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Identification of Novel A-RAF Phosphorylation Sites

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Differences and Similarities in the Regulation of RAF Isoforms: Identification of Novel A-RAF Phosphorylation Sites Dissertation zur Erlangung des naturwissenschaftlichen Doktorgrades der Bayerischen Julius-Maximilians-Universität Würzburg vorgelegt von Angela Baljuls aus Koktschetaw, Kasachstan Würzburg, 2008 Eingereicht am:____________________ Mitglieder der Promotionskommission: Vorsitzender: Prof. Dr. Martin J. Müller Gutachter: Prof. Dr. Ulf R. Rapp Gutachter: Prof. Dr. Ricardo Benavente Tag des Promotionskolloquiums:____________________ Doktorurkunde ausgehändigt am:____________________ Meiner Familie zum Dank für Eure Liebe und Unterstützung Basic research is like shooting an arrow into the air and, where it lands, painting a target. Homer Burton Adkins ___________________________________________________________________Table of Contents TABLE OF CONTENTS 1. SUMMARY 5 2. ZUSAMMENFASSUNG 6 3. INTRODUCTION 7 3.1. MAP kinase signaling pathways 7 3.2. Key components of the classical MAP kinase cascade 11 3.2.1. EGF receptor as an example of receptor tyrosine kinases (RTKs) 11 3.2.2. Ras-GTPase 12 3.2.3. Lck as an example of Src family kinases 15 3.2.4. MEK 17 3.2.5. ERK 19 3.2.6. Scaffolds 22 3.2.7. RAF kinases 24 3.2.7.1. Structure of RAF proteins 26 3.2.7.2. Regulation of RAF activity by 14-3-3 proteins 28 3.2.7.3. Mechanism of Ras–RAF coupling 31 3.2.7.4. Current mechanism of RAF activation 34 3.2.7.5. Regulation of RAF activity by phosphorylation 36 3.3. Isoform-specific properties of A-RAF 37 3.3.1. A-RAF gene 37 3.3.1.1. Localization of A-RAF gene 37 3.3.1.2. Molecular organization of A-RAF gene 38 3.3.2. A-RAF protein 39 3.3.2.1. Splicing variants of A-RAF 39 3.3.2.2. Expression of A-RAF 39 3.3.2.3. Localization of A-RAF protein 41 3.3.2.4. Physiological role of A-RAF 42 3.3.2.5. Interaction partners of A-RAF 43 4. AIM OF THE PROJECT 46 5. MATERIALS AND METHODS 47 5.1. Materials 47 5.1.1. Instruments 47 5.1.2. Chemical reagents and general materials 48 5.1.3. Software 49 5.1.4. Cell culture materials 49 5.1.5. Antibodies used for Western blotting and immuno- precipitation 50 5.1.6. Enzymes 51 5.1.7. Kits 51 1 ___________________________________________________________________Table of Contents 5.1.8. Plasmids 51 5.1.9. Oligonucleotides 52 5.1.10. Cell lines and bacterial strains 54 5.2. Solutions and buffers 55 5.3. Methods 60 5.3.1. Bacterial manipulation 60 5.3.1.1. Preparation of chemocompetent cells (CaCl2 method) 60 5.3.1.2. Transformation of chemocompetent bacteria 60 5.3.2. Methods of molecular biology 61 5.3.2.1. Amplification of DNA fragments by PCR 61 5.3.2.2. Agarose gel electrophoresis of DNA 62 5.3.2.3. Isolation of DNA fragments from agarose gel 63 5.3.2.4. Purification of DNA fragments 63 5.3.2.5. Digestion of DNA with restriction endonucleases 63 5.3.2.6. DNA ligation 64 5.3.2.7. Mini-preparation of plasmid DNA 65 5.3.2.8. Midi-preparation of plasmid DNA 65 5.3.2.9. Determination of DNA concentration and quality 65 5.3.2.10. DNA sequencing (Sanger's Dideoxynucleotide Synthetic Method) 66 5.3.2.11. Site-directed mutagenesis 67 5.3.3. Biochemical methods 69 5.3.3.1. Preparation of cell lysates 69 5.3.3.2. Determination of protein concentration (Bradford Assay) 70 5.3.3.3. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) 70 5.3.3.4. Immunoblotting 71 5.3.3.5. Immunoblot stripping 72 5.3.3.6. Immunoprecipitation 72 5.3.3.7. RAF in vitro kinase assay 73 5.3.3.8. Purification of GST-fusion RAF proteins from Sf9 and COS7 cell lysates 74 5.3.3.9. Purification of GST-fusion 14-3-3 proteins from E. coli 74 5.3.3.10. Purification of His-tagged RAF proteins from Sf9 cell lysates 74 5.3.3.11. Subcellular fractionation 75 5.3.3.12. Mass spectrometry measurements 76 5.3.3.13. BIAcore’s SPR technology 77 5.3.4. Cell culture methods 78 5.3.4.1. Cultivation and passaging of eukaryotic cells 78 5.3.4.2. Cell counting 79 5.3.4.3. Freezing, long-term storage and thawing of cells 80 5.3.4.4. Transfection of mammalian adherent cells 80 5.3.4.5. Infection of insect cells 80 2 ___________________________________________________________________Table of Contents 5.3.5. Bioinformatic methods 84 5.3.5.1. Modeling of the N-region interactions with the catalytic part of RAF kinases 84 5.3.5.2. Modeling of the three-dimensional structure of the IH-segment in A-RAF 84 6. RESULTS 86 6.1. MS analysis of A-RAF phosphorylation 86 6.1.1. Phosphorylation sites within the kinase domain of A-RAF 86 6.1.2. Phosphorylation sites within the regulatory part of A-RAF 88 6.2. A-RAF kinase activity is regulated by 14-3-3 proteins 92 6.2.1. Regulation of A-RAF activity by 14-3-3 in Sf9 cells 93 6.2.2. Regulation of A-RAF activity by 14-3-3 in COS7 cells 93 6.2.3. Mammalian 14-3-3 proteins associate with RAF kinases in isoform-specific manner 95 6.2.4. 14-3-3 binding sites of RAF kinases differ in their binding affinities 98 6.3. Phosphorylation of MEK binding sites is critical for A-RAF kinase activity 100 6.4. Phosphorylation of the activation segment is necessary for maximal activation of A-RAF 101 6.5. N-region determines low basal activity and limited inducibility of A-RAF kinase 102 6.5.1. Substitution of serine 299 to alanine in A-RAF abrogates its activation 104 6.5.2. Phosphorylation of the conserved serine in the N-region is predicted to depend on amino acid at position –3 105 6.5.3. Mutation in the N-region leads to a constitutively active form of A-RAF kinase 106 6.5.4. C-RAF behaves similar to A-RAF: mutations at the positions 335 and 339 in the N-region of C-RAF modulate its kinase activity 111 6.5.5. Active forms of C-RAF mutants are located preferentially at membranes 115 6.5.6. A model derived from the tertiary structure of RAF reveals a tight contact between the N-region of A-RAF and its catalytic domain 118 6.5.7. R398/K399 in C-RAF and analogous residues R359/K360 in A-RAF are indispensable for their activation 121 6.5.8. Substitution of R398 in C-RAF and R359 in A-RAF by alanine induces mobility shift-associated hyperphosphorylation of these proteins 125 6.6. Feedback regulation of A-RAF 129 3 ___________________________________________________________________Table of Contents 6.6.1. Phosphorylation within the IH-domain positively regulates activation process of A-RAF 129 6.6.2. Spatial model reveals charge reversal at the molecular surface of IH-segment of A-RAF upon phosphorylation 133 7. DISCUSSION 136 7.1. Isoform-specific regulation of A-RAF by 14-3-3 proteins 136 7.1.1. Phosphorylation of the N-region may support the association of A-RAF with 14-3-3 dimers 136 7.1.2. Differences in subcellular localization of RAF isoforms may explain the isoform-specific association with 14-3-3 proteins 138 7.2. Maximal activation of A-RAF kinase requires phosphorylation of both MEK binding sites, S432 and T442 139 7.3. Negative charge of the N-region is a prerequisite for phosphorylation of activation segment 140 7.4. Non-conserved residues within the N-region determine activation properties of RAF kinases 141 7.4.1. PKA and RSK are predicted to phosphorylate RAF on the conserved serine within the N-region 142 7.4.2. Tyrosine 296 determines the low activating potency of A-RAF by sterical reasons 144 7.4.3. Disruption of interaction between the N-region and the PABR facilitates binding to lipids 145 7.4.4. Regulation of RAF activity by PABR is similar for A- and C-RAF 146 7.5. ERK-mediated feedback phosphorylation is proposed to participate in A-RAF activation 148 7.6. Molecular modeling of the IH-segment suggests a “switch-of-charge” mechanism for A-RAF regulation 150 8. REFERENCES 153 9. APPENDIX 171 9.1. MascotTM results pages of the phosphopeptide spectra from Table 3 171 9.2. Abbreviations 184 ACKNOWLEDGMENTS 188 CURRICULUM VITAE 189 ERKLÄRUNG 192 4 Summary 1. SUMMARY In mammals, the RAF family of serine/threonine kinases consists of three members, A-, B- and C-RAF. Activation of RAF kinases involves a complex series of phosphorylations. Although the most prominent phosphorylation sites of B- and C-RAF are well characterized, little is known about regulatory phosphorylation of A-RAF. Using mass spectrometry, we identified here a number of novel in vivo phosphorylation sites in A-RAF. The physiological role and the function of these sites were investigated subsequently by amino acid exchange at the relevant positions. In particular, we found that S432 participates in MEK binding and is indispensable for A-RAF signaling. On the other hand, phosphorylation within the activation segment does not contribute to epidermal growth factor-mediated activation. Regarding regulation of A-RAF activity by 14-3-3 proteins, we show that A-RAF activity is regulated differentially by its C-terminal and internal 14-3-3 binding domain.
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