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Functional Study of the Threonine Phosphorylation and the Transcriptional Coactivator Role of P68 RNA Helicase

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Functional Study of the Threonine Phosphorylation and the Transcriptional Coactivator Role of P68 RNA Helicase Georgia State University ScholarWorks @ Georgia State University Biology Dissertations Department of Biology Fall 12-7-2012 Functional Study of the Threonine Phosphorylation and the Transcriptional Coactivator Role of P68 RNA Helicase Heena T. Dey Georgia State University Follow this and additional works at: https://scholarworks.gsu.edu/biology_diss Recommended Citation Dey, Heena T., "Functional Study of the Threonine Phosphorylation and the Transcriptional Coactivator Role of P68 RNA Helicase." Dissertation, Georgia State University, 2012. https://scholarworks.gsu.edu/biology_diss/123 This Dissertation is brought to you for free and open access by the Department of Biology at ScholarWorks @ Georgia State University. It has been accepted for inclusion in Biology Dissertations by an authorized administrator of ScholarWorks @ Georgia State University. For more information, please contact [email protected]. FUNCTIONAL STUDY OF THE THREONINE PHOSPHORYLATION AND THE TRANSCRIPTIONAL COACTIVATOR ROLE OF P68 RNA HELICASE by HEENA T. DEY Under the Direction of Zhi-Ren Liu ABSTRACT P68 RNA helicase is a RNA helicase and an ATPase belonging to the DEAD-box family. It is important for the growth of normal cells, and is implicated in diverse functions ranging from pre-mRNA splicing, transcriptional activation to cell proliferation, and early organ development. The protein is documented to be phosphorylated at several amino-acid residues. It was previously demonstrated in several cancer cell-lines that p68 gets phosphorylated at threonine residues during treatments with TNF-α and TRAIL. In this study, the role of threonine phosphorylation of p68 under the treatment of anti-cancer drug, oxaliplatin in the colon cancer cells is characterized. Oxaliplatin treatment activates p38 MAP-kinase, which subsequently phosphorylates p68 at T564 and/or T446. P68 phosphorylation, at least partially, influences the role of the drug on apoptosis induction. This study shows an important mechanism of action of the anti- cancer drug which could be used for improving cancer treatment. This study also shows that p68 is an important transcriptional regulator regulating transcription of the cytoskeletal gene TPPP/p25. Previous analyses revealed that p68 RNA helicase could regulate expression of genes responsible for controlling stability and dynamics of different cytoskeletons. P68 is found to regulate TPPP/p25 gene transcription by associating with the TPPP/p25 gene promoter. Expression of TPPP/p25 plays an important role in cellular differentiation while the involvement of p68 in the regulation of TPPP/p25 expression is an important event for neurite outgrowth. Loss of TPPP expression contributes to the development and progression of gliomas. Thus, our studies further enhance our understanding of the multiple cellular functions of p68 and its regulation of the cellular processes. INDEX WORDS: P68 RNA helicase, DEAD-box, ATPase, threonine phosphorylation, oxaliplatin, p38 MAP-kinase, transcriptional regulation, TPPP FUNCTIONAL STUDY OF THE THREONINE PHOSPHORYLATION AND THE TRANSCRIPTIONAL COACTIVATOR ROLE OF P68 RNA HELICASE by HEENA T. DEY A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the College of Arts and Sciences Georgia State University 2012 Copyright by Heena T. Dey 2012 FUNCTIONAL STUDY OF THE THREONINE PHOSPHORYLATION AND THE TRANSCRIPTIONAL COACTIVATOR ROLE OF P68 RNA HELICASE by HEENA T. DEY Committee Chair: Dr. Zhi-Ren Liu Committee: Dr. Julia Hilliard Dr. Shyam Reddy Electronic Version Approved: Office of Graduate Studies College of Arts and Sciences Georgia State University December 2012 iv ACKNOWLEDGEMENTS I sincerely thank Dr. Zhi-Ren Liu for his mentorship and support. His enthusiasm, dedication, and encouragement have urged me to strive higher. I also earnestly thank the exceptional committee members, Dr. Julia Hilliard and Dr. Shyam Reddy. I especially thank Dr .Hilliard for critical analysis of my research and for instilling in me a zest for science. I also thank Dr. Reddy for his invaluable suggestions and making me believe in my abilities. I thank Dr. Jenny Yang for her guidance and advice in improving my dissertation work. I would like to acknowledge Debby Walthall and Sonya Young for all their technical assistance and LaTesha Warren for her valuable support and patience in all these years. I also thank Dr. David Blaustein for giving me an opportunity to teach and developing a passion for teaching. Special thanks to all my colleagues, past and present members of the Liu lab for all their support and for creating a great work environment. Thanks Ravi for being a great friend and making my graduate life so enjoyable. Thanks Vaishali and Sushma from Aneja lab for their material and technical support and Mugdha for always helping me, both inside and outside the lab. Finally, I thank my parents for their continuous encouragement, my sister and brother for their support and last but not the least, my husband and my daughter for their love, great support, patience and sacrifices to help me achieve my career goals and for always being there for me. v TABLE OF CONTENTS ACKNOWLEDGEMENTS...............................................................................................iv LIST OF TABLES………………………………..…..………………………...…..…….…...xii LIST OF FIGURES…………………………………………………………………………...xiii CHAPTER 1 GENERAL INTRODUCTION…………….…..……...……………….………..1 1.1 RNA HELICASES…………..………………………………………………………1 1.2 P68 RNA HELICASE…………………………..…………………………….…….2 1.2.1 P68: a DEAD-box RNA helicase……..………..…………………….2 1.2.2 Expression of p68…..……………………………………..…………..4 1.3 FUNCTIONS OF P68 RNA HELICASE………………………...………………..5 1.3.1 Function of P68 in mRNA splicing…………………………………...5 1.3.2 Function of P68 in Transcriptional Regulation…….………………7 1.3.3 Role of P68 in Post-transcriptional Processes……………...…...14 1.3.4 Role of P68 in Differentiation……………………………………..…14 1.3.5 Role of P68 in Cell proliferation and Cancer……………………..16 1.4 MODIFICATIONS OF P68…………………………………...…………………..19 1.4.1 Ubiquitylation and Sumoylation of P68………………...…..……..19 1.4.2 Phosphorylation of P68…………………………………...….………21 1.4.2.1 Phosphorylation of P68 by PKC………………..……...…23 1.4.2.2 Phosphorylation of P68 by Tlk1………………..…….…..25 1.4.2.3 Phosphorylation of P68 by c-abl kinase…….…..….…..26 1.4.2.4 A-kinase-anchoring protein (AKAP95)………………..…28 1.5 COLORECTAL CANCER (CRC)………………………………………....…….29 vi 1.5.1 Chemotherapy for Metastatic Colorectal Cancer………………..30 1.5.1.1 FOLFIRI or FOLFOX Regimen………………………….…30 1.5.1.2 Targeted Therapies…………………………………...…….31 1.5.1.3 Oxaliplatin…………………………………………………….32 1.5.2 Phosphorylation of P68 by P38 MAP-kinase……….………...…..34 1.6 CYTOSKELETON………………………………………...………………………38 1.6.1 Actin……………………………………………………………………...38 1.6.2 Intermediate Filaments……………………………………………….39 1.6.3 Microtubules…………………………………………………………....40 1.7 TPPP/P25………………………………………………………………………….41 1.7.1 Structure and Expression……………………………………………41 1.8 FUNCTIONS OF TPPP/P25………………………………………….………….44 1.8.1 Cellular Functions of TPPP/p25…………………………………….44 1.8.2 Role of TPPP/p25 in Differentiation……………………………...…48 1.8.3 Involvement of TPPP/p25 in Different Diseases…………………49 1.9 AIMS AND SIGNIFICANCES OF THE DISSERTATION…………………….50 1.10 REFERENCES………………………………………………………………..…52 CHAPTER 2 PHOSPHORYLATION OF P68 RNA HELICASE BY P38 MAP-KINASE CONTRIBUTES TO COLON CANCER CELLS APOPTOSIS INDUCED BY OXALIPLATIN…………………………………………………….………………………….103 2.1 ABSTRACT……………………………..……………………………...……..…103 2.2 INTRODUCTION……………………………………………………………...…103 vii 2.3 RESULTS………………………………………………………………..…….…106 2.3.1 Oxaliplatin treatment of colon cancer cells induced p68- threonine phosphorylation……………………..…………………………106 2.3.2 P68 is phosphorylated by p38 MAP-kinase at T564 and T446 upon the drug treatment…………..………………………………………107 2.3.3 Phosphorylation of p68 at threonine mediates the effects of oxaliplatin in induction of apoptosis…………………….…………...…110 2.4 DISCUSSION…………………………………………………………………….111 2.5 MATERIALS AND METHODS………...…………………………..…………..112 2.5.1 Cell Culture and antibodies…………………..…………………….112 2.5.2 Drug Treatment, DNA constructs, Transfections, and siRNA Interference…………………………………………….………………….…113 2.5.3 Protein Expression and Purification………………………….…..114 2.5.4 In Vitro Kinase Assay………………………………………………..115 2.5.5 Cell Viability and Apoptosis Assay……………………………….115 2.6 REFERENCES………………………………………………………………..…116 CHAPTER 3 P68 RNA HELICASE REGULATES THE EXPRESSION OF TUBULIN POLYMERIZATION PROMOTING PROTEIN INVOLVED IN NEURITE OUTGROWTH AND GLIOMA SUPPRESSION………………………………….………………………...129 3.1 ABSTRACT………………………………………………………..………….….129 3.2 INTRODUCTION…………………………………………………………..….…129 3.3 RESULTS……………………………………………………………………..….131 viii 3.3.1 P68 RNA helicase regulates transcription of TPPP/p25 gene.131 3.3.2 P68 RNA helicase regulates the neurite outgrowth of PC12 neuronal cells………………………………………………………………..132 3.3.3 TPPP/p25 expression plays a role in the progression of gliomas …………..………………………………………………………..……………133 3.4 DISCUSSION……………………………….………………………………..…..135 3.5 MATERIALS AND METHODS………………………………………………...137 3.5.1 Reagents and Antibodies………………………………………..…137 3.5.2 Cell Culture, DNA constructs, Transfections, and siRNA Interference…………………………………………………………………..137 3.5.3 Cellular Extract Preparation, Immunoblot Analysis and Immunofluorescence staining…………………………………………....138 3.5.4 RT-PCR………………………………………………………………...139 3.5.5 Chromatin Immunoprecipitation (ChIP)………………………….139 3.5.6 Cell Proliferation Assays……………………………………………140
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