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