Protein phosphorylation is a reversible post-translational modification. It is catalyzed by protein kinases that add phosphate groups to an amino acid residue and reversed by protein phosphatases that remove phosphate groups. Protein phosphorylation was first reported in 1906 by Phoebus Levene at the Rockefeller Institute for Medical Research with the discovery of phosphorylated vitellin. However, the discovery of phosphorylation as a regulatory physiological mechanism arose from the work in 1955 of Edmond H. Fischer and Edwin G. Krebs who showed that the activation of GPb (glycogen phosphorylase b) to GPa (glycogen phosp1horylase a) was dependent on a protein kinase action together with ATP. In 1992, Edmond H. Fischer and Edwin G. Krebs were jointly awarded the Nobel Prize in Physiology or Medicine for their discoveries concerning "reversible protein phosphorylation as a biological regulatory mechanism". They purified and characterized the first enzyme that phosphorylates proteins. Their fundamental finding initiated research in cell regulation, which today is one of the most active and wide-ranging fields of biological research.
Protein phosphorylation is crucial for modulating protein structure, protein localization and the protein-protein interactions that form the basis of many cell-signaling networks. Phosphorylation-based signaling often takes the form of a cascade where sequential protein phosphorylations lead to changes in protein stability, function and localization. Protein kinases, the enzymes that propagate these signals, catalyze the transfer of gamma phosphate from ATP onto serine, threonine or tyrosine residues of substrate proteins. The sites of protein phosphorylation and phosphorylation dynamics are important in determining the biological outcome of a signaling event. For instance, protein phosphorylation drives many of the changes during the cell cycle. During mitosis kinases are activated at precise times to direct the course of chromosome segregation and cell division. For example CDK1 activation at the beginning of mitosis leads to phosphorylation of NUP98 during prophase which in turn promotes nuclear envelope disassembly. Additionally, increased protein phosphorylation rate and constitutive activation of signaling networks due to hyper-activated kinases is considered a hallmark of cancer. Since the rate of substrate phosphorylation is a straightforward readout of kinase activity, there is growing interest in measuring phosphorylation rates to better understand phosphorylation-based signaling networks
and potentially design more effective cancer treatments.
Functions of protein phosphorylation
Addition of a phosphoryl group to serine, threonine, or tyrosine (in eukaryotes and occasionally in prokaryotes) or to histidine or aspartic acid (in prokaryotes) residues confers properties that can have profound effects on protein conformation and function. The phosphoryl group with a pKa of ∼6.7 is predominantly dianionic at physiological pH. The property of a double negative charge (a property not carried by any of the naturally occurring amino acids) and the capacity for the phosphoryl oxygens to form hydrogen-bond networks presents special characteristics. There are two main types of interactions between phosphate and protein. First, the phosphate group frequently interacts with the side chain of one or more arginine residues. The guanidinium group of an arginine residue is well suited for interactions with a phosphate group by virtue of its planar structure and its ability to form multiple hydrogen bonds. Electrostatic interactions between arginine and phosphoryl groups provide tight binding sites that play a dominant role in recognition and conformational response. Theoretical calculations on the strengths of hydrogen bonds have shown that the bidentate interactions available to arginine with phosphate provide much stronger interactions than those that can be formed with -NH3+ groups as in lysine side chains. In addition, a number of other residues are also involved at phospho-recognition sites, and these include lysine, tyrosine, serine, threonine, asparagine, histidine, and metal ions. Second, the phosphoryl group interacts with backbone nitrogens at the start of an α-helix and utilizes the positive charge of the helix dipole for neutralizing charges.
Protein phosphorylation can activate enzyme activity through allosteric conformational changes, as observed for glycogen phosphorylase, the binding of phosphate group converts the activated dimer into the deactivated tetramer. Many protein kinases rely on phosphorylation by upstream kinases. Phosphorylation can inhibit enzyme activity. In isocitrate dehydrogenase, the phosphate group acts as a steric blocking agent and does not promote any conformational change. Phosphorylation can lead to recognition sites for other protein molecules, such as in the phosphotyrosine recognition SH2 domains important for regulation of kinases such as Src, ZAP70 (ζ -chain-associated protein kinase of 70 kDa), Fes and Abl protein. In addition, phosphorylation is 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.
Protein kinases are enzymes that modify protein substrates by catalyzing the transfer of the gamma phosphate group of ATP to serine, threonine, or tyrosine residues (phosphorylation sites). This results in a functional change of the protein substrate that regulates the activity, localization and/or stability. In this way, kinases play a key role in regulating almost all cellular processes. For instance, kinases are involved in signal transduction and are known to regulate the majority of cell processes, such as cell growth, differentiation, metabolism, gene transcription, and so on. The human genome is estimated to contain about 518 protein kinases, representing about 2% of human gene. Importantly, it is estimated that between 30-50% of all eukaryotic cell proteins are modified by kinases. Most serine and threonine kinases can act on either serine or threonine residues whilst tyrosine kinases modify tyrosine residues. Finally, dual specificity kinases are able to modify S, T and Y. In many cases, acidity, basicity or hydrophobicity of the surrounding residues of the phosphorylation residue determines the specificity of a protein kinase.