Overview of Protein Phosphorylation

Bartholomew M. Sefton1

1 The Salk Institute, San Diego, California
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 14.1
DOI:  10.1002/0471143030.cb1401s00
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Phosphorylation is the most common and important mechanism of acute and reversible regulation of protein function. Studies of mammalian cells metabolically labeled with [32P]orthophosphate suggest that as many as one‐third of all cellular proteins are covalently modified by protein phosphorylation. Protein phosphorylation has an important role in essentially all aspects of cell biology. Most polypeptide growth factors (platelet‐derived growth factor and epidermal growth factor are among the best studied) and cytokines (e.g., interleukin 2, colony stimulating factor 1, and γ‐interferon) stimulate phosphorylation upon binding to their receptors. Induced phosphorylation in turn activates cytoplasmic protein kinases, such as Raf, the activators of the mitogen‐activated protein (MAP) kinases SEK and MEK, the MAP kinases ERK, JNK, and p38, the Janus/JAK kinases, the p21 activated kinases (PAKs), and the phosphatidylinsoitil 3'‐kinase‐activated kinase, protein kinase B/Akt. Additionally, in all nucleated organisms, cell cycle progression is regulated at both the G1/S and the G2/M transitions by cyclin‐dependent protein kinases. These kinases regulate the G1/S transition by the phosphorylation of cell cycle regulators such as Rb protein and the G2/M transition through the phosphorylation of nuclear lamins and histones.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • History
  • Labeling Studies
  • Sites of Phosphorylation
  • Detection of Unlabeled Phosphoamino Acids
  • Protein Kinases
  • Protein Phosphatases
  • Literature Cited
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Alessi, D.R. and Cohen, P. 1998. Mechanism of activation and function of protein kinase B. Curr. Opin. Genet. Dev. 8:55‐62.
   Arion, D., Meijer, L., Brizuela, L., and Beach, D. 1988. cdc2 is a component of the M phase‐specific histone H1 kinase: Evidence for identity with MPF. Cell 55:371‐378.
   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.
   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.
   Fauman, E.B. and Saper, M.A. 1996. Structure and function of the protein tyrosine phosphatases. Trends Biochem. Sci. 21:413‐417.
   Furlong, R.A. 1992. The biology of hepatocyte growth factor/scatter factor. Bioessays 14:613‐617.
   Hanks, S.K. and Polte, T.R. 1997. Signaling through focal adhesion kinase. Bioessays 19:137‐145.
   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.
   Kamps, M.P. and Sefton, B.M. 1988. Identification of multiple novel polypeptide substrates of the v‐rsc, v‐yes, v‐fps, v‐ros, and v‐erb‐B oncogenic tyrosine protein kinases utilizing antisera against phosphotyrosine. Oncogene 2:305‐315.
   Lim, L., Manser, E., Leung, T., and Hall, C. 1996. Regulation of phosphorylation pathways by p21 GTPases. The p21 Ras‐related Rho subfamily and its role in phosphorylation signalling pathways. Eur. J. Biochem. 242:171‐185.
   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.
   Perrimon, N., Lu, X., Hou, X.S., Hus, J.C., Melnick, M.B., Chou, T.B., and Perkins, L.A. 1995. Dissection of the Torso signal transduction pathway in Drosphila. Mol. Reprod. Dev. 42:515‐522.
   Peter, M., Nakagawa, J., Doree, M., Labbe, J.C., and Nigg, E.A. 1990. In vitro disassembly of the nuclear lamina and M phase‐specific phosphorylation of lamins by cdc2 kinase. Cell 61:591‐602.
   Reed, S.I. 1997. Control of the G1/S transition. Cancer Surv. 29:7‐23.
   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 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.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library