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5.1. Towards a quantitative understanding of kinase signaling networks in growth and
differentiation.
Background and Significance
Protein phosphorylation affects 30% of the proteome by defining the functional and structural
plasticity of proteins. It is involved in a range of fundamental biological processes and controls the
dissemination of the information carried by hormones and other extracellular signals. Protein
phosphorylation regulates the pathways that transmit, encode and integrate the numerous external
and internal signals in different gene expression patterns and diverse physiological responses. It
determines for example (i) where and when enzymes become active, (ii) how signal transduction
pathways are spatially and temporally organized, (iii) when and where interactions with scaffold
and anchoring proteins occur in distinct subcellular environments, (iv) whether movement of
proteins into and out of these complexes occurs and thereby (v) temporally regulate numerous
signaling events. In response to growth-stimulatory or growth-inhibitory signals, morphogens or
hormones, eukaryotic cells rely upon the timely activation and inactivation of several distinct
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 specification3.
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 and downstream effector
molecules whose activities are controlled by phosphorylation and dephosphorylation (Fig. 1)2,4,5.
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
organisms6-9. A detailed molecular description of such networks using these novel technologies in
combination with mathematical modeling is likely to (i) reduce the complexity of these networks, (ii)
result in an understanding of these networks at a systems level and finally (iii) predict the outcome
of perturbations1.
Members of this consortium are studying the role of phosphorylation in cell specification and
differentiation in animal and plant cells. Animal and plant cells show major differences in the types
of kinases. For example, receptor kinases in plants phosphorylate Ser and Thr residues, whereas
in animals, the predominant receptor kinases phosphorylate Tyr residues. In addition, the two
major types of kinases that decode calcium signals in animals (calmodulin-dependent protein
kinases and protein kinase C) appear to be missing or under-represented in plants. Conversely,
plants contain a number of kinase families that either are not found in animals or yeast or are
highly divergent or largely expanded (e.g. receptor like kinases belong to a large gene family with
more than 600 members in Arabidopsis). Judging from the need for intricate communication
networks and the need to respond to a multitude of environmental factors in multicellular
organisms, the abundance of specific groups of kinases represents specific adaptations for
extracellular signal sensing and propagation.
Canonical kinase signaling pathways that have been studied in animals include among others
different MAP kinase pathways (Benzing, Reth, Baumeister)17-22, PI3 kinase/AKT signaling23,24
(Benzing, Baumeister), tyrosine kinase pathways (Reth, Benzing)25-27 as well as canonical and
non-canonical Wnt signaling pathways (Benzing)28. Palme, Reski and Schäfer have studied
phosphorylation-dependent regulatory mechanisms in plants that will now lead to a dynamic
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characterization of entire phosphoproteomes in differentiating plant cells29-34. Timmer and
Backofen have extensive experience in the data-based derivation of models for dynamics of
cellular processes37-42. Thus, the foundation of this proposal is a highly interdisciplinary
collaboration that brings together developmental and molecular cell biology with biochemistry,
molecular medicine, plant physiology, biophysics and computer science. These investigators have
a proven record of successful collaboration; and integration of their distinct but complementary
disciplines is of central importance to a systems-level, network-biology approach. In this approach
functional proteomics will be an essential component to obtain qualitative and quantitative data on
the identity of the proteins, their levels and the dynamic changes occurring by modification. 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 well-established model systems; identification
of systems properties) will allow to successfully study kinase signaling networks controlling cell
growth in animals and plants and to develop a better understanding of the quantitative
phosphorylation events governing cell differentiation.
Specific Aims
This project will generate an internationally competitive unique research platform for
phosphoproteomics within ZBSA. Support for instrumentation (i.e. advanced, high performance
mass spectrometer) is requested, 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, (3) to test the obtained hypotheses in well-established model
systems (such as cultured cells) and organisms (including Caenorhabditis elegans, Xenopus
laevis, Arabidopsis thaliana, Physcomitrella patens, zebrafish and Drosophila melanogaster), and
(4) to derive the design principles of these networks in terms of regulatory mechanisms,
modularity, hierarchy, and robustness.
These efforts will be combined with a novel phosphopeptide library-based proteomic screening
technology10,11 that has been developed and improved by members of this consortium (Benzing) to
simultaneously (1) reveal specific pSer/pThr-binding domains downstream of kinases involved in
regulating cell cycle progression and cell differentiation, (2) allow determination of the optimal
sequence motifs recognized by the newly-identified domain, (3) provide 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) facilitate bioinformatics and systems biology studies
to identify downstream targets of the domain that mediate cell growth and differentiation.
Specific topics that will be studied include:
1) Hierarchical phosphorylation events in canonical and non-canonical Wnt signaling
networks in vertebrates
Wnt proteins are secreted glycoprotein ligands that regulate critical aspects of development,
including cell proliferation, differentiation, and cell fate43. Wnt signaling regulates these diverse sets
of processes through activation of networks of proteins that together regulate the activity of the
scaffolding protein Dishevelled44,45. Several divergent cellular pathways exist including the
canonical Wnt/beta-catenin signaling46, the non-canonical or planar cell polarity signaling
pathway47,48, and the Wnt-Ca(2+)/cyclic guanosine monophosphate pathway49. All these pathways
seem to require Wnt-mediated activation of its cellular receptor, a member of the superfamily of Gprotein-coupled receptor Frizzled family, heterotrimeric G proteins and the phosphoprotein
Dishevelled. A molecular understanding of the roles of Dishevelled proteins in development is
evolving and most recent observations suggest that Dishevelled proteins act as scaffolds essential
for Wnt signaling providing docking sites for a diverse and interesting set of protein kinases,
phosphatases, adaptor proteins, G proteins, and other proteins such as Axin43. Although the
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components of the canonical Wnt signaling pathway including glycogen synthase kinase 3 (GSK3), casein kinase 1 (CK-1), beta-catenin, and TCF/LEF transcription factors have been studied in
detail, the phosphorylation-dependent signaling networks downstream of Dishevelled in noncanonical pathways are less well understood. The Benzing/Walz labs have recently identified a
family of proteins that serves as a molecular switch to block canonical Wnt signaling, and promote
non-canonical Wnt activation, a pathway that diverges at the level of Dsh28. Non-canonical Wnt
signaling requires activation of c-jun N-terminal kinase (JNK) and Rac/Rho GTPases, and involves
re-localization of Dsh to the plasma membrane. Using phosphoproteomics technologies we will
now study the quantitative changes in protein phosphorylation of downstream components that are
required for both the canonical as well as the non-canonical Wnt signaling pathways. To setup
mathematical models of kinase signaling networks that are activated through Wnt-induced cellular
activation 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
screens10,11. Since non-canonical Wnt signaling has been shown to enforce the asymmetric
distribution of core proteins (including frizzled, Dsh, prickle, strabismus, flamingo, diego) at the
plasma membrane in a plane perpendicular to the apical-basal axis, a process called planar cell
polarity, we will use Drosophila and zebrafish (Driever lab) to test our models derived from the
systems biology approach with a focus on planar cell polarity.
2) Quantitative phosphorylation responses in cell differentiation in the moss Physcomitrella
patens
In the group of Ralf Reski, a highly sensitive method for the isolation and identification of
phosphoproteins has recently been established in the moss Physcomitrella patens29 and is
currently successfully applied to the analysis of signal transduction pathways. The merits of this
experimental system lie within the feasibility of highly efficient gene targeting in Physcomitrella, a
feature being its unique characteristics among plants30. Hence, specific genetic manipulation of
identified or predicted phosphosignaling network systems will elucidate their functional implication
in fundamental cellular processes like cell cycle control, apoptosis and differentiation events.
As stated above kinase signaling is of critical importance in the plant kingdom. These signal
transduction elements, however, appear to be substantially different from those of animals. Thus,
an uncritical transfer of paradigms derived from research on yeast or mammals towards signaling
mechanisms in plants is prone to providing rather inaccurate and misleading results. To date, large
scale phosphoproteomics studies on plants are still exceptionally few. Thus, for example, while the
impact of MAP kinase cascades on numerous stress signal transduction pathways in plants is
widely recognized54, the mechanisms underlying MAP kinase activation are largely unknown,
Furthermore, information about phosphorylated substrates of plant MAP kinases is still lacking and
only two studies have described the identification of their in vivo target proteins55,56. Progress in the
field of phosphorylation-dependent regulatory mechanisms in plants will therefore depend crucially
on the identification and dynamic characterization of entire phosphoproteomes which is part of this
proposed project. In this project we will focus towards understanding of the mechanisms by which
internal and environmental (abiotic and biotic) factors govern Physcomitrella development and
control adaptive responses. This will ultimately path the way towards novel strategies in crop
improvement and protection.
3) Phosphosignaling networks required for cell differentiation and polarization in
Arabidopsis thaliana
In the group of Palme components of phosphosignaling networks have been identified which
regulate asymmetric subcellular localization of proteins at the plasma membrane34,57-63. Kinases of
the widely known AGC family act as a reversible switch that regulate PIN protein localization. In
cells where the PID kinase is above threshold levels, PIN is targeted to the apical membrane,
whereas in cells lacking PID, PIN proteins accumulate in the cellular basal membrane. Induction of
PID in Arabidopsis leads to a quick apical-basal redistribution of PIN, demonstrating the
importance of PID for cellular polarity. The availability of components of the protein complexes,
knowledge on the dynamic recruitment of certain partner proteins into these complexes and
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information on the role of dynamic modifications such as phosphorylation and ubiquitinylation or
hormone signalling will allow to determine the functional relationship amongst them. Through
quantitative determination of concentrations of proteins recruited into the functional complexes in
space and time it will be possible to generate mathematical models of this system. Furthermore, by
simulating and predicting new experiments we will be able to determine the role of certain proteins
in the different signalling outputs.
In this project we plan to determine the dynamic behaviour of molecular interactions of soluble and
membrane located protein kinases, GTPases and other cellular components using a range of
advanced microscopic methods to visualize protein networks without disrupting the cellular
integrity. Beside analysis in planta we will utilize Arabidopsis protoplasts. The protoplast system
will allows expression and analysis of proteins in high-throughput. This is required to build up a
dynamic in vivo regulatory network. In addition we will analyze the quantitative changes in protein
phosphorylation by mass spectrometric analysis. Results from both approaches will be used to
predict and correlate networks with biological function. This will enable us to study the complex
dynamic interactions in situ, at light microscopic resolution and obtain information on rates and
concentrations using biochemical and mathematical techniques.
4) Phosphorylation signalling for light-regulated development in Arabidopsis thaliana
In the group of Schäfer the phytochrome signalling network which controls photomorphogenesis is
studied. Phytochromes show a light-dependent nuclear transport64.This light-regulated intracellular
localization depends on the phosphatase PAP565. After nuclear import of the photoreceptors a
rapid formation of nuclear protein complexes occurs66. These complexes contain besides
phytochromes members of the bHLH transcription factor family. Of those PIF3 and others show a
very rapid proteasome dependent degradation66. Recently we could demonstrate that the
degradation of PIF3 is preceded by a rapid phosporylation, which is dependent on the physical
interaction of PIF3 with the phytochromes67. Not only PIF3 but also the photoreceptor phyA shows
a rapid degradation after activation. The degradation occurs both in the cytosol and the nucleus.
In this project we will analyse the photoreceptor dynamics and PIF3 dynamics( phosphorylation,
localization, complex formation and degradation). Based on the measurement of the single
reaction components the complex reaction model will be simulated and tested for robustness and
additional components
5) Rebuilt kinase signaling pathways for the study of signaling networks in vitro
Numerous intracellular signaling elements have been identified in recent years, but their spatial
and temporal relationship is poorly understood. Inside the cell signals are processed by a multitude
of proteins each of which has its own unique structural and regulatory features as well as different
kinetic parameters for signaling. To cope with this complexity, many approaches exist to generate
mathematical models of signaling networks. However, a model is only as good as it can be used to
make clear predictions about the outcome of a signaling process inside the cell. The Reth
laboratory has developed a method that allows the reconstitution of mammalian receptors and their
signaling cascades in the genetically distant environment of Drosophila S2 Schneider cells. This
synthetic biology approach allows free choice and combination of different components of a
signaling pathway. As most cellular signaling processes require a membrane and a normal cellular
environment for their proper function, reconstitution of kinase signaling in synthetic biology
approaches cannot be done in a test tube. One solution to this problem is the rebuilding of a
mammalian signaling pathway in a cell that lacks most components of this pathway. Drosophila S2
Schneider cells are fulfilling these requirements50. These cells can be efficiently cotransfected in a
transient manner with up to 15 plasmids each of which carrying a different gene of a signaling
pathway51. The transient transfection method used in the S2 reconstitution system allows to
analyze in a short time frame many mutants of given signaling proteins. Another advantage of the
S2 cell system is, that any Drosophila protein that may interfere with the rebuilt signaling pathway,
once identified, can efficiently be eliminated by the RNAi technique, which works very efficiently in
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S2 cells52. The read-outs in this experimental system are protein kinase activation, substrate
phosphorylation, protein-protein interaction and signaling complex assembly53. Furthermore, by
controlling the protein expression and function in an inducible fashion modification of the kinetic
and quantitative parameters of signaling pathways is feasible.
At present most signaling studies by genetic means are done by the generation loss of function
mutants via RNAi or knock-out technology. These approaches are very important to identify the
functional importance of a given signaling element or a given signaling pathway. However, the
mechanistic and quantitative aspects of signaling pathways as well as the critical feedback loops,
which critically determine their outcome, are hard to study by these loss-of-function approaches
alone. Therefore, in this proposed part of the project these studies will be complemented with a
synthetic biology and reverse engineering approach that will also be used to verify or falsify
existing mathematical models of kinase signaling.
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