Identification of Protein Kinase Substrates by the Kinase‐Interacting Substrate Screening (KISS) Approach

Mutsuki Amano1, Tomoki Nishioka1, Yoshimitsu Yura1, Kozo Kaibuchi1

1 Department of Cell Pharmacology, Graduate School of Medicine, Nagoya University, Nagoya, Aichi
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 14.16
DOI:  10.1002/cpcb.8
Online Posting Date:  September, 2016
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Abstract

Identifying the substrates of protein kinases to understand their modes of action has been undertaken by various approaches and remains an ongoing challenge. Phosphoproteomic technologies have accelerated the accumulation of data concerning protein phosphorylation and have uncovered vast numbers of phosphorylation sites in vivo. In this unit, a novel in vitro screening approach for protein kinase substrates is presented, based on protein‐protein interaction and mass spectrometry‐based phosphoproteomic technology. © 2016 by John Wiley & Sons, Inc.

Keywords: kiss; kinase; phosphorylation; phosphoproteomics; mass spectrometry

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation and Phosphorylation of Protein Kinase Substrates
  • Support Protocol 1: Preparation of Rat Brain Lysate by Ammonium Sulfate Precipitation
  • Basic Protocol 2: Tryptic Digestion of Kinase‐Substrate Complexes and Phosphopeptide Concentration for Mass Spectrometric Analysis
  • Support Protocol 2: LC/MS/MS Analysis and Peptide Identification
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Isolation and Phosphorylation of Protein Kinase Substrates

  Materials
  • Glutathione Sepharose 4B (GE Healthcare, cat no. 170756)
  • TED buffer (20 mM Tris·Cl pH 7.5, 1 mM EDTA, 1 mM DTT)
  • 800 pmol of GST‐tagged recombinant protein kinase (GST‐Rho‐kinase‐cat; 6‐553 aa)
  • Tissue or cell lysate (adult rat brain lysate; see protocol 2)
  • Phosphorylation reaction solution (see recipe)
  • ATP
  • Ice

Support Protocol 1: Preparation of Rat Brain Lysate by Ammonium Sulfate Precipitation

  Materials
  • Rat brains (approximately 40 g)
  • Homogenization buffer (see recipe)
  • Solid ammonium sulfate
  • TED buffer (20 mM Tris·Cl pH 7.5, 1 mM EDTA, 1 mM DTT)
  • NaCl
  • Potter‐Elvehjem Teflon‐glass homogenizer
  • Gauze
  • Centrifuge
  • 4°C shaking incubator

Basic Protocol 2: Tryptic Digestion of Kinase‐Substrate Complexes and Phosphopeptide Concentration for Mass Spectrometric Analysis

  Materials
  • Guanidine hydrochloride (Nakalai Tesque, cat. no. 17318‐82)
  • Phosphorylation reaction mixture (see protocol 1)
  • Tris·Cl, pH 8.5
  • Dithiothreitol (DTT)
  • Iodoacetamide (Wako Pure Chemical, cat. no. 093‐02152)
  • Ice
  • Methanol, ice‐cold
  • Chloroform, ice‐cold
  • Urea
  • Ammonium bicarbonate (Sigma‐Aldrich, cat. no. A6141)
  • 1 μg/μl of Trypsin/Lys‐C mix (Promega, cat. no. V5072)
  • Titansphere Phos‐TiO kit (GL Science, cat no. 5010‐21306) containing:
  • Titansphere Phos‐TiO tip column
  • Waste fluid tube with centrifugal adapter
  • Collection tubes
  • Buffer A
  • Buffer B
  • Pyrrolidine solution
  • 25% Ammonia solution
  • Trifluoroacetic acid (TFA) (Wako Pure Chemical Industries Cat No. 204‐02743)
  • Acetonitrile (ACN) (Kanto Kagaku Cat No. 01031‐1B)
  • Centrifuge with swinging‐bucket rotor
  • 1.5‐ml tubes (low protein retention)
  • Vacuum apparatus
  • 37°C incubator
  • SPE C‐TIP (AMR, cat. no. KT‐200‐C18)
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Figures

Videos

Literature Cited

  Amano, M., Hamaguchi, T., Shohag, M.H., Kozawa, K., Kato, K., Zhang, X., Yura, Y., Matsuura, Y., Kataoka, C., Nishioka, T., and Kaibuchi, K. 2015. Kinase‐interacting substrate screening is a novel method to identify kinase substrates. Cell Biol. 209:895‐912. doi: 10.1083/jcb.201412008.
  Biondi, R.M. and Nebreda, A.R. 2003. Signaling specificity of Ser/Thr protein kinases through docking‐site‐mediated interactions. Biochem. J. 372:1‐13. doi: 10.1042/bj20021641.
  Cox, J. and Mann, M. 2008. MaxQuant enables high peptide identification rates, individualized p.p.b.‐range mass accuracies and proteome‐wide protein quantification. Nat. Biotechnol. 26:1367‐1372. doi: 10.1038/nbt.1511.
  Duarte, M.L., Pena, D.A., Nunes Ferraz, F.A., Berti, D.A., Paschoal Sobreira, T.J., Costa‐Junior, H.M., Abdel Baqui, M.M., Disatnik, M.H., Xavier‐Neto, J., Lopes de Oliveira, P.S., and Schechtman, D. 2014. Protein folding creates structure‐based, noncontiguous consensus phosphorylation motifs recognized by kinases. Sci. Signal. 7:ra105. doi: 10.1126/scisignal.2005412.
  Goldsmith, E.J., Akella, R., Min, X., Zhou, T., and Humphreys, J.M. 2007. Substrate and docking interactions in serine/threonine protein kinases. Chem. Rev. 107:5065‐5081. doi: 10.1021/cr068221w.
  Hornbeck, P.V., Kornhauser, J.M., Tkachev, S., Zhang, B., Skrzypek, E., Murray, B., Latham, V., and Sullivan, M. 2012. PhosphoSitePlus: A comprehensive resource for investigating the structure and function of experimentally determined post‐translational modifications in man and mouse. Nucleic Acids Res. 40:D261‐270. doi: 10.1093/nar/gkr1122.
  Ubersax, J.A. and Ferrell, J.E. Jr. 2007. Mechanisms of specificity in protein phosphorylation. Nat. Rev. Mol. Cell Biol. 8:530‐541. doi:10.1038/nrm2203.
  Yamaguchi, H., Kasa, M., Amano, M., Kaibuchi, K., and Hakoshima, T. 2006. Molecular mechanism for the regulation of rho‐kinase by dimerization and its inhibition by fasudil. Structure 14:589‐600. doi: 10.1016/j.str.2005.11.024.
Internet Resources
  https://srpbsg01.unit.oist.jp/index.php?ml_lang=en, https://kanphos.neuroinf.jp/index.php?ml_lang=en
  Information about the candidate substrates and phosphorylation sites detected by KISS approach will be available in the Kinase‐associated neural phospho‐signaling (KANPHOS) database.
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