Characterization of signal transduction pathways by MS-based quantitative proteomics [Elektronische Ressource] : phosphotyrosine interactomics and insulin signaling ; absolute quantitation of proteins by mass spectrometry / Stefan Hanke

Stefan Hanke

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• Gesundheit

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• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2008
• Nombre de lectures: 26
• Poids de l'ouvrage: 10 Mo

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CHARACTERIZATION OF SIGNAL TRANSDUCTION
PATHWAYS BY MS-BASED QUANTITATIVE
PROTEOMICS

PHOSPHOTYROSINE INTERACTOMICS AND INSULIN SIGNALING

ABSOLUTE QUANTITATION OF PROTEINS
BY MASS SPECTROMETRY

Dissertation
Stefan Hanke

aus
Rosenheim










2008 Dissertation zur Erlangung des Doktorgrades
der Fakultät für Chemie und Pharmazie
der Ludwig-Maximilians-Universität München


CHARACTERIZATION OF SIGNAL TRANSDUCTION
PATHWAYS BY MS-BASED QUANTITATIVE
PROTEOMICS

PHOSPHOTYROSINE INTERACTOMICS AND INSULIN SIGNALING

ABSOLUTE QUANTITATION OF PROTEINS
BY MASS SPECTROMETRY


Stefan Hanke

aus

Rosenheim


2008 Erklärung
Diese Dissertation wurde im Sinne von § 13 Abs. 3 der Promotionsordnung vom
29. Januar 1998 von Herrn Prof. Dr. Matthias Mann betreut.


Ehrenwörtliche Versicherung
Diese Dissertation wurde selbständig, ohne unerlaubte Hilfe erarbeitet.



München, am 29. August 2008






……………………………………………..
(Unterschrift des Autors)




Dissertation eingereicht am 01. September 2008
1. Gutachter Prof. Dr. Matthias Mann
2. Gutachter Prof. Dr. Elena Conti
Mündliche Prüfung am 24. September 2008TABLE OF CONTENTS
Table of contents
1 INTRODUCTION ................................................................................... 1
1.1 PHOSPHORYLATION IN CELL SIGNALING .............................................................. 1
1.1.1 Phosphorylation as regulatory switch in proteins ............................................. 1
1.1.2 Tyrosine phosphorylation .................. 2
1.1.3 Interaction modules in phosphotyrosine signaling ............ 2
1.1.3.1 SH2 domains ........................................ 2
1.1.3.2 PTB domains ........ 7
1.1.3.3 Other phosphotyrosine-binding domains............................................................ 8
1.2 INSULIN SIGNALING ....................................................................................... 8
1.2.1 Insulin signaling in health and disease .............................................................. 8
1.2.1.1 Systemic aspects .................................................................................................. 8
1.2.1.2 The intracellular signaling network ................................................................... 10
1.2.2 The insulin receptor family .............................................. 13
1.2.3 The insulin receptor substrate (IRS) family ...................................................... 14
1.2.4 The pivotal role of IRS-1 and IRS-2 in insulin signaling .... 15
1.3 MASS SPECTROMETRY-BASED QUANTITATIVE PROTEOMICS... 18
1.3.1 Modern mass spectrometry applied to protein research ................................ 18
1.3.2 Instrumentation ............................................................................................... 19
1.3.2.1 Linear ion trap .... 20
1.3.2.2 FT-ICR ................................................. 21
1.3.2.3 Orbitrap ............. 22
1.3.3 Stable isotope labeling for quantitative readout ............ 24
1.3.3.1 Relative quantitation ......................................................................................... 25
1.3.3.2 Absolute quantitation ........................................................ 28
1.4 GOALS OF THE STUDY .................................................................................. 29
2 PROFILING OF PHOSPHOTYROSINE-INTERACTION PLATFORMS IN THE
INSULIN SIGNALING PATHWAY ........................................................... 31
2.1 PUBLICATION: THE PHOSPHOTYROSINE INTERACTOME OF THE INSULIN RECEPTOR FAMILY
AND ITS SUBSTRATES IRS-1 AND IRS-2 ............................ 31
3 DEVELOPMENT OF A NOVEL METHOD FOR VERY HIGH ACCURACY IN
PROTEIN QUANTITATION .................................................................... 70
3.1 PUBLICATION: ABSOLUTE SILAC FOR ACCURATE QUANTITATION OF PROTEINS IN
COMPLEX MIXTURES DOWN TO THE ATTOMOLE LEVEL ........... 70
4 MOTIF DECOMPOSITION OF THE PHOSPHOTYROSINE PROTEOME ...... 84
4.1 PUBLICATION: MOTIF DECOMPOSITION OF THE PHOSPHOTYROSINE PROTEOME REVEALS A
NEW N-TERMINAL BINDING MOTIF FOR SHIP2 ................................................... 84
5 MS-ANALYSIS OF INTACT SILAC-LABELED PROTEINS ............................ 97
5.1 PUBLICATION: TOP-DOWN QUANTITATION AND CHARACTERIZATION OF SILAC-LABELED
PROTEINS .................................................................................................. 97

iii TABLE OF CONTENTS
6 FURTHER PUBLISHED AND UNPUBLISHED WORK .............................. 105
6.1 VESICULAR TRANSPORT IN THE GOLGI-APPARATUS ............................................ 105
6.2 TRAFFICKING OF CAVEOLAE .......................................... 105
6.3 CAPSID PROTEINS OF HERPES SIMPLEX VIRUS .................................................. 106
6.4 PHOSPHORYLATION DYNAMICS OF IRS-1 AND IRS-2 ......... 106
7 CONCLUDING REMARKS AND PERSPECTIVES .................................... 108
7.1 SUMMARY AND CONCLUSIONS ..................................... 108
7.2 PERSPECTIVES .......................................................... 110
8 BIBLIOGRAPHY .................................................................................. 112
9 ABBREVIATIONS ................ 119
10 ACKNOWLEDGEMENTS ..................................................................... 122
11 CURRICULUM VITAE .......... 123

iv INTRODUCTION
1 INTRODUCTION
1.1 PHOSPHORYLATION IN CELL SIGNALING
Cells need to react quickly to changes in their environment and to this end they developed
mechanisms to translate extracellular and intracellular stimuli into a biochemical code that
can be read by the protein machinery. As fundamental requirement this often needs to occur
fast and thus cannot only rest upon synthesis of new proteins. A large part of this code is
therefore based on posttranslational modifications (PTMs) that are attached to specific sites
in proteins. In order to maintain flexibility, many types of PTMs can be removed again.
Phosphorylation is one of the most prominent reversible PTMs on proteins and typically
occurs on serine, threonine and tyrosine. In prokaryotes, histidine and aspartate are also
common amino acids for the attachment of phospho-moieties. Apart from proteins, lipids and
sugars can also be phosphorylated, with phosphatidylinositol phosphates and
phosphoinositides constituting important docking sites and second messengers, respectively.
In agreement with the central and manifold role of phosphorylation, the human genome
1, 2harbors more than 500 protein kinases and more than 150 protein phosphatases .
1.1.1 Phosphorylation as regulatory switch in proteins
Phosphorylation can change the functional state of a protein either by triggering a
conformational change or by facilitating or preventing intermolecular interactions. Regulated
protein-protein interactions form the basis of cellular signal transduction, and frequently
posttranslational modifications such as phosphorylation constitute the molecular switch to
enable the association or dissociation of proteins. A phosphorylated motif can serve as a
docking site for a specific binding domain. Prominent domains that recognize phosphoserine
3, 4(pSer) and phosphothreonine (pThr) are the 14-3-3, WW, and FHA domains . 14-3-3
domains prefer motifs with a proline in the +2 position (with respect to the pSer/pThr), and a
serine or aromatic amino acid in the -2 position, preceded by an arginine in the -3 or -4
position. WW domains typically recognize short proline sequences like PPXY, PPLP or PPR.
Only a subset of WW domains binds to pSer/pThr followed by a proline. FHA domains are
dedicated to the recognition of pThr-motifs, and they exploit the +3 position for selectivity.
Phosphotyrosine (pTyr) engages separate domain families for recruitment, which will be
highlighted in detail below.
1 INTRODUCTION
1.1.2 Tyrosine phosphorylation
Phosphorylation is a prominent instrument in the toolbox of signaling, and especially tyrosine
phosphorylation has attracted much attention in the propagation of signals downstream of
receptor tyrosine kinases (RTKs) and cytoplasmic protein tyrosine kinases. The human
genome harbors 90 genes for protein tyrosine kinases and 107 genes for protein tyrosine
5phosphatases . Phosphorylated tyrosine sites serve particularly often as docking interfaces.
Intramolecular interactions with a pTyr-binding module such as an SH2 domain can activate
6the protein (as in the case of Fes) or inactivate it (as in the case of Src and Abl) . In
intermolecular interactions, the mere binding to a phosphorylated motif can also modulate
the enzymatic activity of the protein in some cases. However, the primary goal of the
interaction is usually the effect on the localization of the recruited protein. By increasing its
local concentration and scaffolding it together with its upstream or downstream effectors,
signaling pathways can be initiated, streamlined