH 3T3 fibroblasts correlates with the activity status of Ras and mitogen-activated protein (MAP) kinases. In vitro translated purified Vav activated by phorbol myristate acetate (PMA) or phosphorylation with recombinant p56lck displayed GEF activity against Ras but not against recombinant RacI, RacII, Ral, or RhoA proteins. Expression of vav or proto-vav in stably transfected NIH 3T3 cells led to a approximately 10-fold increase in basal or PMA-stimulated Ras exchange activity, respectively, in total-cell lysates and Vav immunoprecipitates. Elevated GEF activity was paralleled in each case by a significant increase in the proportion of active, GTP-bound Ras. PMA had a minimal effect on the low Ras. GTP level in untransfected control fibroblasts but increased it from 20 to 37% in proto-vav-transfected cells. vav-transfected cells displayed a constitutively elevated Ras. GTP level (35%), which was not increased further by PMA treatment. MAP kinases, known downstream intermediates in Ras-dependent signaling pathways, similarly exhibited increased basal or PMA-stimulated activity in Vav-expressing cells by comparison with normal NIH 3T3 cells. These results demonstrate a physiologic interaction between Vav and its target, Ras, leading to MAP kinase activation.

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