MCB 252 Topic 3 Bio Membranes and Cell Surface Receptors I the RAS MAPK Signaling Pathway Prof David Rivier MCB 252 Spring 2015 Reading for Topic 2 Background review Chapter 10 sections 10 1 10 2 pages 443 464 Reading Figure 10 10 and pages 450 452 Chapter 16 introductory section pages 721 723 Ras MAP kinase pathway section 16 2 Outline Properties of Membranes FRAP technology Properties of Membrane Associated Proteins Protein Farnesylation Signal Transduction 1 The RAS Pathway An example involving a Receptor Tyrosine Kinase RTK Receptor l a gn i S Reception Amplification Transmission Change in gene expression Response How is a signal received and transmitted Biomembranes Structure Properties Membrane receptors and channels Properties and Structure of Membranes Exoplasmic Leaflet Cytoplasmic Leaflet Assymetric semi permeable fluid Phosphatidylserine and apoptosis Membrane Associated Proteins Integral Peripheral Lipid anchored Membrane Associated Proteins Channels Receptors Transporters Enzymes Cell Adhesion Molecules Targets of over 50 of medically relevant drugs 20 30 of genes in genomes code for memb assoc proteins Properties and Structure of Membranes Assymetric semi permeable fluid How do we know they are fluid Laser FRAP curve Fluorescence Recovery After Photobleaching Eric A J Reits Jacques J Neefjes Nature Cell Biology 3 E145 E147 2001 Lateral diffusion of GFP GalTase within Golgi membranes measured by FRAP Lippincott Schwartz Outline Properties of Membranes FRAP technology Properties of Membrane Associated Proteins Protein Farnesylation Signal Transduction 1 The RAS Pathway An example involving a Receptor Tyrosine Kinase RTK Receptor Gly Cys Some proteins are anchored by short hydrophobic groups added post translationally such as GPI anchor or prenyl groups GPI GlycosylPhosphatidylInositol These anchors are added to proteins in the ER Lumen which explains their extra cellular orientation Acylation Addition of a fatty acyl group such as Palmitate or Myristate onto a glycine near the N terminus of a protein Prenylation Addition of an unsaturated fatty acyl group such as Farnesyl or Geranyl group onto a Cys residue near or at the Cterminus of a protein Primary structure AA sequence Nter MVAGMLGLREEKSEDQDLQGLKDKPLKFKKVKKDKKEEKEGKHEPV QPSAHHSAEPAEAGKAETSEGSGSAPAVPEASASPKQRRSIIRDRGPMY DDPTLPEGWTRKLKQRKSGRSAGKYDVYLINPQGKAFRSKVELIAYFE KVGDTSLDPKHRGEGERKDIVSSSMPRPNREEPVDSRTPVTCVLY Cter N terminus Nter NH2 C terminus Cter COOH Tertiary structure Nter Nter Cter Protein Cter Nter Protein CVLY Cter Cys Val Leu Tyr CaaX motif C Cys a aliphatic residue such as Ala Val and Leu X any one of the 20 amino acids Nter Protein Farnesyl diphosphate CVLY SH Farnesyl Transferase Nter Protein CVLY S Pyrophosphate Nter Protein Caax Protease Nter Protein C CVLY S S Methyl Transferase Nter Targeting the Plasma membrane Protein C S CH3 Extra cellular Plasma membrane Cytoplasm Ras Outline Properties of Membranes FRAP technology Properties of Membrane Associated Proteins Protein Farnesylation Signal Transduction 1 The RAS Pathway An example involving a Receptor Tyrosine Kinase RTK Receptor RAS MAP Kinase Pathway Cast of Characters EGF EGFR GRB2 signaling molecule EGF receptor a receptor tyrosine kinase an adaptor protein with SH2 and SH3 domains SOS RAS GEF RAS monomeric G protein a GTPase RAF MEK MAPK kinases in cascade ser thr MAPK the kinase that enters the nucleus 14 3 3 protein RAF inhibitor Growth Factor Extra cellular Ras Cytoplasm Nucleus Ras is a GTPase can hydrolyse GTP into GDP Pi Ras functions in transducing signals from many membrane receptors Ras alternates between an active on state with bound GTP and an inactive off state bound with GDP GTPase Activating Protein OFF ON GAP GEF Guanine Exchange Factor Example of a signaling event that induces a change in gene expression EGF Epithelial Growth Factor R EGF Extra cellular P P P Ras GTP GDP OFF ON P Raf Cytoplasm SOS 14 3 3 2 GRB Nucleus ATP ADP KINASE P Nter Nter Protein Protein Cter Cter PHOSPHATASE P Amino acids that can be phosphorylated Serine Threonine Tyrosine and Histidine Upstream and Downstream Components in Pathway Extra cellular ATP ON P ON Ras RasGAP GTP GDP OFFON Cytoplasm P ATP ON P P ADP Raf ADP MEK MAPK 14 3 3 Phosphorylation of transcription factors Change in gene expression Nucleus ON OFF EGFR SHUTTING OFF THE PATHWAY P P Extra cellular P P Raf Ras OFF GDP P P ON 14 3 3 P MEK OFF ON Cytoplasm P MAPK OFF What if Ras is mutated in such a way that it cannot hydrolyze GTP Extra cellular Ras RasGAP GTP ON P P MEK is ON Raf 14 3 3 MAPK is ON Uncontrolled Cell Growth and Division Gene Families and the Ras Superfamily Ras Superfamily Ras Cell Proliferation 32 family members in humans Rho Cytoskeleton Dynamics Cell Morphology Cell Migration 21 Rab Membrane Trafficking 65 Rap Cellular Adhesion 5 Arf Vesicular Transport 29 Ran Nuclear Transport and Spindle Formation 1 Rheb mTOR Pathway 2 RGK unknown probably blood vessel formation 4 Rit unknown 2 MIROMitochondrial Transport 2 Total of 163 Human Ras Superfamily Members Ras Superfamily Recent evidence suggests but does not prove that the original Ras family members played a role in vesicle transport