Two distinct regions of Ras participate in functional interaction with GDP-GTP exchangers.
We have previously implemented a combined genetic/biochemical approach, for analysis of insertion-deletion mutants, to identify sites of Harvey-Ras participating in the interaction with guanine nucleotide exchangers, using the yeast Cdc25 as a model exchanger. We showed that positions 101-106 may be required for catalyzed exchange. We here present a further improved strategy to define more precisely the residues on Ras participating in this interaction. Non-conservative replacements at positions 103 or 105 abolished response to Cdc25 while substitutions at positions 102 or 104 were partially affected. The same substitutions had no effect on coupling to adenylyl cyclase. Since the strategy enables us to assess Ras functional interaction with both the exchanger and effector simultaneously, we have also examined the effect of substitutions in the distal part of the switch II region (amino acids 69-78). In contrast to other reports, substitutions at positions 69 or 73 prevented Cdc25 response while mutations at position 74 did not prevent this interaction. However, all these substitutions partly affected cyclase activation. These findings establish the crucial role of the 102-105 region in the catalyzed exchange reaction and suggest that the 69-74 area would be required for the functional interaction with both exchangers and effector molecules.
Segal
M
M
Department of Biological Chemistry, Hebrew University of Jerusalem, Israel.
Marbach
I
I
Willumsen
B M
BM
Levitzki
A
A
D000262:Adenylyl Cyclases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000502:physiology*; D011905:Genes, ras / Q000502:physiology*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D017354:Point Mutation; D011506:Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D020692:rab3 GTP-Binding Proteins; D020666:ras-GRF1
D011506:Proteins; D020666:ras-GRF1; D010749:Phosphoprotein Phosphatases; D019204:GTP-Binding Proteins; D020692:rab3 GTP-Binding Proteins; D000262:Adenylyl Cyclases