Transcript Modeling Ganine Nucleotide-Ras Binding in the MAP Kinase

Modeling Guanine NucleotideRas 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 Novo
Sythesis
(R5P)
Xanthosine
or Xanthine
Guanosine
or Guanine
Salvage
Pathways
IMP
IMPDH
XMP
GMP
GDP
GTP
IMPDH is the rate limiting step of De Novo Synthessis
How does Ras Signaling Work?
GEF
Ras
GDP
GDP
Ras
GDP
Pi
GAP
GEF
Ras
Ras
GTP
GEF
Ras
GTP
GTP
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
Percent Ras in GTP-bound state in K562
IMPDH activity and [GTP] in HL-60 cell
35
16
30
14
12
Control
15
Tiazofurin
10
nmol/10
20
10
Tiazofurin
7
cells
25
8
Control
6
4
5
2
0
0
Ras-GTP
IMPDH activity
[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)
Equilibrium Model
[Ras-GDP] + [GEF]
Keq1
[Ras-GDP-GEF]
Keq2
kGAP
[GDP]
[Ras-GEF]
[GAP]
Keq3
[GTP]
Keq1
[Ras-GTP] + [GEF]
[Ras-GTP-GEF]
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]
Keq1= [Ras-GDP][GEF]
Eq (2):
[Ras-GEF][GDP]
Keq2= [Ras-GDP-GEF]
Eq (3):
[Ras-GEF][GTP]
Keq3=
[Ras-GTP-GEF]
=
[Ras-GTP-GEF]
[Ras-GTP][GEF]
Kinetic Equations
d[Ras-GTP]
[Ras-GTP-GEF]*k-1 – [Ras-GTP]*k1
=
0
=
dT
– [Ras-GTP][GAP]*kGAP
algebra
Eq (4):
[Ras-GTP-GEF]*k-1
[Ras-GTP] =
[GEF]*k1 + [GAP]*kGAP
Working Model Equation
[Ras-GTP]
[Ras-GDP]
[GTP] Keq2
[GDP] * Keq3
=
Keq2 = .625 uM-1 *
Keq3 = 3.33 uM-1
35
[Ras-GTP]/[Ras-GDP]
30
[Ras-GTP/[Ras-GDP] = 0.1875[GTP]/[GDP]
slope=Keq2/keq3
25
20
15
10
5
0
0
20
40
60
80
100
120
140
160
180
200
[GTP]/[GDP]
*As determined by Lenzen et al., Biochem 37 7420-7430 (1998)
Model Limitations/ Future Work
Experimental
%[GTP] Change
37 +/- 13 *
Experimental
% Ras-GTP change
35% +/- 13
§
Model prediction for
% Ras-GTP change
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)
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
Joneson, T., Bar-Sagi, D., J. Mol. Med (1997) 75; 587-593
http://www2.hama-med.ac.jp/w1a/bio1/index-j.html
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 Biology 3; 339-348 (2002)
Method of Conditional Expression
TET on/off Expression System
• 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)
www.clontech.co.jp/qa/tet.html
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
Monitoring Cellular State
www.acl.ac.uk/biology/new/admin/pix/astrossm.jpg
www.icnet.uk/axp/facs/davies/brdu1.gif
TET on/off switch
Ligand/RTK Binding
IMP/IMPDH
Regulation
Tiazofurin Inhibition
Phosphoproteomics &
Antibody array
Changing GTP
GEFs
Activation control
Other Ras effectors
Ras activation
Model
GTP sensor & population measurement
Phosphoproteomics
Array/RT-PCR
MAPK Pathway
Transcription
Protein Regulation
Cell Cycle
Apoptosis
Proliferation
Growth kinetics
FACS
Immunofluoresence
Differentiation
Acknowledgements
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Dr. James Sherley
Ali Khademhosseini
BE computer room population
Doug and Paul
References
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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) 1654116548.
Hata, Y., Natsumeda,Y., Weber,G., Tiazofurin decreases Ras-GTP complex in K4562 cells., Oncol Res (1993) 161164.
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