Note to users. If you're seeing this message, it means that your browser cannot find this page's style/presentation instructions -- or possibly that you are using a browser that does not support current Web standards. Find out more about why this message is appearing, and what you can do to make your experience of our site the best it can be.

Subscribe

Logo for

Mol. Cell. Biol. 21 (6): 2107-2117

Copyright © 2001 by the American Society for Microbiology. All rights reserved.

Molecular and Cellular Biology, March 2001, p. 2107-2117, Vol. 21, No. 6
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.6.2107-2117.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Ras Binding Triggers Ubiquitination of the Ras Exchange Factor Ras-GRF2

Carmen L. de Hoog,1,2 Jackie A. Koehler,1,2 Marni D. Goldstein,1 Paul Taylor,3 Daniel Figeys,3 and Michael F. Moran1,2,3,*

Banting and Best Department of Medical Research1 and Department of Molecular and Medical Genetics,2 University of Toronto, Toronto, Ontario M5G 1X5, and MDS Proteomics, Inc., Toronto, Ontario M5G 1V2,3 Canada

Received 3 October 2000/Returned for modification 20 November 2000/Accepted 13 December 2000

Ras is a small GTPase that is activated by upstream guanine nucleotide exchange factors, one of which is Ras-GRF2. GRF2 is a widely expressed protein with several recognizable sequence motifs, including a Ras exchanger motif (REM), a PEST region containing a destruction box (DB), and a Cdc25 domain. The Cdc25 domain possesses guanine nucleotide exchange factor activity and interacts with Ras. Herein we examine if the DB motif in GRF2 results in proteolysis via the ubiquitin pathway. Based on the solved structure of the REM and Cdc25 regions of the Son-of-sevenless (Sos) protein, the REM may stabilize the Cdc25 domain during Ras binding. The DB motif of GRF2 is situated between the REM and the Cdc25 domains, tempting speculation that it may be exposed to ubiquitination machinery upon Ras binding. GRF2 protein levels decrease dramatically upon activation of GRF2, and dominant-negative Ras induces degradation of GRF2, demonstrating that signaling downstream of Ras is not required for the destruction of GRF2 and that binding to Ras is important for degradation. GRF2 is ubiquitinated in vivo, and this can be detected using mass spectrometry. In the presence of proteasome inhibitors, Ras-GRF2 accumulates as a high-molecular-weight conjugate, suggesting that GRF2 is destroyed by the 26S proteasome. Deleting the DB reduces the ubiquitination of GRF2. GRF2 lacking the Cdc25 domain is not ubiquitinated, suggesting that a protein that cannot bind Ras cannot be properly targeted for destruction. Point mutations within the Cdc25 domain that eliminate Ras binding also eliminate ubiquitination, demonstrating that binding to Ras is necessary for ubiquitination of GRF2. We conclude that conformational changes induced by GTPase binding expose the DB and thereby target GRF2 for destruction.


* Corresponding author. Mailing address: MDS Proteomics, Inc., 480 University Ave., Toronto, ON M5G 1V2, Canada. Phone: (416) 644-5103. Fax: (416) 644-5111. E-mail: m.moran{at}mdsproteomics.com.


Molecular and Cellular Biology, March 2001, p. 2107-2117, Vol. 21, No. 6
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.6.2107-2117.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

THIS ARTICLE HAS BEEN CITED BY OTHER ARTICLES:
RasGRF2 Rac-GEF activity couples NMDA receptor calcium flux to enhanced synaptic transmission.
B. Schwechter, C. Rosenmund, and K. F. Tolias (2013)
PNAS 110, 14462-14467
   Abstract »    Full Text »    PDF »
Elmo1 inhibits ubiquitylation of Dock180.
Y. Makino, M. Tsuda, S. Ichihara, T. Watanabe, M. Sakai, H. Sawa, K. Nagashima, S. Hatakeyama, and S. Tanaka (2006)
J. Cell Sci. 119, 923-932
   Abstract »    Full Text »    PDF »
The RAP1 Guanine Nucleotide Exchange Factor Epac2 Couples Cyclic AMP and Ras Signals at the Plasma Membrane.
Y. Li, S. Asuri, J. F. Rebhun, A. F. Castro, N. C. Paranavitana, and L. A. Quilliam (2006)
J. Biol. Chem. 281, 2506-2514
   Abstract »    Full Text »    PDF »
Farnesyl and Geranylgeranyl Transferase Inhibitors Induce G1 Arrest by Targeting the Proteasome.
E. T. Efuet and K. Keyomarsi (2006)
Cancer Res. 66, 1040-1051
   Abstract »    Full Text »    PDF »
Stress induces depletion of Cdc25p and decreases the cAMP producing capability in Saccharomyces cerevisiae.
L. Wang, G. Renault, H. Garreau, and M. Jacquet (2004)
Microbiology 150, 3383-3391
   Abstract »    Full Text »    PDF »
p35/Cyclin-Dependent Kinase 5 Phosphorylation of Ras Guanine Nucleotide Releasing Factor 2 (RasGRF2) Mediates Rac-Dependent Extracellular Signal-Regulated Kinase 1/2 Activity, Altering RasGRF2 and Microtubule-Associated Protein 1b Distribution in Neurons.
S. Kesavapany, N. Amin, Y.-L. Zheng, R. Nijhara, H. Jaffe, R. Sihag, J. S. Gutkind, S. Takahashi, A. Kulkarni, P. Grant, et al. (2004)
J. Neurosci. 24, 4421-4431
   Abstract »    Full Text »    PDF »
Insulin-induced phosphorylation of FKHR (Foxo1) targets to proteasomal degradation.
H. Matsuzaki, H. Daitoku, M. Hatta, K. Tanaka, and A. Fukamizu (2003)
PNAS 100, 11285-11290
   Abstract »    Full Text »    PDF »
rgr Oncogene: Activation by Elimination of Translational Controls and Mislocalization.
I. Hernandez-Munoz, M. Benet, M. Calero, M. Jimenez, R. Diaz, and A. Pellicer (2003)
Cancer Res. 63, 4188-4195
   Abstract »    Full Text »    PDF »
Guanine nucleotide exchange factors for Rho GTPases: turning on the switch.
A. Schmidt and A. Hall (2002)
Genes & Dev. 16, 1587-1609
   Full Text »    PDF »
Targeted Disruption of Ras-Grf2 Shows Its Dispensability for Mouse Growth and Development.
A. Fernandez-Medarde, L. M. Esteban, A. Nunez, A. Porteros, L. Tessarollo, and E. Santos (2002)
Mol. Cell. Biol. 22, 2498-2504
   Abstract »    Full Text »    PDF »
Nedd4 Regulates Ubiquitination and Stability of the Guanine-Nucleotide Exchange Factor CNrasGEF.
N. Pham and D. Rotin (2001)
J. Biol. Chem. 276, 46995-47003
   Abstract »    Full Text »    PDF »
Cloning and Characterization of Mouse UBPy, a Deubiquitinating Enzyme That Interacts with the Ras Guanine Nucleotide Exchange Factor CDC25Mm/Ras-GRF1.
N. Gnesutta, M. Ceriani, M. Innocenti, I. Mauri, R. Zippel, E. Sturani, B. Borgonovo, G. Berruti, and E. Martegani (2001)
J. Biol. Chem. 276, 39448-39454
   Abstract »    Full Text »    PDF »

To Advertise     Find Products


Science Signaling. ISSN 1937-9145 (online), 1945-0877 (print). Pre-2008: Science's STKE. ISSN 1525-8882