Ras binding triggers ubiquitination of the Ras exchange factor Ras-GRF2

Ras is a small GTPase that is activated by upstream guanine nucleotide exch ange factors, one of which is Ras-GRF2, GRF2 is a widely expressed protein with several recognizable sequence motifs, including a Ras exchanger moth ( REM), a PEST region containing a destruction box (DB), and a Cdc25 domain. The Cdc25 domain possesses guanine nucleotide exchange factor activity and interacts,vith Pas. Herein we examine if the DB motif in GRF2 results in pr oteolysis via the ubiquitin pathway. Based on the solved structure of the R EM and Cdc25 regions of the Son-of-sevenless (Sos) protein, the REM may sta bilize the Cdc25 domain during Ras binding. The DB motif of GRF2 is situate d between the REM and the Cdc25 domains, tempting speculation that it may b e exposed to ubiquitination machinery upon Ras binding. GRF2, protein level s 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 imp ortant for degradation. GRF2 is ubiquitinated in vivo, and this can be dete cted using mass spectrometry. In the presence of proteasome inhibitors, Ras -GRF2 accumulates as a high-molecular-weight conjugate, suggesting that GRF 2 is destroyed by the 26S proteasome. Deleting the DB reduces the ubiquitin ation of GRF2. GRF2 lacking the Cdc25 domain is not ubiquitinated, suggesti ng that a protein that cannot bind Pas cannot be properly targeted for dest ruction. Point mutations within the Cdc25 domain that eliminate Pas binding also eliminate ubiquitination, demonstrating that binding to Ras is necess ary for ubiquitination of GRF2. We conclude that conformational changes ind uced by GTPase binding expose the DB and thereby target GRF2 for destructio n.