Identification and characterization of PKD2 substrates [Elektronische Ressource] : CIB1A as a substrate for PKD2 / vorgelegt von Milena Armacki

Sujets

• Gesundheit

Informations

• Publié par: universitat_ulm
• Publié le: 01 janvier 2010
• Nombre de lectures: 25
• Langue: English
• Poids de l'ouvrage: 4 Mo

Extrait

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
11.Introduction
31.1. Cell migration
31.1.1. The migration cycle
51.1.2. Focal adhesion
71.2. Protein Kinase D family (PKD)
71.2.1. Modular structure of PKD enzymes
71.2.2. Regulation of PKD activity
71.2.2.1. Intramolecular regulation of PKD
81.2.2.2. Activation mechanisms of PKDs in various signalling pathways
91.2.3. Biological functions of PKDs
91.2.3.1. PKD substrates
111.2.3.2. Regulation of cell shape, adhesion, motility and invasion
121.2.3.3.on of cell proliferation and differentiation
131.2.3.4. Role of PKDs in Secretion
131.2.3.5. Role for PKD in Angiogenesis
141.3. Ca and Integrin Binding protein (CIB)
141.3.1. CIB structure
161.3.2. Molecular interactions of CIB1
171.3.2.1. CIB 1 and Integrins
181.3.2.2. Role for CIB1 in ischemia-induced pathological and adaptive angiogenesis
181.3.2.3. Role of CIB1 in regulating PAK1 activation and cell migration
201.4.Aim of the study
212.Materials and methods
212.1. Chemicals and other products
212.2. Cell biology
212.2.1. Cell lines
222.2.2. Transient transfection
222.2.2.1. Transfection with Poly Ethylen Imine (PEI)
222.2.2.2.of cells with Nucleofector
222.2.3. Activation of PKD2 in HEK 293T
232.2.4. Inhibition of nuclear export by Leptomycin B( LMB)
232.3. Molecular Biology
232.3.1. Enzymes and ready-to-use kits
242.3.2. Primers for cDNA constructs
242.3.3. Plasmids
252.3.4. Plasmid purification
252.3.5. Computer programs for DNA analysis
262.3.6. Cloning Methods
262.3.6.1. PCR
26.1. Nested PCR
272.3.6.1.2. Overlapping PCR
272.3.6.2. TOPO Cloning
282.3.6.3. Site directed mutagenesis (SDM)
282.3.7. Generation of cDNA-constructs
282.3.7.1. Cloning of the pGEX 4T1-CIB 1a
292.3.7.2. Generation of point CIB 1a mutants
292.3.7.3.e Myc-CIB 1a
292.3.7.4. Generation of the CIB1
302.3.7.5. Cloning of the N- and C- terminal eGFP tagged CIB1a mutants
302.3.7.6. Cloning of the IRES-CIB 1a-GFP
312.3.7.7. Cloning of pcDNA 3-CIB 1a
312.3.7.8.e Cherry-CIB1a
312.3.7.9. Cloning of the PKD2
322.3.8. RNA isolation and Semi-quantitative RT-PCR 332.3.9. Quantitative one step real time PCR
332.3.9.1. Relative quantification
332.3.9.2. Determining amplification efficiencies
342.4. Biochemical technique
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. Expressication 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
443.1. Library screening
463.1.1. Molecular identification of specific clones
483.2. Expression pattern of CIB1/ CIB1a
503.3. In vitro phosphorylation of CIB1a by PKD2
523.3.1. Identification of the phosphorylated Serine in silico method and by computer modelling
533.3.2. PKD2 phosphorylates CIB1a on Ser 118 in vitro
543.3.3. In vivo phosphorylation of CIB1a by PKD2
553.4. PKD2 directly interacts with CIB1a
563.4.1. Identification of the CIB1a-binding site within PKD2
583.5. CIB1a directly stimulates PKD2 activity
593.6. Localisation of CIB1a
613.7. CIB1a interacts with PAX 3
613.8.Phosphorylation of CIB1a on Ser 118 induces its subcelular re-localisation
633.9. Nuclear export of CIB1a
3.10. Phosphorylated CIB 1a colocalises with Vimentin 66
683.10.1. CIB1a physically interacts with Vimentin
693.11. Phosphorylated CIB1a promotes cell migration
3.11.1. Effect of CIB1a phosphorylation by PKD2 on FA formation 70
734.Discussion
795.Summary
6.References 81
97ACKNOWLEDGEMENTS








iiIndex 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 phosphoryl