The story of Src
What Viruses Taught Us About Cancer
What Causes Cancer?
- Random mutations (mistakes at the assembly line)
- Inherited mutations (pre-disposition)
- Viral infections
- Environmental factors (chemical; physical)
Viral infection as a cause for cancer is not common
in humans, but it does exist (human papillomavirus
can cause carcinoma of the uterine cervix)
Viruses cause cancer in animals
The key to our understanding of the
molecular and cellular mechanisms of cancer
in humans
Bacteria and viruses are infectious agents
Infection ?
If infected by a filtrate = a virus
Viruses use the cellular
machinery for replication
and protein synthesis
The Rous Sarcoma Virus (RSV)
A virus can transform a normal cell into a tumor
Peyton Rous
Nobel prize in
Physiology or
Medicine 1966
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
No contact inhibition
on cell division
Changes in cell property and morphology
Normal
Cancer
Normal
Cancer
RSV can transform cells in culture
- RSV can induce cancer
- Cancer is a disease of malfunctioning cells
rather than abnormally developing tissues
- Cancer can be studied relatively easily
at the cellular level
The central dogma
DNA
Transcription
mRNA
Translation
Protein
The problem: RSV contains RNA, not DNA
Howard Temin
Temin: 1964
Nobel prize in physiology
and medicine 1975
David Baltimore
One organism missed
the lecture on the
central dogma
The retrovirus
The viral genome of the normal chicken virus
(ALV) contains 3 viral genes
RSV, a relative of ALV,
contains an additional
sequence: Src
Peter Duesberg
Mutant RSVs, mutated in the src sequence, fail to induce cancer
Conclusions
RSV transforms normal cells into cancerous
cells by infecting them with the src gene
The src gene (and its product, the Src
protein) is probably responsible for the other
viral-associated cancers
Let’s label the src gene and follow its dynamics
inside the host cell, after infection
Bishop and Varmus
Nobel prize in
physiology and
medicine 1989
The cellular src gene is highly conserved
between different species
There is a cellular src that probably has
healthy, normal roles in the cellular machinery
There is a viral src that promotes cancer
We distinguish between
c-src
(cellular)
v-src
(viral)
src was only the first one
Jennifer Harvey found a murine leukemia Virus,
inducing sarcoma in rats: v-ras (later named H-ras)
Other viral transforming genes (14)
showed similar patterns
c-src
(cellular)
Proto-oncogene
v-src
(viral)
Oncogene
v-src is similar, but NOT identical to c-src
How was v-src generated?
RSV exploited a kidnapped cellular gene
v-src is similar, but NOT identical to c-src
It is not all about mutations
Retroviruses can transform cells by altering
gene expression (e.g. amplification)
So, what is src ?
Cells transformed by the v-src oncogene
exhibit various cellular alterations
v-src affects a wide variety of cellular targets
The first BIG step:
using serum to
immunoprecipitate the
v-Src protein
Protein kinases and protein phosphatases add and
remove phosphate groups from target proteins
Lodish et al. Fig. 20-5
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 do we have here?
A _______ blot
These are proteins identified by
antibodies that recognize
phosphorylated tyrosine
Compare c-Src with conserved Src proteins
Src contains three domains that are
shared with other proteins
Binds polyproline motifs
Phosphorylates
other proteins
Binds peptides phosphorylated on Tyr
Src is phosphorylated on its Tyr residue
Phosphorylated Tyr 527 inactivates Src
Tyr 527
RCSB Protein databank
How is Src activated normally?
Martin, Nature Reviews MCB (2001)
- Dephosphorylation of Y527
- Configuration changes (e.g. SH2 binds to
other proteins)
Recent work has provided a more detailed
model of Src activation
Closed = OFF
Cowan-Jacob et al. Structure 13, 861-871 (2005)
Open = ON
Cowan-Jacob et al. Structure 13, 861-871 (2005)
- Myristylation of Src to the cell membrane is the
first step in its activation
And what about v-Src ?
The kidnapped cellular gene was
only a partial gene
Summary
- RSV and other retroviruses can transform cells
into tumors
- Transformed cells exhibit changes in cell shape
and behavior
- A viral oncogene is responsible for transformation
- Viral oncogenes are mutated versions of cellular
proto-oncogenes
- The v-Src protein is a constitutively active tyrosin
kinase
c- Src is a tyrosine kinase
What does it do in the cell?
What are its targets?
Identifying the targets of Src
Myristylation of Src is
essential for transformation
Identifying The Targets of 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
Few Examples
- 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
Cell-cell
junctions
Cell-matrix junctions
Basal lamina
Epithelial cells secrete a special ECM called the
basal lamina
Epithelial cells
Basal Lamina
Connective tissue
Integrins cluster into sites of contact
with the ECM called focal adhesions
Focal adhesions are sites of
intense protein tyrosine phosphorylation
Focal
adhesions
Actin: Green
Phosphotyrosine: Red
Olga Krylyshkina
The ECM serves as a substrate for
migrating cells
Revathi Ananthakrishnan 07
Matrix affects cell
Cells affect matrix
Albert Harris, Biology UNC
Pioneering the studies of forces exerted by cells on the
matrix (e.g. cells can remodel the matrix)
Zoom into the focal adhesions
actin
filaments
focal adhesion
proteins
integrins
Plasma
membrane
FAK is recruited by integrins to the
membrane and is autophosphorylated
- Src binds to
phophorylated 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
Src- FAK also promote cell survival and growth
Summary
- Viruses can act as infectious “cancer agents” via
viral oncogenes
- Viral oncogenes are mutated versions of cellular
proto-oncogenes
- The v-Src protein is a constitutively active tyrosin
kinase, as a result of lack of Tyr at the C-terminus
- c-Src phosphorylates target proteins, including FAK
- FAK and c-Src act in the focal adhesions
- Focal adhesions are sites of cell-matrix interactions
(both ways)
How do we know this?
Rules of evidence
Src is localized at the right place and time
(correlative evidence)
src is sufficient
(gain-of-function evidence)
src is necessary
(loss-of-function evidence)
v-Src promotes adhesion dynamics
and cell migration
focal adhesions
actin
normal cells
v-Src transformed cells
lose actin-membrane attachments
lose adhesion
Frame et al. Nature Review MCB (2002)
How do we know this?
Rules of evidence
Src is localized at the right place and time
(correlative evidence)
src is sufficient
(gain-of-function evidence)
src is necessary
(loss-of-function evidence)
If Src is a critical regulator
of cell adhesion, what happens to
an animal without any Src?
Knockout mice
Oliver Smithies
UNC
Nobel prize in
physiology and
medicine 2007
If Src is a critical regulator
of cell adhesion, what happens to
an animal without any Src?
Knockout mice
- Defects in bone remodeling
- No lethality
Soriano et al. Cell (1991)
9 different murine Src family protein kinases
compensate for each other
Ubiquitous expression:
Src Yes Fyn
How can we test their functions?
src; fyn; yes triple mutant mice die at
embryonic day 9.5 with multiple defects
Triple mutant
Wild-type
Klinghoffer et al. EMBO (1999)
Wound healing/Scratch assay
src; fyn; yes (SYF) triple mutant cells fail to migrate
Scratch assay
How do we know this?
Rules of evidence
Src is localized at the right place and time
(correlative evidence)
src is sufficient
(gain-of-function evidence)
src is necessary
(loss-of-function evidence)
Figure 1
Table 1
Figure 2
Figure 3
Src as a target for anti-cancer drugs
Dasatinib
Regulated Src
Constitutively active Src
Src as a target for anti-cancer drugs
50 clinical trials, 14 types of tumors
Usually, in combination with other drugs
dasatinib
bosutinib
Inhibit Src active site
saracatinib
Before we leave, what do we know?
-Viruses kidnapped a normal proto-oncogene
- During the “kidnapping”, the proto-oncogene
became an oncogene
- A new viral infection inserted an oncogene
into the recipient, leading to cancer