UC San Diego UC San Diego Electronic Theses and Dissertations Title Regulation of PHLPP and Akt signaling Permalink https://escholarship.org/uc/item/1ff6w0r6 Author Warfel, Noel Andrew Publication Date 2011 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California UNIVERSITY OF CALIFORNIA, SAN DIEGO Regulation of PHLPP and Akt signaling A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Biomedical Sciences by Noel Andrew Warfel Committee in charge: Professor Alexandra C. Newton, Chair Professor Steve F. Dowdy Professor Kun-Liang Guan Professor Tony Hunter Professor Jing Yang 2011 The Dissertation of Noel Andrew Warfel is approved, and it is acceptable in quality and form for publication on microfilm and electronically: Chair University of California, San Diego 2011 iii DEDICATION Dedicated to Meredith Warfel for her endless dedication, love, and support and being a constant source of inspiration Dedicated to Stephen and Barbara Warfel for their guidance throughout my life. Without their support and advice this would not have been possible. iv TABLE OF CONTENTS Signature Page……………………………………………………...…………...…….iii Dedication……………………………………………………………………………..iv Table of Contents…………………………………...………………………...………..v List of Figures……………………………………………………………..…..……....vi List of Tables…………………………………...………………………..…..……......ix Acknowledgements…………………………………………………….……..…...….x Vita…………………………………………………………………………....….......xii Abstract of the Dissertation….…………………..…………………………..……....xiv Chapter 1: An Introduction to Akt and PHLPP………………….….....……..……1 Chapter 2: Identification of a Negative Feedback Loop through which Akt Activity Controls the Stability of PHLPP1.……..…….…........................….…19 Chapter 3: Mislocalization of the E3 ligase, β-TrCP1, in Glioblastoma Uncouples Negative feedback between PHLPP1 and Akt......……..…47 Chapter 4: Disruption of the Interface Between the PH and Kinase Domains of Akt is Sufficient for Hydrophobic Motif Phosphorylation Site in the Absence of mTORC2.…………………………………….....……..…89 Chapter 5: Unraveling a Complex Network of Signal Transduction Pathways...120 Appendix A: Regulation of PHLPP Activity and Evidence of PHLPP Phosphorylation……………………………….……..………….…..141 v LIST OF FIGURES Chapter 1 Figure 1.1: Akt activation by mitogen stimulation.………………..………...…....17 Figure 1.2: The PHLPP family of phosphatases.……………………………..…...18 Chapter 2 Figure 2.1: PHLPP1α and PHLPP1β are degraded more rapidly than PHLPP2.....40 Figure 2.2: Chronic inhibition of Akt decreases PHLPP1 protein levels.…...........41 Figure 2.3: PHLPP1β degradation is accelerated in cells lacking mTORC2.…….42 Figure 2.4: Akt activity directly correlates with PHLPP1 stability.…………...….43 Figure 2.5: Regulation of PHLPP1 stability by Akt is dependent on the activity of the proteasome and GSK-3β..…………………………………..…….45 Chapter 3 Figure 3.1: Akt-mediated feedback loop enhancing PHLPP1 levels is preferentially lost in CNS tumors.………………………..………………………….81 Figure 3.2: Loss of the feedback loop in glioblastoma is independent of PHLPP1, CK1, and GSK-3.……..……………………........................................83 Figure 3.3: β-TrCP1 is confined to the nucleus in glioblastoma cell lines and patient samples.…………………………………………………….…84 Figure 3.4: PHLPP1 turnover is slower and mRNA levels are reduced in glioblastoma cell lines..…………………………………………....….85 Figure 3.5: β-catenin turnover is impaired in glioblastoma cell lines……...……..86 vi Figure 3.6: Expression of β-TrCP1 in the cytosol rescues Akt-mediated regulation of PHLPP1 in glioblastoma.…………………………………...….....87 Chapter 4 Figure 4.1: Akt can be phosphorylated at S473 in the absence of mTOR kinase activity.………………………………………………………………113 Figure 4.2: Activation of PI3K is sufficient to restore Akt (S473) phosphorylation in cells lacking mTORC2……………………………………………114 Figure 4.3: Disruption of the PH and kinase domain interface of Akt is sufficient for S473 phosphorylation in the absence of mTORC2…………………………………………………………….115 Figure 4.4: Disruption of the interaction between the PH and kinase domains of Akt overcomes the necessity for mTORC2 for hydrophobic motif phosphorylation.…………………………………..………………....116 Supplemental Figure 4.1: Inhibition of S6K is necessary for rapamycin-induced phosphorylation of S473 in Sin1 -/- MEFs.…………………………117 Supplemental Figure 4.2: Rescue of S473 phosphorylation in Sin1 -/- MEFs is dependent on PI3K activity and the conformation of Akt.…….........118 Supplemental Figure 4.3: Regulation of Akt phosphorylation is specifically mediated by mTORC2.………………………………………………………...119 Chapter 5 vii Figure 5.1: The ability of Akt to regulate PHLPP1 stability is lost in glioblastoma due to the nuclear confinement of β-TrCP1.………………………...139 Figure 5.2: PHLPP expression levels are regulated at multiple levels downstream of PI3K.…………………………………………………………..….140 Appendix A Figure A.1: The phosphatase activity of PHLPP1β and PHLPP2, but not PHLPP1α, is increased in response to EGF stimulation.………………………..153 Figure A.2: Active Ras does not bind to the predicted RA domain of PHLPP1β, but binds to a separate region.…………………………………………...154 Figure A.3: Identification phosphorylation sites on PHLPP1 and PHLPP2 under basal and agonist stimulated conditions.…………………………….155 Figure A.3: PHLPP is phosphorylated in a EGF dependent manner at a site that is recognized by the p-p38 (T180/Y182) antibody.…………………....156 Figure A.4: PHLPP is phosphorylated by its substrates, Akt and PKC.…………157 viii LIST OF TABLES Chapter 3 Supplemental Table 3.1: Numerical values corresponding to each of the NCI60 tumor cell lines analyzed in Figure 3.1..………………..……………….…..89 Supplemental Table 3.2: Experimental identifiers and institutions responsible for generating microarray data made available by the Developmental Therapeutics Program NCI/NIH (http://dtp.cancer.gov/mtargets/mt_index.html)……………………...89 ix ACKNOWLEDGEMENTS I would like to acknowledge Professor Alexandra Newton, Ph.D. for her guidance as my research mentor and chair of my thesis committee. Her sense of optimism and passion for science were a constant source of support and motivation. Also deserving of special recognition for playing a direct role in my training are the members of my thesis committee for their support and insight. I would also like to thank my classmates and friends in San Diego and elsewhere for their support throughout my graduate career. Special thanks to Drs. Maya Kunkel, Matt Niederst, and Charles King for their insight and technical support and the Newton lab, past and present, for making graduate school an enjoyable experience. Chapter 1 is, in part, a review article to be submitted to the Journal of Biological Chemistry, 2011. It was co-authored by Alexandra C. Newton. Figure 5 of Chapter 2 has been previously published as supplemental data in the Journal of Biological Chemistry, 2010. Chapter 3 is, in part, a reprint of the material as it appears in the Journal of Biological Chemistry, 2010. It was co-authored by Matt Niederst, Michael W. Stevens, Paul M. Brennan, Margaret C. Frame, and the research was supported under the guidance of Alexandra C. Newton. I was the primary investigator and author of this research. x Chapter 4 is, in full, a research article accepted for publication in the Journal of Biological Chemistry, 2011. It was co-authored by Matt Niederst and supported under the guidance of Alexandra C. Newton. I was the primary investigator and author of this research. xi VITA 2004 Bachelor of Science, James Madison University 2006 Master of Science, The Johns Hopkins University 2011 Doctor of Philosophy, University of California, San Diego PUBLICATIONS 1. Warfel NA and Newton AC. PH domain Leucine-rich Repeat Protein Phosphatase (PHLPP): Structure, Function, Regulation, and Role in Disease. Manuscript to be submitted. J Biol Chem. 2. N. A. Warfel, M. Niederst, A. C. Newton, Disruption of the interface between the PH and kinase domains of Akt is sufficient for hydrophobic motif site phosphorylation in the absence of mTORC2. J Biol Chem, (2011). 3. Warfel NA, Niederst M, Stevens MW, Brennan PM, Frame MC, Newton AC. Mislocalization of the E3 Ligase, β-Transducin Repeat-containing Protein 1 (β-TrCP1), in Glioblastoma Uncouples Negative Feedback between the Pleckstrin Homology Domain Leucine-rich Repeat Protein Phosphatase 1 (PHLPP1) and Akt. J Biol Chem 2010;286:19777-88. 4. Brognard J, Niederst M, Reyes G, Warfel N, Newton AC. Common polymorphism in the phosphatase PHLPP2 results in reduced regulation of Akt and protein kinase C. J Biol Chem 2009;284:15215-23. 5. Gills JJ, Lopiccolo J, Tsurutani J, Shoemaker RH, Best CJ, Abu-Asab MS, Borojerdi J, Warfel NA, Gardner ER, Danish M, Hollander MC, Kawabata S, Tsokos M, Figg WD, Steeg PS, Dennis PA. Nelfinavir, A lead HIV protease inhibitor, is a broad-spectrum, anticancer agent that induces endoplasmic reticulum stress, autophagy, and apoptosis in vitro and in vivo. Clin Cancer Res 2007;13:5183-94. 6. Gills JJ, Castillo SS, Zhang C, Petukhov PA, Memmott, RM, Hollingshead M, Warfel N, Han J, Kozikowski AP, Dennis PA. Phosphatidylinositol ether lipid analogues that inhibit AKT also independently activate the stress kinase, p38alpha, through MKK3/6-independent and -dependent mechanisms. J Biol Chem 2007;282:27020-9. 7. Granville CA, Warfel N, Tsurutani J, Hollander MC, Robertson M, Fox S, Veenstra TD, Issaq HJ, Linnoila RI, Dennis PA. Identification of a highly xii effective rapamycin schedule that markedly