Profiling Native Kinases by Immuno‐Assisted Activity‐Based Profiling

Eric Okerberg1, Ann Shih1, Heidi Brown1, Senait Alemayehu1, Arwin Aban1, Tyzoon K. Nomanbhoy1

1 ActivX Biosciences, La Jolla, California
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch130084
Online Posting Date:  October, 2013
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Abstract

The protocols in this unit describe efficient and cost‐effective approaches to determine the interaction of small‐molecule inhibitors with native kinases, and also analyze the interactions between kinases and their binding partners in a cellular setting. The combined attributes of activity‐based probes and western blotting procedures provide for quantitative measurement of inhibitor efficacy, isoform selectivity, and post‐translational modifications. We further demonstrate the ability to identify protein‐protein interactions between a probe‐labeled protein and its noncovalent binding partners. Curr. Protoc. Chem. Biol. 5:213‐226 © 2013 by John Wiley & Sons, Inc.

Keywords: activity based protein profiling; immuno‐assisted activity based profiling; native kinases; inhibitors

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Profiling the JNK Inhibitor JNK‐IN‐8 Against Native JNK1 and JNK2 Using the Immuno‐Assisted Activity Based Profiling Method
  • Alternate Protocol 1: Profiling the EGFR Inhibitor Tarceva Against Native EGFR and Phospho‐EGFR in EGF‐Treated A431 Cells Using the Immuno‐Assisted Activity‐Based Profiling Method
  • Basic Protocol 2: Profiling Interactions Between Kinases and their Binding Partners Using the Immuno‐Assisted Activity‐Based Method
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Profiling the JNK Inhibitor JNK‐IN‐8 Against Native JNK1 and JNK2 Using the Immuno‐Assisted Activity Based Profiling Method

  Materials
  • Jurkat cells (ATCC #TIB‐152)
  • RPMI medium with 10% FBS supplemented with 25 mM HEPES, 1× glutamine, and 1× penincillin/streptomycin
  • Phosphate‐buffered saline (PBS; e.g., VWR Scientific), ice cold
  • Kinase buffer (see recipe) with and without phosphatase and protease inhibitors
  • BCA protein assay kit (Thermo Scientific; also see Simonian and Smith, )
  • Manganese chloride (MnCl 2; VWR Scientific, cat. no. JT2540‐4)
  • JNK‐IN‐8 (EMD, cat. no. 420150, http://www.emdmillipore.com/)
  • Dimethylsulfoxide (DMSO; VWR Scientific)
  • ActivX Desthiobiotin‐ATP Probe (Pierce, cat. no. 88311)
  • 8 M urea (VWR Scientific, cat. no. IB72064; see recipe)
  • High‐capacity streptavidin‐agarose (50% slurry; ThermoFisher, cat. no. 20359)
  • 2× gel sample buffer (see recipe)
  • Any kD Mini‐PROTEAN TGX precast gel (BioRad, cat. no. 456‐9033; also see Gallagher, )
  • Nonfat dry milk (grocery store)
  • PBS‐T (see recipe)
  • Antibodies of interest:
    • JNK1 antibody (Cell Signaling, cat. no. 3708)
    • JNK2 antibody (Cell signaling, cat. no. 4672)
    • CDK2 antibody (Santa Cruz, cat. no. SC‐163)
    • Anti‐rabbit DyLight 800 Conjugate (Cell Signaling, cat. no. 5151)
    • Anti‐mouse DyLight 680 Conjugate (Cell Signaling, cat. no. 5470)
  • Odyssey (Li‐COR)
  • IgorPro WaveMetrics (or any software for curve‐fitting)
  • 175‐cm2 (T‐175) flasks
  • Allegra 6R centrifuge (Beckman Coulter)
  • Probe‐type sonicator (VWR)
  • Econo‐Pac 10DG chromatography columns (BioRad, cat. no. 732‐2010)
  • Tube rotator
  • 95°C heat block or water bath
  • BioRad Turbo transfer system (also see Gallagher et al., )
  • Additional reagents and equipment for protein assay (Simonian and Smith, ), SDS‐PAGE (Gallagher, ), and western blotting (immunoblotting; Gallagher et al., )

Alternate Protocol 1: Profiling the EGFR Inhibitor Tarceva Against Native EGFR and Phospho‐EGFR in EGF‐Treated A431 Cells Using the Immuno‐Assisted Activity‐Based Profiling Method

  Additional Materials (also see protocol 1)
  • Cell line of interest: A431 cells (ATCC #CRL1555)
  • DMEM medium (with 4.5 g/liter glucose) supplemented with 10% FBS and 1× glutamine
  • Serum‐free DMEM medium
  • EGF (Calbiochem, cat. no. 324831)
  • Phosphate‐buffered saline (PBS; e.g., VWR Scientific), ice cold
  • Tarceva (Selleck Chem, cat. no. S1023; http://www.selleckchem.com/)
  • Antibodies of interest:
    • EGRF antibody (Cell Signaling, cat. no. 2232)
    • p‐EGFR antibody (Cell Signaling, cat. no. 2231)
  • 15‐cm tissue culture plates
  • Cell scraper (Starstedt)

Basic Protocol 2: Profiling Interactions Between Kinases and their Binding Partners Using the Immuno‐Assisted Activity‐Based Method

  Materials
  • Kinase buffer (see recipe) with and without phosphatase and protease inhibitors
  • BCA protein assay kit (Thermo Scientific; also see Simonian and Smith, )
  • Manganese chloride (MnCl 2; VWR Scientific, cat. no. JT2540‐4)
  • ActivX Desthiobiotin‐ATP Probe (Pierce, cat. no. 88311)
  • 2× gel sample buffer (see recipe)
  • Any kD Mini‐PROTEAN TGX precast gel (BioRad, cat. no. 456‐9033; also see Gallagher, )
  • Nonfat dry milk (grocery store)
  • PBS‐T (see recipe)
  • Antibodies of interest:
    • EGRF antibody (Cell Signaling, cat. no. 2232)
    • p‐EGFR antibody (Cell Signaling, cat. no. 2231)
    • CDK2 antibody (Santa Cruz, cat. no. SC‐163)
    • STAT3 antibody (Cell Signaling, cat. no. 9139)
    • PLCγ antibody (Cell Signaling, cat. no. 2822)
    • p‐SHC antibody (Cell Signaling, cat. no. 2434)
    • SHC antibody (Cell Signaling, cat. no. 2432)
    • Grb2 antibody (Cell Signaling, cat. no. 3972)
    • Anti‐rabbit DyLight 800 conjugate (Cell Signaling, cat. no. 5151)
    • Anti‐mouse DyLight 680 conjugate (Cell Signaling, cat. no. 5470)
  • Dounce tissue grinder
  • Centrifuge
  • Tube rotator
  • Gel‐loading tips
  • 95°C heat block or water bath
  • BioRad Turbo transfer system (also see Gallagher et al., )
  • Odyssey (Li‐COR)
  • Additional reagents and equipment for profiling Tarceva against native EGFR and phospho‐EGFR in EGF‐treated A431 cells ( protocol 2), determining protein concentration (Simonian and Smith, ), SDS‐PAGE (Gallagher, ), and western blotting (immunoblotting; Gallagher et al., )
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Figures

Videos

Literature Cited

Literature Cited
  Adam, G.C., Cravatt, B.F., and Sorensen, E.J. 2001. Profiling the specific reactivity of the proteome with non‐directed activity‐based probes. Chem. Biol. 8:81‐95.
  Adam, G.C., Sorensen, E.J., and Cravatt, B.F. 2002. Proteomic profiling of mechanistically distinct enzyme classes using a common chemotype. Nat. Biotechnol. 20:805‐809.
  Bantscheff, M., Eberhard, D., Abraham, Y., Bastuck, S., Boesche, M., Hobson, S., Mathieson, T., Perrin, J., Raida, M., Rau, C., Reader, V., Sweetman, G., Bauer, A., Bouwmeester, T., Hopf, C., Kruse, U., Neubauer, G., Ramsden, N., Rick, J., Kuster, B., and Drewes, G. 2007. Quantitative chemical proteomics reveals mechanisms of action of clinical ABL kinase inhibitors. Nat. Biotechnol. 25:1035‐1044.
  Cravatt, B.F., Wright, A.T., and Kozarich, J.W. 2008. Activity‐based protein profiling: From enzyme chemistry to proteomic chemistry. Annu. Rev. Biochem. 77:383‐414.
  Gallagher, S.R. 2012. One‐dimensional SDS gel electrophoresis of proteins. Curr. Protoc. Mol. Biol. 97:10.2A.1‐10.2A.44.
  Gallagher, S., Winston, S.E., Fuller, S.A., and Hurrell, J.G. 2008. Immunoblotting and immunodetection. Curr. Protoc. Mol. Biol. 83:10.8.1‐10.8.28.
  Kidd, D., Liu, Y., and Cravatt, B.F. 2001. Profiling serine hydrolase activities in complex proteomes. Biochemistry 40:4005‐4015.
  Liu, Y., Patricelli, M.P., and Cravatt, B.F. 1999. Activity‐based protein profiling: The serine hydrolases. Proc. Natl. Acad. Sci. U.S.A. 96:14694‐14699.
  Okerberg, E.S., Wu, J., Zhang, B., Samii, B., Blackford, K., Winn, D.T., Shreder, K.R., Burbaum, J.J., and Patricelli, M.P. 2005. High‐resolution functional proteomics by active‐site peptide profiling. Proc. Natl. Acad. Sci. U.S.A. 102:4996‐5001.
  Patricelli, M.P., Szardenings, A.K., Liyanage, M., Nomanbhoy, T.K., Wu, M., Weissig, H., Aban, A., Chun, D., Tanner, S., and Kozarich, J.W. 2007. Functional interrogation of the kinome using nucleotide acyl phosphates. Biochemistry 46:350‐358.
  Patricelli, M.P., Nomanbhoy, T.K., Wu, J., Brown, H., Zhou, D., Zhang, J., Jagannathan, S., Aban, A., Okerberg, E., Herring, C., Nordin, B., Weissig, H., Yang, Q., Lee, J.D., Gray, N.S., and Kozarich, J.W. 2011. In situ kinase profiling reveals functionally relevant properties of native kinases. Chem. Biol. 18:699‐710.
  Pawson, T. and Kofler, M. 2009. Kinome signaling through regulated protein‐protein interactions in normal and cancer cells. Curr. Opin. Cell Biol. 21:147‐153.
  Simonian, M. H. and Smith, J. A. 2006. Spectrophotometric and colorimetric determination of protein concentration. Curr. Protoc. Mol. Biol. 76:10.1A.1‐10.1A.9.
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