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. 1991 Jun;10(6):1325–1330. doi: 10.1002/j.1460-2075.1991.tb07651.x

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.

J Serth 1, A Lautwein 1, M Frech 1, A Wittinghofer 1, A Pingoud 1
PMCID: PMC452790  PMID: 2026138

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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Selected References

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  1. Adari H., Lowy D. R., Willumsen B. M., Der C. J., McCormick F. Guanosine triphosphatase activating protein (GAP) interacts with the p21 ras effector binding domain. Science. 1988 Apr 22;240(4851):518–521. doi: 10.1126/science.2833817. [DOI] [PubMed] [Google Scholar]
  2. Calés C., Hancock J. F., Marshall C. J., Hall A. The cytoplasmic protein GAP is implicated as the target for regulation by the ras gene product. Nature. 1988 Apr 7;332(6164):548–551. doi: 10.1038/332548a0. [DOI] [PubMed] [Google Scholar]
  3. Chattopadhyay A., London E. Fluorimetric determination of critical micelle concentration avoiding interference from detergent charge. Anal Biochem. 1984 Jun;139(2):408–412. doi: 10.1016/0003-2697(84)90026-5. [DOI] [PubMed] [Google Scholar]
  4. Downward J., Graves J. D., Warne P. H., Rayter S., Cantrell D. A. Stimulation of p21ras upon T-cell activation. Nature. 1990 Aug 23;346(6286):719–723. doi: 10.1038/346719a0. [DOI] [PubMed] [Google Scholar]
  5. Downward J., Riehl R., Wu L., Weinberg R. A. Identification of a nucleotide exchange-promoting activity for p21ras. Proc Natl Acad Sci U S A. 1990 Aug;87(15):5998–6002. doi: 10.1073/pnas.87.15.5998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Ellis C., Moran M., McCormick F., Pawson T. Phosphorylation of GAP and GAP-associated proteins by transforming and mitogenic tyrosine kinases. Nature. 1990 Jan 25;343(6256):377–381. doi: 10.1038/343377a0. [DOI] [PubMed] [Google Scholar]
  7. Ferguson K. M., Higashijima T., Smigel M. D., Gilman A. G. The influence of bound GDP on the kinetics of guanine nucleotide binding to G proteins. J Biol Chem. 1986 Jun 5;261(16):7393–7399. [PubMed] [Google Scholar]
  8. Gibbs J. B., Schaber M. D., Allard W. J., Sigal I. S., Scolnick E. M. Purification of ras GTPase activating protein from bovine brain. Proc Natl Acad Sci U S A. 1988 Jul;85(14):5026–5030. doi: 10.1073/pnas.85.14.5026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Halenbeck R., Crosier W. J., Clark R., McCormick F., Koths K. Purification, characterization, and western blot analysis of human GTPase-activating protein from native and recombinant sources. J Biol Chem. 1990 Dec 15;265(35):21922–21928. [PubMed] [Google Scholar]
  10. Hall A. ras and GAP--who's controlling whom? Cell. 1990 Jun 15;61(6):921–923. doi: 10.1016/0092-8674(90)90054-i. [DOI] [PubMed] [Google Scholar]
  11. Hannun Y. A., Loomis C. R., Bell R. M. Activation of protein kinase C by Triton X-100 mixed micelles containing diacylglycerol and phosphatidylserine. J Biol Chem. 1985 Aug 25;260(18):10039–10043. [PubMed] [Google Scholar]
  12. Huang Y. K., Kung H. F., Kamata T. Purification of a factor capable of stimulating the guanine nucleotide exchange reaction of ras proteins and its effect on ras-related small molecular mass G proteins. Proc Natl Acad Sci U S A. 1990 Oct;87(20):8008–8012. doi: 10.1073/pnas.87.20.8008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kaplan D. R., Morrison D. K., Wong G., McCormick F., Williams L. T. PDGF beta-receptor stimulates tyrosine phosphorylation of GAP and association of GAP with a signaling complex. Cell. 1990 Apr 6;61(1):125–133. doi: 10.1016/0092-8674(90)90220-9. [DOI] [PubMed] [Google Scholar]
  14. Kazlauskas A., Ellis C., Pawson T., Cooper J. A. Binding of GAP to activated PDGF receptors. Science. 1990 Mar 30;247(4950):1578–1581. doi: 10.1126/science.2157284. [DOI] [PubMed] [Google Scholar]
  15. Leupold C. M., Goody R. S., Wittinghofer A. Stereochemistry of the elongation factor Tu X GTP complex. Eur J Biochem. 1983 Sep 15;135(2):237–241. doi: 10.1111/j.1432-1033.1983.tb07643.x. [DOI] [PubMed] [Google Scholar]
  16. Lichtenberg D., Robson R. J., Dennis E. A. Solubilization of phospholipids by detergents. Structural and kinetic aspects. Biochim Biophys Acta. 1983 May 24;737(2):285–304. doi: 10.1016/0304-4157(83)90004-7. [DOI] [PubMed] [Google Scholar]
  17. Margolis B., Li N., Koch A., Mohammadi M., Hurwitz D. R., Zilberstein A., Ullrich A., Pawson T., Schlessinger J. The tyrosine phosphorylated carboxyterminus of the EGF receptor is a binding site for GAP and PLC-gamma. EMBO J. 1990 Dec;9(13):4375–4380. doi: 10.1002/j.1460-2075.1990.tb07887.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Marshall M. S., Hill W. S., Ng A. S., Vogel U. S., Schaber M. D., Scolnick E. M., Dixon R. A., Sigal I. S., Gibbs J. B. A C-terminal domain of GAP is sufficient to stimulate ras p21 GTPase activity. EMBO J. 1989 Apr;8(4):1105–1110. doi: 10.1002/j.1460-2075.1989.tb03480.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. McCormick F. The world according to GAP. Oncogene. 1990 Sep;5(9):1281–1283. [PubMed] [Google Scholar]
  20. Molloy C. J., Bottaro D. P., Fleming T. P., Marshall M. S., Gibbs J. B., Aaronson S. A. PDGF induction of tyrosine phosphorylation of GTPase activating protein. Nature. 1989 Dec 7;342(6250):711–714. doi: 10.1038/342711a0. [DOI] [PubMed] [Google Scholar]
  21. Newton A. C., Koshland D. E., Jr High cooperativity, specificity, and multiplicity in the protein kinase C-lipid interaction. J Biol Chem. 1989 Sep 5;264(25):14909–14915. [PubMed] [Google Scholar]
  22. Satoh T., Endo M., Nakafuku M., Akiyama T., Yamamoto T., Kaziro Y. Accumulation of p21ras.GTP in response to stimulation with epidermal growth factor and oncogene products with tyrosine kinase activity. Proc Natl Acad Sci U S A. 1990 Oct;87(20):7926–7929. doi: 10.1073/pnas.87.20.7926. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Satoh T., Endo M., Nakafuku M., Nakamura S., Kaziro Y. Platelet-derived growth factor stimulates formation of active p21ras.GTP complex in Swiss mouse 3T3 cells. Proc Natl Acad Sci U S A. 1990 Aug;87(15):5993–5997. doi: 10.1073/pnas.87.15.5993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Trahey M., McCormick F. A cytoplasmic protein stimulates normal N-ras p21 GTPase, but does not affect oncogenic mutants. Science. 1987 Oct 23;238(4826):542–545. doi: 10.1126/science.2821624. [DOI] [PubMed] [Google Scholar]
  25. Tsai M. H., Hall A., Stacey D. W. Inhibition by phospholipids of the interaction between R-ras, rho, and their GTPase-activating proteins. Mol Cell Biol. 1989 Nov;9(11):5260–5264. doi: 10.1128/mcb.9.11.5260. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Tsai M. H., Yu C. L., Wei F. S., Stacey D. W. The effect of GTPase activating protein upon ras is inhibited by mitogenically responsive lipids. Science. 1989 Jan 27;243(4890):522–526. doi: 10.1126/science.2536192. [DOI] [PubMed] [Google Scholar]
  27. Tucker J., Sczakiel G., Feuerstein J., John J., Goody R. S., Wittinghofer A. Expression of p21 proteins in Escherichia coli and stereochemistry of the nucleotide-binding site. EMBO J. 1986 Jun;5(6):1351–1358. doi: 10.1002/j.1460-2075.1986.tb04366.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. West M., Kung H. F., Kamata T. A novel membrane factor stimulates guanine nucleotide exchange reaction of ras proteins. FEBS Lett. 1990 Jan 1;259(2):245–248. doi: 10.1016/0014-5793(90)80019-f. [DOI] [PubMed] [Google Scholar]
  29. Wolfman A., Macara I. G. A cytosolic protein catalyzes the release of GDP from p21ras. Science. 1990 Apr 6;248(4951):67–69. doi: 10.1126/science.2181667. [DOI] [PubMed] [Google Scholar]

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