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Raft nanodomains contribute to Akt/PKB plasma membrane recruitment and activation

Abstract

Membrane rafts are thought to be sphingolipid- and cholesterol-dependent lateral assemblies involved in diverse cellular functions. Their biological roles and even their existence, however, remain controversial. Using an original fluorescence correlation spectroscopy strategy that recently enabled us to identify nanoscale membrane organizations in live cells, we report here that highly dynamic nanodomains exist in both the outer and inner leaflets of the plasma membrane. Through specific inhibition of biosynthesis, we show that sphingolipids and cholesterol are essential and act in concert for formation of nanodomains, thus corroborating their raft nature. Moreover, we find that nanodomains play a crucial role in triggering the phosphatidylinositol-3 kinase/Akt signaling pathway, by facilitating Akt recruitment and activation upon phosphatidylinositol-3,4,5-triphosphate accumulation in the plasma membrane. Thus, through direct monitoring and controlled alterations of rafts in living cells, we demonstrate that rafts are critically involved in the activation of a signaling axis that is essential for cell physiology.

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Figure 1: Monitoring of dynamic nanodomains on both leaflets of the plasma membrane of living DWT6.11 T cells.
Figure 2: Analysis of Akt activation and membrane association in DWT6.11 and Jurkat JA16 cells.
Figure 3: Membrane binding dynamics and nanodomain partitioning of GFP-Akt, GFP-Akt-PH and GFP-GRP1-PH in JA16 cells.
Figure 4: Restoration of raft nanodomain formation and Akt activation in myriocin- and zaragozic acid–treated JA16 cells upon addition of C12-SM.
Figure 5: Akt activation trigger by IGF1 in COS-7 cells.

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Acknowledgements

This research project was supported by institutional grants from Institut National de la Santé et de la Recherche Médicale and Centre National de la Recherche Scientifique, and by specific grants from Agence Nationale de la Recherche, Association pour la Recherche sur le Cancer, European regional development fund, Fondation pour la Recherche Médicale, Institut National du Cancer (PL06-026, PL96-009 and PF108-05), Ligue Nationale Française contre le Cancer, Ministère de l'Éducation Nationale, de la Recherche et de la Technologie and Centre National de la Recherche Scientifique. Part of the work was supported by IFR30 Lipidomics of the Functional Exploration Platform of Toulouse Genopole. R.L. and F.C. were awarded fellowships from the Ministère de l'Éducation Nationale, de la Recherche et de la Technologie. We thank M. Fallet (PICsL imaging core facility) and F. Garçon for technical assistance, P. Chavrier (Institut Curie), J. Downward (Cancer Research UK London Research Institute), H. Lelouard (Centre d'Immunologie de Marseille-Luminy), S. Méresse (Centre d'Immunologie de Marseille-Luminy) and T. Meyer (Stanford University Medical School) for reagents, Y. Xia for chemical structure drawing, K. Simons, G. van Meer and C. Zhu for discussion, P. Golstein, J.P. Gorvel, A.-O. Hueber, L. Leserman, A. Pamidi and T. Reisine for critical reading of the manuscript and E. Witty (AngloScribe) for editing the English.

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R.L., X.-J.G., F.C., Y.H., B.P., D.M. and H.-T.H. conceived and designed the experiments. R.L., X.-J.G., F.C., Y.H., O.H., A.-M.B. and S.M.S. performed the experiments. R.L., X.-J.G., F.C., Y.H., B.P., D.M. and H.-T.H. analyzed the data. P.-F.L., H.R., D.O., G.B., J.A.N. and B.P. contributed reagents, materials and analysis tools. D.M. and H.-T.H. wrote the paper with contributions from R.L., X.-J.G., F.C., Y.H. and B.P.

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Correspondence to Bernard Payrastre or Hai-Tao He.

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Lasserre, R., Guo, XJ., Conchonaud, F. et al. Raft nanodomains contribute to Akt/PKB plasma membrane recruitment and activation. Nat Chem Biol 4, 538–547 (2008). https://doi.org/10.1038/nchembio.103

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