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Identification and Characterization of Pkd2 Substrates -Cib1a As a Substrate for Pkd2

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Identification and Characterization of Pkd2 Substrates -Cib1a As a Substrate for Pkd2 Universitätsklinikum Ulm Zentrum für Innere Medizin Klinik für Innere Medizin I Ärztliche Director: Prof. Dr. G. Adler IDENTIFICATION AND CHARACTERIZATION OF PKD2 SUBSTRATES -CIB1A AS A SUBSTRATE FOR PKD2- Dissertation zur Erlangung des Doktorgrades der Humanbiologie (Dr. biol. hum.) der Medizinischen Fakultät der Universität Ulm vorgelegt von Milena Armacki aus Zrenjanin, Serbien 2010 Amtierender Dekan: Prof. Dr. med. Klaus-Michael Debatin 1. Berichterstatter: Prof. Dr. med Thomas Seufferlein 2. Berichterstatter: Prof. Dr. Johan van Lint Tag der Promotion: 19.02.2010. Table of content i Index of Figures iii Abbreviations iv Table of Content 1.Introduction 1 1.1. Cell migration 3 1.1.1. The migration cycle 3 1.1.2. Focal adhesion 5 1.2. Protein Kinase D family (PKD) 7 1.2.1. Modular structure of PKD enzymes 7 1.2.2. Regulation of PKD activity 7 1.2.2.1. Intramolecular regulation of PKD 7 1.2.2.2. Activation mechanisms of PKDs in various signalling pathways 8 1.2.3. Biological functions of PKDs 9 1.2.3.1. PKD substrates 9 1.2.3.2. Regulation of cell shape, adhesion, motility and invasion 11 1.2.3.3. Regulation of cell proliferation and differentiation 12 1.2.3.4. Role of PKDs in Secretion 13 1.2.3.5. Role for PKD in Angiogenesis 13 1.3. Ca and Integrin Binding protein (CIB) 14 1.3.1. CIB structure 14 1.3.2. Molecular interactions of CIB1 16 1.3.2.1. CIB 1 and Integrins 17 1.3.2.2. Role for CIB1 in ischemia-induced pathological and adaptive angiogenesis 18 1.3.2.3. Role of CIB1 in regulating PAK1 activation and cell migration 18 1.4.Aim of the study 20 2.Materials and methods 21 2.1. Chemicals and other products 21 2.2. Cell biology 21 2.2.1. Cell lines 21 2.2.2. Transient transfection 22 2.2.2.1. Transfection with Poly Ethylen Imine (PEI) 22 2.2.2.2. Transfection of cells with Nucleofector 22 2.2.3. Activation of PKD2 in HEK 293T 22 2.2.4. Inhibition of nuclear export by Leptomycin B( LMB) 23 2.3. Molecular Biology 23 2.3.1. Enzymes and ready-to-use kits 23 2.3.2. Primers for cDNA constructs 24 2.3.3. Plasmids 24 2.3.4. Plasmid purification 25 2.3.5. Computer programs for DNA analysis 25 2.3.6. Cloning Methods 26 2.3.6.1. PCR 26 2.3.6.1.1. Nested PCR 26 2.3.6.1.2. Overlapping PCR 27 2.3.6.2. TOPO Cloning 27 2.3.6.3. Site directed mutagenesis (SDM) 28 2.3.7. Generation of cDNA-constructs 28 2.3.7.1. Cloning of the pGEX 4T1-CIB 1a 28 2.3.7.2. Generation of point CIB 1a mutants 29 2.3.7.3. Cloning of the Myc-CIB 1a 29 2.3.7.4. Generation of the CIB1 29 2.3.7.5. Cloning of the N- and C- terminal eGFP tagged CIB1a mutants 30 2.3.7.6. Cloning of the IRES-CIB 1a-GFP 30 2.3.7.7. Cloning of pcDNA 3-CIB 1a 31 2.3.7.8. Cloning of the Cherry-CIB1a 31 2.3.7.9. Cloning of the PKD2 31 2.3.8. RNA isolation and Semi-quantitative RT-PCR 32 2.3.9. Quantitative one step real time PCR 33 2.3.9.1. Relative quantification 33 2.3.9.2. Determining amplification efficiencies 33 2.4. Biochemical technique 34 2.4.1. SDS-PAGE and Western Blot 34 2.4.1.1. Antisera and Antibodies 35 2.4.2. Coomassie staining 35 2.4.3. Preparation of whole cell lysate 35 2.4.4. (Co-)immunoprecipitation 35 2.4.5. Catch and Release® v2.0 Reversible Immunoprecipitation 36 2.4.6. Production and purification of phospho-specific CIB1a antibody 36 2.4.6.1. Antibody evaluation by ELISA 37 2.4.7. Expression and purification of GST-tagged proteins in bacteria 37 2.4.8. GST-pull down experiments 38 2.4.9. In vitro kinase assays 38 2.4.10. The In Vitro Expression Cloning (IVEC) Method 39 2.4.10.1. Preparation of protein pools 39 2.4.10.2. Identification of phosphorylated proteins by gel mobility assay 40 2.4.10.3. Subdivision of positive pools and Molecular Identification of Specific Clones 40 2.4.11. Computer programs for protein analysis 40 2.5. Imaging techniques 41 2.5.1. Time-lapse microscopy 41 2.5.1.1. Migration Assay 41 2.5.2. Epi-fluorescent and Laser scanning confocal microscopy (LSCM) 41 2.5.2.1. Colocalisation of proteins 42 2.5.2.2. Staining of endogenous proteins 42 2.5.2.3. Quantication of Focal adhesions 43 3.Results 44 3.1. Library screening 44 3.1.1. Molecular identification of specific clones 46 3.2. Expression pattern of CIB1/ CIB1a 48 3.3. In vitro phosphorylation of CIB1a by PKD2 50 3.3.1. Identification of the phosphorylated Serine in silico method and by computer modelling 52 3.3.2. PKD2 phosphorylates CIB1a on Ser 118 in vitro 53 3.3.3. In vivo phosphorylation of CIB1a by PKD2 54 3.4. PKD2 directly interacts with CIB1a 55 3.4.1. Identification of the CIB1a-binding site within PKD2 56 3.5. CIB1a directly stimulates PKD2 activity 58 3.6. Localisation of CIB1a 59 3.7. CIB1a interacts with PAX 3 61 3.8.Phosphorylation of CIB1a on Ser 118 induces its subcelular re-localisation 61 3.9. Nuclear export of CIB1a 63 3.10. Phosphorylated CIB 1a colocalises with Vimentin 66 3.10.1. CIB1a physically interacts with Vimentin 68 3.11. Phosphorylated CIB1a promotes cell migration 69 3.11.1. Effect of CIB1a phosphorylation by PKD2 on FA formation 70 4.Discussion 73 5.Summary 79 6.References 81 ACKNOWLEDGEMENTS 97 ii Index of Figures Figure 1: A schematic of the metastatic process 2 Figure 2: Stages of cell movement: 4 Figure 3: Molecular architecture of focal contacts 6 Figure 4: Molecular structure of members of the protein kinase D family 7 Figure 5: Calmyrin/Ca and integrin binding protein(CIB1) amino acid sequence and transcript expression pattern. 15 Figure 6. Relationship of Ca and integrin binding protein 1(CIB1) with some of its nearest homologs 16 Figure 7. Strategy for performing deletion mutagenesis by PCR Fusion. 27 Figure 8. Phosphorylation of single clones by catalytically active Protein kinase D2 (PKD2se) 45 Figure 9. Sequence analysis of the positive IVEC clone. 47 Figure 10. Expression of CIB1 and CIB1a in human cancer cell lines 48 Figure 11. Expression of CIB and CIB1a proteins. 49 Figure 12. Quantification of the mRNA expression level of the CIB1a gene in various cell lines. 50 Figure 13. In vitro phosphorylation of CIB1a and CIB1 by PKD2. 51 Figure 14. Identification of the protein kinase D2 (PKD2) phosphorylation site in CIB 1a. 53 Figure 15. Protein Kinase D2 (PKD2) phosphorylates CIB1a at Serine 118 and not at Threonine 207 54 Figure 16. Development of site-specific phospho-CIB1a antibody. 55 Figure 17. CIB1a interacts with Protein Kinase D2 (PKD2). 56 Figure 18. Localisation of interaction sites in Protein Kinase D2 (PKD2) 57 Figure 19. Localisation of CIB1a interaction site in PKD1 and PKD3 58 Figure 20. Phosphorylation of CIB1a stimulates Protein Kinase D2 (PKD2) activity 59 Figure 21. Localisation of tagged and untagged CIB1a in HeLa cells 60 Figure 22. CIB1a interacts with Pax 3 independently of CIB1a phosphorylation 61 Figure 23. Catalytically active PKD2 induces phosphorylation and subsequent translocation of CIB1a to the cytoplasm in HeLa cells 62 Figure 24. Catalytically active PKD2 induces phosphorylation and subsequent translocation of CIB1a to the cytoplasm in U 87 cells 63 Figure 25. NetNES 1.1 prediction of the nuclear export signal (NES) in CIB1a protein. 64 Figure 26. Nuclear export of PKD2 is regulated by a CRM-1 dependent mechanism 65 Figure 27. CIB1a interactes with Crm1 independent of the phosphorylation by PKD2 66 Figure 28. Subcelular distribution of wild type or phosphorylated CIB1a and Vimentin in HeLa cells. 67 Figure 29. Subcelular distribution of wild type or phosphorylated CIB1a and Vimentin in U87 cells. 68 Figure 30. CIB1a interacts with Vimentin 69 Figure 31. CIB positively regulates migration as a result of phosphorylation on Serine 118. 70 Figure 32. Effect of the CIB1a phosphorylation on the focal adhesion formation 71 Figure 33. Effect of CIB1a phosphorylation by PKD2 on Focal Adhesions (FA) formation. 72 iii Abbreviations A Adenin aa amino acid AB Antibody Abl Abelson leukaemia AEBSF 4-(2-Aminoethyl)benzylsulfonylfluorid Arg Arginine (three letter code) Ala Alanine (three letter code) Amp Ampicillin ATCC American Type Culture Collection ATP Adenosine Triphosphate APC Antigen Presenting Cell bp base pairs BSA Bovine Serum Albumin BCR B Cell Receptor C Cytosine °C degree Celsius Ca2+ Calcium ion CAMK Ca2+/Calmodulin-depenent protein kinase cAMP cyclic Adenosine Monophosphate CCK-2 Cholecystokinine2 CCK-2R Cholecystokinine2 Receptor CDK Cyclin-dependent protein kinase cDNA mRNA complementary DNA CIB Calcium and integrin binding protein CRD Cysteine Rich Domain Crm1 Chromosomal region maintenance 1 CTP Cytidin Triphosphate Cys Cysteine (three letter code) DAG Diacylglycerol DMEM Dulbecco's modified Eagles medium DNA Desoxyribonucleinacid dNTP desoxy ATP/CTP/GTP/TTP DTT Dithiothreitol ECL Enhanced chemiluminescence ECM Extra Cellular Matrix EDTA Ethylendiamine-N, N, N´, N´- tetra-acetate EF hand Helix-loop-helix structural domain found in family of calcium-binding proteins FA Focal adhesions FAK Focal Adhesion Kinase e.
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