POSITIVE REGULATION OF PKB/AKT KINASE ACTIVITY BY THE VACUOLAR (H+)-ATPASE IN THE CANONICAL INSULIN SIGNALING PATHWAY: IMPLICATIONS FOR THE TARGETED PHARMOCATHERAPY OF CANCER. By Sevag A Kaladchibachi A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Medical Biophysics Graduate Department University of Toronto © Copyright by Sevag Kaladchibachi 2014 Positive regulation of PKB/Akt kinase activity by the vacuolar (H+)-ATPase in the canonical insulin signaling pathway: implications for the targeted pharmacotherapy of cancer. Sevag Kaladchibachi, Doctor of Philosophy, 2014 Department of Medical Biophysics University of Toronto Abstract The canonical PI3K/Akt pathway is activated downstream of numerous receptor tyrosine kinases, including the insulin and insulin-like growth factor receptors, and is a crucial regulator of growth and survival in metazoans. The deregulation of Akt is implicated in the pathogenesis of numerous diseases including cancer, making the identification of modifiers of its activity of high chemotherapeutic interest. In a transheterozygous genetic screen for modifiers of embryonic Akt function in Drosophila, in which the PI3K/Akt signaling pathway is conserved, we identified the A subunit of the vacuolar ATPase (Vha68-2) as a positive regulator of Dakt function. Our characterization of this genetic interaction in the larval stage of development revealed that Vha68-2 mutant phenotypes stereotypically mimicked the growth defects observed in mutants of the Drosophila insulin signaling pathway (ISP). The loss of Vha68-2 function, like Dakt- deficiency, was found to result in organismal and cell-autonomous growth defects, and consistent with its putative role as a positive regulator of Dakt function, both the mutational and pharmacological inhibition of its activity were found to downregulate Akt activation. Genetic epistasis experiments in somatic clones of Vha68-2/dPTEN double mutants demonstrated that the loss of Vha68-2 function suppressed the growth defects ii associated with dPTEN-deficiency, placing Vha68-2 activity downstream of dPTEN in the ISP, while the examination of PI3K activity and PH domain-dependent membrane recruitment in pharmacologically inhibited larval tissues further placed Vha68-2 function downstream of PI3K. These findings were recapitulated in cultured NIH-3T3 cells, whose treatment with bafilomycin A1, a potent and specific inhibitor of V-ATPase, resulted in the downregulation of Akt phosphorylation, particularly in non-cytoplasmic intracellular compartments. Furthermore, cellular subfractionation of bafilomycin-treated NIH-3T3 cells demonstrated a decrease in the localization of Akt to early endocytic structures, and a downregulation in the localization and activation of Akt in the nuclei of both Drosophila and mammalian cells. Finally, the pharmacotherapeutic relevance of V- ATPase inhibition was addressed in two tumor models – multiple myeloma and glioblastoma – and our preliminary findings in these cancers, which are often associated with ectopic PI3K/Akt signaling, showed significant cytotoxic efficacy in vitro, warranting its consideration as a tractable pharmacological option in the treatment of cancer. iii Acknowledgements I am first and foremost greatly indebted to my supervisor, Dr. Armen Manoukian, who has not only been a mentor and teacher, but also a valued friend. I would like to thank my labmates, particularly Dr. Sam Scanga, from whom I learned much, and whose mentorship was crucial to my development as a scientist. I thank Dr. Jim Woodgett, who has been a member of both my Masters’ and Doctoral committees, and whose guidance and advice I have always held in high esteem. I thank Dr. Peter Cheung for serving on my Doctoral committee, generously providing access to his research facilities, and fulfilling his advisory role to a tee. Last but never least, I thank my immediate family – my parents Josephine and Harout Kaladchibachi, my brothers Khajak and Jacques Kaladchibachi, and my maternal aunt Sirouhie Achjian – from the bottom of my heart, and dedicate this thesis to them. Their undying support through the good times and bad was indispensable for the completion of this work, and their incessant faith in me inspires me daily. iv Table of contents Abstract…………………………………………………………………………………....ii Acknowledgements…………………………………………………………...…………..iv Table of Contents………………………………………………………………………….v List of Tables, Figures, and Appendices………………………………………………...vii List of Diagrams…………………………………………………………………………..x List of Abbreviations……………………………………………………………………xiv List of Publications……………………………………………………………………..xvii Chapter 1: General Introduction……………………………………………………….1 Prologue…………………………………………………………………………………...2 1-1 – PIP3-dependent stimulation of class I PI3K effectors………………………………8 1-2 – PTEN-mediated downregulation of PIP3-dependent signaling……………………13 1-2.1 – Structure and biochemical function of the PTEN phosphatase………………17 1-2.2 – Genetic characterization of PTEN function…………………………………..18 1-3 – The Akt kinases……………………………………………………………………20 1-3.1 – Mechanisms of RTK-dependent Akt activation……………………………...22 1-3.2 – PDK1-dependent phosphorylation of the Akt catalytic domain T-loop……...26 1-3.3 – mTORC2-dependent phosphorylation of the Akt hydrophobic motif………..28 1-3.4 – Current mechanistic model of phosphorylation-dependent Akt activation…..40 1-4 – The role of Akt signaling in the mediation of cellular growth and survival ……...46 1-4.1 –The role of Akt substrates in the promotion of cellular growth………………47 1-4.2 – The role of Akt-mediated GSK3 inhibition in cellular growth………………50 1-4.3 – Growth factor-dependent PI3K/Akt-mediated mTORC1 activation………....58 1-4.4 – TSC1/2 integrates multiple modulatory inputs in the regulation mTORC1….66 1-4.5 – Activation of mTORC1 by amino acid/nutrient availability…………………70 1-4.6 – TORC1/S6K1-mediated negative feedback regulation of PI3K/Akt activity..83 1-4.7 – The role of Akt substrates in the promotion of cellular survival……………..88 1-4.8 – Identification of novel Akt interactions and signaling components………….96 1-5 – The vacuolar H+-ATPase…………………….…………………………………...101 1-5.1 – V-ATPase structure and function…………………………………………...102 1-5.2 – V-ATPase-mediated regulation of endomembrane acidification and traffic..106 1-6 – Study rationale……………………………………………………………………111 Chapter 2: Genetic characterization of Vha68-2 as a cell-autonomously acting positive regulator of PKB/Akt signaling Drosophila………...……………………...113 2-1 – Introduction………………………………………………………………………114 2-2 – Materials and Methods…………………………………………………………...136 2-3 – Results……………………………………………………………………………140 2-3.1 – Identification of a genetic interaction between dVha68-2 and Dakt...……...140 2-3.2 – Dakt and Vha68-2 mutants have similar growth phenotypes……………….141 2-3.3 – Genetic or pharmacological inhibition of V-ATPase downregulates Akt…..145 2-3.4 – Vha68-2 mutant clones cell-autonomously phenocopy Dakt deficiency…...148 2-3.5 – Vha68-2P463 suppresses the overgrowth phenotype of dPTEN deficiency….153 2-3.6 – Bafilomycin inhibits Dakt phosphorylation independently of PI3K activity.160 v 2-3.7 – V-ATPase activity cell-autonomously promotes intracellular acidification..170 2-4 – Discussion………………………………………………………………………...174 Chapter 3: Biochemical characterization of bafilomycin as an inhibitor of PKB/Akt signaling in NIH-3T3 fibroblasts……………………………………………………..185 3-1 – Introduction………………………………………………………………………186 3-2 – Materials and Methods…………………………………………………………...199 3-3 – Results……………………………………………………………………………203 3-3.1 – Bafilomycin downregulates Akt phosphorylation and induces apoptosis…..203 3-3.2 – Subcellular fractionation and compartmental enrichment…………………..212 3-3.3 – Bafilomycin-induced changes in the intracellular localization and compartment-specific activation of Akt………………………………………………...218 3-3.4 – Bafilomycin treatment downregulates mTORC1/S6K1 signaling concomitantly with Akt inhibition……………………………………………………...221 3-3.5 – Bafilomycin- induced changes in the compartment-specific localization of selected endomembrane markers……………………………………………………….228 3-3.6 – Bafilomycin diminishes the growth factor-stimulated recruitment of Akt to signaling endosomes……………………………………………………………………236 3-4 – Discussion………………………………………………………………………...249 Chapter 4: In vitro assessment of V-ATPase inhibition as a viable avenue of cancer pharmacotherapy……………………………………………………………………...263 4-1 – Introduction………………………………………………………………………264 4-2 – Materials and Methods…………………………………………………………...287 4-3 – Results……………………………………………………………………………289 4-3.1 – Bafilomycin reduces the viability of cultured human myeloma cell lines….289 4-3.2 – Bafilomycin reduces the viability of cultured human glioma cell lines…….294 4-3.3 – Bafilomycin downregulates Akt phosphorylation in human myeloma and glioblastoma cells……………………………………………………………………….301 4-4 – Discussion ………………………….…………………………………………….307 Concluding Remarks……………………………………………………………….....310 References………………………………………………………...……………………316 Appendices…………………...…………………………….…………………………..411 vi List of Tables, Figures and Appendices Table 2-1. Genetic interaction between Dakt and Vha68-2……………………………143 Table 2-2. Lethal phase of examined mutant alleles of Vha68-2 and Dakt……………143 Figure 2-1. Larval and pupal size phenotypes of Dakt and Vha68-2 mutants…………146 Figure 2-2. Dakt phosphorylation at S505 is downregulated in Vha68-2 mutants…….147 Figure 2-3. Pharmacological inhibition of V-ATPase activity downregulates both endogenous and transgenically expressed Akt