UNIVERSITY OF CALIFORNIA, SAN DIEGO Signaling by PHLPP1 and PKC alpha: function and specificity A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Biomedical Sciences by Audrey Kathleen O’Neill Committee in Charge: Professor Alexandra C. Newton, Chair Professor Joan Heller Brown Professor Kun-Liang Guan Professor Jerrold Olefsky Professor Jing Yang 2012 Copyright Audrey Kathleen O’Neill, 2012 All Rights Reserved The dissertation of Audrey Kathleen O’Neill is approved, and it is acceptable in quality and form for publication on microfilm and electronically: Chair University of California, San Diego 2012 iii DEDICATION Dedicated to my grandparents, Thomas & Kathryn O’Neill and Jack and Nancy Reardan, for their love and support. iv TABLE OF CONTENTS Signature page................................................................................................................................ iii Dedication page ..............................................................................................................................iv Table of Contents.............................................................................................................................v List of Figures .................................................................................................................................vi List of Tables................................................................................................................................. viii Acknowledgements.........................................................................................................................ix Vita..................................................................................................................................................xi Abstract of the Dissertation............................................................................................................ xii Chapter 1: Regulation of signaling by PHLPP and PKC..................................................................1 Chapter 2: Characterization of PHLPP1-null mice.........................................................................40 Chapter 3: PKCα promotes cell migration through a PDZ-dependent interaction with its novel substrate Discs Large homolog (DLG) 1........................................................................................72 Chapter 4: Summary and Conclusions ........................................................................................102 Appendix A: PKCα signaling in invadopodia ...............................................................................114 v LIST OF FIGURES Figure 1.1: Signaling by PHLPP and PKC .....................................................................................25 Figure 1.2: Domain structure of the human PHLPP isozymes.......................................................26 Figure 1.3: PHLPP suppresses the PI3K/Akt signaling pathway...................................................27 Figure 1.4: Domain structure and activator sensitivity of the three classes of PKC isozymes.......28 Figure 1.5: Mechanisms by which scaffolding proteins regulate cPKC signaling ..........................29 Figure 2.1: Expression of PHLPP1 and 2 mRNA in various mouse tissues ..................................61 Figure 2.2: Generation of PHLPP1-null mice.................................................................................62 Figure 2.3: Expression and phosphorylation of various signaling factors in PHLPP1-null mouse tissues............................................................................................................................................63 Figure 2.4: Body weights and blood parameters of PHLPP1-null and heterozygous mice............64 Figure 2.5: Responses of PHLPP-null mice and their wild type littermates to a high fat diet.........65 Figure 2.6: Characterization of pancreatic islets in PHLPP1-null and wild type mice....................66 Figure 2.7: Characterization of biochemical changes and apoptosis in primary and immortalized mouse embryonic fibroblasts (MEFs) lacking PHLPP1 .................................................................67 Figure 2.8: Increased PKC signaling and decreased apoptosis in primary astrocytes isolated from PHLPP1-null mice..........................................................................................................................68 Figure 3.1: PKCα interacts via its PDZ ligand with the PDZ domain scaffold DLG1/SAP97 .........90 Figure 3.2: PKCα promotes cell migration in wound healing assays.............................................91 Figure 3.3. A second shRNA construct targeting PKCα also inhibits wound healing ....................92 Figure 3.4: Wild type PKCα but not PKCα lacking the last three amino acids rescues wound healing in PKCα-depleted H1703 cells ..........................................................................................93 Figure 3.5: Interfering with DLG1 blocks the effects of PKCα knockdown on wound healing .......94 Figure 3.6: A second siRNA targeting DLG1 also inhibits wound healing .....................................95 Figure 3.7: DLG1 contains a conserved conventional PKC phosphorylation site..........................96 Figure 3.8: PKCα phosphorylates DLG1 at T656 ..........................................................................97 vi Figure 3.9: PKCα signaling at the DLG1 scaffold is increased in highly invasive NSCLC lines relative to their less invasive counterparts .....................................................................................98 Figure 4.1: Conclusions ...............................................................................................................111 Figure A.1: DLG1 and PKCα are both present at invadopodia in Src-transformed 3T3 fibroblasts.....................................................................................................................................124 Figure A.2: Conventional PKC activity is necessary for the maintenance and formation of invadopodia in Src-trasformed 3T3 fibroblasts ............................................................................125 Figure A.3: Stimulation of PKC activity is not sufficient to increase invadopodia formation in Src- transformed fibroblasts ................................................................................................................126 Figure A.4: DLG1 localization at invadopodia is dependent on cPKC activity but not presence of the phosphoacceptor at position T656.........................................................................................127 Figure A.5: DLG1 is not necessary for maintenance of invadopodia in Src-transformed fibroblasts.....................................................................................................................................128 vii LIST OF TABLES Table 1.1: Alterations in PHLPP levels in human cancer...............................................................30 viii ACKNOWLEDGMENTS First, I would like to thank my mentor, Alexandra Newton, who has helped bring this dissertation to completion by supporting and guiding my research and by teaching me how to design, perform, and, most importantly, learn from experiments (even those producing negative results). She has been a wonderful leader and inspiration for me, and her relentless positivity and persistence have set an example that I will do my best to follow. I would also like to acknowledge the critical contributions of past and present members of the Newton lab (Hilde Abrahamsen, Corina Antal, Laura Braughton, John Brognard, Lisa Gallegos, Christine Gould, Tianyan Gao, C.C. King, Maya Kunkel, Matt Niederst, Gloria (Ximena) Reyes, Cicely Schramm, Emma Sierecki, Shoana Sikorski, Irene Tobias, Noel Warfel, and Alyssa Wu-Zhang), who provided invaluable technical assistance and helpful discussions of data. I would particularly like to thank Lisa Gallegos, with whom I collaborated on the PKCα/DLG1 project and who was consistently reliable, helpful, and positive; Gloria Reyes, Tianyan Gao, and Shoana Sikorski, who gave me a great deal of assistance with the PHLPP1-null mouse project; and C.C. King and Maya Kunkel, who are inexhaustible sources of wisdom and kindness. Many members of the scientific community in San Diego contributed their time and expertise to helping me learn new techniques and/or gain access to materials I needed for this work. I would like to thank members of the Brown lab, especially Nicole Purcell and Sunny Xiang, as well as Vincenzo Cirulli, Malik Keshwani, Manuela Quintavalle, and Sara Courtneidge, for their assistance. I would also like to acknowledge Jeffrey Morimune for his help in the lab. Finally and most importantly, I would like to note the contributions of my thesis committee, Drs. Joan Heller Brown, Kun-Liang Guan, Jerry Olefsky, and Jing Yang, for their excellent critiques of and suggestions for my research. I would also like to thank my collaborators, who include Satoru Masubuchi and Paolo Sassone-Corsi at UC Irvine; Muhan Chen and Lloyd Trotman at Cold Spring Harbor; Erin Garcia and Randy Hall at Emory University; and Verline Justilien and Alan Fields at the Mayo Clinic, as ix well as Lisa Gallegos and Matt Niederst. In particular, I would like to acknowledge the continuing contributions and critiques from Randy Hall, Verline Justilien, and Alan Fields, who were instrumental in bringing the PKCα/DLG1 project to completion.