Using Phospho‐Motif Antibodies to Determine Kinase Substrates

Colin D. White1, Alex Toker1

1 Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 18.20
DOI:  10.1002/0471142727.mb1820s101
Online Posting Date:  January, 2013
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Abstract

Phosphorylation of substrates by protein kinases regulates a myriad of cellular processes, ranging from proliferation and migration to autophagy, senescence, and apoptosis. Kinase substrate selectivity is largely dependent on the amino acid sequence surrounding the phosphorylation site; therefore, substrate‐directed, phosphorylation‐state‐sensitive, motif‐specific (“phospho‐motif”) antibodies represent powerful tools to identify novel kinase substrates and to investigate mechanisms of substrate phosphorylation in many signaling pathways typically associated with human malignancies. Phospho‐motif antibodies are engineered to recognize proteins that contain a phosphorylated residue in the context of a specific motif. They are raised against a library of phospho‐peptides comprising both the phosphorylated residue and the surrounding residues that determine kinase specificity, with degenerate residues taking up the remaining positions. Currently, several categories of phospho‐motif antibody are commercially available, which may be used to specifically detect Ser, Thr, Ser/Thr, or Tyr residues phosphorylated by different protein kinase families. These antibodies are commonly used in immunoprecipitation and/or immunoblotting protocols to determine kinase‐induced substrate phosphorylation. This unit describes the use of phospho‐motif antibodies to elucidate the kinase(s) responsible for phosphorylating substrate proteins. Curr. Protoc. Mol. Biol. 101:18.20.1–18.20.11. © 2013 by John Wiley & Sons, Inc.

Keywords: phospho‐motif antibody; phosphorylation; protein kinase

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: In Silico Analysis to Identify the Kinase Responsible for Phosphorylating a Known Substrate
  • Basic Protocol 2: Biochemical Validation of Protein Kinase–Induced Substrate Phosphorylation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: In Silico Analysis to Identify the Kinase Responsible for Phosphorylating a Known Substrate

  Materials
  • Ligand(s) for cell stimulation (see protocol 2 introduction)
  • 10‐cm2 dish of 90% to 100% confluent cells
  • Phosphate‐buffered saline (PBS; see recipe), ice‐cold
  • RIPA (radio‐immunoprecipitation assay) lysis buffer (see recipe)
  • Modified Bradford assay kit (Bio‐Rad #500‐0002; also see unit 10.1)
  • SDS sample buffer (see recipe)
  • Antibody raised against the putative protein kinase substrate [Cell Signaling Technology (http://www.cellsignal.com), Abcam (http://www.abcam.com), or Santa Cruz Biotechnology (http://www.scbt.com)]
  • Protein A‐Sepharose slurry (Fisher Scientific 45‐000‐116)
  • Immunoprecipitation (IP) wash buffer (see recipe)
  • Phospho‐motif antibody (see Table 18.20.1)
  • 5% nonfat dry milk/TBS‐T (see recipe)
  • 5% BSA/TBS‐T: 5% bovine serum albumin (Sigma‐Aldrich #A7030) in TBS‐T (see recipe), freshly prepared
  • Protein A‐HRP (horseradish peroxidase) (Millipore #18‐160). Immediately prior to use, dilute 1:5,000 in 5% nonfat dry milk/TBS‐T (see recipe)
  • Aspirator
  • Cell scrapers
  • Polyvinylidene fluoride (PVDF) membranes (optional)
  • Additional reagents and equipment for Bradford assay (unit 10.1), SDS‐PAGE (unit 10.2), and immunoblotting (unit 10.8)
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Figures

Videos

Literature Cited

Literature Cited
   Bain, J., Mclachlan, H., Elliott, M., and Cohen, P. 2003. The specificities of protein kinase inhibitors: an update. Biochem. J. 371:199‐204.
   Bain, J., Plater, L., Elliott, M., Shpiro, N., Hastie, C.J., McLauchlan, H., Klevernic, I., Arthur, J.S., Alessi, D.R., and Cohen, P. 2007. The selectivity of protein kinase inhibitors: a further update. Biochem. J. 408:297‐315.
   Blenis, J., Kuo, C.J., and Erikson, R.L. 1987. Identification of a ribosomal protein S6 kinase regulated by transformation and growth‐promoting stimuli. J. Biol. Chem. 262:14373‐14376.
   Cheek, S., Zhang, H., and Grishin, N. V. 2002. Sequence and structure classification of kinases. J. Mol. Biol. 320:855‐881.
   Davies, S.P., Reddy, H., Caivano, M., and Cohen, P. 2000. Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem. J. 351:95‐105.
   Doppler, H., Storz, P., LI, J., Comb, M. J., and Toker, A. 2005. A phosphorylation state‐specific antibody recognizes Hsp27, a novel substrate of protein kinase D. J. Biol. Chem. 280:15013‐15019.
   Hutti, J.E., Jarrell, E.T., Chang, J.D., Abbott, D.W., Storz, P., Toker, A., Cantley, L.C., and Turk, B.E. 2004. A rapid method for determining protein kinase phosphorylation specificity. Nat. Methods 1:27‐29.
   Johnson, S.A. and Hunter, T. 2005. Kinomics: methods for deciphering the kinome. Nat. Methods 2:17‐25.
   Kane, S., Sano, H., Liu, S.C., Asara, J.M., Lane, W.S., Garner, C.C., and Lienhard, G.E. 2002. A method to identify serine kinase substrates. Akt phosphorylates a novel adipocyte protein with a Rab GTPase‐activating protein (GAP) domain. J. Biol. Chem. 277:22115‐22118.
   Manning, B.D. 2009. Challenges and opportunities in defining the essential cancer kinome. Sci. Signal. 2:pe15.
   Manning, B.D. and Cantley, L.C. 2002. Hitting the target: Emerging technologies in the search for kinase substrates. Sci. STKE 2002:pe49.
   Manning, B.D., Tee, A.R., Logsdon, M.N., Blenis, J., and Cantley, L.C. 2002a. Identification of the tuberous sclerosis complex‐2 tumor suppressor gene product tuberin as a target of the phosphoinositide 3‐kinase/akt pathway. Mol. Cell 10:151‐162.
   Manning, G., Whyte, D.B., Martinez, R., Hunter, T., and Sudarsanam, S. 2002b. The protein kinase complement of the human genome. Science 298:1912‐1934.
   Nishikawa, K., Toker, A., Johannes, F.J., Songyang, Z., and Cantley, L.C. 1997. Determination of the specific substrate sequence motifs of protein kinase C isozymes. J. Biol. Chem. 272:952‐960.
   Obenauer, J.C., Cantley, L.C., and Yaffe, M.B. 2003. Scansite 2.0: Proteome‐wide prediction of cell signaling interactions using short sequence motifs. Nucleic Acids Res. 31:3635‐3641.
   Ross, H., Armstrong, C.G., and Cohen, P. 2002. A non‐radioactive method for the assay of many serine/threonine‐specific protein kinases. Biochem J. 366:977‐981.
   Songyang, Z., Blechner, S., Hoagland, N., Hoekstra, M.F., Piwnica‐Worms, H., and Cantley, L.C. 1994. Use of an oriented peptide library to determine the optimal substrates of protein kinases. Curr. Biol. 4:973‐982.
   Songyang, Z., Carraway, K.L. 3rd, Eck, M.J., Harrison, S.C., Feldman, R.A., Mohammadi, M., Schlessinger, J., Hubbard, S.R., Smith, D.P., Eng, C., et al. 1995. Catalytic specificity of protein‐tyrosine kinases is critical for selective signalling. Nature 373:536‐539.
   Songyang, Z., Lu, K.P., Kwon, Y.T., Tsai, L.H., Filhol, O., Cochet, C., Brickey, D.A., Soderling, T.R., Bartleson, C., Graves, D.J., DeMaggio, A.J., Hoekstra, M.F., Blenis, J., Hunter, T., and Cantley, L.C. 1996. A structural basis for substrate specificities of protein Ser/Thr kinases: Primary sequence preference of. casein kinases I and II, NIMA, phosphorylase kinase, calmodulin‐dependent kinase II, CDK5, and Erk1. Mol. Cell. Biol. 16:6486‐6493.
   Yaffe, M.B., Leparc, G.G., Lai, J., Obata, T., Volinia, S., and Cantley, L.C. 2001. A motif‐based profile scanning approach for genome‐wide prediction of signaling pathways. Nat. Biotechnol. 19:348‐353.
   Yang, X., Hubbard, E.J., and Carlson, M. 1992. A protein kinase substrate identified by the two‐hybrid system. Science 257:680‐682.
   Zhang, H., Zha, X., Tan, Y., Hornbeck, P.V., Mastrangelo, A.J., Alessi, D.R., Pokakiewicz, R.D., and Comb, M.J. 2002. Phosphoprotein analysis using antibodies broadly reactive against phosphorylated motifs. J. Biol. Chem. 277:39379‐39387.
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