A Dissertation The E3 Ligase CHIP Mediates Ubiquitination and Degradation of Mixed Lineage Kinase 3 and Mixed Lineage Kinase 4 Beta by Natalya A. Blessing Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biology _________________________________________ Dr. Deborah Chadee, Committee Chair _________________________________________ Dr. Richard Komuniecki, Committee Member _________________________________________ Dr. Malathi Krishnamurthy, Committee Member _________________________________________ Dr. Frank Pizza, Committee Member _________________________________________ Dr. Don Ronning, Committee Member _________________________________________ Dr. Patricia R. Komuniecki, Dean College of Graduate Studies The University of Toledo May 2015 Copyright 2015, Natalya A. Blessing This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of The E3 Ligase CHIP Mediates Ubiquitination and Degradation of Mixed Lineage Kinase 3 and Mixed Lineage Kinase 4 Beta by Natalya Blessing Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biology The University of Toledo May 2015 The mixed lineage kinases (MLKs) are serine/threonine mitogen-activated protein kinase kinase kinases (MAP3Ks) that modulate the activities of extracellular signal- regulated kinase, c-Jun N-terminal kinase, and p38 signaling pathways. MLK3 plays a pivotal role in cell invasion, tumorigenesis and metastasis. Wild type MLK4 negatively regulates MAPK signaling by possibly inhibiting MLK3 activation, while mutant MLK4 plays in important role in driving colorectal cancer and glioblastomal tumorigenesis (Martini et al., 2013). The mechanisms by which MLK3 and MLK4 protein levels are regulated in cells are unknown. The carboxyl terminus of HSC-70 interacting protein (CHIP) is a U-box E3 ubiquitin ligase that regulates cytosolic protein degradation in response to stress. We examined the regulation of MLK3 and MLK4 protein levels by stresses. Treatment of SKOV3 ovarian cancer cells with the Hsp90 inhibitor, geldanamycin (GA), promotes the dissociation of endogenous MLK3 from Hsp90, and association of MLK3 with Hsp70. GA treatment induces K-48 linked ubiquitination of MLK3, and reduction of endogenous MLK3 and MLK4β protein levels. Heat shock and osmotic stress cause a similar reduction of MLK3 and MLK4β. MLK4β suppresses heat iii and osmotic shock-induced activation of ERK, p38, and JNK. We assessed the role of CHIP in modulating MLK3 and MLK4β protein levels. GA, thermostress, and osmotic shock-induced degradation of MLK3 is abolished in cells with CHIP siRNA knockdown. CHIP overexpression promotes ubiquitination and a proteasome-dependent reduction of MLK3. CHIP interacts with both MLK3 and MLK4β proteins in vitro and with MLK3 in cells; and CHIP ubiquitinates MLK3 and MLK4β (with UbcH5 E2s) in vitro. Furthermore, CHIP-dependent regulation of MLK3 is required for invasion of SKOV3 cells. These findings suggest that CHIP regulates MLK3 and MLK4β protein levels and, in part, cell invasiveness by facilitating ubiquitination and proteasome-dependent degradation of MLK3. iv I would like to dedicate this work to my eccentric and loving family: my amazing mom, Irina Blessing, my wonderful dad, John Blessing, my sweet grandmother, Klaudia Alekseyenko, and my whimsical grandfather, Nikolai Alekseyenko. Their support, encouragement and love have gotten me through my toughest times in graduate school. Special thanks to my mom, Irina, who has been with me and worried for me every step of the way. Mom and dad have been there for me and have gone to all the graduation ceremonies, departmental parties, and defense parties and have always made me feel special and loved. My parents’ fierce protectiveness and unyielding faith in me have given me strength to conquer the world. Grandma and grandpa have encouraged me with their amazing cooking, kind hugs, and heartfelt words. Together, we have earned this degree and I have gotten the best reward out of the process-making my family proud. Acknowledgements I would like to thank my wonderful advisor, Dr. Deborah Chadee, without whom this degree would have been impossible. She has given me an amazing chance to succeed in her lab, invaluable help and guidance on my project, and wonderful life and career advice for which I would always be grateful. I would also like to acknowledge Dr. Richard Komuniecki, who has helped me with his many advices, written documents, and stories about graduate school. Additionally, I would like to thank Dr. Malathi Krishnamurthy for being supportive throughout graduate school and stepping in as a committee member on such short notice for my defense. As well, I would like to thank Dr. John Plenefisch and John Arnold for helping me get through my teaching assignments and putting a smile on my face. Of utmost importance are also the many wonderful friends that have helped me to get through graduate school, including, April Brockman, Widian Abi-Saab, Alan Hammer, Sercan Buyukkomurcu, Evan Zink, Sam Stefl, Adam Pore, Lindsay Schreiner, Adnan Siddiqui, Peter Oladimeji, Jenny Jay and Jeff Zaharatka. Widian has helped me transition into the lab and was a dear friend for most of my graduate school. April and Lindsay made up our wonderful golden trio of close friends for life. Alan and Jeff have been amazing friends, scientific advisors, and terrific seminar companions to me. Together with Peter, Jenny and Alan we have celebrated many wonderful events. Evan was my golden student, and Adam Pore got me through a tough time. I am sincerely grateful. v Table of Contents Abstract .............................................................................................................................. iii Acknowledgements ..............................................................................................................v Table of Contents ............................................................................................................... vi List of Figures .................................................................................................................... ix List of Abbreviations ......................................................................................................... xi 1 Introduction ...............................................................................................................1 1.1 MAPK Pathway Activation and Function ........................................................1 1.2 The ERK Signaling Pathway .............................................................................2 1.3 The P38 Signaling Pathway ...............................................................................5 1.4 The JNK Signaling Pathway ............................................................................10 1.5 The MLK Signaling Pathway ..........................................................................14 1.6 MLK Biological Functions ..............................................................................16 1.7 The Ubiquitin Proteasome System...................................................................20 1.8 Proteasome-Coupled Quality Control ..............................................................21 1.9 The CHIP E3 Ligase ........................................................................................23 1.10 Significance....................................................................................................26 vi 2 Materials and Methods ...........................................................................................27 2.1 Cell Culture ......................................................................................................27 2.2 Expression Vectors ..........................................................................................28 2.3 Plasmid and siRNA Transfections ...................................................................28 2.4 Cell Treatments ................................................................................................30 2.5 Quantitative Real-time PCR (RT-PCR) ...........................................................31 2.6 Protein Purification from Bacterial and Mammalian Cells..............................33 2.7 In Vitro Binding Assays and Immunoprecipitations .......................................34 2.8 Immunoblotting................................................................................................35 2.9 Endogenous MLK3 Ubiquitination..................................................................37 2.10 In vitro Ubiquitination Assay.........................................................................38 2.11 Invasion Assay ...............................................................................................38 3 Results ....................................................................................................................40 MLK3 3.1 GA Reduces the Level of Endogenous MLK3 Protein in SKOV3 .....40 3.2 GA Enhances K48-Linked Ubiquitination and Degradation of MLK3 ..................................................................................................43 3.3 GA Induces Degradation of MLK3 in HEK293 Cells .........................45 3.4 Heat or Osmotic Stresses Cause a Decline in Endogenous MLK3 .....48 3.5 Heat or Osmotic Stresses Accelerate MLK3 Turnover .......................50