125

Chapter 7

MAP Kinase Activation by Receptor Tyrosine Kinases: In Control of Cell Migration

Gabi Tarcic and Yosef Yarden

Abstract

A myriad of cellular processes instigated by growth factors are mediated by cell surface-associated receptor tyrosine kinases (RTKs). Subsequent downstream activation of signaling cascades, as well as their crosstalk, endows specificity in terms of the phenotypic outcome, e.g., cellular proliferation, migration, or differentia-tion. Such signaling diversity is exemplified by the ability of the epidermal growth factor receptor (EGFR) to stimulate different MAPK cascades, especially the ERK1/2 cascade. It has been shown that the ability of the ERK1/2 cascade to specify cell fate, such as cell migration, is dependent on signal duration governed by feedback control. Here we focus on one experimental system, MCF10A human mammary cells, and a phenotypic outcome of cell migration. We present methods to identify key components of underlying cas-cades and their effects on the migratory phenotype. We focus on profiling activation of signaling modules, as well as transcriptional regulation, emphasizing the high-throughput potential of such approaches.

Key words: MAP kinase, ERK1/2, EGFR, Flow cytometry, Real-time PCR, Cell migration

Animal cells constantly exchange information with their tissue envi-ronment by means of signaling molecules (e.g., growth factors; GFs) and structural components (e.g., extracellular matrix; ECM). These molecules harbor essential information, which enables orchestration of key cellular functions leading to proliferation, dif-ferentiation or migration. One important class of environment-sensing molecules are receptor tyrosine kinases (RTKs) (1). RTKs are type I transmembrane proteins with an extracellular ligand-binding domain, a kinase domain, and multiple tyrosine phospho-rylation sites with regulatory functions. Upon ligand binding, receptors dimerize, thus activating their kinase domains and form-ing a signaling complex with auto- and trans-phosphorylation capabilities, thereby allowing recruitment of Src homology 2 (SH2)

1. Introduction

Rony Seger (ed.), MAP Kinase Signaling Protocols: Second Edition, Methods in Molecular Biology, vol. 661,DOI 10.1007/978-1-60761-795-2_7, © Springer Science+Business Media, LLC 2010

126 Tarcic and Yarden

and phosphotyrosine-binding (PTB) containing signaling adaptors (2). One of the most extensively studied subfamily of RTKs is the ErbB group, composed of four family members, each with its own binding partners and signaling capabilities (3, 4).

The prototype of the ErbB family is the epidermal growth factor receptor (EGFR or ErbB-1). EGFR binds not only with EGF, but also with six more ligand growth factors. Upon ligand binding, EGFR is capable of recruiting effector molecules of a variety of signaling cascades, including the mitogen-activated pro-tein kinase cascades (MAPK), phosphatidylinositol 3 kinase (PI3K), signal transducers and activators of transcription (STAT) and phos-pholipase Cg (PLCg) pathways (reviewed in (5)). The activation of these pathways defines the nature of the cellular response, as well as permits signal amplification. One striking feature of this configura-tion is the ability of a canonical linear pathway to generate several distinct cellular outcomes. A well-defined system exemplifying the ability of a linear cascade to generate multiple phenotypes is the proliferation vs. neurite outgrowth example, first exemplified using rat adrenal pheochromocytoma (PC-12) cells (6). In this system, proliferation can be induced by EGF or insulin, while neurite out-growth is induced by the nerve growth factor (NGF), both utilizing the ERK1/2 cascade. Among other parameters, outcome specificity is encoded by the duration of ERK1/2 phosphorylation: transient activation leads to cell proliferation, whereas sustained activation results in neuronal differentiation (6). It was later shown that the topology of the MAPK network enables this dichotomy; only the transient mode of activation can induce negative feedback (7). It has long been realized that equally important in shaping the cellular outcome of GF stimulation is the transcriptional response elicited downstream to MAPK and other pathways (8). Recently it has been shown that the transcriptional activation of a module of negative feedback regulators is able to attenuate growth factor signaling, consequently drive robust cellular outcome (9).

In this chapter, we present several methods to explore the cascaded layers of signal propagation. Potentially, this enables reconstruction of a signaling pathway stemming from GF stimulation, through cytoplasmic signaling pathways, nuclear transcription, and eventually functional output. The methods described can serve as a basis for high-throughput screening strategies of a system of choice, as well as unraveling the regu-latory hubs and novel feedback loops.

1. Dulbecco’s Modified Eagle’s Medium (DMEM) supple-mented with 10% fetal bovine serum (FBS, Gibco/BRL, Bethesda, MD), 1 mM sodium pyruvate (Biological Industries,

2. Materials

2.1. Cell Culture and Stimulation

127MAP Kinase Activation by Receptor Tyrosine Kinases: In Control of Cell Migration

Beit Haemek, Israel) for HeLa cells. DMEM/F-12(HAM) 1:1 supplemented with 10 mg/ml insulin (Biological indus-tries), 5% horse serum (Gibco), 1 mg/ml cholera toxin, 1 mg/ml hydrocortisone, and 10 ng/ml EGF (Sigma, St. Louis, MO) for MCF10A cells (see Note 1).

2. Epidermal growth factor (EGF, Sigma) dissolved at 100 ng/ml in PBS and stored at −20°C. MEK1/2 inhibitor U0126 dissolved at 5 mM in dimethylsulfoxide (DMSO) and stored in single use aliquots at −20°C.

3. Solution of trypsin (0.25%) and ethylenediamine tetraacetic acid (EDTA) (1 mM) from Biological Industries.

1. 3% paraformaldehyde (PFA) dissolved in PBS, stored at −20°C.

2. 100% methanol stored at −20°C. 3. Staining medium: 0.5% bovine serum albumin (BSA), 0.02

NaN3 in PBS, stored at 4°C. 4. Primary antibodies: anti-doubly phosphorylated ERK1/2 conju-

gated to Alexa-488 (Molecular Probes; Leiden, The Netherlands), anti-phosphorylated p38 antibody conjugated to Alexa-647, anti human HLA-A,B,C conjugated to PE (BD, Franklin Lakes, NJ), anti-EGFR (clone 111.6, Thermo-Scientific, UK).

5. Secondary antibody: FITC-conjugated goat-anti-mouse IgG (FITC-GaM, Jackson ImmunoResearch Laboratories, West Grove, PA).

1. RNA extraction: PerfectPure RNA Cultured Cell Kit (5 Prime, Gaithersburg, MD).

2. cDNA synthesis: High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA).

3. Real-time PCR: Power SYBR Green PCR Master Mix (Applied Biosystems).

1. Transwell permeable support with 8 mm polycarbonate mem-brane insert (Corning Incorporated, Corning, NY).

2. Lysis solution: 0.5% Triton X-100 in PBS. 3. Staining solution: 0.3% methyl-violet dissolved in PBS and

stored at 22°C.

Multiple signaling cascades are simultaneously activated upon RTK stimulation to achieve correct signal transduction, leading to various cellular responses. The probing of these activation events,

2.2. Intracellular Flow Cytometry

2.3. RNA Extraction and cDNA Synthesis and qPCR

2.4. Transwell Migration Assay

3. Methods

128 Tarcic and Yarden

mainly exemplified by protein phosphorylation, may be assayed using various techniques, each with its own advantages and draw-backs. There are several main parameters that need to be con-trolled to attain reliable results. First, each sample analyzed might contain several subpopulations of cells that differ in the activation state of different signaling pathways. This phenomenon becomes more important when dealing with cells that have undergone treatments, such as ectopic expression or silencing of specific genes. Second, biological samples sometimes contain very small number of cells, inappropriate for certain biochemical analyses. Third, simultaneous measurements of several protein active states in the same sample are advantageous, as it allows in-depth analysis of the regulation of signaling cascades. One such technique is intracellular flow cytometry developed by Garry Nolan’s lab at Stanford (10, 11). This technique allows simultaneous measure-ments of multiple protein states at a single cell resolution, offering useful tools for reconstructing signaling networks (12, 13).

After activation of cytoplasmic signaling pathways, the signal is relayed into the nucleus where it is translated into a transcrip-tional response (9). The identification of such transcriptional events is enabled by using high-throughput platforms, such as DNA microarrays. After initial characterization of specific genes that underwent alterations, it is possible to quantify and verify the events (up- or downregulation) using quantitative real-time PCR (RT-PCR). This method offers both precise determination of transcript levels and a confirmation of transcript identity. The design of the primer pair for each gene is a key step to accom-plishing efficient and specific PCR reactions. In general, the prim-ers are designed such that they all contain similar properties (with emphasis on annealing temperature and product length), offering a uniform PCR protocol.

Ultimately, the importance of the different signaling event needs to be tested in respect to the phenotypic changes they evoke. The involvement of RTK stimulation, via MAPKs, in cell motility is well established (5), and one such cell system is EGF stimulation of MCF10A cells (14). MCF10A, an immortalized mammary epi-thelial cell line, was shown to migrate in response to activation of various RTKs such as the insulin-like growth factor-I receptor (IGF-IR) (14), or the EGFR (9). While the process of cell migra-tion is executed by an intricate machinery involving many regula-tory and mechanical steps, the final endpoint of cell motility can easily be measured. One of the widely used assays is the Transwell chamber migration assay in which cells are placed on the top part of a polycarbonate membrane. If the cells are motile they can actively move through the pores to the bottom part of membrane. This simple assay is suitable for many cell types and for small num-bers of cells. Further, it can also serve as a chemotactic assay if different media are placed at the two sides of the membrane.

129MAP Kinase Activation by Receptor Tyrosine Kinases: In Control of Cell Migration

1. HeLa cells are grown in 10-cm tissue culture dishes to 80% confluence. At this point, cells are treated with trypsin and plated onto 6-well plates (0.1 × 106 per well) in 2 ml growth medium. Twenty-four hours later the growth medium is replaced with 2 ml DMEM without FBS (starvation medium) for a starvation period of 16 h (see Note 2).

2. On next day: change the starvation medium to fresh starvation medium, thaw 3% PFA and prepare fresh staining medium.

3. Prepare stimulation medium: dilute EGF in starvation medium (2 ml/sample; preheated to 37°C) to 20 ng/ml.

3.1. Preparation of Samples for Intracellular Flow Cytometry Assaying Active MAPK and EGFR Levels (See Fig. 1)

Fig. 1. Time-dependant activation of MAPKs and internalization of EGFR upon stimulation of HeLa cells with EGF. (a) HeLa cells were serum starved for 16 h, then stimulated with EGF (20 ng/ml) for the indicated time intervals. Cells were then fixed, permeabilized and stained with a fluorescently labeled Alexa-488-pERK1/2 antibody, Alexa-647-p-p38 antibody or PE-HLA-A,B,C antibody (control). Fluorescence intensity was measured using a flow cytometer. (b) Quantification of the results presented in (a). Activation of EGFR is followed by strong activation of the ERK1/2 cascade, as reflected by ERK1/2 phosphorylation, and relatively weak activation of p38. The activation of ERK is rapid, yet transient; peaking at 5 min, then slowly decreasing. (c) HeLa cells were serum starved for 16 h, then stimulated with EGF (20 ng/ml) for the indicated time intervals. Thereafter, cells were surface labeled with antibodies to EGFR. The remaining surface fraction of EGFR was quantified by flow cytometry and plotted as a function of time of incubation with EGF.

130 Tarcic and Yarden

4. The cells are treated with the ligands according to the experimental setup. For each primary antibody used, an unstained sample should be included as a background sample for each secondary antibody.

5. Immediately after the appropriate stimulation, cells are placed on ice, washed once with 1 ml ice cold PBS, and incubated for 5 min, on ice, in 1 ml trypsin solution (see Note 3).

6. For each sample prepare a labeled tube containing 500 ml of 3% PFA (see Note 4).

7. Collect suspended cells to the labeled tubes. Incubate 10 min at room temperature (RT).

8. Pellet the cells by centrifugation (5 min, 500 g), then aspirate the medium.

9. Place cells on ice, then resuspend by vigorous vortexing, and add 1 ml 100% methanol drop wise, to avoid clumping. Incubate 10 min at 4°C (see Note 5).

10. Add 3 ml staining medium, let stand for 2–3 min for proper rehydration. Pellet the cells by centrifugation (as aforemen-tioned) and aspirate the medium.

11. Wash once with 3 ml staining medium, centrifuge and aspirate.

12. Resuspend samples in 100 ml staining medium and remove an aliquot (100 ml) to a new tube.

13. Add primary antibody: anti-pERK-488, anti-p-p38-647, anti-HLA-A,B,C-PE (1:100), or anti-EGFR (1:100), then incu-bate for 30 min at RT

14. Wash with 3 ml staining medium; centrifuge, aspirate the medium and resuspend in 100 ml staining medium.

15. Remove an aliquot (100 ml) to a new tube. Add secondary antibody to the anti-EGFR sample: FITC-GaM (1:5,000), incubate for 30 min at RT

16. Wash with 3 ml staining medium, centrifuge and resuspend in 300 ml staining medium. Samples are ready to be analyzed on a FACS instrument.

1. HeLa cells are plated and treated as described above (see Note 6).

2. In the morning of the experiment, change the medium to fresh starvation medium and prepare stimulation medium: dilute EGF to 20 ng/ml and U0126 to 5 mM.

3. Treat the cells according to the experimental design. Cool the cells on ice following the appropriate time intervals.

4. Wash once with ice cold PBS and add 500 ml lysis buffer, sup-plied with the RNA extraction kit.

3.2. Quantitative Real-Time PCR for Validation of Gene Expression Events Regulated upon EGFR Activation (See Fig. 2)

131MAP Kinase Activation by Receptor Tyrosine Kinases: In Control of Cell Migration

5. Proceed with the RNA purification protocol according to the manufacturer’s instructions (see Note 7).

6. After completing the protocol, measure RNA concentration. Make sure that the ratio between 280 and 260 nm is ~2 and

Fig. 2. The transcriptional response induced by EGF depends on MAPK activation. MCF10A (0.1 × 106) cells were seeded in six-well plates. Following a 24-h recovery period, cells were serum starved for 16 h, then stimulated either with EGF alone (10 ng/ml) or with EGF plus a MEK1/2 inhibitor (U0126; 5 mm) for the indicated time intervals. Thereafter, the cells were lysed, total RNA was extracted, and 1 mg was used for cDNA synthesis. (a) EGR1 mRNA levels were measured using the synthesized cDNA and real-time PCR, in both EGF- and EGF+U0126-stimulated cells. (b) c-FOS mRNA levels were measured, essentially as described in (a). As can be seen, both transcription factors (i.e., c-FOS and EGR1), are induced after EGF stimulation and the mRNA levels decrease after 2 h. This induction is mediated by ERK1/2 activation as inhibition of MEK1/2, its direct upstream activator, abolished induction of these genes.

132 Tarcic and Yarden

that the ratio between 260 and 230 nm exceeds 1.8. Values that differ from these limits suggest that a contamination is present in the sample. RNA concentration should be over 0.05 mg/ml, as lower concentrations might interfere with effi-cient cDNA synthesis.

7. Either continue to cDNA synthesis directly or store RNA at −80°C (see Note 8).

8. Use 1 mg of RNA for cDNA synthesis, according to the man-ufacturer’s protocol. Although smaller amounts of RNA may be used, we found that for most purposes 1 mg of RNA is suitable and provides optimal cDNA concentration for PCR reactions.

9. cDNA can be stored at −20°C for long periods of time. 10. Each reaction of RT-PCR should include, besides the genes

of interest, a housekeeping gene whose expression pattern does not change between the different experimental condi-tions. Common genes used are b2-microglobulin or GAPDH. In addition, for each gene probed a non-template control (NTC) is added.

11. Primer design is usually done using online software (such as the Universal ProbeLibrary Assay Design Center provided by Roche Applied Science). The advantage of such software pro-grams is the automatic design of primers to identify exon–exon junctions, specifically amplifying mRNA. Additionally, the standardization of such primers allows usage of a single PCR protocol.

12. According to the RT-PCR kit used, each reaction contains cDNA, primers, and PCR reaction components. Amounts of cDNA samples are calibrated such that the threshold cycle of the control housekeeping gene is between 15 and 20. This allows high sensitivity range for the identification of most transcripts. Primer concentration is 0.5 nM per primer per reaction.

1. Two hours prior to the beginning of the experiment, incu-bate an appropriate number of Transwell chambers, depend-ing on the experimental setup, with MCF10A medium without EGF. Add 600 ml of medium to the bottom part of the well and 100 ml to the top part.

2. Before adding cells to the Transwell chambers, aspirate the medium and add the treatment medium. Each experiment needs to include an EGF-free medium as a negative control and an EGF-containing medium as a positive control.

3. MCF10A cells are grown to 70–80% confluence and then transferred onto the preincubated Transwell chambers. Usually, seeding 0.6 × 105 cells per well results in adequate cell

3.3. Transwell Migration Assay to Assess the Contribution of MAPK Activity to EGF-Induced Cell Migration (See Fig. 3)

133MAP Kinase Activation by Receptor Tyrosine Kinases: In Control of Cell Migration

densities. Make sure to avoid adding more that 100 ml of medium-containing cells as this might cause the medium to spill over the sides of the Transwell to its bottom part. Incubate the cells for 16 h in the Transwell chamber (see Note 9).

4. On next day: Thaw 3% PFA and prepare fresh lysis solution. 5. Aliquot 1 ml of PFA, lysis solution or staining solution per

sample in a 24-well plate. 6. Wash Transwell chambers in PBS (three times) by lifting each

chamber and gently immersing in a vessel containing PBS. 7. Incubate chambers for 15 min at RT in PFA. 8. Wash chambers in PBS three times, as described. 9. Incubate the chambers in lysis solution (15 min at RT). 10. Wash the chambers in distilled water, as described. 11. Incubate the chambers in staining solution (5 min at RT).

Fig. 3. EGF induces migration of MCF10A cells. MCF10A (0.6 × 105) cells were seeded in Transwell chambers and grown for 24 h in medium containing either serum with EGF (FM ), same medium without EGF (Serum), or with either an EGFR kinase inhibitor (AG1478) or a MEK1/2 kinase inhibitor (U0126). Thereafter, the cells were washed, fixed, permeabilized, and incubated with a crystal violet dye. Photos were taken using a light-microscope connected to a CCD-camera (bar = 500 mm). Evidently, EGF induces robust cell migration, which is dependent on EGFR activation and signaling through the ERK1/2 cascade.

134 Tarcic and Yarden

12. Thoroughly wash the chambers in distilled water as described.

13. Using a cotton swab, gently scrape the cells on the upper part of the chamber. It is important to make sure all the cells are removed from the top part of the chamber, as these cells are the nonmigrating cells.

14. Photograph the Transwell chambers.

1. When preparing growth medium for MCF10A cells, add all components to the medium, except for EGF. The medium is then filtered through a 0.45-mm filter, and EGF added.

2. The numbers of cells indicated here are the minimal numbers necessary for analysis; higher cell numbers can be used.

3. This step is applicable only for adherent cells. If using nonad-herent cells skip this step.

4. It is important to use polystyrene tubes in this experiment as fixed cells adhere to polypropylene tubes.

5. If conducting surface staining of cells, such as staining for EGFR, do not perform this step, as permeabilizing the cells is not necessary.

6. The number of cells may vary according to the purpose of the experiment and cell type. Usually smaller numbers of cells also yield adequate amounts of RNA, enough for cDNA synthesis.

7. There are numerous RNA purification kits that can be used. In this protocol we use a kit supplied by 5 Prime (Gaithersburg, MD); however, other kits able to produce high enough amounts of RNA and with high purity, can be used.

8. Given that RNA is liable to degradation, it is best to proceed immediately with cDNA synthesis. Alternatively, RNA sam-ples may be stored for several months at −80°C.

9. To make sure that an excess volume of medium is not added to the top part of the Transwell, count the cells and adjust to roughly 0.6 × 106 per ml. Moreover, when comparing differ-ent cell lines or cells undergoing different treatments, plate in a separate 24-well plate the same amount of cells as that added to the Transwell. This will enable quantification of the num-ber of cells in each Transwell chamber, and allow proper com-parison of migrating cells.

4. Notes

135MAP Kinase Activation by Receptor Tyrosine Kinases: In Control of Cell Migration

Acknowledgments

Our laboratory is supported by research grants from the National Cancer Institute (grant CA72981), the M.D. Moross Institute for Cancer Research and the Willner Family Center for Vascular Biology. Y.Y. is the incumbent of the Harold and Zelda Goldenberg Professorial Chair.

References

1. Hunter T (2000) Signaling–2000 and beyond. Cell 100(1):113–127.

2. Pawson T (2004) Specificity in signal trans-duction: from phosphotyrosine-SH2 domain interactions to complex cellular systems. Cell 116(2):191–203.

3. Citri A & Yarden Y (2006) EGF-ERBB sig-nalling: towards the systems level. Nat Rev Mol Cell Biol 7(7):505–516.

4. Jones RB, Gordus A, Krall JA, & MacBeath G (2006) A quantitative protein interaction net-work for the ErbB receptors using protein microarrays. Nature 439(7073):168–174.

5. Katz M, Amit I, & Yarden Y (2007) Regulation of MAPKs by growth factors and receptor tyrosine kinases. Biochim Biophys Acta 1773(8):1161–1176.

6. Marshall CJ (1995) Specificity of receptor tyrosine kinase signaling: transient versus sus-tained extracellular signal-regulated kinase activation. Cell 80(2):179–185.

7. Santos SD, Verveer PJ, & Bastiaens PI (2007) Growth factor-induced MAPK net-work topology shapes Erk response deter-mining PC-12 cell fate. Nat Cell Biol 9(3):324–330.

8. Treisman R (1996) Regulation of transcription by MAP kinase cascades. Curr Opin Cell Biol 8(2):205–215.

9. Amit I, et al. (2007) A module of negative feedback regulators defines growth factor sig-naling. Nat Genet 39(4):503–512.

10. Perez OD & Nolan GP (2002) Simultaneous measurement of multiple active kinase states using polychromatic flow cytometry. Nat Biotechnol 20(2):155–162.

11. Krutzik PO, Irish JM, Nolan GP, & Perez OD (2004) Analysis of protein phosphoryla-tion and cellular signaling events by flow cytometry: techniques and clinical applica-tions. Clin Immunol 110(3):206–221.

12. Irish JM, et al. (2004) Single cell profiling of potentiated phospho-protein networks in can-cer cells. Cell 118(2):217–228.

13. Sachs K, Perez O, Pe’er D, Lauffenburger DA, & Nolan GP (2005) Causal protein-sig-naling networks derived from multiparameter single-cell data. Science 308(5721):523–529.

14. Irie HY, et al. (2005) Distinct roles of Akt1 and Akt2 in regulating cell migration and epi-thelial-mesenchymal transition. J Cell Biol 171(6):1023–1034.