PHOSPHORYLATION AND MECHANISTIC REGULATION OF A NOVEL IKK SUBSTRATE, ITCH By JESSICA MARIE PEREZ Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Dissertation Advisor: Dr. Derek W. Abbott Department of Pathology CASE WESTERN RESERVE UNIVERSITY January 2018 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Jessica Marie Perez candidate for the Doctor of Philosophy degree*. (signed) Dr. George Dubyak (chair of the committee) Dr. Pamela Wearsch Dr. Theresa Pizarro Dr. Clive Hamlin Dr. Brian Cobb Dr. Derek Abbott (date) October 2, 2017 *We also certify that written approval has been obtained for any proprietary material contained therein. Table of Contents List of Tables ........................................................................................................ v List of Figures ..................................................................................................... vi Acknowledgements .......................................................................................... viii List of Abbreviations .......................................................................................... ix Abstract ............................................................................................................... xi Chapter 1: Introduction ....................................................................................... 1 1.1 Introduction .......................................................................................... 1 1.2 Sarcoidosis pathophysiology and treatment ...................................... 1 1.3 Blau Syndrome is a NOD2-associated disorder ................................. 4 1.4 NOD2 signaling ...................................................................................... 5 1.6 Hypothesis and approach ..................................................................... 9 1.7 Dissertation Rationale ......................................................................... 13 1.7 Specific questions addressed ............................................................ 14 1.8 Works cited in this chapter ................................................................. 17 Chapter 2: ITCH .................................................................................................. 25 2.1 Introduction: C2-WW-HECT (CWH) family of E3 ubiquitin ligases . 25 2.1.1 C2 domains ................................................................................ 27 2.1.2 WW domains .............................................................................. 27 2.1.3 HECT domains ........................................................................... 27 2.2 Ubiquitin and ubiquitination ............................................................... 28 2.3 ITCH as an attenuator of inflammatory signaling ............................. 30 i 2.4 Human/mouse ITCH deficiency .......................................................... 31 2.5 Rationale to study IKK-mediated phosphorylation of ITCH ............ 33 2.6 Works cited in this chapter ................................................................. 34 Chapter 3: IκB Kinases phosphorylate ITCH .................................................. 40 3.1 Introduction .......................................................................................... 42 3.2 Materials and methods ........................................................................ 44 3.2.1 Cell culture, transient transfection, immunoprecipitation, and Western blotting ................................................................................... 44 3.2.2 In vitro kinase assay ................................................................... 46 3.2.3 Mass spectrometry ..................................................................... 46 3.2.4 In vitro ubiquitin ligase assay ...................................................... 46 3.2.5 Luciferase assays ....................................................................... 46 3.2.6 Lentiviral shRNA and retroviral RNAi-Resistant ITCH reconstitution/CRISPR NEMO−/− Cells ................................................ 47 3.2.7 qRT-PCR .................................................................................... 48 3.2.8 Mice and histology scoring ......................................................... 48 3.2.9 Graphs and statistical analyses .................................................. 49 3.2.10 Structural and sequence analyses ........................................... 49 3.3 Results and discussion ....................................................................... 49 3.3.1 IKKs phosphorylate the HECT E3 ubiquitin ligase ITCH on Serine 687 ....................................................................................................... 49 3.3.2 Ubiquitin ligase activity and downstream signaling is modulated in an ITCH S687D phosphomimetic mutant ........................................ 59 ii 3.3.3 ITCH phosphorylation causes increased TNF-induced pro- inflammatory cytokine release, and loss of TNF signaling delays the pulmonary inflammatory phenotype in ITCH−/− mice ........................... 66 3.3.4 Conclusions ................................................................................ 70 3.5 Works cited in this chapter ................................................................. 71 Chapter 4: Phosphorylation of E3 reduces E2-E3 binding ............................ 78 4.1 Introduction .......................................................................................... 79 4.2 Materials and methods ........................................................................ 81 4.2.1 Cell culture, transient transfection and immunoprecipitation/pull- down .................................................................................................... 81 4.2.2 Western blotting .......................................................................... 81 4.2.3 Protein purification and SPR ...................................................... 82 4.2.4 Lentiviral transduction, CRISPR Resistant UbcH7 Construct ..... 82 4.3 Results and discussion ....................................................................... 83 4.3.1 ITCH phosphorylation impairs UbcH7 binding ............................ 83 4.3.2 Genetic deletion of UbcH7 phenocopies phosphorylation of ITCH89 4.3.3 Conclusions ................................................................................ 93 4.5 Works cited in this chapter ................................................................. 93 Chapter 5: Conclusions and Future Directions .............................................. 98 5.1 Introduction: Scope of work ............................................................... 98 5.2 Future directions ................................................................................. 99 5.2.1 In vivo reconstitution with phosphomimetic ITCH ....................... 99 5.2.2 IKK and IKK-related Kinase phosphorylation of other CWD ubiquitin ligases ................................................................................... 99 iii 5.2.3 UbcH7 point mutation to chemically accommodate phosphoITCH S687 .................................................................................................. 102 5.2.4 NDFIP1 recovery of phosphoITCH Ubiquitin ligase activity ..... 105 5.3 Therapeutic implications .................................................................. 107 5.3.1 Targeting the ubiquitin system for inhibition .............................. 108 5.3.2 Phosphorylation of an E3 ligase to inhibit binding of an E2 ...... 109 5.4 Closing remarks ................................................................................. 111 5.5 Works cited in this chapter ............................................................... 112 Copyright permissions .................................................................................... 118 Bibliography ..................................................................................................... 123 iv List of Tables Table 5.1 Potential UbcH7 Mutants to accommodate binding to phosphoITCH 103 v List of Figures Figure 1.1 The IKK complex is a central innate immune signaling hub ................. 7 Figure 1.2 Approach to identify novel IKK substrates .......................................... 11 Figure 2.1 Domain structure of the CWD family of ubiquitin ligases .................... 26 Figure 2.2 A simplified schematic of the HECT E3 ubiquitin ligase cascade ....... 29 Figure 2.3 Overview of ubiquitin chain linkages .................................................. 32 Figure 3.1 Graphical Abstract .............................................................................. 41 Figure 3.2 ITCH Is a Novel Substrate of IKKs ..................................................... 52 Figure 3.2 ITCH Is a Novel Substrate of IKKs (continued) .................................. 53 Figure 3.2.1 Production of recombinant ITCH and requirement of NEMO for ITCH phosphorylation. ........................................................................................... 55 Figure 3.3 ITCH Is Phosphorylated on Ser 687 ................................................... 57 Figure 3.3 ITCH Is Phosphorylated on Ser 687 (continued) ................................ 58 Figure 3.4 Ubiquitin Ligase Activity and Downstream Signaling Is Modulated in ITCH S687D Phosphomimetic Mutant .......................................................... 61 Figure