Spatial Aspects of Ras Signaling

Manipulating the Spatial Organization of Ras proteins

Dr. Nachiket Vartak

Dept. of Systemic Cell Biology

Max Planck Institute for Molecular Physiology , Dortmund

RIKEN-Max Planck Joint Research Center Symposium

Japan, 2013

Outline

The Acylation Cycle and its inhibition by small molecules

The regulation of the Acylation Cycle with feedbacks

General reaction diffusion dynamics and their inhibition

Hope to bring out the confluence of imaging, chemical biology and cell biology

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Ras proteins

Small G-proteins

30% of human neoplasms have H,N,K-Ras activating mutations

GTPase Reactions cycles

Catalysis and signaling extensively studied

Kiyokawa E et al. 2011

Lipidation and Localization

Rocks et al. Science (2005)

Its a small G-protein. GEFs, GAPs,

Prolific oncogene discovered thrice independently

Signals through various effector pathways

Three isoforms in humans : H , N and K

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Monolipidated proteins stain membranes aspecifically

Irreversible lipidations: N-myristoylation, C-prenylation

The presence of a single lipid group can confer the same localization irrespective of the chemical nature of the lipid group.

Monolipidated proteins diffuse fast in the cell

PA-GFP fusion

Photoactivation

Bessel Function based fitting : D = 0.9 mm2/s

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Palmitoylation generates localization

Ras localization on the golgi is dynamic.

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in both directions : Entry and exit

O.Rocks , Science (2005)

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The Golgi is where the action is

DYNAMIC Palmitoylation generates a steady state localization

Palmitoylation

Palmitoylation lacks local substrate specificity

D-Amino Acid

And can accept unnatural substrates

Dictyostelium can palmitoylate Human H-Ras

8 DHHC proteins No homologues of Y/M RasPATs

5-11 Ras isoforms none of which is palmitoylated

HRas Mobility in Dictyostelium is similar to that in human cells

t1/2 in human cells :

HRasC181S,C184S : 10 s

HRas wt : t1/2 = 156 s

Directionality

Vesicular transport transfers enrichment to PM

Depalmitoylation

hAPT1 (PDB: 1FJ2)

Acyl Protein Thioesterases

/-hydrolase

Palmitoyl protein thioesterase

Poor lysophospholipase

Aspecific towards protein

Homologs: APT1, APT2

the only known non-lysosomal PPT activity in cells

Reportedly cytoplasmic

APT1 Inhibitor Design

Dr. Frank Dekker

Dr. Christian Hedberg

Palmostatin B

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FRET-Fluorescence Lifetime Imaging

Grecco et al. Optics InfoBase (2012)

Electron absorbs energy and goes to excited state.

Electron dissipates energy , emits photon to return to ground state.

If there is an (additional) energy sink, average lifetime will reduce.

FRET can be that energy sink.

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TAMRA-Palmostatin B binds APT in cells

Palmostatin B inhibits APT1

Palmostatin redistributes N-Ras to all membranes

Manders' overlap coefficient is based on the Pearson's correlation coefficient with average intensity values being taken out of the mathematical expression (Manders 1992). This new coefficient will vary from 0 to 1, the former corresponding to non-overlapping images and the latter reflecting 100% co-localisation between both images. M1 is defined as the ratio of the "summed intensities of pixels from the green image for which the intensity in the red channel is above zero" to the "total intensity in the green channel" and M2 is defined conversely for red. Therefore, M1 (or M2) is a good indicator of the proportion of the green signal coincident with a signal in the red channel over its total intensity, which may even apply if the intensities in both channels are really different from one another.

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2-Bromopalmitate affects N-Ras localization

KRas is not redistributed

Summary

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APT1 inhibition

Acylation Cycle and Ras activity

DMSO, 30 min + EGF

Palmostatin B inhibits Ras

Palm B Activation 1mM , 30 min + EGF

Phenotypic Reversion

Of EMT

Explain F3 cells

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Acylation Cycles

generic aspecific spatial patterning mechanism

generates asymmetry and counters entropy

Regulation ?

Palmitate Metabolism

CoA, mitochondrial transport, Golgi transport

Protein expression levels

Other protein PTMs

Myristoylation, Prenylation, Phosphorylation, Proline isomerization etc.

Activity and tissue-specific expression of the diverse(Similarity 95%

CSS-Pam 3.0 (Ren et al. 2008)

APTs are palmitoylated

Independently verified (i.e we got scooped on it)

E. Kong, S. Peng, G. Chandra, C. Sarkar, Z. Zhang, M. B. Bagh, A. B. Mukherjee,

Dynamic palmitoylation links cytosol-membrane shuttling of

acyl-protein thioesterase-1 and acyl-protein thioesterase-2 with that of

proto-oncogene H-Ras product and growth associated protein-43,

J. Biol. Chem. (2013), doi:10.1074/jbc.M112.421073.

APT Mobility in cells

Why Palmitoylation ?

Membrane Localization is altered.

Dimerization

occludes active site

Access to substrates on membranes

Cytosolic proteins seem unlikely to act on membrane-embedded lipids

ES Imaging

ES Imaging with APTs

ES Imaging works!

Cytosolic APTs can act on Membrane bound substrates

But substrate moves aroundso we need to catch the moment . Catching the moment when APT reactivates cytosolic APT works too..then why palmitoylation?

Quantification

Counting pixels here. No dimersthe interaction is active site dependent!

Why Palmitoylation ?

Membrane Localization is altered.

Dimerization

occludes active site

Access to substrates on membranes

Cytosolic proteins seem unlikely to act on membrane-embedded lipids

Regulation ?

Active Site

Flexible

N-Terminus

hAPT1 (PDB: 1FJ2)

Conserved Cysteine with high probability of palmitoylation on a flexible N-terminus that can reach its own active site

APTs could be their own substrates!

Why Palmitoylation?

APTs form ES-complexes with APTs

Both APTs interact with each other! Only at the Golgi -> Palmitoylation

Quantification of APT-APT interaction

Simulations

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Compartmental Model Golgi and Cytosol

Michaelis-Menten Kinetics

Simulation predicts a robust steady state

Negative Feedback on the Golgi !

Explain absolute levels and what the perturbation is

KRas has electrostatic interactions with PM

Monolipidated proteins diffuse fast in the cell

PA-GFP fusion

Photoactivation

Bessel Function based fitting : D = 0.9 mm2/s

How?

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The Rapid Diffusive State - Solubilizers

How does a prenylated protein leave membranes?

Ismail et al (2012)

PDEd

The KRas Cycle

Inhibiton of PDEd desolubilizes KRas

Dose Response to find in cell binding constants

Inhibiton of PDEd redistributes KRas

to be continued.

Generic Conclusions

Reaction-diffusion cycles can create complex asymmetry

Interruption of these cycles is a potent tool to modulate the activity of proteins driven by these cycles

Acknowledgements

Dept. of Systemic Cell Biology

Dr. Malte Schmick

Dr. Oliver Rocks

Dr. Philippe Bastiaens

Dr. Anchal Chandra

Dept. of Structural Biology

Dr. Shehab Ismail

Dept. of Chemical Biology

Dr. Christian Hedberg

Dr. Sebastian Koch

Dr. Marc Gerauer

Kristina Goermer

Dr. Herbert Waldmann

Dekker et al. Nat Chem Biol (2010) 6: 449-456

Rocks et al. Cell (2010) 141: 458-471

Vartak N & Bastiaens P EMBO J (2010) 29: 2689-2699

Lorentzen et al. Sci. Signal. (2010) 3: ra68

Zimmerman et al. Nature (2013) in press

Thank you for your attention