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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
2
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
4
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
.
.....................................
....
....
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?
52
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