Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2012 Activation, regulation and functional characterization of class II PI3KC2B Błajecka, Karolina Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-164197 Dissertation Published Version Originally published at: Błajecka, Karolina. Activation, regulation and functional characterization of class II PI3KC2B. 2012, University of Zurich, Faculty of Science. ACTIVATION, REGULATION AND FUNCTIONAL CHARACTERIZATION OF CLASS II PI3KC2B Dissertation zur Erlangung der naturwissenschaftlichen Doktorwürde (Dr. sc. nat.) vorgelegt der Mathematisch-naturwissenschaftlichen Fakultät der Universität Zürich von Karolina Błajecka aus Polen Promotionskomitee Prof. Dr. Alessandro Sartori (Vorsitz) Dr. Mohamed Bentires-Alj Prof. Dr. Josef Jiricny PD Dr. Alexandre Arcaro (Leitung der Dissertation) Zürich, 2012 The experimental work presented in this thesis was performed at the Division of Pediatric Oncology at the Children's University Hospital Zürich and at the Division of Pediatric Hematology/Oncology, Department of Clinical Research, University of Bern. The supervision of the thesis was conducted by PD Dr. Alexandre Arcaro (Division of Pediatric Hematology/Oncology, Department of Clinical Research, University of Bern), Prof. Dr. Alessandro Sartori (Institute of Molecular Cancer Research, University of Zürich) and Dr. Mohamed Bentires-Alj (Friedrich Miescher Institute for Biomedical Research, Basel). TABLE OF CONTENTS ABBREVIATIONS …………...……………………………………………………………………… 1 SUMMARY……………………...……………………………………………………………………. 3 ZUSAMENFASSUNG ……..……………………………………………………………………….. 5 1. INTRODUCTION……………………………………………………………………….…….… 7 1.1. ROLE OF THE KINOME AND TYROSINE PHOSPHORYLATION IN CELL SIGNALING …...... 7 1.2. RECEPTOR TYROSINE KINASES ………………………………………………………… 8 1.2.1. Phosphotyrosine binding motifs in signal transduction ……………………….. 9 1.2.2. Adaptor and docking proteins …………………………………………………… 10 1.3. PHOSPHATIDYLINOSITOL 3-KINASES ………………………………………………….. 11 1.3.1. Classification of the PI3K family members …………………………………….. 13 1.4. CLASS II PI3KS …………………………………………………………………..…….. 18 1.4.1. Identification and expression …………………………………………………..... 18 1.4.2. Substrates preferences in vitro and in vivo ……………………………………. 19 1.4.3. Structural characteristics ………………………………………………………… 20 1.4.4. Mechanisms of activation ……………………………………………………….. 21 1.4.5. Cellular and physiological functions of class II PI3Ks ……………………….. 25 1.4.6. Involvement of class II PI3Ks in cancer ……………………………………….. 27 1.5. CYTOSKELETAL REARRANGEMENTS IN CANCER CELL MIGRATION AND ADHESION ….. 30 1.5.1. Rho GTPases and their regulators ………………………………………………30 1.5.2. The role of class I PI3Ks in the regulation of Rho GTPases in cancer …...… 33 1.5.3. The prototypic Dbl GEF and its oncogenic counterpart ………………………. 34 1.5.4. Dbl’s role in cancer ……………………………………………………………….. 37 2. AIMS OF THE STUDY …………………………………………………………………….… 39 3. RESULTS ……………………………………………………………………………………... 41 3.1. PHOSPHOINOSITIDE 3-KINASE C2B REGULATES RHOA AND THE ACTIN CYTOSKELETON THROUGH AN INTERACTION WITH DBL (PROJECT I) ………………... 41 3.1.1. Summary …………………………………………………………………………... 41 3.1.2. Introduction ……………………………………………………………………...… 42 3.1.3. Results …………………………………………………………………………..… 44 3.1.4. Material and Methods ……………………………………………………….....… 52 3.1.5. Discussion ……………………………………………………………………........ 57 3.1.6. Conclusions and Outlook ………………...…………………………………….... 65 3.2. IDENTIFICATION AND FUNCTIONAL CHARACTERIZATION OF PI3KC2B N-TERMINUS TYROSINE PHOSPHORYLATION SITES (PROJECT II) ………………………………….. 66 3.2.1. Summary …………………………………………………………………………... 66 3.2.2. Introduction ……………………………………………………………………...… 67 3.2.3. Results …………………………………………………………………………….. 73 3.2.4. Material and Methods ……………………………………………………………. 85 3.2.5. Discussion ………………………………………………………………………… 89 3.2.6. Conclusions and Outlook ………………………………………………………… 98 4. REFERENCES ...........................................................................................................100 5. ACKNOWLEDGEMENTS .......................................................................................... 110 6. CURRICULUM VITAE ................................................................................................111 7. APPENDIX .................................................................................................................115 ABBREVIATIONS ACK1 activated Cdc42Hs-associated kinase 1 Akt/PKB murine thymoma viral oncogene homolog 1/ Protein Kinase B ALL acute lymphoblastic leukemia AML acute myeloid leukemia CHIP carboxyl terminus of Hsc70-interacting protein c-Kit/SCFR mast/stem cell growth factor receptor c-Met hepatocyte growth factor receptor Dbl diffuse B-cell lymphoma DH Dbl-homology domain DN dominant-negative EGF epidermal growth factor EGFR epidermal growth factor receptor EMT epithelial-mesenchymal transition Erk extracellular signal-regulated kinase ESCC oesophageal squamous carcinoma FGFR fibroblast growth factor receptor GAP GTPase activating proteins GBM glioblastoma multiform GDI guanine nucleotide dissociation inhibitors GDP guanosine diphosphate GEF guanine nucleotide exchange factor GPCR G protein-coupled receptor Grb2 Growth factor receptor-bound protein 2 GSK3 glycogen synthase kinase 3 GST glutathione S-transferase GTP guanosine-5’-triphosphate GTPase guanosine triphosphate phosphohydrolase Hsc70 heat shock cognate protein Hsc70 Hsp90 heat shock protein Hsp90 IGF-IR insulin-like growth factor receptor INTS intersectin IR insulin receptor JNK/SAPK c-Jun N-terminal kinase/stress actvated protein kinase KD kinase-dead LC-MS Liquid chromatography-mass spectrometry 1 LPA lysophosphatidic acid MAPK mitogen-activated protein kinase MII myosin II MTM1 myotubularin 1 mTORC1 mammalian target of rapamacin-raptor complex 1 mTORC2 mammalian target of rapamacin-rictor complex 2 NB neuroblastoma NPDL nodular poorly differentiated lymphoma NSCLC non-small cell lung cancer onco-Dbl oncogenic Dbl PDGF platelet-derived growth factor PDGFR platelet-derived growth factor receptor PDK1 phosphoinositide-dependent kinase-1 PH Pleckstrin-homology domain PI3K Phosphatidylinositol-3 kinase PI3KC2β Phosphatidylinositol 3-kinase C2 domain containing subunit beta PNET peripheral neuroectodermal tumor proto-Dbl prototype Dbl PTB phosphotyrosine-binding PtdIns phosphatidylinositol PtdIns(3)P PtdIns 3-phosphate PtdIns(3,4)P2 PtdIns 3,4-bisphosphate PtdIns(3,4,5)P3 PtdIns 3,4,5-trisphosphate PtdIns(4,5)P2 PtdIns 4,5-bisphosphate PTEN phosphatase and tensin homologue deleted on chromosome 10 RTK receptor tyrosine kinase S6 ribosomal protein S6 S6K1 ribosomal protein S6 kinase 1 SCF stem cell factor SCLC small cell lung cancer SH2 Src homology-2 SH3 Src homology-3 SHIP SH2-containing phosphatase SNP single-nucleotide polymorphism Sos son of sevenless WT wild-type 2 SUMMARY Class II PI3K (phosphoinositide 3-kinase) isoforms have not been extensively investigated since the whole family of PI3Ks was discovered in the 1980s, and class II PI3K family members are still the least studied among all PI3Ks. To date the interest of the scientific community was mostly focused on class I PI3K enzymes especially due to their well established role in the development of several human disease including diabetes and cancer. However, an increasing amount of data has emerged recently suggesting an important role of class II PI3Ks in physiological and pathological processes. Together with receiving more attention, the understanding of their cellular functions will considerably increase. There is not much known yet about the mechanisms of class II PI3Ks activation and their downstream targets. Interestingly, accumulating data in the literature suggest that due to synthesis of distinct phosphoinositide or through different intracellular localization than other PI3K isoforms, class II PI3Ks possibly regulate biological processes or different steps in the same process distinct from class I and III PI3Ks. The molecular mechanisms underlying their action however still remain unrevealed. PI3KC2β belongs to class II PI3Ks and its cellular functions have been associated with pro-migratory and pro-survival signals, as well as cell proliferation. Following ligand stimulation, the enzyme is recruited to the activated receptor tyrosine kinases (RTKs) via Grb2 or Shc adaptor proteins. At the plasma membrane PI3KC2β generates mostly PtdIns(3)P or PtdIns(3,4)P2 and forms multi-protein complexes, whose assembly lead to activation of the Rho GTPases and regulation of the Akt/PKB signaling pathway. How exactly PI3KC2β contributes to the above-mentioned functions has not been precisely described. However, its mechanism of action seems to be related to the kinase multi-domain structure, which differs much from the structures of class I and class III PI3Ks. The most characteristic features of the enzyme are its lack of association to regulatory subunits, which is accompanied by a high molecular mass and elongated N- and C-terminal extensions, which play a regulatory role for PI3KC2β catalytic activity. The knowledge about PI3KC2β structural features still needs to be translated into a precise mode of action. In my studies I have investigated the regulatory mechanism linking PI3KC2β to the activation of the Rho family of small GTPases through the Dbl guanine exchange factor (GEF) in NIH3T3 mouse fibroblasts, where PI3KC2β over-expression induced marked cell morphology