Overview of Protein Phosphorylation

Bartholomew M. Sefton1, Shirish Shenolikar2

1 The Salk Institute, San Diego, 2 Duke University Medical Center, Durham
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 13.1
DOI:  10.1002/0471140864.ps1301s00
Online Posting Date:  May, 2001
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This overview provides a history of protein phosphorylation research and provides the reader with an understanding of how and why labeling studies are performed. The various sites of protein phosphorylation are described along with the roles of the many kinases and phosphatases that regulate phosphorylation. Methods for detecting unlabeled phosphoamino acids, including high‐voltage electrophoresis on thin‐layer cellulose acetate plates, gel‐shift assays, and the use of anti‐phosphopeptide antibodies.

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Table of Contents

  • History
  • Labeling Studies
  • Sites of Phosphorylation
  • Detection of Unlabeled Phosphoamino Acids
  • Protein Kinases
  • Protein Phosphatases
  • Literature Cited
PDF or HTML at Wiley Online Library


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Literature Cited

Literature Cited
   Alberola‐Ila, J., Forbush, K.A., Seger, R., Krebs, E.G., and Perlmutter, R.M. 1995. Selective requirement for MAP kinase activation in thymocyte differentiation. Nature. 373:620‐623.
   Brunet, A., Pages, G., and Poussegur, J. 1994. Constitutively active mutants of MAP (MEK1) induce growth factor‐relaxation and oncogenicity when expressed in fibroblasts. Oncogene. 9:3379‐3387.
   Charbonneau, H. and Tonks, N.K. 1992. 1002 protein phosphatases. Annu. Rev. Cell Biol. 8:463‐493.
   Cohen, P. 1985. The role of protein phosphorylation in the hormonal control of enzyme activity. Eur. J. Biochem. 15:439‐448.
   Cohen, P. 1989. The structure and regulation of protein phosphatases. Annu. Rev. Biochem. 58:453‐508.
   Cohen, P. 1991. Classification of protein serine/threonine phosphatases: Identification and quantitation in cell extracts. Methods Enzymol. 201:389‐398.
   Cori, G.T. and Cori, C.F. 1945. Enzymatic conversion of phosphorylase a to b. J. Biol. Chem. 158:321‐345.
   Crews, C.M. and Erickson, R.L. 1992. Purification of a murine protein‐tyrosine/threonine kinase that phosphorylates and activates the erk‐1 gene product: Relationship to the fission yeast byr1 gene product. Proc. Natl. Acad. Sci. U.S.A. 89:8205‐8209.
   Czernik, A.J., Girault, J.A., Nairn, A.C., Chen, J., Snyder, G., Kebabian, J., and Greengard, P. 1991. Production of phosphorylation state–specific antibodies. Methods Enzymol. 201:264‐283.
   Doree, M. and Galas, S. 1994. The cyclin‐dependent protein kinases and the control of cell division. FASEB J. 85:1114‐1121.
   Duclos, B., Marcandier, S., and Cozzone, A.J. 1991. Chemical properties and separation of phosphoamino acids by thin‐layer chromatography and/or electrophoresis. Methods Enzymol. 201:10‐21.
   Eisenmann, D.M. and Kim, S.K. 1994. Signal transduction and cell fate specification during Caenorhabditis elegans vulval development. Curr. Opin. Genet. Dev. 4:508‐516.
   Hardie, D.G., Haystead, T.A.J., and Sim, A.T.R. 1991. Use of okadaic acid to inhibit protein phosphatases in intact cells. Methods Enzymol. 201:469‐477.
   Heldin, C.‐H. 1995. Dimerization of cell surface receptors in signal transduction. Cell. 80:213‐223.
   Ihle, J.N., Witthuhn, B.A., Quelle, F.W., Yamamoto, K., Thierfelder, W.E., Kreider, B., and Silvennoinen, O. 1994. Signalling by the cytokine receptor superfamily: JAKS and STATS. Trends Biochem. Sci. 19:222‐227.
   Kamps, M.P. 1991. Determination of phosphoamino acid composition by acid hydrolysis of protein blotted to Immobilon. Methods Enzymol. 201:21‐27.
   Marshall, C.J. 1995. Specificity of receptor tyrosine kinase signalling: Transient versus sustained extracellular signal‐regulated kinase activation. Cell. 80:179‐185.
   Parker, L.L., Atherton‐Fessler, S., and Piwinica‐Worms, H. 1992. p107 wee1 is a dual‐specificity kinase that phosphorylates p34cdc2 on tyrosine 15. Proc. Natl. Acad. Sci. U.S.A. 89:2917‐2921
   Planas‐Silva, M.D. and Means, A.R. 1992. Expression of a constitutive form of calcium/calmodulin‐dependent protein kinase II leads to arrest of the cell cycle in G2. EMBO J. 11:507‐517.
   Ringer, D.P. 1991. Separation of phosphotyrosine, phosphoserine, and phosphothreonine by high‐performance liquid chromatography. Methods Enzymol. 201:3‐10.
   Shenolikar, S. 1994. Protein serine/threonine phosphatases: New avenues for cell regulation. Annu. Rev. Cell Biol. 10:55‐86.
   Shenolikar, S. and Ingebritsen, T.S. 1984. Protein (serine, threonine) phosphate phosphatases. Methods Enzymol. 107:102‐130.
   Shenolikar, S. and Nairn, A.C. 1991. Protein phosphatases: Recent progress. Adv. Second Messenger Phosphoprotein Res. 23:1‐121.
   Simon, M. 1994. Signal transduction during the development of the Drosophila R7 photoreceptor. Dev. Biol. 166:431‐442.
   Spencer, D.M., Wandless, T.J., Schreiber, S.L., and Crabtree, G.R. 1993. Controlling signal transduction with synthetic ligands. Science. 262:1019‐1024.
   Sun, H., Charles, C.H., Lau, L.F., and Tonks, N.K. 1993. MKP‐1 (3CH134), an immediate early gene product, is a dual‐specificity phosphatase that dephosphorylates MAP kinase in vivo. Cell. 75:487‐493.
   Thorburn, J., McMahon, M., and Thorburn, A. 1994. Raf‐1 kinase activity is necessary and sufficient for gene expression changes but not sufficient for cellular morphology changes associated with cardiac myocyte hypertrophy. J. Biol. Chem. 269:30580‐30586.
   Zhou, S., Clemens, J.C., Stone, R.L., and Dixon, J.E. 1994. Mutational analysis of a ser/thr phosphatase. Identification of residues important in phosphatase substrate binding and catalysis. J. Biol. Chem. 269:26234‐26238.
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