Inhibition of mSOS-activity by binding of phosphatidylinositol 4,5-P2 to the mSOS pleckstrin homology domain.
There are several recently reported examples of inositol phospholipids binding to pleckstrin homology (PH) domains of proteins. The PH domain of SOS, a guanine nucleotide exchange factor for Ras, binds to phosphatidylinositol 4,5 bisphosphate (PtdIns4,5P2). We found that binding of PtdIns4,5P2 to 6-his-tagged recombinant mSOS in vitro inhibits the ability of SOS to catalyze the association of GTP on p21RAS. This inhibition was specific for PtdIns4,5P2: a number of other phosphatidylinositols and phosphatidylserine failed to inhibit Ras GTP-association. We confirmed that the specificity of binding of PtdIns's to recombinant GST-SOS-PH domain is the same as the specificity of PtdIns's for inhibition of SOS activity: namely, that only PtdIns4,5P2 binds significantly to the SOS-PH domain. In addition, the inhibition of Ras GTP-binding is not blocked by excess free inositols suggesting that SOS binds to PtdIns4,5P2 with higher affinity than it binds to free inositols. Addition of SOS-PH domain protein prevented the inhibition of SOS by PtdIns4,5P2 as did addition of the high affinity PtdIns4,5P2-binding drug neomycin. This confirmed that SOS inhibition is mediated by the SOS-PH domain binding to the inositol moiety of PtdIns4,5P2. Binding of Grb2 to SOS did not prevent the inhibition of SOS by PtdIns4,5P2 suggesting that there must be another mechanism for regulating this inhibition. These findings show that the phospholipid PtdIns4,5P2 can suppress the activity of an enzyme involved in signal transduction and suggest that this inhibitory effect must be relieved when SOS is activated.
Jefferson
A B
AB
Chiron Corporation, Emeryville, California 94608, USA.
Klippel
A
A
Williams
L T
LT
D048868:Adaptor Proteins, Signal Transducing; D001665:Binding Sites; D001798:Blood Proteins; D051380:GRB2 Adaptor Protein; D020662:Guanine Nucleotide Exchange Factors; D006160:Guanosine Triphosphate / Q000378:metabolism; D009355:Neomycin / Q000494:pharmacology; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000494:pharmacology*; D010750:Phosphoproteins; D011506:Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D020663:ras Guanine Nucleotide Exchange Factors
D048868:Adaptor Proteins, Signal Transducing; D001798:Blood Proteins; D051380:GRB2 Adaptor Protein; D020662:Guanine Nucleotide Exchange Factors; D019269:Phosphatidylinositol 4,5-Diphosphate; D010750:Phosphoproteins; D011506:Proteins; C039077:platelet protein P47; D020663:ras Guanine Nucleotide Exchange Factors; D006160:Guanosine Triphosphate; D016283:Proto-Oncogene Proteins p21(ras); D009355:Neomycin