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A phosphorylated serine residue

Phosphorylation is the addition of a phosphate group (PO43−) to a molecule. Phosphorylation and its counterpart, dephosphorylation, turn many protein enzymes on and off, thereby altering their function and activity. Protein phosphorylation is one type of post-translational modification.

Protein phosphorylation in particular plays a significant role in a wide range of cellular processes. Its prominent role in biochemistry is the subject of a very large body of research (as of March 2015, the MEDLINE database returns over 240,000 articles on the subject, largely on protein phosphorylation).

Protein phosphorylation[edit]


In 1906, Phoebus Levene at the Rockefeller Institute for Medical Research identified phosphate in the protein vitellin (phosvitin),[1] and by 1933 had detected phosphoserine in casein, with Fritz Lipmann.[2] However, it took another 20 years before Eugene P. Kennedy described the first ‘enzymatic phosphorylation of proteins’.[3] The first phosphorylase enzyme was discovered by Carl and Gerty Cori in the late 1930s. Carl and Gerty Cori found two forms of glycogen phosphorylase which they named A and B but did not correctly understand the mechanism of the B form to A form conversion. The interconversion of phosphorylase b to phosphorylase a was later described by Edmond Fischer and Edwin Krebs, as well as, Wosilait and Sutherland, involving a phosphorylation/dephosphorylation mechanism.[4] It was found that an enzyme, named phosphorylase kinase and Mg-ATP were required to phosphorylate glycogen phosphorylase by assisting in the transfer of the γ-phosphoryl group of ATP to a serine residue on phosphorylase b. Protein phosphatase 1 is able to catalyze the dephosphorylation of phosphorylated enzymes by removing the phosphate group. Earl Sutherland explained in 1950, that the activity of phosphorylase was increased and thus glycogenolysis stimulated when liver slices were incubated with adrenalin and glucagon. Phosphorylation was considered a specific control mechanism for one metabolic pathway until the 1970s, when Lester Reed discovered that mitochondrial pyruvate dehydrogenase complex was inactivated by phosphorylation. Also in the 1970s, the term multisite phosphorylation was coined in response to the discovery of proteins that are phosphorylated on two or more residues by two or more kinases. In 1975, it was shown that cAMP-dependent proteins kinases phosphorylate serine residues on specific amino acid sequence motifs. Ray Erikson discovered that v-Src was a kinase and Tony Hunter found that v-Src phosphorylated tyrosine residues on proteins in the 1970s.[5] In the early 1980, the amino-acid sequence of the first protein kinase was determined which helped geneticists understand the functions of regulatory genes. In the late 1980s and early 1990s, the first protein tyrosine phosphatase (PTP1B) was purified and the discovery, as well as, cloning of JAK kinases was accomplished which led to many in the scientific community to name the 1990s as the decade of protein kinase cascades.[6][7] Edmond Fisher and Edwin Krebs were awarded the Nobel prize in 1992 “for their discoveries concerning reversible protein phosphorylation as a biological regulatory mechanism”.[8]


Reversible phosphorylation of proteins is an important regulatory mechanism that occurs in both prokaryotic and eukaryotic organisms.[9][10][11][12] Kinases phosphorylate proteins and phosphatases dephosphorylate proteins. 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, tyrosine and histidine residues in eukaryotic proteins. Histidine phosphorylation of eukaryotic proteins appears to be much more frequent than tyrosine phosphorylation.[13] In prokaryotic proteins phosphorylation occurs on the serine, threonine, tyrosine, histidine or arginine or lysine residues.[9][10][13][14] 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 protein dynamics can induce a conformational change in the structure of the protein via long-range allostery 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[15] 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.[16] 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.[citation needed] 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.[17]
    • 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".[18]
  • 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.[19]
  • 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.
  • Important in glycolysis.

Glycolysis is an essential process of glucose degrading into two molecules of pyruvate, through various steps, with the help of different enzymes. It occurs in ten steps and proves that phosphorylation is a much required and necessary to attain the end products. Phosphorylation initiates the reaction in step 1 of the preparatory step[20] (first half of glycolysis), and initiates step 6 of payoff phase (second phase of glycolysis).[21]

Glucose, by nature, is a small molecule with the ability to diffuse in and out of the cell. By phosphorylating glucose (adding a negatively charged phosphate group), glucose is converted to glucose-6-phosphate and trapped within the cell as the cell membrane is negatively charged. This reaction occurs due to the enzyme hexokinase, an enzyme that helps phosphorylate many six-membered ring structures. Glucose-6-phosphate cannot travel through the cell membrane and is therefore, coerced to stay inside the cell. Phosphorylation takes place in step 3, where fructose-6-phosphate is converted to fructose-1,6-bisphosphate. This reaction is catalyzed by phosphofructokinase.

While phosphorylation is performed by ATPs during preparatory steps, phosphorylation during payoff phase is maintained by inorganic phosphate. Each molecule of glyceraldehyde-3-phosphate is phosphorylated to form 1,3-bisphosphoglycerate. This reaction is catalyzed by GAPDH (glyceraldehyde-3-phosphate dehydrogenase). The cascade effect of phosphorylation eventually causes instability and allows enzymes to open the carbon bonds in glucose.

Phosphorylation functions as an extremely vital component of glycolysis, for it helps in transport, control and efficiency.[22]

Signaling networks[edit]

Elucidating complex signaling pathway phosphorylation events can be difficult. In cellular signaling pathways, 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.[23] They have been successfully used to identify dynamic changes in the phosphorylation status of more than 6000 sites after stimulation with epidermal growth factor.[24] Another approach for understanding Phosphorylation Network, is by measuring the genetic interactions between multiple phosphorylating proteins and their targets. This reveals interesting recurring patterns of interactions – network motifs.[25] Computational methods have been developed to model phosphorylation networks [26][27] and predict their responses under different perturbations.[28]

Protein phosphorylation sites[edit]

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 1/10 to 1/2 of proteins are phosphorylated (in some cellular state).
  3. One study indicates that 30% of proteins in the human genome can be phosphorylated, and abnormal phosphorylation is now recognized as a cause of human disease.[29]
  4. 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.

Phosphorylation sites are crucial for proteins and their transportation and functions. They are the covalent modification of proteins through reversible phosphorylation. This enables proteins to stay inbound within a cell since the negative phosphorylated site disallows their permeability through the cellular membrane. Protein dephosphorylation allows the cell to replenish phosphates through release of pyrophosphates which saves ATP use in the cell.[30] An example of phosphorylating enzyme is found in E. coli bacteria. It possesses alkaline phosphatase in its periplasmic region of its membrane. The outermost membrane is permeable to phosphorylated molecules however the inner cytoplasmic membrane is impermeable due to large negative charges.[31] In this way, the E. coli bacteria stores proteins and pyrophosphates in its periplasmic membrane until either are needed within the cell.

Recent advancement in phosphoproteomic identification has resulted in the discoveries of countless phosphorylation sites in proteins. This required an integrative medium for accessible data in which known phosphorylation sites of proteins are organized. The work done by Shahid Ullah, Shaofeng Lin and team developed a curated database of dbPAF, containing known phosphorylation sites in H. sapiens, M. musculus, R. norvegicus, D. melanogaster, C. elegans, S. pombe and S. cerevisiae. Their database currently holds 294,370 non-redundant phosphorylation sites of 40,432 proteins.[32] Other tools of phosphorylation prediction in proteins include NetPhos [33] for eukaryotes, NetPhosBac [33] for bacteria and ViralPhos [34] for viruses.

Types of phosphorylation[edit]

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 but is at the origin of protein phosphorylation signaling pathways in most of the eukaryotes. Phosphorylation on amino acids, such as serine, threonine, and tyrosine results in the formation of a phosphoprotein, when the phosphate group of the phosphoprotein reacts with the -OH group of a Ser, Thr, or Tyr sidechain in an esterification reaction.[35] 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.[36] In prokaryotes, archea, and some lower eukaryotes histidine's nitrogen act as a nucleophile and binds to a phosphate group.[37] Once histidine is phosphorylated the regulatory domain of the response regulator catalyzes the transfer of the phosphate to aspartate.

Phosphorylation of tyrosine[edit]

Tyrosine phosphorylation requires the presence of protein tyrosine kinases (PTK) as well as protein tyrosine phosphatase (PTP) for activation. Tyrosine phosphorylation is fast to react and the reaction can be reversed. Being one of the major regulatory mechanisms in signal transduction - cell growth, differentiation, migration and metabolic homeostasis are cellular processes maintained by tyrosine phosphorylation. The function of protein tyrosine kinases and protein-tyrosine phosphatase are counterbalances the level of phosphotyrosine on any protein. The malfunctioning of specific chains of protein tyrosine kinases and protein tyrosine phosphatase has been linked to multiple human diseases such as obesity, insulin resistance, and type 2 diabetes mellitus.[38] Phosphorylation on tyrosine doesn't occur in just eukaryotes but has been discovered to occur in a selection of bacterial species and present among prokaryotes. Phosphorylation on tyrosine maintains the cellular regulation in bacteria similar to its function in eukaryotes.[39]

Phosphorylation of serine[edit]

There are a large variety of serine residues, and the phosphorylation of each residue can lead to different metabolic consequences.

  • Protein kinase N1 is responsible for the phosphorylation of the TNF receptor-associated factor (TRAF1) on serine 139 under specific conditions. Murine TRAF1 is also phosphorylated by the same kinase, which leads to the silencing of IKK/NF-κB activity. The elimination of phosphorylation on serine 139 can be achieved by the replacement of TRAF1 with an Alanine residue, which consequently leads to the improved recruitment of TBK1.[40]
  • At the serine 789 residue, FGFR1 is phosphorylated by RSK2 when the kinase is in its active form. The signaling capabilities of FGFR1 at the serine 777 site can be weakened by phosphorylation. Serine 1047 and serine 1048 have been linked to the decreased binding affinity of ubiquitin ligase c-Cbl to EFGR when they are phosphorylated.[41]
  • When serine 349 is phosphorylated, the binding affinity between protein complex p62 and the protein Keap1 is strengthened, which is linked to stress response.[42]
  • When serine 337 is phosphorylated by protein kinase A in vitro, the DNA binding efficiency of the p50 subunit of NF-κB is greatly increased.[43]

Phosphorylation of glucose[edit]

The chemical equation for the conversion of D-glucose to D-glucose-6-phosphate in the first step of glycolysis is given by D-glucose + ATP -> D-glucose-6-phosphate + ATP ΔG° = -16.7 kJ.

Research D. G. Walker of the University of Birmingham determined the presence of two specific enzymes in adult guinea pig liver, both of which catalyze the phosphorylation of glucose to glucose 6 phosphate. The two enzymes have been identified as a specific glucokinase (ATP-D-glucose 6-phosphotransferase) and non-specific hexokinase (ATP-D-hexose 6-phosphotransferase).

Hepatic cell is freely permeable to glucose, and the initial rate of phosphorylation of glucose is the rate-limiting step in glucose metabolism by the liver (ATP-D-glucose 6-phosphotransferase) and non-specific hexokinase (ATP-D-hexose 6-phosphotransferase).[44]

The role of glucose 6-phosphate in glycogen synthase: High blood glucose concentration causes an increase in intracellular levels of glucose 6 phosphate in liver, skeletal muscle and fat (adipose) tissue. (ATP-D-glucose 6-phosphotransferase) and non-specific hexokinase (ATP-D-hexose 6-phosphotransferase). In liver, synthesis of glycogen is directly correlated by blood glucose concentration and in skeletal muscle and adipocytes, glucose has a minor effect on glycogen synthase. High blood glucose releases insulin, stimulating the trans location of specific glucose transporters to the cell membrane.[44][45]

The liver’s crucial role in controlling blood sugar concentrations by breaking down glucose into carbon dioxide and glycogen is characterized by the negative delta G value, which indicates that this is a point of regulation with. The hexokinase enzyme has a low Km, indicating a high affinity for glucose, so this initial phosphorylation can proceed even when glucose levels at nanoscopic scale within the blood.

The phosphorylation of glucose can be enhanced by the binding of Fructose-6-phosphate, and lessened by the binding fructose-1-phosphate. Fructose consumed in the diet is converted to F1P in the liver. This negates the action of F6P on glucokinase,[46] which ultimately favors the forward reaction. The capacity of liver cells to phosphorylate fructose exceeds capacity of metabolize fructose-1-phosphate. Consuming excess fructose ultimately results in an imbalance in liver metabolism, which indirectly exhausts the liver cell’s supply of ATP.[47]

Allosteric activation by glucose 6 phosphate, which acts as an effector, stimulates glycogen synthase, and glucose 6 phosphate may inhibit the phosphorylation of glycogen synthase by cyclic AMP-stimulated protein kinase.[45]

Phosphorylation of glucose is imperative in processes within the body. For example, phosphorylating glucose is necessary for insulin-dependent mechanistic target of rapamycin pathway activity within the heart. This further suggests a link between intermediary metabolism and cardiac growth.[48]

Phosphorylation of histones[edit]

Eukaryotic DNA is organized with histone proteins in specific complexes called chromatin. The chromatin structure functions and facilitates the packaging, organization and distribution of eukaryotic DNA. However, it has a negative impact on several fundamental biological processes such as transcription, replication and DNA repair by restricting the accessibility of certain enzymes and proteins. Post-translational modification of histones such as histone phosphorylation has been shown to modify the chromatin structure by changing protein:DNA or protein:protein interactions.[49] Histone post-translational modifications modify the chromatin structure. The most commonly associated histone phosphorylation occurs during cellular responses to DNA damage, when phosphorylated histone H2A separates large chromatin domains around the site of DNA breakage.[50] Researchers investigated whether modifications of histones directly impact RNA polymerase II directed transcription. Researchers choose proteins that are known to modify histones to test their effects on transcription, and found that the stress-induced kinase, MSK1, inhibits RNA synthesis. Inhibition of transcription by MSK1 was most sensitive when the template was in chromatin, since DNA templates not in chromatin were resistant to the effects of MSK1. It was shown that MSK1 phosphorylated histone H2A on serine 1, and mutation of serine 1 to alanine blocked the inhibition of transcription by MSK1. Thus results suggested that the acetylation of histones can stimulate transcription by suppressing an inhibitory phosphorylation by a kinase as MSK1.[51]

Detection and characterization[edit]

Antibodies can be used as powerful tool 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.[52] They are becoming critical reagents both for basic research and for clinical diagnosis.

Example of posttranslational modification detected on a 2D gel (spot boundaries delimited by analysis software, identification by mass spectrometry, P46462 is the protein ID in Expasy)

Posttranslational modification (PTM) 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.

In some very specific cases, the detection of the phosphorylation as a shift in the protein's electrophoretic mobility is possible on simple 1-dimensional SDS-PAGE gels, as it's described for instance for a transcriptional coactivator by Kovacs et al.[53] Strong phosphorylation-related conformational changes (that persist in detergent-containing solutions) are thought to underlie this phenomenon. Most of the phosphorylation sites for which such a mobility shift has been described fall in the category of SP and TP sites (i.e. a proline residue follows the phosphorylated serine or threonine residue).

More recently large-scale mass spectrometry analyses have been used to determine sites of protein phosphorylation. Over the last 4 years, dozens of studies have been published, each identifying thousands of sites, many of which were previously undescribed.[54][55] Mass spectrometry is ideally suited for such analyses using HCD or ETD fragmentation, as the addition of phosphorylation results in an increase in the mass of the protein and the phosphorylated residue. Advanced, highly accurate mass spectrometers are needed for these studies, limiting the technology to labs with high-end mass spectrometers. However, the analysis of phosphorylated peptides by mass spectrometry is still not as straightforward as for “regular”, unmodified peptides. Recently EThcD has been developed combining electron-transfer and higher-energy collision dissociation. Compared to the usual fragmentation methods, EThcD scheme provides more informative MS/MS spectra for unambiguous phosphosite localization.[56]

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.[57] A relative quantitation can be obtained with a variety of differential isotope labeling technologies.[58] There are also several quantitative protein phosphorylation methods, including fluorescence immunoassays, Microscale thermophoresis, FRET, TRF, fluorescence polarization, fluorescence-quenching, mobility shift, bead-based detection, and cell-based formats.[59][60]

Other kinds[edit]

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.

See also[edit]


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