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

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

3. 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

4. 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

5. 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

6. 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

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

8. Ubiquitinylation – Proteosome Degradation
E3 determines protein substrate

9. 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

10. 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

11. 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

12. Conjugation – Type Bonds Multi-Enzyme ProcessUb
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

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

14. Roles of Ubiquitination

15. Different Types of Ubiquitin Tags

16. 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

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

18. 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)

19. Posttranslational Modification (ubiquitination) and Protein Sorting

20. ESCRT Proteins and the MVB

21. 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)

22. 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)

23. 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

24. 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

25. 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

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

27. Ubiquitin E3 Enzymes
RING finger E3
Really Interesting New Gene

28. Multiple
E3 RING Ligases
Complex RING

29. E2 Ub Transfer vs
E3 Ub Transfer

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

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

32. 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

33. 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

34. 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

35. 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

36. Nedd4 and other Ubiquitin ligases important for protein transport

37. ENaC Subunits

38. Regulation of ENaC Surface Stability

39. Regulation of ENaC Surface Stability

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

41. Ubiquitin-like Proteins:

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

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

44. 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

45. 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