Immune‐Complex Assays for Tyrosine Protein Kinases

Anne L. Burkhardt1, Joseph B. Bolen1

1 Bristol‐Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 11.4
DOI:  10.1002/0471142735.im1104s07
Online Posting Date:  May, 2001
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Abstract

Tyrosine protein kinases (TPKs) represent a diverse group of enzymes that contribute to cellular signal transduction. The generally low abundance of TPKs, coupled with their rapid activation and deactivation, usually precludes their purification through conventional biochemical means. Using immune‐complex protein kinase assays, the presence or absence of a given TPK can be established and an estimation of its functional state obtained. In the of this unit, TPKs are immunoprecipitated, allowed to autophosphorylate in the presence of labeled ATP, run out on an SDS‐PAGE gel, and detected by autoradiography. Alternate protocols are provided for the assessment of the functional state of TPKs by providing a potential substrate along with the labeled ATP in the reaction mixture. In the first alternate protocol, the exogenous substrate is a protein, permitting simultaneous assessment of autophosphorylation and exogenous substrate phosphorylation. The second alternate protocol utilizes a peptide substrate, resulting in a rapid, high‐throughput assay that evaluates only exogenous substrate phosphorylation.

     
 
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Table of Contents

  • Basic Protocol 1: Immunoprecipitation and Autophosphorylation of Tyrosine Protein Kinases
  • Alternate Protocol 1: Phosphorylation of an Exogenous Protein Substrate
  • Alternate Protocol 2: Phosphorylation of an Exogenous Peptide Substrate
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Immunoprecipitation and Autophosphorylation of Tyrosine Protein Kinases

  Materials
  • Cells to be assayed
  • Phosphate‐buffered saline (PBS; appendix 2A), ice cold
  • recipe1× lysis buffer with protease inhibitors (made fresh and stored on ice)
  • Coomassie protein assay reagent (Pierce) or equivalent
  • recipe5× lysis buffer with protease inhibitors (for cell activation; made fresh and stored on ice)
  • Rabbit antiserum to src‐related TPKs, polyclonal or monoclonal ( UBI, Oncogene Science, or Santa Cruz Biotechnology)
  • Control serum (Sigma): normal rabbit serum (if antiserum is polyclonal) or isotype‐matched IgG (if antiserum is monoclonal)
  • Pansorbin (Staphylococcus aureus fixed with 10% formalin; Calbiochem) or protein A– or protein G–Sepharose (Pharmacia Biotech)
  • Rabbit anti‐mouse IgG, affinity‐purified (if antiserum is monoclonal; Organon Teknika Cappel)
  • recipeNP‐40 buffer, ice cold
  • recipeTyrosine protein kinase (TPK) buffer, ice cold
  • 5 mCi/ml [γ32P]ATP (3000 Ci/mmol; Du Pont NEN)
  • recipeATP solution, made fresh
  • recipeModified RIPA buffer, ice cold
  • 1× SDS/sample buffer (unit 8.4)
  • Labquake tube shaker/rotator ( PGC Scientific)
  • Gel dryer
  • Eppendorf model 5432 mixer
  • Additional reagents and equipment for SDS‐PAGE using the Laemmli system (unit 8.4) and autoradiography ( appendix 33)
NOTE: Carry out all procedures except washes on ice or in 4°C cold room.CAUTION: Perform all steps involving 32P behind a plexiglass shield, observing appropriate safety regulations. Provide waste containers for both dry and liquid radioactive waste.

Alternate Protocol 1: Phosphorylation of an Exogenous Protein Substrate

  Additional Materials
  • recipeExogenous substrate (rabbit‐muscle enolase or α‐casein; Reagents and Solutions)
  • recipeATP solution (make fresh and store on ice)
  • 2× SDS/sample buffer (unit 8.4)
  • Gel fixative solution: 3:1:6 (v/v/v) methanol/glacial acetic acid/water
  • Additional reagents and equipment for SDS‐PAGE (unit 8.4) and autoradiography ( appendix 33)

Alternate Protocol 2: Phosphorylation of an Exogenous Peptide Substrate

  Additional Materials
  • 1 mg/ml RR‐SRC peptide (GIBCO/BRL) in recipeTPK buffer
  • recipeATP solution (make fresh)
  • Glacial acetic acid
  • 10% (v/v) trichloroacetic acid
  • 75 mM phosphoric acid
  • Phosphocellulose discs (available in sheets; GIBCO/BRL)
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Figures

Videos

Literature Cited

   Appleby, M.W., Gross, J.A., Cooke, M.P., Levin, S.D., Qian, X., and Perlmutter, R.M. 1992. Defective T cell receptor signaling in mice lacking the thymic form of p59fyn. Cell 70:751‐763.
   Bolen, J.B., Rowley, R.B., Spana, C., and Tsygankov, A.Y. 1992. The src family of tyrosine protein kinases in hemopoietic signal transduction. FASEB J. 6:3403‐3409.
   Burkhardt, A.L., Brunswick, M., Bolen, J.B., and Mond, J.J. 1991. Anti‐immunoglobulin stimulation of B lymphocytes activates src‐related protein tyrosine kinases. Proc. Natl. Acad. Sci. U.S.A. 88:7410‐7414.
   Chan, A.C., Iwashima, M., Turck, C.W., and Weiss, A. 1992. ZAP‐70: A 70 kD protein‐tyrosine kinase that associates with the TCR ζ chain. Cell 71:663‐670.
   Dymecki, S.M., Niederhuber, J.E., and Desiderio, S.V. 1990. Specific expression of a tyrosine kinase gene, blk, in B lymphoid cells. Science (Wash. DC) 247:332‐336.
   Eiseman, E. and Bolen, J.B. 1992. Engagement of the high‐affinity IgE receptor activates src‐related tyrosine protein kinase. Nature (Lond.) 355:78‐80.
   Heyeck, S.D. and Berg, L.J. 1993. Developmental regulation of a murine T‐cell‐specific tyrosine kinase gene, Tsk. Proc. Natl. Acad. Sci. U.S.A. 90:669‐673.
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   Hutchcroft, J.E., Harrison, M.L., and Geahlen, R.L. 1991. B lymphocyte activation is accompanied by phosphorylation of a 72‐kDa protein tyrosine kinase. J. Biol. Chem. 266:14846‐14849.
   Molina, T.J., Kishihara, K., Siderovski, D.P., van Ewijk, W., Narendran, A., Timms, E., Wakeman, A., Paige, C.J., Hartmann, K.‐U., Veillette, A., Davidson, D., and Mak, T.W. 1992. Profound block in thymocyte development in mice lacking p56lck. Nature (Lond.) 357:161‐164.
   Samelson, L.E., Philips, A.F., Luong, E.T., and Klausner, R.D. 1990. Association of the fyn protein tyrosine kinase with the T‐cell antigen receptor. Proc. Natl. Acad. Sci. U.S.A. 87:4358‐4362.
   Siliciano, J.D., Morrow, T.A., and Desiderio, S.V. 1992. ITK, a T cell specific tyrosine kinase gene inducible by interleukin‐2. Proc. Natl. Acad. Sci. U.S.A. 89:11194‐11198.
   Smith, J.A. 1987. Colorimetric methods. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 10.1.1‐10.1.3. Greene Publishing Associates, and John Wiley & Sons, New York.
   Stein, P.L., Lee, H.‐M., Rich, S., and Soriano, P. 1992. pp59fyn mutant mice display differential signaling in thymocytes and peripheral T cells. Cell 70:741‐750.
   Straus, D.B. and Weiss, A. 1992. Genetic evidence for the involvement of the lck tyrosine kinase in signal transduction through the T cell antigen receptor. Cell 70:585‐593.
   Taniguchi, T., Kobayashi, T., Kondo, J., Takahashi, K., Nakamura, H., Suzuki, J., Nagai, K., Yamada, T., Nakamura, S.‐I., and Yamamura, H. 1991. Molecular cloning of a porcine gene syk that induces a 72‐kDa protein tyrosine kinase showing high susceptibility to proteolysis. J. Biol. Chem. 266:15790‐15796.
   Tsukada, S., Saffran, D.C., Rawlings, D.J., Parolini, O., Allen, R.C., Klisak, I., Sparkes, R.S., Kubagawa, H., Mohandas, T., Quan, S., Belmont, J.W., Cooper, M.D., Conely, M.E., Witte, O.N. 1993. Deficient expression of a B cell cytoplasmic tyrosine kinase in human X‐linked agammaglobulinemia. Cell 72:279‐290.
   Tsygankov, A., Bröker, B., Fargnoll, J., Ledbetter, J.A., and Bolen, J.B. 1992. Activation of tyrosine kinase p60fyn following T cell antigen receptor cross‐linking. J. Biol. Chem. 267:18259‐18262.
   Ullrich, A. and Schlessinger, J. 1990. Signal transduction by receptors with tyrosine kinase activity. Cell 61:203‐211.
   Veillette, A., Bookman, M.A., Horak, E.M., Samelson, L.E., and Bolen, J.B. 1989. Signal transduction through the CD4 receptor involves the activation of the internal membrane tyrosine protein kinase p56lck. Nature (Lond.) 338:257‐259.
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   Vetrie, D., Vorechovsky, I., Sideras, P., Holland, J., Davies, A., Flinter, F., Hammarstrom, L., Kinnon, C., Levinsky, R., Bobrow, M., Smith, C.I.E., and Bentley, D.R. 1993. The gene involved in X‐linked agammaglobulinaemia is a member of the src family of protein‐tyrosine kinases. Nature (Lond.) 361:226‐233.
Key Reference
   Hunter, T. and Sefton, B.M. (eds.) 1991. Protein Phosphorylation, Protein Kinases: Assays, Purification, Antibodies, Functional Analysis, Cloning, and Expression. Methods Enzymol. 200.
  This issue of Methods in Enzymology devotes several chapters to various assays of tyrosine protein kinases and substrates, including nonradioactive methods of assay and detection.
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