the story of src what viruses and nobel laureates taught us about cancer
TRANSCRIPT
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