Abstract
We identify a role for the GDI-like solubilizing factor (GSF) PDEδ in modulating signalling through Ras family G proteins by sustaining their dynamic distribution in cellular membranes. We show that the GDI-like pocket of PDEδ binds and solubilizes farnesylated Ras proteins, thereby enhancing their diffusion in the cytoplasm. This mechanism allows more effective trapping of depalmitoylated Ras proteins at the Golgi and polycationic Ras proteins at the plasma membrane to counter the entropic tendency to distribute these proteins over all intracellular membranes. Thus, PDEδ activity augments K/Hras signalling by enriching Ras at the plasma membrane; conversely, PDEδ down-modulation randomizes Ras distributions to all membranes in the cell and suppresses regulated signalling through wild-type Ras and also constitutive oncogenic Ras signalling in cancer cells. Our findings link the activity of PDEδ in determining Ras protein topography to Ras-dependent signalling.
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Change history
25 January 2012
In the version of this article initially published online and in print, the y-axis label for the graph in Figure 6b was incorrect. The correct label for the axis is "Normalized Ras-GTP".
References
Colicelli, J. Human RAS superfamily proteins and related GTPases. Sci. STKE 2004, RE13 (2004).
Goodwin, J. S. et al. Depalmitoylated Ras traffics to and from the Golgi complex via a nonvesicular pathway. J. Cell Biol. 170, 261–272 (2005).
Rocks, O. et al. The palmitoylation machinery is a spatially organizing system for peripheral membrane proteins. Cell 141, 458–471 (2010).
Rocks, O. et al. An acylation cycle regulates localization and activity of palmitoylated Ras isoforms. Science 307, 1746–1752 (2005).
Dekker, F. J. et al. Small-molecule inhibition of APT1 affects Ras localization and signaling. Nat. Chem. Biol. 6, 449–456 (2010).
Camp, L. A. & Hofmann, S. L. Purification and properties of a palmitoyl-protein thioesterase that cleaves palmitate from H-Ras. J. Biol. Chem. 268, 22566–22574 (1993).
Sang, S. L. & Silvius, J. R. Novel thioester reagents afford efficient and specific S-acylation of unprotected peptides under mild conditions in aqueous solution. J. Pept. Res. 66, 169–180 (2005).
Peyker, A., Rocks, O. & Bastiaens, P. I. Imaging activation of two Ras isoforms simultaneously in a single cell. ChemBioChem 6, 78–85 (2005).
Hanzal-Bayer, M., Renault, L., Roversi, P., Wittinghofer, A. & Hillig, R. C. The complex of Arl2-GTP and PDE δ: from structure to function. EMBO J. 21, 2095–2106 (2002).
Nancy, V., Callebaut, I., El Marjou, A. & de Gunzburg, J. The δ subunit of retinal rod cGMP phosphodiesterase regulates the membrane association of Ras and Rap GTPases. J. Biol. Chem. 277, 15076–15084 (2002).
Paz, A., Haklai, R., Elad-Sfadia, G., Ballan, E. & Kloog, Y. Galectin-1 binds oncogenic H-Ras to mediate Ras membrane anchorage and cell transformation. Oncogene 20, 7486–7493 (2001).
Elad-Sfadia, G., Haklai, R., Balan, E. & Kloog, Y. Galectin-3 augments K-Ras activation and triggers a Ras signal that attenuates ERK but not phosphoinositide 3-kinase activity. J. Biol. Chem. 279, 34922–34930 (2004).
Florio, S. K., Prusti, R. K. & Beavo, J. A. Solubilization of membrane-bound rod phosphodiesterase by the rod phosphodiesterase recombinant δ subunit. J. Biol. Chem. 271, 24036–24047 (1996).
Marzesco, A. M., Galli, T., Louvard, D. & Zahraoui, A. The rod cGMP phosphodiesterase δ subunit dissociates the small GTPase Rab13 from membranes. J. Biol. Chem. 273, 22340–22345 (1998).
Zhang, H. et al. Deletion of PrBP/δ impedes transport of GRK1 and PDE6 catalytic subunits to photoreceptor outer segments. Proc. Natl Acad. Sci. USA 104, 8857–8862 (2007).
Wilson, S. J. & Smyth, E. M. Internalization and recycling of the human prostacyclin receptor is modulated through its isoprenylation-dependent interaction with the δ subunit of cGMP phosphodiesterase 6. J. Biol. Chem. 281, 11780–11786 (2006).
Bhagatji, P., Leventis, R., Rich, R., Lin, C. J. & Silvius, J. R. Multiple cellular proteins modulate the dynamics of K-ras association with the plasma membrane. Biophys. J. 99, 3327–3335 (2010).
Chen, Y. X. et al. Synthesis of the Rheb and K-Ras4B GTPases. Angew Chem. Int. Ed. 49, 6090–6095 (2010).
Griesbeck, O., Baird, G. S., Campbell, R. E., Zacharias, D. A. & Tsien, R. Y. Reducing the environmental sensitivity of yellow fluorescent protein. Mechanism and applications. J. Biol. Chem. 276, 29188–29194 (2001).
Shaner, N. C. et al. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat. Biotechnol. 22, 1567–1572 (2004).
Apolloni, A., Prior, I. A., Lindsay, M., Parton, R. G. & Hancock, J. F. H-ras but not K-ras traffics to the plasma membrane through the exocytic pathway. Mol. Cell Biol. 20, 2475–2487 (2000).
Choy, E. et al. Endomembrane trafficking of ras: the CAAX motif targets proteins to the ER and Golgi. Cell 98, 69–80 (1999).
Drisdel, R. C. & Green, W. N. Labeling and quantifying sites of protein palmitoylation. Biotechniques 36, 276–285 (2004).
Farrell, F. X., Yamamoto, K. & Lapetina, E. G. Prenyl group identification of rap2 proteins: a ras superfamily member other than ras that is farnesylated. Biochem. J. 289 (Pt 2), 349–355 (1993).
Winegar, D. A., Molina y Vedia, L. & Lapetina, E. G. Isoprenylation of rap2 proteins in platelets and human erythroleukemia cells. J. Biol. Chem. 266, 4381–4386 (1991).
Gosser, Y. Q. et al. C-terminal binding domain of Rho GDP-dissociation inhibitor directs N-terminal inhibitory peptide to GTPases. Nature 387, 814–819 (1997).
Longenecker, K. et al. How RhoGDI binds Rho. Acta Crystallogr. D Biol. Crystallogr. 55, 1503–1515 (1999).
Wouters, F. S., Verveer, P. J. & Bastiaens, P. I. Imaging biochemistry inside cells. Trends Cell Biol. 11, 203–211 (2001).
Zhang, H. et al. Photoreceptor cGMP phosphodiesterase δ subunit (PDEδ) functions as a prenyl-binding protein. J. Biol. Chem. 279, 407–413 (2004).
Webb, Y., Hermida-Matsumoto, L. & Resh, M. D. Inhibition of protein palmitoylation, raft localization, and T cell signaling by 2-bromopalmitate and polyunsaturated fatty acids. J. Biol. Chem. 275, 261–270 (2000).
Patterson, G. H. & Lippincott-Schwartz, J. A photoactivatable GFP for selective photolabeling of proteins and cells. Science 297, 1873–1877 (2002).
Heo, W. D. et al. PI(3,4,5)P3 and PI(4,5)P2 lipids target proteins with polybasic clusters to the plasma membrane. Science 314, 1458–1461 (2006).
Yeung, T. et al. Membrane phosphatidylserine regulates surface charge and protein localization. Science 319, 210–213 (2008).
Yeung, T. et al. Contribution of phosphatidylserine to membrane surface charge and protein targeting during phagosome maturation. J. Cell Biol. 185, 917–928 (2009).
Yeung, T. et al. Receptor activation alters inner surface potential during phagocytosis. Science 313, 347–351 (2006).
Kim, J., Shishido, T., Jiang, X., Aderem, A. & McLaughlin, S. Phosphorylation, high ionic strength, and calmodulin reverse the binding of MARCKS to phospholipid vesicles. J. Biol. Chem. 269, 28214–28219 (1994).
Bivona, T. G. et al. PKC regulates a farnesyl-electrostatic switch on K-Ras that promotes its association with Bcl-XL on mitochondria and induces apoptosis. Mol. Cell 21, 481–493 (2006).
Lorentzen, A., Kinkhabwala, A., Rocks, O., Vartak, N. & Bastiaens, P. I. Regulation of Ras localization by acylation enables a mode of intracellular signal propagation. Sci. Signal 3, ra68 (2010).
Richards, C. A., Short, S. A., Thorgeirsson, S. S. & Huber, B. E. Characterization of a transforming N-ras gene in the human hepatoma cell line Hep G2: additional evidence for the importance of c-myc and ras cooperation in hepatocarcinogenesis. Cancer Res. 50, 1521–1527 (1990).
Tuveson, D. A. et al. Endogenous oncogenic K-ras(G12D) stimulates proliferationand widespread neoplastic and developmental defects. Cancer Cell 5, 375–387 (2004).
Sarkisian, C. J. et al. Dose-dependent oncogene-induced senescence in vivo and its evasion during mammary tumorigenesis. Nat. Cell Biol. 9, 493–505 (2007).
Hingorani, S. R. et al. Preinvasive and invasive ductal pancreatic cancer and its early detection in the mouse. Cancer Cell 4, 437–450 (2003).
Hingorani, S. R. et al. Trp53R172H and KrasG12D cooperate to promote chromosomal instability and widely metastatic pancreatic ductal adenocarcinoma in mice. Cancer Cell 7, 469–483 (2005).
Skoulidis, F. et al. Germline Brca2 heterozygosity promotes Kras(G12D)-driven carcinogenesis in a murine model of familial pancreatic cancer. Cancer Cell 18, 499–509 (2010).
Gidekel Friedlander, S. Y. et al. Context-dependent transformation of adult pancreatic cells by oncogenic K-Ras. Cancer Cell 16, 379–389 (2009).
Singh, A. et al. A gene expression signature associated with ‘K-Ras addiction’ reveals regulators of EMT and tumor cell survival. Cancer Cell 15, 489–500 (2009).
Alexander, M. et al. Mapping the isoprenoid binding pocket of PDEδ by a semisynthetic, photoactivatable N-Ras lipoprotein. ChemBioChem 10, 98–108 (2009).
van Meer, G., Voelker, D. R. & Feigenson, G. W. Membrane lipids: where they are and how they behave. Nat. Rev. Mol. Cell Biol. 9, 112–124 (2008).
Daleke, D. L. Regulation of transbilayer plasma membrane phospholipid asymmetry. J. Lipid Res. 44, 233–242 (2003).
Williamson, P. & Schlegel, R. A. Back and forth: the regulation and function of transbilayer phospholipid movement in eukaryotic cells. Mol Membr. Biol. 11, 199–216 (1994).
Lorenz, B. et al. Cloning and gene structure of the rod cGMPphosphodiesterase δ subunit gene (PDED) in man and mouse. Eur. J. Hum. Genet. 6, 283–290 (1998).
Ismail, S. A. et al. Arl2-GTP and Arl3-GTP regulate a GDI-like transport system for farnesylated cargo. Nat Chem. Biol. 7, 942–949 (2011).
Schreiber, F. S. et al. Successful growth and characterization of mouse pancreatic ductal cells: functional properties of the Ki-RAS(G12V) oncogene. Gastroenterology 127, 250–260 (2004).
Varga, M. et al. Pancreatic resection for metastatic renal cell carcinoma. Klin. Onkol. 22, 288–290 (2009).
Pfaffl, M. W. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 29, e45 (2001).
Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25, 402–408 (2001).
Politis, E. G., Roth, A. F. & Davis, N. G. Transmembrane topology of the protein palmitoyl transferase Akr1. J. Biol. Chem. 280, 10156–10163 (2005).
Lopez, A., Dupou, L., Altibelli, A., Trotard, J. & Tocanne, J. F. Fluorescence recovery after photobleaching (FRAP) experiments under conditions of uniform disk illumination. Critical comparison of analytical solutions, and a new mathematical method for calculation of diffusion coefficient D. Biophys. J. 53, 963–970 (1988).
Axelrod, D., Koppel, D. E., Schlessinger, J., Elson, E. & Webb, W. W. Mobility measurement by analysis of fluorescence photobleaching recovery kinetics. Biophys. J. 16, 1055–1069 (1976).
Elson, E. L. & Magde, D. Fluorescence correlation spectroscopy. I. Conceptual basis and theory. Biopolymers 13, 1–27 (1974).
Grecco, H. E., Roda-Navarro, P. & Verveer, P. J. Global analysis of time correlated single photon counting FRET-FLIM data. Opt. Express 17, 6493–6508 (2009).
Acknowledgements
We thank D. Vogt and K. Michel for technical support, M. Schmick for assistance with FLAP data analysis, O. Sabet (Department of Systemic Cell Biology, Max Planck Institute for Molecular Physiology, Dortmund, Germany) for providing mTFP–calreticulin, C. Schmees (Tumor Biology Group, NMI Natural Sciences and Medical Institute, Tübingen University, Reutlingen, Germany) for providing hTERT/SV40 cells and A. Krämer for help in preparing the manuscript.
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A.C. carried out and analysed the experiments, H.E.G. analysed the FLIM data, A.C., V.P., D.P. and L.C. carried out colony-formation assays, F.S. provided mPDAC cells, S.A.I. provided intellectual input, C.H. provided palmostatin-B, M.H-B. provided valuable initial experiments, P.I.H.B. and A.W. conceived the project and P.I.H.B. wrote the paper with A.C. and A.R.V.
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Chandra, A., Grecco, H., Pisupati, V. et al. The GDI-like solubilizing factor PDEδ sustains the spatial organization and signalling of Ras family proteins. Nat Cell Biol 14, 148–158 (2012). https://doi.org/10.1038/ncb2394
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DOI: https://doi.org/10.1038/ncb2394
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