The oncogenic protein BCL-3, a member of the IκB family, was originally identified in a subset of human B-cell chronic lymphocytic leukemias that carry a translocation t(14,19), which results in BCL-3 overexpression. BCL-3 is also overexpressed in many solid tumors, such as in breast cancers and in cylindromas. This IκB protein activates or represses gene transcription through binding with the NF-κB proteins p50 and p52. Furthermore, BCL-3 is K63-linked polyubiquitinated, which leads to its translocation into the nucleus and to its target genes expression. BCL-3 is also K48-linked polyubiquitinated after GSK3 phosphorylation, which leads to its subsequent proteasomal degradation. However, the mechanisms underlying both its polyubiquitination and its ability to repress gene transcription remain poorly understood. In order to gain more insight into these BCL-3 functions, parallel screenings involving both yeast-two-hybrid experiments and biochemical purifications led to the identification of BCL-3-interacting partners. Those screenings identified CtBP as a molecule required for the ability of BCL-3 to repress gene transcription. CtBP is also required for the stability, for the oncogenic potential and for the ability of BCL-3 to inhibit UV-mediated cell apoptosis in keratinocytes. We also defined the E3 ligase TBLR1 as a key element involved in BCL-3 polyubiquitination and degradation through a GSK3-independent pathway and the proteasome subunit PSMB1 as a protein required for the GSK3-dependent and -independent proteasomal degradation of polyubiquitinated BCL-3. Importantly, all interactions require unique motifs within the amino-terminal domain of BCL-3. In conclusion, our data define multiple BCL-3-associated proteins that differentially and specifically regulate its function and stability and indicate that a better understanding of the mechanisms underlying the oncogenic properties of this IκB protein could be achieved through similar interactomic studies.

Retinoic acid (RA) has been recognized as a chemotherapeutic agent for various malignances such as lung, skin as well as cervical cancers. It binds to retinoid receptors heterodimers and consequently activates several RA-responsive genes which are involved in many biological processes including vertebrate development, bone growth, vision, haematopoiesis, cell growth, differentiation and apoptosis. These genes are under the control of numerous regulators to ensure their timely ordered activities. Among these regulators, we focused here on the 26S proteasome and ubiquitination. It has been reported that the activity of the ubiquitin/proteasome system (UPS) plays a fundamental role in retinoic acid receptor (RAR)-regulated transactivation. The mechanisms underlying this role, however, remain to be established. Chromatin immunoprecipitation (ChIP) assays in our study demonstrated that the 26S proteasome activity is important for preserving the occupancy of a TATA box-containing RA-responsive promoters by liganded retinoid receptors and thus by their coactivators. Additionally, by using coimmunoprecipitation assays and by measuring the half-life of retinoid receptors, we found that the non-proteolytic function of the proteasome is required for ligand-dependent association between DNA-free RAR-α and both DNA-free RXR-α and coactivators. Moreover, using immunofluorescent staining and in vivo ubiquitination assays, a proteasome inhibition-dependent cytoplasmic localization of RAR-α as well as ligand-enhanced ubiquitination and stabilization of RAR-α were shown. Our findings therefore, define novel mechanisms by which the UPS controls RAR-regulated genes. Furthermore, we shed new light on the regulators of retinoid receptors ubiquitination and subcellular localization.

The p53 tumour suppressor gene is inactivated by mutation in over 50% of all human cancers. The p53 protein is activated and stabilized through several post-translational modifications in response to various stresses and promotes cell cycle arrest and apoptosis. Thus, regulation of p53 is critical for normal cellular function. Pirh2 is a p53-regulated gene recently identified in our laboratory which encodes an E3 RING-finger ubiquitin ligase that binds to p53 and negatively regulates p53 by targeting it for ubiquitin-mediated proteolysis. Pirh2 is similar to another well-characterized E3 RING finger ubiquitin ligase, Mdm2, which also participates in a similar negative feedback loop with p53. At least seven E3 ubiquitin ligases are known to target p53 for degradation and the reason for this functional redundancy is unclear. The purpose of this study is to characterize Pirh2 activity. This study has two aims the first is to identify additional interacting proteins for Pirh2, and the second is to delineate Pirh2 regulation of p53. Using several tandem affinity purification strategies and a GST-pull down approach, we have identified PKC delta as a candidate interacting protein. The second aim is to further characterize Pirh2 regulation of p53. Splenocytes and thymocytes from Pirh2-/- mice demonstrate a subtle increase in total p53 levels after irradiation when compared to wild-type controls. Phosphoserine 15 p53 levels are significantly higher in splenocytes and thymocytes from Pirh2 -/- mice relative to wild-type counterparts. Cells stably transfected with Pirh2 have decreased levels of phosphoserine 15 p53 and decreased induction of p21 relative to vector control and Mdm2 expressing cells. The stability of the p53 protein is primarily regulated through ubiquitin mediated proteolysis, and there are multiple ubiquitin ligases targeting p53 for degradation. Here we are able to address the question of functional redundancy by indicating that Pirh2 can target serine 15 phosphorylated p53 which is reported to not be regulated by Mdm2.

Protein ubiquitination is essential for the proper functioning of many eukaryotic cellular processes. The cleavage of ubiquitin chains from ubiquitinated proteins is performed by deubiquitinating enzymes, of which there are 16 in the Ubp (ubiquitin specific protease) group in the budding yeast Saccharomyces cerevisiae. The goal of my thesis has been to examine the biological roles and molecular functions of these enzymes using a combination of proteomic and molecular genetic approaches. As part of a large collaborative effort, interacting protein partners of the Ubps were isolated through affinity purification of tagged proteins, followed by protein identification by mass spectrometry. Purification of tagged Ubp6 led to the identification of the 19S proteasome complex, along with a novel subunit, Sem1. As the human homologue of Sem1 was previously identified as being associated with a protein involved in the repair of DNA double-strand breaks, I examined the possible role of Sem1 in DNA damage repair. A deletion of Sem1 and other 19S subunits resulted in hypersensitivity to various DNA damaging drugs, implicating the 19S complex in the process of DNA repair. iii I also found that purified Ubp2 interacted stably with the ubiquitin ligase Rsp5 and the protein Rup1. UBP2 interacts genetically with RSP5, indicating a functional relationship, while Rup1 facilitates the physical tethering of Ubp2 to Rsp5. Using the uracil permease Fur4, a Rsp5 substrate, as a model reporter, I found that ubp2Δ cells exhibited a temporal stabilization of Fur4 at the plasma membrane following the induction of endocytosis, implicating Ubp2 in protein sorting, specifically at the multivesicular body. In order to understand the role of Ubp2, I examined the effect of Ubp2 on Rsp5 function. I found that Rsp5, similar to its mammalian homologues, is auto-ubiquitinated in vivo, and that Ubp2 is able to directly deubiquitinate Rsp5 in vitro. Moreover, the presence of a substrate or Rup1 both resulted in increased autoubiquitination, implying an auto-inhibitory mechanism of Rsp5 regulation. Taken together, the data presented in this thesis implicate deubiquitinating enzymes in interesting and varied roles in the cell, and suggest a novel mechanism for the modulation of Rsp5-dependent trafficking processes.

The ubiquitin-proteasome pathway plays vital roles in multiple cellular processes including protein turnover and transcription regulation. The fate of a ubiquitinated protein is determined by the number of ubiquitin molecules added and the site to which they are added. Monoubiquitinated proteins are stabilized and often activated, while polyubiquitinated proteins are rapidly targeted for degradation. Major histocompatibility complex class II (MHC II) molecules are a vital part of the immune response and are responsible for presenting antigens to CD4+ T cells. The class II transactivator (CIITA) is the master regulator of MHC II transcription and has been shown to have increased transactivity when monoubiquitinated. The focus of this thesis is on the impact of ubiquitination on CIITA stability and MHC II gene expression through the identification of an E3 ligase that targets and ubiquitinates CIITA.

The p53 tumour suppressor gene is inactivated by mutation in over 50% of all human cancers. The p53 protein is activated and stabilized through several post-translational modifications in response to various stresses and promotes cell cycle arrest and apoptosis. Thus, regulation of p53 is critical for normal cellular function. Pirh2 is a p53-regulated gene recently identified in our laboratory which encodes an E3 RING-finger ubiquitin ligase that binds to p53 and negatively regulates p53 by targeting it for ubiquitin-mediated proteolysis. Pirh2 is similar to another well-characterized E3 RING finger ubiquitin ligase, Mdm2, which also participates in a similar negative feedback loop with p53. At least seven E3 ubiquitin ligases are known to target p53 for degradation and the reason for this functional redundancy is unclear. The purpose of this study is to characterize Pirh2 activity. This study has two aims the first is to identify additional interacting proteins for Pirh2, and the second is to delineate Pirh2 regulation of p53. Using several tandem affinity purification strategies and a GST-pull down approach, we have identified PKC delta as a candidate interacting protein. The second aim is to further characterize Pirh2 regulation of p53. Splenocytes and thymocytes from Pirh2-/- mice demonstrate a subtle increase in total p53 levels after irradiation when compared to wild-type controls. Phosphoserine 15 p53 levels are significantly higher in splenocytes and thymocytes from Pirh2 -/- mice relative to wild-type counterparts. Cells stably transfected with Pirh2 have decreased levels of phosphoserine 15 p53 and decreased induction of p21 relative to vector control and Mdm2 expressing cells. The stability of the p53 protein is primarily regulated through ubiquitin mediated proteolysis, and there are multiple ubiquitin ligases targeting p53 for degradation. Here we are able to address the question of functional redundancy by indicating that Pirh2 can target serine 15 phosphorylated p53 which is reported to not be regulated by Mdm2.

Protein ubiquitination is essential for the proper functioning of many eukaryotic cellular processes. The cleavage of ubiquitin chains from ubiquitinated proteins is performed by deubiquitinating enzymes, of which there are 16 in the Ubp (ubiquitin specific protease) group in the budding yeast Saccharomyces cerevisiae. The goal of my thesis has been to examine the biological roles and molecular functions of these enzymes using a combination of proteomic and molecular genetic approaches. As part of a large collaborative effort, interacting protein partners of the Ubps were isolated through affinity purification of tagged proteins, followed by protein identification by mass spectrometry. Purification of tagged Ubp6 led to the identification of the 19S proteasome complex, along with a novel subunit, Sem1. As the human homologue of Sem1 was previously identified as being associated with a protein involved in the repair of DNA double-strand breaks, I examined the possible role of Sem1 in DNA damage repair. A deletion of Sem1 and other 19S subunits resulted in hypersensitivity to various DNA damaging drugs, implicating the 19S complex in the process of DNA repair. iii I also found that purified Ubp2 interacted stably with the ubiquitin ligase Rsp5 and the protein Rup1. UBP2 interacts genetically with RSP5, indicating a functional relationship, while Rup1 facilitates the physical tethering of Ubp2 to Rsp5. Using the uracil permease Fur4, a Rsp5 substrate, as a model reporter, I found that ubp2Δ cells exhibited a temporal stabilization of Fur4 at the plasma membrane following the induction of endocytosis, implicating Ubp2 in protein sorting, specifically at the multivesicular body. In order to understand the role of Ubp2, I examined the effect of Ubp2 on Rsp5 function. I found that Rsp5, similar to its mammalian homologues, is auto-ubiquitinated in vivo, and that Ubp2 is able to directly deubiquitinate Rsp5 in vitro. Moreover, the presence of a substrate or Rup1 both resulted in increased autoubiquitination, implying an auto-inhibitory mechanism of Rsp5 regulation. Taken together, the data presented in this thesis implicate deubiquitinating enzymes in interesting and varied roles in the cell, and suggest a novel mechanism for the modulation of Rsp5-dependent trafficking processes.

Retinoic acid (RA) has been recognized as a chemotherapeutic agent for various malignances such as lung, skin as well as cervical cancers. It binds to retinoid receptors heterodimers and consequently activates several RA-responsive genes which are involved in many biological processes including vertebrate development, bone growth, vision, haematopoiesis, cell growth, differentiation and apoptosis. These genes are under the control of numerous regulators to ensure their timely ordered activities. Among these regulators, we focused here on the 26S proteasome and ubiquitination. It has been reported that the activity of the ubiquitin/proteasome system (UPS) plays a fundamental role in retinoic acid receptor (RAR)-regulated transactivation. The mechanisms underlying this role, however, remain to be established. Chromatin immunoprecipitation (ChIP) assays in our study demonstrated that the 26S proteasome activity is important for preserving the occupancy of a TATA box-containing RA-responsive promoters by liganded retinoid receptors and thus by their coactivators. Additionally, by using coimmunoprecipitation assays and by measuring the half-life of retinoid receptors, we found that the non-proteolytic function of the proteasome is required for ligand-dependent association between DNA-free RAR-α and both DNA-free RXR-α and coactivators. Moreover, using immunofluorescent staining and in vivo ubiquitination assays, a proteasome inhibition-dependent cytoplasmic localization of RAR-α as well as ligand-enhanced ubiquitination and stabilization of RAR-α were shown. Our findings therefore, define novel mechanisms by which the UPS controls RAR-regulated genes. Furthermore, we shed new light on the regulators of retinoid receptors ubiquitination and subcellular localization.

No description available.

The class II Transactivator (CIITA) is the master regulator for Major Histocompatibility Class II (MHC II) molecules. CIITA is dynamically regulated by a series of Posttranslational Modifications (PTMs). CIITA is responsible for initiating transcription of MHC II genes, thus allowing peptides derived from extracellular antigens to be presented to CD4+ T cells. CIITA’s PTMs are necessary for regulation of CIITA’s location, activity, and stability. Our work identifies the kinase complex ERK1/2 as being responsible for phosphorylating the previously identified regulatory site, serine (S) 280 on CIITA. Phosphorylation by ERK1/2 of CIITA S280 leads to increased levels of CIITA mono-ubiquitination and overall increases in MHC II activity. We further identify a novel ubiquitin modification on CIITA, lysine (K) 63 linked ubiquitination poly ubiquitination. Our data shows novel crosstalk between K63 ubiquitination and ERK1/2 phosphorylation. K63 ubiquitinated CIITA is concentrated to the cytoplasm, and upon phosphorylation by ERK1/2, CIITA translocates to the nucleus, thus demonstrating that CIITA’s location and activity is regulated through PTM crosstalk. While ubiquitination has been shown to be a critical PTM in the regulation of CIITA, the enzyme(s) mediating this important modification remained to be elucidated. Previous reports implicating the histone acetyltransferase (HAT), pCAF as an ubiquitin E3 ligase were intriguing, as pCAF is also known to participate in the acetylation of both histones at the MHC II promoter and in acetylation of CIITA. We now identify novel roles for pCAF in the regulation of CIITA. We show pCAF acts as an E3 ligase, mediating mono, K63, and K48 linked ubiquitination of CIITA. We therefore demonstrate an additional substrate for the “dual acting” enzyme, pCAF. In sum, our observations identify enzymes involved in both the phosphorylation and ubiquitination of key residues of CIITA, which ultimately regulate CIITA activity. Together our observations contribute to knowledge of CIITA’s growing network of PTMs and their role in regulating the adaptive immune response, and will allow for development of novel therapies to target dysregulated CIITA activity during adaptive immune responses.