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 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 discovered a virus that causes cancer 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 research RSV can transform cells in culture Harry Rubin RSV stock Immortality

Studying cancer at the cellular level RSV infection Changed cells No contact inhibition on cell division

No contact inhibition of cell division Normal RSV infected = Cancer

But how??? Normal RSV infected = Cancer

I HOPE you remember the central dogma

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

Transcription Translation mRNA DNA Protein The central dogma

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

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

NEXT Breakthrough discovery Retroviruses can cause cancer by picking up mutated versions of normal cellular genes Alberts et al. Fig

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 wnt-1 gene exons Transcribe to mRNA 5 kilobases Retroviral insertion sites in different tumors Alberts et al. Fig

Two mechanisms of gene activation by retroviral insertion Lodish et al. Fig

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

in the presence of P 32 -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 Kinase As 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 antibody that recognizes Tyr phosphorylated proteins

The structures of c-src and v-src provided an important clue! Lodish et al. Fig

Binds peptides phosphorylated on Tyr Binds polyproline motifsPhosphorylates 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 :

Tyrosine phosphorylation of the C-terminus creates an intramolecular and inhibitory interaction Lodish et al. Fig

Src is normally inactive due to intramolecular inhibition Lodish et al. Fig

Recent work has provided a more detailed model of Src activation Closed = OFF Open = ON Cowen-Jacob et al. Structure 13, (2005)

v-src lacks the C-terminal Tyr and thus cannot be inactivated! Lodish et al. Fig

From Schwartzenberg, Oncogene 17, (1998) Activation of Src has multiple consequences

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, (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 for Proteins 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 for Proteins 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 junctionsBasal 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

Actin: Green Focal Adhesions (orange) Cells interact with the ECM via Focal adhesions, which also anchor the actin cytoskeleton Alberts et al. Fig

Focal adhesions are linked to the actin cytoskeleton Alberts et al. Fig

A complex network of proteins links the focal adhesion to actin and regulates actin polymerization Alberts et al. Fig

Actin: GreenPhosphotyrosine: 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 matrix Migratory growth factors e.g., EGF, PDGF RTKs Src FAK PI-3- kinase Remodel cell-matrix junctions -> cell motility From Jones et al. Eur J. Cancer 36, (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 : 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

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 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 Active site SU6656

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