1 / 24

Modeling Guanine Nucleotide-Ras Binding and Cell Behavior

Modeling Guanine Nucleotide-Ras Binding and Cell Behavior. Kate Brown Anna Stevens Katy Wack. Project Goals:. Understanding the quantitative relationship between IMPDH, intracellular GTP concentration, Ras mediated signaling and cell behavior

cynthia-warren
Download Presentation

Modeling Guanine Nucleotide-Ras Binding and Cell Behavior

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Modeling Guanine Nucleotide-Ras Binding and Cell Behavior Kate Brown Anna Stevens Katy Wack

  2. Project Goals: • Understanding the quantitative relationship between IMPDH, intracellular GTP concentration, Ras mediated signaling and cell behavior • How does this relationship define a cell’s intracellular state and its decision making processes • Stem cell self renewal or maturation • Cancer cell proliferative capacity

  3. Implications of GTP in cell decisions • Stem Cell Self Renewal/asymmetric kinetics • Inhibition of IMPDH induces differentiation • Addition of guanine nucleotide precursors reverses this and restores exponential growth • Cancer cells-high proliferative/undifferentiated state (lose the ability to mature) • Some Cancer drugs (Tiazofurin), inhibit IMPDH, result in decrease of GTP and change in proliferative capacity, not just proliferative rate

  4. De Novo Nucleotide Synthesis De Novo Sythesis (R5P) Xanthosine or Xanthine Guanosine or Guanine Salvage Pathways IMPDH IMP XMP GMP GDP GTP IMPDH is the rate limiting step of De Novo Synthessis

  5. How does Ras Signaling Work? GEF Ras Ras GDP GDP GEF GDP Pi GAP Ras GEF Ras Ras GTP GTP GTP

  6. Ras effector pathways http://193.175.244.148/maps/ras.html

  7. Influencing the kinetics of Ras-GTP binding • Change intracellular GTP concentration • IMPDH inhibition/stimulation • Change GAP/GEF • GTPase dephosphorylation • Nucleotide binding • Change Ras behavior • oncogenic Ras has different nucleotide binding affinity

  8. Inhibition of IMPDH reduces GTP and Ras-GTP Tiazofurin inhibits IMPDH lowering cellular GTP concentration GMP and GDP concentrations do not change appreciably due to An excess of enzymes converting them to GTP Knight et al. Blood, 69 634-639 (1987) Hata et al. Oncol Res., 5 (4-5) 161-164 (1993)

  9. Equilibrium Model Keq1 [Ras-GDP] + [GEF] [Ras-GDP-GEF] Keq2 [GDP] [Ras-GEF] kGAP [GAP] Keq3 [GTP] Keq1 [Ras-GTP] + [GEF] [Ras-GTP-GEF]

  10. Assumptions • The system is at equilibrium • Pseudo steady state - d[Ras-GTP]/dt = 0 • GEF binds Ras-GTP and Ras-GDP with no bias • The Ras-GEF complex does not bind equally to GTP and GDP Haney et al., J. Bio. Chem 269 (24) 16541-16548 (1994) Lenzen et al., Biochem 37 7420-7430 (1998)

  11. Equilibrium Equations Eq (1): [Ras-GDP-GEF] [Ras-GTP-GEF] [Ras-GDP][GEF] [Ras-GTP][GEF] Eq (2): [Ras-GEF][GDP] [Ras-GDP-GEF] Eq (3): [Ras-GEF][GTP] [Ras-GTP-GEF] = Keq1= Keq2= Keq3=

  12. Kinetic Equations d[Ras-GTP] dT [Ras-GTP-GEF]*k-1 – [Ras-GTP]*k1 = 0 = – [Ras-GTP][GAP]*kGAP algebra [Ras-GTP-GEF]*k-1 [GEF]*k1 + [GAP]*kGAP Eq (4): [Ras-GTP] =

  13. Working Model Equation [Ras-GTP] [GTP] Keq2 [Ras-GDP] [GDP] Keq3 = * * Keq2 = .625 uM-1 Keq3 = 3.33 uM-1 *As determined by Lenzen et al., Biochem 37 7420-7430 (1998)

  14. Model Limitations/ Future Work Experimental %[GTP] Change Experimental % Ras-GTP change Model prediction for % Ras-GTP change § * 37 +/- 13 35% +/- 13 37% • Need to generate more data for better determination of kinetic parameters in order to test model. • Evidence that there is biphasic activation of Ras, so we may want to explore the full time course of Ras activation, and therefore generate a kinetic model using our system. • Would like to incorporate our model into current MAPK signaling models to quantitatively predict the effect of changing GTP pools on the cellular response to extracellular ligands. * Knight et al. Blood, 69 634-639 (1987) § Hata et al. Oncol Res., 5 (4-5) 161-164 (1993)

  15. Experimental Goals • Explore the relationship between IMPDH and GTP • Measure total vs. signaling [GTP] • Explore GTP “sensing” by Ras • Consider both phases of Ras activation • Kinetics of Ras activation • Explore the role of specific Ras effecter pathways in cell cycle and maintaining “stemness” • Characterize changes in cell state with GTP variation • Quantify signaling system • Consider changes in GTP

  16. Experimental Cell Lines • Stem Cell • Putative adult rat liver stem cell line-lig 8 • Cancer Cell • Hepatoma 3924A cell line • Primary Epithelial • Hepatocytes, freshly isolated

  17. Characterization of Ras and GTP dependent cell cycling Joneson, T., Bar-Sagi, D., J. Mol. Med (1997) 75; 587-593 http://www2.hama-med.ac.jp/w1a/bio1/index-j.html

  18. GTP “Sensing” Fluorescence Resonace Energy Transfer • Use FRET to measure signaling GTP • Understand the spatial aspect of Ras activation • Use GTP-sensor to monitor biphasic behavior of Ras activation Cullen, P.J., Lockyer P.J., Nature Reviews Molecular Cell Biology3; 339-348 (2002)

  19. Method of Conditional Expression • Controlled expression of type II IMPDH • Can be modified to use as a reporter gene system • Can be modified to control Ras chimera expression (GTP-sensor) TET on/off Expression System www.clontech.co.jp/qa/tet.html

  20. Tools for defining intracellular state at the Protein level Proteomics and Phosphoproteomics Antibody array for Protein expression www2.mrc-lmb.cam.ac.uk/groups/arrays swehsc.pharmacy.arizona.edu/analysis/images/proteomics.gif

  21. Monitoring Cellular State www.acl.ac.uk/biology/new/admin/pix/astrossm.jpg www.icnet.uk/axp/facs/davies/brdu1.gif

  22. TET on/off switch Ligand/RTK Binding IMP/IMPDH Regulation Tiazofurin Inhibition Phosphoproteomics & Antibody array Changing GTP GEFs Model Ras activation Activation control GTP sensor & population measurement Phosphoproteomics Array/RT-PCR Other Ras effectors MAPK Pathway Transcription Protein Regulation Cell Cycle Differentiation Apoptosis Growth kinetics FACS Immunofluoresence Proliferation

  23. Acknowledgements • Dr. James Sherley • Ali Khademhosseini • BE computer room population • Doug and Paul

  24. References • Sherley, J.L., An Emerging Cell Kinetics Network:Integrated Control of Nucleotide Metabolism and Cancer Gene Function, submitted • Sherley, J.L., Asymmetric Cell Kinetics Genes: The Key to Expansion of adult Stem Cells in Culture, Stem Cells, 2002 • Wright,D.G., A Role for Guanine Ribonucleotides in the Regulation of Myeloid Cell Maturation. Blood, Vol. 69 (1987) 334-337 • Knight,R.D., Mangum,J., Lucas,D.L., Cooney,D.A., Khan,E.C., Wright,D.G., Insoine Monophosphate Dehydrogenase and Myeloid Cell Maturation. Blood, vol. 69 (1987) 634-639 • Collart,F.R., Huberman,E., Expression of IMP Dehydrogenase in Differentiong HL-60 Cells, Blood, vol.75 (3) (1990) 570-576 • Colombo,R.S., Coccetti,P., Martegani,E., Role of guanine nucleotides in the regulation of the Ras/cAMP pathway in Saccharomyces cerevisiae. Biochima Biophys Acta, (2001) 181-189 • Haney,S.A., Broach, J.R., Cdc25p, the guanine Nucleotide Exchange Factor for the Ras Proteins of Saccharomyces cervisiae, Promotes Exchange by stabilizing Ras in a Nucleotide-free State, J. Bio. Chem, vol. 269 (1994) 16541-16548. • Hata, Y., Natsumeda,Y., Weber,G., Tiazofurin decreases Ras-GTP complex in K4562 cells., Oncol Res (1993) 161-164. • Taylor S., Shalloway D., Cell cycle-dependent activation of Ras., Current Biology vol.6 (1996) 1621-1627 • Nature Review Molecular Cell Biology 3; 339-348 (2002) • Gille H., Downward J., Multiple Ras Effector Pathways Contribute to G1 Cell Cycle Progression, J. Biol CChem vol 274 (1999) 22033-22040

More Related