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
• 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
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
De Novo Nucleotide Synthesis
De NovoSythesis(R5P)
IMP XMP GDPGMP GTPIMPDH
IMPDH is the rate limiting step of De Novo Synthessis
Xanthosine or Xanthine
Guanosine or Guanine
SalvagePathways
How does Ras Signaling Work?
Ras
GDP
Ras
GTP
GEF
Ras
GDP
GEF
Ras
GTP
GEF
Ras
GTP
GDP
GAPPi
Ras effector pathways
http://193.175.244.148/maps/ras.html
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
Inhibition of IMPDH reduces GTP and Ras-GTP
Tiazofurin inhibits IMPDH lowering cellular GTP concentration
GMP and GDP concentrations do not change appreciably due toAn 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)
IMPDH activity and [GTP] in HL-60 ce ll
0
2
4
6
8
10
12
14
16
IMPDH activity [GTP]n
mo
l/10
7 c
ells
Tiazofurin
Control
Percent Ras in GTP-bound state in K562
0
5
10
15
20
25
30
35
Ras-GTP
Control
Tiazofurin
Equilibrium Model
[Ras-GTP]
[GEF]
[Ras-GTP-GEF]
[Ras-GDP]
[GEF]
[Ras-GDP-GEF]
[GDP]
[GTP]
[Ras-GEF]
+
+
[GAP]kGAP
Keq3
Keq2
Keq1
Keq1
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)
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=
Kinetic Equations
d[Ras-GTP] dT = 0 = [Ras-GTP-GEF]*k-1 – [Ras-GTP]*k1
– [Ras-GTP][GAP]*kGAP
Eq (4): [Ras-GTP] [Ras-GTP-GEF]*k-1
[GEF]*k1 + [GAP]*kGAP=
algebra
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)
*
[Ras-GTP/[Ras-GDP] = 0.1875[GTP]/[GDP]slope=Keq2/keq3
0
5
10
15
20
25
30
35
0 20 40 60 80 100 120 140 160 180 200
[GTP]/[GDP]
[Ras
-GTP]/[Ras
-GDP]
Model Limitations/ Future Work
• 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.
Experimental %[GTP] Change
Experimental% Ras-GTP change
Model prediction for % Ras-GTP change
37 +/- 13 35% +/- 13 37% * §
* Knight et al. Blood, 69 634-639 (1987)§ Hata et al. Oncol Res., 5 (4-5) 161-164 (1993)
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
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
Characterization of Ras and GTP dependent cell cycling
http://www2.hama-med.ac.jp/w1a/bio1/index-j.htmlJoneson, T., Bar-Sagi, D., J. Mol. Med (1997) 75; 587-593
GTP “Sensing”
• Use FRET to measure signaling GTP• Understand the spatial aspect of Ras activation• Use GTP-sensor to monitor biphasic behavior of Ras
activation
Fluorescence Resonace Energy Transfer
Cullen, P.J., Lockyer P.J., Nature Reviews Molecular Cell Biology 3; 339-348 (2002)
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
Tools for defining intracellular state at the Protein level
Proteomics and Phosphoproteomics
Antibody array forProtein expression
www2.mrc-lmb.cam.ac.uk/groups/arraysswehsc.pharmacy.arizona.edu/analysis/images/proteomics.gif
Monitoring Cellular State
www.acl.ac.uk/biology/new/admin/pix/astrossm.jpgwww.icnet.uk/axp/facs/davies/brdu1.gif
Ligand/RTK Binding
Changing GTP
IMP/IMPDH
Ras activation
MAPK PathwayOther Ras effectors
Transcription
Protein Regulation
Regulation
TET on/off switch
Proliferation
DifferentiationApoptosis
GEFs
GTP sensor & population measurement
Phosphoproteomics
Phosphoproteomics &Antibody array
Activation control
Tiazofurin Inhibition
Model
Array/RT-PCR
Cell Cycle
Growth kineticsFACS
Immunofluoresence
Acknowledgements
• Dr. James Sherley
• Ali Khademhosseini
• BE computer room population
• Doug and Paul
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