Identification of Camk-II Protein Targets in Tissue Culture and Zebrafish Embryos Using Tandem Mass Spectrometry
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Virginia Commonwealth University VCU Scholars Compass Theses and Dissertations Graduate School 2009 Identification of CaMK-II Protein Targets in Tissue Culture and Zebrafish Embryos using Tandem Mass Spectrometry Alexandra Myers Virginia Commonwealth University Follow this and additional works at: https://scholarscompass.vcu.edu/etd Part of the Biology Commons © The Author Downloaded from https://scholarscompass.vcu.edu/etd/10 This Thesis is brought to you for free and open access by the Graduate School at VCU Scholars Compass. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of VCU Scholars Compass. For more information, please contact [email protected]. IDENTIFICATION OF CAMK-II PROTEIN TARGETS IN TISSUE CULTURE AND ZEBRAFISH EMBRYOS USING TANDEM MASS SPECTROMETRY A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science, Biology at Virginia Commonwealth University. by ALEXANDRA LEIGH MYERS B.S., Virginia Commonwealth University, 2006 Director: ROBERT M. TOMBES, PH. D ASSOCIATE PROFESSOR, DEPARTMENT OF BIOLOGY Virginia Commonwealth University Richmond, Virginia August, 2009 Acknowledgement There are several people I would like to acknowledge and thank, as this research over the past two and a half years in general wouldn’t have been possible without them. I would foremost like to thank my advisor Dr. Tombes, who has always been understanding and never failed to support me in all of my endeavors. He has been an inspiration to me in my growth and career as a scientist. Thanks to my committee members who have provided their time and effort in assisting me through this process. Everyone in Dr. Scott Gronert’s lab has been integral to this research, specifically, David Simpson, who has analyzed countless samples and helped however he could along the way. Thanks to Kristina Nelson, and Brad Mangrum of the Mass Spectrometry Core Facility for their help and suggestions. I would also like to thank all of my lab co- workers, Jamie Mcleod, Sarah Chase Rothschild, Ludmilia Francescatto, Bennett Childs and Chas Easley, without whom I would have never been able to complete any of my work. Last but not least, I would like to sincerely and endlessly thank my family and friends for always supporting and having faith in me, no matter what, especially throughout the past year. ii Table of Contents Page List of Figures .................................................................................................................... vi List of Abbreviations ....................................................................................................... viii Abstract................................................................................................................................x Introduction..........................................................................................................................1 Materials and Methods mPCT and HEK Cell Culture........................................................................8 Zebrafish Care and Strains ............................................................................8 Vectors...........................................................................................................8 In vitro translation .........................................................................................9 Transfection...................................................................................................9 cDNA Injections of Zebrafish Embryos........................................................9 Cell and Zebrafish Embryo Harvest............................................................10 FLAG Immunoprecipitation........................................................................10 Cell Sorting by Flow Cytometry .................................................................11 Western Transfer and Immunoblotting .......................................................11 Antibodies ...................................................................................................12 CaMK-II Kinase Assay and Phosphorylation Assay ..................................13 Mass Spectrometry Sample Preparation......................................................14 iii LC-ESI MS/MS...........................................................................................15 Peptide/Protein Identification and Data Analysis .......................................16 Immunolocalization.....................................................................................16 Imaging........................................................................................................17 Results Design, preparation and verification of mutants .........................................18 Point mutations in the catalytic domain of CaMK-II reduce kinase activity...................................................................................................19 Optimization of Immunoprecipitation and tandem mass spectrometry sample preparation ................................................................................20 Proteomic assessment of substrate-trapping mutants verifies known cytosolic binding partners of CaMK-II .................................................22 43 43 Proteomic assessment of FLAG δe K A, δe K G and FLAG δe T287D/K43A CaMK-II identifies p53 as a possible substrate................25 Protein profiles can be resolved and compared using substrate-trapping and tandem mass spectrometry..............................................................26 CaMK-II binds to, but may not phosphorylate p53.....................................28 43 High levels of FLAG δe K A CaMK-II injected into zebrafish embryos induce a split body axis phenotype at 24hpf .........................................31 Proteomic assessment of injected zebrafish embryos at 3dpf yields limited protein profiles ..........................................................................33 Tandem mass spectrometry is sufficient for resolving individual, tissue- specific proteins in 3dpf zebrafish heart tissue .....................................33 Proteomic assessment of GFP-positive zebrafish kidney cells resolves cytosolic and membrane fractions and identifies specific proteins...…34 iv Discussion ..........................................................................................................................36 Figures................................................................................................................................43 Literature Cited ..................................................................................................................62 Appendices Table of Contents for Appendices...........................................................................67 1. MS/MS Results – Mouse Proximal Convoluted Tubule Cells....................68 2. MS/MS Results – Human Embryonic Kidney Cells...................................89 3. MS/MS Results – Zebrafish .....................................................................104 Vita…...............................................................................................................................164 v List of Figures Page Figure 1: Point mutations in CaMK-II alter kinase activity ..............................................44 Figure 2: Sample workflow ...............................................................................................45 Figure 3: Western Blots for GFP in vitro...........................................................................46 Figure 4: Binding partners of CaMK-II constructs in mPCT cells....................................47 Figure 5: Binding partners of CaMK-II substrate-trapping constructs in mPCT cells......48 Figure 6: Binding partner of CaMK-II substrate-trapping constructs in mPCT cells........49 Figure 7: Binding partners of CaMK-II constructs in HEK cells ......................................50 Figure 8: Binding partners of CaMK-II substrate-trapping constructs in HEK cells ........51 Figure 9: Binding partners of CaMK-II substrate-trapping constructs in HEK cells ........52 Figure 10: Comparison of different CaMK-II constructs and cell lines ............................53 Figure 11: CaMK-II binds p53 in cell lysates....................................................................54 Figure 12: CaMK-II autophosphorylates, but may not phosphorylate p53 .......................55 Figure 13: p53 localizes to the nucleus in mPCT cells fixed with methanol.....................56 Figure 14: p53 co-localizes with inactive CaMK-II constructs in the cytosol of mPCT cells fixed with paraformaldehyde ........................................................57 Figure 15: Zebrafish embryos injected with different CaMK-II constructs exhibit different phenotypes..........................................................................................58 Figure 16: Zebrafish embryos injected with different CaMK-II constructs exhibit similar protein profiles.....................................................................................59 vi Figure 17: Tandem mass spectrometry of 3dpf zebrafish hearts is sufficient for resolving proteins in a complex mixture..........................................................60 Figure 18: Proteomic assessment of GFP-positive zebrafish kidney cells resolves cytosolic and membrane fractions and identifies tissue specific proteins .......61 vii List of Abbreviations µg Microgram µL Microliter A Alanine ABC Ammonium Bicarbonate