The story of SrcWhat Viruses and Nobel Laureates

Taught Us About Cancer

Before We Start--remember you’ll be reading and be ready to discuss a paper next week

How to read a Paper

1) What did we already know?2) What is the question addressed here?

3) How did the researchers address this question?

4) What are the results?

5) What are the conclusions?

for the paper, as well as for each figure

Peyton Rous discovereda virus that causescancer in chickens

No thank you!

The Rous Sarcoma Virus (RSV)A virus can transform a normal cell into a tumor

The Rous Sarcoma Virus (RSV)A virus can transform a normal cell into a tumor

Nobel Prize in Physiology or Medicine 1966

The Rous Sarcoma Virus (RSV)A virus can transform a normal cell into a tumor

But what’s a virus???

Yamagiwa

Chemicals can directly induce cancer

Viral Infection Out

Chemical Induction In

1920s

Carcinogens

Howard Temin

30 Years Later: Rebirth of RSV researchRSV can transform cells in culture

Harry Rubin

RSV stock

Immortality

Studying cancer at the cellular level

RSV infectionChanged cells

No contact inhibition on cell division

No contact inhibition of cell division

Normal

Normal

RSV infected= Cancer

RSV infected= Cancer

But how???

Normal

Normal

RSV infected= Cancer

RSV infected= Cancer

I HOPE you remember the central dogma

This is one of those biology factsThat you need to have permanently stored

Transcription

Translation

mRNA

DNA

Protein

The central dogma

RSV is a retrovirusThese viruses reverse the central dogma,

making a DNA copy of their RNA genome and inserting it into your DNA

Alberts et al. Fig. 24-23

Alberts et al. Fig. 24-23

Nobel Prize in Physiology and Medicine 1975

Howard Temin and David Baltimore

Your genome is a retrovirus graveyard:living and dead retroviruses make up

8%of your genome, with ~100,000 whole or partial copies!

Alberts et al. Fig. 24-23

NEXT Breakthrough discoveryRetroviruses can cause cancer by picking up

mutated versions of normal cellular genes

Alberts et al. Fig. 24-23

The paper that createdtwo more Nobel laureatesand founded the modern field ofCancer biology

Let’s take a very short detour

Retroviruses can also cause cancer by inserting next to and thus

activating the expression of proto-oncogenes

wnt-1 gene

exons

Transcribe to mRNA

5 kilobases

Retroviral insertionsites in different tumors

Alberts et al. Fig. 22-24

Two mechanisms of gene activationby retroviral insertion

Lodish et al. Fig. 24-10

OK—Back to src

You know mis-expressing this gene canInitiate cancer

What do you want to know now??

So, what job does the protein encoded by src

do within the cell?

The first BIG step:using antibodies to

immunoprecipitate the v-Src protein

This led to the discovery that Src is post-translationally modified

This led to the discovery that Src is post-translationally modified

What’s translation??

Protein kinases and protein phosphatasesadd and remove phosphate groups from

target proteins

Lodish et al. Fig. 20-5

in the presence of P32-ATP

Src is a kinase

Adding labeled ATP to a precipitated Src showed that Src can phophorylate a substrate

A substrate is phosphorylated

Which amino acids can be phosphorylated?

And Why those amino acids??

Figure 15-18a Molecular Biology of the Cell (© Garland Science 2008)

Src is a Tyrosine KinaseAs a kinase, it can affect many cellular events

Normally, Src kinase intrinsic activity is low

What makes Src so active in transformed cells?

Western Blot with antibodythat recognizes

Tyr phosphorylated proteins

The structures of c-src and v-srcprovided an important clue!

Lodish et al. Fig. 24-17

Binds peptides phosphorylated on Tyr

Binds polyproline motifs Phosphorylates other proteins

Src contains three domains that are shared with other proteins

Scientists have determined the precise 3-dimensional structure of Src

Xu et al. Nature. 1997 385:595-602

Tyrosine phosphorylation of the C-terminuscreates an intramolecular and inhibitory interaction

Lodish et al. Fig. 24-17

Src is normally inactive due tointramolecular inhibition

Lodish et al. Fig. 24-17

Recent work has provided a more detailed model of Src activation

Closed = OFF Open = ON Cowen-Jacob et al. Structure 13, 861-871 (2005)

v-src lacks the C-terminal Tyr and thus cannot be inactivated!

Lodish et al. Fig. 24-17

From Schwartzenberg, Oncogene 17, 1463-1468 (1998)

Activation of Src has multiple consequences

Where is Src within cells?

This is a covalently attached lipidwhat might that mean?

Myristylation of Src is essential for transformation

Recent work has provided a more detailed model of Src activation

Cowen-Jacob et al. Structure 13, 861-871 (2005)

c- Src is a tyrosine kinase

What does it do in the cell?

What are its targets?

Remember, we are still in the late 70s

Bishop and Varmus

V = v-Src transfected cells

2A/V = non-myristylated v-Src transfected cells

Identifying The Targets of Src-look forProteins ONLY modified by biologically active Src

Western blotting with anti-phosphotyrosine antibodies

Reynolds et al. MCB (1989)

V = v-Src transfected cells

2A/V = non-myristylated v-Src transfected cells

Identifying The Targets of Src-look forProteins ONLY modified by biologically active Src

Western blotting with anti-phosphotyrosine antibodies

p120 catenin: modulates cell-cell adhesion

Reynolds et al. MCB (1989)

- p120 catenin: modulates cell-cell adhesion

Identifying the targets of Src

- Cortactin A: regulates actin polymerization

- Focal Adhesion Kinase: involved in cell-matrix interactions

Mike Schaller, ex-UNC

Src modulates both cell-cell and cell matrix adhesion: The basics

Cell-cell junctions

Cell-matrix junctions Basal lamina

Src modulates both cell-cell and cell matrix adhesion: The basics

Lodish et al. Fig. 22-2

Epithelial cells

Basal Lamina

Epithelial cells secrete a special ECM called the basal lamina

Alberts et al. Fig. 19-54

Actin: Green

Focal Adhesions(orange)

Cells interact with the ECMvia Focal adhesions, which also anchor

the actin cytoskeleton

Alberts et al. Fig. 17-42

Focal adhesions are linked to theactin cytoskeleton

Alberts et al. Fig. 16-75

A complex networkof proteins linksthe focal adhesion to actinand regulatesactin polymerization

Alberts et al. Fig. 16-75

Actin: Green Phosphotyrosine: Red

Focal adhesions

Focal adhesions are sites of intense protein tyrosine phosphorylation

An oversimplified model of Src function

Normal skin cell tightly adherent to ECM

Wounding->platelet recruitment->cell migration and proliferation

Alberts et al.

Actin

Adaptors

Integrins

Extracellular matrixMigratory growth factorse.g., EGF, PDGF

RTKsSrc

FAK

PI-3-kinase

Remodel cell-matrix

junctions -> cell motility From Jones et al. Eur J. Cancer36, 1595-1606 (2000)

A less oversimplified model

- Src binds to phosphorylated FAK

FAK is recruited by integrins to the membrane and is autophosphorylated

- Src changes conformation and becomes active

- Src further phosphorylates FAK

- Src-FAK phosphorylate target proteins

Src and FAK act together to regulate other focal adhesion proteins

Src-FAK signals to regulate adhesion turnover

Src-FAK active = less adhesion, more migration

Cell 1991 64:693-702

Targeted disruption of the c-src proto-oncogene leads to osteopetrosis in mice.

Soriano P, Montgomery C, Geske R, Bradley A.

If Src is a critical regulator of cell adhesion, what happens to

an animal without any Src?

Why is this phenotype so modest?

Redundancy!!

Src has two very close relatives: Fyn and Yes

Different Src family kinases

work downstream of different receptors

Alberts et al. Fig. 23-54

Fyn mutant mice are viable but have defects in myelination of brain neurons

Yes mutant mice are viable but with subtle changes in B-cell function

Src; Fyn; Yes triple mutant mice die at embryonic day 9.5 with multiple defects

Wild-type

Triple mutant

However, triple mutant cellsstill make focal adhesions

However src; fyn; yes (SYF) triple mutant cells fail to migrate!

Scratch assay

Scientists have determined the precise 3-dimensional structure of Src

Active site

This aided identification of kinase inhibitorsthat block Src action

Active site

SU6656

In leukemia, adding Src inhibition to inhibition of the related kinase Ablimproves prognosis in phase II trials.

It is approved to help get around drug resistance in CML

dasatinib

Ottmann et al. Blood 110, 2309 (2007)

This same Src inhibitor is inPhase II trials for advanced breast cancer,

melanoma and advanced sarcomas

dasatinib

Ottmann et al. Blood 110, 2309 (2007)

Another Src inhibitor is in Phase I/II trials formetastatic pancreatic, breast, ovarian, and prostate cancers

Active site

AZD0530