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Identification of Crosstalk in Insulin pathway using

Pathway Logic
Azmat Ali Khan1, Jamil Ahmad2
1,2

Research Center for Modeling and Simulation


NUST
Islamabad, Pakistan
azmat.ali@rcms.nust.edu.pk, jamil.ahmad@rcms.nust.edu.pk

AbstractSignaling Pathways regulate several processes in


cells. Analysis of these signaling pathways is difficult because the
number of proteins/chemicals involved in signal transduction is
enormous. Moreover presence of crosstalk makes these pathways
so complex that analysis without some computational framework
is very difficult. Knocking out/mutation of crosstalk may affect
the regulation of some particular pathways. Pathway Logic (PL)
provides a platform where biologist can model and query the
signaling pathway using formal methods. First the known
interactions in Insulin pathway are modeled using PL and then
several crosstalk are identified.
key words Component, formatting, style, styling, insert.

INTRODUCTION
Cell signal transduction occurs when cell responds to some
external stimulus. This process involves number of chemical
modifications that form a cascade of information[9]. This
information propagates downstream through stimulation and
inhibition. This cascade has evolved over the time to show
divergence and the crosstalk between signaling pathways.
These signaling pathways control plethora of processes like
protein synthesis, lipid synthesis, cell growth and proliferation
etc.
Signaling pathways have been modeled using diverse
formalism. Ordinary Differential Equation (ODE) modeling is
widely used to capture the dynamic behavior of the process. A
set of ODEs are solved simultaneously to predict the behavior
in time space. This modeling approach provides very useful
insight in terms of concentrations of chemical species involved
in signaling. On the basis of concentrations, the relative role of
each chemical compound can be ascertained. ODE modeling
involves number of reaction rates and constants. These reaction
rates and constants are usually difficult to find and act as bottle
neck in this type of modeling.
Stochastic modeling involves relative probabilities of
reactions or population of chemical compounds. Main
drawback of this technique is that results vary somewhat with
the change in size of population of chemical compounds. Large
population of chemicals produce better results but at the same
time combinatorial explosion restricts a fairly good population
size.
Hybrid systems techniques are important for modeling
signaling pathways where one wants to capture both

continuous and discrete aspects. Large State space is generated


for hybrid automata thus making it difficult to work with larger
signaling pathways.
In rule base modeling the states are instantiated only when
they are available. This is the main advantage of this approach
as it can avoid the combinatorial explosion problem in large
models [2].
Pathway Logic is an approach to model signaling processes
based on formal methods enabling the symbolic analysis[4].
Pathway Logic is developed with the aim to provide biologist
with tools for computing and analyzing signaling pathways in
the way they informally think about the pathway. In this
approach signaling pathways are modeled using rewriting logic
language Maude [1] and are analyzed by the number of formal
tools like Petri net based query tool Pathway Logic Assistant
(PLA)[5][]. PLA using the Inter Operation Prototype (IOP) and
Maude framework provides a user interface that provides
visualiza8tion and interaction with the models written in
rewriting logic. In particular, PLA uses Petri nets which
provide graphical representations and tools for reachability
analysis interactively, displaying the results in a graphical
form[6].
Signaling pathways expressed in graphical form are
relatively easy to comprehend and appealing than mathematical
equations. These graphs give better insights into the pathway
than traditional textbook diagrams[7]. PLA provides several
ways to compute with a model. One can specify an initial state
to find a possible final state, or search for all states satisfying
some property. To find a pathway satisfying some temporal
logic property the Low Level Petri net Analyzer (LoLA)
model-checker can be used.
A Petri net model is a tuple (T, P, F) consisting of a set of
transitions T, and a set of places P and set of arcs F. Arcs
connect the places and transitions. Arcs are either directed from
the places to transition or from transition to places.
'A Petri subnet is a tuple (T, I ,G,A) consisting of a set of
transitions T, an initial marking I, a goal marking G, and an
avoids set A'[6].
A petri subnet is constructed by finding an initial marking
and reaching a state with the goals marked without marking an

avoid. They cannot be considered as proper Petri net

Fig.1. Sample Petri net


models because they do not have valid starting point for further
analysis.
INSULIN PATHWAY
Insulin is produced in beta cells of pancreas and is essential
for appropriate tissue development, growth, and maintenance
of whole body glucose homeostasis. Insulin regulates glucose
homeostasis by controlling the glucose output from liver and
uptake into muscles and adipose tissue. Insulin also affects
lipid metabolism. Failure to effectively regulate these processes
at normal concentrations of insulin is called insulin resistance.
Insulin resistance is presumably caused by some defect in
insulin signaling pathway.
Insulin signaling is comprised of a complex, integrated
network that control several processes. Insulin receptor are
linked to the activation PI3K-AKT pathway, which is
responsible for most of metabolic actions of insulin, and Rasmitogen-activated protein kinase (MAPK) pathway, which
control cell differentiation and growth. Moreover insulin
pathway includes Ras-related C3 botulinum toxin substrate
1(RAC1) that plays a critical role in processes, such as control
of cell morphology, transcriptional activation, and apoptosis
signaling. The Mammalian target of rapamycin(mTOR) is
another member of this pathway that is involved in a diverse
range of cellular processes.
For such complex system it is very important to identify the
crosstalk because the proteins mediating these pathways
perform diverse roles. Moreover knocking out of these
mediating proteins may affect the regulation of some particular
pathway. The identification of such proteins may also help
scientist prioritize their experiments, as they may focus on
these biological entities.
METHODOLOGY
First step in modeling of signaling pathways is to decide
the level of abstraction. The complexity of problem can be
reduced by including only those chemical entities that are
essential for the analysis. Too much detail makes the model
computationally inefficient as well as difficult to analyze the
results. Such models can take a long time to execute and may
suffer combinatorial explosion problem. For this study a list of

54 chemical entities and 34 relevant chemical modifications is


made. Only those entities are included in the list that are
essential for signal propagation or are essential for the analysis.
Insulin signaling pathway is modeled using MAUDE language.
For this study insulin pathway information provided by
national cancer institute of national Institutes of Health,
MaryLand, USA is combined with KEGG's pathway.
Second step is to express the chemical modifications in the
form of rewriting rules. Rewriting rules are algebraic structures
in which chemical entities are expressed as symbols and
chemical modifications are expressed as equations. It is
necessary that right hand side of equation is simpler than left
hand side. In this way MAUDE makes deductions. The
chemical entities and rules set is jointly called knowledge base.
Insulin receptor gets activated when insulin from outside the
cell gets attach to it. Rewriting rule for this process is of the
form:
[

]
[

[
|

|
[

]
]

This rule describes the binding of an Insulin ligand to


.
The term
is a variable that matches any
cell type.
|
matches any cell membrane
location that contains
, since
is a variable that will
match the rest of the membrane contents.

Fig.2. Insulin attached to insulin receptor


Third step is to convert these rewriting rules to set of
occurrences and transition knowledge base. Then this model
can be read by PLA where goal/avoid analysis can be carried
out. The constituents of the cell already present in the cell are
defined in initial state (dish). PLA construct a Petri Net from
dish and displays it in graphical form. Then goals can be set
and PLA perform a reachability query for the subnet that
contains all the transitions from initial starting point to goal.
Only those transitions are included that are necessary for the
signal propagation from receptor to goal. Other transitions are
ignored. The number of reachability queries can be made
producing relevant subnets. These subnets can be compared.
The results of comparison is also in graphical form where
common portion of two subnets is displayed in single colour

Fig.3. Complete Insulin Signaling Pathway

and distinct parts are shown in two different colours. The


usage of different colours makes the analysis very easy.
RESULTS AND DISCUSSION
To find crosstalk within insulin signaling pathway
following five proteins are taken as goal
A. HRAS
HRAS is involved in regulating cell proliferation and
growth. Always active state of HRAS leads to continuous cell
proliferation process resulting in cancer.
B. mTOR
The mTOR pathway is an intracellular signaling pathway
important in apoptosis and hence cancer. In many cancers this
pathway is overactive reducing apoptosis and allowing
proliferation[10].
C. Protein Kinase C(PKC)
PKC is directly involved in insulin mediated glucose
transfer.

D. RAC1
RAC1 regulate a diverse range of cellular processes,
including the control of cytoskeletal reorganization and protein
activation. It also plays a critical role in control of cell
morphology and apoptosis signaling.
E. Forkhead box (FOXO)
FOXO proteins are a family of transcription factors that
play important roles in regulating cell growth, proliferation,
differentiation, and longevity. Many FOXO proteins are
important to embryonic development.
Subnets are obtained for said goals and a careful
comparison is made. After examination of these comparisons
following crosstalk are identified.

Fig.4. Comparison of mTOR and RAC1 pathway


Fig.6. Comparison of mTOR and HRAS pathway

Fig.5. Comparison of HRAS and FOXO

Fig.7.Comparison of FOXO and mTOR pathway

A. Phosphatidylinositide 3-kinases(PI3K)
PI3K act as a crosstalk between mTOR and RAC1
pathways. Pi3K directly activates RAC1 whereas the activation
of mTOR takes place through two paths. Pi3K activates AKT1
that in turn activates mTOR. In second pathPi3K activates
AKT1 which triggers the activation of TSC and RhEB that
activates mTOR. This shows that AKT1 is involved in both
paths.

primarily in the muscles and generalized defect in body


growth[3] and preadiposytes have defects in differentiation[11]
.
mTOR and RAC1 pathways involve IRS3. IRS3 activates
PI3K/AKT/mTOR pathway directly by activating PI3K. RAC1

Fig.9. Comparison of PKC and FOXO pathway


Fig.8. Comparison of PKC and mTOR pathway
B. IR/IRS
In our study only two IRS proteins IRS1 and IRS3 are
involved. IR/IRS node act as crosstalk for following pairs of
targets.
PKC and RAC1
HRAS and FOXO
HRAS and mTOR
It is interesting to observe that IRS1 is involved in
pathways leading to PKC and HRAS. PKC activation by IRS1
involves CAP, APS, CbL, CRK2, C3G, TC10 and CIP4.
HRAS activation takes through GrB2 and SOS1. PKC is
known to be involved in insulin mediated glucose uptake and
HRAS acts like a switch that triggers several pathways one of
which is MAPK pathway that control the cell growth and
proliferation. Irs1 knockouct mice have defective insulin action

activation also takes place through Pi3K which is activated by


IRS3.
C. AKT (serinethreonine kinase/protein kinase B)
AKT acts as crosstalk between mTOR and FOXO. AKT
mediates
insulin
stimuated
protein
synthesis
by
phosphorylating TSC( tuberous sclerosis complex). RhEB and
mTOR are also involved in this process. FOXO is directly
activated by AKT. AKT is involved in number of actions that
all leads to anti-apoptosis and proliferation of cell.
D. PDK(Phosphoinositide-dependent kinase-1)
PDK acts as crosstalk between PKC and mTOR. It also
mediates PKC and FOXO. PDK directly activates the PKC and
hence play a role in glucose uptake. The activation of FOXO
by PDK takes place through two paths. In first path PDK
activates SGK1 that in turn activates FOXO. On the other hand
it activates AKT which in turn activates FOXO. It is interesting

to observe that IRS is second crosstalk for above mentioned


pair of pathways. The activation path of PKC by IRS1and that
of mTOR by IRS3 is already discussed. The activation of
FOXO by IRS3 takes place through PI3K and AKT1.
E. IRS1
IRS1 is a connection between PKC and HRAS. The
activation paths are already discussed above.
CONCLUSION
Pathway Logic provides an efficient framework for
modeling and analyzing signaling pathways. By using this
approach Biologist can work with signaling pathways with
ease. By using pathway logic, some crosstalk in Insulin
pathway are identified. By including more biological
proteins/chemicals in studied model of insulin pathway, further
insight is possible which may lead to effective treatment for the
ailments associated with insulin resistance. Pathway Logic
framework needs the functionality of identifying crosstalk
automatically.

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