MAMMALIAN UBC6 E2 CONJUGATING ENZYMES AND

STRESS-RELATED PHOSPHORYLATION OF UBC6E

by

Ray Sangin Oh

A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy, Graduate Department of Molecular Genetics University of Toronto

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While these forms may be included Bien que ces formulaires in the document page count, aient inclus dans la pagination, their removal does not represent il n'y aura aucun contenu manquant. any loss of content from the thesis. Canada MAMMALIAN UBC6 E2 CONJUGATING ENZYMES AND

STRESS-RELATED PHOSPHORYLATION OF UBC6E

Doctor of Philosophy (2008)

Ray Sangin Oh

Graduate Department of Molecular and Medical Genetics

University of Toronto

ABSTRACT

Endoplasmic reticulum associated degradation (ERAD) of misfolded membrane proteins

is critical for maintenance of cellular homeostasis. 'ER stress', caused by overload of misfolded

proteins as commonly occurs in many physiological and disease states, activates the Unfolded

Protein Response (UPR) which promotes restoration of homeostasis but also triggers apoptosis

and other physiological responses. Ubc6 is an ER membrane-associated E2 ubiquitin-

conjugating enzyme, implicated in the ERAD of many misfolded proteins in S. cerevisiae.

These studies examine two recently identified human homologs, Ubc6 and Ubc6e, and reveal

stress-related modification of Ubc6e.

These homologues were confirmed to be ER-localized, UPR-regulated, and possess E2

enzyme activity in vitro, which supports their role in mammalian ERAD. Intriguingly, one of

these homologs, Ubc6e, is modified by phosphorylation via a PKR-like ER Kinase (PERK) pathway during pharmalogically induced ER stress. This phosphorylation involves residue Ser-

184, and is associated with inhibited E2 activity, in vitro. These findings suggest that Ubc6e

activity can be regulated by UPR via phosphorylation and highlight diversity in function

between the mammalian homologues.

ii Emerging evidence of cross-talk between UPR and other stress pathways, and the role of

ERAD in maintaining cellular homeostasis, led to investigations of a broader role of Ubc6e in stress response. It was observed that additional and diverse environmental stresses, including hyperosmotic, genotoxic, and pharmacologic stress as well as heat shock and various mitogens also rapidly induce phosphorylation of Ubc6e. A role for p38 MAPK (Mitogen-Activated

Protein Kinase) signaling in Ubc6e modification, with direct involvement of MK2 (MAPK- activated Kinase) was further established. Phosphorylation is triggered via different routes as

PERK involvement was limited to ER stress. Cell lines expressing Ubc6e wild-type, phospho- mutant and phospho-mimic variants revealed that phosphorylation is associated with apoptosis, and involves c-Jun kinase and caspase-12 activation during ER stress. These studies elaborate the central role of the ER in mediating stress signaling by establishing MK2 as a novel regulator of Ubc6e in a wide array of cell stimuli. They further suggest that modulation of the function of this E2 enzyme is critical to stress-related responses and cell survival.

iii ACKNOWLEDGEMENTS

Foremost, I cannot express enough gratitude to my supervisor, Dr. Johanna Rommens, who has been a constant source of support and encouragement over the years. Thank you for your enthusiastic mentorship, patience, and for teaching me to think like a scientist.

I extend thanks to my thesis committee members, Drs. Paul Sadowski, John Glover and

Gergely Lukacs, for providing guidance and the occasional whip-crack to help me get to this point. Thank you also for offering helpful comments on this thesis and various manuscripts.

My numerous labmates have taught me many things and I consider myself fortunate for having worked with such great people. I want to thank them for useful discussions on a 'non- disease'-related research topic, and for making the lab an enjoyable place to work. In particular,

I thank Mark van Oene for patiently showing me the ropes when I was green and Xinli Bai for providing great technical help.

I would also like to express my gratitude to several grad students and post-docs who have helped me in one way or another, and made this odyssey bearable with their friendship. In particular, I wish to thank - Arvin Dar, Kengo Asai, Daniel Suh, Pam Cheung, Mike Szego,

Pleasantine Mill, Janet Atkinson, Darlene Ellenor, Nicole Richards, Siyi Zhang, Lingling Chen,

Isabel Aznarez, Adam Smith, John Cleary, Paul Bradshaw, Rachel Szilard. I am especially grateful to Joy Nishikawa for being an excellent thesis writing partner, and for making the last few years more enjoyable and meaningful.

Lastly, I would like to thank my friends and family for their encouragement and love.

IV TABLE OF CONTENTS

ABSTRACT ii ACKNOWLEDGEMENTS iv LIST OF FIGURES viii LIST OF TABLES x LIST OF ABBREVIATIONS xi INTERNET RESOURCES xiv

CHAPTER I: UBIQUITIN-MEDIATED PROTEOLYSIS, ENDOPLASMIC RETICULUM-ASSOCIATED DEGRADATION AND STRESS RESPONSE

1.1 Ubiquitin-mediated proteolysis 2 1.1.1 Introduction 2 1.1.2 Ubiquitin-conjugating machinery: E1, E2 and E3 enzymes 3 1.1.2.1 El ubiquitin-activating enzyme 3 1.1.2.2 E2 ubiquitin-conjugating enzyme 6 1.1.2.3 E3 ubiquitin ligase 8 1.1.3 Substrate selection and regulation 9 1.1.4 Poly-ubiquitination, de-ubiquitination and proteasomal degradation 11 1.2 ER-quality control and ER-associated degradation 13 1.2.1 Introduction 13 1.2.2 ER protein folding factory 15 1.2.3 ER-quality control 19 1.2.4 ER-associated degradation 21 1.2.4.1 Degradation of ERAD substrates by cytosolic proteasomes 21 1.2.4.2 Selection of ERAD substrates by chaperones 21 1.2.4.3 Retro-translocation 23 1.2.4.4 Ubiquitination of ERAD substrates 23 1.2.4.5 Membrane extraction and delivery to the proteasome 26 1.2.5 ERAD substrates of medical relevance 27 1.3 ER stress and the Unfolded Protein Response 29 1.3.1 Introduction 29 1.3.2 IRE1, PERK and ATF6-sensors of ER stress 30 1.3.3 Reducing ER folding stress: adaptive UPR pathways 34 1.3.3.1 IRE1 34 1.3.3.2 ATF6 35 1.3.3.3 PERK 35 1.3.4 Coordination and regulation of UPR pathways 37 1.3.5 Other strategies to reduce ER folding stress 38 1.3.6 Integration of UPR with other physiological responses and apoptosis 39 pathways 1.3.6.1 Inflammation and immune responses 40 1.3.6.2 Stress-activated MAPK pathways 42 1.3.6.3 Pro-apoptotic transcription factor CHOP/GADD153 42 1.3.6.4 Caspases 43 1.3.6.5 BCL-2 family proteins 44 1.3.7 Balance between survival and death pathways 45 1.3.8 ER stress and disease 45 1.4 Thesis Outline 47

CHAPTER II: HUMAN HOMOLOGS OF UBC6P UBIQUITIN- CONJUGATING ENZYME AND PHOSPHORYLATION OF HSUBC6E IN RESPONSE TO ER STRESS

Summary 50 2.1 Introduction 51 2.2 Experimental procedures 52 2.2.1 Construction of hsUbc6 and hsUbc6e mammalian expression plasmids 52 2.2.2 Cell culture and transfections 54 2.2.3 Protein extract preparation, electrophoresis, immunoblotting 54 2.2.4 Phosphatase assay 55 2.2.5 UPR induction, RNA and protein analysis 55 2.2.6 PERK in vitro kinase assay 56 2.2.7 Metabolic labeling and immunoprecipitation 57 2.2.8 Membrane isolation 58 2.2.9 Immunofluorescence 58 2.2.10 Cloning of Parkin and Pael-R cDNA 59 2.2.11 Co-immunoprecipitation of Parkin and Ubc6e 60 2.2.12 Pulse-chase analysis of Pael-R 60 2.2.13 Recombinant expression and purification of hsUbc6e, hsUbc6esl84D and 61 hsUbc6esl84E 2.2.14 Yeast-two hybrid screening 62 2.2.15 Thiol-ester assay 62 2.3 Results 63 2.3.1 HsUbc6 and hsUbc6e sequence analysis and confirmation of E2 activity 63 by thiol-ester assay 2.3.2 HsUbc6 and hsUbc6e are upregulated with distinct responses during UPR 65 2.3.3 HsUbc6e is phosphorylated in response to ER stress 65 2.3.4 ER stress induced phosphorylation of mmUbc6e requires PERK 70 signaling 2.3.5 HsUbc6e is phosphorylated at Ser-184 73 2.3.6 Phosphorylated hsUbc6e is stable and localized to the ER membrane 75 2.3.7 HsUbc6e phosphorylation has no effect on Parkin E3 interaction and 77 degradation of Pael-R 2.3.8 Identifying putative phospho-regulated protein interactions by yeast 79 two-hybrid screen 2.3.9 Phosphorylation affects formation of ubiquitin-hsUbc6e thiol-esters 82 2.4 Discussion 85

CHAPTER III: PHOSPHORYLATION OF THE UBIQUITIN-CONJUGATING ENZYME UBC6E BY MAPKAPK2 OCCURS IN RESPONSE TO MULTIPLE CELL STRESSORS AND IS ASSOCIATED WITH APOPTOSIS SIGNALING

Summary 91 3.1 Introduction 92 3.2 Experimental Procedures 94 vi 3.2.1 Antibodies and chemicals 94 3.2.2 Cell culture, plasmids and transfections 95 3.2.3 Western blotting, immuno-detection and immunoprecipitation 95 3.2.4 siRNA experiments 96 3.2.5 MK2 kinase assay 97 3.2.6 Cell viability, FACS and apoptosis studies 97 3.2.7 Statistical analysis 98 3.3 Results 99 3.3.1 Phosphorylation of Ubc6e is induced by various stress and mitogen stimuli 99 3.3.2 Stress-induced phosphorylation of Ubc6e by a p38 pathway involving 101 MK2 3.3.3 Phosphorylation of Ubc6e is associated with reduced cell survival and 107 increased caspase activity during stress 3.3.4 Phosphorylation of Ubc6e is implicated in activation of JNK and caspase- 111 12 during ER stress 3.4 Discussion 116

CHAPTER IV: DISCUSSION AND FUTURE DIRECTIONS

4.1. Distinct functionality between the mammalian Ubc6 and Ubc6e homologs: 123 stress response and sequence divergence 4.2 Phosphorylation of mammalian Ubc6e and regulation of E2 activity 125 4.2.1 Thiol-ester formation, substrate conjugation 125 4.2.2 E3 binding 130 4.2.3 Other aspects of E2 activity and regulation: Multi-ubiquitin chain 131 formation, multi-step substrate ubiquitination 4.2.4 A role for Ubc6e phosphorylation in the regulation of protein degradation 135 at the ER 4.3 A role for Ubc6e in regulating stress and apoptosis signaling at the ER 137 membrane 4.4 p38/MK2 regulation of Ubc6e 140 4.4.1 p38 MAPK and ER stress signaling pathways 140 4.4.2 MK2: a stress responsive kinase 140 4.5 Physiological implications of Ubc6e phosphorylation 142

REFERENCES 144

APPENDICES

A.l Supplemental data to Chapter II 182 A.2 Supplemental data to Chapter III 187

vn LIST OF FIGURES

Figure 1.1 Ubiquitin-conjugation pathway leading to proteasomal degradation 4

Figure 1.2 ER-quality control and ER-associated degradation 16

Figure 1.3 Adaptive UPR signal transduction mechanisms mediated by IRE1, 33 ATF6 and PERK

Figure 1.4 Integration of UPR signaling with other physiological responses and 41 apoptosis pathways

Figure 2.1 Human Ubc6 and Ubc6e are active E2 enzymes 64

Figure 2.2 HsUbc6 and hsUbc6e are induced by UPR 66

Figure 2.3 HsUbc6e is phosphorylated during ER stress 68

Figure 2.4 ER stress induced phosphorylation of mmUbc6e requires PERK signaling 71

Figure 2.5 HsUbc6e is phosphorylated at residue Ser 184 74

Figure 2.6 Phosphorylated hsUbc6e is stable and membrane associated 76

Figure 2.7 Phosphorylation does not alter hsUbc6e subcellular localization 78

Figure 2.8 HsUbc6e phosphorylation has no effect on Parkin E3 interaction and 80 degradation of Pael-R

Figure 2.9 HsUbc6e mutants S184D and S184E that mimic phosphorylation show 83 reduced thiol-ester bond formation

Figure 3.1 Phosphorylation of Ubc6e is induced by various stress and mitogen 100 stimuli

Figure 3.2 Stress-induced phosphorylation of Ubc6e can be blocked by the p3 8 102 inhibitor SB203580

Figure 3.3 Ubc6e is phosphorylated by MK2 104

Figure 3.4 Phosphorylation of Ubc6e is associated with reduced viability and 109 apoptosis

Figure 3.5 Phosphorylation of Ubc6e is implicated in activation of JNK and caspase- 113 12 during ER stress

Figure 3.6 Ubc6e is not involved in the regulation of TRAF2 stability during stress 115

vin Figure 3.7 Stress-related phosphorylation of Ubc6e by a p3 8/MK2 pathway is 121 associated with apoptosis

Figure 4.1 Strategy and preliminary results for pulse-chase analysis of total 128 membrane protein turnover during ER stress

Figure 4.2 hsUbc6e predicted secondary structure 129

Figure A. 1.1 Multiple sequence alignment of Ubc6 and Ubc6e family members 182

Figure A. 1.2 Human Ubc6 and Ubc6e are abundantly expressed in various cell lines 183

Figure A. 1.3 ER stress induced phosphorylation of hsUbc6e in various cell lines 184

Figure A. 1.4 Myc-tagged human Ubc6 localizes to ER and peri-nuclear regions 185

Figure A.2.1 Phosphorylation of Ubc6e in response to diverse stressors in various 187 human cell lines

Figure A.2.2 Expression of Ubc6e variants in multiple stable CHO-K1 clones 188

Figure A.2.3 Phosphorylation of Ubc6e is associated with reduced viability in 189 multiple stable cell lines

Figure A.2.4 Phosphorylation of Ubc6e is implicated in activation of JNK and 190 caspase-12 during ER stress in multiple stable cell lines

IX LIST OF TABLES

Table 1.1 Selected ERAD substrates in S. cerevisiae and mammals

Table 1.2 Pathological and physiological conditions where ER stress signaling is observed

Table A. 1 Putative interactor clones obtained from yeast-two hybrid screens using 1 S184A and S184E as bait against a human liver cDNA library LIST OF ABBREVIATIONS AND GENE NAMES (not listed in Tables 1.1 and 1.2)

AR-JP Autosomal recessive juvenile Parkinson's ASK Apoptosis signaling kinase Atg Autophagy related ATF Activating transcription factor BCL-2 B-cell lymphoma 2 BiP Binding protein BrdU Bromodeoxyuridine BSA Bovine serum albumin p-TrCP B-Transducin repeat-containing protein CD Cluster of differentiation Cdc Cell division cycle COP Coat protein CHIP Carboxyl terminus of Hsc70-interacting protein CHO Chinese hamster ovary CHOP C/EBP homology protein Cue Coupling of ubiquitin conjugation to ER degradation CMV Cytomegalo virus Der Degradation in the endoplasmic reticulum Doa Degradation of alpha DMEM Dulbecco's modified Eagle's medium DMSO Dimethyl sulfoxide DTT Dithiothreitol DUB Deubiquitylating enzyme E6AP E6-associated protein EDEM ER-degradation enhancer elF Eukaryotic translation initiation factor ER Endoplasmic reticulum ERAD Endoplasmic reticulum associated degradation ERGIC ER-Golgi intermediate compartment ERQC Endoplasmic reticulum quality control

XI FACS Fluorescence activated cell sorting FBS Fetal bovine serum Fbs F-Box protein that recognizes sugar chains FITC Fluorescein isothiocyanate GADD Growth arrest DNA damage GAPDH Glyceraldehyde-3-phosphate dehydrogenase GFP Green fluorescent protein GRP Glucose-regulated protein HA Hemagglutinin Hac Homologous to Atf/Crebl HECT Homologous to E6-AP carboxy terminus HEK Human embryonic kidney Hrd Hmg-coa reductase degradation Hsp Heat-shock protein IAP Inhibitor of apoptosis

IKB Inhibitor of NFKB IRE1 Inositol-requiring kinase 1 ISG Interferon-stimulated gene JNK Jun N-terminal kinase MAPK Mitogen activated protein kinase MK MAPK activated kinase Mdm Murine double mutant MEF Mouse embryonic fibroblast MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide NEDD Neural precursor cell expressed developmentally down-regulated

NF-KB Nuclear factor-kappa B OASIS Old astrocyte specifically induced substance PBS Phosphate buffered saline PERK PKR-like ER kinase PKR Protein kinase RNA-activated Rad Radiation sensitive RBX Ring box protein RING Really interesting new gene RIPA Radioimmunoprecipitation assay RNAi RNA interference RUB Related to ubiquitin SCF Skpl/Cull/F-box protein complex SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis siRNA Short interfering ribonucleic acid SIC Substrate/subunit inhibitor of cyclin-dependent protein kinase SUMO Small ubiquitin-related modifier TNF Tumour necrosis factor TRAF Tumour necrosis factor receptor associated factor UB Ubiquitin UBA Ubiquitin-Associated UBC Ubiquitin-Conjugating UBE Ubiquitin-conjugating Enzyme UBL Ubiquitin-like protein UBX Ubiquitin regulatory X UFD Ubiquitin fusion degradation protein UGGT UDP-glucose:glycoproteinglucosyltransferase UPR Unfolded protein response UPRE UPR element VCP Valosin containing protein VIMP VCP-interacting membrane protein VPU Virus protein U XBP X-box binding protein Yos Yeast OS-9 homolog

Xlll LIST OF INTERNET RESOURCES

National Center for Biotechnology Information (NCBI)

http://www.ncbi.nlm.nih.gov/

NCBI BLAST Server

http://www.ncbi.nlm.nih.gov/BLAST

NetPhos 2.0 http://www.cbs.dtu.dk/services/NetPhos/

SCANSITE Motif Scan http://scansite.mit.edu/motifscan_seq.phtml

SSPro, University of California, Irvine

http://download.igb.uci.edu/sspro4.html CHAPTER I

UBIQUITIN-MEDIATED PROTEOLYSIS, ENDOPLASMIC RETICULUM-

ASSOCIATED DEGRADATION AND STRESS RESPONSE

1 1.1 Ubiquitin-mediated proteolysis

1.1.1 Introduction

Ubiquitin is a highly conserved 76-amino acid protein that is present in all eukaryotes.

Its covalent attachment to proteins through highly specific and complexly regulated processes is known as ubiquitination (also referred to as ubiquitylation or ubiquitinylation). Target substrates tagged with four or more ubiquitin moieties linked by Lys-48 (K48) of ubiquitin, are targeted for degradation by a multi-subunit ATP-dependent protease complex called the 26 S proteasome. Numerous cellular processes are regulated by ubiquitin-mediated proteolysis, including cell-cycle control [1], inflammatory responses [2], apoptosis [3], and protein quality control in the endoplasmic reticulum (ER) [4]; ER-quality control will be discussed in detail in

Section 1.2. Consequently, aberrations in ubiquitin-mediated proteolysis have been implicated in the pathogenesis of numerous diseases, including various cancers and neurodegenerative disorders [5]. Degradation is not the only fate of ubiquitin-modified proteins; through proteasome-independent mechanisms, mono-ubiquitination or poly-ubiquitination linked through lysines other than K48, most notably K63, can affect processes such as DNA repair, transcription, and trafficking of membrane proteins to the lysosome [6]. There is also a growing list of ubiquitin-like proteins (UBLs), such as SUMO, RUB, ISG15 and ATG8, which contain the conserved ubiquitin fold and are conjugated to other proteins by a pathway similar to ubiquitin conjugation. Through proteasome-independent mechanisms, UBLs regulate numerous cellular processes including transcription, DNA repair, signal transduction, autophagy and cell- cycle control [7]. Proteasome-independent processes mediated by ubiquitin and UBLs will not be discussed in this section, except in descriptions of ubiquitination mechanism.

2 In general, substrate ubiquitination requires the sequential activity of three classes of enzymes (Fig. 1.1;[8]): ubiquitin activation by El, ubiquitin-conjugation by E2 enzymes, and substrate selection by E3 ligases. The organization of the ubiquitin conjugation machinery is more or less hierarchical. A single El activates ubiquitin for all reactions and interacts with multiple E2s, each of which may serve several E3s, and each E3 recognizes a set of substrates that possess similar ubiquitination signals. The existence of multiple E2s and E3s facilitates the highly specific and selective targeting of individual substrates for ubiquitination. In the next sections, I will briefly review how these enzymes achieve their high substrate specificity and how substrate ubiquitination is regulated by a variety of controls, including regulation of the accessibility and activity of conjugating enzymes, post-translational modification of substrates, and also the participation of deconjugating enzymes.

1.1.2 Ubiquitin-conjugating machinery: El, E2 and E3

1.1.2.1 El ubiquitin-activating enzyme

The observation of cell-cycle arrest in a temperature-sensitive mutant of El in S. cerevisiae provided the first evidence of the physiological importance of ubiquitination [9]. It is generally thought that a single highly conserved El enzyme exists in most species, although recent studies have identified novel and divergent Els [10,11]. With regard to ubiquitin-like proteins, their activation requires other distinct and specific Els [12]. To activate ubiquitin, El binds ATP and then ubiquitin, leading to formation of an ubiquitin-adenylate intermediate that serves as the donor of ubiquitin to the catalytic cysteine of the El active site; fully loaded El therefore carries two molecules of ubiquitin, one as a thiol-ester, the other as an adenylate. The active ubiquitin is then transferred to the catalytic cysteine of an E2 [13,14].

3 Figure 1.1 Ubiquitin-conj ligation pathway leading to proteasomal degradation

E2]-S-C-^ O AMP+PP ii E1 •s-c HO-C- (35 rATPA El -SH

K48-linkage Proteasomal M < degradation

Schematic representation of the ubiquitin-conjugation pathway involving El, E2 and E3 enzymes. El ubiquitin-activating enzyme, forms a thiol-ester with the carboxyl Gly-76 of ubiquitin in an ATP-dependent manner; E2 ubiquitin-conj ugating enzyme, accepts the activated ubiquitin in a transthiolation reaction; and E3 ubiquitin-ligase, transfers the activated ubiquitin from E2 to the substrate, either directly (RING E3) or via an additional transthiolation reaction

(HECT E3), resulting in isopeptide linkage between the carboxyl terminus of ubiquitin (Gly-76) and the 8-amino group of a lysine residue of the substrate, or of a ubiquitin already attached to the protein. Although lysines are the most frequent site of ubiquitin attachment, conjugation can sometimes occur on other residues [15-17]. The attachment of ubiquitin often occurs at multiple

4 lysine residues in a random pattern apparently not dependant on amino acid sequence context

[18,19]. The ubiquitin chain is lengthened by repeated action of the E2 and E3, and sometimes with the help of an accessory factor (E4)[20], to at least four attached ubiquitin moieties linked by K48 of previously attached ubiquitins. This poly-ubiquitin chain can then be recognized by the 26S proteasome which degrades the substrate to short peptides and reusable ubiquitin is released by de-ubiquitinating enzymes (DUB)[21].

5 1.1.2.2 E2 ubiquitin-conjugating enzyme

Diversity in enzyme activity and increasing specificity of the ubiquitin-conjugating machinery becomes evident with E2s. In S. cerevisiae, there are 13 E2s, termed Ubc (UBiquitin-

Conjugating) 1-13. Not all of the E2s form thiol-esters with ubiquitin; Ubc9 and Ubc 12 are E2s for ubiquitin-like (UBL) protein modifiers SUMO and Nedd8, respectively [22,23]. All E2 enzymes possess the topologically conserved a/p-fold core domain of ~150 residues [8] that fulfills basic E2 functions of transthiolation (accepting ubiquitin from El), recognition of E3 ligase (see below) and transfer of ubiquitin to substrate (or HECT class E3). Within the core domain are the active site cysteine, and other highly conserved residues that are proposed to catalyze receipt of ubiquitin from El or transfer ubiquitin to substrate [8,24,25]. As well, a limited number of residues within the core domain are critical for binding with E3 ligases, as revealed by X-ray crystallography of E2-E3 complexes [25-27]. Many E2s possess amino- and carboxy-terminal extensions to the core domain, or have insertions within the core domain, that are speculated to facilitate specific interactions ([8,28] and below). E2s are classified by absence or presence of these extra domains; class I E2s possess only the core domain, class II E2s have carboxy-terminal extensions and class III E2s have amino-terminal extensions [28].

The functions of E2s have been most thoroughly studied in S. cerevisiae. The 13 E2 enzymes in S. cerevisiae are involved in a variety of cellular functions, including DNA repair, cell cycle control, protein translocation, stress response, and chromosomal organization [28].

Individual E2s function in the turnover of distinct sets of substrates, with specificity being achieved by specific E2-E3 interactions. For example, Cdc34 (Ubc3) can serve two related but distinct E3s, to facilitate the degradation of multiple phosphorylated substrates involved in cell- cycle regulation, including Gl cyclins and the cyclin-dependent kinase inhibitor Sicl (reviewed in [29]).

6 How E2s pair with specific E3s is influenced by their core domain residues and additional amino- and carboxy-terminal domains. X-ray crystallography of the UbcH7 class I E2 complexed with E3s c-Cbl RING or E6AP HECT revealed that certain E2-E3 contacts may be shared, namely the Phe-63 of UbcH7 fits into a groove of either HECT and RING E3 domains, while other E2-interacting regions with these E3s appear distinct [26,27]. The previously mentioned core domain insertions may influence E2-E3 interaction as well. For example, 12-13 amino acid loop insertion within the core domain of Ubc7 may restrict specific E2-E3 interactions as modeling of the Ubc7 structure on the UbcH7-E3 structures suggests [30,31].

Class II E2s often utilize their carboxyl extensions to facilitate interaction with specific E3s.

For example, interaction between Cdc34 and subunit-components of the SCF E3, Cdc53 and

Cdc4 [32], and between Ubc2 and the Ubrl E3 [33,34] can be abrogated by deletion of carboxy- terminal acidic amino acid segments. E3-E2 pairing can also be regulated by ancillary factors

[35] or E2 post-translational modification [36], or through restricted cellular localization

[37,38]. Cis sequence elements of E3s themselves that occur outside of the RING motif have also been implicated in determining E2-E3 pairing [39].

While E2s possess non-overlapping functions as is reflected in the distinct phenotypes of many S. cerevisiae mutants [28], redundancy is also seen, where multiple E2s are involved in the ubiquitination of the identical substrates. For example, Ubc6, Ubc7, Ubc4 and Ubc5 are all involved in the degradation of short-lived transcriptional repressor Mata2 [40]. This kind of redundancy can be achieved in two ways: several E2s may be utilized by the same E3 [41,42] or the degradation of a particular target involves different combinations of E3s and E2s [43,44].

The use of multiple E2 and E3s may in some cases facilitate poly-ubiquitin chain formation [45]

(discussed further in Section 1.1.4 and 4.3.2).

An increasing number of E2 genes in eukaryotes correlates with increasing developmental complexity: 13 in S. cerevisiae, 20 in C. elegans, 25 in Drosophila, and greater 7 than 30 are known in mammals. Increasing number reflects multiple isoforms of some E2s [46-

48] as well as the evolution of new E2s [49,50]. An expanding E2 repertoire may result in further functional redundancy. Systematic E2 RNAi knock-out analysis in C.elegans revealed similar phenotypes for many E2s [51]. Diversification of roles of individual E2s is also apparent; for example, inactivation of mHR6B (one of the mouse homologues of yeast

Rad6/Ubc2) causes male sterility associated with chromatin modification [52], while deletion of its homologue mHR6A, which shares over 90% sequence identity and is expressed the same tissues as mHR6B, is not associated with reduced fertility [53]. Having many E2s may diversify the specificity of ubiquitination, whereby different E2s could modulate substrate specificity via different E3s. The involvement of multiple E2s may also control the flow of activated ubiquitin to different E3s, which may be important if the pool of El-activated ubiquitin is limited [8].

1.1.2.3 E3 ubiquitin ligase

E3s are responsible for substrate recognition, and dedicated E3-substrate pairings permit independent regulation of the ubiquitination of individual substrates. There are two main families of E3s, known as HECT or RING types [8]. There are -50 HECT E3s known in humans but only 5 in S. cerevisiae. HECT E3s are so named because they bear a -350 amino acid domain that is homologous to E6-AP carboxy terminus. For HECT E3s, ubiquitin is first transferred from an E2 to a cysteine in the active site of it conserved HECT domain before it is transferred to substrate. The amino-termini of HECT E3s possess additional domains that determine subcellular localization and facilitate substrate recognition (reviewed in [31,54]). For example, the C2 domain of NEDD4 recruits this E3 to the plasma membrane, and the WW domain binds to PY motifs of the epithelial sodium channel (ENaC) [55].

The second E3 family is defined by the RING (Really Interesting New Gene) finger, a motif that coordinates a pair of zinc ions [8]. Unlike HECT domain E3s, RING domain E3s (and 8 the structurally related PHD finger and U-box E3s), function, at least in part, as adaptors: they bind E2-ubiquitin and substrate simultaneously, bringing the reactive thiol-ester E2-ubiquitin bond into proximity with a substrate lysine, thereby facilitating transfer of ubiquitin. There are several hundred RING-finger domain proteins in public databases. They have been implicated in myriad cellular processes (reviewed in [56]). There are two varieties of RING E3s: single- member and multi-subunit classes. Single-member RING E3s use their RING domain to bind

E2 and other domains within the same molecule to recognize substrates, while in multi-subunit

E3s the RING domain and substrate recognition domains occur in separate polypeptides. RING and substrate recognition domain subunits are brought together by a scaffold protein called a cullin. There also exist additional adaptor protein families (Skps, Elongins) that link cullins to substrate recognition domains. The modular organization of these multi-subunit or SCF

(Skpl/Cull/F-box protein complex) E3s permits the core ligase activity (RING domain) to be paired with multiple substrate recognition domains (F-boxes, SOCs boxes), cullins and adaptors, resulting in an extremely plastic system that can target numerous substrates for degradation [57].

This is well demonstrated with the RING E3, Rbxl, which can interact with numerous F-boxes that contain WD40 domains that recognize different phosphorylated substrates such as cyclin- dependent kinase inhibitor Sicl, Gl cyclins, IKB, P-catenin and others (reviewed in [29]).

1.1.3 Substrate selection and regulation

The existence of many E3s implies that substrate ubiquitination signals are diverse; this is reflected in the many types of substrate recognition motifs found in E3s that mediate specific protein-protein interactions. In the next section, a few representative examples of the types of ubiquitination signals will be described.

9 Ubiquitination signals are frequently short polypeptide segments. The first ubiquitination signal described was the destruction-box (D-box) found in mitotic cyclins [58] and other substrates of the APC E3 complex. The destruction box sequence, RXALGXIXN, is recognized by substrate recognition F-boxes of the APC, such as Cdc20 and Cdhl. Other sequence regions that signal substrates for APC-mediated degradation have since been described

(e.g. KEN box, A box) (reviewed in [59]).

E3-substrate interaction can also be modulated by substrate post-translational modification, which couples ubiquitin-mediated degradation to various cell processes and signaling events (reviewed in [57]). For example, the SCF E3 complexes target substrates that are phosphorylated by cyclin-dependent kinases at particular points in cell cycle [29].

Hydroxylation is another post-translational modification that triggers substrate recognition: the oxygen dependent hydroxylation of a specific proline residue of hypoxia-induced factor- la induces recognition by the Von-Hippel-Lindau E3 complex; degradation of hypoxia-induced factor-la shuts-off hypoxia induced gene expression [60,61]. In another example, a certain set of N-terminal residues are known to cause protein destabilization (N-end rule) [62,63]. N- terminal asparagine, glutamine, and cysteine, in particular, can be arginylated [64,65] which induces their recognition by 'N-recognin' E3s [62,66]; N-end rule degradation has multiple physiological roles including signaling by nitric oxide [65] and maintenance of chromosome stability [67]. Protein glycosylation can also be linked to ubiquitination (discussed in Section

1.2.4.5)

Protein conformation can also serve as a signal for ubiquitination. This is apparent for proteins denatured by heat-shock or misfolded from amino-acid analogue incorporation are selectively and efficiently degraded by the ubiquitin pathway [68-70], which involves specific

E2s [71]. However, the targeting mechanisms remain incompletely described. It is thought that

10 certain E3s may bind to inappropriately exposed hydrophobic surfaces [72], such as DoalO with its predicted coiled-coil domain [41]. Other E3s, like the U-box CHIP (Carboxyl terminus of

Hsc70-Interacting Protein), may utilize protein chaperones, like Hsp70, as their specificity factors for unfolded proteins [73]. This method of substrate recognition is also seen in the ER, where nascent membrane proteins that misfold are 'triaged' by localized chaperones and folding enzymes to a membrane linked degradation machinery (more on ER-associated degradation in

Section 1.2.4).

Substrate recognition by chaperones is an example of recognition in trans, where an ancillary factor couples the E3 and the substrate. Another example of recognition in trans is viral protein-mediated ubiquitination. In papilloma virus infected cells, viral protein E6 binds to both HECT E3 E6AP and p53, thereby inducing the ubiquitination and degradation of p53 [74].

1.1.4 Poly-ubiquitination, de-ubiquitination and proteasomal degradation

Proteins bearing Lys48-linked chains of at least four ubiquitin molecules are generally good substrates for proteasomal degradation [75]. The molecular mechanisms of ubiquitin-chain assembly are still not well understood. One model involves repeated cycles of E1-E2-E3, the

'sequential addition' model, but there is evidence to suggest alternate pathways where poly- ubiquitin chains are generated prior to attachment to substrate on active sites of E2s or E3s, the

'processive addition' model (reviewed in [76]). In support of this, numerous E2s show the ability to homo-and hetero-dimerize and auto-ubiquitinate [77-79], sometimes enhancing their

E2 conjugation activity [79]. Poly-ubiquitin chain formation at catalytic sites of HECT class E3

E6-AP has also been observed [80]. Proteolysis of certain substrates may require the additional activity of chain elongation factors, or E4s, which are thought to enable long ubiquitin chain

11 synthesis [20]. Although the exact mechanism by which they promote poly-ubiquitin chains is unknown, their RING-like U-box domain permits them to function as E3s in other assays [81].

Another aspect of ubiquitin chain assembly that is poorly understood is the extent to which different E3-E2 combinations contribute to the initiation and elongation phases of synthesis. In the example of Proliferating cell nuclear antigen, which is ubiquitinated during

DNA damage response in S. cerevisiae, initial mono-ubiquitination and subsequent polyubiquitination is performed by different E2-E3 complexes [45]. This case clearly emphasizes that specificity for substrate versus substrate bound by ubiquitin, can be determined by different E2-E3 complexes.

The labeling of substrate with poly-ubiquitin chains can be reversed by the action of deubiquitylating enzymes (DUBs) that specifically cleave ubiquitin-linked molecules after the terminal carbonyl of the last residue of ubiquitin (Gly-76) (reviewed in [82]). The DUBs are a large group of enzymes (-80 predicted in human), and are critical for maintaining ubiquitin and proteasome homeostasis by: salvaging ubiquitin from proteasomal degradation, clearing the proteasome of competing 'free' ubiquitin chains, and processing inactive ubiquitin precursors.

When involved prior to the commitment phase of proteolysis, DUBs can also negatively regulate protein degradation. It is not certain whether there is a general susceptibility of many or most ubiquitinated substrates to deubiquitination; however, numerous DUBs can cleave ubiquitin from specific substrates [82], such as herpes virus-associated ubiquitin specific protease which deubiquitinates p53 in vitro and in vivo resulting in stabilization of p53 even in the presence of overexpressed Mdm2 (a p53 E3) [83].

Poly-ubiquitin chains of four or more ubiquitins are efficiently targeted for degradation by the 26 proteasome, a multicomponent enzymatic complex with a molecular mass of approximately 2,000 kDa (reviewed in [84]). The 26S proteasome consists of two major subcomplexes: a 20S particle, known as the 20S proteasome, which is the catalytic core, and a 12 19S particle, known as PA700, which is the regulatory component. The 20S particle is composed of four heptameric-stacked rings of subunits forming a cylindrical structure, within which lie the peptidase active sites [85]. Substrate access to the lumen is regulated by gated channels, positioned at the ends of the 20S particle, composed of the N-terminal tails of the a- subunits; binding of the 19S particle induces opening of the channel [86]. The 19S particle contains subunits involved in poly-ubiquitin binding [87,88], ATPase-mediated substrate unfolding [89,90], and deubiquitination [91,92]. Unfolded proteins translocate into the 20S particle channel processively from one terminus and are proteolysed by catalytic (3-subunits to short polypeptides [93]. Proteolysis can also be restricted to flexible loop regions of proteins that enter the proteolytic channel [94]. The latter activity is required for partial proteolysis required to release some transcription factors from their inactive states [95,96].

The delivery of polyubiquitinated protein to the proteasome can involve additional mechanisms. Certain E3s have been found to associate with the 19S particle, providing direct delivery of ubiquitinated substrates to the proteasome [97,98]. As well, a growing group of so- called ubiquitin-like (UBL) and ubiquitin-associated (UBA) proteins can simultaneously bind proteasomes and poly-ubiquitin chains, and have been proposed to function as delivery factors

[99].

1.2 ER-quality control and ER-associated degradation

1.2.1 Introduction

13 The endoplasmic reticulum (ER) is a continuous membrane network whose primary function is the import, modification, folding and assembly of all proteins and protein complexes that travel along or reside in the secretory pathway of eukaryotic cells. It is estimated that one third of all proteins in the eukaryotic cell are translocated into the ER [100]. The environment of the ER is highly specialized for protein folding and possesses chaperones and folding enzymes that catalyze formation of tertiary and quaternary protein structure (see below)[101]. The ER is also a major site of membrane lipid synthesis, supplying steroids and phospholipids to other organelles [102], and is the cell's main storage compartment of calcium ions [103].

Protein folding in the cell is subject to quality control, a process whereby chaperones recognize and sort misfolded proteins for degradation [104]. This process is essential for maintaining cellular homeostasis as accumulated misfolded proteins can lead to formation of toxic aggregates, inactivation of functional proteins, and disruption of ER homeostasis, ultimately leading to cell death. In eukaryotic cells, targeted degradation of misfolded proteins proceeds for the most part by the ubiquitin-proteasome pathway [105]. Many cellular events and conditions can result in protein misfolding, such as biosynthetic errors (i.e. errors in translation or transcription) and genetic mutation, as well as cell stresses such as glucose starvation and chemical perturbations that disrupt the homeostasis of the ER. Even in unstressed cells, it has been estimated that up to 30% of all new proteins are degraded due to folding defects and inefficiencies [106].

When misfolding takes place in the ER, localized chaperones and folding enzymes together with translocon components mediate 'retro-translocation' of substrates to the cytosol where membrane-anchored ubiquitin-conjugating enzymes and ligases attach poly-ubiquitin chains to cytosolic substrate segments. Substrate dislocation from the membrane is completed by cytosolic ATPases, followed by proteasomal degradation. The processes of substrate selection, delivery to the cytosol and ubiquitin-mediated proteolysis is referred to as ER- 14 associated degradation (Fig. 1.2). This pathway is essential for maintenance of ER homeostasis and is intimately linked with the 'unfolded-protein response' (UPR) that reprograms the folding and degradative capacity of the ER in the face of accumulated misfolded protein (more on UPR in section 1.3). Finally, it is worth mentioning that degradation of proteins at the ER is not always for quality control purposes, although substrate selection criteria may be similar; ERAD machinery also targets metabolically controlled enzymes that reside in the ER membrane [107] as well as soluble regulatory proteins [41].

In the following section, I will describe the ER folding environment and the mechanisms of ER-quality control (ERQC) and ER-associated degradation (ERAD). There are numerous examples of ERAD substrates that are associated with human disease, some of which are listed in Table 1.1, and I will describe a few in Section 1.2.4.

1.2.2 ER protein folding factory

The translation of proteins entering the secretory pathway is carried out by ribosomes bound to the cytosolic face of the ER [108,109]. Targeting to the ER generally requires a signal sequence at the N-terminus of nascent polypeptides that can be recognized by a cytosolic signal recognition particle [110,111]. The signal recognition particle directs the nascent polypeptide to a proteinaceous pore in the ER membrane, the heterotrimeric Sec61 complex, through which proteins are translocated into the lumen (soluble proteins) or integrated into the membrane of the organelle (integral membrane proteins) [112,113].

Newly synthesized proteins fold with the help of ER resident and cytosolic chaperones during and after translocation into the ER [112]. These include cytosolic Hsp70 and Hsp90 class chaperones, ER-resident Hsp70 class chaperones BiP/GRP78 (Kar2 in S. cerevisiae) [114],

GRP170 [115], various co-chaperones of BiP that stimulate substrate binding cycles [114], and 15 Figure 1.2 ER-quality control and ER-associated degradation

ribosome translocon/Sec61 complex chaperones, folding enzymes I

misfolded folded b

ER lumen cytosol

Proteasomal degradation

Schematic representation of protein quality-control and degradation in the ER. Polypeptides destined for the secretory pathway are translocated into the ER via the translocon. Chaperones and folding enzymes assist newly synthesized proteins attain their proper conformation.

Correctly folded proteins are allowed to transit the ER while misfolded or incompletely folded proteins are retained. Terminally misfolded proteins are selected for reverse-translocation which may or may not involve components of the translocon. Cytosolic segments of misfolded proteins are targeted for ubiquitination by ER-localized E3 and E2 enzymes. Complete extraction of substrates from the ER requires action by the ATPase Cdc48. Poly-ubiquitinated substrates are degraded by cytosolic 26S proteasomes.

16 Table 1.1 Selected ERAD substrates in S. cerevisiae and mammals

ERAD Substrate Topology Description Ste6* Polytopic Mutant form of yeast plasma membrane ABC transporter Ste6; bears a destabilizing C- terminal truncation causing its degradation by ERAD [116,117] CPY* Soluble Mutant form of yeast vacuolar protein Cpy; bears a destabilizing point mutation [118]; widely used ERAD model substrate [119-124] Sec61-2 Polytopic Conditional folding mutant of the major translocon component Sec61; this strain exhibits defective ERAD [122]; itself an ERAD target [125] CFTR (AF508) Polytopic An epithelial CI channel that matures poorly in the ER; -75% of wild-type and >99% of the folding mutant AF508 are degraded by ERAD [126,127]; AF508 is the most common disease allele in cystic fibrosis [128] HMG-CoA reductase Polytopic Catalyzes the rate-limiting step in cholesterol biosynthesis [129]; abundant cellular cholesterol levels induce its ERAD (in yeast, [130]; in humans, [131]) a-1 anti-trypsin - Z Soluble Mutant variant of the secreted trypsin inhibitor; variant bears a destabilizing point mutation that causes protein ERAD and aggregation [132,133]; is linked to liver and lung disease [134] Pael-Receptor Polytopic Putative G-protein coupled receptor that is aggregation prone and degraded at the ER by E3 Parkin [135]; mutant Parkin associated with inherited Parkinsons disease [136]; accumulation of Pael-R in the ER is associated

17 with ER stress and neuronal death [135,137] ApoB Soluble, secreted Major protein component of cholesterol- transporting LDL and VLDL particles [129]; lipid availability regulates degradation in the ER [138,139] TCR, a- and CD3- Single TCR subunits that are degraded by ERAD when subunits transmembrane orphaned at the ER [140,141] segment MHCI Single Presentation of antigens at cell surface; HCMV transmembrane encoding ER glycoproteins US2 and US 11 segment trigger MHCI ERAD [142,143]

Abbreviations: Ste6, STErile; ABC, ATP-binding cassette; CPY, carboxypeptidase Y;

Sec61, SECretory; CFTR, Cystic Fibrosis Transmembrane Conductance Regulator; HMG-CoA,

3-hydroxy-3-methylglutaryl-CoA; Pael, Parkin-associated endothelin; ApoB, apolipoprotein B;

TCR, T-cell antigen receptor; MHC; major histocompatability complex; HCMV, human cytomegalovirus; US, Unique Short.

18 the Hsp90 family member GRP94 [144]. Chaperones bind to hydrophobic patches of incompletely folded proteins, shielding them from other molecules. Proper folding and assembly of protein structures requires burial of amino acid sidechains into a close-packed structure that excludes water from the protein's core. Also present in the ER are peptidyl-prolyl cis-trans isomerases which catalyze isomerization of the proline side chain orientation [145].

The environment of the ER is distinct in several ways that aids protein folding. First, the oxidizing character of the ER lumen (relative to the cytoplasm) facilitates disulfide bond formation between cysteine residues, providing protein stability and oligomer structure.

Oxidative folding is assisted by ER-specific folding enzymes of the protein disulfide isomerase family, such as PDI and ERp57 [146], which are re-oxidized by ER-oxidoreductin 1 proteins

[147]. Another special characteristic of the ER folding environment is the role of TY-(asparagine) linked glycosylation. JV-glycans are added to newly synthesized proteins in the ER lumen by oligosaccharyltransferase. Following the trimming of terminal glucose residues by glucosidases to produce a mono-glucosylated glycan, proteins become substrates for lectin chaperones calnexin and calreticulin. Through association with attached glycans, lectin chaperones are thought to maintain substrate proteins in a folding competent state [148].

The ER folding environment is also characterized by a high ER lumen calcium concentration, which is maintained by Ca2+ pumps and exchangers (reviewed in [103]). Most

ER calcium is bound by the chaperones calnexin, calreticulin, BiP and Grp94 [103] and perturbation of calcium levels disrupt substrate binding by these chaperones, altering folding and secretion (examples [149,150]).

1.2.3 ER-quality control

19 In addition to mediating protein folding, ER chaperones and folding enzymes submit the maturation of newly synthesized ER client proteins to quality control [151]. Proteins that are deemed properly folded, and therefore functional, are permitted to transit the organelle, while incompletely folded or misfolded proteins are retained in the ER through their interaction with

ER chaperones and folding enzymes. For example, BiP, which was originally discovered as binding to immature and orphan immunoglobulin chains [152], can retain the majority of unassembled light chains in the ER of B cells [153].

For newly synthesized glycoproteins, retention of unfolded polypeptides relies on glycan trimming and interactions with lectin chaperones. As described above, calnexin and calreticulin bind monoglucosylated substrates; when the remaining glucose is removed by glucosidase II, calnexin and calreticulin are released [154]. The 'foldedness' of the substrate is then interrogated by UDP-glucose:glycoprotein glucosyltransferase (UGGT), a folding sensor [155].

If the protein is incompletely folded, UGGT reglucosylates the N-glycan allowing the substrate to re-associate with calnexin and calreticulin for additional retention in the ER folding environment. Cycles of deglucosylation and reglucosylation continue until the polypeptide released from calnexin/calreticulum fulfills quality control requirements of UGGT (reviewed in

[156]).

Proteins that are deemed correctly folded are released from their associated chaperones and exit the ER in COPII coated vesicles. Many proteins are non-specifically incorporated into such vesicles, representing 'bulk-flow' from the ER to Golgi, while other proteins require specific export receptors to mediate their exit from the organelle (reviewed in [157]). For example, glycoproteins are recruited to COPII coated vesicles by the lectin ERGIC53 in mammalian cells [158]. Interestingly, certain misfolded proteins can escape chaperone- mediated retention and transit the ER [132,159-162]; why certain misfolded proteins are not

20 retained in the ER is not understood, but this may be due to maintained recognition of an ER exit signal by a COPII receptor in these mutants [160,163] thus favoring exit over retention.

1.2.4 ER-associated degradation

1.2.4.1 Degradation ofERAD substrates by cytosolic proteasomes

Some proteins never attain their proper conformation, either due to mutation or lack of cellular energy required to drive sufficient rounds of chaperone binding, or absence of partner oligomer subunits. As a result, the cell must dispose of these misfolded or unassembled proteins to maintain homeostasis. It was initially believed that degradation took place in the ER lumen

[164-166]. Later, studies of an ER-retained mutant form of polytopic membrane protein CFTR revealed that its degradation was sensitive to proteasome inhibitors and required ubiquitination

[167,168], suggesting the involvement of the cytosolic ubiquitin-proteasome pathway. Around the same time, studies in S. cerevisiae determined that the degradation of a mutated versions of an integral membrane protein, the translocon component protein Sec61 [125,169], and of a soluble mutant luminal protein, vacuolar protein carboxypeptidase Y (CPY*), also required the proteasome [123,170]. These last studies also indicated that soluble substrates had to be exported to the cytosol for degradation. Since then a large number of integral membrane and soluble proteins in the ER of S. cerevisiae and mammals have been identified as substrates for ubiquitin-mediated proteolysis (reviewed [148]). A few examples are listed in Table 1.1.

1.2.4.2 Selection ofERAD substrates by chaperones

Chaperones recognize and bind to incompletely folded proteins; they are also central in distinguishing folding intermediates from terminal aberrantly folded proteins (those that have failed to fold after many attempts) that require disposal. Recognition that a substrate requires 21 degradation generally involves chaperones (reviewed in [171]). Secretory proteins can have defects in their luminal, transmembrane, or cytosolic regions; thus, topology and location of the lesion will invoke the involvement of different chaperones. In general, luminal chaperone

BiP/Kar2 is required for soluble substrate ERAD, while cytosolic chaperones Hsp70/Hsp40 are required for integral membrane protein degradation in S. cerevisiae [116,120,172]. Vashist and

Ng further determined that the lesion location also determined chaperone involvement [173].

Upon examination of the degradation of protein chimeras with folded and misfolded domains oriented either in the ER lumen or cytosol, in various wild-type and chaperone mutant S. cerevisiae, they determined that membrane and soluble proteins with luminal lesions are targeted to a BiP-dependent ERAD-L (L for luminal) pathway, whereas membrane proteins with cytosolic domain lesions use a Hsp70-dependent ERAD-C (C for cytosolic) pathway [173].

How chaperones are able to distinguish terminally misfolded proteins from folding intermediates is not entirely understood. For glycosylated ER lumen or transmembrane proteins, an important signal for folding failure is the state of mannosylation on attached glycans

(reviewed in [174]). As described above, glucosylation status of folding glycoproteins will determine their engagement with the calnexin/calreticulin and UGGT cycle. However, this cycle competes with mannose trimming which appears to target misfolded proteins for ERAD.

ER resident mannosidase I removes a terminal mannose from N-glycans yielding Man8 oligosaccharide [175,176]. This modification regulates degradation of misfolded glycoproteins, as inhibition of mannosidase I retards ERAD of an unsecreted thyroglobulin mutant [177], and its overexpression enhances ERAD of an unsecreted a 1-antitrypsin mutant [178]. As the mannosidase displays relatively slow reaction kinetics [179], it is thought that it serves as a timer for glycoprotein folding [174]. Mannose trimming of proteins trapped in the calnexin/calreticuluin cycle leads to their recognition by lectins (carbohydrate-binding proteins)

Html in S. cerevisiae and EDEM1, 2, and 3 in mammals, which are thought to promote their 22 exit from the calnexin/calreticulin folding cycle, permiting their ERAD [180-183]. Another ER lectin that recognizes Man8 oligosaccharide structures is Yos9, which is required for ER exit and degradation of misfolded glycoprotein CPY* [119,184,185].

1.2.4.3 Retro-trans location

Following quality control assessment by chaperones, misfolded proteins exit the ER to the cytosol. Evidence from yeast Sec61 mutants that exhibit a temperature-sensitive retrotranslocation defect indicates the involvement of the Sec61 import complex ([122,186] others in [4]), and this is supported by studies that demonstrate association of Sec61 subunits with various mammalian ERAD targets [143,187]. Other polytopic ER membrane proteins are suggested to form or contribute to an export channel in the ER, such as mammalian Derlin-1 and its yeast homologue Deri, which are required for retrotranslocation and ERAD of some misfolded proteins in S. cerevisiae [124,188] and mammalian cells [189-192].

How substrates are delivered to the retrotranslocation machinery is not entirely understood. For glycosylated proteins, ER lectins are thought to be a link. Man8 lectin EDEM1 has been found to associate with putative export channel proteins Derlin-2 and -3 in mammalian cells [193], and Yos9 has been found in a complex with several membrane proteins involved in

ER exit and ubiquitination in S. cerevisiae (more in the following section) [194]. BiP was also found in this complex, associated with Yos9, indicating that this chaperone may relay client proteins to Yos9 [194]. BiP, as well, is required for keeping luminal substrates in an ERAD- competent soluble state [195,196]. BiP may also possess some undefined 'gating' function at the translocon during retrotranslocation, as it has been shown to regulate the conformation of

Sec61 during translocation [197].

1.2.4.4 Ubiquitination of ERAD substrates 23 Genetic studies in S. cerevisiae have contributed greatly in identifying the factors involved in targeting misfolded proteins for degradation. In seminal studies that provided some of the first indications that ubiquitin-mediated degradation of misfolded membrane proteins takes place in the cytosol, Sommer and Jentsch found that ubiquitin-conjugating enzymes Ubc6 and Ubc7 were required for degradation of mutant Sec61 [38,125]. Furthermore, it was shown that a misfolded ER-luminal protein, carboxypeptidase Y (CPY*), is also degraded in a Ubc7- dependent manner [123]. Both Ubc6 and Ubc7 localize to the cytoplasmic face of the ER; Ubc6 is an integral membrane protein [38] while Ubc7 is recruited by ER-resident Cuel [198]. The requirement of these cytosolic ubiquitin conjugating enzymes in the degradation of misfolded

Sec61 and CPY* clearly indicated the involvement of the cytosolic ubiquitin-proteasome pathway. Studies of another substrate, membrane protein HMG-CoA reductase, led to the identification of a number of other ERAD components [169], including the E3 ligase

Hrdl/Der3, a multi-spanning membrane protein whose RING-finger domain is cytosolically oriented [199]. This ligase functions with Ubc7 and another ubiquitin-conjugating enzyme,

Ubcl, and can ubiquitinate misfolded proteins in vitro [199]. Hrdl is also required for degradation of CPY*[200]; thus Hrdl targets transmembrane and luminal misfolded proteins.

Another ER membrane protein, Hrd3 is involved in regulating the activity and stability of Hrdl

[121], and possesses a large ER luminal domain that is required for substrate recognition and forming complexes with chaperones [201] (more below). Besides the RING E3 Hrdl, another integral membrane RING E3 has been identified in S. cerevisiae, DoalO, which has been implicated in the degradation of numerous misfolded ER transmembrane proteins and also soluble substrates [41,116,202]. Degradation of one of these, transcriptional repressor MATa2, requires exposure of a hydrophobic patch of an amphipathic helix normally masked by a heterodimer partner [72]; recognition of this 'degron' may involve a predicted coiled-coil

24 domain on DoalO [41]. Like Hrdl, DoalO enlists the activity of at least two E2s, Ubc7 and

Ubc6 [41].

Just as in chaperone-driven quality control steps, the topology of ER degradation substrates appears to determine which E3 will be required for their clearance (reviewed in

[203]). Degradation of substrates with defects in cytosolic domain(s) (ERAD-C), like integral membrane ATPase Ste6 mutants, requires DoalO, while substrates exhibiting luminal domain defects (ERAD-L), like CPY*, require Hrdl and co-factor Hrd3 [116,173]. Supporting this, a

DoalO protein complex has been described which contains cytosolic ERAD factors Ubc7 and its recruiting factor Cuel; in contrast, a Hrdl complex has identified ER luminal co-components

Hrd3, Deri, and ER chaperone BiP and lectin Yos9 [194,204]. An ERAD-M pathway was also proposed, where degradation of substrates with lesions located in transmembrane domains, such as Hmg2, Sec61-2, and Pdr5*, require Hrdl/Hrd3 but not Deri, indicating a different mode of substrate selection and retrotranslocation [204]. Currently, it is not understood how misfolded proteins are delivered to the Hrdl ligase; BiP and Yos9 may first recruit substrate to Hrd3, or

Hrd3 itself may sample the lumen for misfolded proteins [201,204]. Because only a few model

ER-luminal and integral membrane substrates have been used to identify the key ERAD components (example [173] [199]), it remains to be seen whether the ERAD-C/L/M pathways are so well defined. For some ERAD substrates, both DoalO and Hrdl ligases are involved

[202,205]. Such redundancy, which is observed more frequently in mammalian systems

(below), would ensure the degradation of potentially toxic proteins.

Studies in mammalian systems have revealed numerous parallels with ERAD processes of S. cerevisiae, as well as a greater number of ERAD components. There exist two versions of

Ubc6 and Ubc7 in mammals, and these are also associated with the ER membrane [48,206,207] and have been implicated in ERAD processes (example [44,207-210]). The mammalian version of Hrdl is also an ER membrane-resident ubiquitin ligase [211,212], and has been implicated in 25 the degradation of unassembled T-cell receptor subunits TCR-ot and CD3-8 [213]. Mammalian

Hrdl forms a complex with Sell, a mammalian orthologue of Hrd3, which is required for the efficient degradation of virally destabilized major histocompatability complex I and misfolded ribophorin [190,214]. A candidate mammalian Doa 10 homologue is the ubiquitin ligase TEB4

[215,216]. Additional ligases have been described, such as gp78, which combines a Hrdl-like

RING domain with a Cue domain which recruits Ubc7, also to ubiquitinate TCRa [217], as well as apoplipoprotein B [218] and HMG-CoA [219]. RING membrane-associated-1 is another ER membrane resident E3 which targets ubiquitination of misfolded CFTR with the E2 Ubc6e [44].

Studies in mammalian systems have also revealed the involvement of various cytosolic

E3s in ERAD. For example, a new SCF-E3 was identified that can recognize sugar chains of N- glycoproteins with two F-box adaptors, Fbsl and Fbs2, and can facilitate ERAD of model substrates [220,221]; denatured substrates are preferentially recognized and ubiquitinated in vitro [222]. In studies of CFTR degradation, another cytosolic E3 was shown to target this misfolded protein's cytosolic loops via interaction with a cytosolic chaperone. This chaperone,

Hsc70, is recruited to the cytoplasmic face of the ER to promote folding of CFTR [223], and can also interact with the cytosolic U-box E3 CHIP, via an interaction between its C-terminus and the tetratricopeptide repeat of CHIP [224]. Association of a Hsc70-CHIP E3 complex has been shown to promote ubiquitination of a misfolded mutant of CFTR, in vitro [209,225].

As in S. cerevisaie, substrate dislocation and ubiquitination are tightly linked in mammalian cells; putative export channel components Derlins has been shown to interact with membrane associated E3s gp78, Hrdl, Sell (Hrdl co-factor) and RING-membrane associated-1

[44,190,191,214].

1.2.4.5 Membrane extraction and delivery to the proteasome

26 At the point of substrate exit from the ER, ERAD-C and ERAD-L pathways apparently merge and utilize the same factors involved in substrate extraction and delivery to the proteasome. ER-resident Ubx2 (VIMP in mammals) recruits (AAA)-ATPase Cdc48p (p97/VCP in mammals), and its co-factors Npl4 and Ufdl to DoalO and Hrdl ligases at the ER membrane

[194,204,226,227]. Here, Cdc48 releases substrates from the membrane [228-231], presumably by coupling ATP hydrolysis with major conformational change of the protein and unfolding activity [232]. Following dislocation from the membrane, substrates are received by Rad23 and

Dsk2, which bind ubiquitinated substrates with their ubiquitin-associated domain and can recruit proteasomes with a ubiquitin-like domain (reviewed in [233]). Rad23 also interacts with

Peptide N-Glycanase 1, a cytosolic N-glycanase that is thought to remove glycans from dislocated proteins prior to their proteolysis [234]. ATPase activity of the proteasome 19S regulatory particle may also directly unfold certain ERAD substrates as they emerge from the

ER and feed them into the proteolytic 20S particle, in a ubiquitin-independent manner

[235,236].

1.2.5 ERAD substrates of medical relevance

Many medically relevant proteins have been identified as ERAD substrates (Table 1.1).

The underlying disorders fall into two categories. The first results from loss-of-function mutations in ERAD components that result in stabilized misfolded proteins, which in turn accumulate and damage the cell, causing ER stress and cell death (more on ER stress in Section

1.3). An autosomal-recessive form of Parkinson's disease (AR-JP) is caused by mutations in the

Parkin gene [136]. Parkin is a RING E3 and one of its substrates is a putative G protein-coupled

27 transmembrane polypeptide, Pael-Receptor (Parkin-associated endothelin-receptor like receptor

(Pael-R)) [135]. Pael Receptor has a tendency to accumulate and aggregate at the ER membrane in vitro, and in the Drosophila brain [237], and has also been found aggregated in AR-JP patient brains [135,137]. Thus it is thought that inactivating Parkin mutations can lead to accumulation of Pael Receptor in patient brains, causing the disease pathology. Indeed, the neuro-toxicity associated with Pael Receptor overexpression in Drosophila and mammalian neuronal cells is overcome with Parkin expression [135,237].

A second class of disorders is due to premature degradation of a secretory or membrane protein, resulting in their absence from target distal compartments. This is the case with cystic fibrosis, where the major disease-causing mutation, the AF508 mutation in the CFTR gene

[128], produces a protein that exhibits delayed folding and is subsequently sorted for ubiquitin- mediated degradation at the ER by quality control machinery [126,127,168,238,239]. Absence of CFTR function at the cell surface is the cause of mortality in CF patients because it alters the hydration of the mucosal layer of airway epithelia and gives rise to persistent microbial infections with resultant lung fibrosis and failure [240]. Numerous strategies have been devised to increase the cell surface quantities of CFTRAF508 (as well as channel activity and stability)

[241]. Several chemical chaperones and small molecules have been shown to improve

CFTRAF508 folding and, as a result, permit escape from ER-associated degradation and traffic to the cell surface [242-244]. Theoretically, increasing cell surface CFTRAF508 might also be achieved by inhibiting the ubiquitin-degradation machinery involved in CFTRAF508 ERAD or by increasing chaperone activity. In fact, overexpression of calnexin can slow ERAD of

CFTRAF508 and expression of a calnexin fragment can promote trafficking of a small amount of CFTRAF508 to the cell surface [245,246]. In general, though, by-passing ERAD either by chaperones or manipulation of quality control and ERAD components, is not a satisfactory

28 therapeutic approach because it would likely yield increased amounts of other ER proteins, such as Pael-R and HMG-CoA, which could be harmful to the cell.

Additional medically relevant ERAD substrates are two cell-surface antigen presenting proteins that are down-regulated by viral-induced degradation at the ER. Expression of human cytomegalovirus genes US2 and US 11 and human immunodeficiency virus gene Vpu promotes the ERAD of host cell major histocompatibility complex-I and CD4 receptor proteins, respectively. US2 and US 11 are small membrane glycoproteins that select MHC-1 molecules for dislocation from the ER membrane to the cytosol for ubiquitin-mediated proteolysis

[142,143], with involvement of various ERAD factors [17,188,192,214]. Vpu is also a small membrane glycoprotein, and it can recruit the SCF-p-TrCP E3 complex to the ER membrane to ubiquitinate CD4 receptor [247]. By promoting the degradation of these cell-surface immunogenic markers, viruses are able to evade the immune system.

1.3 ER stress and the Unfolded Protein Response

1.3.1 Introduction

Processing of ER 'client' proteins is well-managed by ER-quality control and ERAD machineries. However, the ER must also adapt to changes in folding load imposed by conditions that challenge the folding capacity of the organelle. An early observation of such adaptation was made for BiP and GRP94 chaperones which were up-regulated when mutant misfolded haemagglutinin was expressed in mammalian cells [248]. Numerous pharmacologically induced conditions, as well as physiological and pathological conditions, can

29 lead to accumulation of misfolded protein in the ER and are also observed to induce ER chaperone up-regulation (summarized in Table 1.2).

The imbalance between the load of unfolded proteins and the ERQC and ERAD machineries that handle this load is referred to as ER stress. The cell possesses sensing and response mechanisms that over-see cellular response to ER stress, which are collectively referred to as the unfolded protein response (UPR) pathways. Initial efforts by the cell are aimed at mitigating ER stress, by reducing the unfolded protein load in the ER through attenuation of translation and promotion of folding, secretion and degradation (Figure 1.3). At the same time, apoptotic mechanisms are activated (Figure. 1.4), which prepares the cell for death if homeostasis cannot be restored.

In the following sections I will describe the ER-localized stress sensors and the UPR pathways that mediate adaptive responses, and then describe the pro-apoptotic measures activated in response to ER stress as well as other stress pathways that cross-talk with the ER.

As well, there is growing evidence that many human diseases exhibit ER stress as a characteristic cellular pathology, and some of these will be discussed.

1.3.2 IRE1, PERK and ATF6 - sensors of ER stress

The canonical UPR pathway elucidated in metazoans has three distinct branches mediated by different transmembrane proteins: kinase and endoribonuclease inositol-requiring protein-1 (IRE1), basic leucine-zipper activating transcription factor-6 (ATF6) and protein kinase RNA (PKR)-like ER kinase (PERK) serve as proximal sensors of unfolded protein in the

ER (Figure 1.3). In response to unfolded protein, IRE1 homodimerizes; this can be mediated by binding of unfolded protein to the luminal domain (which is structurally similar to the peptide- binding domains of major histocompatibility complexes [249,250]), or may involve BiP 30 Table 1.2 Pathological and physiological conditions where ER stress signaling is observed

ER stress inducing condition Description of stressors mode of action Secretory cell development B-cell High immunoglobulin secretion demands [251] Pancreatic p-cell High insulin secretion demands [252,253] Altered metabolic state Glucose deprivation Perturbs protein glycosylation; energy deficit [254] Hypoxia Perturbs oxidizing environment of the ER; energy deficit[147,255,256] Chemical insult Thapsigargin Inhibitor of SERCA pumps, disrupts Ca++ dependent folding in the ER [257] Tunicamycin Inhibitor of protein N-glycosylation [258] Dithiothreitol Blocks disulfide bond formation in the ER[259] Disease Diabetes, Type I 'Akita' mouse Insulin with missense mutation is misfolded and accumulates in the ER causing p-cell death [260-262] Wolcott-Rallison disease, Lack of functional PERK disrupts organism's ability to PERK -/- mouse manage insulin demand with translation control leading to 0- cell death [252,253,263] Diabetes, Type II Obesity 'Metabolic stress' of over-nutrition; is partially correctable with chaperones in an obese mouse [264] [265] Neurological disorders Machado-Joseph disease Expanded poly-glutamine repeats in the SCA3 protein form cytoplasmic aggregates which inhibit the proteasome leading to an impaired ERAD [266,267] Juvenile Parkinson's Mutations in E3 Parkin gene leads to impaired ERAD and toxic accumulation of some membrane proteins (eg. Pael-R (Table 1.1)) [136] [135]

31 Marinesco-Sjogren Mutation in SIL1 gene, a nucleotide exchange factor for BiP, Syndrome/woozy mouse leads to impaired ER.QC and protein aggregate formation, purkinje cell death [268,269] Others a-1-antitrypsin deficiency Misfolded mutant variant of a-1-antitrypsin aggregates in the ER causing liver toxicity [133,270] Hepatitis C (HCV) Mechanism unknown; viral glycoprotein production demands have been suggested [271,272] Cancer - solid tumours Mechanism unknown; hypoxia (above) has been suggested [273] [255] Abbreviations: SERCA, sarco/endoplasmic reticulum calcium ATPase; SCA3, spinocerebellar ataxia type 3: SIL1, Suppressor of Irel/Lhsl synthetic lethality

32 Figure 1.3 Adaptive UPR signal transduction mechanisms mediated by IRE1, ATF6 and PERK

AJ6 ER / cytosol /

^ mRNA processing proteolytic cleavage -> 1= at Golgi ID XI XBP-1 mRNA sXBP-1 mRNA I CATF6) @PJ) I general attenuation anti-oxidant response chaperones chaperones of protein synthesis metabolic homeostasis XBP-1 lipid synthesis ERAD proteins

transcriptional activation of UPR genes

ER stress activates three transmembrane proteins, as described in 1.3.1. Activated PERK phosphorylates eIF2a, causing general translational arrest leading to a reduction in ER client protein load, and induced expression of certain genes, like ATF4. ATF4 is a transcriptional activator of genes of the Integrated Stress Response which regulates amino acid transport, anti­ oxidant response and recovery from translational arrest (GADD34). The cleavage fragment of

ATF6 produced by SIP and S2P (site-1 and site-2 proteases) at the Golgi is a transcriptional activator for chaperones and folding enzymes and XBP1. Activated IRE1 splices XBP-1 mRNA leading to translation of a potent transcriptional activator (sXBP-1), which promotes

ERAD of misfolded proteins and ER biogenesis, as well as recovery from translational arrest

(p58IPK).

33 chaperone release of IRE1 monomers in favour of unfolded protein allowing IRE1 oligomerization [274,275], or both [276]. The luminal domain of PERK is structurally similar with that of IRE1 and interchangeable for stress signaling [274], so PERK activation likely follows a similar mechanism. Both IRE1 and PERK possess cytoplasmic serine/threonine kinase domains, that autophosphorylate and activate upon dimerization [274,277,278]. ATF6 and a related group of ER membrane-tethered transcription factors also possess stress-sensing domains in the ER lumen. During ER stress, ATF6 transits to the Golgi, possibly involving BiP chaperone release [279], where it is cleaved by Site 1 and Site 2 proteases, producing a free cytoplasmic fragment that is an active transcription factor [280].

1.3.3 Reducing ER folding stress: adaptive UPR pathways

1.3.3.1 IRE 1

Activated IRE1 has cytosolic endonucleolytic activity, for which there is only one target,

Hacl mRNA in S. cerevisiae [281] and its metazoan ortholog, XBP-1 [282,283]. Excision of an intron from Hacl/XBP-1 mRNA produces an active transcription factor that binds several different response elements, referred to as UPREs, in target genes [258,284]. A study in S. cerevisiae determined the transcriptional reach of the Ire 1/Hacl arm of UPR to include some

400 genes (~5% of the yeast genome) [285]. Genes found to be transcriptionally up-regulated by Ire 1/Hacl included those that code for proteins involved in ER protein folding and modification, ERAD, phospholipid biosynthesis and vesicular transport in post ER- compartments of the secretory pathway [285]. Thus, the transcriptional program mediated by

Ire 1/Hacl increases the capacity of the ER folding machinery, promotes clearance of proteins from the ER via degradation and post-ER transit, and drives expansion of the organelle.

34 For metazoans, a complete inventory of UPR target genes has not yet been described, but it has been shown that, as in yeast, ER folding factors, ERAD components are regulated in the response to ER stress [286-288]. IRE1/XBP-1 signaling in metazoans is also required for the

UPR induction of lipid synthesis enzymes and concomitant expansion of the ER [289,290].

Expansion of the organelle is a feature of developing secretory cells to allow for greater production of secretory protein; XBP-1 has an essential role in these processes in plasma B-cells and pancreatic exocrine cells [251,291], Accordingly, both IRE1 and XBP-1 are essential genes in mice [251,292]

1.3.3.2 ATF6

The transcriptional UPR program in metazoans involves another transcription factor family, ATF6. The transcription factor fragment of activated ATF6 can bind several different promoter elements [293-295], and induces expression of ER chaperones and folding enzymes, as determined by microarray analysis of Hela cell stably expressing the active transcription factor segment of ATF6 [296]. Other ATF6-like proteins have specific tissue expression, such as OASIS and cyclic AMP response element-binding protein, which regulate transcriptional

UPR in astrocytes and hepatocytes, respectively [297,298].

1.3.3.3 PERK

The third branch of metazoan UPR is mediated by PERK. Activated PERK phosphorylates the a subunit of eukaryotic translation initiation factor, eIF2a, inhibiting translation initiation by impairing the eIF2B-catalyzed guanine nucleotide exchange reaction required for recycling of eIF2ato its active GTP-bound form [277,299]. A reduction in available active eIF2ot results in general attenuation of translation, which immediately reduces

35 the burden of nascent proteins entering the ER. Another result of translational attenuation is the rapid decrease in cyclin Dl levels and subsequent Gl cell cycle arrest [300,301]. This would enable the cell to divert energy towards efforts in re-establishing ER homeostasis.

Although phosphorylation of eIF2a represses translation of most mRNAs, several mRNAs possess internal ribosomal entry sites or small upstream open reading frames that allow for selective translated during stress conditions [302]. One of these upregulated genes encodes

Activating Transcription Factor 4 (ATF4) which activates genes predicted to function in amino acid import and metabolism and in protecting cells against oxidative stress [303,304]. This stress response has been conserved from S. cerevisiae where eIF2a phosphorylation by kinase

GCN2 is linked to translation of transcription factor GCN4, which is important for cellular response to nutrient deprivation [305]. In mammals, there are at least 3 eIF2a kinases which couple different upstream stress signals to eIF2a phosphorylation. The translational repression and downstream transcriptional regulation mediated by eIF2ot phosphorylation are referred to as the Integrated Stress Response [304].

PERK signaling is critical for maintaining ER homeostasis and survival of the cell during ER stress. PERK deficient mouse fibroblasts have an impaired ability to survive ER stress [306], and the pancreas of PERK-null mice experience increased cell death leading to progressive diabetes mellitus and hypoinsulinemia [252,263]. It is thought that PERK mediated translation repression is critical for linking insulin production with glucose homeostasis; without this 'brake' on translation, (3-cells become stressed and die [307]. Another important aspect of adaptive PERK signaling is the anti-oxidant response downstream of ATF4; perk-/- cells accumulate reactive oxygen species [304]. As reactive oxygen species are a product of oxidative protein folding in the ER, PERK-mediated translation arrest is critical for maintaining

ER redox homeostasis [304].

36 1.3.4 Coordination and regulation of UPR pathways

Although IRE1, ATF6 and PERK molecules are all activated by the same signal, unfolded protein in the ER lumen, the effects of their activation are observed to have different latencies. First in response to ER stress is translational repression, mediated by PERK phosphorylation of eIF2a [277]. Effects of ATF6 and IRE1 activation on transcriptional reprogramming are, as expected, observed later in the response. Between ATF6 and IRE1- mediated transcriptional activation events, there is a biphasic response, where ATF6, which requires only post-translational processing, first promotes transcription of chaperone and protein folding enzymes [296]. This is followed by production of XBP-1 transcription factor, which requires not only IREl-mediated splicing, but upregulation by ATF6 [308,309]. IRE1 promotes protein folding, but also ERAD, secretion and expansion of the ER [285,287]. These findings have led to a three-phase model of UPR, beginning with reduction in ER load by translational arrest, followed by up-regulation of folding pathways, and lastly the stimulation of protein degradation [310].

There is evidence of functional overlap between the transcriptional effects of the known

UPR branches. Transcriptional profiling in C. elegans indicate that ATF6, IRE1/XBP-1 and

PERK have overlapping targets; these UPR branches are functionally redundant for larval development, as deduced from synthetic lethality of IRE-1 RNAi with RNAi of either ATF6 or

PERK [288,311]. As well, cross-talk exists between pathways, resulting in mutual re­ inforcement, as seen with XBP-1 upregulation by ATF6 [308,309]. It is also clear that different

UPR sub-pathways are particularly critical for tissue-specific developmental processes: IRE1 is essential for differentiation of antibody-producing B cells, while PERK/eIF2a is not required

37 for B-cell maturation [292]; Transcript Induced in Spermiogenesis 40, a testis-specific ATF6- like transcription factor, is required for proper maturation of sperm head nuclei [312].

As with any signaling pathway, UPR signaling is subject to negative regulation and feedback to ensure responses are not hyper-activated. Activation of GADD34, a regulatory subunit of phosphatase PP1, by ATF4 promotes dephosphorylation of eIF2a and terminates

PERK/eIF2a stress signaling [313]. Another protein implicated in the recovery of translational arrest is p58IPK which binds and inhibits PERK [314,315]; p58IPK gene activation relies on

IRE1/XBP-1 [286]. In S. cerevisiae, Hacl transcription factor is rapidly degraded by SCF-Cdc4

E3 complex, minimizing the potent Ire 1-mediated transcriptional response [316]. These negative feedback loops allow the cell to recover from general translational inhibition and cease stress-induced gene expression.

1.3.5 Other strategies to reduce ER folding stress

Along with translational attenuation and transcriptional reprogramming of the ER, several other strategies are employed by the cell to reduce the burden of misfolded proteins on the ER. A few mechanisms appear to pre-emptively remove ERAD targets before they even enter the secretory pathway at the ER membrane. IRE-1 in Drosophila has been found to promote degradation of ER-associated secretory and membrane-protein encoding mRNAs during ER stress, which may involve IRE-1 nuclease activity or the local activation of another nuclease [317]. Co-translational degradation of nascent proteins during stress is another reported quality control mechanism. This involves P58IPK, a J-domain containing co- chaperone found to associate with Sec61 translocon and cytosolic Hsp70 chaperones, which has been shown to have increased association with nascent proteins during ER stress [318]. It is

38 thought that p58IPK/Hsp70 can promote co-translational extraction of misfolded proteins from the translocon (e.g. VCAM), and in such a way, reduce the entry of proteins into the already stressed ER [318]. Translocation of proteins into the ER may itself be regulated during ER stress, as well. Proteins with inefficient signal peptides, such as prion precursor protein, are selectively excluded from the ER during stress, while others (like prolactin or BiP) can continue to be translocated into the ER [319]. Thus, the cell may alleviate the ER load by reducing certain incoming proteins, while at the same time allowing production of required UPR target proteins, like BiP.

Other, non-proteolytic mechanisms have been shown to clear misfolded protein from the

ER. This includes autophagy, which involves engulfing of cytoplasm by a double membrane organelle, followed by digestion of sequestered contents by lysosomes [320]. ER-retained mutant dysferlin and fibrinogen proteins are degraded in part by an autophagy mechanism

[321,322]. This process is linked to UPR, as autophagosome formation can be induced during

ER stress in S. cerevisiae [323] and mammalian cells [324]. Autophagy requires IREla signaling and is protective against cell death induced by ER stress [324]. Besides removing misfolded protein aggregates, autophagy is also important for recycling ER membranes that had been used for expanding the organelle's folding capacity [323]. These studies suggest that by sequestering potentially toxic protein aggregates and recycling expanded ER membranes, the

UPR regulated autophagy helps maintain ER homeostasis.

Studies in S. cerevisiae have also identified degradation pathways during ER stress that require ER-Golgi trafficking; over expressed mutant misfolded Cpy* is thought to 'over-flow' to the lysosome [325] or to an IRE 1-dependent ubiquitin-mediated degradation fate requiring vesicular trafficking [326].

1.3.6 Integration of UPR with other physiological responses and apoptosis pathways 39 Clearly, there are numerous adaptive mechanisms employed by the cell to return the burdened ER to homeostasis, and many of these have protective effects against ER stress induced death that have been demonstrated experimentally. For example, ER chaperone overexpression [327-330] and enforced eIF2a phosphorylation [331], or inhibited dephosphorylation [332] can protect cells against ER stress and other types of environmental stress. As well, up-regulation of ERAD activity is an essential aspect of cellular adaptation to

ER stress [211,285,333]. However, when the adaptive measures of UPR signaling are unable to re-establish ER homeostasis, the stressed cell dies. This is not surprising considering the toxic potential of misfolded and unfolded proteins in the cell. ER stress-induced cell death is observed in cultured mammalian cells experiencing prolonged and severe ER stress induced by pharmacological agents, such as thapsigargin and tunicamycin (for examples [331,334]), and in numerous disease states (Section 1.3, Table 1.2.). Contributing to the cell's ultimate fate is the simultaneous activation of additional physiological responses, such as inflammation and immune responses and apoptosis pathways, which will now be discussed (Figure 1.4).

1.3.6.1 Inflammation and immune responses

The transcription factor NF-kB plays an important role in mounting inflammation and immune reponses, as well as modulating apoptosis and cell growth processes in response to diverse environmental conditions (reviewed in [335]). NF-kB can also be activated by ER stress, as initially observed in cells expressing a viral secretory protein that accumulates in the

ER [336]. eIF2a phosphorylation has been linked to activation of NF-kB transcription factor

[337], which may be due to translational repression of inhibitory protein IkB [338]. NF-kB activation may also involve an IRE 1-mediated process [339,340]. How NF-kB contributes to

40 Figure 1.4 Integration of UPR signaling with other physiological responses and apoptosis pathways

Ca2+ BAX/BA/BAK / BCL-2 fc pi A

vTRAF2^-TRAF2 Ca2+^ 1 <— ^alpairy apoptosome XA formation / + (mitochondria) (GI arrest)

(NFK| (SUN) I executioner f inflammation' / caspases | and immune 1/ I response j [apoptosis]

ER stress activates numerous physiological responses and apoptotic mechanisms.

Phosphorylation of eIF2a leads to cell cycle arrest in Gl and activation of NF-kB that promotes inflammation and immune response. All three UPR signaling branches can up-regulate the pro- apoptotic transcription factor CHOP. IRE1 can initiate MAPK signaling pathways from a

TRAF2 complex, leading to JNK and p38 MAPK activation. ER-localized pro-caspase-12 (p-

C-12) can be activated by TRAF2-mediated clustering and other proteases (calpain and caspase-

7, C-7), leading to the activation of executioner caspases. During ER stress, calcium ions are released from the ER leading to activation of the mitochondrial apoptotic pathway and calpain.

ER calcium levels are regulated by BCL-2 family proteins BAX, BAK and BCL-2.

41 the transcriptional UPR mediated by IRE1/XBP and PERK/eIF2a/ATF4 has not been determined. Another link between ER stress signaling and inflammatory response was revealed recently, when it was shown that an ATF6-related transcription factor found in liver, CREBH, initiates inflammatory response by activating expression of acute phase response genes during

ER stress [298].

1.3.6.2 Stress-activated MAPK pathways

In mammals, activated IRE1 forms a heterotrimeric complex with TRAF2 (tumour necrosis factor receptor-associated factor-2)[341] and apoptosis signal-regulating kinase 1

(ASK1)[267], reminiscent of cell surface tumour necrosis factor receptor-dependent activation of ASK1. ASK1 is a mitogen-activated protein kinase kinase kinase (MAPKKK), and during various environmental stress conditions and cytokine stimulus it activates a MAPK cascade leading to phosphorylation and activation of Jun N-terminal Kinase (JNK) and p38 MAPK

[342,343], The IRE1/TRAF2/ASK1 complex promotes cell death, as ASK1-/- primary neurons are resistant to ER stress-induced JNK activation and cell death [267]. Activated JNK may promote apoptosis by potentiating several transcription factors, particularly c-Jun [344] and by regulating BCL-2 family members (reviewed in [345]; more on BCL-2 proteins below); similarly activated p38 can potentiate several pro-apoptotic transcription factors, such as p53 and CHOP [346,347], the latter which is a key pro-apoptotic factor during ER stress (more below). Overall, however, JNK and p38 can activate both cell survival and pro-apoptotic responses to stress [344,348,349].

1.3.6.3 Pro-apoptotic transcription factor CHOP/GADD153

42 Another player in ER stress-induced apoptosis is CHOP/GADD153 transcription factor, which is regulated by all branches of the UPR, PERK/eIF2a/ATF4, ATF6, and IRE/XBP-1

[296,303,350]. CHOP-deleted mammalian cells are partially protected from ER stress-induced cell death [351]. CHOP is believed to contribute to cell death by multiple mechanisms, including increasing the load and oxidation of client ER proteins via up-regulation of GADD34, which together with protein phosphatase I catalyzes eIF2a dephosphorylation, and ER- oxidoreductin 1, an ER oxidase [304,352]. CHOP was also found to repress expression of anti- apoptotic Bcl-2 [353] and up-regulate the death receptor DR5 [354].

1.3.6.4 Caspases

ER stress also leads to proteolytic cleavage of several caspases. Caspases are a family of cysteine proteases that are critical mediators of apoptosis; caspases are activated by other caspases in a sequential cascade of cleavage (reviewed in [355]). Numerous forms of 'intrinsic' apoptotic stimuli, including ER stress, can promote calcium release from the ER lumen which leads to mitochondria permeabilization, cytochrome c release, and formation of the

'apoptosome' (reviewed in [356]). The apoptosome contains initiator caspase-9, which cleaves and activates executioner caspases including caspase-3 and caspase-7 [357,358]. ER stress specific caspases have also been identified: caspase-12 in mouse [359] and caspase-4 in human

[360], both of which localize to the cytoplasmic face of the ER membrane. Caspase-12-deleted cortical neurons are resistant to amyloid-p protein toxicity, and caspase-12 deleted mice are resistant to tunicamycin-induced renal epithelial cell death [359]. Several different mechanisms of caspase-12 activation have been presented. Caspase-12 may be activated via clustering

(leading to self-cleavage) mediated by IRE1-TRAF2 complexes at the ER membrane [361].

Activation may also be mediated by other proteases, such as caspase-7 [362] and calcium-

43 activated protease calpain [363]. Caspase-12 can cleave procaspase-9 and consequently activate the caspase cascade leading to cell death [364,365].

1.3.6.5 BCL-2 family proteins

BCL-2 family proteins are important players in mitochondrial apoptosis (reviewed in

[366]). Generally, BAX (and its homologue BAK) promote apoptosis by inducing permeabilization of the mitochondrial membrane, thereby releasing cytochrome c and activating the apoptosome and the caspase cascade. Anti-apoptotic BCL-2 proteins inhibit BAX/BAK activation by blocking their translocation into the mitochondrial membrane; BH3-only proteins promote apoptosis by binding and inhibiting BCL-2 family proteins. BAX, BAK and BCL-2 are also localized at the ER membrane [367]. Over-expression of ER-targeted BCL-2 or deletion of BAX and BAK imparts resistance to ER stress [368,369]; mitochondrial-targeted

BAX/BAK can return the apoptotic phenotype in the BAX/BAK-deletion background, indicating that signals from the ER must be communicated to the mitochondria. ER-localized

BCL-2 family proteins regulate steady-state ER calcium levels. Deletion of BAX and BAK reduces the resting calcium ion levels in the ER, and reduces activation of caspase-12 during thapsigargin and tunicamycin induced ER stress [370,371], while BCL-2-deficient cells display elevated ER Ca2+ [372]. BCL-2 family members can regulate ER calcium levels in a mechanism involving type 1 inositol trisphosphate receptor, an ER channel that regulates calcium leak into the cytosol [373]. Maintaining lower resting ER calcium is thought to reduce sensitivity to stress by reducing the amount of released Ca2+ into the cytosol during stress and attendant activation of calcium-dependent proteases and mitochondrial permeabilization [345].

BAX and BAK may also directly modulate apoptotic UPR signaling, as one recent study shows that BAX/BAK double knock-out cells are impaired in IRE1 mediated JNK phosphorylation and that BAX and BAK can form a complex with IRE1 [374]. 44 1.3.7 Balance between survival and death pathways

In the face of excessive ER stress, the balance between adaptive and apoptotic measures tips towards cell death. As genetic ablation of these individual apoptotic pathways confers only a degree of protection (for example, as observed with chop'' mouse embryonic fibroblasts

[375]), it appears that multiple apoptotic pathways need to be engaged for cell-death to occur. It is currently not understood how these multiple pro-death pathways, MAPK and inflammation responses are coordinated with the adaptive UPR to ultimately decide whether the cell will die or survive. Clearly, under certain physiological conditions, some cells can adapt to stress (e.g. during B-cell development). And many human pathological conditions that are associated with

UPR activation, such as diabetes and various neurodegenerative diseases (Section 1.3.7) occur over a course of many years. Thus, how can cells escape cell death in the face of continued stress or when the folding stress can be alleviated? A recent study suggests that cell survival in the face of chronic low level ER stress might be facilitated by the selective instability of pro- apoptotic mRNAs and proteins, like CHOP and GADD34, and simultaneous stability of adaptive mRNAs and proteins, like BiP, GRP94 and p58IPK [376]. In this model, cell death is achieved when levels of pro-apoptotic proteins like CHOP and its target genes, like GADD34, reach a particular apoptotic threshold, as seen during prolonged and severe ER stress. The selective stability of stress-adaptive components may also explain the phenomenon of pre­ conditioning, where transient exposure of one form of stress protects cells against subsequent stresses (example [377,378]). Indeed, BiP overexpression as well as enforced eIF2a phosphorylation can protect cells from other stresses including ER stress [327,329,331].

1.3.8 ER stress and disease

45 As discussed in previous sections, UPR signaling is essential for physiological processes such as secretory cell development and for maintaining cellular homeostasis. Thus, it is not surprising that disrupted UPR and ERAD functionality can lead to various human diseases, such as neurodegenerative disorders and diabetes (reviewed in [307,379] and selected examples in Table

1.2). The prolonged ER stress experienced in these disease states lead to up-regulation of pro- apoptotic markers in the affected tissues and eventually cell death [252,253,263,380]. For many neurodegenerative diseases, like prion-disorders, Huntington's and Machado-Joseph diseases, the misfolded proteins accumulate in the cytosol or nucleus and their ability to disrupt ER function is attributed to an inhibited proteasome, which leads to a disruption of general ERAD

[266,267]. For some other diseases, such as a-1 antitrypsin inhibitor deficiency, the misfolded protein accumulates at the ER and therefore causes ER stress directly [133,270]. Recently, chemical chaperone treatment and inhibitors of ER stress signaling, such as an inhibitor of eIF2a-dephosphorylation, have been demonstrated to be effective at reducing ER stress

[265,332]. Thus, where protein misfolding is the underlying pathology, correction of the folding defect or mitigation of pro-apoptotic aspects of stress signaling are potential therapeutic strategies.

Other diseases, such as certain cancerous tumours and viral infections, are accompanied with an activated UPR, but it is uncertain whether ER stress is a primary event in pathogenesis or an epiphenomenon [381,382]. In the case of cancer, UPR activation, presumably due to the hypoxic tumour environment, contributes to cell proliferation, as transformed perk'1' mice exhibit reduced tumor formation in vivo [383] and xbp-1'1' mice have compromised growth of transplanted tumours [384]. Thus, targeted modulation of UPR signaling represents a potential avenue for cancer therapeutics.

46 1.4 Thesis Outline

In eukaryotes, misfolded proteins of the secretory pathway are degraded by an ERAD process, and many of these are related to human disease. Studies in S. cerevisiae have elucidated many details of substrate selection, retrotranslocation, and ubiquitination, though these processes in mammals are less well understood. To gain further insight into the mammalian ERAD system, I sought to characterize the human homologues of the ER-associated ubiquitin conjugating enzyme Ubc6, which, in S. cerevisiae, has been implicated in the ERAD of numerous substrates [117,123,125,385,386]. Mammalian Ubc6 homologues have been shown by others to participate in ERAD [135,208-210] and also in the degradation of non- membrane proteins [387,388]. However, these homologs have yet to be characterized in detail and it is unknown how they are distinguished functionally. Investigations presented in Chapter

2 describe the characterization of the two known Ubc6 human homologues, including localization and confirmation of UPR up-regulation. Intriguingly, it was observed that one of these homologues, Ubc6e is modified by phosphorylation by an ER stress-induced PERK pathway. This phosphorylation was found to involve residue Ser-184, and is associated with an inhibition of E2 activity, in vitro. These findings represent the first post-translational modification to be identified for an ERAD component.

Given the essential role for ERAD in maintaining cellular homeostasis and the many links between ER stress and other physiological responses, it was of interest to determine the role of

Ubc6e phosphorylation in ER stress as well as its broader role in stress response. Chapter 3 presents investigations to determine the function of this phosphorylation modification. They revealed that Ubc6e phosphorylation is not limited to ER stress, but is a common response to various environmental stressors and mitogen stimulation with PERK involvement restricted to

ER stress. This stress-induced modification requires p38 MAPK and directly involves protein 47 kinase MK2. Interestingly, this modification correlates with cell death and apoptosis signaling at the ER. These results establish a novel link between a MAPK pathway and regulation of an

ERAD component and further suggest that modification of this E2 is implicated with apoptosis signaling at the ER. As Ubc6e is an E2 enzyme, identification of this modification raises the possibility that substrate selectivity via regulated E3 partnership, or aspects of conjugation activity, could be regulated during stress, and these possibilities are discussed in Chapter 4.

48 CHAPTER II

HUMAN HOMOLOGS OF UBC6P UBIQUITIN-CONJUGATING ENZYME AND

PHOSPHORYLATION OF HSUBC6E IN RESPONSE TO ER STRESS

The work in this chapter, with the exception of Section 2.2.9, was presented in the publication:

Oh, R.S., Bai, X., Rommens, J.M. (2006). Human homologs of Ubc6p ubiquitin-conjugating enzyme and phosphorylation of hsUbc6e in response to ER stress. Journal of Biological

Chemistry. 281(30):21480-90.

Contributions:

I organized and led the investigations in this chapter. I thank Xinli Bai for excellent technical assistance: she performed UPR induction and subsequent Northern blot analysis of Ubc6e expression, and cloned the PERK gene by RT-PCR. She also generated the Ubc6e phospho- mutant-DNA binding domain fusion yeast expression vectors and managed the two-hybrid screen. All other work was performed by me.

49 Summary

Ubiquitin-conjugating enzyme Ubc6 is a tail-anchored protein that is localized to the cytoplasmic face of the ER membrane and has been implicated in the degradation of many misfolded membrane proteins in yeast. I have undertaken characterization studies of two human homologs, hsUbc6 and hsUbc6e. Both possess tail-anchored protein motifs, display high conservation in their catalytic domains, and are functional ubiquitin-conjugating enzymes as determined by in vitro thiol-ester assay. Both also display induction by the unfolded protein response (UPR), a feature of many endoplasmic reticulum associated degradation (ERAD) components. Post-translational modification involving phosphorylation of hsUbc6e was observed to be ER-stress related and dependent on signaling by the ER-stress inducible kinase,

PERK. The phosphorylation site was mapped to Serl84, which resides within the uncharacterized region linking the highly conserved catalytic core and the carboxyl terminal transmembrane domain. Phosphorylation of hsUbc6e also did not alter stability, subcellular localization or interaction with a partner E3. To attempt to identify phospho-regulated protein interactions, an unbiased yeast-two hybrid screen was performed. Assays of hsUbc6esl84D and hsUbc6esl84E, which mimic the phosphorylated state, suggest that phosphorylation may reduce capacity for forming ubiquitin-enzyme thiol-esters. The occurrence of two distinct Ubc6 homologs in vertebrates, including one with phosphorylation modification in response to ER stress, emphasizes diversity in function between these E2s during ERAD processes.

50 2.1 Introduction

Targeted protein degradation via the proteasome requires substrate modification with covalent attachment of ubiquitin (Ub) through the sequential action of three classes of enzymes

(reviewed in [389]). First, Ub-activating enzyme (El) in an ATP-dependent step catalyzes the formation of a high-energy thiol-ester bond between its active-site cysteine and the carboxy terminus (G76) of Ub. The activated Ub is then transferred to the active site cysteine of an Ub- conjugating enzyme (E2) in a transthiolation reaction. Target protein ubiquitination can be achieved usually in conjunction with E3 ubiquitin-protein ligases, to regulate substrate specificity. After the initial ubiquitin is attached to an internal Lys residue of the substrate, additional ubiquitins are added sequentially forming a K48-linked poly-ubiquitin chain, which then targets the substrate for degradation by the 26S proteasome.

Ubiquitin-mediated protein degradation is involved in many cellular processes, including

ER-quality control of membrane proteins. In ER-quality control, terminally misfolded or misassembled membrane proteins are disposed of by a multi-faceted process termed ER- associated degradation (ERAD) (reviewed in [390,391]). To become accessible to proteasomes, targeted proteins are retrotranslocated to the cytosol with the involvement of chaperones and components of the protein translocon machinery [122,143,392]. Ubiquitination machinery specifically localized at the cytosolic face of the ER membrane then targets ERAD substrates for proteasomal degradation [125,169,198]. Accumulation of misfolded proteins in the ER, which can be induced in numerous physiological and pathological conditions, activates the unfolded protein response (UPR) signaling pathway (reviewed in [310]). Consequences of UPR activation in mammalian cells include transcriptional induction of UPR target genes, global translational repression and cell cycle arrest (reviewed in [393]). Genetic and biochemical studies in yeast have implicated the Ub-conjugating enzyme Ubc6 in the degradation of numerous ERAD target 51 proteins. These include mutant forms of the translocon pore protein Sec61 [125], vacuolar protein Cpy [123], plasma membrane protein uracil permease [385], as well as wild-type or mutant ABC transporters Ste6 [117] and Pdr5* [386]. Ubc6 is a tail-anchored ER membrane protein with a single transmembrane domain at its extreme C-terminus and an N-terminal catalytic domain oriented toward the cytoplasm [38]. Localization of Ubc6 to the ER membrane is a requirement for the proper turnover of its ERAD substrates [40,125].

In mammals, as in yeast, ERAD target proteins are generally degraded by components of the ubiquitin-proteasome pathway. Mammalian ERAD substrates known to be ubiquitinated prior to degradation include ApoB [394], HMG-CoA reductase mutants [131], unassembled T- cell receptors [140], and the cystic fibrosis transmembrane conductance regulator [167,168].

Recently, two highly conserved subfamilies of Ubc6-related proteins in vertebrates have been identified by sequence alignment [48]. Mammalian members of both subfamilies are bound to the cytosolic face of the ER membrane and are involved in mammalian ERAD [135,207-

210,395]. Their participation in the quality control of non-membrane proteins has also been suggested [387,388]. However, these homologs have yet to be characterized in detail and it is unknown how they are distinguished functionally. I investigated these homologs and have identified a phosphorylation modification of hsUbc6e that occurs in response to ER stress.

2.2 Experimental Procedures

2.2.1 Construction of hsUbc6 and hsUbc6e mammalian expression plasmids

52 The amino acid sequence of yeast Ubc6p (NP_011026) was used to identify human orthologs from the expressed sequence tag (EST) database division of GenBank using

TBLASTN [396]. cDNA clones of the two human homologs, hsUbc6 (HUGO gene name

UBE2J2) (IMAGE: 754549, AA411279, refseq NM_058167) and hsUbc6e (HUGO gene name

UBE2J1) (IMAGE: 2387149, AI798116, refseq NM_016336), were used as PCR templates for construction of pCMV expression plasmids using standard procedures [397]. For hsUbc6e, the

N-terminal myc epitope tag (EQKLISEEDL) was incorporated in frame immediately following the natural start codon with a single round of PCR using forward (5' - ATA AGA ATG CGG

CCG CCC ACC ATG GAA CAA AAA CTC ATC TCA GAA GAG GAT CTG GCT AGC

AGC ACC AGC AGT AAG) and reverse (5' - ATA GTT TAG CGG CCG CCC TGT GCT

GGG CAG TGT) oligonucleotides. The N-terminal myc-tagged version of hsUbc6e lacking the transmembrane domain (hsUbc6eATMD) was generated with the forward sequence primer above and the reverse oligonucleotide (5' - ATA GTT TAG CGG CCG CTT ACC CAC CAT

GAT CAG TGT G), leading to the introduction of the TAA stop codon immediately following amino acid G286, Fig. \A. For hsUbc6, the N-terminal myc epitope was incorporated using the forward (5' - ATA AGA ATG CGG CCC ACC ATG GAA CAA AAA CTC ATC TCA GAA

GAG GAT CTG GCT AGC AGC ACC AGC) and reverse (5' - ATA GTT TAG CGG CCG

CCC TGT GCT GGG CAG TGT GTC CAG) oligonucleotides.

The myc-tagged mutants of hsUbc6e S184A, S184D, S184E, T195A, S251A, S266A plasmid versions were generated using the Quikchange® Site-Directed Mutagenesis Kit

(Stratagene) with the following oligonucleotides: S184A (5' - GGC TAG GCA AAT AGC CTT

TAA GGC AGA AGT CAA TTC ATC TGG), S184D (5' - GAA CTG GCT AGG CAA ATA

GAC TTT AAG GCA GAA GTC AAT TC), S184E (5' - GAA CTG GCT AGG CAA ATA

GAG TTT AAG GCA GAA GTC AAT TC), T195A (5' - CAA TTC ATC TGG AAA GGC TAT CTC TGA GTC AGA CTT AAA CC), S251A (5' - GCT AAG AAT ACC TCC ATG 53 GCC CCT CGA CAG CGC, S266A (5' - GCA GAG TCA GAG AAG GTT GGC TAC TTC

ACC AGA TGT AAT CC). All PCR-generated and coding segments of expression vectors were verified by DNA sequencing.

2.2.2 Cell culture and transfections

HEK293, Panc-1 and SH-SY5Y cells were grown at 37 °C with 5% C02 in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and antibiotics.

Subconfluent HEK293 and Panc-1 cells were transfected according to the ExGen 500 in vitro transfection reagent protocol provided by the manufacturer (MBI Fermentas), using 10 \xg of expression plasmid per 100 mm plate. Cells were harvested 24 h later, or as indicated for individual experiments, perk'1' mutant cells [303] were kindly provided by D. Ron (Skirball

Institute), and were cultured as previously described [304], SH-SY5Y cells were transfected with Lipofectamine 2000 (Invitrogen), using 5 ug of expression plasmid per 60 mm plate.

2.2.3 Protein extract preparation, electrophoresis and immunoblotting

Transfected cells were washed with ice cold PBS and solubilized in RIP A buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.5% deoxycholate, protease inhibitor cocktail (Roche)) supplemented with phosphatase inhibitors (10 mM NaF, 10 mM (3- glycerophosphate, 10 mM sodium orthovanadate, 10 mM glucose-1-phosphate, 10 mM paranitrophenylphosphate), for 5 minutes on ice. Cell debris was removed by centrifugation at

15,000 x g for 15 min at 4 °C, and the supernatant protein extracts were used for immunoblot analysis or immunoprecipitation. Protein samples were separated by SDS-PAGE on 11.5%> gels

54 and transferred to Hybond-C super membranes (Amersham Biosciences). Immunoblot detection of myc-fusion proteins was performed using a 1:5000 dilution of monoclonal anti-myc antibody (clone 9E10; Oncogene). To detect endogenous hsUbc6e, rabbit polyclonal antibodies were raised against the peptide antigen AKNTSMSPRQRRAQQQS and affinity purified

(Washington Biotechnology, Inc.). (3-tubulin, GAPDH, and calnexin antibodies (Santa Cruz

Biotechnology, Inc.) and phospho-eIF-2a (Ser51) (Cell Signaling) were used according to manufacturers' protocols. Immunoreactive protein was detected using enhanced chemiluminescence of horseradish peroxidase-conjugated anti-mouse secondary antibody

(Amersham Biosciences) or anti-rabbit secondary antibody (Bio-Rad) with Hyperfilm™ ECL film (Amersham Biosciences).

2.2.4 Phosphatase assay

Protein extracts from 3xl00-mm plates transfected with hsUbc6e were collected and pooled. Immunoprecipitation of myc-tagged hsUbc6e was performed by incubating the lysates with a 1:100 dilution of anti-myc antibody for 1 h at 4°C, followed by incubation with lOOul of

50% Protein-G Sepharose slurry (Roche) for 4 h at 4°C. The beads were washed three times with ice cold RIPA (1ml) and aliquoted for reaction. Samples were incubated with 5 or 10 units of calf intestinal alkaline phosphatase (NEB) at 37°C in 100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2, 1 mM DTT, in a 30 ul reaction volume, with or without phosphatase inhibitors.

Reactions were stopped with addition of 3x Laemmli sample buffer, and products were separated by SDS-PAGE for immunoblot analysis.

2.2.5 UPR induction, RNA and protein analysis

55 Cultured Panc-1 cells were incubated with 10 ug/ml tunicamycin (Calbiochem) in

DMSO or DMSO alone. RNA was extracted using a QIAshredder™ Homogenizer and

RNeasy® mini kit (Qiagen) according to supplier's protocols. 10 ug of total RNA were resolved on agarose gels containing 1% formaldehyde, followed by blotting to nylon membranes (Hybond™-N, Amersham Pharmacia) and UV cross-linking. Following hybridization with P-labeled hsUbc6e cDNA or hsUbc6 cDNA fragments, membranes were washed and exposed to film. Membranes were then deprobed and sequentially rehybridized with

BiP/GRP78 (IMAGE 878587) and GAPDH cDNA probes [397].

To examine ER stress effects on phosphorylation, cells were incubated with 10 ug/ml tunicamycin (Calbiochem), 1.0 uM thapsigargin (Sigma-Aldrich) or 2.0 mM DTT (Sigma-

Aldrich). For UPR induction experiments with overexpressed hsUbc6e, Panc-1 cells were first transfected using 0.5 ug myc-hsUbc6e plasmid (mixed with pCMV vector to a total of 3 ug

DNA) per 60-mm dish, grown overnight, and then treated. Cell lysates were prepared and protein samples were separated by SDS-PAGE for detection of endogenous or myc-tagged hsUbc6e by immunoblot analysis, as above.

2.2.6 PERK in vitro kinase assay

First-strand cDNA from fetal liver total RNA (Stratagene) was synthesized by reverse transcription with oligo (dT)12-18 primer. Using total first-strand cDNA as template, the cDNA of PERK kinase domain (amino acid residues 589-1114) was generated by standard PCR procedures with the forward (5' - AAT AGA ATG CGG CCG CCC TCA CAG GCA AAG

GAA G) and reverse (5' - CCG CTC GAG CTA ATT GCT TGG CAA AGG) oligonucleotides.

56 The PCR-generated open reading frame segment was inserted into the pET-28a bacterial expression vector (Novagen) and verified by sequencing. Bacterial transformation, induction of protein expression and protein purification were carried out as described below. For the in vitro kinase assay, purified PERK kinase domain (2.5 uM) or mock (vector control), was pre- incubated with kinase buffer (50 mM Hepes, 10 mM MgCl2, 5 mM EDTA, 100 mM NaCl, 30 uM ATP) for 10 min at 30°C. Kinase reactions were initiated with addition of substrates, purified eIF-2a(100uM) (a gift from F. Sicheri) or His-Ubc6eATMD (50uM) (described below), and 2.0 uCi of [y- P]ATP, with incubation for 20 min at 30°C. Reactions were terminated with addition of Laemmli sample buffer and products were resolved by SDS-PAGE, followed by autoradiography. Purified PERK and Ubc6e proteins were detected by immunoblot analysis with 1:7500 dilution of monoclonal anti-T7 antibody (Novagen) and enhanced chemiluminescence using Hyperfilm™ ECL film (Amersham Biosciences) as above.

2.2.7 Metabolic labeling and immunoprecipitation

Transfected cells from 100-mm plates were split 24 h post-transfection onto 60-mm plates and grown for an additional 24 h. For [32P]-phosphate labeling, cells were incubated in phosphate-free DMEM (Cellgro) supplemented with 10% dialyzed FBS (Invitrogen) for 2 h at

37°C. The cells were then pulse-labeled by addition of 0.3 mCi/ml [32P]-phosphate for 2 h at

37°C, followed by a chase period of 2 h with addition of complete DMEM containing 10% FBS.

For metabolic labeling with [35S]-methionine and [35S]-cysteine, cells were incubated in methionine- and cysteine-free a-MEM for 30 min at 37°C. The cells were then pulse-labeled by addition of 140 uCi/ml [35S]-methionine and [35S]-cysteine (Pro-mix L-[35S] in vitro cell

57 labeling mix; Amersham Biosciences), 7 min, followed by chase periods with addition of complete DMEM containing 10% FBS.

Cells were collected and lysate volumes for immunoprecipitation were normalized for total labeled protein, as determined by scintillation counting of trichloracetic acid (TCA) precipitated material. Immunoprecipitation of labeled extracts was performed as described above and immunoreactive products were separated by SDS-PAGE. Gels were then fixed in

10% acetic acid and 40% ethanol, soaked for 30 minutes in Amplify (Amersham Biosciences), dried and exposed to film. Labeled proteins were visualized by exposure to Kodak BIOMAX

MR film for 1-3 days.

2.2.8 Membrane isolation

Transfected HEK293 cells were washed with PBS and scraped into ice cold suspension buffer (0.25 M sucrose, 10 mM triethanolamine, 1 mM EDTA, pH 7.4) supplemented with protease inhibitor cocktail and phosphatase inhibitors. The cell suspension was passed 15 times through a 27-gauge needle and then centrifuged at 1000 x g for 5 minutes at 4°C. The clarified extract was then centrifuged at 100,000 x g for 1 h at 4°C. The supernatant was collected and subjected to TCA precipitation. Precipitated materials and the 100,000 x g pellet were resuspended in an equal volume of RIP A buffer and then analysed by immunoblot.

2.2.9 Immunofluorescence

COS-7 cells transfected with expression vectors were split 24 h post-transfection and cultured for an additional 24 h in 4-chamber slides (BD Falcon). The cells were fixed and permeabilized in 4% paraformaldehyde and 0.1% Triton X-100 treatments for 30 min durations 58 at room temperature. Following blocking with 1% BSA in phosphate-buffered saline (PBS-

BSA) for 30 min, cells were incubated with anti-Grp94 antibody (Santa Cruz Biotechnology,

Inc.), diluted 1:1000 in PBS-BSA, for 1 h at room temperature. Cells were washed twice with

PBS-BSA and incubated with 1:1000 anti-goat rhodamine-conjugated secondary antibody

(Jackson ImmunoResearch Laboratory, Inc) for 1 h at room temperature. Cells were again washed and incubated with anti-myc antibody, diluted 1:1000, for 1 h, then washed again and incubated with 1:300 anti-mouse FITC-conjugated secondary antibody (Jackson

ImmunoResearch Laboratory, Inc) for 1 h at room temperature. Following antibody treatments, cells were washed twice with PBS-BSA, once with PBS, and mounted with Vectashield mounting medium (Vector Laboratories). Indirect immunofluorescence was examined with a

Nikon El000 fluorescence microscope. Images were captured using a CCD camera and

OpenLab Spectrum Software (Scanalytics).

2.2.10 Cloning of Parkin and Pael-R cDNA

First-strand cDNA from human fetal brain total RNA (Clontech) was synthesized by reverse transcription with oligo (dT)12-18 primer. Using total first-strand cDNA as template, open reading frame cDNA of Parkin was generated by standard PCR procedures with the forward (5' - ATA GTG TTT GTC AGG TTC) and reverse (5' - CTA CAC GTC GAA CCA

GTG) oligonucleotides. Open reading frame cDNA of Pael-receptor (Pael-R) was generated with two overlapping PCR products using the forward (5' - GCA GCA GGT GTC GAT CCT A

-3') and reverse (5' - TGA TGA GAA AGT CCC AGA AGG - 3'), and the forward (5' -

GGA TTC TTG GGT GAA GGA AT - 3') and reverse (5' - CAA GTA CTGTCC TTC AGC A

-3') oligonucleotides. The full length open reading frame of Pael-R was then generated by overlap extension of the two PCR fragments. 59 Open reading frame cDNAs were used as PCR templates for construction of pCMV expression vectors. N-terminal FLAG-tagged Parkin with start site Kozak consensus sequence was generated with the forward (5' - ATA AGA ATG CGG CCG CCC ACC ATG GAT TAC

AAG GAT GAC GAC GAT AAG GCT ATA GTG TTT GTC AGG TTG - 3') and reverse (5'

- ATA GTT TAG CGG CCG CCT ACA CGT CGA ACC AGT G - 3') oligonucleotides. C- terminal hemagglutinin (HA)-tagged Pael-R with start site Kozak consensus sequence was generated with the forward (5' - ATA AGA ATG CGG CCG CCC ACC ATG CGA GCC CCG

GGC GCG CTT - 3') and reverse (5' - ATA GTT TAG CGG CCG CTC ATG CAT AGT CCG

GTA CAT CGT ATG GGT AAG CGC AAT GAG TTC CGA CA) oligonucleotides. All PCR- generated segments and reading frames of expression vectors were verified by DNA sequencing.

2.2.11 Co-immunoprecipitation of Parkin and hsUbc6e

Protein extracts were prepared from transfected HEK293 in lysis buffer (50 mM Tris-

HC1, 150 mM NaCl, 1% Triton X-100, 5 mM EDTA, ImM DTT, protease inhibitor cocktail

(Roche)) supplemented with phosphatase inhibitors. Immunoprecipitation of FLAG-tagged

Parkin was performed by incubating the lysates with a 1:1000 dilution of anti-FLAG antibody

(Sigma) for 1 h at 4 °C, followed by incubation with 40ul of 50% Protein-G Sepharose slurry

(Roche) for 4 h at 4 °C. The beads were washed three times with ice cold lysis buffer (1ml).

Immunoprecipitated products were separated by SDS-PAGE for immunoblot analysis.

2.2.12 Pulse-chase analysis of Pael-R

60 SH-SY5Y cells were cultured in 60-mm plates and transfected with various expression vectors using Lipofectamine 2000 (Invitrogen) according to manufacturer's directions. 24 h post-transfection, cells were pulse-labeled with [35S]-methionine and [35S]-cysteine (as described above) for 30 min. Cells were collected in lysis buffer (50 mM Tris-HCl, 150 mM

NaCl, 1% Triton X-100, 5 mM EDTA, ImM DTT, protease inhibitor cocktail (Roche)) supplemented with phosphatase inhibitors. Immunoprecipitation of labeled HA-tagged Pael-R was performed using 1:1000 dilution of anti-HA antibody (Covance) for 1 h at 4 °C, followed by incubation with 40ul of 50% Protein-G Sepharose slurry (Roche) for 4 h at 4 °C. The beads were washed five times with ice cold lysis buffer (1ml) and immunoprecipitated products were separated by SDS-PAGE. Gels were then fixed, dried and exposed to film as described above.

The radioactivity associated with the immunoprecipitated Pael-R bands was quantified using a

Phosphorlmager with ImageQuant software (Molecular Dynamics).

2.2.13 Recombinant expression and purification of hsUbc6e, hsUbc6esl84D and hsUbc6esl84E

Bacterial expression vectors encoding 6xHis-tag fused hsUbc6eATMD and variants were generated by PCR with forward (5' - CGC GGA TCC ATG GAG ACC CGC TAC AAC CTG) and reverse (5' - CCG CTC GAG TTA CCC ACC ATG ATC AGT GTG) oligonucleotides, leading to the introduction of the TAA stop codon following amino acid G286 (Fig. 1A). To generate S184D and S184E variants, the corresponding full length pCMV expression vectors were used as templates in PCR. The transmembrane coding regions were excluded to promote solubility of the fusion proteins. PCR-generated open reading frame segments were verified by sequencing after insertion into the pET-28a expression vector (Novagen).

61 E. coli cells (BL21DE3) transformed with expression plasmids were induced with 0.2 mM isopropyl-p-thiogalactopyranoside for 4 h at 37°C and collected and lysed with B-PER®

Bacterial Protein Extraction Reagent according to manufacturer's protocol (Pierce). Expressed proteins were purified by affinity chromatography using Ni-NTA His-Bind® Resin (Novagen) according to manufacturer's protocol.

2.2.14 Yeast-two hybrid screening

S184A and S184E (ATMD)-DNA binding domain fusion yeast expression vectors were generated and transformed into the appropriate yeast strain. Expression of fusion proteins was confirmed by western blot and absence of selection marker gene autoactivation was also confirmed. A human liver Gal4 activation domain cDNA library (BD Biosciences Clontech) was screened following manufacturer's instructions. Approximately 5 x 106 clones were screened. In mating experiments, yeast carrying Gal4-activation domain and Gal4-DNA binding domain plasmids were grown overnight together in yeast extract-peptone-dextrose-adenine.

Diploid yeasts were selected on synthetic dropout (SD)/-Leu/-Trp plates and subsequently streaked onto SD/-Leu/-Trp/-His/-Ade/+5-bromo-4-chloro-3-indolyl-B-D-galactopyranoside (X- a-Gal) plates to detect interacting proteins. cDNA plasmids of positive clones were isolated (BD

Biosciences Yeastmaker Yeast Plasmid Isolation Kit) and sequenced for identification.

2.2.15 Thiol-ester assay

Thiol-ester E2 complex formation with ubiquitin was measured for hsUbc6e and the

S184D and S184E variants in a two-step assay. Ubiquitin activating enzyme, El, was pre-

62 incubated as follows: 0.3 uM rabbit El (Boston Biochem), 1 mM ubiquitin (Sigma, U6253), 2 mM ATP, 20 mM Tris, pH 7.5, 50 mM NaCl, 15mM MgCl2 at 37°C for 10 min. After cooling to 7°C, hsUbc6e was added to a final concentration of 0.2 uM and incubation was continued as indicated. Reactions were stopped with Laemmli sample buffer and products were resolved by

SDS-PAGE, followed by immunodetection with a 1:7500 dilution of monoclonal anti-T7 antibody (Novagen) and enhanced chemiluminescence using Hyperfilm™ ECL film

(Amersham Biosciences). Purified hsUbc6e was used as a standard for densitometry measurement (FluorChem 9900, Alpha Innotech).

2.3 Results

2.3.1 HsUbc6 and hsUbc6e sequence and expression analysis and confirmation of E2 activity by thiol-ester assay

Human homologs display high sequence conservation with yeast Ubc6 in their catalytic cores, with 64% and 46% identity for hsUbc6 and hsUbc6e, respectively, and their catalytic cysteine positions are invariant (Fig. 2.1 A). As well, both homologs possess hydrophobic C- terminal transmembrane domains and lack N-terminal signal sequences indicating that they are tail-anchored proteins. However, with the exception of the transmembrane domain, the sequences in regions C-terminal to the catalytic core have been poorly conserved. Both Ubc6 and Ubc6e subfamilies are represented in all eukaryotic kingdoms, including fungi, protists, plant and metazoa with Ubc6e subfamily members exhibiting divergent and longer C-terminal segments, as illustrated with hsUbc6e and in Figure A. 1.1.

63 Figure 2.1 Human Ubc6 and Ubc6e are active E2 enzymes

scUbc6 rfjTKi hsUbc6 MSSTSSKR0PTTSTi hsUbc6e --METRYMLKS

139 146 IEGAHIILDYTPEEI BDFCCEGCGSAMKDV 159

scUbc6 ISYfjiTFQNVRraKLIG EDAEHJGDETEjBPFTKAAKEK 193 hsUbc6 IfiFJS-LJaDKVraCEI IKQ- -KQKA333 LSSRPQTLPjjPJHWPDGETHI. 197 hsUbc6e LLPLKSGSDSSQSDQEAJgELARQIS SGKBlSESDa5HSFSLTDLQDDIPTTFQEATESBSyGf£QNSSAASFHQP 240

SCUbc6 ^SLEEIj^--@EDBIRAEl«iAj«agSEimSKKDEKEPNDSSSt«YlElBiraLFI^GffFMK 250 hsUbc6 fflMGIOlfllNGHAPGAVPNLAglBBlANRHHGLllmALAN--LFBlVEFgaBAYTBKYVilRSIAOE 259 hsUbc6e ^PVAKMTSMSRR0gRAO(^SOfpLSTSPDVI0^0PRDNHT5HGFS^LIVILTiiAfllAALIFRRIYLAMEYIFDFEL 318

B T R hsUbc6e hsUbc6 XSXK PKC C91S C94S ATP - + + + + CaMKII hsObc6e 176 inmUbc6e 176 47.5- •«. ggObc6e 176 xtUbc6e 159 «•» *---

A, Comparison of scUbc6, hsUbc6 and hsUbc6e amino acid sequences by CLUSTALW alignment. The conserved catalytic cores are boxed and the invariant active-site cysteine positions are indicated with an asterisk. C-terminal transmembrane domains are underlined.

Putative hsUbc6e phosphorylation sites, predicted by NetPhos [398] and Scansite [399], are indicated by arrowheads. S. cerevisiae (sc); H. sapiens (hs).

B, HsUbc6eATMD and hsUbc6ATMD are active E2s by their ability to accept ubiquitin from

El in non-reducing conditions. The in vitro thiol-ester assay demonstrated E2-ubiquitin thiol- ester bond formation that is dependent on ATP and the catalytic cysteines as predicted by alignment.

C, Alignment of sequence surrounding Serl84 in Ubc6e subfamily members. Serl84 and flanking predicted CaMKII and PKC consensus sequences are conserved in H. sapiens (hs), M. musculus (mm), G. gallus (gg), X. tropicalis (xt) but not in D. rerio (dd) or C. elegans (ce).

64 Mammalian Ubc6e and Ubc6 are broadly expressed, according to the expressed sequence tag (EST) data of GenBank. Additionally, it was confirmed that expression of Ubc6 and Ubc6e was abundant and similar under non-stress conditions in various human cell lines

(Fig. A. 1.2).

To demonstrate that the homologs are bona fide E2s, their activity was assessed by in vitro thiol-ester assay. As recombinant proteins, hsUbc6e and hsUbc6 accept ubiquitin from El in an ATP-dependent manner and with involvement of their catalytic cysteines (Fig. 2. IB).

2.3.2 HsUbc6 and hsUbc6e are upregulated with distinct responses during UPR

Given their predicted role in the ERAD of misfolded proteins, I examined whether the

Ubc6 homologs are up-regulated by the unfolded protein response (UPR) in Panc-1 secretory cells. Cells were treated with tunicamycin, a potent inhibitor of N-linked glycosylation, and then subjected to mRNA analysis (Fig. 2.2). The ER chaperone Grp78 (also known as BiP) showed sustained increased expression confirming that UPR had been invoked. HsUbc6e showed strong induction with a 7-fold increase in expression following 24 h of tunicamycin treatment (average of two trials). HsUbc6 was also induced significantly; both -1.3 kb and ~2.3 kb messages showed 2.5 fold increases following 8 hours of treatment (average of two trials).

Peak inductions were distinct; hsUbc6e expression increased slowly and was sustained at 24 h, very much like Grp78, whereas hsUbc6 expression increased rapidly, peaking at ~8 h and then dissipated even with continued drug treatment. Overall, up-regulation of both genes is only observed later during ER stress (> 4 h of treatment), typical of other ERAD components [287].

2.3.3 HsUbc6e is phosphorylated in response to ER stress

65 Figure 2.2 HsUbc6 and hsUbc6e are induced by UPR

tunicamycin DMSO tunicamycin DMSO

treatment (h): 0 4 8 24 24 0 4 8 24 24

28S" -^. **"#«* --•#

hsUbc6e hsUbc6

Grp78

Bl GAPDH

Panc-1 cells were treated with tunicamycin (10 fig/ml in DMSO) or DMSO for the times indicated leading to increased hsUbc6 and hsUbc6e mRNA expression. Total RNA blots were probed with hsUbc6e and hsUbc6 cDNAs. Representative results are shown. The two messages observed for hsUbc6 are consistent with different polyadenylation signals as evident from two major classes of clones in dbEST of GenBank. Grp78 was used as a control for UPR induction and GAPDH was used as the RNA quantitation control.

66 To characterize the human Ubc6 homologs, N-terminal c-myc epitope fusion plasmids were constructed for transient expression in HEK293 cells. Immunoblotting of whole cell lysates using anti-myc antibody identified protein products consistent with calculated sizes (Fig.

2.3A). Interestingly, a prominent doublet was observed for hsUbc6e. Sensitivity to phosphatase treatment indicated that the upper band (b-form) likely represented a phosphorylated form of the lower band (a-form) (Fig. 2.3B). To confirm that the endogenous hsUbc6e is also susceptible to phosphorylation, I raised polyclonal antibodies against a peptide sequence in the less conserved carboxyl-terminal region. Specificity of the antibody was confirmed by the detection of a band in whole cell extracts that co-migrated with the over-expressed hsUbc6e (Fig. 2.3C), and the absence of immunoreactivity with the pre-immune serum (data not shown). Endogenous hsUbc6e in unstressed Panc-1 cells was detected as a single band; however when protein expression was induced by tunicamycin treatment, the upper band appeared. This upper form was confirmed to be phosphorylated by phosphatase treatment (Fig. 2.3D). With short durations of drug treatment, it was evident tunicamycin induced the phosphorylation of endogenous hsUbc6e, indicating that this modification is ER stress regulated (Fig. 2.3E). This stress induced modification was also observed in Caco-2, HEK293, and COS-7 cell lines (Fig. A. 1.3).

Phosphorylation was also induced by other known activators of ER stress: thapsigargin which promotes release of intracellular Ca2+ stores, and DTT, a reducing agent that causes protein unfolding in the ER. Tunicamycin elicited a modest and slow increase in phosphorylated hsUbc6e while thapsigargin and DTT induced stronger and more rapid responses. In tunicamycin and thapsigargin treated cells, phosphorylated hsUbc6e was still present after 8 h of incubation, accounting for -15 and -25% of total protein, respectively, as measured by densitometry. A known responder to ER stress is translation initiation factor eIF-2a whose phosphorylation during UPR results in translation attenuation [400]. By comparison, strong elF-

67 Figure 2.3 HsUbc6e is phosphorylated during ER stress

B incubation alkaline phosphatase s yy control kDa 5U 10U Pi 83- incubation (h): 0 0.5 2 0.5 0.5 2 47.5- 62- _^. - hsUbc6eb ^•-hsllbc6ea 47.5- 32.5-*"* ^*^" -hsUbc6eb • hsUbc6ea 32.5- -hsUbc6 25-

<£• tunicamycin DMSO j& alkaline phosphatase Oh 24h 24h

47.5 47.5 - hsUbc6eb • hsUbc6eb •hsUbc6ea 32.5- • hsUbc6ea 32.5-

25-

p-tubulin

tunicamycin thapsigargin DTT

(h): 0 12 4 8 0 12 4 8 12 4 8 47.5- - hsUbcSeb • - hsUbceea 32.5-

elF-2a~p

• p-tubulin

A, Expression of myc-tagged hsUbc6e and hsUbc6 in transiently transfected HEK293 cells. The anti-myc antibody detected two bands for hsUbc6e, referred to as forms a and b.

B, Myc-tagged hsUbc6e immunoprecipitated from HEK293 transfection lysates were treated with alkaline phosphatase (5 or 10 units) for the indicated times. The b-form decreased with incubation but could be sustained in presence of phosphatase inhibitors (pi).

68 C, Detection of endogenous hsUbc6e in tunicamycin treated Panc-1 cells using polyclonal antibodies. Panc-1 cells were treated for the times indicated with either tunicamycin (10 |xg/ml) or DMSO. Control lane, Panc-1 cell transfected with myc-tagged hsUbc6e.

D, Endogenous hsUbc6e is phosphorylated. Lysates of tunicamycin treated Panc-1 cells were treated with alkaline phosphatase (10 units) for 30 min. The b-form was sustained in presence of phosphatase inhibitors. Control lane, untreated.

E, Endogenous hsUbc6e is phosphorylated during ER stress, upper panel. The phospho-specific antibody to eIF-2a confirms the stress response was elicited, middle panel. P-tubulin was used as loading control, lower panel. Panc-1 cells were treated with tunicamycin (10 fig/ml), thapsigargin (1.0 uM), and DTT (2.0 mM) for up to 8 h.

69 2a phosphorylation occurs only during early UPR [401]; hsUbc6e phosphorylation was temporally different, being maintained throughout tunicamycin and thapsigargin induced ER stress (Fig. 2.3E). Interestingly, DTT treatment elicited a distinct response with a peak amount of phosphorylated hsUbc6e at 1-2 h, accounting for up to ~25% of total protein, with subsequent decrease. The same response was also observed at lower DTT levels (0.5 mM, data not shown).

The decrease in the level of phosphorylated hsUbc6e was not a result of DTT clearance, as hsUbc6e and ER chaperone Grp94 protein levels continue to increase up to at least 24 h confirming continued ER stress (data not shown). The observed differences in phosphorylation induced by the different ER stressors likely reflect the distinct nature of each applied stress and the cellular responses elicited. Together, these data demonstrate that hsUbc6e is phosphorylated during the ER stress response in a manner dependent on the nature of the stressor.

2.3.4 ER stress-induced phosphorylation of mmUbc6e requires PERK signaling

It was considered that hsUbc6e may be a target of PERK, an ER-membrane localized serine/threonine protein kinase that inhibits global protein synthesis via eIF-2a phosphorylation during ER stress [277,402]. To examine the involvement of PERK in murine Ubc6e phosphorylation, wild-type and perk'1" mouse embryonic fibroblasts (MEFS) were stressed with thapsigargin (1.0 uM) and DTT (2.0 mM) (Fig. 2.4A). In wild-type cells, increased phosphorylation of endogenous mmUbc6e occurred within 1 h of thapsigargin or DTT treatment, from -8% to -25% of total protein (Fig. 2.4B). In perk'1' cells, no significant change was observed. Further, while eIF-2a showed modest levels of phosphorylation with sustained stressor treatment for 6 h in perk'1' cells, likely due to activities of other stress responsive eIF-2a kinases [403], mmUbc6e did not. Induction of the ER chaperone, Grp78, confirmed that wild-

70 Figure 2.4 ER stress induced phosphorylation of mmUbc6e requires PERK signaling

TG DTT TG DTT

(h): 0 16 16 0 16 16

wild-type perlc/-

B r^ no treatment • thapsigargin mock PERK •1 DTT elF2a Ubc6e elF2a Ubc6e 35i 0 47.5 CO o 30- £1 32.5 => 25" 32 E r E 20- 25 •a a> 15- ID­ I f Ubc6e S' PERK 0' wild-type perlcl

Wild-type W4 and perk''' mouse embryonic fibroblasts (MEFS) were treated with thapsigargin

(1.0 uM) or DTT (2.0 mM) for up to 6 h.

A, Representative data from three trials are shown. Endogenous mmUbc6e was detected by immunoblot, upper panel. Grp78 and phospho-specific eIF-2a antibodies confirm the stress response was elicited, middle panels. GAPDH was used as loading control, lower panel.

B, The proportions of mmUbc6e that are phosphorylated at 1 h with or without treatment with thapsigargin or DTT in wild-type versus perk7" MEFS were calculated by densitometry and

71 differed significantly (p=0.0208, TG; p=0.0081, DTT). Results are expressed as the means ±

S.E. for three independent trials.

C, Recombinant purified proteins, hsUbc6eATMD and eIF-2cc, were incubated with the kinase domain of PERK, or vector control (mock), in the presence of radio-labeled ATP. Reaction products were resolved by SDS-PAGE and visualized by autoradiography revealing phosphorylation of the well known substrate, eIF-2a, but not of hsUbc6eATMD (upper panel).

Purified PERK and hsUbc6eATMD proteins were readily detected by T7 antibody (lower panels).

72 type and perk" MEFS were under ER stress. To test whether PERK may be directly phosphorylating Ubc6e, in vitro kinase assays were performed; however recombinant purified

PERK displayed no detectable activity towards hsUbc6e (Fig. 2.4C) under the conditions used.

This, together with the observation of low amounts of phosphorylated mmUbc6e in perk'1' cells

(Fig. 2.4B), suggests that, while PERK may not be directly responsible, Ubc6e phosphorylation requires PERK signaling during ER stress.

2.3.5 HsUbc6e is phosphorylated at Ser-184

HsUbc6e protein sequence analysis identified several putative phosphorylation sites

(Fig. 2.1 A), of which S184, T195, S251, S266 were selected as candidate sites based on sequence conservation with the mouse ortholog. Expressed versions with individual mutagenized sites then revealed that the S184A mutant showed complete absence of the b-form, while the other mutants retained their doublet patterns (Fig. 2.5A). To confirm that the b-form results from phosphorylation of Serl84 alone, metabolic [32P]-phosphate labeling of wild-type and S184A hsUbc6e was performed in transiently transfected HEK 293 cells (Fig. 2.5B). By

^9 immunoprecipitation and SDS-PAGE, the [ P]-phosphate labeled b-form of wild-type hsUbc6e was readily detected. A corresponding band was not detected with the S184A mutant and only very minor alternate labeled species were observed. Thus, the b-form of hsUbc6e, migrating as a major discrete band as detected by immunoblot (Fig. 2.5B, lower panel), is the SI84 phosphorylated form of hsUbc6e. To establish that the UPR-regulated phosphorylation of hsUbc6e involved SI84, Panc-1 cells were transfected with wild-type and SI84A hsUbc6e, and then treated with tunicamycin, thapsigargin or DTT. Immunoblots show that the amount of b- form increased during treatment with various ER stress inducers while the S184A mutant remained unchanged. 73 Figure 2.5 HsUbc6e is phosphorylated at residue Serl 84

47.5- a-myc «••» - hsUbc6eb S» «• - hs(Jbc6ea c wild-type S184A treatment: TM - TG DTT - TM TG DTT 47.5 -

A, Western detection of myc-tagged hsUbc6e, wild-type and T159A, S184A, S266A and S251A predicted phosphorylation site mutants in transiently transfected HEK293 cells. Only the S184A mutant showed an altered immunoreactive pattern.

B, Metabolic orthophosphate labeling of HEK293 cells transfected with wild-type and S184A hsUbc6e. The arrowhead indicates the Ser-184 phosphorylated form of hsUbc6e, corresponding to b-form identified by the anti-myc antibody shown in the lower panel.

C, Myc-tagged hsUbc6e is phosphorylated during ER stress at Ser-184. Transfected Panc-1 cells were treated with tunicamycin (8 h), thapsigargin (2 h) or DTT (1 h).

74 Residue Serl84 is located between the catalytic core domain and the transmembrane domain, within a highly charged segment. The serine and flanking predicted kinase consensus sequences are completely conserved within mammalian members of the Ubc6e subfamily, including M. musculus and G. gallus as well as with amphibian X. tropicalis, but not with lower vertebrates, D. rerio, or invertebrates, C. elegans (Fig. 2.1C), or with Ubc6 subfamily members.

2.3.6 Phosphorylated hsUbc6e is stable and localized to the ER membrane

I investigated the stability of hsUbc6e by metabolic [35S]-methionine and -cysteine pulse-chase labeling in HEK 293 cells transfected with wild-type hsUbc6e (Fig. 2.6A).

Following a short pulse, the phosphorylated form appeared early, being readily evident at the earliest chase periods (30 min or less), and by -12 h accounted for as much as -40% of the labeled protein (phosphorimage analysis). The observed constitutive phosphorylation of Ubc6e did not appear to be related to ER stress, as Grp78 expression and phosphorylated eIF-2a protein levels remain unchanged with Ubc6e over-expression (Fig. 3.5B and Fig. A.2.2A).

Overall, both forms were remarkably stable with the b-form persisting with a half-life equal to the a-form (ty2 - 20h), indicating that phosphorylation did not significantly affect the stability of hsUbc6e.

I also examined whether phosphorylation regulated or modified the ER membrane localization of hsUbc6e. Like the yeast ortholog, hsUbc6e has no known ER- retention/retrieval signal and a basis for ER retention is not completely understood, although the hydrophobic tail- segment of the yeast ortholog is thought to contain some targeting information [404]. Isolation of crude membrane fractions revealed that both full-length phosphorylated and unphosphorylated forms of hsUbc6e are membrane-associated (Fig. 2.6B). Consistent with the involvement of stress signaling at the ER membrane, it was also evident that phosphorylation 75 Figure 2.6 Phosphorylated hsUbc6e is stable and membrane associated

chase (h): 0 0.5 6 12 24 48

47.5- hsUbcgeb hsUbcoea 32.5-

B Sup Pellet c wt 4^ *N

hsUbcgeb hsUbcgeb 47.5- •hsUbcoea hsUbcoea •ATMD

32.5-

» calnexin

A, Phosphorylated hsUbc6e appears soon after translation and is long-lived. HEK293 cells transiently transfected with myc-tagged hsUbc6e were pulse-labeled with [35S]-methionine and

[ S]-cysteine (10 min) and chased as indicated.

B, Fractionated cell preparations from HEK293 cells transiently transfected with myc-tagged hsUbc6e reveal that both a and b forms are localized to membranes (pellet). The ER membrane marker calnexin was used as control for fraction preparation. Sup, supernatant.

C, Phosphorylation of hsUbc6e requires membrane localization. Transiently expressed myc- tagged hsUbc6eATMD is not modified, comparable to S184AATMD, as detected by immunoblot.

76 requires hsUbc6e membrane localization, as a truncated version lacking the transmembrane domain (ATMD, deletion of amino acid residues 287-318) was not modified, as detected by immunoblot (Fig. 2.6C). Immunofluorescence was then used to evaluate whether phosphorylation of hsUbc6e functioned as a targeting determinant. By indirect immunofluorescence of transiently transfected COS-7 cells, myc-tagged hsUbc6e with an intact carboxyl transmembrane domain displayed dense perinuclear and extended lace-patterned staining characteristic of ER membrane localization and strongly co-localized with the ER marker protein, Grp94 (Fig. 2.7B). In contrast, hsUbc6eATMD exhibited diffuse staining throughout the entire cell (Fig. 2.7A). Single site mutants S184D and S184E, which possess negatively charged amino acid side chains at position 184, were generated to assess the effect of phosphorylation on hsUbc6e localization. These phosphorylation mimicking variants localized similarly to wild-type, also displaying an ER- patterned staining (center and right panel, Fig.

2.7C). Moreover, S184A, which cannot be phosphorylated, also displayed similar ER-patterned staining (left panel, Fig. 2.7C), further demonstrating that phosphorylation has no direct role in determining hsUbc6e ER localization. The homologue, hsUbc6, was found to have similar ER- patterned staining (Fig. A. 1.4).

2.3.7 HsUbc6e phosphorylation has no effect on Parkin E3 interaction and degradation ofPael-R

I next examined whether phosphorylation of hsUbc6e could modulate interaction with a reported partner E3 or influence turnover of an ERAD substrate. Interaction between an E2 and

E3 is known to be directly mediated by E2 phosphorylation, thereby enhancing substrate degradation [36]. E3 ubiquitin-ligase Parkin can directly interact with hsUbc6e alone, and

77 Figure 2.7 Phosphorylation does not alter hsUbc6e subcellular localization

Representative images of COS-7 cells transfected with myc-tagged A, wild-type hsUbc6eATMD, B (left panel), wild-type hsUbc6e, and various mutants as indicated, C. Indirect immunofluorescence indicated that ER localization required the transmembrane domain but not phosphorylation. The endogenous Grp94 marker and its merged image with hsUbc6e confirmed the ER localization as shown in the middle and right panels of B.

78 together with hsUbc7 has been shown to ubiquitinate the Pael receptor (Pael-R), in vitro [135].

This receptor is a transmembrane protein prone to aggregation causing ER stress in a neuronal cell model [135]. With transiently transfected HEK293 cells, both phosphorylated and unphosphorylated forms co-immunoprecipitate with Parkin (Fig. 2.8A), indicating that phosphorylation does not modify this interaction. Next, I examined whether phosphorylation altered hsUbc6e E2 activity on Pael-R. In transiently transfected SH-SY5Y neuroblastoma cells, the turnover of metabolically labeled Pael-R was measured with co-expression of wild-type hsUbc6e and phosphorylation mimicking variants in pulse-chase experiments (Fig. 2.8B).

However, it was apparent that hsUbc6e variants had no effect on Pael-R half-life (Fig. 2.8C). In fact, the unaltered half-life with the C91S variant indicated that other E2s play more prominent roles in vivo, despite the ability of hsUbc6e to bind Parkin.

2.3.8 Identifying putative phospho-regulated protein interactions by yeast two-hybrid screen

The C-terminal extension where the phosphosite is located may be involved in mediating an E3 interaction, as other E2s utilize their respective C-terminal extensions in such a manner

[32,34,405]. Although I have seen that phosphorylation does not affect Ubc6e interaction with a known E3, Parkin, it may regulate interaction with other E3s or ERAD factors present at the ER membrane (for example [188,215]). To assess these possibilities, I used hsUbc6e S184A and

S184E variants, which contain the Gal4 DNA binding domain fused in frame to the N-terminus of Ubc6e, as bait in a yeast-two hybrid screen of a human liver cDNA library, as an unbiased approach to identify proteins interacting with hsUbc6e. This approach has previously been used to uncover E2-E3 interactions [34,406,407]. I obtained multiple putative interactor clones from both SI 84 A and S184E screenings, some of which encoded El ubiquitin-activating 79 Figure 2.8 HsUbc6e phosphorylation has no effect on Parkin E3 interaction and degradation ofPael-R

B |T .# # y ^ 4* & & chase (min): 15 30 60 120 Flag-Parkin + + + pCMV 175- MM 83- Wt

62- **$#*&<$& *"& - S184E 47.5- hsUbc6eb hsUbc6ea 32.5-

25- wMNI 601 _50. a-Flag ip, a-hsUbc6e ib c 1.40 62- .9? *? 30 47.5- - Parkin ^ •c 20 - hsUbc6eb 32.5- -hsUbc6ea 10 wee, a-Flag + a-myc # *>*^«&\^cf * 83-

a-Flag ip, a-Flag ib

A, Phosphorylated and unphosphorylated hsUbc6e co-immunoprecipitate with Parkin. HEK293 cells were transiently transfected with FLAG-tagged Parkin and wild-type or SI84A myc- tagged hsUbc6e. Anti-FLAG immunoprecipitates from cells expressing Parkin and hsUbc6e contain both phosphorylated and unphosphorylated Ubc6e, as detected by oc-hsUbc6e immvmoblot.

80 B, Pulse-chase analysis of Pael-R in HEK293 cells transiently transfected with HA-tagged Pael-

R and myc-tagged hsUbc6e, wild-type or S184E, or vector control, pCMV. Cells were pulse- labeled and chased as indicated.

C, Pulse-chase results for Pael-R half-life (min) with co-transfection of pCMV, myc-tagged hsUbc6e, wild-type, C91S, S184A or S184E, are presented as means ± S.E. for 3 independent trials.

81 enzyme polypeptide fragments (Table A.l). El is expected to physically interact with E2 enzymes, and therefore provided positive confirmation of the technical aspects of the procedures used. Unfortunately, no clones containing putative or known E3 or ERAD component encoding sequences were detected. As well, the positive hits returned (Table A.l) were considered not interesting or significant as the encoded proteins were not ER-localized (e.g. MTA2) or displayed no difference in interaction strength between S184A or S184E screenings (e.g.

MTA2, El), or were especially weak interactors (e.g. eukaryotic translation elongation factor 1 alpha).

2.3.9 Phosphorylation affects formation of ubiquitin-hsUbc6e thiol-esters

A thiol-ester assay was employed to examine whether phosphorylation of hsUbc6e alters enzymatic activity as measured by E2 bond formation with ubiquitin. Single end-point trials revealed the capacity of both recombinant wild-type and phosphomimicking variants lacking their transmembrane domains to form thiol-ester complexes by accepting ubiquitin from El in the presence of ATP (results not shown). To detect subtle differences in enzymatic activity, I examined the rate of ubiquitin-enzyme formation over a short time course and at reduced temperature. As only the transfer of ubiquitin from El to the ubiquitin-conjugating enzyme was being measured, I first pre-incubated El with ubiquitin and ATP to generate El-ubiquitin thiol- ester intermediates. This pre-incubation step was also necessary because the initial formation of

El-ubiquitin thiol-ester intermediate was found to be rate-limiting under the reaction conditions

(results not shown). Purified wild-type, S184D and S184E hsUbc6e were incubated for short periods with the pre-formed El-ubiquitin complexes. At early time points (1,2 min), reaction products of the phosphorylation mimicking mutants S184D and S184E exhibited reduced ubiquitin-E2 formation compared to wild-type (Fig. 2.9). Under identical conditions, the 82 Figure 2.9 HsUbc6e mutants S184D and S184E that mimic phosphorylation show reduced thiol-ester bond formation

wild-type S184D S184E min: 0 12481248 1248 — — - E2-Ub - E2

B 1 min 0.75n 2 min

•o £ 0.50' > c o o J 0.25' o IS 0.00' a .& V & a$> V *? *

Recombinant wild-type, S184D, and S184E hsUbc6eATMD proteins were incubated with excess activated El protein. Transfer of ubiquitin to recombinant E2 proteins was then monitored by immunoreaction with T7 antibody and enhanced chemiluminescence. The linear range of the film was determined and a standard curve was used to correlate densitometry values to arbitrary units, from which converted fractions were calculated.

A, A representative immunoblot of thiol-ester bond formation between wild-type, S184D, or

S184E hsUbc6e and ubiquitin.

83 B, The fractions of converted hsUbc6e were calculated at 1 and 2 minutes of reaction. The results are presented as means ± S.E. for 3 independent trials. Values for wild-type and S184D hsUbc6e differ significantly (p=0.0458, 1 min; p=0.0303, 2 min). Values for wild-type and

S184E hsUbc6e differ significantly (p=0.0100, 1 min; p =0.0003,2 min).

84 converted fractions were significantly reduced compared to wild-type, suggesting that phosphorylation at Serl84 inhibited acceptance of ubiquitin from El.

2.4 Discussion

All Ubc6 homologs possess the characteristic catalytic core and membrane-spanning domain, however, the joining segments within and between Ubc6 and Ubc6e subfamily members have diverged significantly. Particularly, the Ubc6e subfamily members that are found only in higher eukaryotes exhibit notably extended segments. I have determined that hsUbc6e is phosphorylated in vivo during ER stress at Serl84 which is positioned in this poorly characterized and divergent segment. This modification distinguishes the Ubc6e and Ubc6 vertebrate subfamilies, and its absence in lower eukaryotic Ubc6e members indicates that it corresponds to a recent adaptation.

I have shown that both genes are UPR inducible, consistent with their involvement in

ERAD processes. Their transcriptional responses, however, appear independent as the up- regulation of hsUbc6e occurs later with tunicamycin treatment, and for more prolonged periods compared to hsUbc6. This indicates that expression of each gene is uniquely regulated under

UPR and suggests that the encoded proteins may play distinct roles in ERAD during ER stress.

Further, the observation of phosphorylation at Serl84 reveals additional modulation of hsUbc6e that appears to be dependent on the ER stressor.

I have shown that ER stress induced phosphorylation of Ubc6e requires signaling by the

ER resident transmembrane kinase PERK. PERK belongs to a family of protein kinases that couple distinct upstream stress signals to phosphorylation of the a subunit of the translation initiation factor eIF-2 (reviewed in [408]), which in turn invokes immediate global translational 85 repression, thereby reducing the load of client proteins on the ER during ER folding stress

[277,402]. Phosphorylated eIF-2a also enhances the translation of transcriptional regulator

ATF4 which augments expression of additional transcription factors, ATF3 and

CHOP/GADD153 that contribute to the regulation of genes important for stress recovery and apoptosis [303,304,403]. The importance of PERK signaling in maintaining cellular homeostasis is demonstrated in perk" "mice which exhibit defects in glucose homeostasis and post-natal lethality [252,263,409]. Involvement in early ER stress response is evident with the early appearance (1 h) of the phosphorylated Ubc6e form during both DTT and thapsigargin treatments. Ubc6e phosphorylation displays similar kinetics of eIF-2a phosphorylation in early stress, thus I examined whether PERK is directly phosphorylating Ubc6e. In vitro kinase assays and observations of residual phosphorylated Ubc6e in perk~f~ MEFs indicate that PERK may not be the direct kinase, suggesting the involvement of other downstream kinases. Localization or recruitment of involved kinases to the ER membrane appears to be required as mislocalized

Ubc6eATMD is not modified. Analysis of the sequence surrounding Serl84 of hsUbc6e does reveal Ca2+/calmodulin-kinase type II and protein kinase C consensus motifs (Fig. 1C), but treatment of cells with corresponding kinase inhibitors, including bisindolylmaleimides and KN-

62, appeared not to block hsUbc6e phosphorylation during chemically induced ER stress.

Additionally, kinase inhibitor treatment of pulse-labeled hsUbc6e transfected cells appeared to have no effect (data not shown).

The implication of PERK in the phosphorylation of an ER component raises the exciting possibility that PERK signaling regulates ERAD events during UPR. In a role complementary to global translational repression, phosphorylation of Ubc6e could alleviate ER stress by modulating its activity thereby altering the clearance of ERAD target proteins during UPR.

Phosphorylation does not alter stability, membrane association or ER subcellular localization of

86 hsUbc6e, which suggests a continued role for the modified enzyme in ERAD. The observation that hsUbc6e both in phosphorylated or unphosphorylated forms are very stable (t'/2~20 h), is in contrast to the yeast ortholog which is a rapidly degraded ERAD substrate (fr/2~ 1 h) [410].

The E2 activity of the phosphorylated enzyme was assessed with in vitro thiol-ester assays. Thiol-ester assays performed with the phosphorylation mimicking variants, hsUbc6esl84D and hsUbc6esl84E, indicated that modification reduces the efficiency at which the enzyme forms thiol-ester linkage with ubiquitin, suggesting that phosphorylation may negatively regulate hsUbc6e activity. It is not apparent, how or why downregulation would manifest during ER stress. Based on observations of other E2 enzymes, a role for phosphorylation in regulating hsUbc6e ability to accept ubiquitin could be considered and is supported by the relative carboxyl terminal position of Serl84 in hsUbc6e. For example, the removal of the carboxyl terminal region of the E2, Ubcl, altered its pattern of multi-ubiquitin chain assembly [411]. In another E2 example, the alkylation rate of the catalytic cysteine of E2-

25K was increased with the removal of its carboxyl terminal segment, suggesting that this region may be blocking access to the critical cysteine [412]. Further, and more recently, interaction of the Ubcl carboxyl terminal region with the catalytic region has been directly implied by the NMR-determined structure, suggesting that the interacting surfaces may protect the catalytic cysteine [413]. It should be cautioned, however, that these examples may not be directly comparable as the structural constraints imposed by the membrane anchor of hsUbc6e may limit interaction between the carboxyl terminal region and the catalytic core.

I have also investigated a putative role for phosphorylation in modulating protein interactions of this E2. The carboxyl terminal regions of E2s are frequently involved in protein complex interactions including E3 partners [32,34,405] so another plausible role for phosphorylation of the acidic tail region of hsUbc6e may be to modulate interactions with its own cognate E3s, permitting, if not signaling, substrate ubiquitination events. Phosphorylation 87 mediated E2-E3 regulated interaction has been reported; UBC3/Cdc34 in its C-terminal domain induces its interaction with the F-box protein p-TrCP, the substrate recognition subunit of an

SCF ubiquitin ligase, and as a result, enhances (3-catenin degradation [36]. Co- immunoprecipitation experiments with the E3 Parkin, however, indicate that phosphorylation of

Ubc6e does not modify this particular E2-E3 interaction, as both phosphorylated and unphosphorylated forms interacted with the E3. Alternatively, phosphorylation of hsUbc6e may also modulate its specific interaction with other known or putative E3s or ERAD components.

The yeast-two-hybrid screen using mutant S184A and phospho-mimic S184E, however, did not uncover such presumed interactors. While El ubiquitin-activating enzyme was identified as an interactor, the transient nature of some partner interactions may limit detectection. Also, the classical yeast two-hybrid system utilized may not be optimal for identifying interactions between Ubc6e and other membrane-bound or associated proteins, as expressed protein portions must translocate to the nucleus to activate the reporter genes. Other gene reporter systems that preserve native membrane localization may yield such interactions [414]. The effect of phosphorylation was also examined in co-expression studies with the ERAD substrate Pael- receptor. Although participation with Parkin in Pael-receptor elimination has been shown [135],

I found no affect of Ubc6e over-expression on Pael-receptor turnover rate. The activity of other

E2s involved in ERAD, such as Ubc7 [207,210,395] or the homologue Ubc6, may be redundant to Ubc6e in vivo.

With the former yeast studies and numerous examples of ERAD, the implication of roles for mammalian Ubc6 and Ubc6e in ubiquitination and degradation are apparent. However, it is unclear as to how, in higher eukaryotes, Ubc6 and Ubc6e are distinguished in their ERAD functions, particularly substrate targeting events. The identification of the ER stress related mammalian Ubc6e phosphorylation site entices new studies towards elucidating these

88 distinctions. With implication of PERK in Ubc6e phosphorylation during ER stress, studies examining the role of phosphorylated Ubc6e as an effector of PERK cell survival signaling can now also be pursued.

89 CHAPTER III

PHOSPHORYLATION OF THE UBIQUITIN-CONJUGATING ENZYME UBC6E BY

MAPKAPK2 OCCURS IN RESPONSE TO MULTIPLE CELL STRESSORS AND IS

ASSOCIATED WITH APOPTOSIS SIGNALING

The work in this chapter has been submitted for publication.

Contributions:

I organized and led the investigations in this chapter. I thank Xinli Bai for technical assistance; she generated the CHO-K1 stable cell lines expressing Ubc6e. All other work was performed by me.

90 Summary

The ER membrane-associated ubiquitin-conjugating Ubc6 enzymes have been implicated in the degradation of misfolded proteins. I previously observed that a mammalian homologue,

Ubc6e, is phosphorylated in response to ER stress involving PKR-like ER kinase (PERK). I now report that additional and diverse environmental stresses including hyperosmotic, genotoxic, and pharmacologic stress as well as heat shock and various mitogens, also rapidly induce phosphorylation. Inhibitor studies implicated the p38 MAPK pathway, and siRNA and in vitro analysis indicated the direct involvement of MAPKAPK2 (MK2).

Phosphorylation is triggered via different routes as PERK involvement was limited to ER stress. Inducible cell lines expressing Ubc6e wild-type, phospho-mutant, and phospho- mimic variants, revealed that stress-induced phosphorylation is associated with increased apoptosis, and involves c-Jun kinase and caspase 12 activation during ER stress. Together, these findings indicate that Ubc6e is an early response to a wide array of cell stressors and that Ubc6e is a novel target of the p38 MAPK/MK2 pathway. They further suggest that modulation of the function of this ubiquitin-conjugating enzyme is critical to stress-related responses and cell survival.

91 3.1 Introduction

Ubiquitin-mediated protein degradation is involved in the regulation of many eukaryotic cellular processes, including ER-quality control and associated degradation (ERAD)

(reviewed in [4]). Selective protein degradation by the proteasome requires the covalent attachment of ubiquitin to target proteins through three sequential reactions (reviewed in [389]): ubiquitin activation by El, ubiquitin-conjugation by E2 enzymes, and substrate selection by E3 ligases. Ubc6 is a tail-anchored E2 enzyme localized to the cytoplasmic face of the ER membrane and has been implicated in the degradation of numerous misfolded membrane and secretory proteins in S. cerevesiae (examples [123,125,386]). There are two readily recognizable mammalian homologs, referred to as Ubc6 and Ubc6e in this study, that are similarly localized to the ER membrane and are also involved in ERAD [44,135,207,208,210]. Ubc6e differs from the Ubc6 homologue by the presence in the former of an extended linking domain between the highly conserved catalytic core and the carboxy-terminal transmembrane domains. Ubc6e has been shown to be phosphorylated within this extension at the serine residue at position 184

(Section 2.3.5), but distinction in the functions of the two homologs have not yet been fully elucidated.

When the capacity of the ER to process newly synthesized proteins is exceeded, the resulting accumulation of unfolded proteins triggers 'ER stress'. ER stress can be caused by numerous pathological and physiological states, and can also be induced experimentally, by chemical inhibition of N-linked glycosylation in the ER, reductive stress, and depletion of ER calcium stores (reviewed in [310]). To maintain homeostasis of the organelle, the mammalian cell responds to ER stress by activating the Unfolded Protein Response (UPR), which is mediated by three classes of ER-resident transmembrane proteins that serve as proximal sensors

92 of misfolded proteins in the ER lumen - the protein kinases PERK and IRE1, and the membrane bound transcription factor ATF6 (reviewed in [393]). Upon activation, PERK phosphorylates eIF-2a which mediates the adaptive attenuation of general translation initiation [277,306,402], and induction of cytoprotective genes in a signaling pathway called the integrated stress response [304]. IRE1 and ATF6 promote transcriptional up-regulation of ER chaperones, folding enzymes and ERAD components to increase the ER's ability to handle unfolded proteins by expanding folding and degradation capacity (reviewed in [393]). In studies of the Ubc6 and

Ubc6e mammalian homologs, I previously observed that both homologs are UPR inducible, consistent with their roles in ERAD (Section 2.3.2). Ubc6e is rapidly phosphorylated during ER stress, and this response is not observed in PERK-deficient cells, suggesting that the activity of this E2 may be specifically regulated during ER stress (Section 2.3.3 and 2.3.4). As this modification did not alter protein stability or subcellular localization, direct roles in degradation processes involving specific effects on ubiquitination conjugation abilities or on the recognition of binding partners are anticipated (Section 2.3.6, 2.3.9 and 2.4).

In addition to UPR-activated adaptive mechanisms, the cell experiencing ER stress simultaneously integrates and relays signals for apoptosis and cell growth processes (reviewed in [415,416]. These include: PERK-eIF2a mediated activation of nuclear transcription factor KB and pro-apoptotic transcription factor CHOP [303,337], and cell-cycle arrest [300]; activation of

JNK and ER-localized caspases, which involve IREl-mediated processes [341,361]; and other pro-apoptotic events such as apoptosome formation [417] and regulation of BCL-2 family protein stability and localization (reviewed in [345]). In the face of excessive ER stress, the balance between adaptive and apoptotic measures tips towards cell death.

Although numerous ERAD factors have been shown to be transcriptionally regulated by

UPR [211,287,418], the phosphorylation of Ubc6e is the first example of UPR regulated post-

93 translational modification of an ERAD component. Since up-regulation of ERAD activity is an essential aspect of cellular adaptation to ER stress [285,333], and phosphorylation of Ubc6e can involve PERK (Section 2.3.4), it was of interest to determine the role of Ubc6e and its phosphorylation in ER stress response. As well, given the well documented cross-talk between

UPR signaling and other stress pathways, I sought to examine the broader role of this E2 in cellular stress response. In the present study, I have observed that Ubc6e phosphorylation is not limited to ER stress, but is a common response to various environmental stressors and mitogen stimulation, and PERK involvement is restricted to ER stress. This stress-induced modification requires p38 MAPK and directly involves protein kinase MK2. Interestingly, this modification correlates with cell death and apoptosis signaling at the ER. These results indicate that Ubc6e is a novel target of the p38MAPK/MK2 pathway and further suggest that selection and degradation of substrates via this ubiquitin-conjugating enzyme may be critical to stress-related responses and cell survival.

3.2 Experimental Procedures

3.2.1 Antibodies and chemicals

Anti-Ubc6e polyclonal antibody for detection of endogenous Ubc6e in human and mouse cell lines was described previously (Section 2.2.3). Antibodies against phospho-eIF2a

(Ser51), MK2, phospho-ERK (phospho-p44/42 MAP kinase, Thr202/Tyr204), c-Jun, phospho- c-Jun (Ser63), caspase-12, TRAF2 and c-IAPl were obtained from Cell Signaling Technology.

Anti-BiP/Grp78 antibody was obtained from StressGen. Anti-myc (9E10), GAPDH, and p-

94 tubulin antibodies were obtained from Calbiochem. Thapsigargin, methyl methanesulfonate, staurosporine, insulin, fibroblast growth factor, and phorbol 12-myristate 13-acetate were obtained from Sigma. MAPK inhibitors SB203580, SP600125, and U0125 were obtained from

Calbiochem.

3.2.2 Cell culture, plasmids and transfections

W4 and perk'A mutant mouse embryonic fibroblast cells (kindly provided by D. Ron,

Skirball Institute) were cultured as previously described [304]. HEK293 cells were grown at

37°C with 5% CO2 in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum

(Invitrogen). For transient expression, HEK293 cells were transfected with myc-tagged hsUbc6e or hsUbc6 pCMV expression plasmids (see Section 2.2.2) with ExGen 500 in vitro transfection reagent (MBI Fermentas). To establish stable inducible cell lines, the cDNAs encoding myc- tagged hsUbc6e wild-type, S184A and S184E were further subcloned into the pTRE2 expression plasmid (BD Biosciences), and then used to transfect BD CHO-K1 Tet-On cells (BD

Biosciences) with ExGen 500. Selection of stable clones was performed essentially according to the manufacturer's protocols. Clones were screened for myc-Ubc6e protein induction in the

Tet-ON cells in the presence of doxycycline (Invitrogen), as judged by immuno-detection on

Western blots. Cell lines were cultured and passaged in Dulbecco's modified Eagle's medium containing 10% Tet-approved fetal bovine serum (Invitrogen), with G418 and hygromycin

(Invitrogen). For cell survival and apoptosis studies, cells were initially cultured in the presence or absence of 2 ug/ml doxycycline for 24 hours and maintained as such during subsequent treatments.

3.2.3 Western blotting, immuno-detection and immunoprecipitation 95 Following treatments indicated in the experiments described below, cells were lysed in

RIPA buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% SDS, 1% Triton, 0.5% deoxycholate, protease inhibitor cocktail tablets (Roche), phosphatase inhibitors (10 mM NaF,

10 mM (3-glycerophosphate, 10 mM sodium orthovandate, 10 mM glucose-1-phosphate, 10 mM para-nitrophenol phosphate), or for immunoprecipitation, cells were lysed in 50 mM Tris-HCl pH 7.5, 120 mM NaCl, 1% Triton, 5 mM EDTA, protease inhibitor cocktail tablets (Roche), phosphatase inhibitors, for 5 min on ice. Cell debris was removed by centrifugation at 15,000 x g for 15 min at 4 °C and the supernatant fluid was used for immunoblot analysis or immunoprecipitation. Total protein concentration was determined by Lowry Assay (Bio-Rad

Labs) and equal amounts of protein from each sample were separated by 11.5 % SDS-PAGE gels and transferred to nylon-supported nitrocellulose membranes (Amersham Biosciences).

Protein detection on Western blots was achieved with the indicated antibodies using enhanced chemiluminescence of horseradish peroxidase-conjugated anti-mouse (Amersham Biosciences) or anti-rabbit (Bio-Rad Labs) secondary antibodies with Hyperfilm™ ECL film (Amersham

Biosciences). For immunoprecipitations, anti-myc antibody was added to equal amounts of lysate (-1.5 mg total protein) and incubated for 2 h, at 4 °C, followed by incubation with

Protein-G Sepharose slurry (Roche), overnight at 4°C, followed by three washes with lysis buffer. Immunoprecipitates were separated by SDS-PAGE gels and subjected to immunoblot analysis, as above.

3.2.4 siRNA experiments

siRNA oligonucleotide pools to MK2, MK3, MK5 and non-targeted controls were obtained from Dharmacon. W4 mouse embryonic fibroblasts were plated at a density of 1 x 105

96 cells per 35 mm plate and transfected with the indicated siRNA oligonucleotides (50 nM) with

Oligofectamine (Invitrogen). After 48 hours, the transfections were repeated with Dharmafect 1 transfection reagent (Dharmacon). Following an additional 48 hours, cells were treated as described below, and collected for total protein and RNA extraction (RNeasy, Qiagen).

Expression of MK3 and MK5 was measured by standard reverse-transcriptase PCR protocols and MK2 protein levels were measured by immuno-detection.

3.2.5 MK2 kinase assay

Active MK2 kinase (Cell Signaling), 50 nM, was incubated with MK2 substrate, Hsp27

(StressGen), or His-tagged Ubc6eATMD (see Section 2.2.13), luM, in reaction buffer (50 mM

Tris-Cl, pH 7.5, 2.5 mM p-glycerophosphate, 1.0 mM EGTA, 0.4 mM EDTA, 4 mM MgCl2, 1 mM DTT, 20 ng/ul bovine serum albumin). Kinase reactions were initiated with addition of 200 uM ATP and 0.1 uCi/ul [y-32P]ATP (GE Healthcare) and incubated for 30 min, 30°C.

Reactions were terminated with addition of Laemmli sample buffer, and products were resolved by SDS-PAGE, followed by autoradiography.

3.2.6 Cell viability, FACS and apoptosis studies

Stress-challenged CHO-K1 inducible cell lines were seeded at a density of 2 x 104 cells per well of a 24-well plate and cultured 24 h with or without doxycycline (2 ug/ml). Cells were subjected to stressors as described, and then incubated with (3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide substrate (MTT; Sigma) for an additional 4 h at a concentration of

5 mg/ml. Formazan crystals produced by mitochondrial dehydrogenase activity were then

97 solubilized in 0.1 N HC1 in anhydrous isopropanol, followed by spectrophotometric measurement of absorbance at a wavelength of 570 nm (SpectraMax microplate reader,

Molecular Devices).

To study cell cycle effects in Ubc6e expressing CHO-K1 cells during stress, cells were seeded and cultured as above, and then pulse-labeled with BrdU in the final 1 h of treatment.

Cells were collected in trypsin and fixed with 4% Para-formaldehyde in PBS. Incorporated

BrdU was stained with anti-BrdU-FITC conjugated antibody, according to manufacturer's protocol (BD Biosciences), followed by flow cytometry analysis (BD FACScan).

To study the activation of caspase-3 and -7 in CHO-K1 cells during stress, cells were seeded at a density of 9 x 10 cells per well of a 48-well plate. Cells were treated as described below, and then lysed with Apo-ONE® Homogeneous Caspase-3/7 Buffer containing the pro fluorescent substrate Z-DEVD-R110 (Bio-Rad Labs). Fluorescence was measured according to the manufacturer's instructions after incubation at room temperature for 12 h (SpectraMAX

Gemini Fluorometer, Molecular Devices).

For caspase-12 cleavage and JNK activation detection, cell lines were seeded at a density of 3 x 10 cells per 60 mm plate and cultured as described above. Cells were subjected to stressors as described, and then collected for immuno-detection with anti-pro-caspase-12 and anti-phospho-c-jun (JNK substrate) antibodies.

3.2.7 Statistical analysis

Statistical analysis was assessed with Prism 4 software (GraphPad Software, Inc.) using the Student's t-test to compare means of three or four trials as indicated. A two-sided p-value of

<0.05 was considered to be statistically significant.

98 3.3 Results

3.3.1 Phosphorylation of Ubc6e is induced by various stress and mitogen stimuli

Phosphorylation of Ubc6e is induced by ER stress, as previously described (Section

2.3.3). To investigate whether this modification is limited to ER stress, or is part of a more general stress response, diverse stress conditions were tested in mouse embryonic fibroblasts

(MEF) (Fig. 3.1 A). Along with typical ER stressors thapsigargin and DTT, oxidative stress

(H2O2), hyper-osmotic shock (NaCl), genotoxic stress (methyl methane sulfonate, UV), and staurosporine (a pan-kinase inhibitor) induced robust and rapid (1 h) phosphorylation of Ubc6e.

Heat-shock only induced a modest response. In addition to environmental stress, certain mitogens including fibroblast growth factor, fetal bovine serum and phorbol myristate acetate, but not insulin, were also able to induce rapid phosphorylation (Fig. 3.1C). Phosphorylation was most robust during early stress or mitogen stimulation (1 h), and decreased over time (6 h), although under certain stimuli (hyper-osmotic shock, genotoxic stress) phosphorylation was sustained for at least several hours (6 h). Phosphorylation of Ubc6e in response to ER stress, genotoxic stress, and hyper-osmotic shock was also observed in Panc-1, HEK293, MCF-7, and

HeLa cell lines (Fig. A.2.1). These cell lines display a relatively muted response compared to mouse embryonic fibroblasts in the extent of Ubc6e phosphorylation (for example, to hyper­ osmotic conditions), which likely is indication of their intrinsic resistance to stress.

While modification of Ubc6e during thapsigargin and DTT treatment requires the activity of the ER stress kinase PERK (Section 2.3.4), it was observed that phosphorylation under other stress conditions was not disrupted in PERK-deficient cells (Fig 3.IB). Further, while ER stress could not induce high levels of eIF-2a phosphorylation in PERK-deficient cells

99 Figure 3.1 Phosphorylation of Ubc6e is induced by various stress and mitogen stimuli

A C

UT TG DTT H,02 NaCI UT HS UV MMS STS (h) 16 16 16 16 16 16 16 16 UT FGF Ins UT FBS PMA — =—fiS— = —»* -UbSfe -"»—«- -a*»a»^ (h)_ J. 6_ 1 J, 16 16 ___ _d__ __ j—UffTT '... . r|-Ubc6e~p-i~'~'''~-i~ Tl

— ... -_ - _ — Grp78 —— » «—' i—ii '• • «— —i .II mmmm — i— — *" GADPH — _ _ _ .~ __~

B D UT DTT TGH2OaNaCIHS UVSTS UT TG DTT H202 NaCI UT HS UV MMS STS "'—»—._ — ^ -Ser184~P 16161616 16161616 —--^ «——— — -*" \ _ — —«" Ubc6e~p - « == «_ __ ^

_ — e|F2a~p — - — — —. —-*• ,. . . Grp78 -i. —• ..___ GADPH — _ -- — _____-'

A, Mouse embryonic fibroblasts (MEFS) were cultured with thapsigargin (TG, 1.5 uM), DTT (2

2 mM), H202 (450 uM), NaCI (300 mM, 600 mOsm), UV (50 J/m ), heat shock (42°C, 0.5 h), methyl methanesulfonate (MMS, ImM), staurosporine (STS, 0.5 uM), and collected after 1 or 6 h. Ubc6e phosphorylation was monitored by western blot (see Section 2.2.3). Phospho-eIF-2cc and Grp78 were markers of general and ER stress, respectively. GAPDH was a protein loading control.

B,perk-/- MEFS were treated with stressors, as in A.

C, MEFS were starved of growth serum for 12 h, then incubated with fibroblast growth factor

(FGF, 100 ng/ml), insulin (Ins, 1 uM), fetal bovine serum (FBS, 10%), and phorbol 12- myristate 13-acetate (PMA, 0.1 uM), and collected after 1 or 6 h.

D, Stress-induced phosphorylation at residue Ser-184. HEK 293 cells were transfected with either wild-type or S184A myc-tagged Ubc6e, and then treated with stressors for 1 h. Ubc6e was detected with anti-myc antibody. The faint upper band detected in S184A transfected cells represents an uncharacterized isoform of Ubc6e (Section 2.3.5).

100 observed in wild-type cells (Fig IB, TG and DTT), other stress conditions induced high levels, indicating that other eIF-2ct kinases were being activated. These other stress conditions did not lead to activation of UPR as indicated by normal protein levels of the chaperone and marker for

ER stress, Grp78 (also known as BiP). Only the thapsigargin and DTT treatments (6 h) affected

Grp78 levels under the conditions used. Stress-induced phosphorylation of Ubc6e invariably involves residue Serl84, which had previously been implicated in ER stress (Section 2.3.5), since there was no detectable shift to the phosphorylated form for the myc-tagged Ubc6e phospho-mutant, S184A in transfected HEK293 cells, regardless of the environmental stress conditions tested (Fig. 3.ID). Together, these data indicate that phosphorylation of Ubc6e is an early response to a wide range of stress and extracellular stimuli. Further, although pertinent for thapsigargin and DTT induced ER stress, PERK signaling is not required for the alternate stress conditions.

3.3.2 Stress-induced phosphorylation of Ubc6e by a p38 MAPK/MK2 pathway

Stress-activated protein kinases c-Jun N-terminal kinase (JNK) and p38 MAPK are broadly activated by environmental stressors, including ER stress and extracellular stimuli

(reviewed in [347]). Furthermore, examples of cross-talk between ER stress-pathways and

MAPK responses have been described [341,346,419,420]. Thus, to assess the involvement of these stress-activated protein kinases, MEFs were treated with specific inhibitors of JNK and p38 MAPK and then exposed to hypertonic or ER stress (Fig. 3.2A, NaCl, thapsigargin).

Specific inhibition of p38a/p isoforms with the compound SB203580 [421] at a concentration of 10 uM, blocked the Ubc6e phosphorylation modification during stress, while treatments with

JNK inhibitor, SP600125, or ERK inhibitor, U0126, did not appear to have any effect.

101 Figure 3.2 Stress-induced phosphorylation of Ubc6e can be blocked by the p38 inhibitor

SB203580

A B C

CON SP SB UO CON H202 HS UV CON NaCI FBS FGF (nM) 0 2 20 1 10 1 10 SB: .+ . + .+ SB: .+ + . + .

A, MEFS were pre-treated with (as indicated) or without (0) specific inhibitors of JNK

(SP600125, SP), p38 MAPK (SB203580, SB) or ERK (U0126, UO), for 30 min, then co- incubated with NaCI (300 mM, 600 mOsm) or thapsigargin (1.5 uM) for 1 h. Extracts from cells that were not treated (CON) are shown for comparisons.

B, MEFS pre-treated without (-) or with (+) SB203580 were incubated with either H202 (450 uM), heat shock (42°C, 0.5 h) or UV (50 J/m2) and collected after 1 h.

C, MEFS were starved of growth serum for 12 h, then pre-treated with SB203580, and then incubated with fetal bovine serum, FBS (10%), or fibroblast growth factor, FGF (100 ng/ml), for 1 h. NaCI treatment was performed as in A. Mitogenic stimulation with FBS and FGF was confirmed by monitoring ERK phosphorylation. Phosphorylation of eIF2a was an indicator of general cell stress. GAPDH was a protein loading control.

102 Phosphorylation induced by hydrogen peroxide, heat shock, and UV was also blocked effectively by p38 MAPK inhibition (Fig. 3.2B). Inhibition of p38 MAPK also blocked Ubc6e phosphorylation during FBS and FGF stimulation (Fig. 3.2C). Thus, phosphorylation of Ubc6e requires p38 MAPK signaling with the wide array of stressors tested and with mitogen stimulation.

Activated p38 MAPK phosphorylates numerous cellular targets, including several protein kinases, such as the mitogen-activated protein kinase-activated protein kinases

(MAPKAPKs) [422]. The MK2, MK3, and MK5 MAPKAPKs are activated by environmental stressors and cytokines and relay MAPK signals to diverse cellular processes (reviewed in

[423]). MK2 and MK3 are directly activated by p38a/p [424,425] and are known to share many of the same substrates (reviewed in [423]). MK5, on the other hand, is primarily activated by atypical MAPK ERK3 and ERK4 and does not share substrates [426-428], despite significant homologies with MK2 and MK3. RNA interference with short-interfering RNAs was used to directly examine the role of MKs in Ubc6e phosphorylation in mouse embryonic fibroblasts.

Transfection with siRNA oligonucleotides targeted against MK2 achieved -80% reduction in

MK2 protein levels while non-targeted oligonucleotide treatment had no effect (Fig. 3.3A).

Because reliable commercial antibodies to MK3 and MK5 were not available, knock-down of the expression of these genes was evaluated by RT-PCR, which revealed that MK3 and MK5 siRNA treatment reduced mRNA levels -75% (Fig. 3.3B). Following MK gene silencing by siRNA, cells were stressed with thapsigargin, H2O2, or NaCl, and then analyzed for Ubc6e phosphorylation. MK2 siRNA treated cells blocked Ubc6e phosphorylation during stress treatments, most readily evident with NaCl treatment, diminishing the ratio of phosphorylated to unphosphorylated Ubc6e by -50% (Fig. 3.3A, D). As expected, siRNA reduction of MK5 did not reduce Ubc6e phosphorylation significantly, nor did it provide additive reduction in MK2 siRNA treated cells (Fig. 3.3A). MK3 directed siRNA also did not diminish phosphorylation of 103 Figure 3.3 Ubc6e is phosphorylated by MK2

A B treatment CON TG H202 NaCI siRNA NT MK2 MK5 MK2 NT MK2 MK5 MK2 NT MK2 MK5 MK2 NT MK2 MK5 MK2 SiRNA NT MK5 NT MK3 +MK5 +MK5 +MK5 +MK5 1 .5 .25 1 1 .5 .25 1 Ubc6e~p — - Ubc6e MK5 MK3 —-

| MK2 GAPDH » — »»-—.

GAPDH

D

siRNA NT MK3 treatment UT TGNaCIH202UT TG NaCIH202 icge~p

TG NaCI H202 treatment

I T ^w F S pCMV Ubc6 Ubc6e pCMV Ubc6 Ubc6e LXRQLSHyd 62 MK2 +SB +SB T R 47.5 XSXK PKC ISR? < MK2 T 32 5 i IIHL *<— RXXSX CaMKII ^P#K^^ ™ hsubc6e 176 jjjsran 192 25 mmUbcSe 176 H!EE 192 | Ubc6e | ggllbc6e 176 fflH^ 192 xtUbc6e 159 fflSp? 175 - Ubc6- drUbc6e 174 poRBHoMmnprn jssgB 190 Autoradiogram cetlbcSe 175 BrJBA^KiJQDIj 3SVVK 191 WCE myc-IP WT S184A L W E L W E 47.5- 32.5- «•» _ m Ubc6eAC Coomassie stain

A, MEFS were transfected with non-targeting (NT), MK2, MK5 and combined MK2 and MK5 targeted siRNA oligonucleotides, and then incubated with thapsigargin (TG, 1.5 uM), H2O2

(450 uM), NaCI (300 mM, 600 mOsm), or without further treatment (CON) for lh. Shown is a representative blot of three independent siRNA experiments. GAPDH was used as a protein loading control.

104 B, RT-PCR of non-targeted, MK3 and MK5 siRNA treated cells. Serial dilutions of the non- targeted RT-PCR product (1, 0.5, 0.25) were used to estimate the reduction of MK3 or MK5 by densitometry analysis (Fluorochem, Alpha-Innotech).

C, MEFS were transfected with non-targeting or MK3 targeted siRNA oligonucleotides, and then treated with stressors as in A. The band indicated with the asterisk (*) corresponds to a non- specific protein.

D, The ratios of phosphorylated to unphosphorylated protein from three independent MK2 siRNA experiments were estimated by densitometry analysis. Stress-induced phosphorylation of Ubc6e is significantly reduced in MK2 versus non-targeting (NT) siRNA treated MEFS (for

TG, p=0.0239; for NaCl, p=0.0227; for H202, p=0.0118).

E, Alignment of amino acid sequence surrounding Ser-184 in Ubc6e subfamily members. Ser-

184 and flanking sequences predicted MK2, CaMKII and PKC consensus sites that are conserved in Homo sapiens (hs), Mus musculus (mm), Gallus gallus (gg), and Xenopus tropicalis (xt), but not in Danio rerio (dr) or Caenorhabditis elegans (ce).

F, In vitro MK2 kinase assay. Hsp 27, purified His-tagged wild-type, or SI84A Ubc6e (all at 1 uM) were incubated with MK2 (50 nM) and [y- P]ATP. The asterisk in the upper panel indicates the radio-labeled phosphorylated form of Ubc6e, not evident for the SI84A variant.

Phosphorylated Hsp27 is indicated with a double-arrowhead. The single-arrowhead indicates a weaker phosphorylated form of Ubc6e (Section 2.3.5). The lower panel shows a Coomassie

Blue stained PAGE-gel of the recombinant His-tagged Ubc6e protein purifications by Ni- column chromatography (L, total lysate; W, column wash fraction; E, column eluate).

G, Myc-tagged Ubc6e co-immunoprecipitates endogenous MK2. HEK293 cells were transiently transfected with pCMV (vector control), myc-tagged Ubc6 and myc-tagged Ubc6e expression vectors. Immunoprecipitation with anti-myc antibody, followed by anti-MK2 immuno­ detection, indicates specific MK2 binding to Ubc6e that was reduced when cells were treated 105 with the p38 inhibitor (SB), right panel. Expression of endogenous and transfected proteins were confirmed by anti-MK2 and anti-Myc immuno-detection of whole cell extracts (WCE), respectively, left panels.

106 Ubc6e during stress (Fig. 3.3C). These findings indicate that MK2 is the primary kinase involved in Ubc6e phosphorylation in stressed mouse embryonic fibroblasts.

Examination of the sequence flanking the Ser-184 phospho-site of Ubc6e revealed a conserved consensus for MK2 [429], (Fig. 3.3E), further supporting that Ubc6e is a substrate of

MK2. Direct involvement of MK2 was confirmed by an in vitro kinase assay (Fig. 3.3F) which demonstrated that MK2 could phosphorylate purified recombinant His-tagged Ubc6e on residue

Ser-184. Ubc6e was phosphorylated less efficiently than the MK2 substrate Hsp27, however, this version lacked the C-terminal transmembrane domain and native-membrane conditions may be required for optimal reaction conditions. To further confirm the direct involvement of MK2, co-immunoprecipitation experiments were performed revealing that endogenous MK2 can form a complex with myc-tagged Ubc6e in transfected HEK293 cells (Fig. 3.3G). This complex is specific for Ubc6e, as the closely related homologue Ubc6 did not show binding to MK2. In addition, inhibition of p38 with SB203580 reduced the amount of MK2 co-immunoprecipitated with myc-Ubc6e, consistent with the reduction in Ubc6e phosphorylation (Fig. 3.3G, right lower panel). Together these data indicate that Ubc6e is a direct target of MK2 phosphorylation during stress due to a variety of ER, environmental or mitogen stimuli.

3.3.3 Phosphorylation of Ubc6e is associated with reduced cell survival and increased caspase activity during stress

To elucidate the function of the modification, I examined whether phosphorylation of

Ubc6e was associated with an adaptive or pro-apoptotic response during cell stress. Cloned

CHO-K1 cell lines with inducible myc-tagged wild-type, S184A or phosphomimic S184E

Ubc6e variants were generated. Upon induction with doxycycline (DOX), the amount of protein produced for each cell line was similar (Fig. 3.4A), and, at least five-fold greater than 107 endogenous Ubc6e levels (Fig. A.2.2). Importantly, I verified that the over-expression of the

Ubc6e forms themselves does not induce stress in this cell culture system. Levels of the ER stress marker Grp78 remain unchanged with induced expression (see Fig. 3.5B, and compare 0 h time points for each cell line). Further, cell lines with induced Ubc6e expression readily showed eIF2a phosphorylation with stressor treatment, indicating these cells were able to respond to stress (Figure A.2.4).

I examined the effect of expression of wild-type or SI84A variants on cell viability during ER stress (thapsigargin) and hyper-osmotic shock (NaCl). Osmotic shock was examined as a stress condition because it induced the most robust and sustained Ubc6e phosphorylation

(Fig. 3.1 A), and is known to induce classical apoptotic events [430]. As assessed by MTT assay, wild-type and S184A cell lines displayed no differences in viability in untreated cultures (Fig.

3.4B, UT) and no significant changes were elicited with DOX-induced expression of the Ubc6e variants. Overall, treatments with both thapsigargin (lOuM) and NaCl (600 mOsm) for 10 h reduced the growth or cell viability (Fig. 3.4B, UT versus TG, NaCl) of all cell lines. Induced expression of wild-type Ubc6e with either ER- or hyper-osmotic stresses was accompanied with an additional modest reduction in cell survival (Fig. 3.4B, uninduced versus induced, for TG and NaCl treatments). Conversely, induced expression of S184A Ubc6e revealed a significant relative increase in cell survival (Fig. 3.4B, uninduced versus induced, in TG and NaCl treatments). This observed protective effect of the induced S184A mutant and the reduced viability of induced wild-type Ubc6e was also observed with alternate CHO-K1 cloned cell lines, as shown in Figure A.2.3.

Response to ER stress and hyper-osmotic shock can include cell cycle arrest [301,430], and MK2 has been shown to be involved in cell cycle control [429]. Thus, effects of over-

108 Figure 3.4 Phosphorylation of Ubc6e is associated with reduced viability and apoptosis

B WT S184A 1.1 1.1 1.0 1.0 0.9 0.9 0.8 0.8 -K1 cell line: WT £ 184A S184E 0.7- 0.7 0.6- 0.6 DOX: - + + + 0.5 0.5 0.4 0.4 , \ Ubc6e < 0.3- 0.3- am — 0.2 0.2- 0.1 0.1- *mm*mm-m »«» mm-mm p-tubulin 0.0 0.0> UT TG NaC1I UT TG NaCII

D

WT S184A WT

6500-1 6500 6000- 6000 JL 5500- 5500 5000- 5000 WT+DOX 4500- 4500 4000- 4000 I 3500- 3500 3000- 3000 2500- 2500 S184A 2000- 2000 1500- J 1500 1000- 1 1000 500- nl 500 ».d 74.5 EoT 36.41 ».q 50.6 1 0- I IIB I 0 m. 1 S184A+DOX UT TG1 , NaCI UT TG NaCI 4- *Sr M-* 83.1

7-AAD-

A, Inducible myc-tagged Ubc6e (wild-type, S184A, S184E) expression in stable CHO-K1 cells.

Following culturing of cell lines with doxycycline (DOX, 2 |ig/ml) for 20 h, expression of

Ubc6e variants was readily observed by anti-myc immuno-dectection. P-tubulin was a protein loading control.

B, Individual CHO-K1 cell lines with induced expression (grey bars) or uninduced (white bars) were exposed to ER stress (thapsigargin, 10 uM), or hyper-osmotic shock (300 mM, 600 mOsm), for 10 h and cell viability was assessed by colorimetric metabolic enzyme activity assay

(MTT assay). Absorbance corresponds to cell survival. Expression of wild-type Ubc6e reduces

109 cell survival during stress while presence of the S184A variant improves cell viability.

Averages of three trials ± S.E. are shown. Differences in survival with or without expression of wild-type Ubc6e or the SI84A variants are statistically significant (for TG treatments: WT uninduced vs. WT induced, p=0.015; S184A uninduced vs. S184A induced, p=0.0347, for NaCl treatments: WT uninduced vs. WT induced, p=0.01; S184A uninduced vs. S184A induced, p=0.0015).

C, Stress-induced cell cycle arrest or delay is unchanged with Ubc6e expression. Cells were incubated as for B, except that thapsigargin and NaCl treatments were for 12 and 4 h, respectively. Cells were pulsed with BrdU in the final hour of treatment and prepared for flow cytometry analysis. Shift in the percentage of S-phase, BrdU+ cells (upper right quadrant) to Gl or G2/M, BrdU" cells (lower right quadrant) during thapsigargin and NaCl treatments is unaffected with induced Ubc6e expression (DOX).

D, As in B, except that thapsigargin and NaCl treatments were 6 and 3h, respectively. Caspase activity was assessed by cleavage of a caspase-3 and -7 pro-fluorescent substrate and expressed in relative fluorescence units (RFUs). Induced expression of wild-type Ubc6e increases caspase activity during stress while presence of S184A mutant decreases caspase activity. Averages of three trials ± S.E. are shown. Differences in caspase activity produced by expression of the wild-type Ubc6e or SI84A variant are statistically significant (for TG treatments: WT uninduced vs. WT induced, p=0.0002; S184A uninduced vs. S184A induced, p=0.0004, for

NaCl treatments: WT uninduced vs. WT induced, p=0.0061; S184A uninduced vs. S184A induced, p=0.0022). It was noted in untreated cells, that the background caspase activity levels were higher for the wild-type than for the S184A cell lines, which may be reflecting leaky expression of the respective Ubc6e genes from their inducible promoters.

110 expressing Ubc6e phospho-variants on survival during stress may be due to direct or pleiotropic consequences of response. I examined these possibilities by using BrdU-pulse labeling to quantify the fraction of actively dividing cells (as determined by cells in S phase) in normal and stress conditions, with and without induced Ubc6e expression (Fig. 3.4C). The percentage of

BrdU+ cells for each cell line (WT and S184A) was similar with or without Ubc6e over- expression (DOX). During stress, a sharp shift in the percentage of S-phase (BrdU+) to Gl or

G2/M (BrdU") cells was observed for each cell line, indicative of cell cycle arrest or delay, regardless of induction status. Thus, while the stressors themselves showed effects, cell cycle responses were largely unaffected by induced over-expression of Ubc6e variants.

To confirm that Ubc6e phosphorylation is associated with apoptosis, stress-induced caspase activation was monitored in the inducible cell lines. Following thapsigargin and NaCl treatment (for 6 and 3 h, respectively), cells were harvested and the cleavage of a pro- fluorescent caspases-3 and -7 consensus substrate was measured. The activity of caspases-3 and

-7 were markedly increased with thapsigargin or NaCl treatments overall, as expected, and consistent with the results from the MTT assay (Fig. 3.4B). I observed that caspase activity was increased with wild-type Ubc6e expression (Fig. 3.4D, WT uninduced vs. WT induced), but conversely, that induced expression of the S184A mutant resulted in diminished activation of caspases during stress treatments (Fig. 3.4D, S184A uninduced vs. S184A induced). These findings indicate that phosphorylation of Ubc6e is associated with increased apoptosis during stress.

3.3.4 Phosphorylation of Ubc6e is implicated in activation of JNK and caspase-12 during

ER stress

111 I next asked whether the association between phosphorylation and increased apoptosis during stress was linked with specific pro-apoptotic events at the ER membrane, given that

Ubc6e is located here, regardless of phosphorylation status (Section 2.3.6). I examined the effects of Ubc6e phosphorylation on specific apoptosis events that are proximal to ER stress signaling, specifically Jun N-terminal Kinase (JNK) activity, because its activation requires the

ER stress-sensor inositol-requiring enzyme, isoform la (IRE1) [341], and the ER-membrane- localized caspase-12. The effects of wild-type, S184A and phosphomimic S184E variant expression on JNK and caspase-12 activation were assessed in thapsigargin treated CHO-K1 cells. To assess JNK activation, the phosphorylation of the transcription factor c-Jun was measured directly with phospho-specific and total antibodies (Fig. 3.5A). c-Jun phosphorylation, which typically peaks early, was significantly reduced with induced expression of S184A (Fig. 3.5A, middle panel) even with up to 60 min of thapsigargin treatment. In contrast, c-Jun phosphorylation was not significantly altered with induced Ubc6e expression in either wild-type and phosphomimic S184E cell lines, (Fig. 3.5A, left panel and right panels). In the same cell lines, thapsigargin could influence the cleavage and activation of caspase-12, as correlated with the reduction in detectable pro-caspase-12. Induced expression of the phosphomimic S184E variant strongly promoted caspase cleavage as almost no pro-caspase-12 remained detectable with prolonged treatment (Fig. 3.5B, right panel, 8 and 16 h). Conversely, pro-caspase 12 was not reduced with the induction of the S184A variant (Fig. 3.5B, center panel, 16h). These effects on JNK and caspase-12 activation were confirmed in other S184A and S184E expressing CHO-K1 cell line clones (Figure A.2.4). Thus, phosphorylation of

Ubc6e is associated with activation of JNK and caspase-12 during ER stress.

The activation of JNK and caspase-12 have been linked to IRE1 via the adaptor TNFa receptor associated family 2 (TRAF2) protein [341,361]. TRAF2 can be destabilized during stress and various other stimuli by ubiquitin-mediated degradation [339,431]. Signaling through 112 Figure 3.5 Phosphorylation of Ubc6e is implicated in activation of JNK and caspase-12 during ER stress

A WT S184A S184E DOX DOX DOX TG time (h): 0 .5 1 0 .5 1 0 .5 1 0 .5 1 0 .5 1 0 .5 1 myc-Ubc6e ~ — — —-«

phospho-Jun **» IB «a» *• S — - - * "

ill |» ^A ^^ ^ ^ Jun ^^ "W VR 4Hb 4tMflfc wMi «*—**— — — —

P-Tubulin ™mTP*

R WT S184A S184E B DOX DOX DOX TGtime(h): 0 8 16 0 8 16 0 8 16 0 8 16 0 8 16 0 8 16 myc-Ubc6e |Mk ^ — —— *^

Individual CH0-K1 cell lines were pre-incubated without or with doxycycline (DOX) to induce wild-type, SI84A, or S184E variant expression and then treated with thapsigargin (10 mM), for the times indicated.

A, To assess JNK activation, phosphorylation of the JNK substrate c-Jun was monitored with anti-c-jun and anti-phospho-c-jun antibodies. JNK activation is suppressed with expression of the S184A variant (*), but unaffected with wild-type and S184E expression, p-tubulin is a protein loading control.

B, Caspase-12 activation was monitored by reduction of pro-caspase 12 with anti-pro-caspase antibody. Expression of the S184A variant maintained pro-caspase 12 levels (<), while S184E led to reduced levels indicating increased caspase-12 cleavage («) most apparent at the 16 h time-point. Grp78 was used as an indicator of ER stress.

113 tumour necrosis factor receptor-2 induces TRAF2 translocation to the ER membrane where it becomes ubiquitinated by the E3 ligase, c-IAPl, and the Ubc6e homologue, Ubc6 [388]. Thus,

I asked if Ubc6e could mediate TRAF2 ubiquitination during stress in a phosphorylation- dependent manner. It is known that E2-E3 interactions can be mediated by E2 phosphorylation

[36], so Ubc6e may be able to modulate its interaction with c-IAPl. In this scenario, unphosphorylated Ubc6e and c-IAPl may promote TRAF2 turnover, in turn preventing JNK and caspase-12 activation (see above). Upon ER stress, phosphorylation may disrupt this E2-E3 binding leading to increased TRAF2, thus promoting increased caspase-12 activation. To test these possibilities, I looked at whether Ubc6e phosphorylation is associated with a change in

TRAF2 stability, which is typically reduced during prolonged stress and cytokine stimulation

[339,388,431]. TRAF2 protein levels in thapsigargin treated CHO-K1 cell lines were not significantly changed with induced expression of the S184A Ubc6e variant (Fig. 3.6A, S184A with or without DOX). TRAF2 levels were also unchanged with expression of wild-type or the

S184E variant (data not shown). Finally, co-immunoprecipitation experiments with lysates of transfected HEK293 cells revealed that the homologue Ubc6 is clearly the preferred E2-binding partner of c-IAPl (Fig. 3.6C), giving further indication that Ubc6e, phosphorylated or nonphosphorylated, is not involved in the regulation of TRAF2 stability during stress.

Together, these investigations point to a role for Ubc6e phosphorylation in promoting apoptosis in response to various cells stressors, and also implicate the activation of JNK and caspase-12 in this process. These effects appear to be independent of TRAF2, likely with involvement of alternate binding partners and/or ubiquitination substrates of Ubc6e that are modulated by the phosphorylation modification.

114 Figure 3.6 Ubc6e is not involved in the regulation of TRAF2 stability during stress

POX TGtime(h): 0 8 16 0 8 16 TRAF2 *•» mm mm —» •—•"

S184A •* ~ —* P-Tubulin *pb> mmiKmm«••»«•

B

Ubc6e Ubc6e

—. - Ubc6 - —»

WCE myc-IP

A, TRAF2 levels are not significantly changed with Ubc6e variant expression. Cells were cultured and treated as in Figure 5B. Endogenous TRAF2 levels were not affected with induced

SI84A expression and prolonged thapsigargin treatment.

B, Phophorylation of Ubc6e does not regulate interaction with the E3, c-IAPl. HEK293 cells were transiently transfected with pCMV (vector control), myc-tagged Ubc6 and myc-tagged

Ubc6e variant expression vectors. Immunoprecipitation with anti-myc antibody, followed by anti-cIAPl immuno-detection, indicates specific c-IAPl binding to Ubc6, but not to the Ubc6e variants, right panel. Expression of endogenous and heterologous proteins were confirmed by anti-cIAPl and anti-Myc immuno-detection of whole cell extracts (WCE), respectively, left panels.

115 3.4 Discussion

My observations that diverse environmental stresses, such as ER-, oxidative-, hyperosmotic-, pharmacologic- (staurosporine) and genotoxic-stress agents, as well as heat shock treatment, and certain mitogens, could promote rapid phosphorylation of Ubc6e in mouse embryonic fibroblasts indicates that modification of this E2-conjugating enzyme is not limited to ER folding stress, but is a common feature of multiple stress pathways. That certain stressors could elicit especially robust and sustained phosphorylation (genotoxic agents, hyperosmotic shock) compared to the relatively mild response of ER stressors, gives further indication that regulation of ER homeostasis is not the only or even primary role of this modification. The short latency and robustness of the event (maximum phosphorylation

Inhibitor studies revealed that Ubc6e phosphorylation, under stress or mitogen stimulation, proceeds largely via p38 MAPKa/p. These kinases mediate response to a wide variety of environmental stress conditions, cytokines and mitogenic agents (reviewed in [432]).

Depending on the stimulus and cell-type, different upstream MAPKKKs can activate MKK3 and MKK6 (MAPKKs), which directly phosphorylate and activate p38 MAPK. For example, the mammalian MAPKKK, MEKK3, activates MKK3 leading to p38 activation during hyperosmotic shock [433]. Another MAPKKK, ASK1, promotes p38 and JNK activation in response to cytokines and oxidative stress (reviewed in [434]), and during ER stress induced by polyglutamine [267]. I observed that modification of Ubc6e is PERK-dependent during thapsigargin and DTT treatment (Fig. 3.IB, Section 2.3.4), suggesting that PERK is required for p38 MAPK activation during ER stress. Supporting this notion, Liang and co-workers have 116 recently demonstrated that PERK is involved in p38 MAPK activation, by monitoring p38 activation during thapsigargin and DTT induced ER stress in perk'A MEFS [435]. Whether

PERK achieves this by activating upstream ER stress-related MAPK activators, like ASK1, by either eIF2a-dependent or independent mechanisms, has not been demonstrated.

I have found that MK2, a target kinase of p38, is the primary and direct kinase involved in stress-related phosphorylation of Ubc6e. MK2 is known to play an essential role in cytokine production during inflammation [436,437] and is also involved in the regulation of the cell- cycle at p53 and Cdc25 check-points [429,438]. Further, MK2 has been implicated in stress- resistance, as MK2-deficient mice exhibit increased resistance to endotoxic shock, impaired production of cytokines such as TNF [436,437], and also increased resistance to oxidative stress and heat shock [439]. Consistent with roles in stress response, I observed that phosphorylation of Ubc6e is associated with a pro-apoptotic outcome while the Ubc6e S184A variant affords some protection from this fate. From my cell culture model system with inducible high levels of expression of the Ubc6e variants (over endogenous levels), it was evident that individual features of the stressors themselves also participate in outcome, or extent of outcome (e.g. NaCl treatment induced greater cell death than thapsigargin, Fig. 3.4). However, it is notable that the levels of phosphorylation seen for endogenous Ubc6e in treated cells (Fig. 3.1), paralleled the levels of cell death seen; high levels of death and sustained Ubc6e phosphorylation were observed with hyper-osmotic shock, and the relatively low levels of cell death during ER stress correlated with modest phosphorylation levels and abbreviated duration. I further observed that this phosphorylation is directly associated with pro-apoptotic ER stress signaling, as expression of S184A Ubc6e could dampen the activation of JNK, whereas expression of the phosphomimic

S184E variant enhanced caspase-12 activation. Processed caspase-12 is reported to cleave pro- caspase-9, and consequently activate a caspase cascade leading to cell death [364,365].

Activation of JNK is a common response to many stress stimuli and can influence cell fate by 117 activating pro-apoptotic transcription factors and via regulation of BCL-2 family proteins [349].

Together, these data indicate that MK2-mediated phosphorylation of Ubc6e can promote apoptosis, which is characterized by activation of JNK and caspase-12 during ER stress.

Post-translational modification of the enzymes in the ubiquitin-proteasome pathway and their substrates provides specificity in the regulation of protein degradation. In some situations, phosphorylation can direct interaction between E3 ligases and substrates or modulate E2 and E3 interactions [36,440,441]. E2s often possess unique amino- or carboxyl-terminal segments outside of their core catalytic domains that afford specific E3 binding [32,34], and there is at least one example where this interaction is directly mediated by E2 phosphorylation [36]. Ubc6e is located at the ER membrane by its C-terminal transmembrane domain (Section 2.3.6), and in collaboration with membrane localized E3s, catalyzes ubiquitination of misfolded membrane proteins [44,135]. I previously asked whether phosphorylation of Ubc6e could affect its ER membrane localization, stability or interaction with a known E3 partner, Parkin [135], and found that it did not alter these properties (Section 2.3.6 and 2.3.7). Ubc6e cooperates with at least one other E3 [44], and possibly several others involved in mammalian ERAD [213,215], so it is possible that phosphorylation regulates one or more of these specific E2-E3 interactions in

ERAD of misfolded membrane proteins. However, a role for this modification must now be broadened since it can be induced under conditions that are distinct from ER folding stress, including a variety of environmental stresses as well as certain mitogenic stimulation, and is associated with apoptosis, presumably under severe stress situations. Yeast Ubc6 is known to participate in E3 DoalO-mediated degradation of soluble and membrane regulatory proteins

[202], so stress-specific targets of Ubc6e may include similar cytosolic or nuclear targets.

Generally, the targeted degradation of stress signaling molecules is a common mechanism contributing to cell fate (reviewed in [3]). The endoplasmic reticulum is emerging as an important stage for pro-apoptotic signaling, as evidenced by the numerous factors and 118 signaling pathways that have been shown to be involved in ER stress-induced cell death

[415,416]. The broad nature of the stressors that appear to be linked to Ubc6e phophorylation further highlight the importance of the ER in physiological and stress responses. As Ubc6e resides at the ER membrane (Section 2.3.6), putative substrates targeted for degradation are likely located or recruited to the ER. The association of Ubc6e phosphorylation with a pro- apoptotic fate would also be consistent with the targeting of specific stress signaling molecules for degradation.

The apparent link between JNK and caspase-12 activation with phosphorylation of

Ubc6e implicates the degradation of upstream mediators of these pathways. I considered that

Ubc6e and its phosphorylation may regulate JNK and caspase-12 initiated apoptotic pathways via TRAF2 degradation, which has been shown to be essential for JNK activation during ER stress and a contributing factor in caspase-12 activation [341,361]. Examination of the role of

Ubc6e and its phosphorylation in regulating TRAF2 stability revealed that this E2 is likely not involved, and is a relatively poor binding partner E2 for the TRAF2 E3, cIAPl, compared to its paralog, Ubc6. Considering that Ubc6e has been shown to interact with cIAPl, in vitro [388], it is possible that Ubc6e and its phosphorylation may facilitate or regulate degradation events mediated by this or other members of the related IAP (Inhibitor of Apoptosis) family of proteins. The IAP family of proteins can suppress apoptosis by binding directly to caspases and

IAP antagonists or by targeting these pro-apoptotic proteins for ubiquitin-mediated proteolysis

(reviewed in [442]). As well, degradation of TRAF proteins by IAPs can influence downstream signaling events such as activation of NF-KB and JNK (reviewed in [442]). Several IAPs, including c-IAPl, C-IAP2, and XIAP, are involved in suppression ER stress-induced apoptosis

[443,444], so it will be of interest to investigate whether Ubc6e mediates any IAP-mediated degradation processes proximal to the ER membrane, including down-regulation of IAPs by auto-ubiquitination. Other attractive candidates for Ubc6e-mediated degradation are the 119 membrane localized BCL-2 family members. Different BCL-2 proteins exert pro-apoptotic and anti-apoptotic effects by regulating the release of apoptotic mitochondrial factors into the cytosol and coordinating Ca dynamics of the ER which in turn affect mitochondrial integrity and many other cellular processes, including caspase activation (reviewed in [345]). Pro- apoptotic Bax and Bak have also been shown to be required for IRE1 signaling to JNK [374].

Several anti-apoptotic and pro-apoptotic BCL-2 family proteins localize to the ER and are regulated by ubiquitin-mediated proteolysis [3,445], but so far, the enzymes responsible for their ubiquitination have not been identified.

In summary, I have discovered that phosphorylation of Ubc6e, an ERAD E2, is induced by a variety of ER and non-ER stressors and mitogen stimuli, indicating that multiple pathways can converge to modify the activity of this E2 conjugating enzyme (Figure 3.7). During ER stress, PERK signaling in involved, but Ubc6e modification is primarily controlled by p38

MAPK pathways, and directly involves the protein kinase MK2. My studies of Ubc6e phospho- variants have indicated that this modification is implicated in apoptosis signaling during stress.

The promotion of apoptotic events initiated at the ER membrane further suggests that Ubc6e may be involved in regulating the stability of key apoptosis regulators at the ER. Investigation of these putative degradation events will be critical to understanding how phosphorylation of this E2 by a MAPK pathway influences cellular fate during stress.

120 Figure 3.7 Stress-related phosphorylation of Ubc6e by a p38/MK2 pathway is associated with apoptosis

J Diverse stress and j apoptosis I mitogen stimulation J i t

caspase cascade fc-JUNp) t y

1 *ASK>^ ^ \ -• p> (£) TRAF2 Nil IRE1

ER cytosol

Schematic representation of MAPK regulation of Ubc6e activity and association with apoptotic signaling at the ER. Diverse stress and mitogen stimulation induces phosphorylation of Ubc6e by a p38 MAPK/MK2 pathway. During ER stress, PERK is required for activation of p38

MAPK by an undefined mechanism. Phosphorylation of Ubc6e is associated with apoptosis and this can involve caspase-12 and JNK activation during ER stress. This is presumed to involve modulation of Ubc6e activity and its ERAD processes (discussed in Section 4.2). Central mediators of stress signaling from the ER are putative targets of Ubc6e (discussed in Section

4.3), but TRAF2 does not appear to be one of these.

121 Chapter IV

DISCUSSION AND FUTURE DIRECTIONS

122 4.1 Distinct functionality between the mammalian Ubc6 and Ubc6e homologs: stress

response and sequence divergence

The studies presented in Chapters 2 and 3 now show that mammalian Ubc6 and Ubc6e are regulated in unique ways during ER stress and emphasize diversity in function between the

Ubc6 and Ubc6e subfamilies of E2s. Firstly, their transcriptional responses are distinctive, where induction of hsUbc6e occurs later and for longer periods during tunicamycin treatment than hsUbc6. This suggests that up-regulation of Ubc6e expression may be more important in

ERAD during sustained ER stress, whereas Ubc6 induction is involved in a more immediate response. How each gene is regulated by the transcriptional arm of UPR is not known, though

ATF6 and/or IRE 1-mediated induction is likely. Interestingly, the yeast orthologue is not UPR- inducible [410], indicating that transcriptional regulation of Ubc6 and Ubc6e has evolved to adapt to ER stress, and possibly other stresses, experienced in higher eukaryotes. Secondly, the observation of phosphorylation at Ser-184 reveals additional modulation of hsUbc6e that is apparently a common response to diverse forms of stress. This suggests that higher eukaryotes that possess the conserved MK2 phosphosite regulate Ubc6e activity, not only in response to ER stress, but to many environmental stimuli.

A certain level of redundancy between the E2s has been apparent from many studies and consequently, it was not appreciated how mammalian Ubc6 and Ubc6e homologues may be functionally distinct. Both mammalian members have been shown to participate in ER- associated degradation of misfolded and naturally short-lived membrane proteins, and soluble regulatory proteins [44,135,205,207-210,395]. The functions of these two homologs appear to overlap, as demonstrated in the examples of ERAD targets they apparently share (e.g. CFTR and T-cell receptor a-subunit [44,208,209]). Both are broadly expressed and are similarly localized to ER membrane and perinuclear regions (see Section 2.3; [207,208,395]). 123 The functional overlap observed between the Ubc6 homologues may be achieved through the utilization of common E3s. However, this has not been formally demonstrated, either with direct interaction or in vitro ubiquitination experiments. Alternatively, as ERAD substrates can be targeted by multiple E3s [44,135,205,446], distinct E3s may recruit Ubc6 and

Ubc6e to target the same substrate. Either way, Ubc6 homologues in concert with the various

ERAD E3s, with contributions from other E2s such as Ubc7, may act redundantly.

Concommitant action of redundant ubiquitination paths clearly complicates heterologous expression studies (for example, see Section 2.3.7), but are likely critical for ensuring degradation of potentially toxic misfolded proteins, such as Pael-R.

Although both mammalian Ubc6 and Ubc6e are involved in the degradation of several

ERAD substrates, it was not clear whether, or how, specific responsibilities may be divided.

From sequence analysis ([48]; Section 2.3.1), the mammalian homologues of S. cerevisiae Ubc6 can readily be identified. However, neither human Ubc6 nor Ubc6e, singly or together, can complement the ERAD deficiency of the yeast Ubc6 mutant [208]. Clearly, gene duplication and sequence divergence has given complexity to Ubc6 function and perhaps an aspect of function achieved with phosphorylation of Ubc6e at Ser-184 is required for successful complementation.

In addition to providing back-up in certain degradation pathways, the evolution of E2s has allowed for nuance and expansion in substrate repertoire. Certainly, it would appear that for some degradation pathways, one Ubc6 homologue may play a predominant role. For example, it was clearly observed that Ubc6, and not Ubc6e, is the preferred binding partner for the E3 c-

IAP1 (see Section 3.3.4). Cooperation with different E3s is also suggested from amino acid sequence analysis. Loop 1 and loop 2 regions of the catalytic domains of Ubc6 and Ubc6e differ in those particular residues that are predicted from X-ray crystallography studies to be critical in determining specificity of E3 interaction (Fig. A.l.l)[26,27]. Specifically, Phe-62 124 (loop 1) and Ala-108 (loop 2) of human Ubc6 are substituted to Met-62 and Ser-108 in human

Ubc6e. While these changes appear conservative, Phe-62 and Ala-108 are conserved in lower eukaryotes and are predicted to make extensive contacts with RING and HECT domains to impart E3 specificity [26,27]. Supporting this notion, loop 1 Phe-62 is conserved in all E2s known to support c-Cbl and HECT-E3-dependent ubiquitination to date [27,447], and when mutated, can confer the functions of other E2s [448]. Additional indication that Ubc6 and

Ubc6e may participate in distinct ubiquitination events comes from their highly dissimilar C- terminal extensions linking the catalytic and transmembrane domains (Section 2.3.1). In some cases, C-terminal extension of E2 is required to mediate E3 interaction [32-34]. As the Ser-184 phosphorylation site is in this region of Ubc6e, an attractive putative function of this modification could be regulation of E3 interaction specifically during stress, possibly directing substrate ubiquitination events. Other possible functions of the C-terminal extension and SI 84- phosphorylation are discussed below.

4.2 Phosphorylation of mammalian Ubc6e and regulation of E2 activity

4.2.1 Thiol-ester formation and substrate conjugation

To examine any effect of phosphorylation on E2 activity of Ubc6e, I evaluated both the capacity to form thiol-esters with ubiquitin and the effect on activity against a putative substrate

(Section 2.3.7 and 2.3.9). Phosphomimic variants had a reduced capacity to from thiol-esters, suggesting that phosphorylation may attenuate Ubc6e activity. Why overall activity of this E2 would be negatively regulated during stress, including ER stress, is not clear, considering its presumed importance in ERAD. An important limitation to the interpretation of these results is 125 that the Ubc6e variants in these assays were truncated prior to their transmembrane domains to enable solubility. Conformational changes induced by truncation may influence E2 activity and the native membrane environment may be required for the E2 to adopt its proper fold.

Nevertheless, these assays do suggest that phosphorylation affects some aspect of catalytic activity (discussed further in Section 4.3.2). It is also worth noting that these studies utilized phosphomimicking variants which may not be perfect models of phosphorylation. With the identification of the responsible kinase, MK2, in vitro studies can now be improved by utilization of purified phosphorylated Ubc6e protein.

When the activity of Ubc6e was tested against a reported substrate, Pael-R, it was observed that phosphorylation mutant expression did not change the turnover of this protein; in fact, even expression of the catalytic mutant did not perturb Pael-R turnover, indicating that other E2s, such as Ubc6 and Ubc7, play more prominent or at least redundant roles in the ubiquitination of this protein.

Continued investigations of how E2 activity may be modulated by phosphorylation will be essential for understanding the physiological relevance of this E2 in ERAD and stress response. This could involve evaluating Ubc6e activity on substrate degradation, with in vivo and in vitro approaches. Examination of in vivo target substrate degradation with heterologous systems like the one employed in Section 2.3.7 are generally complicated by the activities of endogenous redundant E2s and, as well, are susceptible to dominant-negative effects, as seen in yeast and mammalian cells [38,208]. siRNA technology should avoid these pitfalls in investigating the ERAD contribution of this E2, as was recently demonstrated [44]; knock-down of Ubc6e expression could stabilize CFTRAF508. However, such an approach would not address the function of phosphorylation. For this, the 'knock-in' of Ubc6e variants by gene targeting in cells or a mouse would be useful, where degradation of any number of suggested

126 Ubc6e ERAD substrates [44,135,205,207-210,395] could be examined in embryonic fibroblasts, and intact organs, respectively.

The absence of a well-characterized, in vitro substrate of Ubc6e remains a major impediment at assessing Ubc6e conjugation activity. Other groups have implicated Ubc6 homologues in the ERAD of various substrates by over-expression of a catalytic mutant, but my heterologous studies of Pael-R (Section 2.3.7) and CFTR (data not shown), have not shown a significant role for Ubc6 homologues. Examination of the degradation of total cellular membrane proteins may be a more global approach to identify changes in Ubc6e-mediated

ERAD, as was attempted (Figure 4.1), but such a system still possesses the pitfalls mentioned above (i.e. activities of endogenous E2s). Alternatively, with the recent identification of mammalian ERAD E3s, such as Gp78 and Hrdl (Hrdl homologs), Teb4 (DoalO homologue) and Rmal, the use of in vitro substrate ubiquitination reconstitution systems is now possible.

Such systems would allow for detailed analysis of Ubc6e involvement, and the effects of the presence and absence of phosphorylation. If Ubc6e has conserved some of the yeast orthologue's functions, then examination of its activity and phospho-mutant effects against known Ubc6 targets in S. cerevisiae may be also be considered.

As suggested from the in vitro thiol-ester results, the C-tail region of Ubc6 homologues may influence the catalytic activity. The change in thiol-ester formation is not entirely

surprising as studies have shown that, in other E2s, C-terminal extensions can modulate aspects

of ubiquitin chain assembly and access to the catalytic cysteine ([411-413]; Section 2.4). For one E2, its C-tail region can interact with its catalytic region as indicated by NMR-determined

structure, possibly protecting the catalytic cysteine [413]. For some E2s, their C-tails are

important for non-covalent binding of ubiquitin, which can have effects on poly-ubiquitin chain

formation (discussed below). The C-terminal extension of Ubc6 E2s contain a transmembrane

domain and highly positively charged segments ([25,48], Figure 4.2, A. 1.1), which are features 127 Figure 4.1 Strategy and preliminary results for pulse-chase analysis of total membrane protein turnover during ER stress

B A Wt C91S S184A DOX + -- + -- + TG + + - ++ - + + 1. Express Wt, S184A,C91S (Tet-on CHOK1 cells) + Flag-ubiquitin 2. Pre-treat cells with proteasome inhibitors (MGI32, lactacystin) 3. Stress cells with thapsigargin Flag IP of 4. Pulse labeling with S-35 Met and Cys: P100 fraction pulse 30min, chase 30min 5. Collect cells, normalize for incorporated radioactive label 6. Fractionate cells to enrich for membrane fraction 7. Immunoprecipitate Ubiquitin (Flag) 8. SDS-PAGE, 4-12% gradient

-— - -calnexin

P100 fraction - p-tubulin

mm -— myc-Ubc6e

S100 fraction «^t«|«rai««WGFP

A, Protocol for an in vivo ubiquitination assay. This approach was used to examine the effect of

Ubc6e variant expression on the ubiquitination of total cellular membrane proteins during thapsigargin treatment.

B, Preliminary results suggested that Ser-184 phosphorylation of Ubc6e was necessary for ubiquitination of certain membrane proteins (arrowheads). Enrichment of membrane fraction

(PI00) was confirmed with immunoblot detection of ER resident protein calnexin and Ubc6e and absence of P-tubulin. GFP was a transfection control.

128 Figure 4.2 hsUbc6e predicted secondary structure (SSpro, UC Irvine)

METRYNLKSPAVKRLMKEAAELKDPTDHYHAQPLEDNLFEWHFTVRGPPDSDFDGGVYHGRIVLPPEYPMKPPSIIL CCCCCCCCCHHHHHHHHHHHHHCCCCCCEEECCCCCCCEEEEEEEECCCCCCCCCCEEEEEEEECCCCCCCCCCEEE

LTANGRFEVGKKICLSISGHHPETWQPSWSIRTALLAIIGFMPTKGEGAIGSLDYTPEERRALAKKSQDFCCEGCGS ECCCCCCCCCCCEEEEHCCCCCCCCCHHHHHHHHHHHHHHHHCCCCCCCCCCHHHHHHHHHHHHHHHHHHHHHHCCC + * AMKDVLLPLKSGSDSSQADQEAI^ELARQISFKABVNSSGKTISESDLNHSFSLTDLQDDIPTTFQGATASTSYGLQN CCCCCCCCCCCCCCCCHHHHHHHHHHHHHHHHHHHCCCCCEEEHHHCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCC

SSAASFHQPTQPVAKNTSMSPRQRRAQQQSQRRLSTSPDVIQGHQPRDNHTDHGGSAVLIVILTLALAALIFRRIYL CCCCCCCCCCCCCCCCCCCCHHHHHHHHHHHHHCCCCCCCCCCCCCCCCCCCCCHHHHHHHHHHHHHHHHHHHHHHH

ANEYIFDFEL HHHHHHHCCC

Beginning with Q170 (+), this stretch of residues (in red) appears to be an amphipathic helix, as charged and aliphatic residues are spaced from other charged and aliphatic residues by 3-4 residues. The phosphorylation at Serl84 (*) would appear to introduce negative charge near the predicted hydrophobic face of this helix. C, coil; H, helical; E, sheet.

129 unique to this E2 family. Phosphorylation at Ser-184 of Ubc6e introduces negative charge and based on secondary structure prediction would occur near the hydrophobic face of a predicted amphipathic helix (Fig. 4.2). This could disrupt packing of this domain into the hydrophobic core, or perhaps lead to disrupted association with membrane. Thus, phosphorylation may alter the activity of this E2 by inducing a conformational change. Determining the protein structure of Ubc6e would help to understand how this E2's C-tail and phosphorylation influences its catalytic activity. Because membrane proteins are generally not amenable to crystallization, a transmembrane deleted version could be studied initially, although this may not be ideal (i.e. truncation itself could induce conformational changes ).

4.2.2 E3 binding

If the repertoire or general activity of Ubc6e is altered during stress by Ser-184 phosphorylation, then modulation of interactions with partner E3s would be an anticipated consequence of this modification. Regulated interactions with a particular E3 or multiple E3s, may lead to a modified repertoire of target substrates, including the E3 itself. The C-terminal extensions of other E2s have been shown to be required for E3 binding [32-34].

Phosphorylation had no apparent effect on binding to one known partner E3, Parkin (Section

2.3.7), but other ERAD-related E3s such as Hrdl and DoalO homologs have not been tested.

Given the stress-specific nature of this modification, it is also possible that Ubc6e can interact with a non-traditional ERAD E3 to mediate degradation of a specific stress-related target or targets. An attempt to identify E3s by yeast two- hybrid interaction screens with phosphomimicking variants did not return significant or plausible partners. As discussed in

Section 2.4, the inability to uncover such interactions may be due to the inherent limitations of the system, where native membrane localization can not be re-created. Furthermore, 130 interactions are anticipated to be regulated only during conditions activating p38/MK2, and perhaps other post-translational modifications, of E3s for example, may be required; these may not be conserved in yeast. Other gene reporter systems [414] or column affinity approaches with mammalian cell extracts (representing stressed and unstressed conditions) may uncover relevant interactions.

Generally speaking, productive E2-E3 binding events probably involve more than the known interactions, such as those described in structural studies ([26,27] and Section 1.1.2.2) and simple in vitro binding assays. Although many E2s can interact with many E3s in vitro, it has been shown that binding of E2 is not necessarily sufficient for successful E3 directed substrate ubiquitination. UbcH5 and UbcH7 demonstrate equal binding affinity for the RING domain of BRCA1, but only UbcH5 supports ubiquitination [449]. Also, there is recent evidence that RING E3s may induce subtle conformational changes in a bound E2, stimulating the release of ubiquitin from the E2 cysteine and transfer to substrate [450]. Thus, it is possible that binding to a specific E3 induces activation of the conjugation activity of Ubc6e. However, it would appear that phosphorylation modification of Ubc6e is not required for its conjugation activity, as endogenous (and presumably unphosphorylated) Ubc6e has been implicated in the degradation of CFTR under non-stress conditions [44]. The binding of an E2 to specific E3s may, however, influence other aspects of conjugation, such as poly-ubiquitin chain formation by organizing E2 self-association (next section).

4.2.3 Other aspects of E2 activity and regulation: Multi-ubiquitin chain formation,

multi-step substrate ubiquitination

The mechanisms of substrate conjugation and poly-ubiquitination chain formation are aspects of E2 activity that remain to be completely elucidated [76,451]. It is generally thought 131 that a single E2 is brought by E3s into proximity with substrate. This is readily demonstrated in modelling of the UbcH7-c-Cbl interaction onto the SCFskp2 structure which reveals that the F-

Box Skp2 positions the substrate in a favourable orientation to accept ubiquitin from the E3- bound E2 [452,453]. As discussed in Section 1.1.2.2, El binding, formation of the ubiquitin thiol-ester bond, E3 binding and catalysis of ubiquitin transfer to substrate are essential properties of the E2 catalytic core. Currently, it is uncertain how ubiquitin moieties are attached to the growing end of a poly-ubiquitin chain or how the type of linkage between ubiquitin moieties is determined. These processes may be determined by the individual E2. For example, certain E2s clearly function in monoubiquitination while others tend towards polyubiquitination

[454,455] leading to a coordinated mechanism of chain elongation (discussed below). Other

E2s appear to support both initial attachment of ubiquitin and chain elongation [456]. Also, certain E2s apparently function only in the synthesis of specific ubiquitin chain linkages; the

Ubcl3-Mms2 heterodimer [78,457] and E2-25K [412] direct the synthesis of Lys-63- or Lys-

48-linked polyubiquitin chains, respectively. As Ubc6 is only known to participate in ubiquitin- mediated proteolysis, it is assumed that this E2 directs exclusively Lys48-linked polyubiquitin chains, although this has not been formally demonstrated (and has rarely been confirmed for any in vivo ubiquitination substrate). At least in vitro, it is known that the linkage type of ubiquitin chains can be determined by the E3 [458], and thus it cannot be ruled out whether Ubc6 may function to form alternate types of ubiquitin linkages when partnered with particular E3s.

Poly-ubiquitin chain synthesis can be observed to be highly processive (see examples

[456,459]), where high molecular weight ubiquitination products rapidly accumulate with little evidence of lower molecular weight intermediates. Some E2s, including yeast Ubc6 and Ubc7, can build multi-ubiquitins either on themselves [410,411,460,461], or as unanchored chains

[462]. Recently, it was observed that poly-ubiquitin chains can be pre-assembled directly on the catalytic cysteine of Ubc7, in vivo, and its mammalian homologue, in vitro [460,461]. These 132 studies indicated that Ubc7-linked ubiquitin chains are assembled by multiple rounds of transfer reactions between E2 pairs, with each member bound by ubiquitin, in a process mediated by an

E3. This poly-ubiquitin chain was then shown to be transferred directly to a substrate [461], or could remain attached to signal the degradation of the E2 itself [460]. Other mechanisms of multi-ubiquitin chain assembly involve the synthesis on HECT E3s and RING-like domain proteins called E4s (reviewed in [76]).

In vitro ubiquitin thiol-ester assays have not indicated that Ubc6 or Ubc6e, in its unphosphorylated or phosphomimic forms, can acquire multiple ubiquitins (Figure 2.1 and 2.9, and data not shown). However, since the in vivo poly-ubiquitination of yeast Ubc6 is dependent on the E3, DoalO, and fellow ER-associated E2, Ubc7 [41], the presence of E3 (such as Teb4, a putative DoalO homologue), and Ubc7 homologue in the in vitro reaction may be required for ubiquitin chain synthesis. Interestingly, the catalytic mutant of Ubc6 is not observed to be poly- ubiquitinated, but rather, is mono- or di-ubiquitinated [410]. These data suggest that Ubc6 may build ubiquitin chains on itself, in conjunction with DoalO, and perhaps only does so after Ubc7 administers an initial mono- or di-ubiquitin. Thus, for some substrates that require both Ubc6 and Ubc7 for their ERAD, the E2s may act in a concerted manner to initially produce polyubiquitin chains on Ubc6 (or Ubc7, which is also capable of extending poly-ubiquitin chain synthesis on itself in an E3-dependent manner [460,461]).

The cooperative synthesis of ubiquitin chains may explain why in both yeast and mammalian systems, the reduction or over-expression of catalytic mutant Ubc6 or Ubc7 can delay ERAD of a particular substrate [123,125,205,210]. In other degradation pathways, the use of multiple E2s has been shown to comprise a two-step chain assembly mechanism, where one

E3-E2 complex places the initial ubiquitin, after which, an alternate E3-E2 complex extends the chain [45,455]. For example, Ubcl and Ubc4 are both required for degradation of APC-targeted substrates; Ubc4 can promote rapid cyclin monoubiquitination and Ubcl catalyzes K48-linked 133 chain synthesis on attached ubiquitins [455]. Cooperativity between Ubc6 and Ubc7 can readily be envisioned since both are ER-localized and can be utilized by the same E3 for certain substrates [41,205]. The redundancy observed between the E2s may also arise out of separate

E3-E2 pairings that form ubiquitin chains independently.

Further supporting the notion of E2 cooperativity, is the observation that Ubc7 can interact with itself, as well as with Ubc6, as was determined by a yeast two-hybrid interaction

[40]. It is not currently known whether Ubc6 and Ubc7 association is required for multi- ubiquitin chain synthesis in vitro or is relevant to ERAD. Numerous E2 enzymes have been reported to self-associate, in vitro (for example [463-465]), and dimer formation of ubiquitin-E2 thiol-esters can be a pre-requisite to multi-ubiquitin formation [79]. Structural studies suggest that non-covalent binding of ubiquitin by E2s is required to orient donor ubiquitin moieties to allow poly-ubiquitin chain formation by these dimer complexes [457,466,467]. Moreover, analysis of various E2s and their ability to mediate auto-ubiquitination of an E3, has shown that poly-ubiquitination activity correlates with ability to bind ubiquitin non-covalently at a site distal to the active site [454].

Considering these lines of evidence, it is conceivable that phosphorylation of Ubc6e may affect chain formation by modulating interaction with other E2-ubiquitin thiol-esters, or perhaps with a complex-organizing E3. Phosphorylation may also modulate some intrinsic chain assembly property of this E2. Other type II E2 enzymes utilize their C-terminal extensions to modulate their ability to bind E3s [32-34], ubiquitin chain assembly [411,412,468] and self- association [464]. Poly-ubiquitin chain forming activity of Ubc6e could be evaluated in an in vitro ubiquitination assay of an ERAD substrate, such as human deiodinase [210,469], whose targeting E3s are now known [202,470], and the role of Ser-184 phosphorylation in the modulation of Ubc6e activity, by studying phospho-variants, could be examined. The

134 requirement for cooperative E2 activity between Ubc6e and Ubc7, or Ubc6e and Ubc6, could be examined in this in vitro system.

4.2.4 A role for Ubc6e phosphorylation in the regulation of protein degradation at the

ER

Observations of long half-lives and continued presence at the ER membrane in unphosphorylated and phosphorylated states suggest that modified Ubc6e can still participate in

ER-localized protein degradation (Section 2.3.6). The in vitro ubiquitination assays proposed in the previous sections would address E2 conjugation activity on substrate, as well as function for poly-ubiquitin chain assembly. If phosphorylation can regulate some aspect of Ubc6e's function in ubiquitin conjugation and chain assembly, either intrinsically, or by modulating protein interactions, this could have effects on processivity and efficiency of poly-ubiquitination of ERAD substrates. The requirement of PERK signaling during ER protein folding stress suggested a possible role for this modification in maintaining ER homeostasis, for example, by enhancing general ERAD. The answer of whether and how phosphorylation of Ubc6e affects

ERAD will be answered by examining its activity against specific substrates, or in total membrane fraction assays that analyse global membrane protein ERAD (as described in Figure

4.1)

Besides Ubc6e, only a few other examples of regulation of E2s by phosphorylation have been reported. Phosphorylation of the cell-cycle E2 Cdc34 in its C-terminal extension by protein kinase caseine kinase-2, can apparently alter a number of aspects of function, including localization [471], in vitro binding with an SCF E3 [36], and in vitro conjugation activity on a

GST-Ub pseudo-substrate (E3-independent) [472]. This modification is associated with enhanced E3-dependent in vitro ubiquitination of Sicl [472] and degradation of P-catenin [36]. 135 Phosphorylation of other E2s at specific sites in their catalytic domains are known to increase or decrease activities against a substrate [473,474], or reduce their ability to form a thiol-ester with ubiquitin [473].

Post-translational modifications play important roles in regulating ubiquitin-conjugation pathways. Rapid and tight modulation of specific ubiquitin-conjugation events by these modifications can couple extracellular stimuli and cellular processes, such as cell division, with degradation of select targets. As described in Section 1.1.3, phosphorylation and other forms of substrate modification can induce recognition by E3 ligases. The stress-related nature of this modification would suggest that substrates ubiquitinated by this E2 could be involved in stress- response, and their degradation may be an important regulatory event. Furthermore, that phosphorylation of Ubc6e can be induced by numerous stress and mitogen conditions that do not cause classical ER stress, and is associated with MAPK signaling and apoptosis, suggests that substrates of this E2 may include stress-signaling proteins (discussed further in Section 4.5).

Phosphorylation could possibly enhance or diminish binding to the E3 that targets a stress- signaling molecule, depending on the role of the target in promoting survival or cell-death. For example, in un-stressed conditions, an E3 and Ubc6e may mediate substrate ubiquitination from the ER at a steady rate, but reduced binding or dissociation due to phosphorylation of Ubc6e during stress would lead to increased stabilization of the target substrate. With this scenario in mind, binding to the E3 c-IAPl and degradation of pro-apoptotic TRAF2 was examined

(Section 3.3.4). And the converse could also occur, where in un-stressed conditions, a pro- survival factor is stable, but stress-induced formation of the phosphorylated Ubc6e-E3 complex targets its degradation. In either case, modification of the substrate during stress, such as phosphorylation, or a conformational change to a 'misfolded state' as seen with all Ubc6 targets, may occur to enable substrate recognition by an E3. Mediating auto-ubiquitination of the E3 is another way Ubc6e and its phosphorylation could regulate substrate degradation. Yeast and 136 mammalian Ubc6 are known to participate in the degradation of soluble regulatory proteins

[388,460], so stress-specific targets of Ubc6e may include cytosolic or nuclear targets. This would require substrate (and possibly E3) translocation to the ER membrane, as has been reported with c-IAPl and TRAF2 [388], to enable proximity to Ubc6e

4.3 A role for Ubc6e in regulating stress and apoptosis signaling at the ER membrane

Consistent with their role in ERAD, I have observed that the Ubc6 homologues are up- regulated during ER stress (Section 2.3.2) and this likely contributes to the enhanced ERAD required to maintain homeostasis. Numerous yeast E2s are required for surviving conditions that promote protein misfolding such as cadmium toxicity [475], or heat-shock and amino acid analog incorporation [71]. As well, other ERAD factors are required for maintenance of ER homeostasis, and their UPR induced expression during ER stress is often essential to cell survival (see Section 1.3.6).

Interestingly, stress-induced phosphorylation of Ubc6e is not associated with increased survival, but rather, a pro-apoptotic fate, as indicated by increased cell death with elevated caspase levels in CHO-K1 cells (Section 3.3.3, 3.3.4). Furthermore, during ER stress, this is characterized by INK phosphorylation and caspase-12 cleavage, suggesting that degradation of substrate or substrates by Ubc6e likely contribute to the regulation of these signaling events.

These findings should be confirmed with knock-down studies that remove the endogenous

Ubc6e gene (e.g. by siRNA). Possibly, knock-down of endogenous Ubc6e would suppress stress-induced apoptosis, a phenotype that should be rescued by wild-type but not S184A expression. Of course, Ubc6e has a presumed role in maintaining ER homeostasis by mediating

137 quality control related ERAD, thus knock-down could itself cause ER stress and sensitize the cells.

The association of Ubc6e phosphorylation with JNK and caspase-12 activation during

ER stress suggests that an ER proximal event, such as TRAF2 signaling, could be regulated by

Ubc6e. Study of phospho-mutant over-expression revealed that Ubc6e is likely not involved in the degradation of TRAF2, and compared to homologue Ubc6, Ubc6e is not a preferred binding partner E2 for the TRAF2 E3, cIAPl (Section 3.3.4). These studies only assessed steady-state levels of TRAF2 from whole cell protein extracts; it is possible that effects on TRAF2 turnover occurring specifically at the ER membrane would not be measurable in this analysis.

Considering that Ubc6e has been shown to interact with cIAPl, in vitro [388], it is possible that

Ubc6e and its phosphorylation may facilitate or regulate degradation events mediated by this or other members of the related IAP (Inhibitor of Apoptosis) family of proteins (as discussed in

Section 3.4).

The regulated release of calcium ions from the ER is another event that is known to lead to JNK and caspase-12 activation. In response to ER stress, caspase-12 can be activated by numerous other mechanisms (Section 1.3.5.4, see Figure 1.4), including direct cleavage and activation by calpains [363], which are Ca -dependent cysteine proteases [476]. Genetic deletion of a calpain regulatory subunit, capn4, from mouse embryonic fibroblasts resulted in defective caspase-12 activation, as well as JNK activation [477]. Caspase-12 activation also relies on calcium release from the ER mediated by BAX and BAK BCL-2 family proteins

(Section 1.3.5.5 and [370,371]). Thus, release of calcium from the ER appears to initiate certain

ER-related apoptotic processes, such as calpain activation. Calcium ions that are released from the ER are also taken up by the mitochondria triggering cytochrome c release and apoptosome formation (reviewed in [356]). Downstream of apoptosome formation is activation of effector caspases 3 and 7, which can also cleave and activate caspase-12 [362,478]. 138 I have seen that Ubc6e is phosphorylated in response to various stimuli, including ER-, oxidative-, genotoxic-, hyper-osmotic stresses, heat shock and mitogens (Section 3.3.1). These stimuli are known to lead to the release of calcium ions from the ER lumen via inositol 1,4,5- triphosphate receptors at the ER membrane (reviewed in [479]). BCL-2 family proteins BAX,

BAK and BCL-2 are key regulators of calcium release. Interestingly, various BCL-2 family proteins have been shown to be targeted for ubiquitin-mediated proteolysis from the ER in response to different stimuli [445,480], although the ramifications on calcium ion release and apoptosis have not been studied. To date, the enzymes responsible for their ubiquitination have not been identified. It is tempting to speculate that Ubc6e and its stress-induced modification may affect turnover of such central regulators of ER-related calcium signaling, thereby modulating the activation of pro-apoptotic events like JNK activation and caspase-12 activation.

The fact that Ubc6e phosphorylation can be induced by so many different stimuli does suggest that a central regulator in apoptosis signaling that is ER-proximal, such as BCL-2 proteins, may be a target of this E2. An analysis of BCL-2 family protein stability in Ubc6e knock-down and cDNA rescue experiments (wild-type and SI84A) would provide initial indication of the involvement of this E2 and the role of its phosphorylation.

Generally, the targeted degradation of stress signaling molecules is a common feature of pathways contributing to cell fate [3]. Tumour suppressor protein p53 and IkB are well documented examples of exquisitely regulated substrates with immense influence on cell fate

[2,481]. The apparent links between Ubc6e phosphorylation and apoptosis further elaborate the role for the endoplasmic reticulum as an important stage for pro-apoptotic signaling, as discussed in the Section 1.3.6. and [415,416]. Degradation of apoptosis signaling proteins at the

ER membrane, as has been reported for BCL-2 proteins (above) and also p53 [482], may represent an important regulatory aspect of apoptotic signaling.

139 4.4 p38/MK2 regulation of Ubc6e

4.4.1 p38 MAPK and ER stress signaling pathways

The identification of the kinase responsible for Ubc6e phosphorylation, the p38 MAPK regulated kinase MK2 (Section 3.3.2), draws compelling connections between MAPK signaling and ER homeostasis, and further illustrates the importance of the ER in physiological and stress responses. From these studies (Figure 3.1) and others [435], the signaling from PERK appears to be a necessary upstream component in activation of the p38 MAPK/MK2 pathway during ER stress. PERK is considered essential for maintaining ER homeostasis but can also initiate pro- apoptotic and inflammation response (Section 1.3.3 and 1.3.6). PERK-mediated activation of p38 MAPK, by a presently undefined mechanism (discussed in Section 3.4), would transmit ER stress signals to a plethora of other signaling pathways.

The role of p38 MAPK in ER stress and ER stress-induced apoptosis is unclear. p38

MAPK signaling is known to target several signaling molecules during ER stress, and including pro-apoptotic transcription factor CHOP [346,419,483], the UPR signaling component ATF6

[420], immediate early genes c-myc and c-jun [435]. My findings now indicate that this stress regulated kinase may regulate ERAD-processes via modification of Ubc6e, with an apparent net decrease in cell survival.

4.5.2 MK2: a stress-responsive kinase

As the p38 MAPK inhibitor can block Ubc6e phosphorylation robustly under all stress and mitogen conditions tested, it would appear that only p38 MAPK regulated kinases are involved (Section 3.3.2). I have further seen that, at least in mouse embryonic fibroblasts and in 140 response to ER and oxidative stress and hyperosmotic shock, Ubc6e is phosphorylated primarily by MK2 (Section 3.3.3). MK2 is likely to play a primary role in Ubc6e phosphorylation in most tissue and under most conditions, as MK2 is broadly expressed [484], and is known to be activated by p38 MAPK in response to various environmental stresses, such as heat shock, osmotic stress, UV irradiation, sodium arsenite, anisomycin, bacterial lipopolysaccharide (LPS), or when stimulated with cytokines interleukin-1 or tumour necrosis factor (TNF), and certain mitogens (reviewed in [423,432], [485]). It remains a possibility that in certain cell-types and under certain stimuli, p38 MAPK-regulated kinases other than MK2 may be involved, such as the structurally related MK3 [424,425] or the MSK and MNK family of kinases (reviewed in

[422]). MK3 shares many of the same substrates as MK2 (reviewed in [423]), but has lower activity in vivo, likely due to lower expression [486]. MSKs and MNK kinases are both activated by ERK and p38, and are known to regulate gene transcription during inflammation and general translation, respectively (reviewed in [422]).

MK2 is known to relay p38 MAPK signaling to numerous cellular targets that are involved in cytokine production, cell-cycle checkpoint, actin remodeling and motility (reviewed in [423], discussed in Section 3.4). MK2 has a clear role in stress-resistance, as MK2-deficient mice have increased resistance to endotoxic shock [436,437], probably due to impaired production of cytokines, such as TNF, and also increased resistance to oxidative stress and heat shock [439], which is believed to be related to modulation of protective chaperone properties

[439,487]. Phosphorylation of Ubc6e has now also been associated with a pro-apoptotic fate whereas overexpression of the unphosphorylated version leads to increased survival, providing a novel component of the p38-MK2 pathway during stress.

It will be interesting to examine how this modification mediates any of the known MK2- dependent processes. In particular, MK2 is a key mediator of p38 MAPK regulation of pro­ inflammatory response (as discussed above). Study of immune cells (e.g. macrophages, 141 lymphocytes) of Ubc6e mice (knock-out or knock-in phosphovariants) may reveal a role for this

E2 in the survival of these cells in their differentiation or in face of infection. It is known that

ER stress signaling events are well connected with inflammatory response, such as PERK and

IRE-mediated NF-KB induction (discussed in Section 1.3.6.1), and caspase-12 and CHOP activities have been linked to apoptosis during bacterial and lipopolysaccharide-induced infection [488,489].

4.6 Physiological implications of Ubc6e phosphorylation

Although cell culture models of phospho-mutants have revealed some details regarding general stress survival and signaling events, it will be required to examine the functional relevance of Ubc6e phosphorylation at an organ or model organism level to evaluate the physiological ramifications of this modification. Sequences currently available from public databases indicate that only tetrapod vertebrates (air breathing, four-legged animals), including amphibians, reptiles, birds, and mammals, but not fish, possess the Ser-184 phosphorylation site

(Figure A. 1.1). This suggests that phosphorylation at this site evolved as an adaptation to the terrestrial environment, which would impose new physiological demands and cellular stress responses. This would also appear to preclude the capacity of certain lower eukaryotic model organisms, such S. cerevisiae and C. elegans, for the study of this modification's function.

Gene-targeted mice that carry the phospho-mutant variants S184A and S184A, however, would be extremely useful for investigations of the significance of the modification in development, general tissue homeostasis, and for modeling of various stress responses. For example, from the perspective of ER stress, it will be interesting to see how constitutive or blocked Ubc6e phosphorylation will affect the development and homeostatic regulation of organs susceptible to 142 folding stress, like the pancreas or B-cells. As this modification appears to be linked to apoptosis, it will be interesting to examine whether a phospho-mimic expressing mouse would lead to, for example, pancreatic p-cell death, and concomitant diabetes, and conversely, whether a phospho-mutant expressing mouse would protect comparable cells from pharmacologically induced ER stress-related death. Contributions to MK2-dependent processes could also be examined (as discussed above).

An ever increasing number of disease states are being reported to exhibit enhanced ER stress signaling, often with the up-regulation of apoptotic pathways, and these may play significant roles in the disease pathology (Section 1.3.8, Table 1.2). Ubc6e phosphorylation may represent a significant cellular determinant in cell survival in such disease states. Further elucidation of the Ubc6 E2s during stress should be pursued and will offer insight into these various diseases.

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180 APPENDICES

A.1 Supplemental data to Chapter II

A.2 Supplemental data to Chapter III

181 Figure A.1.1 Multiple sequence alignment of Ubc6 and Ubc6e family members

MmUbc6e 1 METRYHLKSPHVEg HsUbc6e 1 METRYHLKSPHVEE GgObc6e 1 MEARYMLK3PHVKE ItDbc«« 1 DrDbc6« 1 MEBKYNLKSP0VK CeObcSe 1 HSBQYNTKNAGVR NcI7bc6« 1 —XATPKFNSKSPTIR AtDbcSe 1 HADERYHRKNPH X10bc6 1 --HSHHSHERAPTT TQ GgUbc6 1 --MSHH6HKRAPTT TQ HmObc6 1 --KSHNSHERAPTT TQ BsObc6 1 - -KSSTSSKRAPTI TQ DmDbc 6 1 H3S9STSGGRKQPT VS ScObc6 1 - MATXQQBE AtDbcS 1 MAEKACIK L2 MmUbc6e 91 TP! EXHQDHCCEGCGSAMEDVLLPLESGSGSSQADQEAEBLARQISF BsUbc€e 91 TP1 KKHQDRACCEGCGSAMKDVLLPLKSASDSSOADQEAKELARQISF GgUbc6e 91 IYTP1 KKLQDG|CCEHCGTSHKTALLPLTSGSGS5QADKEAKEI.ARQ]SF XtUbc6e 76 JYTPI KRHQDYFCEVCGVNMKCTLLPLSS- -SS3QADVEARELARQI|S1F DrUbc6e 91 9YTPEJ3J KKGQD0CCEACSSCMRBALLPLSP--DSDLRPADPQRLAQQINF CeUbc6e 91 |YPP1 ELHCEHKCEECGCVMETAIII.PITG-DGQLEQTEEAKTLAAQLKF HcUbc6e 93 TEA1 JRAHKCQACGKTHEEII»BCEGAAKEBQGBGQAEVBVPSDIIIC AtUbc6e 93 |YPXI IEGRETPPEYGSPERQEXIDEIBQYIIISEATVVPEPLPI.ECSQA XlDbc6 94 IETSDFT] TH-I.EDEVFCELFPEVVBjaiEQEQR- Ggt7bc6 94 IET3EPA] JH-LICDKVFCELFPKVVB llEQKQE MmUbc6 94 IETSDFT 1EKQ[ M-LEDKVFCELFPEVVE BIKQICQK SeUbc6 94 IBTSDFTKQQI H-LEDXVFCELFPEVVE |lEQKQE DmUbc 6 96 IESENYDKQHF H-LRNTHFCELFPEIVESiEQRLRGTQAA ScUbe6 87 ITTSDHQEETJ RHHlSYHTFQHVRFELIFPEVVQ@HVETLEK AtUbc6 87 HTSVA@KQR| ES@LA0N-CESVTFRELFPEYVEEYSQQQV- --

MmtJbc6e 186 EAEVNSSGETIAE SDLNQCFSLHDSQDDLPTTFQ3ATASTSYGAQNPSGAPLPQPTQPAPKglTSMSPRQRRAQQQSQRRPSTSPDVLQGQP HsUbcSe 186 KAEVHS8GKTI3E SD&NHSFSIiTDLQDDIPTTFQGATASTSYGLQHSSAASFBQPTQPVAEfflTBlISPRQRRAQQQSQRRLSTSPDVIQGHQ GgUbc6e 186 XAEVHSSRESEAEPSHSAGLHCSATSPEPQQDGATQAFRGPASAMSETQPSSTAPGQERAP8VPTfflTSI.SPRQRRAQQEBQRWAPASTDFHRVQQ XtObc6e 169 EAEVHPPGSAQGAE NPPHQSPETMEPHABTBBETEAADASTSAGQETSPPTP STJJJVSRSPRQRRAQQ RRIPASTGLIQVQQ DrUbc6e 184 KAESSSSAADSSS SBSAAAEVFBNTSESSETADQDTSDTSAAEQTE88SVAPQTSAEDPSVSAAPSPRQRRSQHQSPRSPVFPASPAP--P CeUbc6e 185 QDESVVEEEVEAAN HQEHPTETEPSEETSSVPTENVEESEEDADEREEGTTVNVNSSEVPDVAQPAVQPIRDPQPLVFQHBAPRL NcUbc6e 188 HGHRDEHDKHSSG AVAASRSGAQQDEEDAESAELAEGFVQTVFLPPAPQPRSVOVTSSSSTPAQPGQGVPQPTRTIPI.PSQFSPHPP AtUbc6e 188 PSIV3EAB3QVEPQ EAITVVEERSIATTDTIVDDQIIEETAEAVNTAA3WPAAAPLPAVEVWKASVSGEQR1JARRAAQKPVDDR XlDbc6 173 - - AQEELNHRPQTLPLPDVVPETEAEIOQgJGAALLNS EVPLAAANBPGLQQPHRH GgUbc€ 173 AQEELNSRPPSLPLPDVVPDaEAHYGQJjJGIPLLHG HVPLAPANHPGLQQAHRN HnObc 6 173 AQDBIiSHRPQHLPLPDVVPDGBEjHRGQBGIQX.IiHG HAFAAGPH1.G»LFQAHRE HBUbc6 173 AQDELSSRPQTLPLPDVVPDGETHLVQHGIQLLNG BAFGAVPNLAGLQQANRB DmUbe 6 180 AAVDGPAAHGVKRSBGLAKaASLASADGHLAVGGlAHPlflAADAIDaGAAGQADtASGaGAAVQHSARB ScObcS 168 RELDEGDAANTGDETEDPFTEAAEEKVISIiEBILDP EDRIRAEQALRQSENNS AtUbc6 166 AEEEAATQQTTTSEHQDFPQKDHAKVESEKSVGLK KESIQEVGLXE

HmUbc 6 « 277 PRAHHTEBGVSAVL III LT LAI.AALI FRRIY YIFDFEL HsUbc6e 277 PRDNHTDHGGSAVL BILTLALAALIFRRIY YIFDFEL GgUbe6e 281 PRAH-PHBTGSTVL HLLTFALAALIFRRIC JYIF XtUbc6e 250 HAVNGSNTG-SAVL HVLTLALAALIFRRIYI DYIFDYEL DrUbc6« 273 BLPQDSSRTGSAII BLLTLALAALIFRRIYI |H0YKFDYEL — CeUbc6e 270 ASTNFDYTLLYKIPVIALCFAIFFTLLARRFI DNLTSPKDGSEL NcUbc6e 275 PXHQERRPQQVRAFHHAVWFCRYHYSVTPRAQPYEGIEV AtUb=6e 274 LFTWAAVGLTIAIHVLLLEKFIKSHGYSTGFMDDQS xinbee 226 HGLLGG-ALAHVFVQ9GFAAFAYTVKYVI.RSIQQ GgUbc6 226 BGLLGG-ALAHLFV BGFAAFAYTVEYVLRSir HmUbc 6 226 BGLLGG-ALANLFV JGFAAFAYTVKYVLRSII HsUbc6 226 HGLLGG-ALAHLFV DOFAAFAYTVKYVLRSI! DmUbc 6 249 SYLHWQSVYSNLVlEJlCFAIFALIVNYVIKNjS 9cube6 221 EXDGEEPNDSSSHVYXGIAIFLFIIVOLFMK- - Atubes 212 RRRNKKEALPGWIVLLLVSIVGVVMALPLLQU

Ubc6 and Ubc6e subfamilies possess distinct sequence features, including changes in the

predicted E3-interacting loops (LI and L2), the dissimilar C-terminal region linking the catalytic

(boxed) and hydrophobic C-terminal domains, and now, the stress-related phosphosite Ser-184

of Ubc6e found only in higher eukaryotes. Mus musculus (Mm), Homo sapiens (Hs), Gallus

gallus (Gg), Xenopus tropicalis (Xt), Drosophila melagonaster (Dm), Caenorhabditis elegans

(Ce), Neurospora crassa (Nc), Arabidopsis thaliana (At), Saccharomyces cerevisiae (Sc).

182 Figure A. 1.2 Human Ubc6 and Ubc6e are abundantly expressed in various cell lines

IB3 HEK293 Panc-1 CFPAC-1 Caco-2 IB3 HEK293 Panc-1 CFPAC-1 Caco-2 -28S Iff^-immm Mmm-28 S -18S -18S

hsUbc6e hsUbc6

GAPDH

HL-60 JRT30 K-562 CFPAC-1 Caco-2 BxPC3 AR42J ARIP Caco-2

' ^•JBr' '•(MP ,*$IH0I$ hsUbc6e

mm

-28S ^^^^w ^^^^ g^p^f ^PBPs w^/^^ ^^^^^

tome* «•»,« -, • *. -18S

RNA analysis of human Ubc6 and Ubc6e transcripts in various cultured cell lines was performed essentially as described in Section 2.2.5. HL-60, human peripheral blood, promyeloid leukemia; JRT30, T lymphocyte cells; K-562, human bone marrow, promyeloid leukemia; Panc-1; CFPAC-1, human pancreas, ductal, epithelioid carcinoma; BxPC-3, human pancreas, adenocarcinoma; AR42J, rat pancreas, exocrine carcinoma; ARIP, rat pancreas, exocrine carcinoma; Caco-2; IB3, human lung, epithelial; HEK293.

183 Figure A. 1.3 ER stress induced phosphorylation of hsUbc6e in various cell lines

Panc-1 Caco-2

TG (h) 0 14 8 0 14 8 hsUbc6e~p hsUbc6e

HEK293 CHOK1

TG(h) 0 14 8 0 14 8 hsUbc6e~p hsUbc6e

Panc-1, Caco-2, HEK293 and CHOK1 cells were treated with thapsigargin, TG (1 uM), for the indicated times. Panc-1, human pancreas, ductal, epithelioid carcinoma; Caco-2, human colon, epithelial adenocarcinoma; HEK293, human kidney; CHO-K1, hamster ovary. Immunoblot detection of endogenous hsUbc6e was performed as described in Section 2.2.3.

184 Figure A. 1.4 Myc-tagged human Ubc6 localizes to ER and peri-nuclear regions

myc-hsUbc6 Grp94 merge

Representative images of COS-7 cells transfected with myc-tagged human Ubc6. Indirect immunofluorescence (Section 2.2.8) indicated peri-nuclear and ER-localization of the fusion protein (left panels). ER-localization is confirmed in the merged image with ER-marker protein

Grp94 (red) and myc-Ubc6 (green).

185 Table A.1 Putative interactor clones obtained from yeast-two hybrid screens using SI84A and

S184E as bait against a human liver cDNA library (Section 2.3.8)

SD/-Trp/-Leu/-His/- SI84A screen S184E screen Ade//B-gal+ clone (number) 1 El ubiquitin El ubiquitin activating enzyme activating enzyme 2 MT2A El ubiquitin activating enzyme metallothionein 2 3 MT2A MT2A metallothionein 2 metallothionein 2 4 MT2A MT2A metallothionein 2 metallothionein 2 5 - MT2A metallothionein 2 6 - Albumin

7 - Cytochrome c oxidase subunit I

8 - Cytochrome c oxidase subunit I

9 - NADH dehydrogenase subunit I

10 - Human microsomal aldehyde dehydrogenase

11 - aarF domain containing kinase 1

12 - Eukaryotic translation elongation factor 1 alpha

186 Figure A.2.1 Phosphorylation of Ubc6e in response to diverse stressors in various human cell lines

TG DTT NaCI UV UT 1 6 6 1 6 1 6

- hsUbc6e Panc-1 hsUbc6e

t- hsUbc6e MCF-7 * - hsUbc6e

- hsUbc6e - hsUbc6e HEK293

hsUbc6e HeLa - hsUbc6e

Panc-1, MCF7 (breast epithelial carcinoma), HEK293 and HeLa (cervical carcinoma) cells were treated with thapsigargin, TG (1 uM), DTT (1 mM), NaCI (300 mM, 600 mOsm), or UV (50

J/m2) and incubated for the times indicated. Immunoblot detection of endogenous hsUbc6e was performed as described in Section 2.2.3. Asterisk (*) represents a non-specific protein.

187 Figure A.2.2 Expression of Ubc6e variants in multiple stable CHO-K1 clones

A

WT-1 WT-2 S184A-1 S184A-2

POX POX POX POX UT TG NaCI UT TG NaCI UT TG NaCI UT TG NaCI UT TG NaCI UT TG NaCI UT TG NaCI UT TG NaCI

B

WT-1 WT-2 S184A-1 S184A-2 POX: - + - + - + - +

JK — fli . mm •""*

27 7 9 5 fold increase

Immunoblot analysis of wild-type and S184A variant Ubc6e expression in stable CHO-K1 cell lines. A, Myc-antibody detection of doxycycline (DOX) induced expression of Ubc6e variants in four clones. To assess the effect of Ubc6e expression on stress response, cells were treated with thapsigargin (10 uM), NaCI (300 mM, 600 mOsm) for 30 min, or untreated (UT).

Phosphorylated eIF2a was a cell stress marker, p-tubulin was a protein loading control. B,

Fold increases in expression of total Ubc6e (unphosphorylated and phosphorylated) over endogenous protein levels were estimated from anti-Ubc6e immunoblots using densitometry measurement (FluorChem 9900, Alpha Innotech). Values were normalized to p-tubulin.

188 Figure A.2.3 Phosphorylation of Ubc6e is associated with reduced viability in multiple stable cell lines

WT-2 S184A-2 1.1- 1.2i 1.0- 0.9- g 0.8- £ 0•7^ |0.6- .a 0.4" < 0.3- 0.2- 0.H 0.0- UT TG NaCI UT TG NaCI

Additional CHO-K1 stable cell lines displaying the association of phosphorylated Ubc6e expression and reduced cell survival during hyper-osmotic shock and ER stress, as determined by MTT assay. (A complete experimental description is provided in Section 3.2.6). Absorbance corresponds to cell survival. Averages of three trials ± S.E. are shown. These results are comparable to results obtained for clones lWT-l and S184A-V presented in Figure 3.4.

189 Figure A.2.4 Phosphorylation of Ubc6e is implicated in activation of JNK and caspase-12 during ER stress in multiple stable cell lines

S184A-2 S184E-2 S184E-2 DOX DOX DOX

TGtime(h): 016016 04 16 04 16 01601 16

Additional CHO-K1 stable cell lines displaying the association of phosphorylated Ubc6e expression (DOX) and JNK and caspase-12 activation during ER stress. (A complete experimental description is provided in Section 3.2.6). JNK activation (observed as Jun phosphorylation) is suppressed with expression of the S184A variant (*), but unaffected with

S184E expression. S184E activation led to reduced levels of pro-caspase-12 indicating increased caspase-12 cleavage (<). These findings are comparable to the 'S184A-1 and 'S184E-

1' clone results presented in Figure 3.5.

190