Mechanisms of Caspase-3 Regulation in the Execution of Cell Death DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Yadira Malavez Graduate Program in Molecular, Cellular and Developmental Biology The Ohio State University 2012 Andrea I. Doseff, Adviser Dissertation Committee: Dr. Clay B. Marsh Dr. Harold A. Fisk Dr. Tsonwin Hai Copyright by Yadira Malavez 2012 Abstract Apoptosis is an evolutionarily conserved mechanism necessary for the homeostasis in multicellular organisms. The cysteine protease caspase-3 has a key role in apoptosis for its central role in the execution of the apoptotic cascade. The molecular mechanisms that regulate caspase-3 activation are not completely understood. Previously, our laboratory demonstrated that caspase-3 is phosphorylated by PKCδ in human monocytes. However, the role of caspase-3 phosphorylation during apoptosis has not been elucidated. In this investigation, it was observed that PKCδ is necessary for caspase-3 phosphorylation. An interaction motif in caspase-3 was identified to be necessary for PKCδ interaction. Five PKCδ phosphorylated sites in caspase-3 were mapped utilizing mass spectroscopy. Phosphorylation of specific sites promoted caspase- 3 autocatalytic cleavage and apoptosis, in vitro and in vivo. Caspase-3 phosphorylation acts in a positive feedback mechanism to amplify the apoptotic cascade. These results suggest a novel regulatory mechanism to control caspase-3 apoptotic activity and execution of cell death. Furthermore, the apoptotic and immunological heterogeneity in CD14+CD16+ and CD14+CD16- monocytes was analyzed. It was observed that CD14+CD16+ cells were more susceptible to undergo spontaneous apoptosis, in part due to upregulation of the activity of the caspases. CD14+CD16+ monocytes release more TNF-α compared to the ii CD14+CD16- counterparts. Furthermore, the expression of the members of the PKC family was characterized in monocyte subsets. These results suggested that elevated expression of PKCε may play a role in the pro-inflammatory role of CD14+CD16+ monocytes. The current investigation highlights the importance of caspase-3 and the members of the PKC family as important regulators of cell death and survival pathways. iii Dedication This document is dedicated to my mother, Mayra Acevedo Ponce, for her love and support. iv Acknowledgements I would like to thank Dr. Andrea I. Doseff for giving me the opportunity to be part of her laboratory and for her guidance and advice throughout these years. I would like to express my sincere appreciation to Drs. Clay B. Marsh, Harold A. Fisk and Tsonwin Hai, for accepting being in my committee and for their guidance. Next, I would like to thank the members of the Doseff laboratory, especially Drs. Oliver Voss, Martha E. Gonzalez- Mejia, Arti Singh, and Mr. Daniel Arango. It was a pleasure to work with each of them and I am grateful for all the memories that I will take with me throughout my life. Special thanks to Hassan Kamran and Justin Tossey, for their great help in the cloning of the phospho-mutants, and Maria Belen Federico for her help with the development of the cell free system protocol. I am deeply grateful with Dr. Wei Huang and Dr. Prabakaran Nagarajan for their incredible help providing the PKCβ-/- and guidance for the isolation of mouse embryonic fibroblast. I also would like to thank Dr. Nancy Lill and Nurettin Sever for allowing me to utilize their equipment necessary for the transfection of monocytes. Thanks to Lizanel Feliciano, Fabiola Jara, Greetchen Diaz, and Dr. Rebecca Tirado-Corbala, for your friendship and support all these years. Thanks to my family for always believing in me and giving me their love and support. Special thanks to my husband, Esbal Jimenez, for his support and faith in me. Lastly, thanks God for giving me strength to achieve this goal. v Vita November, 6th 1979…………………………………San Juan, PR. 1997 – 2002………………………………………... B.S., Microbiology. University of Puerto Rico, Humacao, PR. 2002 – 2005………………………………………... M.S., Food Microbiology. University of Puerto Rico, Mayaguez, PR. 2006 – Present……………………………………… Graduate Research Associate Working towards Ph.D., Molecular Cellular and Developmental Biology. The Ohio State University, Columbus, OH. Publications Malavez Y., Voss O.H., Gonzalez-Mejia M.E. and Doseff A.I. PKCδ phosphorylation of caspase-3 regulates cell death and survival through a complex regulatory network (In preparation, 2011). Malavez Y., Voss O.H., Gonzalez-Mejia M.E. and Doseff A.I. Characterization of cell fate and immune response of CD14+CD16- and CD14+CD16+ monocytes (In preparation, 2011). Malavez Y., Gonzalez-Mejia M.E. and Doseff A.I. 2008. Protein Kinase C Delta (PRKCD). PKC. Atlas of Genetics and Cytogenetics in Oncology and Haematology. URL: http://AtlasGeneticsOncology.org/Genes/ Fields of Study Major Field: Molecular, Cellular and Developmental Biology vi Table of Contents Abstract……………………………………………………….………………….... ii Dedication…………………………………………………………….……………. iv Acknowledgments……………………………………………….…………………. v Vita……………………………………………………………………..…………... vi List of Figures………………………………………………………………………. xii List of Tables…………………………………………………..…….……………... xiv Abbreviations………………………….………………………….………………… xv Chapter 1…………………………………..……………………….………..…….. 1 1.1 Introduction………….………………………………………………….….…… 1 1.2 Apoptosis…………………………………………..………….………….…...... 2 1.3 Caspase family…………………………………………..…….………………... 4 1.4 Apoptotic pathways ….………………………………………….….………….. 8 1.4.1 Caspase substrates ………………………………..……….……...………. 11 1.5 Regulation of caspase activity….……………..…………...……..……….……. 13 1.5.1 Inhibitors of apoptosis (IAP)……………………………………...………. 14 1.5.2 Regulation of caspase activity by phosphorylation....………………..…… 15 1.6 Caspase-3…………………………………………………..…………..……….. 17 vii 1.6.1 Caspase-3 structure and activation……………………………………….. 19 1.6.2 Caspase-3 functions……………..……………………..…………..…….. 22 1.6.3 Potential role of caspase-3 in human diseases…………...……………….. 23 1.6.4 Regulation of caspase-3 activity……………………….…………..…….. 25 1.6.4.1 Ubiquitination……………………………………………..……… 26 1.6.4.2 Nitrosylation……………………..………………………....…….. 27 1.6.4.3 Heat shock proteins …………………..………….…….…….…... 27 1.7 Protein kinase C delta (PKCδ) .…………….…..…………....…….…….….…. 29 1.7.1 The protein kinase C (PKC) family……………………….……..…..…… 29 1.7.2 PKCδ structure…..…………………………..……………..….…..……… 30 1.7.3 Role of PKCδ in apoptosis ..……….……………………..…..………..… 30 1.7.4 Substrates of PKCδ during apoptosis....…………………..….……..…… 32 1.8. Regulation of cell death in the immune response……….…………....………... 34 1.8.1 Innate and adaptive immune response.……………………………….….. 34 1.8.2 Monocytes and Macrophages ………………………..……..…….……... 35 1.8.3 The role of monocytes in the immune response........................................ 37 1.8.3.1 Toll-like receptors…………………………..……..……..…...….. 37 1.8.4 Monocyte subsets……….………………………………..….…………... 40 1.9 Summary………………………………………………………..……..………... 42 Chapter 2 Materials and methods………………………………….……….…… 58 2.1 Reagents and chemicals…………………………………………….…………... 58 2.2 Cloning and mutagenesis………………………………………….……………. 60 viii 2.3 Tissue culture …………………………………………..…………….….……... 66 2.4 Isolation of mouse embryonic fibroblasts (MEF)………………….……….…... 68 2.5 Extract preparation and immunoblotting……………………………..……….... 69 2.6 Protein quantitation and Western blot analysis………………………….…….... 69 2.7 Immunoprecipitation and in vitro kinase assays………………….……............ 71 2.8 Protein expression and purification………………………….……….……….... 72 2.9 Activation of caspase-3 by recombinant caspase-9…………….……….…….... 74 2.10 Activation of caspase-3 by MCF-7 cell extracts …………………..…………. 74 2.11 Caspase-3 activity assays ……………………………………………….…….. 75 2.12 In vitro kinase assays with recombinant caspase-3 ……………….…….…….. 76 2.13 Monocyte subset isolation ……………………….………………..…….…….. 78 2.14 Inhibition of caspase activity …………………………………….………..….. 79 2.15 Electroporation of monocyte subsets…………………………………….……. 80 2.16 IgG clustering ……………………………………………………….………... 80 2.17 Detection of TNF-α by ELISA …………………………………...………….. 81 2.18 Detection of PKC isoforms in monocyte subsets ………………….……….... 82 2.19 Flow cytometry ……..…………………………………………………..…..... 82 2.22 Statistical analysis……………………………………………………….….…. 83 Chapter 3 PKCδ phosphorylation of caspase-3 regulates the execution of 84 apoptosis……………………………………………………………………………. 3.1 Abstract……………………………………………………………………...….. 84 3.2 Introduction………………………………………………………………..…..... 85 3.3 Results…………………………………………………………………..……..... 87 ix 3.3.1 PKCδ is necessary for caspase-3 phosphorylation during cell death..… 87 3.3.2 Identification of caspase-3 domains phosphorylated and involved in 88 the interaction with PKCδ…………….………………..……….…………… 3.3.3 Identification of PKCδ interaction motif in caspase-3……………….... 89 3.3.4 Identification of PKCδ phosphorylation sites in caspase-3……..…...... 91 3.3.5 Ser12 is important for caspase-3 phosphorylation..………….……….... 94 3.3.6 The phosphorylation of Ser36 is necessary for the PKCδ dependent 95 phosphorylation of caspase-3………………………………….…………….. 3.3.7 Caspase-3 phosphorylation modulates its protease activity…….…....... 96 3.3.8 Role of Ser12 and Ser36 Phosphorylation of in the autocatalytic 97 cleavage of caspase-3….…………………………………....……………….. 3.3.9 Role of caspase-3-Ser36 phosphorylation in apoptosis…..…….……… 100 3.3.10 Phosphorylation of Ser36 is important for caspase-3 cleavage during 102 apoptosis……………………….…………………………………………….. 3.4