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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}

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.

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