Differential Regulation of c-FLIP Isoforms Through Post-translational Modifications

9 INTRODUCTION.10 REVIEW OF THE LITERATURE.11 1 Post-translational regulation of proteins . 11 2 Protein phosphorylation enables versatile protein modification . 11 2.1 The classification and regulation of the protein kinase C.12 2.2 PKCs as targets for cancer therapy.14 3 Post-translational modification of proteins by ubiquitin . 15 3.1 Ubiquitin and ubiquitin-like proteins . 15 3.2 Non-covalent ubiquitin binding . 16 3.3 Three enzymatic steps of ubiquitin conjugation.17 3.4 The deubiquitylating enzymes . 20 3.5 The diverse outcomes of ubiquitylation.20 3.5. 1 Monoubiquitylation . 21 3.5. 2 Polyubiquitylation . 21 3.6 Proteasomal degradation of ubiquitylated proteins . 22 3.6. 1 Structure of the 26S proteasome . 22 3.6. 2 Mechanisms of proteasomal degradation. 23 4 Multiform regulation of programmed cell death. 24 4.1 Execution of apoptosis through the intrinsic and extrinsic pathways.24 4.2 Bcl-2 proteins regulate the intrinsic pathway and mitochondrial apoptotic signaling.25 4.3 Apoptotic initiator and effector caspases . 28 4.3.1 The classification and structure of caspases. 29 4.3.2 Caspase-9 is activated in the apoptosome holoenzyme. 30 4.3.3 Activation of the effector caspases. 31 4.3.4 Regulation of caspase activity. 31 4.3.5 Processing of caspase substrates. 33 4.4 The death receptor systems and the extrinsic apoptotic pathway.34 4.4.1 The TNF superfamily of death ligands and receptors. 34 4.4.2 The TNF receptor system in cell death and survival signaling . 35 4.4.3 The CD95 and TRAIL receptor systems. 37 4.4.4 Activation and biological roles of caspase-8 and caspase-10 . 40 4.5 Regulation of death receptor signaling by c-FLIP.41 4.5. 1 c-FLIP is recruited to diverse signaling complexes. 43 4.5. 2 Transcriptional regulation of c-FLIP. 45 4.5. 3 Post-translational regulation of c-FLIP. 46 4.5. 4 Dynamic regulation of c-FLIP levels affects a multitude of physiological processes. 47 4.6 PKC as a regulator of cell death.49 OUTLINE OF THE STUDY . 51 MATERIALS AND METHODS.52 1 Cell culture and treatments (I-III) . 52 2 Plasmid constructs (I-III) . 53 3 Transient transfections and generation of stable cell lines (I-III) . 53 4 Ubiquitylation assay (I-III) . 53 5 FLAG immunoprecipitation (III) . 54 6 CD95 and TRAIL DISC analysis (I-III) . 54 7 SDS-PAGE and Western blotting (I-III) . 55 8 In vivo P labeling, Edman sequencing and mass spectrometry (III). 55 9 Preparation and use of the anti-pS193 phosphopeptide antibody (III). 56 10 Caspase-3 activity assay (I, II). 56 11 Caspase-8 activity assay (III) . 56 12 Detection of apoptosis (II) . 57 13 Surface expression analysis of CD95 (II) . 57 14 RNase protection assay (II) . 57 15 Metabolic labeling by S (III) . 57 16 Quantification, densitometry and statistical analysis of c-FLIP half-lives and caspase-8 activation (I-III) . 57 17 Molecular modeling of c-FLIPS (I) . 58 RESULTS AND DISCUSSION.59 1 Increased death receptor sensitivity during differentiation and hyperthermia is due to c-FLIP downregulation (I, II) . 59 1.1 Death receptor sensitivity is adjusted by c-FLIP downregulation during differentiation and hyperthermia (I, II) . 59 1.2 c-FLIP proteins are ubiquitylated and degraded in the proteasome following erythroid differentiation of K562 cells and upon fever-like hyperthermia of Jurkat T cells (I, II) . 60 2 Mechanisms of isoform-specific ubiquitylation and degradation of the c-FLIP splice variants (I, II) . 62 2.1 c-FLIP isoforms display different half lives (I). 62 2.2 K192 and K195 are the primary ubiquitylated lysines in c-FLIPS during K562 erythroid differentiation (I). 64 2.3 C-terminal regions determine the half-lives of c-FLIP proteins (I, II) . 65 3 Regulation of TRAIL-mediated apoptosis by PKC-mediated c-FLIP phosphorylation (III) . 67 3.1 Regulation of death receptor signaling by protein kinase C (III). 67 3.2 c-FLIP is phosphorylated on serine 193 (III) . 67 3.3 Serine 193 phosphorylation is mediated by PKC and PKC (III) . 68 3.4 Serine 193 mutations have isoform-specific implications on c-FLIP ubiquitylation and stability (III) . 69 CONCLUDING REMARKS . 75 ACKNOWLEDGEMENTS.76 REFERENCES.78 List of Ori