Evidence for distinct mechanisms of transition state stabilization of GTPases by fluoride.
GTPase-activating proteins (GAPs) function by stabilizing the GTPase transition state. This has been most clearly demonstrated by the formation of a high-affinity complex between various GAPs and GDP-bound GTPases in the presence of aluminum tetrafluoride, which can mimic the gamma-phosphate of GTP. Herein, we report that p190 RhoGAP forms a high-affinity complex with Rho GTPases in the presence of fluoride ions, suggesting that p190 also functions to stabilize the GTPase transition state. However, this Rho-p190 complex does not require aluminum ions or even guanine nucleotide, indicating a distinct role for fluoride that is not consistent with the gamma-phosphate-mimicking hypothesis. These results indicate that it is necessary to reconsider the assumed role of fluoride in stabilizing a variety of other GTPase-GAP interactions where the requirement for aluminum or guanine nucleotide has not yet been addressed.
Vincent
S
S
Massachusetts General Hospital Cancer Center and Harvard Medical School, Building 149, 13th Street, Charlestown, MA 02129, USA.
Brouns
M
M
Hart
M J
MJ
Settleman
J
J
D016475:3T3 Cells; D000818:Animals; D019556:COS Cells; D002460:Cell Line; D004795:Enzyme Stability; D005347:Fibroblasts; D020558:GTP Phosphohydrolases / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D020690:GTPase-Activating Proteins; D005982:Glutathione Transferase / Q000096:biosynthesis; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006153:Guanosine Diphosphate / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D051379:Mice; D014176:Protein Biosynthesis; D011506:Proteins / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D012969:Sodium Fluoride / Q000494:pharmacology*; D014162:Transfection
D020690:GTPase-Activating Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; C085379:rho GTPase-activating protein; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D012969:Sodium Fluoride; D005982:Glutathione Transferase; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins