Receptor Tyrosine Kinases: Methods and Protocols [1 ed.] 9781493917884, 1493917889

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Methods in Molecular Biology 1233

Serena Germano Editor

Receptor Tyrosine Kinases Methods and Protocols

METHODS

IN

MOLECULAR BIOLOGY

Series Editor John M. Walker School of Life Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK

For further volumes: http://www.springer.com/series/7651

Receptor Tyrosine Kinases Methods and Protocols

Edited by

Serena Germano Necker Hospital, Paris, France

Editor Serena Germano Necker Hospital Paris, France

ISSN 1064-3745 ISSN 1940-6029 (electronic) ISBN 978-1-4939-1788-4 ISBN 978-1-4939-1789-1 (eBook) DOI 10.1007/978-1-4939-1789-1 Springer New York Heidelberg Dordrecht London Library of Congress Control Number: 2014952319 © Springer Science+Business Media New York 2015 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. Exempted from this legal reservation are brief excerpts in connection with reviews or scholarly analysis or material supplied specifically for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work. Duplication of this publication or parts thereof is permitted only under the provisions of the Copyright Law of the Publisher's location, in its current version, and permission for use must always be obtained from Springer. Permissions for use may be obtained through RightsLink at the Copyright Clearance Center. Violations are liable to prosecution under the respective Copyright Law. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. While the advice and information in this book are believed to be true and accurate at the date of publication, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein. Printed on acid-free paper Humana Press is a brand of Springer Springer is part of Springer Science+Business Media (www.springer.com)

Preface Receptor tyrosine kinases (RTKs) are a widespread family of single-pass membrane proteins. They all share a similar structure, comprising an extracellular ligand-binding domain, a single-pass transmembrane helix, and an intracellular kinase domain that differentiates them from all other receptors. Given their central role in several cellular functions and in a variety of human pathologies, RTKs are one of the most widely studied of the protein classes. Extensive research efforts on the genetics, cellular, molecular, and structural biology of this protein family have led to quite a complete picture of how these receptors work. Moreover, the current availability of several modulators of RTK activity and expression represents an additional incentive at elucidating their roles in disease, opening the possibilities to administrate more targeted treatments. As a consequence of the widespread interest on RTKs in the scientific community, a large body of protocols and techniques has been developed by researchers to extend our knowledge of their expression, biological functions, positive and negative regulation, as well as mechanistic insights into their aberrant activation in diseases. The aim of this book is to give an up-to-date overview of the most relevant and widely used methods employed in this field, providing detailed protocols that molecular and cellular biologists could easily adopt in their research programs on RTKs. Paris, France

Serena Germano

v

Contents Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

PART I

RTKS ACTIVATION AND SIGNALLING

1 Analysis of Receptor Tyrosine Kinase (RTK) Phosphorylation by Immunoblotting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Martina McDermott and Norma O’Donovan 2 Analysis of Changes in Phosphorylation of Receptor Tyrosine Kinases: Antibody Arrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sweta Rani and Lorraine O’Driscoll 3 Analysis of Epidermal Growth Factor Receptor Dimerization by BS3 Cross-Linking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Harmony F. Turk and Robert S. Chapkin 4 Single-Molecule Optical Methods Analyzing Receptor Tyrosine Kinase Activation in Living Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . Inhee Chung and Ira Mellman 5 Evaluation of the Dimerization Profiles of HER Tyrosine Kinases by Time-Resolved Förster Resonance Energy Transfer (TR-FRET) . . . . . . . . . Evelyne Lopez-Crapez, Alexandre Ho-Pun-Cheung, Patrick Garnero, and Hervé Bazin 6 Applying the Proximity Ligation Assay (PLA) to Mouse Preimplantation Embryos for Identifying Protein-Protein Interactions In Situ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ivan Bedzhov and Marc P. Stemmler

PART II

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3

15

25

35

45

57

RTKS ABERRANT EXPRESSION

7 Analysis of Receptor Tyrosine Kinase Gene Amplification on the Example of FGFR1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Diana Boehm, Anne von Mässenhausen, and Sven Perner 8 Quantification of the Effects of Mutations on Receptor Tyrosine Kinase (RTK) Activation in Mammalian Cells . . . . . . . . . . . . . . . . . . Lijuan He and Kalina Hristova

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Contents

PART III

RTKS TRAFFICKING AND NEGATIVE REGULATION

9 Cell Surface Biotinylation of Receptor Tyrosine Kinases to Investigate Intracellular Trafficking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mathieu J.F. Crupi, Douglas S. Richardson, and Lois M. Mulligan 10 Studying N-Linked Glycosylation of Receptor Tyrosine Kinases. . . . . . . . . . . . Harri M. Itkonen and Ian G. Mills 11 Identification of Receptor Protein Tyrosine Phosphatases (RPTPs) as Regulators of Receptor Tyrosine Kinases (RTKs) Using an RPTP siRNA-RTK Substrate Screen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hojin Lee and Anton M. Bennett 12 Downregulation of Receptor Tyrosine Kinases Through Ubiquitination: Analysis by Immunodetection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Noriaki Shimokawa and Noriyuki Koibuchi 13 Regulation of Receptor Tyrosine Kinases by miRNA: Overexpression of miRNA Using Lentiviral Inducible Expression Vectors . . . . XiangDong Le, Andrew T. Huang, Yunyun Chen, and Stephen Y. Lai

PART IV

91 103

111

121

135

RTKS AS PHARMACOLOGICAL TARGETS

14 Human Tumor Xenografts in Mouse as a Model for Evaluating Therapeutic Efficacy of Monoclonal Antibodies or Antibody-Drug Conjugate Targeting Receptor Tyrosine Kinases . . . . . . . . . . . . . . . . . . . . . . . Liang Feng, Wei Wang, Hang-Ping Yao, Jianwei Zhou, Ruiwen Zhang, and Ming-Hai Wang 15 Receptor Tyrosine Kinase Targeting in Multicellular Spheroids . . . . . . . . . . . . Susan Breslin and Lorraine O’Driscoll 16 Receptor Tyrosine Kinases and Drug Resistance: Development and Characterization of In Vitro Models of Resistance to RTK Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Claire Corcoran and Lorraine O’Driscoll Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

151

161

169 181

Contributors HERVÉ BAZIN • Cisbio Bioassays, Codolet, France IVAN BEDZHOV • Department of Molecular Embryology, Max-Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany; Gurdon InstituteUniversity of Cambridge, Cambridge, UK ANTON M. BENNETT • Department of Pharmacology, Yale University School of Medicine, New Haven, CT, USA; Program in Integrative Cell Signaling and Neurobiology of Metabolism,Yale University School of Medicine, New Haven, CT, USA DIANA BOEHM • Department of Prostate Cancer Research, Institute of Pathology, University Hospital of Bonn, Bonn, Germany SUSAN BRESLIN • School of Pharmacy & Pharmaceutical Sciences and Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin, Ireland ROBERT S. CHAPKIN • Program in Integrative Nutrition and Complex Diseases and the Center Translational Environmental Health Research, Texas A&M University, College Station, TX, USA; Department of Nutrition & Food Science, Texas A&M University, College Station, TX, USA YUNYUN CHEN • Department of Head and Neck Surgery, The University of Texas MD Anderson Cancer Center, Houston, TX, USA INHEE CHUNG • Research Oncology, Genentech Inc., South San Francisco, CA, USA CLAIRE CORCORAN • School of Pharmacy & Pharmaceutical Sciences and Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin, Ireland MATHIEU J.F. CRUPI • Division of Cancer Biology and Genetics, Cancer Research Institute, Queen’s University, Kingston, ON, Canada; Department of Pathology & Molecular Medicine, Queen’s University, Kingston, ON, Canada LIANG FENG • Cancer Biology Research Center, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX, USA; Department of Biomedical Sciences, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX, USA PATRICK GARNERO • Cisbio Bioassays, Codolet, France LIJUAN HE • Department of Materials Science and Engineering, Johns Hopkins University, Baltimore, MD, USA ALEXANDRE HO-PUN-CHEUNG • ICM, Institut du Cancer Montpellier, Montpellier, France KALINA HRISTOVA • Department of Materials Science and Engineering, Johns Hopkins University, Baltimore, MD, USA ANDREW T. HUANG • Department of Head and Neck Surgery, The University of Texas MD Anderson Cancer Center, Houston, TX, USA HARRI M. ITKONEN • Prostate Cancer Research Group, Centre for Molecular Medicine Norway, Nordic EMBL Partnership, University of Oslo and Oslo University Hospital, Oslo, Norway

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Contributors

NORIYUKI KOIBUCHI • Department of Integrative Physiology, Gunma University Graduate School of Medicine, Gunma, Japan STEPHEN Y. LAI • Department of Head and Neck Surgery, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; Department of Molecular and Cellular Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA XIANGDONG LE • Department of Head and Neck Surgery, The University of Texas MD Anderson Cancer Center, Houston, TX, USA HOJIN LEE • Department of Pharmacology, Yale University School of Medicine, New Haven, CT, USA EVELYNE LOPEZ-CRAPEZ • ICM, Institut du Cancer Montpellier, Montpellier, France ANNE VON MÄSSENHAUSEN • Department of Prostate Cancer Research, Institute of Pathology, University Hospital of Bonn, Bonn, Germany MARTINA MCDERMOTT • Translational Cancer Therapeutics Program, Department of Drug Discovery and Biomedical Sciences, South Carolina College of Pharmacy, University of South Carolina, Columbia, SC, USA IRA MELLMAN • Cancer Immunology, Genentech Inc., South San Francisco, CA, USA IAN G. MILLS • Prostate Cancer Research Group, Centre for Molecular Medicine Norway, Nordic EMBL Partnership, University of Oslo and Oslo University Hospital, Oslo, Norway; Department of Cancer Prevention, Oslo University Hospital, Oslo, Norway; Department of Urology, Oslo University Hospital, Oslo, Norway LOIS M. MULLIGAN • Division of Cancer Biology and Genetics, Cancer Research Institute, Queen’s University, Kingston, ON, Canada; Department of Pathology & Molecular Medicine, Queen’s University, Kingston, ON, Canada NORMA O’DONOVAN • Molecular Therapeutics for Cancer Ireland, National Institute for Cellular Biotechnology, Dublin City University, Dublin, Ireland LORRAINE O’DRISCOLL • School of Pharmacy & Pharmaceutical Sciences and Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin, Ireland SVEN PERNER • Department of Prostate Cancer Research, Institute of Pathology, University Hospital of Bonn, Bonn, Germany SWETA RANI • Regenerative Medicine Institute (REMEDI), National University of Ireland, Galway, Ireland; School of Pharmacy & Pharmaceutical Sciences and Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin, Ireland DOUGLAS S. RICHARDSON • Division of Cancer Biology and Genetics, Cancer Research Institute, Queen’s University, Kingston, ON, Canada; Department of Pathology & Molecular Medicine, Queen’s University, Kingston, ON, Canada; Center for Biological Imaging, Harvard University, Cambridge, MA, USA NORIAKI SHIMOKAWA • Department of Integrative Physiology, Gunma University Graduate School of Medicine, Gunma, Japan MARC P. STEMMLER • Department of Molecular Embryology, Max-Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany; Department of Visceral SurgeryUniversity Medical Center Freiburg, Freiburg, Germany HARMONY F. TURK • Program in Integrative Nutrition and Complex Diseases and the Center Translational Environmental Health Research, Texas A&M University, College Station, TX, USA MING-HAI WANG • Cancer Biology Research Center, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX, USA; Department of Biomedical Sciences, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX, USA

Contributors

xi

WEI WANG • Cancer Biology Research Center, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX, USA; Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX, USA HANG-PING YAO • State Key Laboratory for Diagnosis & Treatment of Infectious Diseases at First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China RUIWEN ZHANG • Cancer Biology Research Center, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX, USA; Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX, USA JIANWEI ZHOU • Department of Molecular Cell Biology and Toxicology, School of Public Health, Nanjing Medical University, Nanjing, China

Part I RTKs Activation and Signalling

Chapter 1 Analysis of Receptor Tyrosine Kinase (RTK) Phosphorylation by Immunoblotting Martina McDermott and Norma O’Donovan Abstract Immunoblotting for phosphorylated forms of receptor tyrosine kinases (RTKs) has been the mainstay of investigations on RTK signaling for the past two decades. Despite the development of quantitative mass spectrometry, reverse-phase protein array, and multiplex technologies, immunoblotting with phosphospecific antibodies is still used in parallel with these technologies and remains a powerful, and reproducible, method for interrogating signaling networks involving RTKs. Key words Phosphoprotein, HER2, EGFR, HER3, IGF1R, c-Met, Immunoblotting

1

Introduction Reversible phosphorylation of receptor tyrosine kinases plays a key role in regulating the activity of RTKs. In general, ligand binding to the extracellular domain of the receptor triggers autophosphorylation of tyrosine residues in the kinase domain resulting in activation of the kinase. Additional phosphorylation sites, on the intracellular portion of the receptor, can negatively and/or positively regulate the activity of the RTK and can create docking sites for downstream cytoplasmic targets [1–3]. Early studies on phosphorylation of tyrosine kinases used immunoprecipitation with an antibody targeting the specific tyrosine kinase followed by immunoblotting with an antiphosphotyrosine antibody [4, 5]. There is now increasing availability of phospho-specific antibodies for individual RTKs, particularly the well studied RTKs such as members of the EGFR family. Phospho-specific antibodies bind to and detect the phosphorylated form of the protein and do not bind to the unphosphorylated protein [6]. The phospho-specific antibodies can be applied to a variety of antibody-based approaches to detect changes

Serena Germano (ed.), Receptor Tyrosine Kinases: Methods and Protocols, Methods in Molecular Biology, vol. 1233, DOI 10.1007/978-1-4939-1789-1_1, © Springer Science+Business Media New York 2015

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Martina McDermott and Norma O’Donovan

in the phosphorylation state of the RTK, including ELISAs, immunofluorescence, flow cytometry, and immunoblotting [7, 8]. Immunoblotting for phosphorylated RTKs is a semiquantitative method which can be used to examine changes in phosphorylation and activation of RTKs following stimulation (e.g., with growth factors), or inhibition (e.g., with inhibitors), and to examine cross talk between RTKs. Time course experiments can be performed to examine the duration of the response, and dose response assays can be performed to determine the potency of a stimulant or inhibitor. This chapter details a protocol for immunoblotting for RTKs in protein lysates prepared from cell lines. Immunoblotting can also be performed on tissue samples but presents additional challenges with regard to the stability of the phospho-groups on the proteins and the processing of the tissue samples [9, 10]. Novel fixation and storage buffers have recently been developed to address these issues [11, 12]. The original method of immunoblotting was based on an overnight wet transfer system for transfer of proteins to the membrane. Semidry blotting allowed faster transfer times of approximately 45–60 min [13, 14]. More recently, new systems, such as the iBlot [15], have been developed which allow very rapid transfer of proteins to a membrane in 7–10 min. The protocol outlined here describes both semi-dry transfer and the iBlot transfer procedure.

2

Materials Prepare all solutions using ultrapure water (prepared by purifying deionized water to attain a sensitivity of 18 MΩ cm at 25 °C) and analytical grade reagents. Prepare and store all reagents at room temperature (unless otherwise indicated).

2.1 Protein Extraction

1. RIPA buffer: 50 mM Tris–HCl, pH 8.0, 150 mM sodium chloride, 1.0 % igepal CA-630 (NP-40), 0.5 % sodium deoxycholate, 0.1 % sodium dodecyl sulphate. Store at 4 °C. 2. 100× Protease Inhibitor cocktail (Millipore Corporation, Billerica, MA, USA). Store at −20 °C. 3. 0.2 M phenylmethylsulfonyl fluoride (PMSF): Dissolved in isopropanol to make a 100× solution. Store at −20 °C. 4. 0.1 M sodium orthovanadate: Dissolved in ultrapure water, pH adjusted to 10, boiled for 5 s, cooled on ice and pH readjusted to 10. Store at −20 °C. 5. 21-gauge needle with a 1 mL syringe. 6. Bicinchoninic acid (BCA) protein assay (Thermo Fisher Scientific, Rockford, IL, USA).

RTKs Immunoblotting

2.2 SDS Polyacrylamide Gels

5

1. Resolving gel buffer: 1.5 M Tris–HCl, pH 8.8 (see Note 1). 2. Stacking gel buffer: 0.5 M Tris–HCl, pH 6.8. 3. Acrylamide: 30 % (29:1) acrylamide:bis-acrylamide. Store at 4 °C (see Note 2). 4. Ammonium persulfate (APS): 10 % solution in ultrapure water (see Note 3). 5. N,N,N,N′-Tetramethyl-ethylenediamine (TEMED). 6. SDS-PAGE running buffer: 0.025 M Tris, 0.192 M glycine, 0.1 % SDS, pH 8.3 (see Note 4). 7. 5× sample buffer: 0.125 M Tris–HCl, pH 6.8, 4 % SDS, 20 % glycerol, 10 % beta-mercaptoethanol, 0.004 % bromophenol blue. Store at −20 °C (see Note 5). 8. Pre-stained molecular weight protein marker.

2.3

Transfer

2.3.1 Semidry Transfer

1. Semidry transfer unit. 2. Nitrocellulose membrane. 3. Filter paper. 4. Transfer buffer: 0.048 M Tris, 0.039 M glycine, 0.375 % SDS, 20 % methanol (see Note 6).

2.3.2 iBlot® Transfer

1. iBlot® gel transfer device (Life Technologies, Carlsbad, CA, USA) (see Note 7). 2. iBlot® nitrocellulose transfer stack (Life Technologies). 3. iBlot® filter paper. 4. Ultrapure water (dH2O).

2.4

Immunoblotting

1. Ponceau S solution: 0.1 % in 5 % acetic acid. 2. Phosphate-buffered saline (PBS) (see Note 8). 3. PBS containing 0.1 % Tween-20 (PBS-T). 4. Blocking and antibody solution: 2.5 % milk powder in PBS-T (see Note 8). 5. Primary antibodies: A list of antibodies for RTKs and their phosphorylated forms is reported in Table 1. 6. Control antibody: Anti-tubulin (Sigma-Aldrich, St Louis, MO, USA) (see Note 9). 7. Secondary antibodies: HRP-conjugated anti-rabbit and antimouse secondary antibodies. 8. Enhanced chemiluminescent substrate. 9. Film or digital imaging system.

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Martina McDermott and Norma O’Donovan

Table 1 Antibodies for specific receptor tyrosine kinases Antibody

Phospho-site

Anti-HER2 Anti-phospho-HER2

Tyr1221/1222

Anti-EGFR Anti-phospho-EGFR

Tyr1173

Anti-HER3 Anti-phospho-HER3

Tyr1289

Anti-IGF1Rβ Anti-phospho-IGF1Rβa

Thr1131

Anti-MET Anti-phospho-MET a

Tyr1234/1235

Company

Cat no.

Calbiochem

OP15

CST

#2249

Neomarkers

MS-665-P

CST

#4407

CST

#4754

CST

#4791

SCB

sc-713

CST

#3021

CST

#8198

CST

#3077

This antibody also detects insulin receptor β (Tyr1146)

3

Methods

3.1 Preparation of Cell Lysates ( See Note 10)

1. Seed cells at approx. 5 × 105 to 1 × 106 cells per petri dish (10 cm) with 10 mL of medium (see Note 11). 2. Incubate cells and allow to grow until approx. 80 % confluent (feed every 2–3 days as necessary). 3. To examine the effects of a growth factor with or without inhibitors on signaling it is advisable to serum starve the cells for 24 h prior to stimulation and treatment (see Note 12). 4. Following treatment, remove the medium and wash once with ice-cold PBS. 5. Add 500 μL cold RIPA buffer with protease inhibitor cocktail, PMSF, and sodium orthovanadate (10 μL each per mL of RIPA buffer). 6. Incubate on ice for 20 min (see Note 13). 7. Remove lysed cells, using cell scraper if necessary and transfer to a sterile eppendorf. 8. Pass the sample through a 21-gauge needle (with a 1 mL syringe) 3/4 times to shear. 9. Centrifuge at 16,000 × g for 5 min at 4 °C. 10. Transfer the lysate (supernatant) to labeled Eppendorfs in 100–200 μL aliquots and store at −80 °C (see Note 14). 11. Determine protein concentration using the bicinchoninic acid (BCA) protein assay (see Note 15).

RTKs Immunoblotting

7

Table 2 Antibody concentrations, blotting conditions, and recommended positive control for a range of receptor tyrosine kinases, based on the antibodies listed in Table 1 Antibody

Dilution

Blotting conditions

Secondary

+ve control

Anti-HER2

1:1,000

5 % milk/0.1 % PBS-T

Mouse

BT474

Anti-phospho-HER2

1:1,000

2.5 % milk/0.1 % PBS-T

Rabbit

BT474

Anti-EGFR

1:250

2.5 % milk/0.1 % PBS-T

Mouse

MDA-MB-468

Anti-phospho-EGFR

1:1,000

2.5 % milk/0.1 % PBS-T

Rabbit

MDA-MB-468

Anti-HER3

1:1,000

2.5 % milk/0.1 % PBS-T

Rabbit

BT474

Anti-phospho-HER3

1:1,000

2.5 % milk/0.1 % PBS-T

Rabbit

BT474

Anti-IGF1R

1:500

5 % milk/0.1 % PBS-T

Rabbit

BT474

Anti-phospho-IGF1R

1:1,000

2.5 % milk/0.1 % PBS-T

Rabbit

BT474

Anti-MET

1:1,000

2.5 % milk/0.5 % PBS-T

Rabbit

A549

Anti-phospho-MET

1:1,000

2.5 % milk/0.5 % PBS-T

Rabbit

A549 + HGF

See Fig. 2 for examples of western blot results with these antibodies

3.2 SDS Gel Electrophoresis

1. To prepare a 7.5 % resolving gel, mix 3.8 mL of acrylamide, 3.75 mL of resolving buffer, 7.3 mL dH2O, and 150 μL 10 % SDS in a 20 mL container. Add 60 μL of APS and 10 μL of TEMED and pour the gel immediately into a mini-gel cassette (10 × 12 cm). This will be sufficient for two mini-gels. Allow sufficient space for the stacking gel and overlay the resolving gel with isopropanol or water. Allow to set for 45–60 min at room temperature (see Note 16). 2. To prepare a 4 % stacking gel, mix 840 μL of acrylamide, 1.25 mL of stacking gel buffer, 2.84 mL of dH2O and 50 μL of 10 % SDS. Add 20 μL of APS and 5 μL of TEMED and pour immediately. Insert a 10- or 12-well comb, without introducing bubbles, and allow to set for 45–60 min at room temperature. 3. Prepare the protein samples, including positive controls (Table 2; see Note 17), by adding 5× sample buffer (4 μL) to 30 μg (in 16 μL) of protein. Heat the samples at 95 °C for 5 min. Centrifuge briefly. 4. Assemble gel rig and fill tank with SDS-PAGE running buffer. Rinse out wells with buffer using a syringe. Load 20 μL of each sample and 5 μL of pre-stained molecular weight protein marker. 5. Run the gel at a constant voltage of 100–150 V for approximately 1.5 h (until the dye front has reached the bottom of the gel).

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Martina McDermott and Norma O’Donovan 4 sheets of blotting paper

Gel Membrane 4 sheets of blotting paper

Fig. 1 Semidry transfer assembly

6. Following electrophoresis, separate the plates using a spatula and transfer the gel carefully to a dish containing transfer buffer (for semidry transfer) or water (for iBlot transfer) (see Note 18). 3.3 Electrophoretic Transfer 3.3.1 Semi-dry Transfer

1. Cut the nitrocellulose membrane to approximately the size of the gel and soak in transfer buffer. Soak eight pieces of filter paper per blot in transfer buffer. 2. Place the membrane on top of four pieces of pre-soaked filter paper on the semidry blotter (Fig. 1). 3. Carefully transfer the gel on top of the membrane. Gently roll the gel (e.g., with a plastic pipette wetted with transfer buffer) to remove any air bubbles. 4. Place four sheets of pre-soaked filter paper on top of the gel and roll it again. 5. Close the transfer unit and transfer at 250–300 mA for 50 min. 6. After transfer is complete mark the position of the standards with a syringe needle (see Note 19) and disassembly the transfer stack. Check the transfer efficiency by staining the membrane with Ponceau S solution for approximately 1 min, then rinse in dH2O (see Note 20).

3.3.2 iBlot® Transfer

1. Place the iBlot® anode stack into the iBlot gel transfer device, aligned with the gel barrier to the right. 2. Lightly wet the nitrocellulose membrane with ultrapure water, then place the membrane on top of the anode stack. 3. Transfer the gel onto the membrane and overlay with a sheet of pre-wet (ultrapure water) iBlot® filter paper. Remove any air bubbles using the blotting roller (supplied with the iBlot®). 4. Place the cathode stack on top of the filter paper with the electrode side facing up and aligned to the right. Remove air bubbles again using the blotting roller. 5. Place the disposable sponge, with the metal contact on the upper right corner of the lid.

RTKs Immunoblotting

9

6. Close the lid and secure the latch. The red light should come on. 7. Select the correct programme and time and press the Start button. The red light should change to green. 8. When the run is finished, open the lid and discard the sponge and cathode stack. 9. Mark the position of the standards with a syringe needle (see Note 19). 10. Carefully remove and discard the filter paper and gel. Then carefully remove the membrane using a forceps. 11. Check the transfer efficiency by staining the membrane with Ponceau S solution for approximately 1 min, then rinse in dH2O (see Note 20). 3.4 Immunoblotting and Detection

1. Prior to probing, the membrane can be cut to probe for multiple proteins, if their molecular weights are not too close; for example, HER2 (185 kDa), IGF1R (95 kDa) and α-tubulin (55 kDa) could be probed for simultaneously on the same membrane (see Note 21). 2. Incubate the membrane in 10 mL of blocking solution for 1 h at room temperature, shaking (see Note 22). 3. Incubate in 10 mL of blocking solution containing primary antibody (Table 2) overnight at 4 °C, shaking (see Note 23). When examining phosphorylation of a receptor tyrosine kinase it is standard practice to immunoblot for both the phosphoprotein and the total protein (see Note 24). See Fig. 2 for examples of western blots obtained using some of the antibodies listed in Table 2. 4. Wash three times for 10 min in 1× PBS-T, at room temperature shaking (see Note 8). 5. Incubate in 10 mL of blocking solution containing secondary antibody (Table 2) for 1 h at room temperature, shaking. 6. Wash three times for 10 min in 1× PBS-T. 7. Incubate in detection solution at room temperature for 1–5 min with enhanced chemiluminescent substrate, remove substrate, and expose using film or digital imaging (see Note 25). 8. Detection of protein bands can be performed using conventional X-ray film (see Note 26) or using a digital imaging system. 9. Densitometry may be performed using image analysis software (e.g., Image J software, NIH) and densitometry readings for the RTKs can be normalized to α-tubulin, the loading control. Densitometry values for phospho-RTKs can be expressed relative to the corresponding total protein (after normalisation).

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Martina McDermott and Norma O’Donovan

Fig. 2 Examples of blotting for RTKs and phospho-RTKs. (a) Decreased pHER2 in SKBR3 cells treated with lapatinib (L); (b) decreased pHER3 and increased total HER3 protein in SKBR3 cells treated with lapatinib; (c) decreased pEGFR in BT20 cells treated with gefitinib, measured by immunoprecipitation with an EGFR antibody followed by immunoblotting with an anti-phosphotyrosine antibody; (d) increased pIGF1R and IGF1R detected in a lapatinib resistant breast cancer cell line (R) compared to the parental cell line (P); (e) increased pMet in A549 cells treated with the c-Met ligand HGF

10. Membranes can be re-probed for different proteins if they are at different size or a membrane can be blotted for a phosphoprotein, then re-probed for the total protein if the primary antibodies are from different species (see Note 27). 11. Figure 3 outlines some of the common problems encountered.

4

Notes 1. Resolving and stacking gel buffers, running and transfer buffers can be purchased commercially or can be prepared from stock solutions. If preparing the buffers from stock solutions use concentrated hydrochloric acid to adjust the pH of the Tris buffers. 2. 30 % acrylamide solution can be purchased commercially or prepared by combining 29.2 g of acrylamide and 0.8 g of Bis in a final volume of 100 mL ultrapure water. Store at 4 °C

RTKs Immunoblotting

11

Fig. 3 Troubleshooting guide to common problems encountered in western blotting

protected from light for up to 1 month. As unpolymerized acrylamide is a neurotoxin, a protective mask should be worn when weighing it. 3. Ammonium persulfate can either be prepared fresh or aliquoted and stored at −20 °C for up to 6 months. 4. The running buffer can be prepared as a 10× solution, stored at room temperature and diluted to 1× with ultrapure water for use.

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5. Beta-mercaptoethanol is extremely pungent. Addition of 5× sample buffer to protein samples should be performed in a fume hood. 6. The transfer buffer can be prepared as a 10× solution without methanol. To prepare 1× solution dilute 100 mL of 10× buffer to 800 mL with water and add 200 mL of methanol. Methanol is only necessary for nitrocellulose membranes. It can be omitted if you are using PVDF membrane (however, PVDF membrane requires pre-activation with methanol prior to transfer). To facilitate transfer of very large proteins it may be beneficial to reduce the methanol concentration and add SDS to a final concentration of 0.1 %. 7. The iBlot® Gel Transfer Device performs western blotting transfer in seven min without buffers. 8. Tris buffered saline (TBS) can also be used instead of PBS. If difficulties are experienced in detecting phosphoprotein, TBS may improve results. Also replacing milk powder with bovine serum albumin may help, as milk contains phosphoproteins. 9. A control antibody is included to ensure equal loading of proteins and to facilitate normalisation of densitometric measurements. Antibodies for other “housekeeping” proteins can also be used, such as GAPDH or beta-actin. 10. Triplicate independent lysates should be prepared for all experiments. Triplicate densitometry values can then be used for statistical analysis. 11. Lysates can also be prepared from cells grown in 6-well plates or 6 cm petri dishes. The quantity of protein obtained will be smaller but may be sufficient for 3–4 gels. 12. To serum starve cells, remove the medium containing serum, wash the cells three times with either PBS or serum-free medium (SFM), then incubate in SFM for 24 h. 13. Once the RIPA buffer is added to the cells ensure that the lysate is kept on ice. 14. It is essential to aliquot your protein samples in small volumes for storage at −80 °C to minimize repeated freeze-thawing of samples. 15. It is usually necessary to dilute the protein samples 1:10 prior to assay to achieve absorbances within the range of the BSA standard curve. Also due to the high concentration of Igepal 630 (1 %) in the RIPA buffer, samples have to be diluted prior to assay if using the Bradford assay. 16. The resolving gels can be stored overnight at 4 °C. To ensure that the gels do not dry out cover with buffer and wrap with film. 17. It is essential to include a reliable positive control on each gel to confirm that your antibody is detecting the correct protein.

RTKs Immunoblotting

13

Positive controls can also be used to normalise samples to facilitate comparison between samples on different gels. 18. Prior to transferring the gel to the transfer buffer or water, it is recommended to cut off the lanes at the top of the gel as it helps to ensure full contact between the gel and the membrane for the transfer. 19. Using a syringe needle gently punch a hole through the gel and membrane in the centre of each of the molecular weight standard bands. This will facilitate alignment of the developed X-ray film with the membrane. It is also advisable to cut the top left hand corner of the membrane following transfer, in order to maintain the correct orientation of the membrane throughout the blotting procedure. 20. Ponceau S is a rapid and reversible stain for detecting proteins on a Western blot, which facilitates quality control for efficient transfer of proteins, equal loading and degradation of protein, which would be visible as a smear. 21. To ensure that the membrane is cut in the correct position it may be helpful to load molecular weight markers in the first and last lanes on the gel. 22. Ensure that the membrane is completely covered by blocking buffer. Do not allow the membrane to dry at any time during the blotting procedure as this will lead to high background. 23. Antibodies, in blocking solution, can be stored at −20 °C and reused 3/4 times. 24. If a phospho-specific antibody is not available for the receptor tyrosine kinase of interest, it may be possible to immunoprecipitate the protein and immunoblot with a phospho-tyrosine antibody. Immunoprecipitation may also be useful in cases where the level of expression of the protein is low and/or it is difficult to detect the phosphoprotein (Fig. 2c). Furthermore, immunoprecipitation can be used to examine dimerisation between RTKs: by performing an immunoprecipitation with an antibody for one RTK (e.g., HER2) followed by immunoblotting with an antibody for a second RTK (e.g., IGF1R) (see 16). 25. Allow the detection reagents to equilibrate at room temperature for approximately 20 min prior to use. 26. The exposure time will depend on signal-to-noise ratio. Initially perform a short exposure, such as 1 min, and then repeat for longer or shorter as required, keeping in mind that the signal will fade with time. 27. If you are blotting for multiple phospho- and total proteins, we suggest running duplicate gels and probe for the phosphoproteins first, and then probe for the total proteins on the opposite gels. For example, if you blot for pHER2 on gel 1

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and pAkt on gel 2, the re-probe gel 1 for total Akt and reprobe gel 2 for total HER2. This eliminates the possibility of carryover of signal from the phosphoprotein to the total protein.

Acknowledgements This work was supported by the Irish Research Council, Science Foundation Ireland (08SRCB410), the Health Research Board (CSA/2007/11), and the Cancer Clinical Research Trust. References 1. Hubbard SR, Miller WT (2007) Receptor tyrosine kinases: mechanisms of activation and signaling. Curr Opin Cell Biol 19:117–123 2. Lemmon MA, Schlessinger J (2010) Cell signaling by receptor tyrosine kinases. Cell 141:1117–1134 3. Pawson T (2002) Regulation and targets of receptor tyrosine kinases. Eur J Cancer 38(Suppl 5):S3–S10 4. Kamps MP, Sefton BM (1988) Identification of multiple novel polypeptide substrates of the v-src, v-yes, v-fps, v-ros, and v-erb-B oncogenic tyrosine protein kinases utilizing antisera against phosphotyrosine. Oncogene 2:305–315 5. Freed E, Hunter T (1992) A 41-kilodalton protein is a potential substrate for the p210bcrabl protein-tyrosine kinase in chronic myelogenous leukemia cells. Mol Cell Biol 12:1312–1323 6. Czernik AJ, Girault JA, Nairn AC et al (1991) Production of phosphorylation state-specific antibodies. Methods Enzymol 201:264–283 7. Wandell JW (2003) Phosphorylation statespecific antibodies: applications in investigative and diagnostic pathology. Am J Pathol 163:1687–1698 8. Brumbaugh K, Johnson W, Liao WC et al (2011) Overview of the generation, validation, and application of phosphosite-specific antibodies. Methods Mol Biol 717:3–43 9. Espina V, Edmiston KH, Heiby M et al (2008) A portrait of tissue phosphoprotein stability in

10.

11.

12.

13.

14.

15. 16.

the clinical tissue procurement process. Mol Cell Proteomics 7:1998–2018 Gündisch S, Hauck S, Sarioglu H et al (2012) Variability of protein and phosphoprotein levels in clinical tissue specimens during the preanalytical phase. J Proteome Res 11:5748–5762 Mueller C, Edmiston KH, Carpenter C et al (2011) One-step preservation of phosphoproteins and tissue morphology at room temperature for diagnostic and research specimens. PLoS One 6:e23780 Burns JA, Li Y, Cheney CA, Ou Y et al (2009) Choice of fixative is crucial to successful immunohistochemical detection of phosphoproteins in paraffin-embedded tumor tissues. J Histochem Cytochem 57:257–264 Kyhse-Andersen J (1984) Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose. J Biochem Biophys Methods 10:203–209 Tovey ER, Baldo BA (1987) Comparison of semi-dry and conventional tank-buffer electrotransfer of proteins from polyacrylamide gels to nitrocellulose membranes. Electrophoresis 8:384–387 http://www.lifetechnologies.com/order/catalog/product/IB1001 Browne BC, Crown J, Venkatesan N et al (2011) Inhibition of IGF1R activity enhances response to trastuzumab in HER-2-positive breast cancer cells. Ann Oncol 22:68–73

Chapter 2 Analysis of Changes in Phosphorylation of Receptor Tyrosine Kinases: Antibody Arrays Sweta Rani and Lorraine O’Driscoll Abstract Tyrosine kinases are mainly classified into two groups, as receptor tyrosine kinase (RTK) and non-receptor tyrosine kinase (NRTK). The RTK family of transmembrane ligand-binding proteins are important mediators of the signaling cascade and includes EGFR, PDGFR (platelet-derived growth factor receptors), FGFR (fibroblast growth factor receptor) and the IR (insulin receptor). RTKs comprise 59 members and their structure includes an extracellular ligand-binding domain, a transmembrane domain, and an intracellular domain possessing the tyrosine kinase activity. This chapter focuses on antibody arrays that are basically used to analyse phosphorylation and dephosphorylation of RTKs. Antibody arrays include well-characterized antibodies for profiling, changes in RTK expression, and comparison between normal, diseased, or treated samples. Key words Kinase activity, Cell lysis, Protein arrays, Phosphorylation, Receptor tyrosine kinase

1

Introduction RTK signaling pathways have been associated with various functions including regulation of cell cycle, proliferation, differentiation, and migration [1–3]. Activation of RTKs initiates the signal transduction pathways [4]. This chapter focuses on antibody arrays that are basically used to analyze phosphorylation and dephosphorylation of RTKs. Most of the antibody arrays available comprise either nitrocellulose membranes or transparent 96-well polystyrene plates containing capture and control antibodies. The respective phosphorylated and non-phosphorylated RTKs in sample lysate bind to these antibodies through their extracellular domain and unbound material is washed off. The arrays are then incubated with anti-phosphotyrosine-HRP that sandwiches with phosphorylated RTKs captured on the array. Following second wash these complexes are then visualized by chemiluminescence. Signal generated at each

Serena Germano (ed.), Receptor Tyrosine Kinases: Methods and Protocols, Methods in Molecular Biology, vol. 1233, DOI 10.1007/978-1-4939-1789-1_2, © Springer Science+Business Media New York 2015

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1. Isolate total protein from cell lines

Cell pellet

Lysed protein

2. Prepare the array and reagent for profiling Apply proteins onto the array

3. RTKs in sample lysate are captured by antibodies

4. Anti-Phospho-TyrosineHRP Detection Antibody is incubated for 2 hours

5. Protein phosphorylation is determined using signal density

Fig. 1 Flow chart of the phosphoprotein array procedure

array spot is proportional to the amount of phosphoprotein bound by each capture antibody. A flow chart is included to guide the readers (Fig. 1).

2 2.1

Materials Cell Culture

1. SKBR3 cells (American Type Tissue Collection, Rockville, MD, USA). 2. Trastuzumab-resistant SKBR3 cells: Established by continuous exposure to 1.4 μM trastuzumab for 9 months [5]. 3. Culture medium: RPMI-1640 medium supplemented with 1 mM L-Glutamine and 10 % foetal bovine serum (FBS). 4. Tissue culture-grade vented flasks (e.g., 175 cm2). 5. Trastuzumab (Roche, Penzberg, Germany).

2.2

Cell Lysis

1. Lysis buffer (Life Technologies, Carlsbad, CA, USA). Store at −20 °C (see Note 1). 2. Phenylmethylsulfonyl fluoride (PMSF, Sigma-Aldrich, St Louis, MO, USA): Prepare 0.3 M stock in DMSO and store at −20 °C.

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17

3. Purified deionized water (dH2O). 4. 20× Protease inhibitor cocktail (Sigma-Aldrich). Store at −20 °C. Dissolve the contents of the vial in 10 mL of dH2O, and then transfer to another container for dilution to 100 mL. 5. Microcentrifuge. 2.3 Protein Quantification

1. Bradford Protein Assay (Bio-Rad, Hercules, CA, USA). Store at 4 °C. 2. Bovine serum albumin (BSA). Store at 4 °C. Once in solution store at −20 °C. 3. 96-Well plates. 4. Spectrophotometer microplate reader.

2.4 Phosphoprotein Arrays

1. Human Phospho-RTK array (R&D Systems, Minneapolis, MN, USA): The kit includes array buffer 1, array buffer 2, wash buffer, chemi-reagent 1, chemi-reagent 2, anti-phosphotyrosine-HRP detection antibody, 4-well multi-dish, and transparency overlay template. Store the reagents as recommended by the manufacturer (see Note 2). 2. Flat-tip tweezers. 3. Rocking platform shaker. 4. Plastic container with the capacity to hold 50 mL (for washing the arrays). 5. Plastic transparent sheet protector (trimmed to 10 cm × 12 cm and opened on three sides). 6. Plastic wrap, paper towels, absorbent wipes. 7. X-ray film and autoradiography film cassette. 8. Flatbed scanner with transparency adapter capable of transmission mode.

2.5

Immunoblotting

1. 4× Laemmli buffer: 8 % sodium dodecyl sulfate (SDS), 40 % glycerol, 0.25 M Tris–HCl, pH 6.8, 0,04 % (w/v) bromophenol blue (BPB). Store at −20 °C. 2. Precast polyacrylamide electrophoresis gels. Store at 4 °C. 3. 10× running buffer: 0.25 M Tris-Hcl, 1.92 M glycine, 1 % SDS, pH 8.3. Store at room temperature. 4. Prestained molecular weight markers. 5. Mini-slab size electrophoresis system. 6. 1× Transfer buffer: 23 mM Tris, 175 mM glycine, 20 % methanol. 7. PVDF membrane and blot paper. 8. 10× Tris-buffered saline (TBS, 10×): 100 mM Tris–HCl, pH 7.5, 1.5 M NaCl. Store at room temperature.

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9. Blocking buffer: 1× TBS containing 5 % low-fat dry milk or BSA. 10. Washing buffer (TBS-T): 1× TBS solution supplemented with 1 % Tween-20. Store at room temperature. 11. Antibody dilution buffer: 1× TBS solution supplemented with non-fat milk/BSA and 1 % Tween-20. Single aliquots frozen at −20 °C. 12. HRP-conjugated IgG secondary antibodies. 13. Semidry electroblotting system. 14. Enhanced chemiluminescent substrate. 15. X-ray film or digital imaging system.

3

Methods

3.1 Cell Culture and Treatment

1. SKBR3 and their resistant variant are grown in 75 cm2 flask till 60–70 % confluence. 2. Cells are washed twice with PBS. 3. SKBR3 cells and their resistant variant are serum-starved by incubation in serum-free medium for 24 h. 4. Cells are synchronized after serum starvation. 5. To analyze the effect of trastuzumab on RTKs, the resistant cell line is incubated with 2 μM trastuzumab in serum-depleted medium for further 24 h. 6. SKBR3 cells are incubated in serum-depleted medium only for further 24 h.

3.2

Cell Lysis

1. Scrape the cells from culture flasks (adherent) or collect cells in PBS by centrifugation (non-adherent). 2. Wash the cells twice with cold PBS. 3. Remove and discard the supernatant and collect the cell pellet. It can either be stored at −80 °C or used straight away. 4. Prepare lysis buffer (thawed on ice) by adding PMSF to a final concentration of 1 mM (see Note 3). 5. Add reconstituted protease inhibitor cocktail (250 μL per 5 mL) to cell lysis buffer. 6. Lyse cell pellet in lysis buffer for 30 min on ice and vortex at 10 min intervals. 7. Transfer the lysates to 1.5 mL microcentrifuge tubes and centrifuge at 16,000 × g for 10 min at 4 °C. 8. Aliquot the clear lysates to clean microcentrifuge tubes. These samples can be used immediately or they can be stored at −80 °C. Avoid multiple freeze/thaw of cell lysates.

Changes in Phosphorylated RTKs – Antibody Arrays

3.3 Protein Quantification

19

1. Prepare the stock solution of BSA at a concentration of 1 mg/ mL using dH2O. 2. Dilute the stock solution further to 0.1, 0.2, 0.3, 0.4, and 0.5 mg/mL with dH2O for protein standards (see Note 4). 3. Dilute the protein lysates with dH2O up to a volume of 20 μL at a ratio of 1:10. 4. Diluted lysates and standards (10 μL each) are pipetted in duplicate into each well of a 96-well plate. 5. Dilute the Bio-Rad protein assay reagent at a ratio of 1:5 using dH2O. 6. Pipette 200 μL of diluted reagent into each well containing protein and standards. 7. Measure the absorbance at 595 nm. 8. Use the values from the standards to plot a standard curve and to determine the protein content of the samples.

3.4 Phosphoprotein Arrays

1. Bring all reagents to room temperature before use. Keep samples on ice (see Note 5). 2. Pipette 2.0 mL of array buffer 1 into each well of the 4-well multi-dish (provided with the kit). Array buffer 1 is used as a blocking buffer. 3. Using a flat-tip tweezers, carefully remove each array from the protective sheets. 4. Place each array into each well of the 4-well multi-dish with array number facing upward (see Note 6). 5. Place the 4-well multi-dish on a rocking platform shaker and incubate for 1 h at room temperature, rocking the array from end to end in its well. 6. While the arrays are blocking, prepare 50 μg of whole-cell lysate by diluting it with array buffer 1 to a final volume of 1.5 mL. The amount of protein needed varies with the cell line used and the level of expression of the protein of interest; therefore optimization is required before using the array. 7. Array buffer 1 is aspirated from the 4-well multi-dish. Samples are then added onto the 4-well multi-dish and covered with lid. 8. Incubate the samples overnight at 2–8 °C on a rocking platform shaker (see Note 7). 9. Wash the membrane by carefully removing each array and placing them into individual plastic containers with 2 0 mL of 1× wash buffer (see Note 8). Thoroughly rinse the 4-well multidish with dH2O and dry. 10. Wash each array twice using 1× wash buffer for 10 min on a rocking platform shaker (total of three washes).

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11. Prepare fresh 1× array buffer 2 (see Note 9) and anti-phosphotyrosine-HRP detection antibody (see Note 10). Pipette 2.0 mL of diluted detection antibody into each well of the 4-well multi-dish. 12. Carefully remove each array from its wash container, draining the excess wash buffer from the array. Return the arrays to the 4-well multi-dish containing the diluted anti-phosphotyrosine-HRP detection antibody and cover with the lid. 13. Incubate for further 2 h at room temperature on a rocking platform shaker. 14. Wash each array as described in steps 9 and 10. 15. Complete all the subsequent steps without interruption. 16. Carefully remove each membrane from its wash container. Drain excess wash buffer from the membrane by blotting the lower edge onto paper towels. Place each membrane on the bottom sheet of the plastic sheet protector with the identification number facing up. 17. Pipette 1 mL of the prepared chemi-reagent mix evenly onto each membrane (see Note 11). 18. Cover the membrane with the top sheet of the plastic sheet protector and gently smoothed out of any air bubbles. Carefully ensure that chemi-reagent mix was spread evenly to all corners of each membrane. 19. Incubate the membrane for 1 min in chemi-reagent mix. 20. Take care to squeeze out the excess chemi-reagent mix by positioning the paper towels on the top and sides of the plastic sheet protector containing the membranes. 21. After removing the top plastic sheet protector carefully blot out any remaining chemi-reagent mix by laying an absorbent wipe on top of the membranes. 22. Leave the membrane at the bottom of plastic sheet protector, and wrap it using plastic wrap to cover the membranes, taking care to gently smooth out any air bubbles. 23. Place the membranes with the identification numbers facing up in an autoradiography film cassette (see Note 12). 24. Then expose the membranes using X-ray film for multiple exposure time ranging between 1 and 10 min (see Note 13). 3.5

Data Analysis

1. The positive signals on developed films are identified by placing the transparent overlay template on the array image and aligning it with the pairs of reference spots in three corners of each array. The stamped identification number on the array must be placed on the left-hand side (see Note 14).

Changes in Phosphorylated RTKs – Antibody Arrays

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2. Transmission mode scanner and image analysis software are used to collect and analyze the pixel densities on developed X-ray film. 3. A template is created to analyze pixel density in each spot of the array. 4. The signals (pixel density) of the pair of duplicate spots representing each RTK are averaged. 5. For the background value a signal from a clear area of the array or the PBS-negative control spots is selected. The averaged background signal is subtracted from each RTK signal. 6. Corresponding signals on different arrays are compared to determine the relative change in tyrosine phosphorylation of specific RTKs between samples. 7. Fold change are calculated and proteins with highest difference in expression are selected for further validation using immunoblotting. 3.6

Immunoblotting

Selected differentially regulated proteins are validated in protein lysates isolated from SKBR3 cells and their resistant variant after treatment. 1. Prepare samples by diluting the protein lysates in 4× Laemmli buffer and heat them on a heating block at 95 °C for 5 min. 2. Place the precast gels in electrophoresis device used. 3. Remove the comb and wash the wells to get rid of any trapped bubbles. 4. Load wells with the samples and the molecular markers. 5. Set up the unit and run at constant voltage up to 130 V to carry samples through the stacking gel, and then increase to 150 V to run through the resolving gel. 6. Turn off the power supply immediately after the Lamelli buffer has run off the gel. 7. Cut a sheet of PVDF paper to the size of the gel size and activate the membrane for 1 min using methanol. Transfer the activated PVDF membrane in another tray filled with transfer buffer. Blot papers are soaked in transfer buffer. 8. Hold the cassette in one hand and use the comb to separate the plates. Loosen the gel and carefully cut and remove the stacking gel with a blade. Transfer the gel into a tray containing transfer buffer. 9. Transfer cassette is assembled by laying the PVDF membrane on the top of the blot paper and then the gel on top of the membrane. Place another blot paper on top of the gel, roll with a roller to make sure that no air bubbles exist between gel and the membrane.

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10. Close the transfer cassette, connect to the power supply and begin transfer, with a constant current of 200 mA for 1 h. 11. Disconnect the power supply after an hour and disassemble the transfer cassette. Remove the PVDF membrane and check for the prestained markers bands clearly visible on the membrane. 12. Block the membrane by incubating it in blocking buffer for 1 h at room temperature on a rocker with gentle shaking. 13. Wash the membrane three times using TBS-T. 14. Incubate the membrane for 1 h at room temperature or overnight at 4 °C (depending on the manufacturer’s instructions) with primary antibody by gentle shaking. 15. Wash the membrane three times with TBS-T for 10 min with vigorous shaking. 16. Freshly prepared secondary antibody solution is added to the membrane for 1 h at room temperature with gentle shaking. The secondary antibody solution can be reused if stored appropriately. 17. Wash by vigorous shaking three times with TBS-T for 10 min. 18. Mix the ECL reagents together at a ratio of 1:1 immediately before use and evenly add the mix to the blot for 5 min. 19. Remove excess ECL. 20. Expose the membrane using film or digital imaging.

4

Notes 1. Lysis buffer should be thawed on ice and working aliquots stored at −20 °C to avoid repeat freezing and thawing. 2. Return unused membranes to the foil pouch containing the desiccant pack. Reseal along entire edge of the zip-seal. May be stored for up to 3 months at 2–8 °C and used within expiry date. 3. PMSF is very unstable and must be added just prior to use. 4. BSA stock and dilution can be reused if stored properly in −20 °C and thawed on ice. 5. To avoid contamination, wear gloves while performing the procedures. 6. When array membrane comes in contact with array buffer 1 the blue dye will disappear from the spots. The capture antibodies are retained in their specific locations. 7. Incubation time can be optimized depending on the abundance of protein present in the samples. 8. Bring the 25× wash buffer concentrate to room temperature and if crystals are formed shake gently till all the crystals have

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completely dissolved. Dilute 40 mL of 25× wash buffer concentrate into 960 mL of dH2O. 9. Prepare this buffer fresh before each use by adding 2 mL of concentrated array buffer 8 mL of dH2O. 10. Detection antibody must be diluted using 1× array buffer 2 immediately before use. Prepare only as much detection antibody as needed to run each experiment. 11. Chemi-reagent mix is prepared by mixing chemi-reagents 1 and 2 in equal volumes within 15 min of use. The mixture must be protected from light. 1 mL of the resultant mixture must be prepared, as using less than 1 mL of chemi-reagent mix per membrane may result in incomplete membrane coverage. 12. Use clean autoradiography cassette. 13. Multiple exposure time is recommended to capture the low signal as well. 14. Reference spots are included to align the transparency overlay template and to demonstrate that the array has been incubated with the anti-phospho-tyrosine-HRP during the assay procedure.

Acknowledgements Science Foundation Ireland’s funding of MTCI [08/SRC/ B1410]; the Higher Education Authority’s PRTLI Cycle 5 support of TBSI; Irish Cancer Society’s support of Breast-PREDICT [CCRC13GAL]; and the Health Research Board [HRA_POR/ 2013/342]. References 1. Linger RM, Keating AK, Earp HS et al (2008) TAM receptor tyrosine kinases: biologic functions, signaling, and potential therapeutic targeting in human cancer. Adv Cancer Res 100:35–83 2. Grassot J, Gouy M, Perriere G et al (2006) Origin and molecular evolution of receptor tyrosine kinases with immunoglobulin-like domains. Mol Biol Evol 23:1232–1241 3. Lemmon MA, Schlessinger J (2010) Cell signaling by receptor tyrosine kinases. Cell 141: 1117–1134

4. Koytiger G, Kaushansky A, Gordus A et al (2013) Phosphotyrosine signaling proteins that drive oncogenesis tend to be highly interconnected. Mol Cell Proteomics 12: 1204–1213 5. Konecny GE, Pegram MD, Venkatesan N et al (2006) Activity of the dual kinase inhibitor lapatinib (GW572016) against HER-2-overexpressing and trastuzumab-treated breast cancer cells. Cancer Res 66: 1630–1639

Chapter 3 Analysis of Epidermal Growth Factor Receptor Dimerization by BS3 Cross-Linking Harmony F. Turk and Robert S. Chapkin Abstract Dimerization of receptor tyrosine kinases is a well-characterized process. It is imperative for the activation of many receptors, including the epidermal growth factor receptor (EGFR). EGFR has been shown to be regulated by a number of factors, including lipid raft localization. For example, alteration of the lipid raft localization of EGFR has been demonstrated to modify receptor dimerization. This protocol describes an assay to quantify EGFR dimers using BS3 cross-linking. BS3 cross-linking is well suited for this purpose because of its length, water solubility, and membrane impermeability. Although this protocol is written specifically for EGFR, the assay can be extrapolated in order to characterize dimerization of other receptor tyrosine kinases. Key words Epidermal growth factor receptor, Dimerization, Lipid rafts, BS3, Cross-linking, DHA

1

Introduction Receptor dimerization is often an indispensable step in receptor tyrosine kinase activation. For example, the epidermal growth factor receptor (EGFR) is a prototypical dimerization-activated receptor tyrosine kinase. Following ligand binding to EGFR, the receptor undergoes conformational changes that lead to the projection of the dimerization loop which facilitates the interaction with another ligand bound EGFR or other EGFR-family dimerization partner [1]. Dimerization is essential to enable the intracellular kinase domain of EGFR to become activated. Signaling of many receptor tyrosine kinases is known to be dependent on the localization of these receptors to specific membrane domains, such as lipid rafts or caveolae. EGFR has been demonstrated to localize to lipid rafts (nanometer sized heterogeneous cholesterol enriched mesodomains), and the signaling capacity of EGFR is regulated by its lipid raft localization [2–4]. Perturbations to lipid rafts can alter EGFR localization and activity. It has been shown that disruption of lipid rafts increases EGFR

Serena Germano (ed.), Receptor Tyrosine Kinases: Methods and Protocols, Methods in Molecular Biology, vol. 1233, DOI 10.1007/978-1-4939-1789-1_3, © Springer Science+Business Media New York 2015

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clustering prior to ligand stimulation [5, 6], as well as increases EGFR dimerization upon ligand stimulation [2, 3]. Therefore, receptor dimerization status is a central part of studies on the lipid raft localization of EGFR. Lipid rafts can be disrupted in a variety of different ways. A commonly used technique is to extract cholesterol, a major component of lipid rafts, from the membrane using methyl-β cyclodextrin (MβCD). This is a very harsh treatment and has very strong effects on lipid rafts. In many of the studies conducted in our laboratory, we assess the effects of long-chain omega-3 fatty acids on lipid raft-mediated processes. Docosahexaenoic acid (DHA) is an omega-3 fatty acid consisting of 22 carbons and six double bonds. Due to the length and high degree of unsaturation of DHA, it is sterically incompatible with cholesterol [7]. Unlike MβCD, DHA subtly alters the size, composition, and function of lipid rafts without completely disrupting them. We have previously demonstrated that DHA shifts the localization of EGFR from lipid rafts into the bulk membrane [3]. The altered localization of EGFR upon DHA treatment leads to increased receptor dimerization upon stimulation with the EGFR-specific ligand, EGF. Interestingly, although EGFR dimerization and phosphorylation, which are considered hallmarks of receptor activation, are increased in DHA-treated cells, downstream signaling from the receptor is suppressed. These data suggest a central role for lipid rafts in regulating receptor tyrosine kinase activity. Herein, we describe a method to assess receptor dimeri zation at the plasma membrane of adherent cells using bis[sulfosuccinimidyl] suberate (BS3) to cross-link dimers. BS3 contains two N-hydroxysulfosuccinimide (NHS) ester active groups on each end, connected by a 11.4 Å spacer arm of eight carbons. The NHS groups of BS3 rapidly react with primary amines on lysine residues and the N-terminal region of proteins. BS3 is water soluble up to approximately 100 mM at a pH of 7–9, which makes it ideal for use in live cell receptor cross-linking assays. Furthermore, the intermediate-length spacer arm works well for cross-linking EGFR dimers formed upon ligand stimulation. BS3 is noncleavable, so it cannot be utilized for reversible cross-linking. BS3 is well suited for studies on plasma membrane lipid rafts because it is membrane impermeable and will only label proteins on the cell surface.

2 2.1

Materials Cell Culture

1. Cell incubator (33 °C and 5 % CO2). 2. 150 mm culture dishes. 3. Young adult mouse colonocytes (YAMC); this is a nonmalignantly transformed, conditionally immortalized cell line

Analysis of EGFR Dimerization

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[8]. The cells express a temperature-sensitive mutant of the SV40 large T-antigen that is inducible with interferon (IFN)-γ. The temperature-sensitive protein is active at 33 °C but inactive at 37 °C. Therefore, unlike most mammalian cell lines, this cell line is maintained at 33 °C. This protocol is equally applicable for cell lines grown at 37 °C. 4. RPMI 1640 complete medium: To a 500 mL bottle add 532 μL insulin, transferrin, selenous acid (ITS) Premix (BD Biosciences, CA, USA), 26.6 mL fetal bovine serum (FBS), and 5.3 mL Glutamax (Gibco, Grand Island, NY, USA). Immediately prior to use, add 1 μL IFNγ (Gibco) per 10 mL medium. 5. RPMI 1640 serum deprivation medium: To serum-free, 1 % Glutamax RPMI 1640 medium, add FBS to a final concentration of 0.5 %. Also add 1 μL IFNγ per 10 mL medium. This medium should not have ITS. 2.2

EGFR Stimulation

1. Murine EGF. 2. Ligand stimulation medium: Serum-free RPMI 1640 medium supplemented with 25 ng/mL EGF. This medium should not have any serum or ITS.

2.3 BS3 CrossLinking

1. BS3 (Thermo Scientific, Rockford, IL, USA): Prepare 3 mM BS3 immediately prior to use by adding 8.58 mg BS3 to 5 mL of 1× Ca2+-, Mg2+-free PBS. Prepare 5 mL per sample. Maintain on ice until ready to use. 2. 250 mM glycine (can be prepared in advance): Add 1.88 g of glycine to 100 mL 1× PBS. Store at 4 °C. 3. 1× Ca2+-, Mg2+-free PBS.

2.4 Cell Lysate Harvest

1. Rubber policeman.

2.5 Protein Quantification

1. Coomassie Plus assay kit (Thermo Fisher Scientific, Rockford, IL, USA).

2. Homogenization buffer (to be prepared the day of use): 50 mM Tris–HCl, pH 7.2, 250 mM sucrose, 2 mM EDTA, 1 mM EGTA, 50 mM sodium fluoride, 1 % triton X-100, 100 μM activated sodium orthovanadate, 10 mM β-mercaptoethanol, Protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA) diluted 1:25. Maintain on ice until ready to use.

2. Bovine serum albumin (BSA). 3. Disposable borosilicate glass tubes. 4. 96-Well clear polystyrene plate. 5. Spectrophotometer plate reader.

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2.6 Western Immunoblotting

1. Heating block. 2. Western blotting apparatus. 3. PVDF membranes. 4. 5× Western blot loading buffer: 0.25 % bromophenol blue, 0.5 M dithiothreitol (DTT), 50 % Glycerol, 10 % sodium dodecyl sulfate (SDS), 0.25 M Tris–HCl, pH 6.8. 5. Wash buffer: 0.1 % Tween-20 in 1× PBS (PBST). 6. Blocking solution and antibody diluent solution: 5 % BSA in PBST. 7. Enhanced chemiluminescent substrate. 8. High-molecular-weight protein marker (preferably prestained). 9. Rabbit monoclonal anti-EGFR antibody (Cell Signaling Technology, Danvers, MA, USA). 10. HRP-conjugated goat anti-rabbit IgG antibody. 11. Film or digital imaging system.

3

Methods Prior to beginning the experiment: It is necessary to have healthy cells growing in culture. Cells should be maintained in the log phase of growth by passaging the cells when the dish is approximately 70 % confluent. It is best to use early-passage cells to avoid any changes that could occur from prolonged culturing which might result in irreproducible results.

3.1

Day 1

1. Seed YAMC cells onto 150 mm dishes at 5.0 × 105 cells per dish in 15 mL of complete (full serum) media (see Note 1). 2. Incubate cells overnight in a 33 °C incubator with 5 % CO2.

3.2

Day 2

1. On the following day, aspirate the media from the dishes and wash the cells one time with room temperature 1× PBS. 2. Add 15 mL of serum deprivation media (0.5 % FBS) to the dishes (see Note 2). 3. Incubate cells at 33 °C with 5 % CO2 overnight (16–18 h).

3.3

Day 3

1. The following morning, remove the media and wash cells twice with room temperature 1× PBS. 2. Aspirate the PBS, place the dishes on ice, and add 15 mL ligand stimulation media, serum-freed media supplemented with 25 ng/mL mouse EGF (see Note 3). 3. Incubate cells for 1 h on ice (see Note 4). 4. Wash the dishes three times with ice-cold 1× Ca2+-, Mg2+-free PBS while maintaining the cells on ice (see Note 5).

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5. Add 5 mL of 3 mM BS3 (in ice-cold 1× Ca2+-, Mg2+-free PBS) to each dish and incubate for 20 min on ice (see Note 6). 6. Quench the excess BS3 with 10 mL of 250 mM glycine in 1× PBS for 5 min at 4 °C. 7. Wash the dishes three times with ice-cold 1× PBS. Completely remove all of the PBS after the final wash. 8. Add 300 μL of homogenization buffer to each plate. Thoroughly scrape the entire cell layer with a rubber policeman. 9. Collect the scraped cell homogenate into 1.5 mL Eppendorf microcentrifuge tubes. Place the tubes on ice. 10. Lyse the cell homogenates by passing the cells through a 29G needle once. Flush the suspension very hard through the needle while maintaining the tube on ice to sheer the cells. 11. Incubate the total cell suspension on ice for 30 min. 12. Centrifuge at 16,000 × g at 4 °C for 20 min. 13. Transfer the supernatant (total cell lysate) to a new 1.5 mL microcentrifuge tube being careful not to disturb the pellet. 14. Mix the total cell lysate by pipetting up and down approximately five times. 15. Aliquot the lysate into 20–30 μL aliquots in 0.5 mL microcentrifuge tubes. Also, prepare one aliquot with only 10 μL to use for protein quantification. 16. Store the aliquots at −80 °C until ready to use. 3.4 Protein Quantification

1. Prepare samples in disposable borosilicate glass tubes by combining 2.5 μL of total cell lysate, 497.5 μL of double distilled water, and 500 μL of Coomassie Plus reagent. 2. Using BSA prepare standards of 0, 0.5, 1, 2, 4, 10, and 20 μg in a volume of 497.5 μL with double distilled water. Add 2.5 μL of homogenization buffer and 500 μL of Coomassie Plus reagent. Prepare samples and standards in triplicate in order to obtain a highly accurate protein concentration. 3. Vortex the samples for 5 s. 4. Add 300 μL of each sample to a designated well on a 96-well clear polystyrene plate. Use a plate reader spectrophotometer and quantify the absorption of the standards and samples at 595 nm. 5. Use the standards to calculate a standard curve and quantify the protein concentration of the samples.

3.5

Western Blotting

1. Prepare protein samples with 25 μg of protein. Bring all samples to a final volume of 20 μL with homogenization buffer or water. Add 5 μL of 5× loading buffer. Heat samples on a heating block at 98 °C for 10 min.

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2. Assess receptor dimerization by immunoblotting for EGFR. Follow a standard protocol for immunoblotting with the following slight alterations. 3. Using a 4–10 % tris-glycine gradient gel, SDS-PAGE should be run for approximately 4–5 h at 125 V (see Note 7). 4. Incubate the gel in transfer buffer for ~15 min before beginning the transfer. 5. Transfer onto a PVDF membrane overnight at 400 mA at 4 °C with a magnetic stirrer set to low speed (see Note 8). 6. Block the membrane with blocking buffer at room temperature for 1 h with slight agitation. 7. Probe the membrane with anti-EGFR rabbit monoclonal antibody at a dilution of 1:1,000 in antibody dilution buffer. Incubate with slight agitation overnight at 4 °C. 8. Wash the membrane three times for 10 min each with PBST and moderate agitation. 9. Incubate the membrane with HRP-conjugated goat anti-rabbit IgG at a dilution of 1:10,000 in antibody dilution buffer at room temperature with slight agitation for 1 h. 10. Wash the membrane three times for 10 min each with PBST and moderate agitation. 11. Incubate blot for 5 min with enhanced chemiluminescent substrate, remove substrate, and expose using film or digital imaging. 12. Quantify the intensity of the bands using image analysis software, such as Image J (NIH). Homodimers on the Western blot should be twice the molecular weight of the single receptor. The molecular weight of heterodimers can be calculated by adding the molecular weight of the individual receptors involved. Serum starved, unstimulated cells should be used as a control to determine the amount of dimer formation in unstimulated conditions.

4

Notes 1. The number of cells seeded on day 1 should enough for the dish to be approximately 90–95 % confluent after 48 h. It is important to determine the effect of serum starvation on the cell growth to calculate the number of cells to be seeded. 2. The serum deprivation step will reduce receptor signaling and increase sensitivity of cells to stimulation the following day. Additionally, serum deprivation allows for the detection of effects that are specific to stimulation with the ligand of interest

Analysis of EGFR Dimerization

31

since FBS contains many growth factors that stimulate receptor tyrosine kinases, including EGFR. It may be necessary to reduce further the amount of serum or to increase the length of time that cells are serum deprived depending on the cell line utilized and the experimental design. 3. The amount of EGF utilized in this protocol is an intermediate dose of EGFR ligand for stimulation of mouse colonic epithelial cells. In the literature the concentration of EGF utilized to stimulate EGFR ranges from less than 1 ng/mL to above 100 ng/mL. The amount of ligand should be adjusted according to the cell line and experiment. It is also necessary at this step to have an unstimulated control in order to observe ligand-dependent changes in receptor dimerization and to determine the status of EGFR dimers prior to stimulation. 4. Incubation on ice during ligand stimulation allows for ligand binding and receptor dimerization but inhibits receptor endocytosis. This is important due to the membrane impermeability of BS3. 5. It is important to thoroughly remove the amine-containing culture media from the dishes by washing before cross-linking with BS3. 6. This volume of 5 mL of 3 mM BS3 solution is enough to just cover the bottom of the dish. Ensure that the entire dish is covered by the BS3 solution and that the dish is flat on the ice so that the solution does not collect on the side of the dish. 7. The 4–10 % gradient gel works well for separation of highmolecular-weight proteins/complexes. The separation is further improved by the long (4–5 h) migration time. It is required to have efficient separation of the monomers and dimers. It is best to have a colored molecular weight marker to ensure separation at high molecular weights. 8. The long transfer time ensures efficient and complete transfer of proteins at high molecular weights. 9. This protocol describes an assay to assess dimerization of EGFR by cross-linking the dimers after ligand stimulation. As seen in Fig. 1, EGFR in unstimulated samples is observed as a monomer at 170 kDa. Upon stimulation with EGF, EGFR dimers are detected at a molecular weight of 340 kDa. Although this protocol is written specifically for EGFR, it can be modified in order to analyze other membrane receptor tyrosine kinases. For each receptor, tests are required to determine the optimal amount of FBS for serum deprivation conditions, the duration of serum deprivation, the concentration of ligand, and the duration of stimulation.

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Fig. 1 Representative Western immunoblot. In unstimulated samples, no EGFR dimers are observed. Upon stimulation with EGF, a band of EGFR dimers (340 kDa) is observed at a molecular weight of twice the EGFR monomers (170 kDa)

10. Many receptor tyrosine kinases have been demonstrated to form homodimers and/or heterodimers. This protocol can be utilized to assess both homodimerization and heterodimerization. To assess heterodimerization, it will be necessary to perform Western blots for each receptor in the dimer. Since this protocol uses a PVDF membrane, the same membrane can be utilized for analysis of each individual receptor by stripping the membrane between probing for each receptor. Additionally, immunoprecipitation prior to Western blotting may be suitable. Oligomers can also be cross-linked using this method; however, alterations might be required depending on the size of the oligomers. Furthermore, this protocol utilizing BS3 can be used to cross-link receptor-ligand interactions. The described protocol is very versatile and can be easily adapted to assess numerous aspects of receptor tyrosine kinase function at the plasma membrane. 11. This protocol is specific for cross-linking proteins at the plasma membrane because BS3 is membrane impermeable. BS3 has been used to cross-link intracellular EGFR dimers [9], but the cells must be permeabilized during the time of BS3 incubation. Alternatively, disuccinimidyl suberate (DSS), a membranepermeable equivalent to BS3, can be substituted. The drawback of DSS usage is that DSS is not water soluble. Therefore, it must first be dissolved in an organic solvent. Overall, alterations to the protocol would be required in order to assess intracellular dimers.

Analysis of EGFR Dimerization

33

Fig. 2 Effect of fatty acid treatment on EGFR dimerization. YAMC cells were treated for 72 h with no fatty acid, linoleic acid (LA-18:2∆9,12), or DHA (22:6∆4,7,10,13,16,19) and serum deprived for the final 24 h. Cells were then stimulated with 25 ng/mL EGF and subjected to BS3 chemical cross-linking as described by the protocol. Cell lysates were assessed by Western blotting for EGFR. Figure reproduced with permission from Turk et al. 2012 [3]

12. Receptor dimerization can be largely affected by alterations to lipid rafts. An important aspect of lipid raft function is the fatty acid composition of the membrane. Treating cells with fatty acids, like DHA, changes the fatty acid composition of the membrane. DHA is incorporated into the plasma membrane of mouse colonocytes where it has been shown to perturb lipid raft mesodomains. In order to assess the effect of fatty acid treatment on receptor tyrosine kinase dimerization, cells must be pretreated with fatty acid(s) for 0–72 h prior to the dimerization experiment. For example, DHA can be complexed to fatty acid-free BSA and added to the cell culture at a concentration of 50 μM as described previously [10]. DHA is maintained in the culture media during the serum deprivation step to prevent its loss from the membrane, but DHA is not utilized during ligand stimulation to avoid non-membrane effects the fatty acid could potentiate. As seen in Fig. 2, cells treated with DHA have increased dimer formation compared to untreated cells [3]. However, linoleic acid (LA), an omega-6 fatty acid, does not perpetrate the same effect on EGFR dimerization as DHA. This data highlights the pivotal role of the fatty acid composition of the membrane on receptor tyrosine kinase activity.

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Acknowledgments This work was supported by RO1 CA168312 and P30ES023512-01. References 1. Zhang X, Gureasko J, Shen K, Cole PA, Kuriyan J (2006) An allosteric mechanism for activation of the kinase domain of epidermal growth factor receptor. Cell 125:1137–1149 2. Ringerike T, Blystad FD, Levy FO, Madshus IH, Stang E (2002) Cholesterol is important in control of EGF receptor kinase activity but EGF receptors are not concentrated in caveolae. J Cell Sci 115:1331–1340 3. Turk HF, Barhoumi R, Chapkin RS (2012) Alteration of EGFR spatiotemporal dynamics suppresses signal transduction. PLoS One 7:e39682 4. Roepstorff K, Thomsen P, Sandvig K, van Deurs B (2002) Sequestration of epidermal growth factor receptors in non-caveolar lipid rafts inhibits ligand binding. J Biol Chem 277:18954–18960 5. Saffarian S, Li Y, Elson EL, Pike LJ (2007) Oligomerization of the EGF receptor investigated by live cell fluorescence intensity distribution analysis. Biophys J 93:1021–1031

6. Chen X, Resh MD (2002) Cholesterol depletion from the plasma membrane triggers ligand-independent activation of the epidermal growth factor receptor. J Biol Chem 277: 49631–49637 7. Wassall SR, Stillwell W (2008) Docosahexaenoic acid domains: the ultimate non-raft membrane domain. Chem Phys Lipids 153:57–63 8. Whitehead RH, VanEeden PE, Noble MD, Ataliotis P, Jat PS (1993) Establishment of conditionally immortalized epithelial cell lines from both colon and small intestine of adult H-2Kb-tsA58 transgenic mice. Proc Natl Acad Sci U S A 90:587–591 9. Sorkin A, Carpenter G (1991) Dimerization of internalized epidermal growth factor receptors. J Biol Chem 266:23453–23460 10. Kim W, Chapkin RS, Barhoumi R, Ma DW (2009) A novel role for nutrition in the alteration of functional microdomains on the cell surface. Methods Mol Biol 579:261–270

Chapter 4 Single-Molecule Optical Methods Analyzing Receptor Tyrosine Kinase Activation in Living Cells Inhee Chung and Ira Mellman Abstract Receptor tyrosine kinase activity is typically measured by diverse biochemical methods detecting the amount of phosphorylation of proteins within a cell lysate. In this chapter, we present biophysical methods that allow for studying the activation process of single receptors, in particular the human epidermal growth factor receptor (EGFR) family, in live cells. We describe optical tracking of quantum dot (QD)-labeled single receptors using the total internal reflection fluorescence microscopy (TIRFM), and initial steps of data analysis to identify the time-dependent variation of single-receptor diffusion, which can be widely applied to studying activation of various cell surface receptors. Key words EGFR receptor tyrosine kinase family, Single-molecule tracking, Quantum dots (QDs), Live-cell imaging, Diffusion analysis, Total internal reflection fluorescence microscopy (TIRFM)

1

Introduction Receptor tyrosine kinases (RTK) are cell surface receptors with transmembrane domains. They can receive external cues (by ligand binding) and transmit signals (by receptor activation), which results in diverse cellular responses such as cell growth and differentiation. Often, these receptors play crucial roles in cancer, and extensive efforts have been made to develop therapeutic strategies that perturb their abnormal activations [1–4]. Thus it is crucial to better understand mechanistic details of RTK activation. Generally, the activation involves receptor oligomerization that enables transactivation of receptors and ligand binding [5]. For the EGFR, one of the most actively studied RTKs, the activation is mediated by reversible dimerization dynamics [6, 7] and ligand-bindinginduced asymmetric interactions between adjacent kinase domains [7–10]. Phosphorylation on the tyrosine residues in the regulatory domains of the receptors results in recruitments of various adaptor molecules and proteins for further downstream signaling. We have shown that the amount of EGFR activation is spatially asymmetric

Serena Germano (ed.), Receptor Tyrosine Kinases: Methods and Protocols, Methods in Molecular Biology, vol. 1233, DOI 10.1007/978-1-4939-1789-1_4, © Springer Science+Business Media New York 2015

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in a live cell [6], and this spatial heterogeneity has important implications for cellular regulation of the receptor signaling. We obtained the molecular and cellular details of the EGFR activation using single-molecule tracking methods to detect the receptor activation in real time on live cells. We will describe the basic parts of these methods that can be generally applied to studies of other RTK activations.

2

Materials Our receptor tagging methods for optical tracking of single molecules make use of (1) a bright chromophore, the semiconductor (CdSe) quantum dot (QD) [11], and (2) Fab fragments of neutral antibodies (non-agonistic and non-antagonistic) against the extracellular domains (ECD) of receptors, followed by chemical conjugation of a single Fab to a single QD (Fab:QD).

2.1 Selection and Preparation of Fab Fragments

1. Identify an antibody that recognizes the receptor ECD but does not perturb the receptor signaling. The latter can be tested by comparing the phosphorylation levels of the receptor before and after the addition of excess antibodies (e.g., ten times higher concentration than the dissociation constant of the antibody). 2. The neutral antibody is then digested either by pepsin to yield F(ab′)2 (F(ab′)2 preparation kit; Thermo Fisher Scientific, Rockford, IL, USA), and then reduced to Fab fragments, or by papain to directly produce Fab’s (Fab preparation kit; Thermo Fisher Scientific).

2.2 Fab Conjugation Components

1. Sulfo-SMCC (Thermo Fisher Scientific). 2. QD605 stock solution (Life Technologies, Carlsbad, CA, USA). 3. DTT solution. 4. HEPES/NaCl buffer at pH 7.2. 5. NAP-5 desalting columns (GE Healthcare, Buckinghamshire, UK) 6. 2-Mercaptoethanol. 7. Pierce Protein Concentrators (Thermo Fisher Scientific). 8. Superdex G200 (GE Healthcare).

2.3 Cell Preparation and Receptor-Labeling Components

1. Glass-bottom dishes. 2. Full RPMI medium (or other recommended cell culture media): RPMI 1640 with 10 % fetal bovine serum (FBS), 1 % L-glutamine, 10 mM HEPES, and 1 % penicillin/streptomycin.

RTKs Activation in Living Cells

2.4 Optical Tracking Components

37

1. Wide-field microscope backbone (such as Nikon Eclipse TE2000 inverted microscope). 2. 100×/1.49 NA Plan Apo objective (Nikon, Tokyo, Japan). 3. Solid state diode laser (Andor Technology, Belfast, UK). 4. Back-illuminated EMCCD camera (Andor Technology). 5. Imaris (BitPlane) or ImageJ (NIH) software.

3

Methods

3.1 Conjugation of Fab to QD of 1:1 Ratio

Chemical reactions that yield Fab:QD conjugates are based on the Qdot antibody conjugation kit (Life Technologies) instructions, except some modifications placed for producing conjugates of 1:1 ratio and prolonged stability. 1. Add 1.75 μL (35.0 nmol) of sulfo-SMCC solution to 62.5 μL (0.5 nmol) of QD605 stock solution. Incubate the mixture at room temperature (RT) for 1 h. 2. In parallel, reduce 100 μg of Fabs in 300 μL PBS by adding 6.1 μL of the 1.0 M of DTT solution at RT for 30 min. 3. Separate the sulfo-SMCC-derivatized QDs from excess unreacted sulfo-SMCC by passing the solutions over NAP-5 desalting columns pre-equilibrated in HEPES/NaCl buffer. 4. Similarly, pass the reduced Fab fragments over NAP-5 columns pre-equilibrated in the same HEPES/NaCl buffer to remove DTT. 5. Mix the derivatized QDs and reduced Fab fragments and allow them to couple at RT for 2 h. 6. Inactivate remaining maleimides by adding 2-mercaptoethanol (final concentration of 10 mM) at RT for 30 min. 7. Concentrate the Fab:QD conjugates by ultrafiltration (Pierce Protein Concentrators), and separate them from unconjugated Fab fragments by gel filtration (Superdex G200). 8. For prolonged usage of the conjugates, the final conjugate pool in PBS can be brought to 50 % glycerol and kept at 4 °C. 9. The ratio of the Fab:QD in the conjugate can be determined by measuring the relative amounts of Fabs and QDs, estimated from the radioactivity of I125 that labeled Fabs at 1:1 ratio and the absorbance the QDs (at the absorption peak), respectively. 10. It is recommended to test the neutrality of the final Fab:QD conjugate in the receptor signaling by western blotting (WB) or other relevant methods.

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3.2 Cell Preparation and Receptor Labeling with Fab:QDs

Cells are plated on glass-bottom dishes 1 or 2 days before use and maintained in 2 mL of full RPMI medium at 37 °C. At the time of imaging, cells are usually ~70 % confluent.

3.2.1 Cell Preparation in the Glass-Bottom Dish 3.2.2

Receptor Labeling

Receptor labeling in cells is preferably done before imaging. 1. Add 0.1–1 μL of ~1 mM Fab:QD mixture (the amount needs to be adjusted for each study) directly to the medium (2 mL) of a cell plated glass-bottom dish. 2. Gently shake the dish a few times and leave it for less than 10 min at RT (see Note 1). 3. Rinse the cells three times with PBS or RPMI. 4. Replenish the dish with the full RPMI medium (10 % FBS can be removed or reduced to 0.1 % if the experiments need serum starvation) that is pre-warmed at 37 °C.

3.3 Optical Tracking of Single Receptors in Live Cells

Here, we discuss the imaging modalities for single-molecule tracking in live cells and the basic data acquisition procedure.

3.3.1 Basic Background Concept

For real-time single-receptor tracking in live cells, it is crucial to use bright chromophores that label individual receptors that can be well resolved in each image frame. To this end, CdSe QDs are an excellent tagging system because of their very large extinction coefficients and high quantum yields [12]. In addition, total internal reflection fluorescence microscopy (TIRFM), and an electron multiplying charge coupled device (EMCCD) are employed as an imaging platform that reduces the background scattering level, and captures fluorescence images with high sensitivity, respectively. The TIRFM utilizes an optical phenomenon called total internal reflection occurring at the interface between two media when the light propagates from the high refractive index (n1) side to the low refractive index (n2) side. When the incidence angle (θ) of the light is greater than the critical angle θc = sin− 1(n2/n1), from the Snell’s law), all the light is reflected back, while creating an evanescent field that extends into the medium of a low index of refraction (n2) (Fig. 1). For n1 (glass) = 1.52 and n2 (water) = 1.33, the critical angle is 61°. The penetration depth dp of the evanescent field is

(

d p = l / 2p n1 sin 2 q - (n2 / n1 )

2

) , where λ is the excitation light

wavelength. For example, the penetration depth is ~150 nm, when λ = 488 nm and θ = 70°. Since the intensity of the evanescent field exponentially decays with the depth in z, ∝ exp(−z/dp), labeled cell surface receptors located in basal cell membranes can be very effectively illuminated.

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Fig. 1 A schematic of an objective-based TIRFM. TIR occurs when light enters from oil to medium at an angle (θ) between the light beam (green line) and the optical axis (vertical orange line), greater than the critical angle (θc). Not to scale 3.3.2 Microscope Configuration

3.3.3 Image Acquisition Procedure

An example microscopic configuration has a wide-field microscope with a 100×/1.49 NA Plan Apo objective. The lateral position of the incidence light to the objective can be motor controlled (ASI) so that an optimal depth of the evanescent field is created. A solidstate diode laser, and a back-illuminated EMCCD camera are used as a light source, and a detector, respectively. The glass-bottom dish is placed on a motorized stage with controls of x and y positions (ASI), and the stage and microscope objectives are contained in a chamber, in which the air temperature and CO2 level are maintained at 37 °C and 5 %, respectively. 1. Equilibrate an environmental chamber to ensure optimal ambient conditions (e.g., temperature at 37 °C and CO2 level at 5 %) for cells during imaging. 2. Turn on lasers, and start the software that controls the camera. 3. Add a drop of oil with the same index of refraction of the glass on the front tip of the objective lens. 4. Check that the laser beam that passes the objective is collimated, which ensures a well-formed fluorescence image of single QDs. 5. Run the receptor Subheading 3.2.2.

labeling

procedure

described

in

6. Place the prepared glass-bottom dish in the dish holder of the stage and make sure that it touches the oil on the objective lens. 7. Use a level to confirm that the surface of the dish is perpendicular to the optical axis of the objective (Fig. 1). 8. Adjust the gain of the CCD and laser intensity (often manipulated by acousto-optic tunable filters (AOTF)) (see Note 2).

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Fig. 2 A snapshot fluorescence image of single QDs on live cells. Fluorescence images of single QDs (red; αEGFR-Fab:QD605) that labeled a small subset of EGFRs on BT20 cells (a human breast cancer cell line) by TIRFM. A laser excitation at 488 nm was used. Each image was acquired for 90 ms in a frame transfer mode (http://www.andor.com/learning-academy/trigger-modes-ixon-ultra-andixon3-trigger-modes)

9. Set an appropriate frame rate and total acquisition time in the software such that all important receptor dynamics will be captured. 10. Acquire a movie of 2D fluorescence images of individual QDs in the basal cell surface (Fig. 2) (see Note 3). 3.3.4 Initial Procedure of Image Rendering

From the raw data, one can now track the center locations of individual fluorescence images of single QDs over time using various software tools, such as the tracking module in Imaris or various tracking plug-ins in ImageJ. The following describes the initial steps for basic characterization of the receptor diffusion and its temporal variation. One can proceed with further analysis schemes according to observed characteristics of various receptor systems. 1. Record positions of individual molecules over time (Fig. 3). The raw data contains the positions (xi, yi) of each molecule in each frame, where i = 1, …, n (the last frame number). 2. Calculate the square displacement (SD), ri2 of a single-receptor location (xi, yi) between subsequent frames, over the course of the trajectory: ri2 = (xi − xi − 1)2 + (yi − yi − 1)2 3. Accumulate ri2 over time to construct cumulative square displacement (CSD), C(j) = ∑ ij= 2ri2 (Fig. 4).

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Fig. 3 Single EGFR trajectories in live cells. Time-dependent positions of single EGFRs are indicated by a false color scheme (from violet to red as time proceeds; image acquisition for 90 ms per frame in a frame transfer mode)

Fig. 4 An overlay of SD and CSD calculated from a single-EGFR trajectory. SD values (black outlined circles) between subsequent frames and accumulation of SD values (CSD; blue line) are overlaid. Variations of slops are visually detected in the CSD plot, as indicated by green vertical lines

Approximately, a linear increase of C(j) during a certain time window implies that the receptor underwent diffusion with a constant diffusion coefficient (D). Therefore by plotting a CSD, one will be able to get a rough idea about whether the diffusion dynamics of receptors of interest were constant or changed over time. Figure 4 shows an example of a diffusion

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Fig. 5 An example MSD plot of a single EGFR. A segment of a single-EGFR trajectory shown in Fig. 4 was used to construct an MSD vs. τ plot (red circles). The plot is fitted by two functions for free diffusion (green line) and directional + free diffusion (magenta line). From the fits, D = 0.16 ± 0.01 μm2/s (free), and D = 0.13 ± 0.01 μm2/s and v = 0.41 ± 0.09 μm/s (free + directional)

dynamics of a single EGFR, which varied over time.In order to more objectively segregate the time windows of different diffusion dynamics, it is recommended to devise further analysis schemes using various statistical methods. 4. Assess the diffusion characteristic in each time window of the C(j) plot and estimate the diffusion coefficient (D). Depending on the diffusion characteristics [13–16], different equations can be employed, which relate mean square displacement (MSD; 〈ri2〉) to diffusion coefficient (D), velocity (v), and confinement size (L). Several equations that describe various 2D lateral diffusions are listed as below. For free diffusion, 〈ri2〉 = 4Dτi. For free + directional diffusion, 〈ri2〉 = 4Dτi + v2τi2. L2 æ æ 12D init For confined diffusion, ri 2 = ç 1 - exp ç L2 3 è è Dini is the initial diffusion coefficient.

öö ÷ ÷ , where øø

For anomalous diffusion, 〈ri2〉 = Γτα, where Γ is a diffusion parameter and α is a constant that characterizes the diffusion (typically, α > 1 or