The inhibition of the GTPase activating protein-Ha-ras interaction by acidic lipids is due to physical association of the C-terminal domain of the GTPase activating protein with micellar structures.

Abstract

The effects of fatty acids and phospholipids on the interaction of the full‐length GTPase activating protein (GAP) as well as its isolated C‐terminal domain and the Ha‐ras proto‐oncogene product p21 were studied by various methods, viz. GTPase activity measurements, fluorescence titrations and gel permeation chromatography. It is shown that all fatty acids and acidic phospholipids tested, provided the critical micellar concentration and the critical micellar temperature are reached, inhibit the GAP stimulated p21 GTPase activity. This is interpreted to mean that it is not the molecular structure of acidic lipid molecules per se but rather their physical state of aggregation which is responsible for the inhibitory effect of lipids on the GTPase activity. The relative inhibitory potency of various lipids was measured under defined conditions with mixed Triton X‐100 micelles to follow the order: unsaturated fatty acids greater than saturated acids approximately phosphatidic acids greater than or equal to phosphatidylinositol phosphates much greater than phosphatidylinositol and phosphatidylserine. GTPase experiments with varying concentrations of p21 and constant concentrations of GAP and lipids indicate that the binding of GAP by the lipid micelles is responsible for the inhibition, a finding which was confirmed by fluorescence titrations and gel filtrations which show that the C‐terminal domain of GAP is bound by lipid micelles.

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“…Considering the first hypothesis, the critical micelle concentration (CMC) lies in the range of 6-60 μM for most unsaturated fatty acids, depending on pH and the counter-cation used (31)(32)(33)(34), which is consistent with the inflection point of 20 μM (Fig. 2A).…”

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“…The mechanism recently proposed for the arachidonicacid-induced inhibition of GAP activity involves the physical interaction of fatty acid micelles with the protein [24]. This could also be the case for the LysoPtdIns-induced inhibition, since its CMC, evaluated by DPH fluorescence measurements (see Materials and Methods) in three independent sets of recordings was =75 pM, therefore, in the range of concentrations affecting GAP activity (Fig.…”

“…The critical micellar concentration (CMC) value for LysoPtdIns was determined using the fluorescent probe 1,6-diphenyl 1,3,5-hexatriene (DPH) as described [24]. Briefly, increasing amounts (1 -200 pg) of LysoPtdIns were dried and dispersed by sonication in 1 ml HBSS; DPH, dissolved in tetrahydrofuran, was then added at a final concentration of 0.25 pM.…”

Structure, Dynamics, Lipid Binding, and Physiological Relevance of the Putative GTPase-binding Domain of Dictyostelium Formin C

“…Besides phosphoinositides, lysobisphosphatidic acid was also suggested to accumulate at phagosomes (35). An inhibition of the interaction of a GTPase-binding protein with its target GTPase due to interactions with micellar structures containing acidic or unsaturated phospholipids was, for example, described for the interaction of GTPase-activating protein with the Ha-ras proto-oncogene product p21 (66). For GTPase-activating protein, a significant inhibitory effect was only observed if the total lipid concentration was above the CMC (66).…”

“…An inhibition of the interaction of a GTPase-binding protein with its target GTPase due to interactions with micellar structures containing acidic or unsaturated phospholipids was, for example, described for the interaction of GTPase-activating protein with the Ha-ras proto-oncogene product p21 (66). For GTPase-activating protein, a significant inhibitory effect was only observed if the total lipid concentration was above the CMC (66). Similarly, the ForC GBD showed significant spectral changes only if the DPC concentration was well above the CMC, and the presence of the negatively charged lipids PIP345 and PIP45 lowered the micelle concentration necessary to induce structural rearrangements.…”