Investigations into the Role of Protein Phosphatases in the EGFR
Signaling Network
jrUTE
MASSACHUSETTS INST
OF TEOHNOLOG-N
By
SEP 10 2008
Kuojung Gordon Lu
B.A. Molecular Cell and Biology
Universitv of California. Berkeley. 2002
I
LIBRARIES
Submitted to the Department of Biology in Partial Fulfillment of the Requirements for the
Degree of
Master of Science in Biology
at the
Massachusetts Institute of Technology
September 2008
© 2008 Massachusetts Institute of Technology
All rights reserved
Signature of Author:
Department of Biology
September 9, 2008
Certified by:
Visiting Prfessor of Biol
Accepted by:
-V
--
Pete K Sorger
ngineering
Thesis Supervisor
Stephen P Bell
Professor of Biology
Chairman, Graduate Committee
Aic gVE8 S
Investigations into the Role of Protein Phosphatases in the EGFR Signaling
Network
by
Kuojung Gordon Lu
Submitted to the Department of Biology on September 9, 2008 in Partial Fulfillment of the
Requirements for the Degree of Master of Science in Biology
ABSTRACT
Protein phosphatases regulate the phosphorylation state of intracellular signaling molecules in
conjunction with protein kinases. In order to better understand the role of phosphatases in the
ErbB signaling network, experimental studies were carried out to validate assays and
phosphatase inhibitors for gathering dynamic, system-wide data of phosphatase activity.
We verified the use of In-Cell Westerns and phospho-ErbB ELISAs for these studies, as well as
the use of different cell lines and both general and specific phosphatase inhibitors in system-wide
experiments. The phosphatase inhibitors we used perturbed cellular systems in complex ways
that can help elucidate the regulation and activity of specific phosphatases in the ErbB signaling
pathway in the future.
Thesis Supervisor: Peter Sorger
Title: Visiting Professor of Biological Engineering
Table of Contents
I.
Introduction....................................................
............................................... 4
1. M otivation ................................................................................................
...... 4
2. Background .............................................................................................
4........
3. Assay and Experim ental Design ................................................................ 10
II.
Materials and Methods .................................................. 14
1. Reagents ........................................................................................................
2. In-Cell W esterns ...................................................
............. 14
3. ELISA - Phospho-ErbB fam ily ..................................................................
15
4. Tim e Courses and IC50 Experiments......................................................
17
5. Cell Culture.................................................
III.
............
14
............................................ 18
Results .................................................................................................................
19
1. Optimization of using phosphatase inhibitors..........................19
2. Co-treating cells with phosphatase inhibitors together with protein
synthesis or kinase inhibitors ......... ....................................................... 26
3. Phosphatase inhibitors in phospho-ErbB ELISA Assays.....................29
IV.
Discussion ............................................................................................................ 31
1. Tools developed for studying phosphatase activity.............................. 31
2. Future work ................................................................................................. 37
V.
References .......................................................................................................... 38
VI.
Figure captions ................................................................................................... 43
VII.
Figures................................................................................................................. 45
I. Introduction
1. Motivation
Protein phosphorylation plays an important role in a variety of cellular functions. This
is especially true in the ErbB signaling network, where protein phosphorylation is involved in
signal transduction leading to various cellular outcomes such as proliferation, survival,
motility, and differentiation. The level of phosphorylation is regulated in part by the
opposing enzymatic activities of protein kinases and protein phosphatases. Thus, the activity
of protein phosphatases is crucial in the regulation of ErbB network signals. In order to
advance our understanding of the ErbB network, it is important to obtain quantitative
information of the specificity and regulation of various phosphatases at the receptor and
downstream levels. This is especially true in the context of aberrant signaling involved in
diseases such as cancer. A combination of specific inhibitors, cancer cell lines, and system
wide signaling assays can help elucidate the role of phosphatases in the ErbB signaling
network. Assays and tools were developed and validated for this purpose.
2. Background
System-wide experiments measuring intracellular signals will be used to investigate the
effect of perturbing the activities of various phosphatases with small molecule inhibitors.
These experiments will be conducted in a panel of tumor cell lines that represent different
human cancers, in order to capture similarities and differences in cellular behavior that can
then be explored in a computational model. Treatment of the cell lines with different ErbB
receptor ligands will stimulate downstream response pathways, and co-treatment with
phosphatase inhibitors will inhibit specific phosphatase nodes to help elucidate the role of
phosphatases in ErbB signaling pathways. The project will initially focus on the behavior of
the Erk and Akt pathways downstream of the ErbB signaling network, and the roles of the
protein tyrosine phosphatase (PTP) and protein phosphatase (PP) families of phosphatase
proteins.
The PTP and PP family of proteins have been shown to be crucial in many types of
cellular signaling systems (Hunter 2007). Phosphorylation and dephosphorylation of proteins
mediate many signal transduction events involved in ErbB signaling (Tiganis 2002; Tonks
2006). PTPs and PPs are groups of structurally diverse and complex enzymes that act in
collaboration with protein tyrosine kinases (PTKs) to modulate a broad range of
physiological processes (Alonso et al. 2004). Recent findings suggest that deregulation of
protein phosphatases are linked to various human disease states including cancer and diabetes
(Easty et al. 2006; Ostman et al. 2006; Zhang and Lee 2003). Thus, it should be interesting to
investigate the importance of these phosphatase families in cellular signaling networks. The
initial focus of the project will be on the PTP family member PTP1B and PP family member
PP2, as they are thought to be intimately involved in growth factor signaling networks and
have been to shown to be linked to human disease processes (Lee and Wang 2007; Perrotti
and Neviani 2008).
Protein Tyrosine Phosphatase 1B (PTP1B)
The PTPs are a large family of enzymes, over 100 of which has been characterized
thus far. They possess a 230-amino acid common catalytic domain, and a signature active site
motif sequence (H/V)C(X)SR(S/T) (Alonso et al. 2004; Mauro et al. 1994). PTPs work
together with PTKs to catalyze the addition and removal of phosphate groups on specific
substrates to regulate cell signaling, and contain a number of functional subunits that help
regulate their subcellular activity and function (Mauro et al. 1994; Yudushkin et al. 2007).
Some PTPs, such as phosphatase and tensin homolog (PTEN), are considered products of
tumor suppressor genes as they reverse the effects of RTKs and are inactivated by mutation
in multiple types of cancers (Li et al. 1997). Other PTPs, however, can also potentiate the
actions of PTKs, leading to cellular transformation. Examples of these include SHP2,
involved in growth factor-mediated, Ras-dependent cellular transformation, and PTPa,
involved in Src activation (Bentires-Alj et al. 2004; Zheng et al. 1992). This demonstrates
that PTPs are involved in the complex regulation of cellular networks and are potential drug
targets in aberrant signaling (Jiang and Zhang 2008).
PTP1B is a widely expressed family member that is a 435 amino acid, 50 kDa protein
localized to the endoplasmic reticulum (Chernoff et al. 1990). It has the ability to
dephosphorylate the EGFR, PDGFR, and insulin receptor (Bourdeau et al. 2005). PTP1B has
been shown to be involved in insulin receptor mediated pathways, and is being investigated
as a potential target in treating type 2 diabetes and obesity (Klaman et al. 2000). More
recently, genetic studies of PTP1B have linked it to breast and colon cancers (Julien et al.
2007; Bentires-Alj and Neel 2007; Zhu et al. 2007). PTP1B is overexpressed along with
HER2 in 90% of all breast tumors (Wiener et al. 1994). It has been found that PTP1B-' mice
are protected against HER2-induced breast tumors and have down-regulated pERK, while
PTP1B /' fibroblasts show slightly up-regulated pEGFR and pERK following EGF
stimulation (Bentires-Alj and Neel 2007; Haj et al. 2003).
As there are a number of findings establishing PTP1B as a potential therapeutic
target, there has been an effort to develop specific and potent PTPIB small molecule
inhibitors. However, this has been difficult due to the conserved nature of the PTP active site
motif (Jiang and Zhang 2008). One strategy involves the binding of an allosteric site away
from the common PTP motif, stabilizing an inactive conformation of PTP1B. This resulted in
a compound that is highly selective for PTP1B, potent (IC50 = 8 uM), and cell permeable
(Wiesmann et al. 2004). This compound is commercially available from Calbiochem (see
materials section) and is used to perturb and investigate PTP1B function in the current study.
There have also been additional, more recent efforts to develop even more potent PTP1B
inhibitors, using a strategy involving bidentate ligands that bind both the active site and a
unique adjacent peripheral site. This has resulted in cell permeable compounds with both
high PTP1B selectivity and IC50s in the nM range (Boutselis et al. 2007; Combs et al. 2006;
Lee and Wang 2007). These compounds could be interesting for future investigations of
PTP1B function in ErbB signaling.
Protein Phosphatase 2A (PP2A)
Serine/threonine protein phosphatases are grouped into two families, the
phosphoprotein phosphatases (PPP) and metal-dependent protein phosphatases (PPM). The
PPPs and PPMs share a common ancestor gene, and are classified based on their metal-ion
dependence, substrate sensitivity, and sensitivity to inhibitory molecules (Lechward et al.
2001). PP2A belongs to the PPP family, and is widely expressed in all eukaryotic organisms.
It is a hetero-multimeric holoenzyme consisting of a common catalytic subunit and a large
group of regulatory and adaptor proteins (Virshup 2000). Upon association with different
subunits, PP2A can dephosphorylate a wide range of substrates and is involved in many
cellular processes, including cell cycle regulation, DNA replication and transcription,
survival, and the MAPK, Akt, and mTOR pathways (Zolnierowicz 2000).
PP2A regulates and mediates processes downstream of growth factors and is involved
in the ErbB signaling network. PP2A can dephosphorylate MEKi and ERK, down-regulating
mitogenic signals (Gomez and Cohen 1991). It can also activate Raf-1 through
dephosphorylation of an autoinhibitory residue and may be involved in ERK-induced
feedback on Raf-1 (Dhillon et al. 2007). The PP2A holoenzyme, depending on regulatory
unit composition, can also directly dephosphorylate Akt or act downstream of PI3K/Akt by
inhibiting the mTOR pathway (Ivaska et al. 2002; Peterson et al. 1999). In addition, mutation
or aberrant expression of PP2A subunits have been found in a number of human cancers,
including breast cancer and leukemia (Perrotti and Neviani 2008). Thus, PP2A is an
important target of study for better understanding the ErbB network in the context of aberrant
signaling. For the perturbation of PP2A function, there are also many cell permeable PP2A
inhibitors available commercially, such as okadaic acid, microcystin, and endothall
(Zolnierowicz 2000).
Erk and Akt
For initial characterization of the output of the ErbB network following ligand
stimulation, we will be measuring the levels of phosphorylated Erk (pERK) and
phosphorylated Akt (pAkt). Aberrant signaling in the Erk and Akt pathways have been
implicated in the growth and proliferation of many types of tumors (Roberts and Der 2007;
Dillon et al. 2007). Erk and Akt pathways have also been used as downstream output
readouts in modeling cellular behavior (Schoeberl et al. 2002; Hatakeyama et al. 2003).
Mitogen-activated protein kinases (MAPKs) are involved in a wide range of key
cellular processes such as differentiation, cellular growth, adaptation to stress, and survival,
in response to stimuli such as growth factors, cytokines, adherence, and stress (Roux and
Blenis 2004). The p44/42 MAPK ERK(1/2) is the downstream MAPK in the three-kinase
pathway consisting of Raf, MEK, and ERK (Rubinfeld and Seger 2005). Growth factors such
as EGF and PDGF can activate ERK through the MAPK kinases MEKI and MEK2, and
through ERK effect cellular responses such as proliferation and differentiation (Chang and
Karin 2001). MEK1 and MEK2 activate ERK through dual phosphorylation of activation
loop residues Thr182 and Tyr184, and phosphorylation of these residues can be used as a
readout of ERK activity (Widmann et al. 1999; Murphy and Blenis 2006).
The phosphatidyl inositol 3-kinase (PI3K) pathway is also involved in a number of
cellular processes such as cell growth, proliferation, and survival (Franke et al. 1995). Protein
kinase B, or Akt, are a 3-member serine-threonine kinase family that is the primary
downstream mediator of the PI3K pathway and affects various downstream processes by
phosphorylation of downstream substrates (Franke 2008). The PI3K pathway can be
activated by insulin and various growth factors such as IGF and EGF, or the integrin receptor
pathway, to promote cell survival and proliferation by inhibiting apoptosis through the
activity of Akt (Zhou et al. 2000). Dysregulation of the PI3K pathway, through mutation of
its components such as PTEN, is often found in human cancers (Dilton et al. 2007). Akt can
be activated by recruitment to the membrane via various scaffolding proteins and
phosphorylation of activating residues Thr308 and Ser473 by phosphoinositide dependent
kinase 1 (PDK1) and mTOR, and phosphorylation of those residues can be used as a
functional readout of Akt activity (Yang et al. 2004; Song et al. 2005).
3. Assay and Experimental Design
In the current project, we will focus on using phosphatase inhibitors in parallel with
ligand stimulation to perturb the activity of PTP1B and PP2A, to deduce quantitative
information about their function in the ErbB network. In order to obtain a large dataset for
the purpose of modeling and predicting the role of these phosphatases, we will develop
further an existing In-Cell Western (ICW) based assay system that will be able to
quantitatively measure signaling dynamics in a high-throughput and reproducible manner.
We will also use this assay system to study the signaling dynamics specifically involved in
cellular response to phosphatase inhibitors and collect a dataset for future extension and
construction of a predictive dynamic model using existing ODE models.
The signaling assays used will look at molecules that are known to be important in
system behavior from previous studies and computational models, specifically phosphoErbBs 1-4, phospho-Erk, and phospho-Akt. The phosphorylation status of receptors will be
measured using total- and phospho-receptor ELISAs, while downstream and intracellular
molecules will be measured using ICW, on the LiCor Odyssey infrared imaging system. The
ICW assay allows quantitative, dynamic signaling data to be gathered in a high throughput
fashion. ICW requires smaller reagent quantities and less biological samples compared to
traditional assays. It is also a relatively simple protocol that requires almost no pre-
processing of the samples and consists of few wash and staining steps, when compared to
kinase activity assays or traditional western blots. The ICW protocol allows cells to be
seeded, grown, treated with cue, stained, and visualized in the same 96 well plate and reduces
the number of steps required for processing samples. Finally, the use of dyes in the far-red
spectrum allows for visualization with low signal overlap and high intensity, and a DNA
stain is used to normalize for cell number variation in each well.
The phosphorylation status of receptors and downstream molecules will serve as a
proxy for their activation state. Since it is not clear what the best metric for measuring
receptor activation is, both fractional phosphorylation as a functional of all receptors and
absolute phosphorylated receptor numbers will be measured. Intracellular signaling assays
will initially use previously worked out protocols in the lab for the measurement of phosphoErk and phospho-Akt using ICW.
In order to investigate and capture differential cell signaling behavior, a combination of
different ErbB ligands, phosphatase inhibitors, downstream inhibitors, tumor cell lines, and
experimental designs will be used.
We will perform time course experiments of co-treating available cell lines, such as
H1666, H3255, A431, SKBR3, and HeLa with ligand and phosphatase inhibitors to look at
dynamic activity of phosphatases. The following types of time course experiments can be
informative. A) Treating cells with ligand in parallel with general phosphatase inhibitors,
such as vanadate, will demonstrate the overall contribution of phosphatases to immediate
signaling following ligand stimulation. B) Using specific phosphatase inhibitors, like okadaic
acid, PTP1B inhibitor, endothall, dephostatin, and menadione, will demonstrate the
specificity and activity of a particular phosphatase on specific network nodes such as MEK
and ERK. This will be more informative in the context of the current ErbB model. C) Cotreatment with phosphatase inhibitors and specific inhibitors of particular nodes, such as
tyrosine kinase inhibitors (TKIs), a MEK inhibitor, or a PI3K inhibitor, will stop additional
signaling downstream of that node and demonstrate how a particular phosphatase regulate
that node. An example of this could be to treat cells with heregulin (HRG), then add a PI3K
inhibitor and a PTP1B inhibitor 10 minutes post ligand stimulation, and follow the Akt time
course to see the effect of PTP1B on the PI3K/Akt pathway. Adding phosphatase inhibitors
at different times (Pre-incubate, co-treat with Ligand, post-ligand stimulation) and in
different combinations (phosphatase inhibitor alone, phosphatase inhibitor plus ligand,
phosphatase inhibitor plus ligand plus node inhibitor) can help identify the contributions of
different phosphatases on particular network nodes.
Another type of experiments could be using different concentrations of phosphatase
inhibitors to find the IC50s of phosphatases in an intracellular context. Comparing the IC50s
of phosphatase inhibitors on basal versus ligand-stimulated cells demonstrates the basal
activity of phosphatases and can help parse out the contribution of each phosphatase to each
network node. This is analogous to an EC50 experiment, but instead of ligand stimulation,
the stimulus is the inhibition of endogenous phosphatase down-regulation of a particular
network molecule.
Phosphatases acting on a node reduce activated node species and can lower the in
vivo IC50 compared to in vitro measurements (Knight and Shokat 2005). Measuring TKI
IC50s in the presence of phosphatase inhibitors can confirm the specificity of a phosphatase
for the TKI target. This can be done with combinations of TKIs specific for different ErbB
family members to show the regulation of receptor dephosphorylation. A specific example
for this type of experiment could be elucidating the functional mechanism for erlotinib
inhibition of HRG-stimulated signaling. In Schaefer 2007, it is proposed that erlotinib is
binding directly to ErbB2 and inhibiting signaling from ErbB2 homo- and hetero-dimers.
This is unexpected given that in vitro experiments suggest erlotinib is 40-3000 fold more
specific for ErbB1 than ErbB2. One hypothesis is that ErbB 1 and ErbB2 have differential
phosphatase regulation in vivo, resulting in a higher apparent Ki of erlotinib for ErbB2. This
can be tested with IC50 experiments looking at the effect of using phosphatase inhibitors in
parallel with HRG stimulation on the phosphorylation states of ErbB1 and ErbB2.
II. Materials and Methods
1. Reagents
Antibodies and Stains, ICW:
Phospho-Erk, Thr202/Tyr204, Rabbit monoclonal 197G2, Cell Signaling Technology,
#4377, 1:100
Phospho-Akt, Ser473, Rabbit monoclonal 193H12, Cell Signaling Technology, #4058, 1:100
Phospho-EGFR, Tyr1068, Rabbit polyclonal, Cell Signaling Technology, #2234, 1:100
800CW IR-Dye Goat anti-Rabbit, Rockland, 1:800
Topro-3 Iodide DNA stain, Molecular Probes, 1:5000
Ligands:
Epidermal growth factor, recombinant human, PeproTech, #100-15
Heregulin, recombinant human HRG1b, PeproTech, #100-03
Amphiregulin, recombinant human, R&D Systems, #262-AR-100
Inhibitors:
PTP1B Inhibitor, Calbiochem, cat. no. 539741
Okadaic Acid, Prorocentrumsp., InSolution, Calbiochem, cat. no. 495609
Sodium Orthovanadate, Calbiochem, cat. no. 567540
2. In-Cell Westerns
The goal of our work is to quantitatively monitor the activity of proteins that contribute to the ErbB signaling cascade. For this purpose, In-Cell Western (ICW) assays were
previously developed to monitor the phosphorylation of phosphor-ERK and phospho-AKT.
The ICW assay provides a straightforward means of collecting multiple time or treatment dependent data points.
The ICW assay involves the following steps. Cells are seeded in 96 well optical plates
at 200 uL/well and grown overnight. The time course or inhibitor experimental treatments are
performed on the cells in the same plate. Cells are then fixed with 100 uL/well of 4%
formaldehyde at room temperature for 20 minutes. The formaldehyde is then aspirated, and
the cells are permeabilized with 4 washes of 0.1 % Triton in PBS, at 5 minutes for each
wash, on a rocker. The plates are then blocked with 100ul/well of Odyssey Blocking Buffer,
for I hour, with shaking. The Odyssey Blocking Buffer is then aspirated, and 50 uL of the
appropriate primary antibody diluted in Odyssey Blocking Buffer is added to each well. The
plates are sealed with parafilm and placed at 4oC overnight.
The next day, the primary antibodies are aspirated, and the plates are washed with 200
uL/well of 0.1 % Tween in PBS, for 5 washes of 5 minutes each, on a rocker. The final wash
is aspirated and replaced with secondary antibody at 1:800 dilution plus Topro-3 DNA stain
at 1:5000 dilution in 100 uL of Odyssey Blocking Buffer. The plates are covered with foil to
protect from light and are incubated for I hour at room temperature with rocking. The plates
are then washed again with 200 uL/well of 0.1 % Tween in PBS, for 5 washes of 5 minutes
each, on a rocker. The last wash is aspirated thoroughly to ensure each well has no liquid remaining. The bottoms of the optical plates are then cleaned with 70% ethanol, and the plates
are scanned on an Odyssey Infrared Scanner immediately. The resulting images were ana-
lyzed using Odyssey software. The phospho-signal is read out as a ratio to the DNA stain intensity to normalize for cell number.
3. ELISA - Phospho-ErbB family
For the measurement of phosphorylated receptor levels, we used the Phospho-EGFR,
Phospho-ErbB2, Phospho-ErbB3, and Phospho-ErbB4 DuoSet ELISA kits from R&D
Systems (DYC1095, DYC1768, DYC1769, and DYC2115). In separate wells, immobilized
capture antibodies specific for EGFR, ErbB2, ErbB3, and ErbB4 bind both phosphorylated
and unphosphorylated forms of the proteins, and an horseradish peroxidase (HRP)
conjugated detection antibody for phosphorylated tyrosine is used to detect the
phosphorylated form of the proteins.
For the ELISA, cells are plated and grown overnight. Time course or IC50
experiments are performed, and then stopped with PBS at 4VC. The cells are rinsed with PBS
and solubilized with lysis buffer #9 (R&D systems), to a concentration of 1x10 7 cells/mL.
The cells are then incubated at 4VC on a rocker. The lyrsates are then microcentrifuged at
14,000g for 5 minutes, and the supernatant is transferred and frozen at -20 0 C until use.
To prepare the ELISA plates, a 96-well plate was coated with 100 uL/well of capture
antibody at 0.8 ug/mL (pEGFR), 4.0 ug/mL (pErbB2), 4.0 ug/mL (pErbB3), or 1.0 ug/mL
(pErbB4) in PBS. The plates are sealed and incubated overnight at room temperature. The
following day, the wells are aspirated and washed 5 times with 0.05% PBS-Tween. The
plates are then blocked with 300 uL/well of 1% BSA in PBS for 1 hour. The wells are then
washed again, as before. 100 uL of sample lysate in IC Diluent #12 buffer (R&D Systems,
DYC002) was added and incubated for 2 hours at room temperature, with appropriate blanks.
The wash step is repeated, and 100uL of HRP-conjugated detection antibody is added and
incubated for 2 hours at room temperature. The plates are washed again, and 100uL of a 1:1
mixture of H20 2 + Tetramethylbenzidine (R&D Systems, DY999) is added as substrate and
incubated for 20 minutes at room temperature. 50uL of 2N H2SO 4 is then added to stop the
reaction, and the ELISA plates are read with a fluorescent plate reader (Spectramax Gemini)
at 450 nm, with wavelength correction set at 540 nm. IC Diluent #12 buffer was used as
blank and subtracted from the readings from each well.
4. Time Courses and IC50 Experiments
Optical 96 well plates (for ICW) or tissue culture treated 12 well plates (for ELISA)
were seeded with cells (see tissue culture for numbers) in 200uL total volume of media. Edge
wells were not seeded and 200 uL PBs was added to those wells to maintain uniform plate
conditions. Semi-permeable membranes were used to seal plates to prevent evaporation, and
the cells were incubated overnight at 37 0C. Prior to starting the experiments, the growth
media was aspirated and the cells were serum-starved with 100 uL of media containing 0.1%
fetal bovine serum for 12-14 hours.
For experiments that included inhibitors, the media from the wells was aspirated and
replaced with 100 uL of serum-free media containing the correct concentration of the
particular inhibitor, and returned to the incubator for the inhibitor pre-treatment period (see
results section for pre-treatment times). For time course experiments, plates were removed
from the incubator 15 seconds before each time point. 100 uL of serum-free media
containing ligand at 2x concentration was added to the corresponding time point wells such
that the final concentration of ligand is lx. The plates were then returned to the incubator.
The longest time points were treated first, then the next longest time point, and so on until the
zero time point, at which time the entire plate was stopped at once. For ELISA experiments
the plates were stopped with 40 C PBS and placed on ice while the other plates are being
treated, and for ICW experiments the media from all wells was replaced with 150 uL 4%
formaldehyde in PBS and incubated for 20 minutes at room temperature. For IC50
experiments, the wells were treated with different concentrations of inhibitor for the same
amount of time, and then stopped at the same time.
5. Cell culture
HeLa cells were maintained in DMEM supplemented with 10% fetal bovine serum
and glutamine. For ICW experiments HeLa cells were plated at 15,000 cells/well in 96 well
plates. For ELISA experiments HeLa cells were plated at 50,000 cells/well in 12 well
plates. H1666 cells were maintained in ACL-4 medium supplemented with 10% fetal bovine
serum. For ICW experiments H1666 cells were plated at 15,000 cells/well in 96 well plates.
For ELISA experiments H1666 cells were plated at 50,000 cells/well in 12 well plates.
H3255 cells were maintained in ACL-4 medium supplemented with 10% fetal bovine serum.
For ICW experiments H3255 cells were plated at 10,000 cells/well in 96 well plates. A431
cells were maintained in DMEM supplemented with 10% fetal bovine serum and glutamine.
For ICW experiments A431 cells were plated at 15,000 cells/well in 96 well plates. Cell
culture conditions and optimal seeding numbers were determined from previous experiments
(data not shown).
III. Results
1. Optimization of using phosphatase inhibitors
PTP1B Inhibitor, Endothall, Okadaic Acid - Time course
In a first-pass experiment, we measured the response of inhibitor pre-treated H1666
cells following EGF and HRG ligand stimulus, over a time course of 60 minutes, with time
points at 0, 5, 10, 15, 30, and 60 minutes (see methods section for time course experimental
design). The cells were pre-treated for 5 minutes with the PTP1B Inhibitor at 100uM,
Okadaic acid at 100nM, and Endothall at 100uM. The H1666 cells were then stimulated at
time zero with saturating ligand concentrations, with EGF at 100ng/mL, and HRG at 50
ng/mL. We sampled the response using intracellular signals for phospho-EGFR, phosphoERK, and phospho-AKT (Figure 1).
The control H1666 cells were treated with either EGF or HRG ligand at time zero,
without pre-treatment by inhibitor. The phospho-signals for the control H1666 cells exhibited
trends similar to previous experiments on these cells. Phospho-EGFR was up-regulated in
EGF-stimulated but not HRG-stimulated cells, with a transient response in EGF-stimulated
cells that peaked around 15 minutes and then down-regulated over time. Phospho-ERK had a
transient response that mostly dropped back to baseline at 30 minutes for both EGF- and
HRG-stimulated cells, and phospho-AKT signals had a small transient peak in EGFstimulated cells and a much stronger, more sustained peak in HRG-stimulated cells.
No data was available for Okadaic acid treated cells because the treatment seemed to
have killed most of the cells and detached them from the well, with very few cells remaining
in Okadaic acid treated wells at the end of the time course. This seemed to be a time
dependent effect, with more and more of the cells falling off the plate at the later time points.
Endothall, a PP2A inhibitor, did not seem to have much of an effect on the
intracellular signals when compared with control cells, at the concentration and treatment
protocol used. Most of the signals measured for endothall treated cells are very close to those
for control cells.
The PTP1B inhibitor, however, seemed to have a potent effect on HRG-stimulated
H1666 cells. It inhibited phospho-ERK and phospho-AKT signaling when the cells are
stimulated with HRG, but not when the cells are stimulated with EGF. The phospho-ERK
signal in inhibitor-treated, HRG-stimulated cells was basically flat over the entire time
course, while the phospho-AKT response was much reduced compared to control, with a
much smaller up-regulation (less than half of the control signal) over the first 30 minutes,
with a peak signal at 60 minutes that was still less than the control signal. The PTP1B
inhibitor also seemed to inhibit the initial peak of the phospho-EGFR signal in EGFstimulated cells, as can be seen at the 5 and 10 minute time points. It also seemed to slightly
up-regulate the phospho-ERK signal compared to control in EGF-stimulated cells, as can be
seen at the 5 and 10 minute time points.
PTPIB Inhibitor on Additional Cell Lines - Time course
In order to compare and confirm the results from the PTP1B inhibitor in the previous
experiment, we used the PTP1B inhibitor to pre-treat 3 additional cell lines for a time course
experiment that included duplicate data points for each condition. A431, H1666, H3255, and
HeLa cells were pre-treated with PTP1B Inhibitor for 1 hour at 250uM, and then the response
of these cells following saturating EGF (100ng/mL) or HRG (50ng/mL) treatment was
measured at 0, 5, 15, 30, 60, and 120 minutes. Only the phospho-signals for ERK and AKT
were measured for these cells, because the phospho-EGFR signal for HRG stimulated cells
was too small to be compared using In-cell Westerns. The cells were seeded at an appropriate
density for each cell line, with the data normalized by DNA content for each well of cells
(see methods section for cell seeding for each line). The phospho-signals for each treatment
condition were compared by each cell line for phospho-ERK (Figure 3A) and phospho-AKT
(Figure 3B).
In H1666 cells, the PTP1B inhibitor results are qualitatively very similar to those of
the first experiment they were used in (Figures 1 and 3 - H1666 pERK panels). As in the first
experiment, the inhibitor down-regulated phospho-ERK and phospho-AKT signaling when
the cells are stimulated with HRG, but not when the cells are stimulated with EGF. The
phospho-ERK signal in inhibitor-treated, HRG-stimulated cells was much reduced compared
to untreated controls, while the phospho-AKT response was down-regulated compared to
control, with a much smaller up-regulation (less than half of the control signal) over the first
30 to 60 minutes. The inhibitor-treated cells also seems to have a reduced baseline for
phospho-ERK compared to untreated cells (Figure 3A, H1666 panel), with the inhibitortreated cells having half the signal of untreated cells at time zero. This effect was not
observed for phospho-AKT in H 1666 cells.
In A431 cells, the PTP1B inhibitor treated cells had much decreased phospho-ERK
signals compared to untreated cells over the entire 120 minute time course for HRG
stimulation, while inhibitor treated cells had only a slight decrease in signal (about 15%)
compared to untreated cells for EGF stimulation (Figure 3A, A431 panel). The inhibitor
abolished HRG-stimulated phospho-ERK signal in A431 cells almost completely. Unlike in
H1666 cells, the phospho-ERK signal baseline at time zero was the same between inhibitortreated and untreated cells. The phospho-AKT signal in A431 cells was slightly elevated for
PTPIB inhibitor treated cells compared to untreated cells for both the EGF and HRG cases
(Figure 3B, A431 panel). However, the baseline signal at time zero was slightly elevated for
phospho-AKT in inhibitor-treated cells compared to control.
In H3255 cells, for phospho-ERK, inhibitor treatment abolished much of the HRGstimulated signal compared to control (Figure 3A, H3255 panel). The activation peak for
inhibitor-treated, EGF-stimulated signal was also reduced in duration compared to untreated
cells, dropping to a plateau at 15 minutes, while untreated cells had a sustained phosphoERK signal that decreased over the entire 120 minute time course. The drop in the baseline
phospho-ERK signal at time zero seen in H1666 cells was also observed for H3255 cells.
Like in H1666 cells, the phospho-AKT signal for H3255 cells was down-regulated in
inhibitor treated cells compared to untreated cells for the HRG ligand (Figure 3B, H3255
panel). Unlike in H1666 cells, the EGF ligand stimulated signal was increased in inhibitor
treated cells compared to untreated cells. However, this could have been due to the elevated
baseline signal in inhibitor-treated H3255 cells for phospho-AKT, which was not seen in
H1666 cells.
For HeLa cells, like in H1666 cells, the phospho-ERK signal was reduced in PTP1B
inhibitor-treated cells compared to untreated cells for HRG-stimulated cells but not for EGFstimulated cells (Figure 3A, HeLa panel). The phospho-ERK signal baseline for HeLa cells,
however, was increased in inhibitor-treated cells, not decreased like in H1666 cells. Also
unlike in H1666 cells, the phospho-AKT signal was increased for inhibitor treated cells
compared to untreated cells, not decreased as in other cell lines (Figure 3B, HeLa panel).
Both the peak signal and duration were increased for HRG-stimulated, inhibitor-treated cells
compared to control, while just the peak signal was increased for EGF-stimulated, inhibitortreated cells compared to control. The baseline signal for phospho-AKT was about the same
between inhibitor-treated and untreated HeLa cells.
PTP1B Inhibitor, Okadaic Acid, Vanadate - Time course
We used additional inhibitors in a first-pass experiment, to increase the panel of
available phosphatase inhibitors for perturbing the ErbB system. Okadaic acid and vanadate
were the two general phosphatase inhibitors used in this experiment, and the PTP1B inhibitor
was included as it had been used in previous experiments and demonstrated an interesting
perturbation of cellular signals. The phospho-signals for ERK and AKT were tracked at 0, 5,
15, and 30 minutes post-EGF stimulation, for the A431, H1666, H3255, and HeLa cell lines
(Figure 4). We also included amphiregulin (AR), an EGFR ligand that we have had
experience using in other signaling experiments in the lab. We used saturating amounts of
each ligand for this experiment, with EGF at 100 ng/mL, HRG at 50 ng/mL, and AR at 50
ng/mL. The following protocol was used for those wells that included phosphatase inhibition.
Cells were pre-treated with PTP1B Inhibitor for 1 hour at 250uM. Okadaic acid was used at 1
uM. Pervanadate, the activated form of inorganic vanadate, was added at luM. Okadaic acid
and pervanadate were added together with the ligand, at time zero, to each well with that
experimental condition.
As this experiment only had singlet data points for each experimental condition, we
used data from previous PTP1B inhibitor treated, EGF and HRG stimulated phospho-ERK
and phospho-AKT signaling time courses as a baseline comparison, and only noted large
differences in time course trends between inhibitor treated and untreated cells for Okadaic
acid and pervanadate. In all four cell lines looked at, the PTP1B inhibitor-treated cells
behaved similarly to previously described trends when compared to control untreated cells
for EGF and HRG stimulation, with similar differences in signaling profiles and up- or
down- regulation of baselines.
In all four cell lines, EGF- and HRG- stimulation resulted in phospho-ERK and
phospho-AKT signaling profiles similar to those previously described (Figure 4A-D).
Amphiregulin, as another EGFR-specific ligand, resulted in similar signaling profiles to EGF
stimulation for phospho-ERK signals for all four cell lines. However, AR stimulation
resulted in differences in phospho-AKT signal profiles compared to EGF stimulation. H1666
cells had almost no phospho-AKT signals when stimulated with AR compared to a low but
detectable phospho-AKT peak when stimulated with EGF (Figure 4B, EGF and AR panels).
H3255 cells had a shorter phospho-AKT peak that dropped back to baseline by 15 minutes
when stimulated with AR, compared to a peak that was sustained past 15 minutes and only
dropped back to baseline by 30 minutes when stimulated with EGF (Figure 4C, EGF and AR
panels). HeLa cells also had almost no detectable phospho-AKT signal when stimulated with
AR, compared to a definite signal peak for EGF-stimulated cells (Figure 4D, EGF and AR
panels). A431 cells, however, had similar EGF and AR activation profiles for phospho-AKT
(Figure 4A, EGF and AR panels). Additionally, AR-stimulated cells had similar sensitivities
to PTP1B inhibitor pre-treatment with HRG-stimulated cells, with similar differences in
signaling profiles and baseline differences between inhibitor-treated and untreated cells.
Most of the Okadaic acid pre-treated cells behaved similarly to control untreated
cells. The only large differences were a slight decrease in phospho-ERK signals for Okadaic
acid treated H1666 cells compared to control H1666 cells, for EGF, HRG and AR
stimulation (Figure 4B, ERK panels), and an earlier drop in signaling duration for phosphoERK for all three ligands and an earlier drop in phospho-AKT signal for EGF-stimulation, in
HeLa cells (Figure 4D, ERK and AKT panels).
The pervanadate, at the concentration used (1 uM), had a large effect on signaling
profiles for all four cell lines, all three ligands, and both phospho-ERK and phospho-AKT
signals. In all the conditions used in this experiment, it increased both the signal peak and
duration for both phospho-ERK and phospho-AKT. The up-regulated signaling profiles are
similar between each ligand, for each of the individual cell lines.
2. Co-treating cells with phosphatase inhibitors together with protein synthesis or
kinase inhibitors
Cycloheximide - Time course
In a recent paper from Amit and Yarden et al. (Amit et al. 2007), they demonstrated
that cycloheximide co-treatment of HeLa cells resulted in an up-regulation of EGF stimulated
phospho-ERK, compared to HeLa cells treated with only EGF, as soon as the first hour post
ligand stimulation. Amit et al. showed that this could have been due to a translational upregulation of phosphatases, possibly DUSPs, in response to ligand stimulation. We wanted to
confirm this in our cell lines, so we did an experiment where we used EGF to stimulate HeLa
and H1666 cells, with or without cycloheximide co-treatment, over a time course sampled at
0, 5, 15, 30, 60, and 120 minutes. We measured phospho-EGFR, phospho-ERK, and
phospho-AKT signals, with 2 repeats for each condition [Figure 2]. The lines between each
data point are best-fit spline approximations generated using Origin software. The figure
from Amit et al. showing their phospho-signal time course was included for comparison.
The EGF-stimulated phospho-EGFR, phospho-ERK, and phospho-AKT signals we
obtained for this time course are similar to those of previous experiments. H1666 cells
showed a low but detectable phospho-EGFR signal that peaked around 5 minutes and
dropped over 30 to 60 minutes, a phospho-ERK signal that peaked at 5 minutes and then
dropped quickly to a plateau, and a low but detectable phospho-AKT peak. HeLa cells, by
comparison, showed phospho-EGFR signals that were almost not detectable by this In-cell
Western protocol, a strong phospho-ERK signal that peaked around 10 to 15 minutes, and a
low but detectable phospho-AKT signal that peaked around 5 minutes.
When Amit et al. co-treated HeLa cells with EGF and cycloheximide, they saw a
large and sustained increase in their phospho-ERK signal, as well as a small increase in their
phospho-EGFR signal. However, cycloheximide co-treatment, in our experiments, had only a
small effect on our HeLa and H1666 cells. Cycloheximide co-treated HeLa cells had a small
increase in their phospho-ERK and phospho-AKT signals (less than 15%) past the signal
peaks, from the 30 minute time point until the 120 minute time point, and the phospho-EGFR
signals were too small to be compared for HeLa cells. On the other hand, cycloheximide cotreated H1666 cells actually had decreased phospho-ERK and phospho-AKT signals
compared to H1666 cells only stimulated with EGF ligand, with a decrease in signal of about
10-20% for phospho-ERK at all time points past 5 minutes, and a decrease in signal of about
10% for phospho-AKT between the 5 and 60 minute time points. The phospho-EGFR signal
was about the same between cycloheximide co-treated and untreated H1666 cells.
Iressa and Vanadate - Time course
We performed an Iressa-chase experiment where we initially stimulated H1666 and
H3255 cells with EGF at 100 ng/mL, then added either a saturating amount of Iressa (16
uM), 1 uM of pervanadate, or both, at 10 minutes post EGF-stimulation. Phospho-EGFR,
phospho-ERK, phospho-SHC, and phospho-AKT signals were sampled at 0 (ligandstimulation), 5, 10 (treatment with or without Iressa and pervanadate), 15, 30, and 60
minutes. Each condition was duplicated in a second identically treated well of cells, and the
results for the four phospho-signals are graphed for H1666 (Figure 6A) and H3255 (Figure
6B). Because the phospho-signals from cells treated with pervanadate at 10 minutes are much
greater in magnitude than cells untreated with pervanadate, the results for pervanadate treated
cells are graphed separately for H1666 (Figure 6C) and H3255 (Figure 6D), with the control
cells and Iressa-only cells there as well for comparison.
For both H1666 and H3255 cells, addition of Iressa at 10 minutes decreased the
amount of phospho-signals almost immediately, at the next time point sampled (15 minutes).
With the exception of phospho-ERK and phospho-AKT in H1666 cells, phospho-signals
decreased to either baseline levels or below baseline and stayed at that level until 50 minutes
post addition of Iressa, at the last time point sampled. For H1666 cells, addition of Iressa
corresponded with dropping of the phospho-EGFR and phospho-SHC signals to baseline
levels, while the phospho-ERK signal only decreased slightly compared to control cells, and
the phospho-AKT signal was very similar in H1666 cells treated or untreated with Iressa at
10 minutes.
In both H1666 and H3255 cells, addition of luM pervanadate alone at 10 minutes
resulted in a large increase of all phospho-signals, with the exception of phospho-ERK
signals in H3255 (Figure 6C and 6D). The phospho-signals either plateaued at a high level
(higher than peaks normally seen with saturating amount of ligand), or continued to rise until
the last time point sampled, 60 minutes. Co-addition of pervanadate with Iressa resulted in
decreased signal up-regulation compared with addition of pervanadate alone for phosphoEGFR and phospho-AKT in H1666s and H3255s, while the phospho-ERK and phospho-SHC
signals were similar for pervanadate-treated alone and co-treated cells in both cell lines.
3. Phosphatase inhibitors in phospho-ErbB ELISA assays
PTP1B Inhibitor - Time course
In this experiment, we used phospho-ErbB ELISA kits to measure phospho-ErbB
signals in a time course experiment. We followed identical protocols for ligand-stimulation
and phosphatase inhibitor treatment of the cells as the In-cell Western experiments, and
measured the response of ErbB1-4 phosphorylation following 100 ng/mL of EGF or 50
ng/mL HRG stimulation of H1666 and HeLa cells, with or without PTP1B inhibitor pretreatment (Figure 5). After incubation for 5 minutes with 100 uM of PTP1B inhibitor, the
time course was sampled at 0, 5, 15, 30, 60, and 120 minutes post ligand-stimulation. Each
treatment had a single data point; however, this experiment was repeated on a separate day,
with similar results (data not shown).
For H1666 cells, EGF stimulation resulted in a sustained phospho-EGFR response
which only dropped off at the 120 minute time point, and transient peaks for phospho-ErbBs
2, 3, and 4, that peaked at 5 minutes post-stimulation and then decreased over the time course
(Figure 5A). HRG stimulation of H1666s resulted in transient peaks for phospho-ErbB2 and
phospho-ErbB3 that peaked at 5 minutes and then decreased to a plateau above baseline over
a period of approximately 25 minutes, but did not increase phospho-EGFR or phosphoErbB4 levels. Pre-incubation with the PTP1B inhibitor decreased the transient peak for EGFstimulated phospho-ErbBs 2, 3, and 4, and also abolished almost completely the transient
peak seen for HRG-stimulated phospho-ErbB2 and phospho-ErbB3. Pre-incubation with the
inhibitor also seemed to prolong EGF-stimulated phospho-EGFR signaling, but this was only
seen at the 120 minute data point. The PTP1B inhibitor also increased the baseline of
phospho-EGFR and phospho-ErbB4 signals in pre-treated cells.
For HeLa cells, EGF stimulated time courses showed a large signal peak for phosphoEGFR and phospho-ErbB2 that peaked at 5-15 minutes, and a smaller signal peak for
phospho-ErbB4 that peaked at 5 minutes, while phospho-ErbB3 did not seem to be
stimulated by EGF (Figure 5B). HRG stimulation of HeLas resulted in a small transient peak
for phospho-ErbB2 and phospho-ErbB3 that peaked at 5 minutes and dropped to about
baseline over 25 minutes, while HRG stimulation of HeLas did not seem to stimulate
phospho-EGFR or phospho-ErbB4. Pre-incubation with 100 uM of PTP1B inhibitor did not
seem to affect either EGF or HRG stimulated phospho-ErbB signals in HeLa cells, except for
a small decrease in the phospho-ErbB3 signal in HRG-stimulated, PTP1B inhibitor-treated
cells. The PTP1B inhibitor also resulted in a slightly elevated baseline like in H1666 cells,
but only for phospho-ErbB4 cells.
IV. Discussion
1. Tools developed for studying phosphatase activity
In this project a set of assays, cell lines, and reagents were tested and verified for the
purpose of studying phosphatase activity in the context of EGFR signaling. Specifically, we
validated In-cell Western and ELISA protocols for the study of intracellular signals
resulting from ligands specific for ErbB family members, and tested both specific and
general inhibitors for phosphatases that are known to play important roles in EGFR
downstream signaling. We also tested and confirmed kinase-specific and translational
inhibitors, different ways for using the inhibitors, and various ErbB ligands. These protocols
will allow us to distinguish the contributions of phosphatase activity to different layers of
the EGFR signaling network.
Validation of In-Cell Western and ELISA assays
We demonstrated that In-Cell Westerns and commercial phospho-ErbB ELISA kits
could gather informative data for the purpose of elucidating the role of phosphatases in the
ErbB network. Through these two techniques we were able to gather data on the
phosphorylation states of the phospho-ErbB receptors and downstream activation of the Erk
and Akt molecules. In-Cell Western assays were used in experiments that showed similar
pEGFR, pERK, and pAKT trends to previous experiments for the same cell lines and
conditions. In addition, we also gathered informative data using ICW assays for pERK and
pAKT.
It was seen in previous experiments that it was difficult to compare the response of
ligand-stimulated EGFR phosphorylation for some of our cell lines such as HeLa's, due to
the low signal of the In-cell Western assay for phospho-EGFR for those lines. Also, we did
not have In-cell Western assays for phospho-ErbB2, phospho-ErbB3, or phospho-ErbB4, due
to difficulties in finding suitable phospho-antibodies for those receptor moieties. However,
we wanted to be able to measure the states for all phospho-ErbB receptors, in order to obtain
a more complete signaling dataset for the receptor level following ligand stimulation. This
was especially true in experiments involving ligands specific for other ErbB receptors than
EGFR, such as heregulin, a ligand with high affinity for ErbB3, or ligands that involved
different receptor heterodimerization and trafficking, such as amphiregulin. Therefore, in
order to sample receptor-level signals, we used commercially available phospho-ErbB
ELISA kits to measure phospho-ErbB signals in time course experiments that can
complement and expand the In-cell Western signaling dataset. We demonstrated that it was
possible to use phospho-ErbB ELISAs in identical experimental treatments as those of ICW
assays, and gather information on the phosphorylation state of the receptors in parallel with
phosphatase inhibitor treatment.
Validation of Phosphatase Inhibitors
In order to determine the combination of phosphatase inhibitors and experimental
protocols that will demonstrate measurable effects and allow us to gather reproducible data
in our cell lines, we tried available phosphatase inhibitors, both specific (PTP1B Inhibitor
and endothall) and broad spectrum (okadaic acid and vanadate) inhibitors. For this purpose,
time course experiments were used. Time courses can be used to show the effects of a
perturbation on different signaling events occurring over a specific period of time following
treatment or cell stimulation. In-cell Western was used for these experiments because of
availability of existing verified reagents and protocols, in addition to its reproducibility.
In our experiments we saw efficacy of a specific inhibitor for PTP1B, the allosteric
inhibitor for PTPIB from Calbiochem. The PTPIB inhibitor we used had an effect on the
receptor layer, which was both ligand and cell line dependent (Figure 5, also results PTPIB Inhibitor Time course). In addition, it had a large effect on downstream pERK and
pAKT signals, which was again ligand and cell line dependent (Figure 3, also results PTP1B Inhibitor on Additional Cell Lines Time course). It is interesting that PTP1B
inhibition resulted in up- or down-regulation of pERK and pAKT, depending on the ligand
and cell line used. For example, the effect of the PTP1B inhibitor was much smaller in
EGF-stimulated cells compared to HRG-cells. This inhibitor could be used, in combination
with additional experimental conditions, to inform the ErbB network model about the
differences in phosphatase regulation between different cell lines and receptor
configurations.
We wanted especially to find a general phosphatase inhibitor that can be used as a
comparative baseline against specific phosphatase inhibitors such as the PTP1B inhibitor. It
would be interesting to compare inhibition of much of the phosphatase activity in the cell
over a short time course versus acute inhibition of just one specific phosphatase.
Vanadate is another broad-spectrum inhibitor that has been shown to inhibit general
protein tyrosine phosphatase activity through competitive inhibition in many systems
(Shisheva and Shechter 1993; Krady et al. 1998). It had not been used in previous
experiments because it had not shown effective perturbation of signals when used
previously in our lab in kinase experiments with similar cells and assays. However, upon
further investigation of literature we discovered that vanadate needs to be activated with
peroxide in a manner that allows it to be cell-permeable and active in cell culture
experiments (Huyer et al. 1997). We then attempted to use pervanadate, the activated form
of inorganic vanadate, in order to see if it provides an interesting comparison to specific
PTP1B inhibition. We saw that pervanadate co-treatment with EGF, HRG, or AR increased
the pERK and pAKT signal intensity and duration, for all four cell lines. However, as the
pervanadate was used to co-treat the cells along with each ligand, it was possible that the
similarity between the three ligand-stimulated signaling profiles for each cell line was a
result of the pervanadate treatment. Further investigation of the effects of pervanadate along
with specific node inhibitors can help elucidate the role of the PTPs in the Erk and Akt
pathways downstream of ErbBs 1-4.
Another potent broad-spectrum phosphatase inhibitor we tried was okadaic acid.
Okadaic acid has been shown to exert an inhibitory effect on many phosphatases of the PPP
family, including PP2A (Zolnierowicz 2000; Perrotti and Neviani 2008). The first
experiment we tried okadaic acid in, we pre-treated the cells for 5 minutes with it, and it
seemed to kill most of the cells and no data was available because most of the cells detached
from the plates. The second time we tried okadaic acid, at a lower concentration (100 nM),
with no pre-incubation time, we did not see much of an effect on downstream molecules
(data not shown). We tried okadaic acid in a third experiment, while it had not shown much
efficacy in our hands, due to its demonstrated effects in the literature. We used a new aliquot
of this reagent, at a higher concentration, in order to make sure that it does not perturb the
signals we are measuring in a significant manner. Although we used ten times the
concentration previously used (1 uM in this experiment, versus 100 nM in the previous
experiment), it still did not seem to have a significant effect on phospho-ERK and phosphoAKT signaling profiles at the conditions we used in this experiment, for all four cell lines and
EGF, HRG, or AR stimulation. This could be due to incorrect pre-incubation times, or that
okadaic acid in the form we used was not cell permeable for the lines we used.
Reagents and protocols for different ErbB network layers
In previous experiments, co-treating with ligand and phosphatase inhibitors resulted
in signaling responses that are a combination of the effects of ligand-stimulated receptor
phosphorylation and perturbation of steady-state phosphatase activity. In order to
distinguish between the contributions of ligand-stimulation and phosphatase inhibition, we
used an Iress-chase experiment where we stimulated cells with ligand, then added a
saturating amount of Iressa to shut down receptor phosphorylation, with or without
pervanadate, to co-inhibit phosphatase function. We saw that adding Iressa decreased
phospho-signals almost immediately. In addition, co-addition of pervanadate with Iressa
resulted in up-regulation of almost all phospho-signals. This type of experiment is
interesting because it could be used to deduce dynamic information about the downregulation of pERK and pAKT by relevant phosphatases, following a pulse of signaling
from the receptor layer.
We also wanted to sample signals following stimulation for another EGFR ligand
that's reported to have different effects on receptor-trafficking following ligand stimulation.
For this purpose we used amphiregulin (AR), an EGFR ligand that has been reported to
have different heterodimerization and trafficking effects compared to EGF, even though
both ligands' favored receptor is EGFR (Salomon et al. 1995; Berasain et al. 2007). We saw
that when cell lines are stimulated with AR, it resulted in distinct pERK and pAKT
signaling profiles when compared to signaling profiles from cell lines stimulated with either
EGF or HRG. Additionally, AR-stimulated cells had similar sensitivities to PTP1B inhibitor
pre-treatment with HRG-stimulated cells, with similar differences in signaling profiles and
baseline differences between inhibitor-treated and untreated cells. This could be due to the
effects of differential receptor trafficking for AR-stimulated cells, and possibly help
elucidate the cellular localization of PTP1B in ErbB signaling.
In addition, we have previously used cycloheximide in EGF-stimulated signaling
experiments, to show that translational responses do not play a big role in EGFR
downstream signaling, for the cell lines we are interested in, and at the time scales we are
looking at. A recent report (Amit et al. 2007) showed that in HeLa cells, the ligandstimulated translational up-regulation of phosphatases, such as DUSPs, could be significant
in the signaling response even as soon as the first 30-60 minutes post ligand addition. We
showed that this result is probably not valid in our cell lines and experimental conditions
(Figure 2).
2. Future work
In the future, to help gain a quantitative and mechanistic understanding of the role of
phosphatases in the cellular system we can employ a combined approach with computational
modeling. A current ordinary differential equations (ODE) model of the ErbB receptor
pathway being developed in our lab can be used to help analyze and understand the data from
these experiments. Further model characterization and calibration can be done as well with
the new experimental results. In addition, model generation and characterization using other
methods than the existing ODE model can also be investigated with the phosphatase
experimental results, with model generation methods being investigated by other
collaborators.
To further calibrate the model we can also use quantitative total- and phospho-ErbB
ELISAs to measure receptor dynamics, and high-throughput Westerns for absolute
quantitation of species in the model. This will enable us to: 1) Validate current model results
and 2) Make the model more predictive by encoding behaviours from additional ligandreceptor interactions. The panel of measurements can also be expanded later to include more
receptor-proximal signaling molecules such as PIP3K and phospho-MEK, or further
downstream readouts such as transcription factor activation, using the Luminex antibodybead based system. We can also expand our panel of phosphatase inhibitors, and look at
other interesting phosphatases such DUSPs. Finally, we can also use different
concentrations of different phosphatase inhibitors in parallel, to demonstrate dose-dependent
effects in IC50 experiments.
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VI. Figure captions
For all time course In-Cell Western experiments, phospho-intensity as a ratio of the 800 nm
(phospho-signal) channel versus the 700 nm (DNA stain) channel is plotted on the y-axis,
and time in minutes on the x-axis.
Figure 1. PTP1B Inhibitor Time course - ICW
Left panels: pEGFR (top), pERK (middle), pAKT (bottom) time courses following 100 ng/
mL EGF treatment. Each colored line is for a different inhibitor treatment.
Right panels: pEGFR (top), pERK (middle), pAKT (bottom) time courses following 50 ng/
mL HRG treatment. Each colored line is for a different inhibitor treatment.
Figure 2. Cycloheximide comparison Time course - ICW
(A) H1666 cells, pEGFR, pERK, and pAKT signals time course, following 100 ng/mL EGF
treatment, with or without cycloheximide (CHX) pre-treatment. Each colored line is for a
different phospho-signal, with or without CHX.
(B) HeLa cells, pEGFR, pERK, and pAKT signals time course, following 100 ng/mL EGF
treatment, with or without cycloheximide (CHX) pre-treatment. Each colored line is for a
different phospho-signal, with or without CHX.
(C) HeLa cells signal time course from Amit et al. 2007 for comparison.
Figure 3. PTP1B Inhibitor Time course for Erk and Akt - ICW
(A) Phospho-Erk signal profiles for A431 (upper left), H1666 (upper right), H3255 (lower
left), and HeLa (lower right), following 100 ng/mL EGF (black and red lines) or 50 ng/mL
HRG (blue and green lines) treatment. The red and green lines represent data from wells
pre-treated with 250 uM of PTP1B inhibitor for 1 hour.
(B) Phospho-Akt signal profiles for A431 (upper left), H1666 (upper right), H3255 (lower
left), and HeLa (lower right), following 100 ng/mL EGF (black and red lines) or 50 ng/mL
HRG (blue and green lines) treatment. The red and green lines represent data from wells
pre-treated with 250 uM of PTP1B inhibitor for 1 hour.
44
Figure 4. PTP1B Inhibitor, Okadaic Acid, and Vanadate - Time course - ICW
Phospho-ERK (top panels) and phospho-AKT (bottom panels) signal profiles. The cells
were treated with 100 ng/mL EGF (left panels), 50 ng/mL HRG (middle panels), or 50
ng/mL AR (right panels). Each colored line represent a different inhibitor treatment:
untreated (black), 250 uM PTP1B inhibitor (red), Okadaic acid (blue), and pervanadate
(green).
(A) A431 cells
(B) H1666 cells
(C) H3255 cells
(D) HeLa cells
Figure 5. PTP1B ELISA for Phospho-ErbB1-4, Time course - ELISA
Phospho-ErbB1-4 signals were plotted as the intensity reading, corrected for background, on
the y-axis, and time in minutes on the x-axis.
(A) H1666 cells, phospho-signals for EGFR (upper left), ErbB2 (upper right), ErbB3 (lower
left), and ErbB4 (lower right), following 100 ng/mL EGF (black and red lines) or 50 ng/mL
HRG (blue and green lines) treatment. The red and green lines represent data from wells
pre-treated with 250 uM of PTP1B inhibitor for 1 hour.
(B) HeLa cells, phospho-signals for EGFR (upper left), ErbB2 (upper right), ErbB3 (lower
left), and ErbB4 (lower right), following 100 ng/mL EGF (black and red lines) or 50 ng/mL
HRG (blue and green lines) treatment. The red and green lines represent data from wells
pre-treated with 250 uM of PTP1B inhibitor for 1 hour.
Figure 6. Iressa then Vanadate-chase Time course - ICW
Cells were treated with 100 ng/mL of EGF at time = 0, and then with or without 16 uM
Iressa at 10 minutes. The error bars represent standard error from duplicate experimental
wells. Phospho-signals were measured for EGFR (upper left), ERK (upper right), AKT
(lower left), and SHC (lower right). Each colored line represent a different inhibitor
treatment: untreated (black), 16 uM Iressa at 10 minutes (red), pre-treated with pervanadate
+ 16 uM Iressa at 10 minutes (blue), and pervanadate pre-treatment alone (green). Since the
pervanadate treated cells resulted in much higher signals, each cell line is plotted twice, with
or without the pervanadate pre-treated signal profiles, to better show the difference between
untreated and Iressa at 10 minute treated signals.
(A) H1666
(B) H3255
(C) H1666 replot, with the pervanadate treated signals
(D) H3255 replot, with the pervanadate treated sig als
VII. Figures
Fig. 1 - PTP1B Inhibitor Time course
HRG
0.55-
0.550.50EGF
0.45-
0.400.450.50-.
OAS.
0640-
0.200.30025-
0.20-
0.20.
0
1o
2o
3•
40
50
60
0
1o
20
lime (mini.)
3o
40
5o
60
rime (min.)
EGF
I--PTpSlki1
1.e.
1.0-
HRG
Enota
1.6.
1.6-
1A.
1A-
12-
12-
1.0.
1.0-
0.60.6-
o.,-
0.4U.,.
0
1o
20
0i
r
0
Time (mmin.)
0
60
Mz
0
1o
2o
io
ai0
rime (min.)
50
0
EGF
. .....
*
..
1.6.
PT~~I~u
HRG
16.
1.4
1.4-
12.
1.2-
1.0
1.0
I-
I
IS0.8.
0.6
0.6.
0.4OA
02
I
0.2.
0
10
20
30
40
50
40
0
Time (min.)
10
20
30
Time(min.)
48
40
50
60
TP1
Elnowalw
Fig. 2 - Cycloheximide comparison Time course
A. B.
HeLa
H1666
--- EGFR
-v- EGFR +CHX
--- Erk
HX
"1)
"I,
2.2-
2.0.
2.0-
1.8-
1.8-
1.6.
1.6-
1.4.
1.4-
~1.2-
C 1.2-
1.0.
I
-X
1.0-
0.8-
0.8-
0.6-
0.6-
0.4-
0.4
0.2-
027
0
20
40
60
80
lime (mins)
100a 120
0
20
SEGF
fIm
40
60
80
100
120
LcbhedmKide
r.
6.n0 A4Da
pErk
pJNK
pp3 8
pEGFR
pSIATS
pMTOR
Jun
0.1
Amit, Yarden et al 2007
Fig. 3 - PTP1B Inhibitor Time course for Erk and Akt
A. phospho-Erk
pErk
A431
-u-EGF
--
EGF.Inh
Inh = w/PTP1B Inhibitor, 250uM, 1hr pre-treat
H1666
E
--
HRG
HRG.InhI
3.0-
EGF.Inh
HRG
-
G In.h
.......... I
2.5
2.0-
1.5.
l 1.5
1.
1.0.
0.5.
O.S
0
20
40
60
80
100
120
0
20
Time (mins)
40
60
80
100
120
Time (mins)
H3255
HeLa
EGF
EGF
EGF.Inh
EGF.Inh
-- HRG
HRG
6.0-
IRG.Inh
5.5-
RG.Inh
5.0-
2.0
454.0-
1.5
S1
,"3.5
a'
3.0.
2)
0 1.0
0.5.
1.5.
1.00
20
40
60
Time (mins)
0•
100
120
0
20
40
60
Time (mins)
80
100
120
B. phospho-Akt
pAkt
A431
EGF
Inh = w/PTP1B Inhibitor, 250uM, 1hr pre-treat
H1666
EGF.Inh
2.0-
-OEGF.Inh
ý RGý
RG.In_
HRG
-q- HRG.Inh
2.0-
|
1.5
1.0-
1.0.
0.
0.5-
·
0
·
20
_·_·····_··
40
60
w0
100
0
120
20
40
60
80
100
120
Time (mins)
Time (mins)
H3255
EGF
EGF.Inh
HRG
IRG.Inhl
5.5.
5.0.
HeLa
1.2.
--
EGF
EGF.Inh
HRG
IRG.Inh
1.0-
4.5.
4.0.
0.8-
3.53.0.
a 0.6-
C.2.5-
2.0-
0.4-
1.5.
1.0.
0.2-
U.320
40T
Time (mins)
k
1;0
120
0
2
40T0
0
Time (mins)
100
120
Fig. 4 - PTP1B Inhibitor, Okadaic Acid, and Vanadate - Time course
A. A431
A431
ERK
EGF
S-4- EGF.PTP1B.Inh
SHRG
S-4- HRG.PTP1Binh
-HRG.OA
5.0- -v- HRG.Vanadate
5.5-
5.0
4.5
45.
4.0
4.0.
3.5
3.5.
3.0
3.0-
~2-3
2.0
7-0-
1.50
15-
1.0.
1.0.
0.5
0.0
03.
,
'
0
5
10
15
'
20
~ '
25
'
'
30
0
5
Time (mins)
10
15
20
25
30
Time (mins)
Time (mins)
AKT
-*-EGF
25
EGFFTPTBInh
is5
-9--
EGF.Vadate
EGF.Vanadate
-
--
HRG
HRG.PTP1B.Inh
--
AR
--- AR.PTP1B.Inh
5
- HRGOA
.-
AR.OA
-v-
AR.Vanadate
I
0 5
0
15i 20 25 3
rime(mins)
0
s
'~'~'~''
10 I
Time (mins)
1
20 25
30
0
5
10
I5
Time (mins)
20
25
3
B. H1666
H1666
ERK
-
ARPT B.
-~AR PTP1B.Inh
0
1 ,-
0
5
10 i (mins)
3
S
rume(mins)
5
Time (mins)
10
15Tim
ins)20
rime (mins)
25
AKT
-0-AR
-,- AR.PTPIB.Inh
-- AR.OA
-,- AR.Vanadate
0
Time (mins)
5
10
15
Trme (mins)
20
25
30
0
5
10
15
20
Time (mins)
25
30
C. H3255
H3255
ERK
- -EGF
EGF.PTP1B.Inh
12. --I- EGF.OA
--- EGF.Vanadate
-U-HRG
--I nn
h.T
- HRG.OA
-rHRG Vanadate
12-
-v-
10.
10.
10-
8.
8-
8-
6.
6.
S6-
4.
4.
4-
2.
5
10
15
20
25
3
0
5
10 15 20
lime (mins)
Time (mins)
AR
AR.PTP1B.Inh
AR.OA
AR.Vanadate
2-
2-
d
0
-----
30
o
me10
15
rime (mins)
AKT
0-
-0-
EGF
EGF.PTP1B.Inh
--
EGF.Vanadate
I--EGF.OA
7-
0
5
10
15
Time (mins)
20
25
30
Time (mins)
Time (mins)
20
2
3
D. HeLa
HeLa
ERK
5.04.5-
----9-
EGF
EGF.PTP1B.Inh
EGF.OA
EGF.Vanadate
------
--
HRG
HRG.PTPIB.Inh
HRG.OA
HRG.Vanadate
5.0.
---
4.5
4.0-
4.0-
4.0-
3.5-
3.5-
3.5.
3.0-
3.0-
3.0
w 2.5-
wL 2.5-
w 2.5
10
2.0-
2.0-
2.0
1.5-
1.5-
1.5-
1.0-
1.0-
1.0-
V..'ý
0
5
10
15
20
25
0.5
30
0
5
Time (mins)
10
---
15
20
25
0
30
AR
AR.PTPIB.Inh
AR.OA
AR.Vanadate
5
Time (mins)
1o 15 20
25 30
Time (mins)
AKT
3.0.
-e--
-V-
EGF
EGF.PTP1B.Inh
EGF.OA
EGF.Vanadate
-*
-&-
-,-
HRG
HRG.PTP1B.Inh
HRG OA
HRG.Vanadate
-U-AR
--
*-4.AR.PTP1Binh
--- AR.OA
---
2.5
AR.Vanadate
25-
2.0.
2.0
4 1.5.
< 1.5
1.0-
1.0*
O.S
05"
0
5
10
15
Time (mins)
20
25
30
2.0-
0
5
10
15
Time (mins)
20
25
30
0
5
10
15 20 25 30
Time (mins)
Fig. 5 - PTP1B ELISA for Phospho-ErbB1-4, Time course
A. H1666
08-29-07 PTP1B Inhibitor ELISA
H1666
EGFR
ErbB2
-,ar.•-
5
2.02.0-
-
0
EGF
--EGFInh
-A- HRG
v- HRGInh
1.5-
1.5-
--
EGFF
.-..-
EGFInh
HRG
HRGInh
W 1.0-
01.0-
05
0.5.
U.U
0
10
20
30
40
50
60
120
0
0
Time (mins)
20
30
40
50
60
120
Time (mins)
ErbB3
ErbB4
1.4.
1.2-*-----
1.0-
S0.6
EGF
EGFInh
HRG
HRGInh
-e- EGF
--EGFInh
0.3-
- HRG
-w-
.0.2-
0.40.1 0.2an
0
10
20
30
Time (mins)
40
50
60
120
0
10
20
30
Time (mins)
40
50
60
120
HRGInh
B. HeLa
08-29-07 PTP1B Inhibitor ELISA
HeLa
EGFR
ErbB2
2.0
U- EGF
4- EGFInh
- HRG
Pw-HRGInh
--
EGF
EGFInh
-A-HRG
-v- HRGInh
m
W 1.0.
0.
0
10
20
30
40
50
60
Y·V_
120
0
10
Time (mins)
, ,• ,• ., - , • , • , / ----- r
20
30
40
50
60
120
Time (mins)
ErbB3
ErbB4
- EGF
- EGFInh
6- HRG
P- HRGInh
m
CL
0.
--.--v-
0.3-
.0.2-
··
UU
0
Time (mins)
0
10
20
30
Time (mins)
40
50
60
m
120
EGF
EGFInh
HRG
HRGInh
Fig. 6 - Iressa then Vanadate-chase Time course
A.
H1666
EGFR
-
0.40-
ERK
EGF
-
ar-C-
0.350.30 -
S0.251.0-
0.20 -
0
5
10
15
20
25
30
60
0
Time (mins)
5
10
15
20
AKT
SHC
rr-r
-a
0.6.
S0.5
U
x
V.
CL
0.40.3.
L•L__
0
5
10
15
20
Time (mins)
30
25
30
-,-
-- L
0.7-
n • .I
25
60
Time (mins)
60
Time (mins)
EGF
I-----
~nl
H3255
EGFR
SEGF
--
1.2.
1-'
..
ERK
-GF
15
25
inc
1.0-
US0.8
w
0.6-
0.4-
U.Lz
"`
0
5
10
15
20
25
30
60
0
5
10
Time (mins)
30
60
Time (mins)
AKT
SHC
-- a
FCF:
U
) 3.02.52.00
5
10
15
20
Time (mins)
25
30
60
1•231
0
5
10
15
20
Time (mins)
25
30
60
fl"
H1666
61
h.I
ERK
EGFR
-EGF
1.4-
3.5-
1.2-
3.0-
1.0-
2.5-
a 8-
2.02_O-
• 0.6-
1.5-
0.4
0.2
o
1.0
.
0.0
.
on
b
O1
.
5
.
10
Time (mins)
.
1'5
,
20
Time (mins)
SHC
AKT
u
x
I-
in
CL
0
Time (mins)
5
10
1
Time (mins)
60
-
,
23
"
i
jz m
06
cz___•
H3255
EGFR
ERK
.
U.
CL
Time (mins)
Time (mins)
AKT
SHC
-0-EGF
1
-*-&-9-
1
Iressa.10'
Iressa.Vi.10'
Vi.lO
I)
M.
U-
0
Time (mins)
5
10
15
20
Time (mins)
25
30
60