Protein phosphorylation

Model of a phosphorylated serine residue

Protein phosphorylation is a post-translational modification of proteins in which an amino acid residue is phosphorylated by a protein kinase by the addition of a covalently bound phosphate group. Phosphorylation alters the structural conformation of a protein, causing it to become activated, deactivated, or modifying its function. The reverse reaction of phosphorylation is called dephosphorylation, and is catalyzed by protein phosphatases. Protein kinases and phosphatases work independently and in a balance to regulate the function of proteins. The amino acids most commonly phosphorylated are serine, threonine, and tyrosine in eukaryotes, and histidine in prokaryotes, which play important and well-characterized roles in signaling pathways and metabolism. However, many other amino acids can also be phosphorylated, including arginine, lysine, and cysteine.^[1] Protein phosphorylation was first reported in 1906 by Phoebus Levene at the Rockefeller Institute for Medical Research with the discovery of phosphorylated vitellin.^[2] However, it was nearly 50 years until the enzymatic phosphorylation of proteins by protein kinases was discovered.^[3]

History

In 1906, Phoebus Levene at the Rockefeller Institute for Medical Research identified phosphate in the protein vitellin (phosvitin),^[2] and by 1933 had detected phosphoserine in casein, with Fritz Lipmann.^[4] 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.^[5] 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.^[6] 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.^[7][8] Edmond Fisher and Edwin Krebs were awarded the Nobel prize in 1992 “for their discoveries concerning reversible protein phosphorylation as a biological regulatory mechanism”.^[9]

Functions of phosphorylation

Phosphorylation introduces a charged and hydrophilic group in the side chain of amino acids, possibly changing a protein's structure by altering interactions with nearby amino acids. Some proteins such as p53 contain multiple phosphorylation sites, facilitating complex, multi-level regulation. Because of the ease with which proteins can be phosphorylated and dephosphorylated, this type of modification is a flexible mechanism for cells to respond to external signals and environmental conditions.^[10]

Regulatory roles of phosphorylation include:

Biological thermodynamics

• 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.

Protein degradation

• Arginine phosphorylation by McsB kinase marks proteins for degradation by a Clp protease. The arginine phosphorylation system, which is widely distributed across Gram-positive bacteria, appears to be functionally analogous to the eukaryotic ubiquitin–proteasome system.^[11]

Enzyme regulation (activation and inhibition)

• The first example of protein regulation by phosphorylation was glycogen phosphorylase. Eddie Fisher and Ed Krebs described how phosphorylation of glycogen phosphorylase b converted it to the active glycogen phosphorylase a. It was soon discovered that glycogen synthase, another metabolic enzyme, is inactivated by phosphorylation.^[12]

• Phosphorylation of the enzyme GSK-3 by AKT (Protein kinase B) as part of the insulin signaling pathway.^[13]
• Phosphorylation of Src tyrosine kinase (pronounced "sarc") by Csk (C-terminal Src kinase) inactivates Src by inducing a conformational change which masks its kinase domain.^[14]

Protein-protein interactions

• 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.^[15]
• 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.

Receptor tyrosine kinases

The AXL receptor tyrosine kinase, showing the symmetry of the dimerized receptors

While tyrosine phosphorylation is found in relatively low abundance, it is well studied due to the ease of purification of phosphotyrosine using antibodies. Receptor tyrosine kinases are an important family of cell surface receptors involved in the transduction of extracellular signals such as hormones, growth factors, and cytokines. Binding of a ligand to a monomeric receptor tyrosine kinase stabilizes interactions between two monomers to form a dimer, after which the two bound receptors phosphorylate tyrosine residues in trans. Phosphorylation and activation of the receptor activates a signaling pathway through enzymatic activity and interactions with adaptor proteins.^[16] Signaling through the epidermal growth factor receptor (EGFR), a receptor tyrosine kinase, is critical for the development of multiple organ systems including the skin, lung, heart, and brain. Excessive signaling through the EGFR pathway is found in many human cancers.^[17]

Cyclin-dependent kinases

Cyclin-dependent kinases (CDKs) are serine-threonine kinases which regulate progression through the eukaryotic cell cycle. CDKs are catalytically active only when bound to a regulatory cyclin. Animal cells contain at least nine distinct CDKs which bind to various cyclins with considerable specificity. CDK inhibitors (CKIs) block kinase activity in the cyclin-CDK complex to halt the cell cycle in G1 or in response to environmental signals or DNA damage. The activity of different CDKs activate cell signaling pathways and transcription factors that regulate key events in mitosis such as the G1/S phase transition. Earlier cyclin-CDK complexes provide the signal to activate subsequent cyclin-CDK complexes.^[18]

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 1/10 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.^[19]
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.^[20] 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.^[21] 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. A curated database of dbPAF was created, containing known phosphorylation sites in H. sapiens, M. musculus, R. norvegicus, D. melanogaster, C. elegans, S. pombe and S. cerevisiae. The database currently holds 294,370 non-redundant phosphorylation sites of 40,432 proteins.^[22] Other tools of phosphorylation prediction in proteins include NetPhos ^[23] for eukaryotes, NetPhosBac ^[23] for bacteria and ViralPhos ^[24] for viruses.

Serine

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.^[25]
• 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.^[26]
• When serine 349 is phosphorylated, the binding affinity between protein complex p62 and the protein Keap1 is strengthened, which is linked to stress response.^[27]
• 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.^[28]

Tyrosine

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.^[29] 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.^[30]

Arginine

Arginine phosphorylation in many Gram-positive bacteria marks proteins for degradation by a Clp protease.^[11]

Evolution

Protein phosphorylation is common among all clades of life, including all animals, plants, fungi, bacteria, and archaea. The origins of protein phosphorylation mechanisms are ancestral and have diverged greatly between different species. In eukaryotes, it is estimated that as many as 30% of all proteins may be phosphorylated, with tens of thousands of distinct phosphorylation sites.^[31] Some phosphorylation sites appear to have evolved as conditional "off" switches, blocking the active site of an enzyme, such as in the prokaryotic metabolic enzyme isocitrate dehydrogenase. However, in the case of proteins that must be phosphorylated to be active, it is less clear how they could have emerged from non-phosphorylated ancestors. It has been shown that a subset of serine phosphosites are often replaced by acidic residues such as aspartate and glutamate between different species. These anionic residues can interact with cationic residues such as lysine and arginine to form salt bridges, stable non-covalent interactions that alter a protein's structure. These phosphosites often participate in salt bridges, suggesting that some phosphorylation sites evolved as conditional "on" switches for salt bridges, allowing these proteins to adopt an active conformation only in response to a specific signal.^[32]

There are hundreds of known eukaryotic protein kinases, making them one of the largest gene families. Most phosphorylation is carried out by a single superfamily of protein kinases that share a conserved kinase domain. Protein phosphorylation is highly conserved in pathways central to cell survival, such as cell cycle progression relying on Cyclin-dependent kinases (CDKs), but individual phosphorylation sites are often flexible. Targets of CDK phosphorylation often have phosphosites in disordered segments, which are found in non-identical locations even in close species. Conversely, targets of CDK phosphorylation in structurally defined regions are more highly conserved. While CDK activity is critical for cell growth and survival in all eukaryotes, only very few phosphosites show strong conservation of their precise positions. Positioning is likely to be highly important for phosphates that allosterically regulate protein structure, but much more flexible for phosphates that interact with phosphopeptide-binding domains to recruit regulatory proteins.^[33]

Comparisons between Eukaryotes and Prokaryotes

Protein phosphorylation is a reversible post-translational modification of proteins. In eukaryotes, protein phosphorylation functions in cell signaling, gene expression, and differentiation. It is also involved in DNA replication during the cell cycle, and the mechanisms that cope with stress-induced replication blocks. Compared to eukaryotes, prokaryotes use Hanks-type kinases and phosphatases for signal transduction. Whether or not the phosphorylation of proteins in bacteria can also regulate processes like DNA repair or replication still remains unclear.^[34]

Compared to the protein phosphorylation of prokaryotes, the studies done in the protein phosphorylation in eukaryotes of yeast and human cells have been more extensive. It is known that eukaryotes rely on the phosphorylation of the hydroxyl group on the side chains of serine, threonine, and tyrosine. These bases are the main regulatory post-translational modifications in eukaryotic cells but the protein phosphorylation of prokaryotes are less intensely studied. While serine, threonine, and tyrosine are phosphorylated in eukaryotes, histidine and aspartate is phosphorylated in prokaryotes. In bacteria, histidine phosphorylation occurs in the phosphoenolpyruvate-dependent phosphotransferase systems (PTSs), which are involved in the process of internalization as well as the phosphorylation of sugars.^[35]

Protein phosphorylation by protein kinase was first obtained by E. coli and Salmonella typhimurim and has since been demonstrated in many other bacterial cells.^[36] It was found that bacteria use histidine and aspartate phosphorylation as a model for bacterial signaling transduction but in the last few years there has been evidence that has shown that serine, threonine, and tyrosine phosphorylation are also present in bacteria. It was shown that bacteria carry kinases and phosphatases similar to that of their eukaryotic equivalent but they have also developed unique kinases and phosphatases not found in eukaryotes.^[35]

Pathology

Abnormal protein phosphorylation has been implicated in a number of diseases, such as Alzheimer's Disease, Parkinson's Disease, and other degenerative disorders.

Tau protein belongs to a group of Microtubule Associated Proteins (MAPs) which, among several things, help stabilize microtubules in cells, including neurons.^[37] Association and stabilizing activity of tau protein depends on its phosphorylated state. In Alzheimer's Disease, due to misfoldings and abnormal conformational changes in tau protein structure, it is rendered ineffective at binding to microtubules and thus unable to keep the neural cytoskeletal structure organized during neural processes; in fact abnormal tau inhibits and disrupts microtubule organization and disengages normal tau from microtubules into cytosolic phase.^[38] The misfoldings lead to the abnormal aggregation into fibrillary tangles inside the neurons, the hallmark of Alzheimer's Disease. There is an adequate amount that the tau protein needs to be phosphorylated to function, but hyperphosphorylation of tau protein is thought to be one of the major influences on its incapacity to associate.^[38] Kinases PP1, PP2A, PP2B, and PP2C dephosphorylate tau protein in vitro, and their activities have found to be reduced in areas of the brain in Alzheimer patients.^[38][39] Tau phosophoprotein is three to fourfold hyperphosphorylated in an Alzheimer patient compared to an aged non-afflicted individual. Alzheimer Disease tau seems to remove MAP1 and MAP2 (two other major associated proteins) from microtubules and this deleterious effect is reversed when dephosphorylation is performed, evidencing hyperphosphorylation as the sole cause of the crippling activity.^[38]

Parkinson's disease

α-Synuclein is a protein that is associated with Parkinson’s disease. This protein is coded by the PARRK1 gene and in its native form, α-Synuclein is involved in the recycling of the synaptic vesicles that carry neurotransmitters and naturally occurs in an unfolded form. Elevated levels of α-Synuclein are found in patients with Parkinson’s disease, and there seems to be a positive correlation between the amount of the α-Synuclein protein present in the patient and the severity of the disease.

Phosphorylation of the amino acid Ser¹²⁹ in the α-Synuclein protein has a profound effect on the severity of the disease. There seem to be correlation between the total α-Synuclein concentration (unphosphorylated) and the severity of the symptoms in Parkinson’s disease patients. Healthy patients seem to have higher levels of unphosphorylated α-Synuclein than patients with Parkinson’s disease. Moreover, the measurement of the changes in the ratio of concentrations of phosphorylated α-Synuclein to unphosphorylated α-Synuclein within a patient could be a potential marker of the disease progression

Phosphorylation of Ser¹²⁹ is associated with the aggregation of the protein and further damage to the nervous system. Furthermore, the aggregation of phosphorylated α-Synuclein can be enhanced if a presynaptic scaffold protein Sept4 is present in insufficient quantities. It is important to note that direct interaction of α-Synuclein with Sept4 protein inhibits the phosphorylation of Ser¹²⁹.^[40][41][42]

References