In 1906, Phoebus A. Levene at the Rockefeller Institute for Medical Research identified phosphate in the protein Vitellin (phosvitin), and by 1933 had detected phosphoserine in Casein, with Fritz Lipmann. However, it took another 20 years before Eugene P. Kennedy described the first ‘enzymatic phosphorylation of proteins’.
Reversible phosphorylation of proteins is an important regulatory mechanism that occurs in both prokaryotic and eukaryotic organisms. 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 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 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
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.
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
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.
PTM (Posttranslational Modification) isoforms are easily detected on 2D gels. 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 (Gerber et al., 2003). A relative quantitation can be obtained with a variety of differential isotope labeling technologies (Gygi et al., 2002, Goshe et al., 2003).
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.
Phosphorylation of Ser.sup.78 .sup.of Hsp27 correlated with HER-2/ neu status and lymph node positivity in breast cancer.(Research)(Clinical report)
Aug 14, 2007; Authors: Daohai Zhang (corresponding author) [1,2]; Lee Lee Wong ; Evelyn SC Koay [1,2]BackgroundHeat shock proteins (Hsp's)...
Phosphorylation of the type I regulatory subunit of cAMP-dependent protein kinase in rat tissues. (Collegiate Communications--Undergraduate).(Brief Article)
Apr 01, 2000; INTRODUCTION cAMP-dependent protein kinase (PKA) is a multi functional enzyme regulating processes such as signaling, gene...