Activation of ras p21 transforming properties associated with an increase in the release rate of bound guanine nucleotide

1986 ◽  
Vol 6 (12) ◽  
pp. 4214-4220
Author(s):  
J C Lacal ◽  
S A Aaronson
Keyword(s):  
Slow Rate ◽  
Active Form ◽  
Biologically Active ◽  
Regulatory Proteins ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Activating Mutations ◽  
Temperature Requirements ◽  
Nucleotide Release ◽  
Ras P21

An Ala-to-Thr substitution at position 59 activates the transforming properties of the p21ras protein without impairment of GTPase activity, a biochemical alteration associated with other activating mutations. To investigate the basis for the transforming properties of the Thr-59 mutant, we characterized guanine nucleotide release. This reaction exhibited a slow rate and stringent temperature requirements. To further dissect the release reaction, we used monoclonal antibodies directed against different epitopes of the p21 molecule. One monoclonal specifically interfered with nucleotide release, while others which recognized different regions of the molecule blocked nucleotide binding. Mutants with the Thr-59 substitution exhibited a three- to ninefold-higher rate of GDP and GTP release than normal p21 or mutants with other activating lesions. This alteration in the Thr-59 mutant would have the effect of increasing its rate of nucleotide exchange. In an intracellular environment with a high GTP/GDP ratio, this would favor the association of GTP with the Thr-59 mutant. Consistent with knowledge of known G-regulatory proteins, these findings support a model in which the p21-GTP complex is the biologically active form of the p21 protein.

scholarly journals Activation of ras p21 transforming properties associated with an increase in the release rate of bound guanine nucleotide.

1986 ◽  
Vol 6 (12) ◽  
pp. 4214-4220 ◽  
Author(s):  
J C Lacal ◽  
S A Aaronson
Keyword(s):  
Slow Rate ◽  
Active Form ◽  
Biologically Active ◽  
Regulatory Proteins ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Activating Mutations ◽  
Temperature Requirements ◽  
Nucleotide Release ◽  
Ras P21

An Ala-to-Thr substitution at position 59 activates the transforming properties of the p21ras protein without impairment of GTPase activity, a biochemical alteration associated with other activating mutations. To investigate the basis for the transforming properties of the Thr-59 mutant, we characterized guanine nucleotide release. This reaction exhibited a slow rate and stringent temperature requirements. To further dissect the release reaction, we used monoclonal antibodies directed against different epitopes of the p21 molecule. One monoclonal specifically interfered with nucleotide release, while others which recognized different regions of the molecule blocked nucleotide binding. Mutants with the Thr-59 substitution exhibited a three- to ninefold-higher rate of GDP and GTP release than normal p21 or mutants with other activating lesions. This alteration in the Thr-59 mutant would have the effect of increasing its rate of nucleotide exchange. In an intracellular environment with a high GTP/GDP ratio, this would favor the association of GTP with the Thr-59 mutant. Consistent with knowledge of known G-regulatory proteins, these findings support a model in which the p21-GTP complex is the biologically active form of the p21 protein.

scholarly journals Neutralizing monoclonal antibody against ras oncogene product p21 which impairs guanine nucleotide exchange.

1987 ◽  
Vol 7 (5) ◽  
pp. 1999-2002 ◽  
Author(s):  
S Hattori ◽  
D J Clanton ◽  
T Satoh ◽  
S Nakamura ◽  
Y Kaziro ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Exchange Reaction ◽  
Dissociation Rate ◽  
Guanine Nucleotides ◽  
Guanine Nucleotide ◽  
Ras Oncogene ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Neutralizing Monoclonal Antibody ◽  
Ras P21

The neutralizing monoclonal antibody Y13-259 severely hampers the nucleotide exchange reaction between p21-bound and exogenous guanine nucleotides but does not interfere with the association of GDP to p21. These results suggest that the nucleotide exchange reaction is critical for p21 function. Interestingly, the v-ras p21 has a much faster dissociation rate than the p21 of the c-ras proto-oncogene.

scholarly journals Mammalian Mon2/Ysl2 regulates endosome-to-Golgi trafficking but possesses no guanine nucleotide exchange activity toward Arl1 GTPase

2013 ◽  
Vol 3 (1) ◽  
Author(s):  
Divyanshu Mahajan ◽  
Boon Kim Boh ◽  
Yan Zhou ◽  
Li Chen ◽  
Tobias Carl Cornvik ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Active Form ◽  
Small Gtpases ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Vitro Assay ◽  
Guanine Nucleotide Exchange ◽  
And Function ◽  
Golgi Network

Abstract Arl1 is a member of Arf family small GTPases that is essential for the organization and function of Golgi complex. Mon2/Ysl2, which shares significant homology with Sec7 family Arf guanine nucleotide exchange factors, was poorly characterized in mammalian cells. Here, we report the first in depth characterization of mammalian Mon2. We found that Mon2 localized to trans-Golgi network which was dependent on both its N and C termini. The depletion of Mon2 did not affect the Golgi localized or cellular active form of Arl1. Furthermore, our in vitro assay demonstrated that recombinant Mon2 did not promote guanine nucleotide exchange of Arl1. Therefore, our results suggest that Mon2 could be neither necessary nor sufficient for the guanine nucleotide exchange of Arl1. We demonstrated that Mon2 was involved in endosome-to-Golgi trafficking as its depletion accelerated the delivery of furin and CI-M6PR to Golgi after endocytosis.

scholarly journals COPI Recruitment Is Modulated by a Rab1b-dependent Mechanism

2003 ◽  
Vol 14 (5) ◽  
pp. 2116-2127 ◽  
Author(s):  
Cecilia Alvarez ◽  
Rafael Garcia-Mata ◽  
Elizabeth Brandon ◽  
Elizabeth Sztul
Keyword(s):  
Active Form ◽  
Brefeldin A ◽  
Small Gtpase ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Inactive Form ◽  
Exact Function ◽  
Guanine Nucleotide Binding

The small GTPase Rab1b is essential for endoplasmic reticulum (ER) to Golgi transport, but its exact function remains unclear. We have examined the effects of wild-type and three mutant forms of Rab1b in vivo. We show that the inactive form of Rab1b (the N121I mutant with impaired guanine nucleotide binding) blocks forward transport of cargo and induces Golgi disruption. The phenotype is analogous to that induced by brefeldin A (BFA): it causes resident Golgi proteins to relocate to the ER and induces redistribution of ER-Golgi intermediate compartment proteins to punctate structures. The COPII exit machinery seems to be functional in cells expressing the N121I mutant, but COPI is compromised, as shown by the release of β-COP into the cytosol. Our results suggest that Rab1b function influences COPI recruitment. In support of this, we show that the disruptive effects of N121I can be reversed by expressing known mediators of COPI recruitment, the GTPase ARF1 and its guanine nucleotide exchange factor GBF1. Further evidence is provided by the finding that cells expressing the active form of Rab1b (the Q67L mutant with impaired GTPase activity) are resistant to BFA. Our data suggest a novel role for Rab1b in ARF1- and GBF1-mediated COPI recruitment pathway.

Neutralizing monoclonal antibody against ras oncogene product p21 which impairs guanine nucleotide exchange

1987 ◽  
Vol 7 (5) ◽  
pp. 1999-2002 ◽  
Author(s):  
S Hattori ◽  
D J Clanton ◽  
T Satoh ◽  
S Nakamura ◽  
Y Kaziro ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Exchange Reaction ◽  
Dissociation Rate ◽  
Guanine Nucleotides ◽  
Guanine Nucleotide ◽  
Ras Oncogene ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Neutralizing Monoclonal Antibody ◽  
Ras P21

The neutralizing monoclonal antibody Y13-259 severely hampers the nucleotide exchange reaction between p21-bound and exogenous guanine nucleotides but does not interfere with the association of GDP to p21. These results suggest that the nucleotide exchange reaction is critical for p21 function. Interestingly, the v-ras p21 has a much faster dissociation rate than the p21 of the c-ras proto-oncogene.

scholarly journals Relationship among guanine nucleotide exchange, GTP hydrolysis, and transforming potential of mutated ras proteins.

1988 ◽  
Vol 8 (6) ◽  
pp. 2472-2478 ◽  
Author(s):  
L A Feig ◽  
G M Cooper
Keyword(s):  
Biological Activity ◽  
Exchange Rates ◽  
Fold Increase ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Activating Mutations ◽  
Guanine Nucleotide Exchange ◽  
Ras Activation ◽  
Fold Reduction ◽  
Activating Mutation

The effect of a series of mutations on the transforming potential of normal human rasH has been compared with their effects on GTPase and guanine nucleotide exchange rates of p21. The mutation Val-146 resulted in partial activation of transforming potential which could be attributed to a greater than 1,000-fold-increased rate of nucleotide exchange in the absence of an effect on GTPase. In contrast, the more modest enhancement of exchange rate (approximately 100-fold) which resulted from the mutation Met-14 did not affect biological activity. The partially activating mutation Thr-59 was found to result in both a 5-fold reduction in GTPase and a 10-fold increase in nucleotide exchange. However, the nontransforming mutant Ile-59 displayed a comparable decrease in GTPase without an effect on nucleotide exchange. The activating effect of the Thr-59 mutation may thus represent a combined effect of reduced GTPase and increased exchange. Similarly, the strongly activating mutation Leu-61 resulted in a fivefold increase in nucleotide exchange in addition to decreased GTPase, whereas weakly activating mutations at position 61 (Trp and Pro) resulted only in decreased GTPase without affecting nucleotide exchange rates. Finally, combining the two mutations Met-14 and Ile-59, which alone had no effect on biological activity, yielded a double mutant with a 20-fold increased transforming potential, demonstrating a synergistic effect of these two mutations. Overall, these results indicate that large increases in nucleotide exchange can activate ras transforming potential in the absence of decreased GTPase and that relatively modest increases in nucleotide exchange can act synergistically with decreased GTPase to contribute to ras activation.

Relationship among guanine nucleotide exchange, GTP hydrolysis, and transforming potential of mutated ras proteins

1988 ◽  
Vol 8 (6) ◽  
pp. 2472-2478
Author(s):  
L A Feig ◽  
G M Cooper
Keyword(s):  
Biological Activity ◽  
Exchange Rates ◽  
Fold Increase ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Activating Mutations ◽  
Guanine Nucleotide Exchange ◽  
Ras Activation ◽  
Fold Reduction ◽  
Activating Mutation

The effect of a series of mutations on the transforming potential of normal human rasH has been compared with their effects on GTPase and guanine nucleotide exchange rates of p21. The mutation Val-146 resulted in partial activation of transforming potential which could be attributed to a greater than 1,000-fold-increased rate of nucleotide exchange in the absence of an effect on GTPase. In contrast, the more modest enhancement of exchange rate (approximately 100-fold) which resulted from the mutation Met-14 did not affect biological activity. The partially activating mutation Thr-59 was found to result in both a 5-fold reduction in GTPase and a 10-fold increase in nucleotide exchange. However, the nontransforming mutant Ile-59 displayed a comparable decrease in GTPase without an effect on nucleotide exchange. The activating effect of the Thr-59 mutation may thus represent a combined effect of reduced GTPase and increased exchange. Similarly, the strongly activating mutation Leu-61 resulted in a fivefold increase in nucleotide exchange in addition to decreased GTPase, whereas weakly activating mutations at position 61 (Trp and Pro) resulted only in decreased GTPase without affecting nucleotide exchange rates. Finally, combining the two mutations Met-14 and Ile-59, which alone had no effect on biological activity, yielded a double mutant with a 20-fold increased transforming potential, demonstrating a synergistic effect of these two mutations. Overall, these results indicate that large increases in nucleotide exchange can activate ras transforming potential in the absence of decreased GTPase and that relatively modest increases in nucleotide exchange can act synergistically with decreased GTPase to contribute to ras activation.

scholarly journals A Histidine pH sensor regulates activation of the Ras-specific guanine nucleotide exchange factor RasGRP1

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Yvonne Vercoulen ◽  
Yasushi Kondo ◽  
Jeffrey S Iwig ◽  
Axel B Janssen ◽  
Katharine A White ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Active Form ◽  
Ph Sensor ◽  
Structural Features ◽  
Structural Basis ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Aberrant Expression ◽  
Membrane Recruitment ◽  
Guanine Nucleotide Exchange

RasGRPs are guanine nucleotide exchange factors that are specific for Ras or Rap, and are important regulators of cellular signaling. Aberrant expression or mutation of RasGRPs results in disease. An analysis of RasGRP1 SNP variants led to the conclusion that the charge of His 212 in RasGRP1 alters signaling activity and plasma membrane recruitment, indicating that His 212 is a pH sensor that alters the balance between the inactive and active forms of RasGRP1. To understand the structural basis for this effect we compared the structure of autoinhibited RasGRP1, determined previously, to those of active RasGRP4:H-Ras and RasGRP2:Rap1b complexes. The transition from the autoinhibited to the active form of RasGRP1 involves the rearrangement of an inter-domain linker that displaces inhibitory inter-domain interactions. His 212 is located at the fulcrum of these conformational changes, and structural features in its vicinity are consistent with its function as a pH-dependent switch.

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