MCB 252 Topic 5 Other Major Signaling Pathways 1 GPCR signaling 2 NFKB Pathway Prof David Rivier MCB 252 Spring 2015 Reading GPCR section 15 3 pages 687 693 NFKB pages 757 759 Ubiquitin pages 85 89 Outline GPCR brief look at signal reception buy the largest family of receptors NFKB a look at a separate pathway that displays several regulatory features worth talking about Examples of Roles of GPCR Family Members Photoreption vision Olfaction smell Neurotransmitters Serotonin dopamine GABA glutamate Immune system Inflammatory response Histamine Autonomic nervous system control of blood pressure heart rate digestion GPCR Structure GPCR Structure GPCR act through trimeric G proteins Trimeric G proteins have lipid attached ala RAS Outline GPCR brief look at signal reception buy the largest family of receptors NFKB a look at a separate pathway that displays several regulatory features worth talking about NFKB a master regulator of txn in cells of the immune system Several Receptors impinge on IKK by different paths The paths are the same from activation of IKK forward NFKB a master regulator of txn in cells of the immune system Extra cellular New question How is a transcription factor which is sequestered in the cytoplasm informed to enter the nucleus at a specific time Cytoplasm TF Regulation of its target genes Nucleus Extra cellular Signal Inhibitor TF Import Cytoplasm Regulation of its target genes Nucleus Extra cellular Signal P Inhibitor TF Import Cytoplasm Regulation of its target genes Nucleus Positively or Negatively regulated P Kinase Inhibitor TF Inhibitor Phosphatase TF Positively or Negatively regulated Remains in the cytoplasm Enters the nucleus Extra cellular Signal Ubiquitin Inhibitor TF Import Cytoplasm Regulation of its target genes Nucleus Positively or Negatively regulated Inhibitor TF Inhibitor Ubiquitination Degradation Activation of the inhibitor coding gene Transcription of the inhibitor mRNA Nuclear export of the inhibitor mRNA Translation of the inhibitor mRNA Remains in the cytoplasm TF Enters the nucleus Extra cellular Signal P50 NF kB Import I kB P65 Cytoplasm Regulation of its target genes Nucleus Exterior Activated TNF receptor I kB kinase cytoplasm Regulation of Target genes Degradation of I kB Nucleus Exposed NLSs New Questions How is a protein poly ubiquitinated How is a poly ubiquitinated protein degraded How does a protein enter the nucleus More generally how is the nucleo cytoplasmic trafficking regulated How is a protein poly ubiquitinated Ubiquitin is a small protein 76 amino acids that folds up into a compact globular structure Target Protein Poly ubiquitination regulates protein turnover in a cell by closely regulating protein degradation How is a protein poly ubiquitinated Ubiquitin Ub is conjugated to a target protein through a covalent bond between the glycine at the Cterminal end of Ub and the side chain of a lysine K on the target protein Ubiquitinyl protein is a target for further ubiquitination the additional ubiquitin units added to the previous ubiquitin to form a poly ubiquitin chain K Target Protein Signal for degradation How is a protein poly ubiquitinated The conjugation of ubiquitin requires ATP and a three enzyme system referred to as E1 Ubiquitin activating enzyme E2 Ubiquitin conjugating enzyme E3 Ubiquitin protein ligase How is a protein poly ubiquitinated Step 1 Ubiquitin is activated at its C terminal end by E1 The product is a high energy thioester between the C ter carboxylate of ubiquitin and a specific Cys residue of E1 Ub Ub ATP Ub E1 PP Ub E1 AMP How is a protein poly ubiquitinated Step 2 Ubiquitin is transferred as a thioester to E2 enzyme Ub E1 E1 SH Ub E2 E2 How is a protein poly ubiquitinated Step 3 Substrate recognition involves an E3 ligase Ub E1 Ub Ub E3 E2 t ge in r Ta te o pr How is a protein poly ubiquitinated There is 1 E1 more than 30 E2 and several hundreds of E3 the list of known E3 is ever increasing E3 provides specificity for substrate recognition The E2s are broadly grouped into four classes The different E2s are able to interact with overlapping sets of E3s How is a protein poly ubiquitinated Proteins that are ubiquitinated display some form of signal that is recognized by an E3 Several short amino acid sequences have been identified as signals for ubiquitination Examples are Destruction Box or D box R XX L XXXX N KEN box KEN XXX N PEST sequences sequences rich in Proline Glutamic acid Serine and Threonine Hydrophobic patch 8 15 hydrophobic residues How is a protein poly ubiquitinated In some cases the signal is always displayed by the target protein A short half life of a regulators allows for rapid adjustment of its concentration A protein can also be conditionally unstable A protein can be short lived or long lived depending on the state of the cell Conditionally short lived regulators are often part of signaling networks How is ubiquitination regulated Exposure masking of a signal recognized by an E3 Activation inhibition of an E3 ligase How is a protein poly ubiquitinated Sig n al Exposure masking of a signal recognized by an E3 Ligase Target protein E3 How is a protein poly ubiquitinated Target protein Target protein Masked signal Exposed signal phosphorylation interaction with a ligand etc Inhibitory subunit How is a protein poly ubiquitinated Target protein Target protein Masked signal Exposed signal Hydrophobic patch Denaturation Misfolded protein Well folded protein How is a protein poly ubiquitinated Activation inhibition of an E3 ligase P Exposed signal Target protein E3 activeE3E3 Inactive How is a protein poly ubiquitinated Activation inhibition of an E3 ligase Exposed signal Target protein Inhibitory subunit Signal E3 active E3 Exterior Activated TNF receptor I kB kinase cytoplasm Regulation of Target genes Degradation of I kB Nucleus Exposed NLSs How is a protein poly ubiquitinated Similar strategies are used to regulate many aspects of the cell cycle CDK Cyclin Dependent Kinase How is a protein poly ubiquitinated Regulates the events of M Phase APC Anaphase Promoting Complex APC has an E3 ligase activity but it is active only when in interaction with Cdc20 or Cdh1 How is a protein poly ubiquitinated Sister chromatids Degradation of securin Inactive Active Cohesin complex How is a protein poly ubiquitinated Regulates the events of M Phase APC Anaphase Promoting Complex APC has an E3 ligase activity but it is active only when in interaction with Cdc20 or Cdh1 How is a protein poly