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Deciphering Human Cytoplasmic Protein Tyrosine Kinase Phosphorylation Specificity in Yeast

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Deciphering Human Cytoplasmic Protein Tyrosine Kinase Phosphorylation Specificity in Yeast DECIPHERING HUMAN CYTOPLASMIC PROTEIN TYROSINE KINASE PHOSPHORYLATION SPECIFICITY IN YEAST THOMAS GEORG CORWIN BERLIN 2015 DISSERTATION ZUR ERLANGUNG DES GRADES EINES DOKTORS DER NATURWISSENSCHAFTEN EINGEREICHT IM FACHBEREICH BIOLOGIE, CHEMIE, PHARMAZIE DER FREIEN UNIVERSITÄT BERLIN Max Planck Institut für molekulare Genetik Otto Warburg Laboratory - Molecular Interaction Networks Freie Universität Berlin Fachbereich Biologie, Chemie und Pharmazie ERSTGUTACHTER: DR. ULRICH STELZL (MAX PLANCK INSTITUT FÜR MOLEKULARE GENETIK) ZWEITGUTACHTER: PROF. DR. MARKUS WAHL (FREIE UNIVERSITÄT BERLIN) TAG DER DISPUTATION: 05.11.2015 Zusammenfassung Die Weitergabe von Signalen mittels Protein Tyrosin Kinasen (PTK) ist eine Eigenschaft von multizellulären Organismen und deren Deregulierung kann zur Entstehung komplexer Krankheiten wie Krebs im Menschen beitragen. Es gibt 90 humane PTKs unterteilt in 58 Rezeptor-PTKs und 32 nicht-Rezeptor PTKs (NRTKs). Es ist von essenzieller Bedeutung zu verstehen, wie jede einzelne NRTK ihre Substratproteine mit gegebener Spezifität erkennt und unterscheidet. Dies ist eine Herausforderung aufgrund überlappender Substratspezifität, enzymatischer Aktivität unterschiedlicher Größenordnungen und zelltypabhängiger Expressionsstärke zwischen NRTKs. Saccharomyces cerevisiae (Bäckerhefe) hat keine PTKs und es wurde gezeigt, dass aktive, menschliche NRTKs einzeln und schwach in Hefe exprimiert werden können ohne einen Hefe- Phänotyp zu erzeugen. Dabei werden Hefeproteine phosphoryliert, was mittels moderner massenspektrometrischer Methoden hintergrundfrei gemessen werden kann. Die Hälfte aller 32 NRTKs, mit Repräsentanten aus allen NRTK Familien, zeigten Aktivität in Hefe. Durch 60 massenspektrometrische Messungen in einem einzelnen Versuchsaufbau, wurden 1433 Phosphorylierungsstellen auf 900 Hefeproteinen gemessen. Die Messungen ermöglichten die Analyse der überlappenden Spezifität zwischen NRTKs und die Definierung linearer Aminosäuresequenz- Motive für jede einzelne NRTK, welche die Zuordnung von NRTKs zu 1388 aus 13240 bekannten menschlichen Phosphorylierungsstellen ermöglichte. Die Konservierung von Modifizierungsstellen zwischen Mensch und Hefe ermöglichte die Zuordnung von NRTKs zu 63 orthologen Modifizierungsstellen und führte zur Entdeckung, dass (aerobe) Glykolyse und Signalwege onkogener NRTKs enger verknüpft sind als bisher angenommen. Für NRTKs wurde individuelles und gemeinsames Modifizieren von einzelnen Substratproteinen und Substratfamilien vorhergesagt, was exemplarisch mittels eines in vitro Kinasen-Testverfahren experimentell überprüft wurde. Die Dechiffrierung menschlicher NRTK-Spezifität in Hefe resultierte in einem neuen Datensatz, welcher die Basis für weitere experimentelle Analysen sein könnte, um die systemweite Signalweitergabe von NRTKs in normalwachsenden und wuchernden Zellen zu entschlüsseln. Schlagwörter: Protein Tyrosine Kinase, PTK, zytoplasmisch, NRTK, homo sapiens, Mensch, Saccharomyces cerevisiae, Hefe, Spezifität, lineares Sequenzmotiv, Motiv Scoring, Protein-Protein Interaktions Netzwerk, PPI Netzwerk I Abstract Protein tyrosine kinase (PTK) signaling can be regarded as a hallmark of multi-cellular organisms and its deregulation causes a variety of complex diseases in human including cancer. Therefore, it is essential to understand how each individual tyrosine phosphorylating enzyme is able to recognize and distinguish its target proteins. This task still remains challenging due to overlapping substrate specificity, magnitude differences in enzymatic activity, and cell type dependent expression levels of PTKs. There are 90 human PTKs with 58 receptor-type PTKs and 32 non-receptor type PTKs (NRTKs) thereof. Saccharomyces cerevisiae is lacking PTKs and it was shown that, when individually expressed at low levels, active human NRTKs phosphorylate yeast proteins - in a background-free environment, in intact cells with high specificity and without a growth phenotype. Half of all 32 NRTKs representing all NRTK families showed activity in yeast. Hence, individual NRTK specificity on both linear amino acid motif- and structural level was determined by 60 measurements in a single experimental set-up recording 1433 tyrosine phosphorylation sites on 900 proteins in yeast using state-of-the-art phospho-proteomics. The mass-spectrometric measurements enabled analysis of NRTK specificity overlap and the determination of linear amino acid sequence motifs for individual NRTKs. Motif- based scoring of 13240 reported human pY-sites enabled kinase inference for 1388 pY-sites. Modification site conservation between yeast and human enabled kinase inference for 63 orthologous pY-sites and led to the discovery that aerobic glycolysis and oncogenic NRTK signaling may be more highly inter-linked than previously assumed. Individual and common NRTK targeting of single and families of substrate proteins was predicted and exemplary experimentally verified by an in vitro kinase assay. Deciphering human NRTK specificity in yeast provided a novel data set which may aid further experimental analysis to unravel system-wide NRTK signaling in normal and proliferating cells. Keywords: Protein Tyrosine Kinase, PTK, NRTK, cytoplasmic, homo sapiens, human, Saccharomyces cerevisiae, yeast, specificity, linear sequence motif, motif scoring, Protein-protein interaction network, PPI network II Contents Zusammenfassung .................................................................................................................................... I Abstract ................................................................................................................................................... II Abbreviations ........................................................................................................................................ VII 1. Introduction ..................................................................................................................................... 1 1.1. Protein phosphorylation.......................................................................................................... 1 1.2. Regulation of protein phosphorylation ................................................................................... 1 1.3. Protein tyrosine kinases .......................................................................................................... 3 1.4. Determination of NRTK substrates and specificity .................................................................. 7 1.4.1. In-vitro kinase assays: from single to thousand proteins .............................................. 10 1.4.2. System-wide characterization of in vivo phosphorylation events ................................ 11 1.4.3. Mass spectrometry based determination of kinase substrates .................................... 13 1.4.4. Determination of PTK specificity ................................................................................... 18 1.5. Thesis aims ............................................................................................................................ 22 2. Material and methods ................................................................................................................... 23 2.1. General molecular biology methods ..................................................................................... 23 2.1.1. Gateway cloning ............................................................................................................ 23 2.1.1.1. BP-reaction ............................................................................................................ 23 2.1.1.2. LR-reaction............................................................................................................. 23 2.1.2. Polymerase Chain Reaction ........................................................................................... 24 2.1.3. Determination of DNA concentration ........................................................................... 24 2.1.4. DNA sequencing ............................................................................................................ 25 2.1.5. Restriction digest ........................................................................................................... 25 2.1.6. Agarose Gel-electrophoresis ......................................................................................... 25 2.1.6.1. Buffers and Reagents ............................................................................................. 25 2.1.6.2. Gel-electrophoresis and gel staining ..................................................................... 25 2.1.7. SDS Gel-electrophoresis ................................................................................................ 26 2.1.7.1. Reagents and Solutions ......................................................................................... 26 2.1.7.2. Method .................................................................................................................. 27 2.1.8. Western-blotting ........................................................................................................... 27 2.1.8.1. Reagents and Solutions ......................................................................................... 27 2.1.8.2. Blotting Methods ................................................................................................... 28 2.1.9. Protein staining ............................................................................................................
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