Protein Targeting and Function:Ubiquitin Modifications

Jim CollawnOctober 17th, 2006

References

1. 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 proteinsAPF-1 identified as ubiquitin

Protein Adduct Protein Degradation Ubiquitin Found Function Found

Avram Hershko – Nobel Lecture

HershkoCiechanover (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

1978Hershko

“APF factor”

2004

Ubiquitinylation – Proteosome Degradation

E3 determines protein substrate

E1-Ub~1000 aa, Cysteine-636 Ub attachment site

E2: TransferUb Conj

Conserved cysteineActive “thio ester”

E3: LigaseSpecificw/E2

Usually do not conjugateHold complex together

19S and 20S Proteasome Subunits

Characteristics• 20S Subunit

– Barrel– Contains 6

proteolytic sites• 2x Tryptic• 2x Chymotryptic• 2x Peptidylglutamyl-

peptidase

– Linearized protein required

• 19S Subunit– Base and Lid– Contains subunits

with known and unknown functions

• Tetra-Ub (K48) recognition

• Deubiquitination activity

• Protein unfolding activity (Chaperone function)

Ubiquitin

– 76 Amino Acids• 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

UbUb

Conjugation – Type BondsMulti-Enzyme Process

E1

Ub RGG

C

ATP ADP

E1 CUb RGG

High-energythioester bond

E2 C

E2 CUb RGG

High-energythioester bond

Protein

Protein

E2 CUb RGG

E3K

GGR

UbIsopeptide bonds

Protein

UbUb

Ub

USP

Ubiquitin AA Sequence

MQIFVKTLTG KTITLEVEPS DTIENVKAKI

QDKEGIPPDQ QRLIFAGKQL EDGRTLSDYN

IQKESTLHLV LRLRGG

48

63

6

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)

2-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)

2-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 receptorEGFRMacrophage colony-stimulating factorPDGFRHepatocyte growth factor receptorTransforming growth factor receptor2-adrenergic receptor

Immune response:IL-2 receptorT cell receptorCXCR4IgG receptor (Fcg receptor II)Pre-T cell receptorB7-2

Transmembrane Proteins Regulated by Ub-dependent Sorting

In metazoans:Neurotransmission: Ion channels:AMPA glutamate receptors ENaCGlycine receptors ClC-5

Cell-cell contacts: Immune molecules E-cadherin downregulated by

viruses:Occludin MHC class I

B7-2Developmental patterning: ICAM-1Delta CD4NotchRoundabout

E3 Ligases

Bridging factors: E2 + substrate Belong to three families

• Homologous E6AP Carboxy TerminusHECT (Rsp5/Nedd4)

• Really Interesting New GeneRING; major family (Cbl)

• Homology (U-box) proteinsUFD2; small family

HECT domain E3

HomologousE6-APC terminus

Substrate specificity

Ubiquitin E3 Enzymes

E6-AP: papilloma virus E6 oncoprotein – associate protein

RING finger E3

Really Interesting New Gene

Ubiquitin E3 Enzymes

MultipleE3 RING Ligases

Complex RING

E2 Ub Transfer vsE3 Ub Transfer

E3 – Substrate Recognition (RING E3)

Cbl protein

Internalization of EGFR, CSF-1, Hepatocyte Growth FR

Ubiquitin Conjugation Deubiquitination

UbUb

UbUb

Ub

Protein

UbUb

Ub

UbUb

UbUbUb

Protein

Ub

USP E1

E2

E3

De-ubiquitination

Deubiquitinating Activity

•Ub C-terminal hydrolase familyUCH“Cysteine protease”

• Ub-specific processing protease UBP

“Cysteine protease”• Others• Ub reutilized

Biological Function

Proteosome regulation

Cell GrowthDevelopmentOncogenesisNeuronal DiseasesChromatin Structure/transcription

Largest family enzymes in Ub system

Poly-Ub Chains

Ub

K

K

Ub

UbUb

Ub

UbUb

Ub

Ub

K48 Linkage

K63 Linkage

K63

K48

Peters, J.M. 1998Ubiquitin and the Biology of the Cell

Signal to proteosome

K48, Ub4

Cell Signaling

K63

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 – blueSUMO-1 – greenNEDD8 - red

UB αβ roll suprfold

SUMO• SUMO

– SUMO-1 & SUMO-2/3

• Shared characteristics– C-terminal -GG essential for conjugation– Affix to lysine residues in target

• NOT directly associated with proteasomal degradation

Competition/RegulationSUMO

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

• 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

PROTEIN SUMO Effect