Biochemical and functional characterization of Fyn-PAG association and its role in T-cell anergy [Elektronische Ressource] / von Michal Smida

Smida

Sujets

• Gesundheit

Informations

• Publié par: otto-von-guericke-universitat_magdeburg
• Publié le: 01 janvier 2008
• Nombre de lectures: 23
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

Biochemical and functional characterization of Fyn-PAG
association and its role in T-cell anergy


Dissertation

zur Erlangung des akademischen Grades


doctor rerum naturalium
(Dr. rer. nat.)


genehmigt durch die Fakultät für Naturwissenschaften
der Otto-von-Guericke-Universität Magdeburg



von Dipl.-Biol. Michal Smida

geb. am 01.02.1976 in Boskovice, Tschechien


Gutachter: Prof. Dr. Burkhart Schraven
Prof. Doreen Cantrell



eingereicht am: 27. September 2007
verteidigt am: 27. Februar 2008
ACKNOWLEDGEMENT
Acknowledgement

I am deeply thankful to my mentor, Dr. Jonathan A. Lindquist, for his invaluable scientific
advice and professional guidance that lead me throughout the whole project. He was more than a
simple supervisor of my PhD thesis; he really took care of my life, not only professionally but
also privately. Not only did he gave me all the knowledge what was in his might, but he also had
that rare gift of providing me with the necessary motivation and encouragement. He always
infected me with his never-ending enthusiasm and found a word of assurance when I was loosing
my last hope.

I would never have been able to perform this work without the intense support of Prof.
Burkhart Schraven. His constant objective view on our experiments and inestimable suggestions
helped me to walk the right way and not to get lost in unlikely explanations. I shall always be
indebted for his excellent scientific input that has broadened my scientific background. His
expectations and trust were the driving forces that motivated me to develop further.

Many thanks should also be dedicated to Anita Posevitz-Fejfar, my colleague “in arms”. It
was always entertaining to work together with her, to support each other and to discuss any
problems in the lab. Thanks for finishing things in the lab on that occasional Fridays when I was
in a hurry to catch my train home. It was her and Vilmos Posevitz who showed me that there is
also another life besides work and did not allow me to feel lonely in my free time. Together with
Xiaoqian Wang and the other companions in our office, they always took care of the lovely
atmosphere in our room.

I will always appreciate the company of Dr. Mauro Togni for his unceasing sense of humor
and his perpetual support and willing to help me at any time. I will also never forget many
valuable discussions in the hallways with Drs. Luca Simeoni, Stefanie Kliche and Roland Hartig.
I would never neglect to mention the wonderful help I received from all of the technicians in our
institute and their assistance with any technique. I feel I must give thanks to all the members of
the Institute of Immunology, since they have made my stay here more enjoyable, by always being
nice, friendly, and making me feel like at home.
ACKNOWLEDGEMENT
I especially want to thank my parents for always believing in me and supporting me in my
research career and in all of my choices. I will also remember the many new and wonderful
friends I have made in Magdeburg, who regularly provided for a beautiful distraction.
Additionally, I must acknowledge my old friends at home, mainly for still staying my friends
despite the long distance between us. Thanks for always being on my side.

Finally, I wish to express my tribute to Pavlina, for her unbeatable tolerance, patience and
loving support in whatever I intended to do. Her belief in me and eternal encouragement has
given me the strength and motivation to move on. I shall always respect her for this.
TABLE OF CONTENTS i
Table of contents

1. Introduction…………………………………………………………………………………..1
1.1. T-cell development..……………………………………………………………………...2
1.2. Peripheral T-cell tolerance……………………………………………………………...3
1.2.1. AICD………………………………………………………………………………...3
1.2.2. Anergy………………………………………………………………………………4
1.2.2.1. Models of anergy……………………………………………………………4
1.2.2.2. Clinical applications of anergy……………………………………………...7
1.2.2.3. Biochemical characteristics of anergic cells………………………………...7
1.2.3. Regulatory T cells…………………………………………………………………...9
1.2.4. T-cell ignorance……………………………………………………………………..9
1.3. Ras proteins……………………………………………………………………………..10
1.3.1. Ras structure and localization……………………………………………………...10
1.3.2. Ras regulation……………………………………………………………………...11
1.3.3. RasGEFs…………………………………………………………………………...12
1.3.4. RasGAPs…………………………………………………………………………..12
1.3.5. Ras effectors……………………………………………………………………….13
1.4. T-cell signaling………………………………………………………………………….14
1.4.1. T-cell receptor……………………………………………………………………...14
1.4.2. Lipid rafts…………………………………………………………………………..15
1.4.3. T-cell signaling pathways………………………………………………………….16
1.5. Adaptor proteins………………………………………………………………………..18
1.5.1. Transmembrane adaptor proteins………………………………………………….19
1.6. PAG……………………………………………………………………………………...20
1.6.1. Structure and expression of PAG.………………………………………………….20
1.6.2. Interacting partners of PAG………………………………………………………..21
1.6.3. The PAG phosphatase……………………………………………………………...22
1.6.4. Function of PAG…………………………………………………………………...23
1.7. Src family kinases (SFKs)……………………………………………………………...26
1.7.1. Structure and localization of Src family kinases…………………………………..26
1.7.2. Regulation and activation of Src family kinases…………………………………..27
TABLE OF CONTENTS ii
1.7.3. Function of Lck and Fyn…………………………………………………………...29
1.8. Csk (C-terminal Src kinase)…………………………………………………………...31
1.9. Aim of the project………………………………………………………………………33
2. Methods……………………………………………………………………………………...34
2.1. Antibodies used in this study…………………………………………………………….34
2.2. General reagents for cell culture…………………………………………………………36
2.3. T cell isolation and purification………………………………………………………….36
2.4. Anergy induction………………………………………………………………………...37
2.5. Proliferation assay……………………………………………………………………….37
2.6. Stimulation of T cells……………………………………………………………………38
2.7. Cell lysis, immunoprecipitation and Western blot analysis……………………………..38
2.8. Mass spectrometry……………………………………………………………………….41
2.9. Subcellular fractionation…………………………………………………………………41
2.10. Flowcytometry………………………………………………………………………….42
2.11. Lipid raft preparation…………………………………………………………………...43
2.12. In vitro kinase assay……………………………………………………………………44
2.13. cAMP measurement……………………………………………………………………44
2.14. Transfection…………………………………………………………………………….45
2.15. Ras activation assay……………………………………………………………………46
2.16. Scanning and quantification……………………………………………………………47
3. Results………………………………………………………………………………………48
3.1. Proximal alterations within Anergic T cells…………………………………………..48
3.1.1. Induction of Anergic T cells…..…………………………………………………...48
3.1.2. Increased Fyn activity and expression within Anergic T cells…………………….51
3.1.3. Altered phosphorylation profile in Anergic T cells………………………………..53
3.1.4. Defective proximal signaling in Anergic T cells…………………………………..54
3.2. Alterations within PAG-associated complex in Anergic T cells……………………..55
3.2.1. PAG-associated kinase activity is enhanced in Anergic T cells…………………...55
317
3.2.2. PAG is hyperphosphorylated at Y in Anergic T cells…..……………………….56
3.2.3. Hyperphosphorylated PAG recruits more Csk in Anergic T cells…………………57
3.2.4. Elevated levels of the PAG-Csk complex create an inhibitory environment in
Anergic cells..………………………………………………………………………57
TABLE OF CONTENTS iii
364 3.2.5. Increased cAMP level and pSer -Csk in Anergic T cells.………………………..59
529 215
3.2.6. Fyn is dually phosphorylated on its Y and Y in Anergic T cells……………..60
3.2.7. Increased Fyn kinase activity and inhibitory tyrosine phosphorylation within the
lipid rafts of Anergic T cells.………………………………………………………61
3.3. PAG forms a novel multiprotein complex, which regulates Ras activation………...65
3.3.1. Increased expression of Sam68 and p120RasGAP………………………………...65
3.3.2. PAG forms a novel multiprotein complex consisting of PAG, Fyn, Sam68 and
p120RasGAP..……………………………………………………………………...66
3.3.3. PAG negatively regulates Ras activation…………………………………………..68
3.3.4. Basic characterization of Y317F mutant of PAG………………………………….71
3.3.5. PAG negatively regulates Ras activation independently of Csk binding………….72
181 3.3.6. Y of PAG is the p120RasGAP binding site……………………………………..74
3.3.7. PAG negatively regulates Ras activation also in the absence of p120RasGAP
binding..…………………………………………………………………………….74
3.3.8. Both Csk and p120RasGAP binding contribute to the block in Ras activation…...76
3.3.9. PAG downregulation leads to enhanced and sustained SFK and Ras activation….77
3.4. Identification and characterization of IGAP………………………………………....80
3.4.1. Expression of DGKs is unchanged in Anergic T cells…………………………….80
3.4.2. DGK alpha antibody cross-reacts with p120 protein………………………………81
3.4.3. Identification of IGAP……………………………………………………………..83
3.4.4. IGAP is phosphorylated in vivo……