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Identification of an Adaptor for the Drosophila Class IA

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Identification of an Adaptor for the Drosophila Class IA Identification of an adaptor for theDrosophila Ciass iA phosphoinositide 3-kinase and the study of its functionIn vivo and In vitro David Weinkove A thesis submitted to the University of London for the Degree of Doctor of Philosophy May 1999 Ludwig Institute for Cancer Research 91 Riding House Street London WIP 8BT Department of Biochemistry and Molecular Biology University College London Gower Street London WCIE 6BT MRC Laboratory for Molecular Cell Biology University College London Gower Street London WCIE 6BT ProQuest Number: 10010362 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest 10010362 Published by ProQuest LLC(2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Abstract Class Ia phosphoinositide 3-kinases (PI3Ks) regulate several cellular processes in response to receptor tyrosine kinase (RTK) activation. This response is mediated by the SH2 domain-containing adaptors for Class Ia PI3Ks, which, by binding to phosphotyrosines on activated RTKs or their substrates, bring Class Ia PI3Ks close to their lipid substrates at the membrane. This thesis describes the identification of p60, the adaptor for the Drosophila Class Ia PI3K, Dpi 10. p60 was isolated by affinity purification with a phosphopeptide derived from a human RTK, and the corresponding cDNA was cloned using peptide sequence data. Like the mammalian Class Ia PI3K adaptors, p60 possesses two SH2 domains, which enable receptor binding, and an inter-SH2 domain, which facilitates Dpi 10 binding. Biochemical analyses showed that the D pll0/p60 complex is widely expressed and possesses lipid and protein kinase activity. After determining the genomic structure of the p60 region and characterising candidate mutations, two p60 mutants were generated. Zygotic p60 mutants, like D pllO mutants, showed a lethal phenotype resulting from defective larval development. Clones ofpSO or DpllO mutant cells survived and proliferated during imaginai disc development, but were reduced in both size and number. To address its in vivo function further, p60 was ectopically expressed in eye and wing imaginai discs. Previously, it was shown that ectopically-expressed DpllO promotes imaginai disc growth. In contrast, p60 ectopic expression resulted in small wings and eyes. Expressed together, DpllO and p60 had little effect on growth, even though the proteins were present at higher levels when coexpressed than when expressed alone. Together, these results demonstrate a specific function for Dpll0/p60 in imaginai disc development. Models for the regulation of DpllO by p60 and the control of organ growth by DpllO/p60 are proposed, and their implications for mammalian Class I a PI3K function are discussed. Table of Contents Title P age ...............................................................................................................................1 Abstract ..................................................................................................................................2 Table of Contents.................................................................................................................3 List of Figures and Tables ................................................................................................. 8 Chapter 1: Introduction................................................................................................... 10 1.1 Overview...........................................................................................................12 1.2 The three classes of phosphoinositide 3-kinases..................................... 12 1.3 PI3Ks may mediate many of the functions of receptor tyrosine kinases......................................................................................................15 1.3.1 Signalling downstream of RTKs via proteins that recognise and bind phosphotyrosine................................................... 15 1.3.2 Class Ia PI3Ks associate with activated RTKs through SH2 domain-containing adaptors..........................................16 1.3.2.1 The adaptors for Class Ia PI3Ks ................................... 17 1.3.2.2 The SH2 domains of the adaptors for Class Ia PI3KS..........................................................................................19 1.3.3 The activation of Class Ia PI3Ks .................................................. 19 1.3.4 RTK specificity in the activation of Class Ia PI3Ks .................22 1.3.5 Other means of activation of Class Ia PI3Ks .............................23 1.3.6 Downregulation of Class Ia PI3K activity .................................24 1.4 Downstream effectors of Class Ia PBKs ...................................................25 1.4.1 The protein serine/threonine kinases, PKB and P D K l............................................................................................................26 1.4.2 Guanine nucleotide exchange factors ........................................ 28 1.4.3 PLCy................................................................................................... 29 1.4.4 Other downstream effectors......................................................... 30 1.5 The cellular functions of Class Ia PI3Ks .................................................... 30 1.5.1 Methods of investigation of Class Ia PI3K function...............31 1.5.2 Stimulation of proliferation by Class Ia PBKs ...........34 1.5.3 Protection from apoptosis by ClassI a PBKs ............................35 1.5.4 Effects of Class Ia PBKs on glucose and lipid metabolism................................................................................................. 36 1.5.5. Regulation of protein synthesis by Class Ia PBKs ..............37 1.5.6 Regulation of membrane trafficking by Class Ia P B K s............................................................................................................38 1.5.7 Cytoskeletal rearrangements mediated by Class Ia PI3KS............................................................................................................39 1.5.8 Class Ia PI3Ks and cell transformation ...................................... 40 1.5.9 Other roles of Class Ia PI3Ks ........................................................ 41 1.6 The role of Class Ia PI3Ks in the whole organism..................................42 1.6.1 Genetic analysis of Class Ia PI3K function in C. elegans .......................................................................................................... 43 1.6.2 Analysis of Class Ia PI3Ks in m ice..............................................46 1.7 Using Drosophila to study Class Ia PI3Ks ..................................................46 1.7.1 The Drosophila Class Ia PI3K, DpllO, plays a role in growth control...........................................................................................47 1.8 Imaginai disc growth ..................................................................................... 47 1.8.1 Imaginai disc developm ent .......................................................... 47 1.8.2 The regulation of growth during development........................51 1.8.2.1 Intrinsic and extrinsic controls of imaginai disc growth.................................................................................... 51 1.8.2.2. The involvement of proliferation, apoptosis and protein synthesis in imaginai disc development................................................................................. 52 1.8.2.3 The coordination of growth and patterning during imaginai disc development .......................................... 55 Chapter 2: Experimental procedures ............................................................................ 57 2.1 Culture of cells and flie s............................................................................... 58 2.1.1 Cell culture........................................................................................58 2.1.2 Fly culture.........................................................................................58 2.2 Biochemical techniques................................................................................. 58 2.2.1 Preparation of lysates .....................................................................58 2.2.2 Affinity purification........................................................................59 2.2.3 Immunoprécipitation......................................................................59 2.2.4 SDS polyacrylamide gel electrophoresis.................................... 59 2.2.4.1 Staining SDS PAGE gels .................................................60 2.2.5 Western blotting.............................................................................. 60 2.2.6 Peptide sequencing .........................................................................61 2.2.7 Making antisera ............................................................................... 61 2.2.8 Protein kinase assays ......................................................................61 2.2.9 Lipid kinase assays
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