Ras binding triggers ubiquitination of the Ras exchange factor Ras-GRF2.
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.
Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario M5G 1X5, Canada.
C L
CL
de Hoog
J A
JA
Koehler
M D
MD
Goldstein
P
P
Taylor
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D
Figeys
M F
MF
Moran
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D002478:Cells, Cultured; D003546:Cysteine Endopeptidases; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D007976:Leupeptins / Q000494:pharmacology; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors; D009154:Mutation; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex; D017434:Protein Structure, Tertiary; D020664:Son of Sevenless Proteins / Q000378:metabolism; D014452:Ubiquitins / Q000378:metabolism*; D020663:ras Guanine Nucleotide Exchange Factors / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D018631:ras Proteins / Q000378:metabolism*; D020666:ras-GRF1 / Q000235:genetics / Q000378:metabolism
D015853:Cysteine Proteinase Inhibitors; D007476:Ionophores; D007976:Leupeptins; D009097:Multienzyme Complexes; C504490:RASGRF2 protein, human; D020664:Son of Sevenless Proteins; D014452:Ubiquitins; D020663:ras Guanine Nucleotide Exchange Factors; D020666:ras-GRF1; D015759:Ionomycin; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D018631:ras Proteins; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde