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Intersectin: a Regulator of Cell Signaling by KATY A

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Intersectin: a Regulator of Cell Signaling by KATY A Intersectin: A Regulator of Cell Signaling BY KATY A. WONG B.S. California Polytechnic State University at San Luis Obispo, 2005 THESIS Submitted as partial fulfillment of the requirements for the degree of Doctor of Philosophy in Pharmacology in the Graduate College of the University of Illinois at Chicago, 2012 Chicago, Illinois Defense Committee: John P. O’Bryan, Chair and Advisor Richard Ye Richard Minshall Graeme Carnegie Karen Colley, Biochemistry ii This thesis is dedicated to my parents, Ann and Ron Wong, without their love and support who knows where I would be. ii ii iii ACKNOWLEDGEMENTS I would first like to acknowledge my advisor, Dr. John O’Bryan, without his guidance and training I would not have been able to accomplish my doctorate degree. Secondly, I would like to thank my thesis committee members, Dr. Richard Ye, Dr. Richard Minshall, Dr. Karen Colley, and Dr. Graeme Carnegie. I appreciate all the time, energy, and insightful comments they have contributed to aid me in the completion of my doctoral degree. I would also like to thank members of the O’Bryan lab past and present. When I started in the lab Sara Dunn and Dr. Yun-Ju Chen were invaluable in my training. I am also especially grateful to Joseph Sennello who was an enjoyable lab mate and friend. I am also grateful for all the friends I made during my graduate school career. Emily Welch, Brendan Quinn, Jackson Hoffman, and Chris O’Donnell who all made graduate school fun and exciting. Thank you for the summer of 2008, turtle races, La Bamba’s, Hawkeye’s lunches, and all other fun times. I am especially thankful to my better half, Erica Southgate. She is truly the greatest friend that anyone could ask for. I would also like to give a special thank you Adam Wieschhaus. Thank you for putting up with me and taking care of me. Graduate school is long and challenging, thank you all for also making it fun. Finally, I would like to thank my family. It is a true gift to have such a strong support system and unconditional love. Thank you is not enough to describe the gratitude I feel for my parents, Ann and Ron Wong. Thank you for your patience, support, love, and guidance. My parents raised three daughters; my older sister Kari who received her doctorate degree from the University of Chicago, and my younger sister Keely who received her nursing degree from California State at Chico. Without them there is no way I would be where I am today. iii iii iv TABLE OF CONTENTS CHAPTER PAGE I. ITSN, PI3K-C2, RAS: THE THREE AMIGOS OF CELL SIGNALING . 1 A. Introduction . 1 B. Intersectin . 1 1. ITSN Genes . 1 2. ITSN and Disease . 2 3. ITSN and Signal Regulation . 5 C. Phosphoinositide 3’ Kinases . 11 1. Three classes of Phosphoinositide 3’ Kinases . 12 2. PI3K Inhibitors . 14 3. Class II . 16 4. Class II PI3K-beta . 16 D. Ras . 19 1. Ras Genes and Structure . 19 2. Ras GTPase . 20 3. Ras Modification and Trafficking . 22 4. Ras Signaling . 23 5. Compartmentalized Signaling of Ras . 26 6. Nucleotide-free Ras Signaling . 28 II. A NEW DIMENSION TO RAS FUNCTION: A NOVEL ROLE FOR NUCLEOTIDE-FREE RAS IN CLASS II PHOSPHOINOSITIDE 3- KINASE BETA (PI3K-C2) REGULATION . 30 A. Introduction . 30 B. Materials and Methods . 30 1. Cell lines, Transfection, and Reagents . 30 2. DNA Constructs and Generation of mutants . 31 3. Protein Purification . 32 4. In vitro Pulldowns . 33 5. Nucleotide-free Assays . 34 6. Biochemical analyses . 34 7. In vitro Kinase Assays . 34 C. Results . 35 1. PI3K-C2 and Ras interact and co-localize with ITSN on intracellular vesicles . 35 iv iv v TABLE OF CONTENTS (continued) CHAPTER PAGE II. A NEW DIMENSION TO RAS FUNCTION: A NOVEL ROLE FOR NUCLEOTIDE-FREE RAS IN CLASS II PHOSPHOINOSITIDE 3- KINASE BETA (PI3K-C2) REGULATION (continued) C. Results . 35 2. Ras is necessary for ITSN activation of AKT . 38 3. PI3K-C2 forms a complex with Ras on intracellular vesicles . 38 4. The PI3K-C2 RBD directly interacts with nucleotide-free Ras . 41 5. Nucleotide-free Ras inhibits PI3K-C2 activity . 47 6. Point mutations in the effector domain of Ras or the RBD of PI3K-C2 disrupt Ras-PI3K-C2 interaction . 47 7. PI3K-C2 alters Ras signaling . 51 D. Discussion . 54 III. COMPARTMENTALIZED SIGNALING: THE RIGHT PLACE AT THE RIGHT TIME . 58 A. Introduction . 58 B. Compartmentalized Signaling . 58 C. Rabs . 59 1. Rab5 . 60 2. Rab7 . 62 D. ITSN's Role in Rab Trafficking . 63 E. PI3Ks Regulate Membrane Trafficking . 63 IV. INTERSECTIN IS LOCATED ON MULTIPLE INTRACELLULAR COMPARTMENTS . 65 A. Introduction . 65 B. Materials and Method s . 65 1. Cell lines, Transfection, and Reagents . 65 2. DNA Construts . 66 v v vi TABLE OF CONTENTS (continued) CHAPTER PAGE IV. INTERSECTIN IS LOCATED ON MULTIPLE INTRACELLULAR COMPARTMENTS (continued) B. Materials and Methods (continued) . 65 3. Western Blot Analyses . 66 4. Immunofluorescence and Imaging . 66 5. Calculation of Co-localization . 67 C. Results . 67 1. ITSN is localized to an early endosomal compartment . 67 2. PI3K-C2 localizes to an early endosomal compartment . 72 3. EEA1 is not recruited to ITSN:PI3K-C2 positive vesicles . 72 4. Different pools of ITSN interact with Ras and PI3K-C2 . 76 D. Discussion . 76 V. FUTURE DIRECTIONS . 80 VI. APPENDIX . 86 VII. CITED LITERATURE . 87 vi vi vii LIST OF TABLES TABLE PAGE I. INTERSECTIN GENES AND ORTHOLOGS . 8 II. ISOFORM SPECIFIC PI3K INHIBITORS . 15 III. PERCENT CO-LOCALIZATION . 71 vii vii iii LIST OF FIGURES FIGURE PAGE 1. Intersectin isoforms and domains . 3 2. ITSN silencing does not inhibit transferrin receptor or bulk internalization . 6 3. Phosphoinositide-3-kinases . 13 4. Ras . 21 5. General schematic of Ras regulated signaling pathways . 24 6. Ras, PI3K-C2, and ITSN co-localize on intracellular vesicles . 36 7. Ras is necessary for ITSN activation of AKT . 39 8. Ras forms a complex with PI3K-C2 . 40 9. PI3K-C2 does not interact with oncogenic Ras . 42 10. ITSN and Ras form a BIFC complex . 43 11. PI3K-C2 does not interact with Ras15A . 44 12. Binding of PI3K-C2 RBD to Ras . 46 13. Nucleotide-free Ras inhibits PI3K-C2 activity . 48 14. Mutations in the effector region of Ras disrupts PI3K-C2 binding . 50 15. Biological activity of PI3K-C2-RBD versus Raf-RBD . 52 16. Model for ITSN-Ras-PI3K-C2 pathway . 56 17. Rab mediated trafficking . 61 18. Myc-Rabpatin5 and endogenous ITSN co-localize . 68 19. Endogenous Rab5 and endogenous ITSN co-localize . 69 20. Endogenous Rab7 and endogenous ITSN co-localize . 70 21. A pool of ITSN is on early endosomes . 73 viii iv LIST OF FIGURES (continued) FIGURE PAGE 22. PI3K-C2 localizes with early and late endosomes . 74 23. ITSN 1and PI3K-C2 complex does not localize with EEA1 . ..
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