Transcript The src
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