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