The story of Src
What 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
for the paper, as well as for each figure
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?
No thank you!
Peyton Rous
discovered
a virus that causes
cancer in chickens
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???
Carcinogens
Chemicals can directly induce cancer
1920s
Viral Infection Out
Yamagiwa
Chemical Induction
In
30 Years Later: Rebirth of RSV research
RSV can transform cells in culture
RSV stock
Howard Temin
Harry Rubin
Immortality
Studying cancer at the cellular level
RSV infection
Changed 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 facts
That you need to have permanently stored
The central dogma
DNA
Transcription
mRNA
Translation
Protein
RSV is a retrovirus
These 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
Nobel Prize in
Physiology and
Medicine 1975
Howard
HowardTemin
Teminand
andDavid
DavidBaltimore
Baltimore
Alberts et al. Fig. 24-23
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 discovery
Retroviruses can cause cancer by picking up
mutated versions of normal cellular genes
Alberts et al. Fig. 24-23
The paper
that created
two more
Nobel laureates
and founded the
modern field of
Cancer 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
Retroviral insertion
sites in different tumors
Transcribe to mRNA
5 kilobases
Alberts et al. Fig. 22-24
exons
wnt-1 gene
Two mechanisms of gene activation
by retroviral insertion
Lodish et al. Fig. 24-10
OK—Back to src
You know mis-expressing this gene can
Initiate 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 phosphatases
add and remove phosphate groups from
target proteins
Lodish et al. Fig. 20-5
Adding labeled ATP to a precipitated Src showed
that Src can phophorylate a substrate
Src is a kinase
in the presence of
P32-ATP
A substrate is
phosphorylated
Which amino acids can be phosphorylated?
And Why those amino acids??
Src is a Tyrosine Kinase
As a kinase, it can affect many cellular events
Figure 15-18a Molecular Biology of the Cell (© Garland Science 2008)
Normally, Src kinase intrinsic activity is low
What makes Src so active in transformed cells?
Western Blot with antibody
that recognizes
Tyr phosphorylated
proteins
The structures of c-src and v-src
provided an important clue!
Lodish et al. Fig. 24-17
Src contains three domains
that are shared with other proteins
Binds polyproline motifs
Phosphorylates
other proteins
Binds peptides phosphorylated on Tyr
Scientists have determined
the precise 3-dimensional structure of Src
Xu et al. Nature. 1997 385:595-602
Tyrosine phosphorylation of the C-terminus
creates an intramolecular
and inhibitory interaction
Lodish et al. Fig. 24-17
Src is normally
inactive due to
intramolecular
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
Activation of Src
has multiple consequences
From Schwartzenberg, Oncogene 17, 1463-1468 (1998)
Where is Src within cells?
This is a covalently attached lipid
what 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
Identifying The Targets of Src-look for
Proteins ONLY modified by biologically active Src
Western blotting with antiphosphotyrosine antibodies
V = v-Src transfected cells
2A/V = non-myristylated v-Src
transfected cells
Reynolds et al. MCB (1989)
Identifying The Targets of Src-look for
Proteins ONLY modified by biologically active Src
Western blotting with antiphosphotyrosine antibodies
V = v-Src transfected cells
2A/V = non-myristylated v-Src
transfected cells
p120 catenin: modulates cellcell adhesion
Reynolds et al. MCB (1989)
Identifying the targets of Src
- p120 catenin: modulates cell-cell adhesion
- 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 secrete a special ECM
called the basal lamina
Epithelial cells
Basal Lamina
Alberts et al. Fig. 19-54
Cells interact with the ECM
via Focal adhesions, which also anchor
the actin cytoskeleton
Focal
Adhesions
(orange)
Actin: Green
Alberts et al. Fig. 17-42
Focal adhesions are
linked to the
actin cytoskeleton
Alberts et al.
Fig. 16-75
A complex network
of proteins links
the focal adhesion to actin
and regulates
actin polymerization
Alberts et al.
Fig. 16-75
Focal adhesions are sites of
intense protein tyrosine phosphorylation
Focal
adhesions
Actin: Green
Phosphotyrosine: Red
An oversimplified model of Src function
Normal skin cell tightly adherent to ECM
Wounding->platelet recruitment->
cell migration and proliferation
Alberts et al.
A less oversimplified model
Migratory growth factors
e.g., EGF, PDGF
RTKs
Src
FAK
PI-3kinase
Extracellular matrix
Integrins
Adaptors
Actin
Remodel cell-matrix
junctions -> cell motility
From Jones et al. Eur J. Cancer
36, 1595-1606 (2000)
FAK is recruited by integrins to the
membrane and is autophosphorylated
- Src binds to
phosphorylated FAK
- 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
If Src is a critical regulator
of cell adhesion, what happens to
an animal without any Src?
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.
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
Triple mutant
Wild-type
However, triple mutant cells
still 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 inhibitors
that block Src action
SU6656
Active
site
In leukemia, adding Src inhibition
to inhibition of the related kinase Abl
improves 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 in
Phase 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 for
metastatic pancreatic, breast, ovarian, and prostate cancers
Active
site
AZD0530