5. Stand der Wissenschaft und Technik, bisherige eigene Arbeiten,
Patentlage, Wirtschaftliche Bedeutung
A. Title of the project
Towards a quantitative understanding of kinase signaling networks in growth
and differentiation.
B. Short Summary
To build tissue, organs or an organism from cells complex intracellular signal
transduction pathways are interconnected in a network of multifunctional, redundant
and non-redundant molecules. These proteins elicit a set of phenotypic responses
that subsequently impact function at the cell, tissue, and organ level. Basic principle
is that the interaction of several pathways in the network can exhibit a new emergent
state as a consequence of their interactions. It is this property of biological systems
that makes signaling networks in growth and differentiation difficult to understand
based on investigating linear pathways. Therefore, in this proposed collaborative
effort we will study quantitative changes of the phosphorylation status of defined
proteomes that are required for cell growth and differentiation.
C. Specific Aims
In response to growth-stimulatory or growth-inhibitory signals, morphogens or
hormones, eukaryotic cells rely upon the timely activation and inactivation of several
families of protein kinases and phosphatases1,2. These enzymes regulate
progression through the cell cycle or cell cycle arrest, cell growth and differentiation,
polarization and cell specification. Catastrophic human diseases such as cancer,
degenerative disorders or developmental defects often result, in part, from mutations
in proteins involved in these signaling pathways leading to the selective proliferation
of mutant cells or lack of cell differentiation. Key molecules are the protein tyrosine
(Tyr) or serine/threonine (Ser/Thr) kinases or phosphatases themselves and
downstream effector molecules whose activities are controlled by phosphorylation
and dephosphorylation (Fig. 1)2-4. Although many of the downstream target proteins
have been identified we have only a rudimentary understanding of the assembly of
signaling networks controlling growth and differentiation; and the details about how
Tyr as well as Ser/Thr phosphorylation confer their unique biochemical functions to
downstream effector molecules are almost entirely unknown. This is mainly due to
the fact that the quantitative assessment of overall changes in protein
phosphorylation has not been possible until recently. However, obtaining knowledge
about the quantitative phospho-proteome is vital for a better understanding of the
mechanisms controlling cell growth, differentiation and specification, and for the
identification of new molecular targets against which specific therapies can be
designed in human disease. Recently developed mass spectrometry-based
technologies (MS) are now available that allow for the quantitative assessment of the
phospho-proteome from living cells and organisms. Therefore, the major goals of the
proposed project are (1) to develop and improve MS-based phospho-proteomics that
can be applied to analyze the dynamic and reversible phosphorylation
/dephosphorylation events that are required for cell growth and differentiation in
different model systems, (2) to use these data to model and predict signaling
networks required for growth and differentiation in silico, and (3) to test the obtained
hypotheses in well-established model systems (such as cultured cells) and
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organisms (including Caenorhabditis elegans, Xenopus laevis, zebrafish and
Drosophila).
These efforts will be combined with a novel phosphopeptide library-based
proteomic screening technology5,6 that simultaneously: (1) reveals specific
pSer/pThr-binding domains downstream of kinases involved in regulating cell cycle
progression and cell differentiation; (2) allows determination of the optimal sequence
motifs recognized by the newly-identified domain; (3) provides reagents for
biophysical, cell biological, and structural studies of the function of the newly
identified domain in the context of cell signaling pathways, as well as reagents for
high-throughput screening of the domain for discovery of small molecule inhibitors;
and (4) facilitates bioinformatics and systems biology studies to identify downstream
targets of the domain that mediate cell growth and differentiation.
Input: cell stimulation
(Wnt, Shh, …)
Dephosphorylation
Phosphorylation
Functional Outputs:
Cell growth, differentiation
Fig. 1: In response to growth-stimulatory or growth-inhibitory signals, morphogens or
hormones, eukaryotic cells rely upon the timely activation and inactivation of several
families of protein kinases and phosphatases. Through mediating phosphorylation/
dephosphorylation of target proteins these enzymes regulate progression through the cell
cycle or cell cycle arrest, cell growth and differentiation, polarization and cell specification.
D. Background and Significance
Phosphorylation-Dependent Formation of Signaling Complexes. There is an
increasing appreciation that many critical cell signaling events occur within large
multi-molecular complexes. I.e., it has been estimated that about 500 protein
complexes contain all proteins of the yeast proteome. Both the assembly and
disassembly of these complexes, as well as their activity, most often appears to be
regulated by specific protein phosphorylation events. Historically, the
phosphorylation-dependent formation of multi-molecular signaling complexes was
first elucidated for growth factor tyrosine kinase receptors3,7,8. Following binding of
their respective ligands, the intracellular tyrosine kinase domains of these dimeric
receptors autophosphorylate the opposite receptor in trans, generating
phosphotyrosine-containing sequence motifs within the cytoplasmic non-kinase
segment. These phosphotyrosine motifs, in turn, recruit the phosphoTyr-binding SH2
2
domains of other intracellular proteins, forming large multi-protein signaling
complexes at the cytoplasmic tails of these receptor tyrosine kinases8. For many
years it was thought that phosphoprotein-binding domains such as SH2 domains
were unique to phosphoTyr, and that signaling events mediated by protein Ser/Thr
phosphorylation resulted from global conformational changes in the phosphorylated
protein. However, it is now more and more appreciated that Ser and Thr
phosphorylation, like Tyr phosphorylation, can directly assemble signaling complexes
through protein-protein interactions involving association of the pSer/pThr-containing
sequence motif in one protein with a pSer/pThr-binding domain in another protein9,10.
Some well-recognized phosphoserine/threonine-binding modules include 14-3-3
proteins, the proline isomerase domain and the WW domain of the mitotic regulatory
protein Pin1, Forkhead Associated (FHA) domains and WD40 repeats 9,10. In many
cases, these pSer/pThr-binding domains are specifically involved in regulating cell
cycle progression and the response of cells to morphogens. Members of this
consortium of investigators have extensive experience in the analysis of protein
function and phosphorylation in cell specification and differentiation (Baumeister,
Benzing, Walz, Driever, others). Canonical kinase signaling pathways that they have
studied in detail include among others different MAP kinase pathways11-13, PI3 kinase
signaling14 as well as canonical and non-canonical Wnt signaling pathways15 and the
activation of atypical protein kinase C16. Furthermore, the groups of Timmer and
Backofen have extensive experience in the data-based derivation of models for the
dynamics of cellular processes. This includes methods for experimental design and
estimation of parameters, testing of model and inference of system properties. In
order to setup mathematical models of kinase signaling networks that are activated
through morphogens and growth factors and in cell differentiation quantitative data
on the phosphorylation status of the proteome will be supplemented with kinetic
parameters for the individual interactions derived from the phospho-peptide library
screens5,6.
Thus, the foundation of this proposal is a highly interdisciplinary collaboration
that brings together developmental and molecular cell biology with biochemistry,
molecular medicine, biophysics and computer science. The investigators have a
proven record of successful collaboration; and integration of these distinct but
complementary disciplines is of central importance to our systems-level, networkbiology approach. As described above, we will attack the major goals of the proposed
project (development of MS-based phospho-proteomics that can be applied to
analyze the dynamic and reversible phosphorylation /dephosphorylation events; data
analysis to model and predict signaling networks required for growth and
differentiation in silico; experimental testing of the obtained hypotheses in wellestablished model systems) in direct interaction of these disciplines at the Center for
Systems Biology (ZBSA) in Freiburg. We expect that this unique combination of
methodologies will certainly allow us to successfully address kinase signaling
networks controlling cell growth and to develop a better understanding of the
quantitative phosphorylation events governing cell differentiation.
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