Protein Targeting and Function: Ubiquitin Modifications Jim Collawn October 17th, 2006

References Hicke and Dunn (2003) Regulation of membrane protein transport by ubiquitin and ub-binding proteins. Annu. Rev. Cell Dev. Biol. 19: 141-172. 2. Minireview: Regulation of Epithelial Na+ Channel Trafficking. Endocrinology 146: 5079-5085.

Outline History and Evolution of Ub-pathway Sites where ubiquitination regulates protein trafficking/function Regulation of sodium channel stability at the cell surface (ENaC) Other ubiquitin-like molecules

Cellular Protein Degradation Lysosomal Nonspecific Endocytosis Foreign proteins Energy favorable to degrade proteins Non-lysosomal Specificity, requires ATP Conditions of stress Ubiquitin-proteosomal pathway 26S proteosome Role in cellular processes/signaling

Non-lysosomal Protein Degradation 1977 – Etlinger and Goldberg (PNAS) Protein degradation in reticulocytes But ----- no lysosomes ATP dependent 1980 – Wilkinson et al (JBC) Identify protein degradation system in reticulocyte Two fractions – (ATP-dependent Proteolytic Factor; APF) APF-1 fraction conjugates to proteins APF-1 identified as ubiquitin Protein Adduct  Protein Degradation Ubiquitin Found Function Found

Avram Hershko – Nobel Lecture Ciechanover (grad student 76-81; major credit) Irwin Rose (Fox Chase Cancer Center) “The Ubiquitin System for Protein Degradation and some of its Roles in the Control of the Cell Cycle” December 8, 2004

APF-1 Protein  Ubiquitin-Proteosomal Pathway “APF factor” 1978 Hershko 2004

Ubiquitinylation – Proteosome Degradation E3 determines protein substrate

E1-Ub E2: Transfer Ub Conj E3: Ligase Specific w/E2 ~1000 aa, Cysteine-636 Ub attachment site E2: Transfer Ub Conj Conserved cysteine Active “thio ester” E3: Ligase Specific w/E2 Usually do not conjugate Hold complex together

19S and 20S Proteasome Subunits Characteristics 19S Subunit Base and Lid Contains subunits with known and unknown functions Tetra-Ub (K48) recognition Deubiquitination activity Protein unfolding activity (Chaperone function) 20S Subunit Barrel Contains 6 proteolytic sites 2x Tryptic 2x Chymotryptic 2x Peptidylglutamyl- peptidase Linearized protein required

Ubiquitin 76 Amino Acids Post-translational modification on proteins Compact, globular superfold protein, 9 kD Highly conserved (3 aa difference from yeast to man) Expressed in EVERY eukaryotic cell C-terminus extends away from protein Post-translational modification on proteins Attaches on Lysine residues Functions: Marks protein for proteasomal degradation Can “activate” protein → trigger cellular effect Endocytosis Modify the activity of protein transport machinery

Conjugation – Type Bonds Multi-Enzyme Process Ub RGG C ATP ADP High-energy thioester bond E2 Protein C E2 Ub RGG K E3 Protein G R Protein Ub USP Ub Ub Ub Isopeptide bonds Ub

Ubiquitin AA Sequence MQIFVKTLTG KTITLEVEPS DTIENVKAKI QDKEGIPPDQ QRLIFAGKQL EDGRTLSDYN IQKESTLHLV LRLRGG 6 48 63

Roles of Ubiquitination

Different Types of Ubiquitin Tags

Modulalar Ub-binding Domains (endocytic proteins): Ubiquitin associated (UBA) or interacting motif (UIM) UVE (ub conjugating enzyme E2 variant CUE These domains bind to Ile44 on Ub Examples Espsin (Eps15-interacting protein) Eps15 (epidermal growth factor receptor substrate 15

Modulalar Ub-binding Domains Lipid binding preference: ENTH domain: PI-4,5-P (PM) FYVE domain: PI-3-P

Endocytic Machinery that is monoubiquitinated CIN85 (Cbl-interacting protein of 85 kDa) required for activated growth factor receptors Epsins Eps15 Hrs Rabex Numb (binds to Eps15 and AP-2) and neg regulates Notch (promotes Notch endocytosis)

Posttranslational Modification (ubiquitination) and Protein Sorting

ESCRT Proteins and the MVB

Ubiquitin and Protein Sorting In yeast, Ub is used for endocytosis and delivery into internal vesicles of the MVB In higher eukaryotes, it is used for the same purpose for regulated transport For regulated internalization and/or lysosomal targeting, an example is the signal transducing receptors: G protein-coupled receptors (GPCRs) b2-adrenergic and CXCR4 (MVB only) Receptor tyrosine kinases (RTKs)

Ub Signal for Internalization and Sorting into the MVB In yeast, Ub is used for endocytosis and delivery into internal vesicles of the MVB In higher eukaryotes, it is used for the same purpose for regulated transport For regulated internalization and/or lysosomal targeting, an example is the signal transducing receptors: G protein-coupled receptors (GPCRs) b2-adrenergic and CXCR4 (MVB only) Receptor tyrosine kinases (RTKs)

Transmembrane Proteins Regulated by Ub-dependent Sorting In yeast: Ste2, Ste3, Ste6, Uracil permease, Carboxypeptidase S, etc In metazoans: Regulation of growth/differentiation: Growth hormone receptor EGFR Macrophage colony-stimulating factor PDGFR Hepatocyte growth factor receptor Transforming growth factor b receptor b2-adrenergic receptor Immune response: IL-2 receptor T cell receptor CXCR4 IgG receptor (Fcg receptor II) Pre-T cell receptor B7-2

Transmembrane Proteins Regulated by Ub-dependent Sorting In metazoans: Neurotransmission: Ion channels: AMPA glutamate receptors ENaC Glycine receptors ClC-5 Cell-cell contacts: Immune molecules E-cadherin downregulated by viruses: Occludin MHC class I B7-2 Developmental patterning: ICAM-1 Delta CD4 Notch Roundabout

E3 Ligases Bridging factors: E2 + substrate Belong to three families Homologous E6AP Carboxy Terminus HECT (Rsp5/Nedd4) Really Interesting New Gene RING; major family (Cbl) Homology (U-box) proteins UFD2; small family

Ubiquitin E3 Enzymes Substrate specificity HECT domain E3 Homologous E6-AP C terminus E6-AP: papilloma virus E6 oncoprotein – associate protein

Ubiquitin E3 Enzymes RING finger E3 Really Interesting New Gene

Multiple E3 RING Ligases Complex RING

E2 Ub Transfer vs E3 Ub Transfer

E3 – Substrate Recognition (RING E3) Cbl protein Internalization of EGFR, CSF-1, Hepatocyte Growth FR

Ubiquitin Conjugation Deubiquitination Protein Protein Ub Ub Ub Ub Ub Ub Ub Ub Ubiquitin Conjugation Deubiquitination E1 USP E2 E3

Deubiquitinating Activity Largest family enzymes in Ub system Ub C-terminal hydrolase family UCH “Cysteine protease” Ub-specific processing protease UBP Others Ub reutilized Biological Function Cell Growth Development Oncogenesis Neuronal Diseases Chromatin Structure/transcription Proteosome regulation

Poly-Ub Chains K48 Linkage K63 K K63 Linkage K48 K Signal to proteosome K48, Ub4 K63 Linkage K48 K Ub Ub Cell Signaling K63 Ub Ub Peters, J.M. 1998 Ubiquitin and the Biology of the Cell Ub

ENaC function Major ion channel that controls salt and fluid resorption in the kidney Mutations in the PPXY motif cause accumulations of channels at the cell surface and result in Liddle’s syndrome, and inherited form of hypertension

ENac surface Stability Nedd 4 (HECT ligase)-negatively regulates ENaC surface stability Nedd4 WW domains bind PPXY motif of ENaC subunits Nedd4 also interacts with serum and glucocorticoid-regulated kinase (SGK) SGK contains two PPXY motifs that bind to Nedd4 WW domains SGK-dependent Nedd4 P inhibits the Nedd4-ENaC interaction therefore, Nedd4 P increases ENaC at the cell surface

Nedd4 and other Ubiquitin ligases important for protein transport

ENaC Subunits

Regulation of ENaC Surface Stability

Regulation of ENaC Surface Stability

Ub-like Proteins SUMO-1 (sentrin, smt-3) 1996 – covalent modification – RanGAP1 RanGAP1 nearly quantitative modified Cytosolic RanGAP1 to nuclear pore Activate shuttling factor

Ubiquitin-like Proteins:

Ubiquitin Superfold and Ubiquitons UB αβ roll suprfold Ub – blue SUMO-1 – green NEDD8 - red

SUMO SUMO Shared characteristics C-terminal -GG essential for conjugation Affix to lysine residues in target NOT directly associated with proteasomal degradation

Competition/Regulation SUMO Reactive Oxygen Species: Oxidizes reactive thiols on SUMO enzymes Uba1/Aos1- S – S – Ubc9 Thus: SUMO can not attach and proteins not Sumoylated

Examples of SUMO function PROTEIN SUMO Effect RanGAP IkB c-Jun p53 and mdm2 Causes nuclear translocation Blocks Ub-conjugation site, prevents degradation Inhibits transcriptional activity Blocks mdm2 self-ubiquitination, prevents degradation SUMO-p53 in DNA binding domain   apoptotic activity