B-Raf regulation by the dimer interface and activation loop phosphorylation:
Gespeichert in:
| 1. Verfasser: | |
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
2012
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| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | XI, 158 Bl. Ill., graph. Darst. |
Internformat
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| 100 | 1 | |a Röring, Michael |e Verfasser |4 aut | |
| 245 | 1 | 0 | |a B-Raf regulation by the dimer interface and activation loop phosphorylation |c vorgelegt von Michael Röring |
| 264 | 1 | |c 2012 | |
| 300 | |a XI, 158 Bl. |b Ill., graph. Darst. | ||
| 336 | |b txt |2 rdacontent | ||
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Datensatz im Suchindex
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| adam_text | IMAGE 1
I. TABLE OF CONTENTS
I. TABLE OF CONTENTS VI
II. ABBREVIATIONS IX
III. SUMMARY XI
1 INTRODUCTION 1
1.1 CELLULAR SIGNALING 1
1.2 MITOGEN-ACTIVATED PROTEIN KINASE (MAPK) SIGNALING PATHWAYS 1 1.2.1
THE MAMMALIAN ERK PATHWAY 3
1.2.2 THE RAF PROTEINS 5
1.2.2.1 DISCOVERY O F RAF KINASES 5
1.2.2.2 STRUCTURE AND REGULATION O F RAF PROTEINS 6
1.3 PHYSIOLOGICAL AND PATHOLOGICAL ASPECTS O F RAF SIGNALING 10
1.4 TARGETING RAF IN CANCER USING SMALL MOLECULE INHIBITORS 13
1.5 PARADOXICAL ERK ACTIVATION AND DRUG-RESISTANCE IN MELANOMA 14 1.6
CONDITIONAL GENE TARGETING 18
2 AIM O F THE STUDY 21
3 RESULTS 22
3.1 THE DIMER INTERFACE IS A MAJOR DETERMINANT OF RAF SIGNALING 22
3.1.1 KSR1 CO-EXPRESSION PROMOTES HUMAN B-RAF KINASE ACTIVITY 23 3.1.2
B-RAF E586K RELIES ON MANY REGULATORY REQUIREMENTS ESTABLISHED FOR B-RAF
VT 23 3.1.3 DIMER INTERFACE MUTATIONS HARDLY AFFECT B-RAF V600E
SIGNALING 26 3.1.4 THE RESILIENCE O F B-RAF V600E TO DIF MUTATIONS IS
NOT BASED ON SATURATION... 27 3.1.5 B-RAF NST AND B-RAF 469A DISPLAY
HIGH RESISTANCE TO R509H DIF MUTATION. 28
3.1.6 B-RAF V600E DIF MUTANTS DO NOT RELY ON ENDOGENOUS RAF PROTEINS 31
3.1.7 THE R509H DIF MUTATION REDUCES THE IN VITRO KINASE ACTIVITY O F
B-RAF V600E 32 3.1.8 B-R A F V600E/R509H/S729A STILL POSSESSES
REMARKABLE CELLULAR SIGNALING POTENTIAL36 3.1.9 B-RAF V600E AND B-RAF
469A FORM MORE STABLE HOMO-DIMERS THAN B-RAF ... 36 3.1.10 THE V600E
MUTATION INDUCES LARGER B-RAF CONTAINING PROTEIN COMPLEXES.. 38
3.1.11 THE V600E MUTATION ENLARGES THE CONTACT ZONES BETWEEN B-RAF
PROTOMERS40 3.1.12 DIF MUTATIONS HARDLY AFFECT THE TRANSFORMATION
POTENTIAL O F B-RAF V600E 41 3.1.13 THE ROLE O F THE DIF FOR
HETERO-DIMER FORMATION WITH KSR AND RAF-1 43 3.1.14 THE ROLE O F THE DIF
FOR PARADOXICAL ERK ACTIVATION 44
3.1.15 THE ROLE O F THE DIF FOR DRUG-INDUCED B-RAF DIMER FORMATION 47
3.1.16 K-RAS G12V INDUCED RAF-1 ACTIVATION IS HIGHLY DIF-DEPENDENT 52
3.2 GENERATION O F A CRE-INDUCIBLE B-RAF^ 1 ^ KNOCK-IN MOUSE MODEL 54
3.2.1 RATIONALE FOR TARGETING THE B-RAF ACTIVATION LOOP BASED
TVKS-MOTIF. 54
3.2.2 CONSIDERATIONS FOR THE B - R A F ^ 1 ^ KNOCK-IN TARGETING VECTOR
56
3.2.3 DESIGN O F THE B - R A F ^ 1 ^ KNOCK-IN TARGETING VECTOR 58
3.2.4 HOMOLOGOUS RECOMBINATION IN ES CELLS AND GERMLINE TRANSMISSION 59
3.2.5 FIRST PHENOTYPIC ANALYSIS O F THE B - R A F ^ 1 ^ MICE 63
B-RAF REGULATION BY ACTIVATION LOOP PHOSPHORYLATION A N D T H E DIMER
INTERFACE VI
HTTP://D-NB.INFO/1031390561
IMAGE 2
4 DISCUSSION 67
4.1 THE ROLE O F KSR IN B-RAF ACTIVATION AND ERK SIGNALING 67
4.2 THE REGULATORY REQUIREMENTS O F THE B-RAF* 586 ONCOPROTEIN 69
4.3 THE RAF SIDE-TO-SIDE DIF - A NEW THERAPEUTIC TARGET? 71
4.3.1 WILDTYPE AND MUTANT B-RAF ARE DIFFERENTIALLY AFFECTED BY DIF
MUTATIONS... 71 4.3.1.1 THE ROLE O F THE ACTIVE CONFORMATION FOR B-RAF
V600E HOMO-DIMERIZATION 76
4.3.1.2 POTENTIAL BIOLOGICAL IMPLICATIONS O F ENHANCED B-RAF V600E
HOMO-DIMERIZATION 77
4.3.2 RAS-DRIVEN (PARADOXICAL) RAF ACTIVATION IS HIGHLY DIF-DEPENDENT 78
4.4 SUCCESSFUL ESTABLISHMENT O F AN INDUCIBLE B-RAF^ 1 ^ KNOCK-IN MOUSE
MODEL ...82 4.5 CONCLUSION 85
5 MATERIAL AND METHODS 86
5.1 MATERIAL 86
5.1.1 LABORATORY EQUIPMENT 86
5.1.2 CONSUMABLES 87
5.1.3 CHEMICALS 88
5.1.4 E. COLI STRAINS 90
5.1.5 CELLS 91
5.1.6 CELL CULTURE 91
5.1.6.1 MEDIA 91
5.1.6.2 REAGENTS 92
5.1.6.3 ANTIBIOTICS 93
5.1.6.4 INHIBITORS 93
5.1.7 MOUSE STRAINS 94
5.1.8 PLASMIDS 94
5.1.8.1 CDNA PLASMIDS 9 4
5.1.9 BUFFERS AND SOLUTIONS 96
5.1.10 KITS 99
5.1.11 OLIGONUCLEOTIDES 100
5.1.12 ENZYMES 102
5.1.13 ANTIBODIES 102
5.1.13.1 PRIMARY ANTIBODIES 102
5.1.13.2 SECONDARY ANTIBODIES 103
5.2 METHODS 104
5.2.1 MOLECULAR BIOLOGY 104
5.2.1.1 GENERATION, CULTIVATION AND CRYOCONSERVATION O F CHEMOCOMPETENT
E. COLI 104 5.2.1.2 TRANSFORMATION O F CHEMOCOMPETENT E. COLI 104
5.2.1.3 PLASMID-DNA PREPARATION 104
5.2.1.4 ISOLATION O F GENOMIC DNA 105
5.2.1.5 MEASURING D N A CONCENTRATIONS 106
5.2.1.6 RESTRICTION ENDONUCLEASE DIGESTS OFPLASMID AND GENOMIC D N A 106
5.2.1.7 AGAROSE-GEL ELECTROPHORESIS 106
5.2.1.8 D N A EXTRACTION FROM AGAROSE GELS 106
5.2.1.9 DEPHOSPHORYLATION O F DNA FRAGMENTS 106
5.2.1.10 LIGATION O F D N A FRAGMENTS 107
5.2.1.11 T/A CLONING O F PCR AMPLICONS 107
5.2.1.12 PURIFICATION O F TOTAL RNA 107
5.2.1.13 CDNA FIRST STRAND SYNTHESIS 107
5.2.1.14 POLYMERASE CHAIN REACTION (PCR) 108
5.2.1.15 DNA SEQUENCING 112
5.2.1.16 SOUTHERN BLOTTING 112
5.2.2 CELL BIOLOGICAL METHODS 115
5.2.2.1 CULTIVATION O F CELLS 115
5.2.2.2 CRYOCONSERVATION O F CELLS 115
IMAGE 3
5.2.2.3 TRANSFECTION O F PLAT-E CELLS 115
5.2.2.4 RETROVIRAL INFECTION 116
5.2.2.5 ISOLATION O F MEFS 116
5.2.2.6 IMMORTALIZATION O F MEFS 117
5.2.2.7 4-HT INDUCIBLE CRE-MEDIATED RECOMBINATION O F THE MURINE
B-RA/ LOCUS IN VITRO 117 5.2.2.8 4-HT INDUCIBLE ACTIVATION O F THE H-RAS
GI2V ONCOPROTEIN 117
5.2.2.9 ELECTROPORATION AND G418-SELECTION O F MURINE ES CELLS 118
5.2.2.10 ISOLATING INDIVIDUAL G418-RESISTANT MURINE ES CELL CLONES 118
5.2.2.11 INJECTION O F ES CELL CLONES INTO C57B1/6 BLASTOCYSTS 118
5.2.2.12 MOUSE BREEDING 119
5.2.2.13 IN VIVO DELETION O F THE G418-RESI STANCE GENE CASSETTE ( NEO K
) 119
5.2.2.14 CROSSING B-RQF 0!TAWKA MICE TO CRE-DELETER STRAINS 119
5.2.3 ISOLATION O F MURINE ORGANS 119
5.2.4 FLOW CYTOMETRY 120
5.2.4.1 FACS ANALYSIS O F MEFS AND DT40 B CELLS 120
5.2.5 PROTEIN BIOCHEMISTRY 120
5.2.5.1 CELL LYSIS AND SAMPLE PREPARATION 120
5.2.5.2 (CO-) IMMUNOPRECIPITATION O F PROTEIN COMPLEXES 121
5.2.5.3 SDS-PAGE AND WESTERN BLOTTING 121
5.2.5.4 B-RAF IN VITRO KINASE ASSAY (IVK) 122
5.2.5.5 LAMBDA PHOSPHATASE TREATMENT 122
5.2.5.6 BLUE-NATIVE PAGE 122
5.2.6 PROTEIN STRUCTURE ANALYSIS 123
6 APPENDIX 124
6.1 CLONING O F THE PFLOXAVKA-MG-NEO TARGETING VECTOR 124
6.2 INCOMPLETE B-RA/LOCUS RECOMBINATION IN VIVO USING A CMV-CRE DELETER
STRAIN 127
6.3 SUPPLEMENTARY TABLES 131
7 REFERENCES 135
8 DANKSAGUNG 158
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| illustrated | Illustrated |
| indexdate | 2024-07-10T00:33:18Z |
| institution | BVB |
| language | English |
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| physical | XI, 158 Bl. Ill., graph. Darst. |
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| spelling | Röring, Michael Verfasser aut B-Raf regulation by the dimer interface and activation loop phosphorylation vorgelegt von Michael Röring 2012 XI, 158 Bl. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Freiburg i. Br., Univ., Diss., 2012 Phosphorylierung (DE-588)4174459-7 gnd rswk-swf Dimerisierung (DE-588)4251822-2 gnd rswk-swf Ras (DE-588)4291111-4 gnd rswk-swf Signaltransduktion (DE-588)4318717-1 gnd rswk-swf Proteinkinasen (DE-588)4175996-5 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Signaltransduktion (DE-588)4318717-1 s Proteinkinasen (DE-588)4175996-5 s Dimerisierung (DE-588)4251822-2 s Ras (DE-588)4291111-4 s Phosphorylierung (DE-588)4174459-7 s DE-604 DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025738035&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Röring, Michael B-Raf regulation by the dimer interface and activation loop phosphorylation Phosphorylierung (DE-588)4174459-7 gnd Dimerisierung (DE-588)4251822-2 gnd Ras (DE-588)4291111-4 gnd Signaltransduktion (DE-588)4318717-1 gnd Proteinkinasen (DE-588)4175996-5 gnd |
| subject_GND | (DE-588)4174459-7 (DE-588)4251822-2 (DE-588)4291111-4 (DE-588)4318717-1 (DE-588)4175996-5 (DE-588)4113937-9 |
| title | B-Raf regulation by the dimer interface and activation loop phosphorylation |
| title_auth | B-Raf regulation by the dimer interface and activation loop phosphorylation |
| title_exact_search | B-Raf regulation by the dimer interface and activation loop phosphorylation |
| title_full | B-Raf regulation by the dimer interface and activation loop phosphorylation vorgelegt von Michael Röring |
| title_fullStr | B-Raf regulation by the dimer interface and activation loop phosphorylation vorgelegt von Michael Röring |
| title_full_unstemmed | B-Raf regulation by the dimer interface and activation loop phosphorylation vorgelegt von Michael Röring |
| title_short | B-Raf regulation by the dimer interface and activation loop phosphorylation |
| title_sort | b raf regulation by the dimer interface and activation loop phosphorylation |
| topic | Phosphorylierung (DE-588)4174459-7 gnd Dimerisierung (DE-588)4251822-2 gnd Ras (DE-588)4291111-4 gnd Signaltransduktion (DE-588)4318717-1 gnd Proteinkinasen (DE-588)4175996-5 gnd |
| topic_facet | Phosphorylierung Dimerisierung Ras Signaltransduktion Proteinkinasen Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025738035&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT roringmichael brafregulationbythedimerinterfaceandactivationloopphosphorylation |