Phosphorylation

Phosphorylation is the addition of a phosphate (PO₄) group to a protein molecule or a small molecule. It can also be thought of as "the introduction of a phosphate group into an organic molecule". Its prominent role in biochemistry is the subject of a very large body of research (as of February 2008, the Medline database returns nearly 148,000 articles on the subject, largely on "protein" phosphorylation).

Protein phosphorylation

History

In 1906, Phoebus A. Levene at the Rockefeller Institute for Medical Research identified phosphate in the protein Vitellin (phosvitin), [P.A. Levene and C.L. Alsberg, The cleavage products of vitellin, "J. Biol. Chem." 2 (1906), pp. 127–133.] and by 1933 had detected phosphoserine in Casein, with Fritz Lipmann. [F.A. Lipmann and P.A. Levene, Serinephosphoric acid obtained on hydrolysis of vitellinic acid," J. Biol. Chem." 98 (1932), pp. 109–114.] However, it took another 20 years before Eugene P. Kennedy described the first ‘enzymatic phosphorylation of proteins’. [G. Burnett and E.P. Kennedy, The enzymatic phosphorylation of proteins, "J. Biol. Chem." 211 (1954), pp. 969–980.]

Function

Reversible phosphorylation of proteins is an important regulatory mechanism that occurs in both prokaryotic and eukaryotic organisms.A.J. Cozzon (1988) Protein phosphorylation in prokaryotes "Ann. Rev. Microbiol." 42:97-125] J.B. Stock, A.J. Ninfa and A.M. Stock (1989) Protein phosphorylation and regulation of adaptive responses in bacteria. "Microbiol. Rev.", p. 450-490 ] [ C. Chang and R.C. Stewart (1998) The Two-Component System. "Plant Physiol." 117: 723-731] [D. Barford, A.K. Das and MP. Egloff. (1998) The Structure and mechanism of protein phosphatases: Insights into Catalysis and Regulation "Annu Rev Biophys Biomol Struct." Vol. 27: 133-164 ] Enzymes called kinases (phosphorylation) and phosphatases (dephosphorylation) are involved in this process. Many enzymes and receptors are switched "on" or "off" by phosphorylation and dephosphorylation. Reversible phosphorylation results in a conformational change in the structure in many enzymes and receptors, causing them to become activated or deactivated. Phosphorylation usually occurs on serine, threonine, and tyrosine residues in eukaryotic proteins. In addition, phosphorylation occurs on the basic amino acid residues histidine or arginine or lysine in prokaryotic proteins. The addition of a phosphate (PO₄) molecule to a polar R group of an amino acid residue can turn a hydrophobic portion of a protein into a polar and extremely hydrophilic portion of molecule. In this way it can introduce a conformational change in the structure of the protein via interaction with other hydrophobic and hydrophilic residues in the protein.

One such example of the regulatory role that phosphorylation plays is the p53 tumor suppressor protein. The p53 protein is heavily regulated [ M. Ashcroft, M.H.G. Kubbutat, and K.H. Vousden (1999). Regulation of p53 Function and Stability by Phosphorylation. "Mol Cell Biol" Mar;19(3):1751-8.] and contains more than 18 different phosphorylation sites. Activation of p53 can lead to cell cycle arrest, which can be reversed under some circumstances, or apoptotic cell death [ S. Bates, and K. H. Vousden. (1996). p53 in signalling checkpoint arrest or apoptosis." Curr. Opin. Genet. Dev." 6:1-7. ] This activity occurs only in situations wherein the cell is damaged or physiology is disturbed in normal healthy individuals.

Upon the deactivating signal, the protein becomes dephosphorylated again and stops working. This is the mechanism in many forms of signal transduction, for example the way in which incoming light is processed in the light-sensitive cells of the retina.

Regulatory roles of phosphorylation include
*Biological thermodynamics of energy-requiring reactions
**Phosphorylation of Na⁺/K⁺-ATPase during the transport of sodium (Na⁺) and potassium(K⁺) ions across the cell membrane in osmoregulation to maintain homeostasis of the body's water content.

*Mediates enzyme inhibition
**Phosphorylation of the enzyme GSK-3 by AKT (Protein kinase B) as part of the insulin signaling pathway. [P.C. van Weeren, K.M. de Bruyn, A.M. de Vries-Smits, J. Van Lint, B.M. Burgering. (1998). "Essential role for protein kinase B (PKB) in insulin-induced glycogen synthase kinase 3 inactivation. Characterization of dominant-negative mutant of PKB. "J Biol Chem" 22;273(21):13150-6.]
**Phosphorylation of src tyrosine kinase (pronounced "sarc") by C-terminal Src kinase (Csk) induces a conformational change in the enzyme, resulting in a fold in the structure, which masks its kinase domain, and is thus shut "off". [Cole, P.A., Shen, K., Qiao, Y., and Wang, D. (2003) Protein tyrosine kinases Src and Csk: A tail's tale, "Curr. Opin. Chem., Biol". 7:580-585.]

* Important for protein-protein interaction via "recognition domains."
**Phosphorylation of the cytosolic components of NADPH oxidase, a large membrane-bound, multi-protein enzyme present in phagocytic cells, plays an important role in the regulation of protein-protein interactions in the enzyme. [Babior, B.M., (1999). NADPH oxidase: an update. "Blood" 93, pp. 1464–1476]

* Important in protein degradation.
**In the late 1990s, it was recognized that phosphorylation of some proteins causes them to be degraded by the ATP-dependent ubiquitin/proteasome pathway. These target proteins become substrates for particular E3 ubiquitin ligases only when they are phosphorylated.

Signaling networks

Elucidating complex signaling pathway phosphorylation events can be difficult. In a cellular signaling pathways, a protein A phosphorylates protein B, and B phosphorylates C. However, in another signaling pathway, protein D phosphorylates A, or phosphorylates protein C. Global approaches such as phosphoproteomics the study of phosphorylated proteins, which is a sub-branch of proteomics combined with mass spectrometry-based proteomics, have been utilised to identify and quantify dynamic changes in phosphorylated proteins over time. These techniques are becoming increasingly important for the systematic analysis of complex phosphorylation networks.J.V. Olsen, B.Blagoev, F. Gnad, B. Macek, C. Kumar, P. Mortensen, and M. Mann. (2006) Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. "Cell". 3;127(3):635-48.] They have been successfully used to identify dynamic changes in the phosphorylation status of more than 6000 sites after stimulation with epidermal growth factor. [Y. Li-Rong , H.J. Issaq and T.D. Veenstra. (2007) Phosphoproteomics for the discovery of kinases as cancer biomarkers and drug targets. "Proteomics Clin. Appl." 1, 1042–1057 ]

Protein phosphorylation sites

There are thousands of distinct phosphorylation sites in a given cell since:1) There are thousands of different kinds of proteins in any particular cell (such as a lymphocyte).2) It is estimated that 1/10th to 1/2 of proteins are phosphorylated (in some cellular state).3) Phosphorylation often occurs on multiple distinct sites on a given protein.

Since phosphorylation of any site on a given protein can change the function or localization of that protein, understanding the "state" of a cell requires knowing the phosphorylation state of its proteins. For example, if amino acid Serine-473 ("S473") in the protein AKT is phosphorylated, AKT is, in general, functionally active as a kinase. If not, it is an inactive kinase.

Types of phosphorylation

"See also kinases for more details on the different types of phosphorylation"

Within a protein, phosphorylation can occur on several amino acids. Phosphorylation on serine is the most common, followed by threonine. Tyrosine phosphorylation is relatively rare. However, since tyrosine phosphorylated proteins are relatively easy to purify using antibodies, tyrosine phosphorylation sites are relatively well understood. Histidine and aspartate phosphorylation occurs in prokaryotes as part of two-component signaling and in some cases in eukaryotes in some signal transduction pathways [http://jcs.biologists.org/cgi/reprint/113/18/3141.pdf] .

Detection and characterization

Antibodies can be used as powerful tools to detect whether a protein is phosphorylated at a particular site. Antibodies bind to and detect phosphorylation-induced conformational changes in the protein. Such antibodies are called phospho-specific antibodies; hundreds of such antibodies are now available. They are becoming critical reagents both for basic research and for clinical diagnosis.

. Indeed, phosphorylation replaces neutral hydroxyl groups on serines, threonines, or tyrosines with negatively-charged phosphates with pKs near 1.2 and 6.5. Thus, below pH 5.5, phosphates add a single negative charge; near pH 6.5, they add 1.5 negative charges; above pH 7.5, they add 2 negative charges. The relative amount of each isoform can also easily and rapidly be determined from staining intensity on 2D gels.

A detailed characterization of the sites of phosphorylation is very difficult, and the quantitation of protein phosphorylation by mass spectrometry requires isotopic internal standard approaches ( [http://dx.doi.org/10.1073/pnas.0832254100 Gerber et al., 2003] ). A relative quantitation can be obtained with a variety of differential isotope labeling technologies ( [http://dx.doi.org/10.1021/pr015509n Gygi et al., 2002] , [http://dx.doi.org/10.1016/S0958-1669(02)00014-9 Goshe et al., 2003] ).

Other kinds

ATP, the "high-energy" exchange medium in the cell, is synthesized in the mitochondrion by addition of a third phosphate group to ADP in a process referred to as oxidative phosphorylation. ATP is also synthesized by substrate-level phosphorylation during glycolysis.ATP is synthesized at the expense of solar energy by photophosphorylation in the chloroplasts of plant cells.

Phosphorylation of sugars is often the first stage of their catabolism. It allows cells to accumulate sugars because the phosphate group prevents the molecules from diffusing back across their transporter.

External links

* [http://mpr.nci.nih.gov/MPR/ Mammalian Phosphorylation Resource] , which integrates information on available phospho-specific antibodies
* [http://www.detectorvision.com/deltaMasses deltaMasses] detection and localization of phosphorylations after mass spectrometry
* [http://www.natureprotocols.com/2007/01/10/functional_analyses_for_sitesp.php Functional analyses for site-specific phosphorylation of a target protein in cells (A Protocol)]

References