Transcript lin-15
Why would constitutively active Ras lead to cancer ? How does Ras act in our body, in vivo ? From cell culture to model organisms Model organisms teach us about ourselves Mus musculus Xenopus laevis Caenorhabditis elegans Drosophila melanogaster 5 Key Signal transduction pathways Are critical for development and homeostasis All are involved in Cancer 5 Key Signal transduction pathways Critical for development and homeostasis All are involved in Cancer RTK- Ras (me) Wnt (me) TGF-ß (you) Hedgehog (you) Notch (you) Signal transduction pathways RTK= Receptor tyrosine kinases Src=non-receptor tyrosine kinase RTKs We’ll start by walking through the RTK pathway and then talk about how we got to this knowledge RTKs In the absence of Ligand RTKs are monomers with an inactive kinase Ligand binding activates RTKs by dimerization Lodish et al. Fig. 20-21 RTKs are their own substrates-i.e., they autophosphorylate Remember SH2 domains? What did they bind?? SH2 domains allow “effector” proteins to bind activated receptors One adaptor with an SH2 domains is Grb2 It also has SH3 domains--what do they bind? One adaptor with an SH2 domains is Grb2 It also has SH3 domains--what do they bind? Ras Step by step--think dominos Ras Ras Remember-Ras is anchored to the membrane through a lipid And SOS is a GEF--remember them?? Each activated protein activates the next Figure 15-60 Molecular Biology of the Cell (© Garland Science 2008) Each activated protein activates the next MAPK/ERK enters nucleus Figure 15-60 Molecular Biology of the Cell (© Garland Science 2008) Each activated protein activates the next MAPK/ERK enters nucleus Figure 15-60 Molecular Biology of the Cell (© Garland Science 2008) Each activated protein activates the next MAPK/ERK enters nucleus Figure 15-60 Molecular Biology of the Cell (© Garland Science 2008) The RTK pathway We made it to the 90s 10 years-old Britney Spears Travolta is still dancing A new graduate from Harvard Kurt Cobain Model organisms teach us about ourselves Mus musculus Xenopus laevis Californias governorus Caenorhabditis elegans Drosophila melanogaster Model organisms teach us about ourselves Mus musculus Xenopus laevis Caenorhabditis elegans Drosophila melanogaster The eye of a fly: One Key to learning how Ras and RTKs work Lodish et al. Fig. 20-24 Did you say flies? Did you say flies? This is all I need to know But wait--flies Helped us Understand The single Most important Human oncogene Lodish et al. Fig. 20-24 I told you the RTK pathway is key in MANY developmental decisions Each ommatidium contains all the cells needed to see the world Including the eight photoreceptors These cells choose fate one by one, each telling the next what fate to adopt sevenless mutants lack an R7 photoreceptor Wild-type sevenless mutant Sevenless encodes an RTK Lodish et al. Fig. 20-25 To get a cellular response, There is a threshold level of pathway activity R7 present sev threshhold R7 absent wild type sev- sevts sevts 22.7o C 24.3o C sevts; enh*/+ 22.7o C Gian Garriga Scientists figured out how to tune RTK activity using a temperature sensitive mutant R7 present sev threshhold R7 absent wild type sev- sevts sevts 22.7o C 24.3o C sevts; enh*/+ 22.7o C Gian Garriga They then looked for mutants In other genes that would drop Pathway activity below the threshold R7 present sev threshhold R7 absent wild type sev- sevts sevts 22.7o C 24.3o C sevts; enh*/+ 22.7o C Gian Garriga The mutations identified were candidates to encode things in the RTK pathway Wow--Ras, the adapter Grb2 and a GEF are all in the RTK pathway! Sos Grb2 Ras Further, Ras acts downstream of the RTK Sevenless Lodish et al. Fig. 20-25 The signal transduction pathway should look familiar Grb2 Alberts et al. Fig. 15-53 Because that’s how we figured it out! Grb2 Alberts et al. Fig. 15-53 Flies were bad enough, but worms!? Caenorhabditis elegans Luckily the Nobel Committee thinks they are cool Caenorhabditis elegans Nobel Prize lineage and programmed cell death Physiology and Medicine 2002 Bob Horvitz John Sulston Sydney Brenner Chemistry 2008 Physiology and Medicine 2006 GFP RNAi Marty Chalfie Andy Fire Craig Mello A simple model for organogenesis Formation of the vulva in C. elegans (“ask Gidi Shemer what he did in graduate school?”) early oocytes embryos sperm vulva Only 22 cells! oocytes The Key Players One gonadal anchor cell (AC) 6 vulval precursor cells (VPCs) The anchor cell induces vulval fates Sherwood and Sternberg (2003) Dev Cell Cell Induction The AC signals the VPCs to adopt vulval fates Only 3 VPCs will actually form the vulva How can we figure out how to build a vulva? How can we figure out how to build a vulva? GENETICS! wild type No vulva induction Mutants with no vulval signaling vulvaless (Vul) Mutants with too much vulval signaling multivulvae (Muv) Loss of function mutants in the signaling pathway vulvaless (Vul) Gain of function mutants in the signaling pathway or loss-of-function mutations in pathway negative regulators multivulvae (Muv) A Screen for Vul and Muv mutants some examples Bob Horvitz Nobel Prize physiology and medicine 2002 Vul Muv let-23 lin-15 lin-3 let-60 Next step: cloning and sequencing the genes The first two vul mutations identify the anchor cell signal and its receptor let-23 is an EGFR homologue = RTK lin-3 is an EGF homologue Where do they function? Lin-3 is expressed in the AC Let-23 is expressed in all the VPCs Alberts et al. 21-44 Muv lin-15 lin-15 let-60 lin-15 was found to be an negative regulator of vulval induction (loss of function mutation) let-60 was found to be a gain of function mutation that promoted vulval induction Next step: clone the let-60 gene First evidence: Ras has an in vivo role as part of the RTK pathway The ras gain-of-function mutation = Glycine 13 Glutamine Sound familiar? The ras gain-of-function mutation = Glycine 13 Glutamine Sound familiar? The gain-of-function mutation = Glycine 13 Glutamine Constitutively active Ras All the VPCs make vulvae G13Q How can we find the rest of the proteins in the pathway? lin-15 Other mutations?of ras ? Suppressor and enhancer screens [suppressors (or enhancers) of the mutated phenotype] For example, second mutations that turn Muv mutants into normal or Vul worms Vul = “bag of worms” Normal vulva” Muv This worked GREAT! lin-15 The fly and worm work allowed cell biologists and biochemists to return to mammalian cells to identify the ways these new proteins worked as machines The RTK-Ras pathway also offers drug targets for cancer treatment Alberts et al. Fig. 21-44 The RTK-Ras pathway offers drug targets for cancer treatment e.g., the Raf kinase inhibitor sorafenib (also inhibits the RTKs VEGFR, PDGFR, and Kit) The RTK-Ras pathway offers drug targets for cancer treatment e.g., the Raf kinase inhibitor sorafenib (also inhibits the RTKs VEGFR, PDGFR, and Kit) Approved for treatment of advanced renal cell carcinoma (Jan. 2006) and approved for inoperable hepatocellular carcinoma (Nov. 2007) And radioactive iodine resistant thyroid cancer (Nov. 2013) The RTK-Ras pathway offers drug targets for cancer treatment e.g., or the Raf kinase inhibitor Vemurafenib Approved for treatment of Late stage melanoma (August 2011) Summary - Cellular oncogenes = viral oncogenes Summary - Cellular oncogenes = viral oncogenes - Ras, as one of these genes, encodes a small GTPase, acting as a molecular switch Summary - Cellular oncogenes = viral oncogenes - Ras, as one of these genes, encodes a small GTPase, acting as a molecular switch - Ras is a major component of the RTK pathway Summary - Cellular oncogenes = viral oncogenes - Ras, as one of these genes, encodes a small GTPase, acting as a molecular switch - Ras is a major component of the RTK pathway - Basic and Clinical Science provide a VERY powerful partnership