Systematic identification and quantification of substrate specificity determinants in human protein kinases (Identificación y cuantificación sistemática de determinantes de la especificidad por sustrato en las proteínas quinasas de humano) Manuel Alejandro Alonso Tarajano ADVERTIMENT. La consulta d’aquesta tesi queda condicionada a l’acceptació de les següents condicions d'ús: La difusió d’aquesta tesi per mitjà del servei TDX (www.tdx.cat) i a través del Dipòsit Digital de la UB (diposit.ub.edu) ha estat autoritzada pels titulars dels drets de propietat intel·lectual únicament per a usos privats emmarcats en activitats d’investigació i docència. No s’autoritza la seva reproducció amb finalitats de lucre ni la seva difusió i posada a disposició des d’un lloc aliè al servei TDX ni al Dipòsit Digital de la UB. No s’autoritza la presentació del seu contingut en una finestra o marc aliè a TDX o al Dipòsit Digital de la UB (framing). 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Tesi Doctoral Universitat de Barcelona Facultat de Farmàcia Departament de Bioquímica i Biologia Molecular Programa de Doctorat en Biomedicina 2013 Systematic identification and quantification of substrate specificity determinants in human protein kinases (Identificación y cuantificación sistemática de determinantes de la especificidad por sustrato en las proteínas quinasas de humano) Memòria presentada per Manuel A. Alonso Tarajano, per optar al títol de doctor per la Universitat de Barcelona This thesis was realized under the supervision and tutorship of ICREA research professor Dr. Patrick Aloy Calaf and under the co-supervision of Dr. Roberto Mosca, in the Structural Bioinformatics and Network Biology group of the Institute for Research in Biomedicine (IRB) Barcelona. Manuel A. Alonso Tarajano Dr. Patrick Aloy Calaf Dr. Roberto Mosca Contents Table of Contents iii List of Figures v List of Tables vii List of Acronyms ix 1 Resumen global 1 1.1 Introducción . .1 1.2 Objetivos . .2 1.3 Materiales y Métodos . .2 1.4 Resultados y discusión . .5 1.5 Conclusiones . .9 2 General introduction 11 2.1 Protein phosphorylation . 11 2.2 Protein kinases . 11 2.2.1 Kinases are responsible for protein phosphorylation . 11 2.2.2 The kinase catalytic domain is highly conserved . 12 2.2.3 Phylogeny and diversity of human protein kinases . 12 2.3 Known mechanisms of protein kinases for substrate identification . 15 2.3.1 Role of the kinase catalytic site . 15 2.3.2 Distal docking sites . 15 2.3.3 Regulatory and targeting domains . 16 3 Objectives 19 4 Sequence logos and position-specific scoring matrices 21 4.1 Introduction . 21 4.1.1 Contribution of residues neighboring the phosphorylation sites . 21 4.1.1.1 Residues commonly phosphorylated in eukaryotes . 21 4.1.1.2 Positioning and orientation of the phospho-acceptor residue 21 4.1.1.3 From phosphorylation sequences to phosphorylation motifs . 22 4.1.2 Graphical representation of phosphorylation motifs . 22 4.1.3 Mathematical representation of phosphorylation motifs . 23 4.1.3.1 Probabilistic models . 23 i 4.1.3.2 Prediction of phosphorylation sites . 24 4.2 Materials and methods . 26 4.2.1 Phylogenetic classification of human protein kinases . 26 4.2.2 Phosphorylation data for human protein kinases . 26 4.2.3 Set of ‘unphosphorylated’ human proteins . 26 4.2.4 Generation of sequence logos . 27 4.2.5 Position-specific scoring matrices . 27 4.2.5.1 Generating PSSMs . 27 4.2.5.2 Selection of a score threshold . 27 4.2.5.3 Statistical significance of the PSSMs . 28 4.2.5.4 Performance of the PSSMs . 29 4.2.6 Quantification of the kinase specificity encoded in the PSSMs . 29 4.2.6.1 Sequence motifs most commonly recognized by kinases . 29 4.3 Results and discussion . 30 4.3.1 Motifs recognized by kinases and kinases families . 30 4.3.2 Strong specificity-determinant residues from kinase families . 30 4.3.2.1 Proline-directed kinase families . 30 4.3.2.2 Glutamine-directed kinase families . 32 4.3.2.3 Basophilic kinase families . 34 4.3.2.4 Acidophilic kinase families . 39 4.3.3 Statistical analysis of the PSSMs . 42 4.3.3.1 Independent kinases . 42 4.3.3.2 Kinase families . 48 4.4 Concluding remarks . 54 5 Contribution of adaptor and scaffold proteins 57 5.1 Introduction . 57 5.1.1 Adaptors and scaffolds are multidomain spatio-temporal regulators . 57 5.1.1.1 Adaptors and scaffolds of the MAPK/ERK cascade . 58 5.1.1.2 The IQGAP scaffolds regulate cytoskeleton dynamics . 60 5.1.2 Adaptors and scaffolds contribute to kinase substrate specificity . 62 5.1.2.1 The biological roles of A-kinase anchoring proteins . 63 5.1.2.2 mAKAP assembles a signalosome at the nuclear envelope . 64 5.1.3 Computational identification of signaling scaffold proteins . 64 5.2 Materials and methods . 67 5.2.1 Protein-protein interactions for human . 67 5.2.2 Statistical enrichment of Pfam families . 67 5.2.3 Statistical enrichment of Gene Ontology terms . 67 5.2.4 Automatic collection of known adaptor/scaffold proteins . 67 5.2.5 Gold standard set of kinase–adaptor/scaffold pairs . 67 5.2.6 Identification of potential adaptors/scaffolds from substrates partners 68 5.2.6.1 Data . 68 5.2.6.2 Collection of PIN for randomization . 68 5.2.6.3 Generating background distributions . 68 5.2.6.4 Testing the classification method . 69 5.2.6.5 Algorithm for the identification of potential adaptors/scaffolds 69 5.2.6.6 Evaluating the performance of the method . 69 5.2.6.7 Co-annotation of substrates and potential adaptors/scaffolds 69 5.2.7 Identification of known adaptors/scaffolds interacting with signifi- cantly large sets of substrates . 70 5.2.7.1 Data . 70 5.2.7.2 Generating background distributions . 70 5.2.7.3 Estimating the statistical significance . 71 5.2.7.4 Statistical likeliness substrate-adaptor/scaffold interactions . 71 5.2.8 Identification of kinases sharing significantly large sets of substrates . 71 5.2.8.1 Data . 71 5.2.8.2 Generating background distributions . 72 5.2.8.3 Estimating the statistical significance . 72 5.3 Results and discussion . 73 5.3.1 Set of known adaptors/scaffolds collected for human kinases . 73 5.3.2 Gold standard set of known adaptors/scaffolds . 76 5.3.3 Potential adaptors/scaffolds identified from substrates partners . 77 5.3.4 Adaptors/scaffolds binding to significantly large numbers of substrates 86 5.3.5 Association to adaptors/scaffolds diminish cross-specificity of kinases 88 5.4 Concluding remarks . 90 6 General discussion 91 6.1 Analysis of phosphorylation sites and their adjacent residues . 91 6.1.1 Sequence logos . 91 6.1.2 Specificity-determinant residues . 91 6.1.3 Position-specific scoring matrices . 92 6.2 Analysis of the association of kinases to adaptors and scaffolds . 93 6.2.1 Identification of adaptors and scaffolds . 93 6.2.2 Colocalization of adaptors and scaffolds with substrates . 94 6.2.3 Adaptors and scaffolds diminish kinase cross-specificity . 95 7 General conclusions 97 Bibliography 101 Appendices 111 A1 SDRs from kinase families . 113 A2 Sequence logos from kinase families . 118 A3 List of known adaptors/scaffolds . 119 A4 List of potential adaptors/scaffolds . 125 A5 Cellular compartment annotation of potential adaptors/scaffolds . 127 A6 Submitted article . 130 List of Figures 2.1 Protein phosphorylation reaction. 12 2.2 Eukaryotic kinase catalytic domain. 13 2.3 Modular domain composition of protein kinases. 14 2.4 Distal docking sites in MAPKs. 16 4.1 Frequencies of the SDRs from two families of Pro-directed kinases. 33 4.2 Frequencies of SDRs from four families of basophilic kinases.