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Open FINAL GRAD SCHOOL.Pdf The Pennsylvania State University The Graduate School Eberly College of Science PHOSPHOPROTEOMIC ANALYSIS OF RIBOSOMAL PROTEINS: IMPLICATIONS IN TRANSLATION AND APOPTOSIS A Dissertation in Biochemistry, Microbiology, and Molecular Biology by Jennifer Lynn Miller © 2009 Jennifer Lynn Miller Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy May 2009 The dissertation of Jennifer Lynn Miller was reviewed and approved* by the following: Emine C. Koc Assistant Professor Biochemistry and Molecular Biology Dissertation Advisor Chair of Committee Robert A. Schlegel Professor of Biochemistry and Molecular Biology Wendy Hanna-Rose Assistant Professor Biochemistry and Molecular Biology Ming Tien Professor of Biochemistry Erin D. Sheets Assistant Professor of Chemistry Richard J. Frisque Professor of Molecular Virology Head of the Department of Biochemistry and Molecular Biology *Signatures are on file in the Graduate School. ABSTRACT Mammalian mitochondrial ribosomes synthesize thirteen proteins that are essential for oxidative phosphorylation. Besides having a major role in ATP synthesis, mitochondria also contribute to biochemical processes coordinating apoptosis, mitochondrial diseases, and aging in eukaryotic cells. This unique class of ribosomes is protein-rich and distinct from cytoplasmic ribosomes. However, mitochondrial ribosomes (55S) share a significant homology to bacterial ribosomes (70S), particularly in size, the general mechanism of translation, and ribosomal protein content. Due to the overall resemblance between the two systems and the earlier reports of post-translational modifications, we investigated how phosphorylation of ribosomal proteins from bacteria and mitochondria regulates translation and other acquired roles. Identification of twenty- four phosphorylated 70S and 55S ribosomal proteins as well as the potential endogenous kinase was achieved using 2D-gel electrophoresis and tandem mass spectrometry. Specific detection of phosphorylation was performed by [γ-32P] ATP-labeling, ProQ staining, or the use of phospho-specific antibodies. Many of the phosphorylated proteins are located at the polypeptide exit tunnel, mRNA binding path, and the sarcin-ricin loop of the ribosome, implying the functional role of this post-translational modification in translation. By immobilized metal affinity and strong cation exchange chromatography, enrichment of phosphopeptides resulted in the mapping of phosphorylation sites that were evolutionarily conserved and correlated to ribosomal function. Moreover, we have demonstrated, in the presence of in vitro phosphorylation, the inhibition of protein synthesis by mitochondrial kinases PKA, PKCδ, and Abl Tyr kinase. From these phosphoproteomic analyses of 70S and 55S ribosomal proteins, a mitochondrial specific protein DAP3 and the conserved L7/L12 protein were chosen for functional studies. Death Associated Protein 3 (DAP3), also referred to as mitochondrial ribosomal protein S29 (MRPS29), is a GTP-binding apoptotic small subunit protein. DAP3 is phosphorylated at Ser215 or Thr216, Ser220, Ser251 or Ser252, and Ser280. Interestingly, most of the phosphorylation sites are dispersed around the GTP-binding motifs and may alter protein conformation and activity. Site-directed mutagenesis studies on selected phosphorylation sites were performed to determine the effect of phosphorylation on cell viability and PARP cleavage as an indication of caspase iii activation. On the other hand, L7/L12, a large subunit protein, is essential for translation by binding and stimulating several GTPases. E. coli L7/L12 is phosphorylated at Ser15, Ser33, and Thr52. Sites of phosphorylation are strategically located at different functional domains of the protein, inducing conformational changes to the stalk. Site-directed mutagenesis studies coupled to in vitro translation assays were performed, stimulating or inhibiting protein synthesis due to the charge repulsion of neighboring acidic residues in various arrangements. In addition, we detected bovine mitochondrial ribosomal L12 to be phosphorylated as determined by 2D-gel analysis and the use of phospho-specific antibodies. Overall, these proteomic studies identified novel phosphorylated targets and demonstrated the importance of post-translational modifications to translation and apoptosis by maintaining the delicate balance between these two opposing processes in the cell. iv TABLE OF CONTENTS LIST OF FIGURES……………………………………………………………………..ix LIST OF TABLES……………………………………………………………………....xi ABBREVIATIONS……………………………………………………………………..xii ACKNOWLEDGEMENTS…………………………………………………………….xvi CHAPTER 1. Introduction………………………………………………………………1 1.1 Mitochondria…………………………………………………………………1 1.2 Structural Features of Bacterial Ribosomes………………………………….2 1.2.1 Small Subunit of the Bacterial Ribosome………………………….4 1.2.2 Large Subunit of the Bacterial Ribosome………………………….9 1.3 Structural Features of Mitochondrial Ribosomes……………………………10 1.3.1 Small Subunit of the Mitochondrial Ribosome……………………11 1.3.2 Large Subunit of the Mitochondrial Ribosome……………………13 1.3.3 Additional Functions of Mitochondrial Ribosomal Proteins………15 1.4 Translation in Bacterial and Mitochondrial Ribosomes……………………..17 1.4.1 Prokaryotic and Mitochondrial Initiation Factors…………………19 1.4.2 Prokaryotic and Mitochondrial Elongation Factors……………….20 1.5 Phosphorylation of Ribosomal Proteins……………………………………..20 1.6 Phosphorylation of Apoptotic Mitochondrial Ribosomal Protein - DAP3….22 1.6.1 Identification and History of DAP3……………………………….23 1.6.2 Post-Translational Modifications of DAP3………………………..24 1.7 Phosphorylation of the L7/L12 Stalk………………………………………...27 1.7.1 Structural Implications of the L7/L12 Stalk……………………….27 1.7.2 Post-Translational Modifications of L7/L12………………………29 1.7.3 Identification of L12 in Mitochondria……………………………..30 1.8 Research Aims……………………………………………………………….30 1.9 References……………………………………………………………………32 CHAPTER 2. Phosphorylated proteins of the mammalian mitochondrial ribosome: implications in protein synthesis and apoptosis…………………………..45 2.1 Rationale……………………………………………………………………..45 2.2 Abstract………………………………………………………………………46 2.3 Introduction………………………………………………………………….47 2.4 Results and Discussion………………………………………………………49 2.4.1 Phosphorylated Proteins of Bovine 55S Mitochondrial Ribosome…………………………………………………………..49 2.4.2 Mapping the Phosphorylation Sites of Bovine 55S Mitochondrial Ribosome...........…………………………………...55 v 2.4.3 Determining the Effect of Phosphorylation on Mitochondrial Translation…………………………………………………………56 2.4.4 Identification of Potential Endogenous Kinases Associated with the Mitochondrial Ribosomes………………………………..60 2.4.5 Summary…………………………………………………………..63 2.4.6 Phosphorylated Bovine Mitochondrial 28S Subunit Proteins with E. coli Homologs……………………………………………..67 2.4.7 Phosphorylated Bovine Mitochondrial 39S Subunit Proteins with E. coli Homologs…….……………………………………….71 2.4.8 Phosphorylated Bovine Mitochondrial 28S and 39S Subunit Proteins without E. coli Homologs………………………………...78 2.5 Conclusion…………………………………………………………………...79 2.6 Materials and Methods………………………………………………………80 2.6.1 Preparation of 55S Bovine Mitochondrial Ribosomes…………….80 2.6.2 NEPHGE Electrophoresis and Phosphoprotein Staining of Mitochondrial Ribosomes………………………………………….80 2.6.3 In vitro Phosphorylation of 55S Mitochondrial Ribosomes……….81 2.6.4 In-Gel Digestions…………………………………………………..82 2.6.5 Enrichment of Phosphorylated Peptides of Mitochondrial Ribosomes………………………………………………………….82 2.6.6 Mass Spectrometric Analysis of Phosphorylated Proteins of Mitochondrial Ribosomes………………………………………….83 2.6.7 Polymerization Assays……………………………………………..84 2.6.8 Enrichment and Identification of Bovine Mitochondrial Kinases…85 2.7 References……………………………………………………………………85 CHAPTER 3. Identification of phosphorylation sites in mammalian mitochondrial ribosomal protein DAP3…………………………………………………94 3.1 Rationale……………………………………………………………………..94 3.2 Abstract………………………………………………………………………94 3.3 Introduction…………………………………………………………………..95 3.4 Results………………………………………………………………………..97 3.4.1 DAP3 is Phosphorylated on 55S Mitochondrial Ribosomes………97 3.4.2 Ectopically Expressed DAP3 is Serine and Threonine Phosphorylated in HEK293T Cells……………………………….101 3.4.3 In Vitro Phosphorylation of Recombinant DAP3 by PKA and PKCδ……………………………………………………………...104 3.4.4 Effects of DAP3 Mutations on Cell Viability and PARP Cleavage…………………………………………………………...105 3.5 Discussion…………………………………………………………………..110 3.6 Materials and Methods……………………………………………………..112 3.6.1 Purification and In vitro Phosphorylation of Recombinant DAP3……………………………………………………………..112 3.6.2 Isolation and Detection of Phosphorylated Mitochondrial Ribosomal DAP3…………………………………………………113 vi 3.6.3 Enrichment of Phosphorylated Peptides………………………….113 3.6.4 Mass Spectrometric Mapping of DAP3 Phosphorylation Sites…..114 3.6.5 Site-Directed Mutagenesis and Transient Transfection…………..115 3.6.6 Pull Down Assay…………………………………………………116 3.6.7 Western Blot Analysis……………………………………………117 3.6.8 Cell Viability Assay………………………………………………117 3.7 References…………………………………………………………………..117 CHAPTER 4. Role of E. coli ribosomal L7/L12 phosphorylation in translation………121 4.1 Rationale……………………………………………………………………121 4.2 Abstract……………………………………………………………………..122 4.3 Introduction…………………………………………………………………122 4.4 Results………………………………………………………………………124 4.4.1 E. coli L7/L12 is Phosphorylated in 70S Ribosomes…………….124 4.4.2 Mapping the Phosphorylation Sites of E. coli L7/L12 in 70S Ribosomes………………………………………………………...125 4.4.3 Reconstitution of the 70S
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