how does ras act in our body, in vivo ? why would constitutively active ras lead to cancer ? from...
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How does Ras act in our body, in vivo ?
Why would constitutively active Ras lead to cancer ?
From cell culture to model organisms
Model organisms teach us about ourselves
Drosophila melanogasterCaenorhabditis elegans
Xenopus laevis
Mus musculus
Californias governorus
5 Key Signal transduction pathwaysCritical for development and homeostasis
All are involved in Cancer
5 Key Signal transduction pathways
RTK- Ras (me)
Critical for development and homeostasisAll are involved in Cancer
Wnt (you)
TGF-ß (you)
Hedgehog (you)
Notch (not this time)
Signal transduction pathways
RTK
Receptor tyrosine kinases
Src RTKs
In the absence of Ligand RTKs are monomers
with an inactive kinase
Ligand binding activates RTKs by dimerization
Lodish et al. Fig. 20-21
RTKs are their own substrates--i.e., they autophosphorylate
Remember SH2 domains?
What did they bind??
SH2 domains allow “effector” proteins to bind activated receptors
One adaptor with an SH2 domains is Grb2It also has SH3 domains--what do they bind?
One adaptor with an SH2 domains is Grb2It also has SH3 domains--what do they bind?
Step by step--think dominos
Remember-Ras is anchored to the membrane through a lipid
And SOS is a GEF--remember them??
Figure 15-60 Molecular Biology of the Cell (© Garland Science 2008)
Each activated protein activates the next
Figure 15-60 Molecular Biology of the Cell (© Garland Science 2008)
MAPK/ERK enters nucleus
Each activated protein activates the next
Figure 15-60 Molecular Biology of the Cell (© Garland Science 2008)
MAPK/ERK enters nucleus
Each activated protein activates the next
Figure 15-60 Molecular Biology of the Cell (© Garland Science 2008)
Each activated protein activates the next
MAPK/ERK enters nucleus
The RTK pathway
We made it to the 90s
10 years-old Britney Spears
Kurt Cobain
A new graduate from Harvard
Travolta is still dancing
Model organisms teach us about ourselves
Drosophila melanogasterCaenorhabditis elegans
Xenopus laevis
Mus musculus
Californias governorus
The eye of a fly:One Key to
learning howRas and RTKs work
Lodish et al. Fig. 20-24
Did you say flies?
Did you say flies?
This is allI need to know
But wait--flies Helped us
UnderstandThe single
Most importantHuman
oncogene
Lodish et al. Fig. 20-24
I told you the RTK pathway is key in MANY developmental decisions
Each ommatidium contains the cells needed to see the world
These cells choose fate one by one, each telling the next what fate to adopt
Wild-type sevenless mutant
sevenless mutants lack an R7 photoreceptor
Sevenless encodes an RTK
Lodish et al. Fig. 20-25
To get a cellular response, There is a threshold level of pathway activity
Gian Garriga
sev pathway activity
wild type sev- sevts sevts sevts; enh*/+ 22.7o C 24.3o C 22.7o C
sev threshhold
R7 present
R7 absent
Scientists figured out how to tune RTK activity
using a temperature sensitive mutant
Gian Garriga
sev pathway activity
wild type sev- sevts sevts sevts; enh*/+ 22.7o C 24.3o C 22.7o C
sev threshhold
R7 present
R7 absent
They then looked for mutantsIn other genes that would drop
Pathway activity below the threshold
Gian Garriga
sev pathway activity
wild type sev- sevts sevts sevts; enh*/+ 22.7o C 24.3o C 22.7o C
sev threshhold
R7 present
R7 absent
The mutations identified were candidatesTo encode things in the RTK pathway
Wow--Ras, an adapter and a GEFare in the RTK pathway!
Sos
Ras
Grb2
Further, Ras acts downstream ofthe RTK Sevenless
Lodish et al. Fig. 20-25
The signal transduction pathwayshould look familiar
Grb2
Alberts et al. Fig. 15-53
Because that’s how we figured it out!
Grb2
Alberts et al. Fig. 15-53
Caenorhabditis elegans
Flies were bad enough, but worms!?
Caenorhabditis elegans
Luckily the Nobel Committee thinks they are cool
lineage and programmed
cell deathBob Horvitz John Sulston Sydney Brenner
Physiology and Medicine 2002
RNAi
Physiology and Medicine 2006
Andy Fire Craig MelloGFP
Chemistry 2008
Marty Chalfie
Nobel Prize
A simple model for organogenesis
Formation of the vulva in C. elegans(“ask Gidi what he did in graduate school?”)
vulva
Only 22 cells!
spermoocytes
embryos
early oocytes
The Key Players
One gonadal anchor cell (AC)
6 vulval precursor cells (VPCs)
The anchor cell induces vulval fatesSherwood and Sternberg (2003) Dev Cell
Cell InductionThe AC signals the VPCs to adopt vulval fates
Only 3 VPCs will actually form the vulva
How can we figure out how to build a vulva?
GENETICS!
No vulva induction
wild type
multivulvae (Muv)
vulvaless (Vul)
Mutants with no vulval signaling
Mutants with too much vulval signaling
multivulvae (Muv)
vulvaless (Vul)
Loss of function mutants in the signaling pathway
Gain of function mutants in the signaling pathway
some examples
A Screen for Vul and Muv mutants
Vul Muvlet-23
lin-3
lin-15
let-60
Next step: cloning and sequencing the genes
Bob Horvitz
physiology and medicine 2002
Nobel Prize
The first two vul mutations identify
the anchor cell signaland its receptor
let-23 is an EGFR homologue = RTK
Alberts et al. 21-44
lin-3 is an EGF homologue
Where do they function?
Lin-3 is expressed in the AC
Let-23 is expressed in all the VPCs
Vul
lin-3let-23
- lin-15 was found to be an inhibitor of vulval induction (loss of function mutation)
Muvlin-15
let-60
lin-15
- let-60 was found to promote vulval induction (gain of function mutation)
Next step: clone the let-60 gene
First evidence: Ras has an in vivo role as part of the RTK pathway
The gain-of-function mutation = Glycine 13 Glutamine
All the VPCs make vulvae
G13QConstitutively active Ras
Suppressor and enhancer screens[suppressors (or enhancers) of the mutated phenotype]
Other mutations of ras
lin-15
How can we find the rest of the proteins in the pathway?
??
Vul = “bag of worms”
Muv
Normal vulva”
For example, second mutations that turnMuv mutants into normal Vul worms
lin-15
This worked GREAT!
lin-15
This worked GREAT!
The fly and worm work allowed cell biologists and biochemists to return to mammalian cells
to identify the ways these new proteins worked as machines
The RTK-Ras pathway also offersdrug targets for cancer treatment
Alberts et al. Fig. 21-44
The RTK-Ras pathway offersdrug targets for cancer treatment
e.g., the Raf kinase inhibitor sorafenib(also inhibits the RTKs VEGFR, PDGFR, and Kit)
The RTK-Ras pathway offersdrug targets for cancer treatment
e.g., the Raf kinase inhibitor sorafenib(also inhibits the RTKs VEGFR, PDGFR, and Kit)
Approved for treatment of advanced renal cell carcinoma (Jan. 2006)
Increased survival in Phase III liver cancer trialand approved for inoperable form (Nov. 2007)
In clinical trials for melanoma and lung cancer
Summary
- Cellular oncogenes = viral oncogenes
Summary
- Cellular oncogenes = viral oncogenes
- Ras, as one of these genes, encodes a small GTPase, acting as a molecular switch
Summary
- Cellular oncogenes = viral oncogenes
- Ras, as one of these genes, encodes a small GTPase, acting as a molecular switch
- Ras is a major component of the RTK pathway
Summary
- Cellular oncogenes = viral oncogenes
- Ras, as one of these genes, encodes a small GTPase, acting as a molecular switch
- Ras is a major component of the RTK pathway
- Basic and Clinical Science provide a VERY powerful partnership