Modeling guanine nucleotide ras binding and cell behavior
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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

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Project goals
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
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 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


How does ras signaling work
How does Ras Signaling Work?

GEF

Ras

Ras

GDP

GDP

GEF

GDP

Pi

GAP

Ras

GEF

Ras

Ras

GTP

GTP

GTP


Ras effector pathways
Ras effector pathways

http://193.175.244.148/maps/ras.html


Influencing the kinetics of ras gtp binding
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
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)


Equilibrium model
Equilibrium Model

Keq1

[Ras-GDP]

+

[GEF]

[Ras-GDP-GEF]

Keq2

[GDP]

[Ras-GEF]

kGAP

[GAP]

Keq3

[GTP]

Keq1

[Ras-GTP]

+

[GEF]

[Ras-GTP-GEF]


Assumptions
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
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
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]

=


Working model equation
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)


Model limitations future work
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)


Experimental goals
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
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
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
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)


Method of conditional expression
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
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
Monitoring Cellular State

www.acl.ac.uk/biology/new/admin/pix/astrossm.jpg

www.icnet.uk/axp/facs/davies/brdu1.gif


Modeling guanine nucleotide ras binding and cell behavior

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


Acknowledgements
Acknowledgements

  • Dr. James Sherley

  • Ali Khademhosseini

  • BE computer room population

  • Doug and Paul


References
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