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UC San Francisco Electronic Theses and Dissertations UCSF UC San Francisco Electronic Theses and Dissertations Title Chemical genetic approaches to study protein kinase signaling pathways Permalink https://escholarship.org/uc/item/0sn1j4nk Author Hertz, Nicholas T. Publication Date 2013 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California Copyright 2013 by Nicholas T. Hertz ii Acknowledgements I would like to thank Kevan for his support, insight, encouragement and enthusiasm for science. I would also like to thank Al for all of his support and encouragement. My parents were instrumental in me getting to this point and I want to thank them for their continued support and my wife Janel for everything. Part of this thesis is a reproduction of material previously published, and contains contributions from collaborators listed therein. Chapter 1 is reproduced in part with permission from: Hertz, N.T., Wang, B.T., Allen, J.J., Zhang, C., Dar, A.C., Burlingame, A.L., and Shokat, K.M., Chemical genetic approach for kinase-substrate mapping by covalent capture of thiophosphopeptides and analysis by mass spectrometry, Current Protocols in Chemical Biology. 2010, February; 2:(15-36). Chapter 2 is reproduced in part with permission from: Ultanir, S.K.*, Hertz, N.T.*, Li, G., Ge1, W.P., Burlingame, A.L., Pleasure, S.J., Shokat, K.M., Jan, L.Y., and Jan, Y., Chemical genetic identification of NDR1/2 kinase substrates AAK1 and Rabin8 uncovers their roles in controlling dendrite arborization and synapse maturation. Neuron. 2012, March 22; 73:1127-42. Chapter 3 is reproduced in part with permission from: Hengeveld, R.C.C.*, Hertz, N.T.*, Vromans, M.J.M., Zhang, C., Burlingame, A.L., Shokat, K.M., Lens, S.M.A., Development of a chemical genetic approach for human Aurora B kinase identifies novel substrates of the chromosomal passenger complex. Molecular and Cellular Proteomics. 2012, May 1; 11:47-59. Chapter 4 is reproduced in part with permission from: Hertz, N.T., Berthet, B., Sos, M.L., Thorn, K.S., Burlingame, A.L., Nakamura, K., Shokat, K.M., A neo-substrate that amplifies catalytic activity of Parkinson’s disease related kinase PINK1, Cell. 2013, August 15; 154:737–747 iii Chemical genetic approaches to study protein kinase signaling pathways Nicholas T. Hertz Kevan M. Shokat Alma L. Burlingame Abstract Protein kinase signaling is fundamental to regulating cellular homeostasis in the in every aspect of life. Protein kinases act by placing a phosphate group on a very specific subset of cellular proteins. The addition of a negative charged group on the surface of a protein can dramatically affect the folding, subcellular localization and activity of substrate proteins. Understanding the kinase responsible for a specific phosphorylation event has proven difficult as there are more than 500 mammalian kinases and greater than 50,000 phospho sites. The Shokat lab has pioneered a technique in which a particular kinase is engineered to accept either a specific inhibitor or modified substrate, which is normally not used by any other kinase. In this thesis we demonstrate the utility of this technique, by demonstrating its application to two important kinases. One of these kinases, NDR, is shown to be important in normal brain development, and we identify novel substrates that act directly downstream of NDR. Additionally, we identify novel targets of the kinase Aurora B, a kinase that is critical to normal chromosome segregation during cell division. In addition to these well-behaved kinases, we investigate a kinase, PINK1,that proves an exception to normal kinases. We show that PINK1 has the ability to utilize a iv kinetin triphosphate (KTP) catalytically better than its endogenous substrate, which we therefore call a neo-substrate. The development of a neo-substrate or new substrate for the kinase PINK1 enhances the cellular activity of the disease-associated allele of PINK1 (G309D), effectively reversing the deleterious effect of the mutation. Since PINK1 is a key element in protection against mitochondrial damage and is genetically linked to Parkinson's Disease, our approach also has immediate therapeutic implications. Typically, overactive kinases such as oncogenic kinases provide the only opportunity for drug development because inhibitors of kinases are the only modality available for regulation of kinases. Therefore we believe this may represent a novel modality to regulate the activity of kinases. v Table of Contents Chapter 1: Chemical genetic approach for kinase-substrate mapping by covalent capture of thiophosphopeptides and analysis by mass spectrometry 1.1 Abstract 2 1.2 Introduction 2 1.3 Strategic Planning 6 1.3.1 Engineering the kinase 6 1.3.2 Lysate optimization 9 1.3.3 Generating positive control peptides and protein 11 1.6 Commentary 13 1.6.1 Background information 13 1.6.2 Critical parameters 14 1.6.3 Troubleshooting 17 1.6.4 Anticipated results 17 1.6.5 Time considerations 17 Chapter 2: Chemical genetic identification of NDR1/2 kinase substrates AAK1 and Rabin8 uncovers their roles in controlling dendrite arborization and synapse maturation 2.1 Abstract 19 2.2 Introduction 19 vi 2.3 Results 23 2.3.1 NDR1/2 are necessary and sufficient to reduce dendrite branching 23 2.3.2 NDR1/2 control dendritic spine maturation 24 2.3.3 Development and characterization of an analog specific NDR1 construct 28 2.3.4 Chemical genetic identification of NDR1 substrates 31 2.3.5 AAK1 controls dendrite branching in a similar way to NDR1/2 35 2.3.3 Rabin8 is necessary for spine maturation 37 2.4 Discussion 39 2.4.1 NDR1/2, dendrite pruning and tumor-suppressors 40 2.4.2 AAK1 phosphorylation regulates dendrite branching and length 41 2.4.3 Rab8 GEF Rabin8 regulates spine morphogenesis 43 2.4.4 Other candidate substrates of NDR1/2 44 2.4.5 Chemical genetics for kinase substrate identification 45 2.5 Experimental procedures 45 2.5.1 Kinase assays and covalent capture for phosphorylation site ID 45 Chapter 3: Development of a chemical genetic approach for human Aurora B kinase identifies novel substrates of the chromosomal passenger complex 3.1 Abstract 48 3.2 Introduction 49 3.3 Results 52 3.3.1 Human Aurora B does not tolerate mutation of the Leucine 154 gatekeeper 52 3.3.2 Identification of a unique second-site suppressor for the human as Aurora B 54 vii 3.3.3 as-Aurora B mutants are inhibited by low concentrations of NA-PP1 in cells 57 3.3.4 as-Aurora B mutant can thiophosphorylate multiple proteins in cell extracts 59 3.3.5 Identification of Aurora substrates and site of phosphorylation in cell extracts 61 3.3.6 HMGN2 is a mitotic substrate of Aurora B 65 3.3.7 Nuclear excluded Aurora B to identify additional mitotic substrates: 67 4.4 Discussion 70 Chapter 4: A neo-substrate that amplifies catalytic activity of Parkinson’s disease related kinase PINK1 4.1 Abstract 76 4.2 Introduction 76 4.3 Results 84 4.3.1 PINK1 accepts N6 modified ATP analog kinetin triphosphate (KTP) 84 4.3.2 KTP is produced in human cells upon treatment with KTP precursor kinetin 88 4.3.3 Kinetin increases phosphorylation of PINK1 substrate anti-apoptotic Bcl-xL 90 4.3.4 Kinetin accelerates Parkin recruitment to depolarized mitochondria 92 4.3.5 Kinetin blocks mitochondrial motility in axons in a PINK1 dependent manner 96 4.3.6 Kinetin decreases apoptosis due to oxidative stress in human derived neurons 98 4.4 Discussion 102 4.5 Experimental Procedures 106 4.5.1 Western blot analysis 106 4.5.2 Expression, purification and enzymatic characterization of PINK1 106 4.5.3 Identification of PINK1 autophosphorylation site by LC/MS/MS 106 viii 4.5.4 Enzymatic production of KMP in vitro 107 4.5.5 HPLC analysis for KTP production in cells 107 4.5.6 Parkin mitochondrial translocation assay 108 4.5.7 Details on automated quantitation of Parkin mitochondrial translocation 108 4.5.8 Mitochondrial motility assay 109 4.5.9 PINK1 shRNA production 110 4.5.10 Dopamine neuron cultures 110 4.5.11 Apoptosis assays and phospho S62 Bcl-xL immunoblot analysis 110 Chapter 5: 5.1 All references in alphabetical order 141 ix List of Figures Figure 1.1 Thiophosphopeptide purification scheme 4 Figure 1.2 Lysate labeling schematic 7 Figure 1.3 Analog specific allele generality 10 Figure 1.4 Generating and testing positive control peptide 12 Figure 1.5 Kinome wide kinase engineering 15 Figure 2.1 NDR1/2 regulates dendritic branching and arborization 21 Figure 2.2 NDR1/2’s role on dendritic spine development 25 Figure 2.3 Design and analysis of NDR AS mutants 27 Figure 2.4 Identification of NDR1’s phosphorylation targets by chemical genetics 30 Figure 2.5 Spectra of as-NDR specific phosphopeptides 34 Figure 2.6 Validation of AAK1 and Rabin8 NDR1 phosphorylation sites 36 Figure 2.7 AAK1 affects dendrite branching and length in hippocampal neurons 38 Figure 2.8 Rabin8 affects spine morphogenesis in dissociated hippocampal neurons 42 Figure 3.1 The H250Y mutation rescues kinase activity of Aurora B as mutants 50 Figure 3.2 The as Aurora B mutants are inhibited by PP1 and utilize N6 modified ATP 53 Figure 3.3 Identification of putative Aurora B substrates by covalent capture 56 Figure 3.4 Analysis of the resulting peptides identified by mass spectrometry 58 Figure 3.5 Validation of potential Aurora B phosphorylation sites and substrate 64 Figure 3.6 Scheme for nuclear excluded Aurora B 66 Figure 3.7 Validation of INCENP expression for nuclear excluded Aurora B 68 Figure 4.1 Alignment of PINK1 to typical kinases reveals several large inserts 77 x Figure 4.2 Neo-substrate KTP amplifies kinase activity
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