The role of tumour suppressor tyrosine kinase SYK in glioblastoma and breast cancer [Elektronische Ressource] / Sushil Kumar

Technische Universität München
Max Planck Institute für Biochemie
Abteilung Molekular Biologie



The Role of Tumour Suppressor Tyrosine Kinase
SYK in Glioblastoma and Breast Cancer


Sushil Kumar


Vollständiger Abdruck der von der Fakultät für Chemie der Technischen Universität
München zur Erlangung des akademischen Grades eines


Doktors der Naturwissenschaften (Dr. rer. nat)


genehmigten Dissertation.



Vorsitzender: Univ.-Prof. Dr. Johannes Buchner

Prüfer der Dissertation: 1. Priv.-Doz. Dr. Nediljko Budisa
2. Hon.-Prof. Dr. Axel Ullrich,
Eberhard-Karls-Universität Tübingen
3. Univ.-Prof. Dr. Horst Kessler







Die Dissertation wurde am 30.05.2007 bei der Technischen Universität München
eingereicht und durch die Fakultät Chemie am 24.07.2007 angenommen.


Erklärung:


Ich erkläre an Eides statt, dass ich die der Fakultät für Chemie der Technischen Universität
München vorgelegte Dissertationsarbeit mit dem Titel:


“The Role of Tumour Suppressor Tyrosine Kinase Syk in Glioblastoma and Breast
Cancer”


angefertigt am Max-Planck-Institut für Biochemie in Martinsried unter der Anleitung und
Betreuung durch Herrn Prof. Dr. Axel Ullrich (MPI für Biochemie, Martinsried) und Herrn PD
Dr. Nediljko Budisa (Molekular Biotechnologie, Max Planck Institute für Biochemie) ohne
sonstige Hilfe verfasst und bei der Abfassung nur die gemäß § 6 Abs. 5 angegebenen Hilfsmittel
benutzt habe.

München, den
________________
Sushil Kumar






















Dedicated to my Parents




































For Nessy


Contents



1 INTRODUCTION .......................................................................................................... 8
1.1 Receptor Tyrosine Kinases.................................................................................................................................... 9
1.1.1 The Epidermal Growth Factor Receptor (EGFR) Family and their Cognate Ligands ................................ 9
1.2 Cytoplasmic Tyrosine Kinases and their Modular Domains............................................................................ 12
1.2.1 Spleen Tyrosine Kinase (Syk)........................................................................................................................ 14
1.3 G Protein Coupled Receptors.............................................................................................................................. 18
1.3.1 GPCRs in cancer ...................................................................................................................................... 18
1.3.2 Lysophosphatidic acid (LPA) in cancer development...............................................................................20
1.4 EGFR Transactivation......................................................................................................................................... 20
1.4.1 Matrix Metalloproteinases.............................................................................................................................. 22
1.5 Regulation of the Cell Cycle and its Implication in Cancer ............................................................................. 22
1.6 Mechanisms of Metastasis in cancer................................................................................................................... 24
1.7 Aim of the study ................................................................................................................................................... 26
2 MATERIALS AND METHODS ................................................................................... 27
2.1 Materials ............................................................................................................................................................... 27
2.1.1 Laboratory Chemicals and Biochemicals....................................................................................................... 27
2.1.2 Enzymes ......................................................................................................................................................... 29
2.1.3 Radiochemicals .............................................................................................................................................. 29
2.1.4 “Kits" and other Materials.............................................................................................................................. 29
2.1.5 Growth Factors and Ligands .......................................................................................................................... 30
2.1.6 Media and Buffers.......................................................................................................................................... 30
2.1.7 Cell Culture Media ......................................................................................................................................... 31
2.1.8 Stock Solutions for Buffers ............................................................................................................................ 31
2.1.9 Bacterial Strains, Cell Lines and Antibodies.................................................................................................. 33
2.1.10 List of Antibodies......................................................................................................................................... 34
2.2 Plasmids and Oligonucleotides............................................................................................................................ 35
2.2.1 Plasmid Preparation for Analytical Purpose................................................................................................... 35
2.2.2 Plasmid Preparation in Preparative Scale....................................................................................................... 35
2.1.3 Plasmid and Oligonucleotides................................................................................................................... 35
2.2.4 Constructs....................................................................................................................................................... 36
2.2.5 Important Primers ..................................................................................................................................... 37
2.2.6 Primers for RT-PCR....................................................................................................................................... 37
2.2.7 Primers for Methylation specific PCR: .......................................................................................................... 38
2.3 Enzymatic Manipulation of DNA ....................................................................................................................... 38
2.3.1 Digestion of DNA Samples with Restriction Endonucleases......................................................................... 38
2.3.1.2 Dephosphorylation of DNA 5'-Termini with Calf Intestine Alkaline Phosphatase (CIAP) ........................ 38
2.3.1.3 DNA Insert Ligation into Vector DNA ....................................................................................................... 38
2.3.1.4 Agarose Gel Electrophoresis ....................................................................................................................... 39
2.3.1.5 Isolation of DNA Fragments Using Low Melting Temperature Agarose Gels ........................................... 39
2.3.2 Introduction of Plasmid DNA into E.coli....................................................................................................... 39
2.3.2.1 Preparation of Competent Cells................................................................................................................... 39
5 Contents


2.3.2.2 Transformation of Competent Cells ............................................................................................................ 40
2.3.3 Oligonucleotide-Directed Mutagenesis .......................................................................................................... 40
2.3.3.1 Preparation of Uracil-Containing, Single-Stranded DNA Template ........................................................... 40
2.3.3.2 Primer Extension ......................................................................................................................................... 41
2.3.4 Enzymatic Amplification of DNA by Polymerase Chain Reaction (PCR) ............................................... 41
2.3.5 DNA Sequencing......................................................................................................................................