University of Kentucky UKnowledge Theses and Dissertations--Molecular and Cellular Biochemistry Molecular and Cellular Biochemistry 2017 RAD GTPASE: IDENTIFICATION OF NOVEL REGULATORY MECHANISMS AND A NEW FUNCTION IN MODULATION OF BONE DENSITY AND MARROW ADIPOSITY Catherine Nicole Kaminski Withers University of Kentucky, [email protected] Digital Object Identifier: https://doi.org/10.13023/ETD.2017.326 Right click to open a feedback form in a new tab to let us know how this document benefits ou.y Recommended Citation Withers, Catherine Nicole Kaminski, "RAD GTPASE: IDENTIFICATION OF NOVEL REGULATORY MECHANISMS AND A NEW FUNCTION IN MODULATION OF BONE DENSITY AND MARROW ADIPOSITY" (2017). Theses and Dissertations--Molecular and Cellular Biochemistry. 34. https://uknowledge.uky.edu/biochem_etds/34 This Doctoral Dissertation is brought to you for free and open access by the Molecular and Cellular Biochemistry at UKnowledge. It has been accepted for inclusion in Theses and Dissertations--Molecular and Cellular Biochemistry by an authorized administrator of UKnowledge. For more information, please contact [email protected]. STUDENT AGREEMENT: I represent that my thesis or dissertation and abstract are my original work. Proper attribution has been given to all outside sources. I understand that I am solely responsible for obtaining any needed copyright permissions. I have obtained needed written permission statement(s) from the owner(s) of each third-party copyrighted matter to be included in my work, allowing electronic distribution (if such use is not permitted by the fair use doctrine) which will be submitted to UKnowledge as Additional File. I hereby grant to The University of Kentucky and its agents the irrevocable, non-exclusive, and royalty-free license to archive and make accessible my work in whole or in part in all forms of media, now or hereafter known. I agree that the document mentioned above may be made available immediately for worldwide access unless an embargo applies. I retain all other ownership rights to the copyright of my work. I also retain the right to use in future works (such as articles or books) all or part of my work. I understand that I am free to register the copyright to my work. REVIEW, APPROVAL AND ACCEPTANCE The document mentioned above has been reviewed and accepted by the student’s advisor, on behalf of the advisory committee, and by the Director of Graduate Studies (DGS), on behalf of the program; we verify that this is the final, approved version of the student’s thesis including all changes required by the advisory committee. The undersigned agree to abide by the statements above. Catherine Nicole Kaminski Withers, Student Dr. Douglas A. Andres, Major Professor Dr. Trevor P. Creamer, Director of Graduate Studies RAD GTPASE: IDENTIFICATION OF NOVEL REGULATORY MECHANISMS AND A NEW FUNCTION IN MODULATION OF BONE DENSITY AND MARROW ADIPOSITY DISSERTATION A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the College of Medicine at the University of Kentucky By Catherine Nicole Kaminski Withers Lexington, Kentucky Director: Dr. Douglas A. Andres Professor of Molecular and Cellular Biochemistry Lexington, Kentucky 2017 Copyright © Catherine N. K. Withers 2017 ABSTRACT OF DISSERTATION RAD GTPASE: IDENTIFICATION OF NOVEL REGULATORY MECHANISMS AND A NEW FUNCTION IN MODULATION OF BONE DENSITY AND MARROW ADIPOSITY The small GTP-binding protein Rad (RRAD, Ras associated with diabetes) is the founding member of the RGK (Rad, Rem, Rem2, and Gem/Kir) family that regulates voltage-dependent calcium channel function. Given its expression in both excitable and non-excitable cell types, the control mechanisms for Rad regulation and the potential for novel functions for Rad beyond calcium channel modulation are open questions. Here we report a novel interaction between Rad and Enigma, a scaffolding protein that also binds to the E3 ubiquitin ligase Smad ubiquitin regulatory factor 1 (Smurf1). Overexpression of Smurf1, but not of a catalytically inactive mutant enzyme, results in ubiquitination of Rad and down regulation of Rad protein levels. The Smurf1-mediated decrease in Rad levels is sensitive to proteasome inhibition and requires the ubiquitination site Lys204, suggesting that Smurf1 targets Rad for degradation. Rad protein levels, but notably not mRNA levels, are increased in the hearts of Enigma-/- mice, leading to the hypothesis that Enigma may function as a scaffold to enhance Smurf1 regulation of Rad. In addition to ubiquitination, phosphorylation of RGK proteins represents another potential means of regulation. Indeed, Rem phosphorylation has been shown to abolish calcium channel inhibition. We demonstrate that β-adrenergic signaling promotes Rad phosphorylation at Ser39. Rad Ser39 phosphorylation is correlated with a decrease in the interaction between Rad and the CaVβ subunit of the calcium channel and an increase in Rad binding to 14-3-3. Interestingly, Enigma overexpression promotes an increase in Rad Ser39 phosphorylation as well. Despite an interaction between Enigma and the CaV1.2 calcium channel subunit, overexpression of Enigma had no effect on Rad-mediated channel inhibition. Thus, Rad Ser39 phosphorylation alters its association with the calcium channel, but its impact on calcium channel regulation has yet to be determined. Finally, we report a novel function for Rad in the regulation of bone homeostasis. Rad deletion in mice results in a significant decrease in bone mass. Dynamic histomorphometry in vivo and primary calvarial osteoblast assays in vitro demonstrate that bone formation and osteoblast mineralization rates are depressed in the absence of Rad. Microarray analysis revealed that canonical osteogenic gene expression is not altered in Rad-/- osteoblasts; instead robust up-regulation of matrix Gla protein (MGP, +11-fold), an inhibitor of mineralization and a protein secreted during adipocyte differentiation, was observed. Strikingly, Rad deficiency also resulted in significantly higher bone marrow adipose tissue (BMAT) levels in vivo and promoted spontaneous in vitro adipogenesis of primary calvarial osteoblasts. Adipogenic differentiation of WT osteoblasts resulted in the loss of endogenous Rad protein, further supporting a role for Rad in the control of BMAT levels. These findings reveal a novel in vivo function for Rad signaling in the complex physiological control of skeletal homeostasis and bone marrow adiposity. In summary, this dissertation expands our understanding of Rad regulation through identification of a novel binding partner and characterization of post-translational regulatory mechanisms for Rad function. This work also defines a new role for Rad that may not depend upon its calcium channel regulatory properties: regulation of the bone-fat balance. These findings suggest that the regulation of Rad GTPase is likely more complex than guanine nucleotide cycling and that functions of Rad in non-excitable tissues warrant further study. KEYWORDS: Rad GTPase, Ubiquitination, Osteoblast, Adipocyte, Bone Catherine Nicole Kaminski Withers July 18, 2017 RAD GTPASE: IDENTIFICATION OF NOVEL REGULATORY MECHANISMS AND A NEW FUNCTION IN MODULATION OF BONE DENSITY AND MARROW ADIPOSITY By Catherine Nicole Kaminski Withers Douglas A. Andres, Ph.D. Direction of Dissertation Trevor P. Creamer, Ph.D. Director of Graduate Studies July 18, 2017 Date To my husband, Brad Withers, and our children, Sylvia and Bennett ACKNOWLEDGEMENTS I would first like to thank my mentor and the chair of my dissertation committee, Dr. Doug Andres. He has encouraged me to think creatively and has supported me as my project extended into new, unfamiliar fields for our laboratory. I am very grateful for the ways he has pushed and challenged me over the years. I also appreciate all the advice and critiques I have received from the other members of my committee, Dr. Wally Whiteheart, Dr. Craig Vander Kooi, and Dr. Jonathan Satin. The work in this dissertation has benefited greatly from their thoughtful suggestions and feedback, and they have each contributed to my development as a scientist. I would also like to thank my outside examiner, Dr. Chris Norris, for taking the time to read this dissertation and attend my defense. Next, I want to extend my gratitude to past and present members of the Andres lab. Dr. Geng-Xian Shi and Dr. Weikang Cai helped me to get started in the lab and provided an environment where hard work and fun happened simultaneously. Megan Pannell has been by my side since my second year of graduate school, and I appreciate her companionship along the way. Jeff Smith was a source not only of technical assistance in the lab but also of advice about science and life and fun conversations (especially during football season, Go Steelers!). Dr. Carole Moncman provided invaluable assistance (and patience) with microscopy as well as interesting conversation and delicious cookies. I also thank Dr. Janet Manning, Dr. Zhaiyi Zhang, Dr. Sajad Mir, and Landon Simpson for their company in the lab and for helpful scientific discussions. Finally, I had iii the privilege of working with two talented REU students, Linda Castillo and Mariana Sandoval. We had a number of collaborators who were essential to completion of the work described in this dissertation. The microcomputed tomography core facility at Rush University and the bone histology core facility at IUPUI provided bone assays that were unavailable at UK and allowed us to generate