A phosphorylated serine residue

Phosphorylation is the addition of a phosphate (PO₄) group to a protein molecule or a small molecule. Serine (abbreviated as Ser or S) is an Organic compound with the formula H[[oxygen O]]2 CCH NH sub>2CH2OH A phosphate, an Inorganic chemical, is a salt of Phosphoric acid. Proteins are large Organic compounds made of Amino acids arranged in a linear chain and joined together by Peptide bonds between the Carboxyl 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). Biochemistry is the study of the chemical processes in living Organisms It deals with the Structure and function of cellular components such as MEDLINE ( Medical Literature Analysis and Retrieval System Online is a literature database of life sciences and biomedical information

Protein phosphorylation

History

In 1906, Phoebus A. 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]

Function

Reversible phosphorylation of proteins is an important regulatory mechanism that occurs in both prokaryotic and eukaryotic organisms. The prokaryotes (proʊˈkærioʊts singular prokaryote /proʊˈkæriət/ are a group of Organisms that lack a Cell nucleus (= karyon or any other Animals Plants fungi, and Protists are eukaryotes (juːˈkærɪɒt or -oʊt Organisms whose cells are organized into complex ^[4][5][6][7] Enzymes called kinases (phosphorylation) and phosphatases (dephosphorylation) are involved in this process. A protein kinase is a Kinase Enzyme that modifies other Proteins by chemically adding Phosphate groups to them ( Phosphorylation) A phosphatase is an Enzyme that removes a Phosphate group from its Substrate by hydrolysing Phosphoric acid mono Esters into Many enzymes and receptors are switched "on" or "off" by phosphorylation and dephosphorylation. Enzymes are Biomolecules that catalyze ( ie increase the rates of Chemical reactions Almost all enzymes are Proteins Reversible phosphorylation results in a conformational change in the structure in many enzymes and receptors, causing them to become activated or deactivated. Proteins are an important class of biological Macromolecules present in all biological organisms made up of such elements as Carbon, Hydrogen Enzymes are Biomolecules that catalyze ( ie increase the rates of Chemical reactions Almost all enzymes are Proteins Phosphorylation usually occurs on serine, threonine, and tyrosine residues in eukaryotic proteins. Serine (abbreviated as Ser or S) is an Organic compound with the formula H[[oxygen O]]2 CCH NH sub>2CH2OH Threonine (abbreviated as Thr or T) is an α- Amino acid with the Chemical formula HO2CCH(NH2CH(OHCH3 Tyrosine (abbreviated as Tyr or Y) or 4-hydroxyphenylalanine, is one of the 20 Amino acids that are used by cells to synthesize In addition, phosphorylation occurs on the basic amino acid residues histidine or arginine or lysine in prokaryotic proteins^[4][5]. Histidine (abbreviated as His or H) is one of the 20 standard Amino acids present in Proteins In the Nutritional sense in Arginine (abbreviated as Arg or R) is an α- Amino acid. The L-form is one of the 20 most common natural amino acids Lysine (abbreviated as Lys or K) is an α- Amino acid with the Chemical formula HO2CCH(NH2(CH24NH2 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. p53 (also known as protein 53 or tumor protein 53) is a Transcription factor encoded by the TP53 gene The p53 protein is heavily regulated^[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^[9] 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. In Biology, signal transduction refers to any process by which a cell converts one kind of signal or stimulus into another The vertebrate retina is a light sensitive part inside the inner layer of the Eye.

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. Biological thermodynamics is a phrase that is sometimes used to refer to Bioenergetics, the study of Energy transformation in the Biological sciences Biological Osmoregulation is the active regulation of the Osmotic pressure of bodily fluids to maintain the Homeostasis of the body's Water content that is it keeps Homeostasis (from Greek: ὅμος hómos, "equal" and ιστημι istēmi, "to stand" lit

• Mediates enzyme inhibition
  • Phosphorylation of the enzyme GSK-3 by AKT (Protein kinase B) as part of the insulin signaling pathway. Enzyme inhibitors are Molecules that bind to Enzymes and decrease their activity. Glycogen synthase kinase 3 ( GSK-3) is a Serine/threonine protein kinase, which means that it mediates the addition of phosphate molecules on certain serine and threonine Akt1, also known as "Akt" or Protein kinase B (PKB is an important molecule in mammalian cellular signaling ^[10]
  • 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". Src is a family of proto-oncogenic Tyrosine kinases originally discovered by J ^[11]

• Important for protein-protein interaction via "recognition domains. Protein-protein interactions refer to the association of Protein molecules and the study of these associations from the perspective of Biochemistry, Signal transduction A protein domain is a part of protein sequence and structure that can evolve, function and exist independently of the rest of the protein chain "
  • 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. The NADPH oxidase (nicotinamide adenine dinucleotide phosphate-oxidase is a membrane-bound Enzyme complex Phagocytes are cells that are found in the blood bone marrow and other tissues of Vertebrates. ^[12]

• 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. Ubiquitin is a highly-conserved regulatory Protein that is ''ubiquitously'' expressed in Eukaryotes. Proteasomes are large Protein complexes inside all Eukaryotes and Archaea, as well as in some Bacteria. These target proteins become substrates for particular E3 ubiquitin ligases only when they are phosphorylated. Ubiquitin is a highly-conserved regulatory Protein that is ''ubiquitously'' expressed in Eukaryotes.

Signaling networks

Elucidating complex signaling pathway phosphorylation events can be difficult. Cell signaling is part of a Complex system of Communication that governs basic cellular activities and coordinates cell actions In a cellular signaling pathways, a protein A phosphorylates protein B, and B phosphorylates C. Cell signaling is part of a Complex system of Communication that governs basic cellular activities and coordinates cell actions 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. Phosphoproteomics is a branch of Proteomics that identifies catalogs and characterizes proteins containing a phosphate group as a post-translational modification Proteomics is the large-scale study of Proteins particularly their structures and functions. Mass spectrometry is an analytical technique that identifies the chemical composition of a compound or sample based on the Mass-to-charge ratio of charged particles Proteomics is the large-scale study of Proteins particularly their structures and functions. These techniques are becoming increasingly important for the systematic analysis of complex phosphorylation networks. ^[13] They have been successfully used to identify dynamic changes in the phosphorylation status of more than 6000 sites after stimulation with epidermal growth factor. Epidermal growth factor or EGF is a Growth factor that plays an important role in the regulation of Cell growth, Proliferation, and ^[13][14]

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). A lymphocyte is a type of White blood cell in the Vertebrate Immune system. 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. Akt1, also known as "Akt" or Protein kinase B (PKB is an important molecule in mammalian cellular signaling Akt1, also known as "Akt" or Protein kinase B (PKB is an important molecule in mammalian cellular signaling 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. In Chemistry and Biochemistry, a kinase, alternatively known as a phosphotransferase, is a type of Enzyme that transfers Phosphate In Chemistry, an amino acid is a Molecule containing both Amine and Carboxyl Functional groups In Biochemistry, this Phosphorylation on serine is the most common, followed by threonine. Serine (abbreviated as Ser or S) is an Organic compound with the formula H[[oxygen O]]2 CCH NH sub>2CH2OH Threonine (abbreviated as Thr or T) is an α- Amino acid with the Chemical formula HO2CCH(NH2CH(OHCH3 Tyrosine phosphorylation is relatively rare. Tyrosine (abbreviated as Tyr or Y) or 4-hydroxyphenylalanine, is one of the 20 Amino acids that are used by cells to synthesize However, since tyrosine phosphorylated proteins are relatively easy to purify using antibodies, tyrosine phosphorylation sites are relatively well understood. Antibodies (also known as immunoglobulins, abbreviated Ig) are Gamma globulin Proteins that are found in Blood or other Bodily Histidine and aspartate phosphorylation occurs in prokaryotes as part of two-component signaling and in some cases in eukaryotes in some signal transduction pathways[1]. Histidine (abbreviated as His or H) is one of the 20 standard Amino acids present in Proteins In the Nutritional sense in Aspartic acid (abbreviated as Asp or D; Asx or B represent either aspartic acid or Asparagine) is an α- Amino acid The prokaryotes (proʊˈkærioʊts singular prokaryote /proʊˈkæriət/ are a group of Organisms that lack a Cell nucleus (= karyon or any other Animals Plants fungi, and Protists are eukaryotes (juːˈkærɪɒt or -oʊt Organisms whose cells are organized into complex

Detection and characterization

Antibodies can be used as powerful tools to detect whether a protein is phosphorylated at a particular site. Antibodies (also known as immunoglobulins, abbreviated Ig) are Gamma globulin Proteins that are found in Blood or other Bodily 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. Antibodies (also known as immunoglobulins, abbreviated Ig) are Gamma globulin Proteins that are found in Blood or other Bodily 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)

PTM (Posttranslational Modification) isoforms are easily detected on 2D gels. Two-dimensional gel electrophoresis, abbreviated as 2-DE or 2-D electrophoresis, is a form of Gel electrophoresis commonly used to analyze proteins 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).

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. Adenosine-5'-triphosphate ( ATP) is a multifunctional Nucleotide that is most important as a " molecular currency" of intracellular Energy In Cell biology, a mitochondrion (plural mitochondria) is a membrane-enclosed Organelle found in most eukaryotic cells. Adenosine diphosphate, abbreviated ADP, is a Nucleotide. It is an Ester of Pyrophosphoric acid with the Nucleoside Adenosine Oxidative phosphorylation is a Metabolic pathway that uses energy released by the oxidation of Nutrients to produce Adenosine triphosphate (ATP ATP is also synthesized by substrate-level phosphorylation during glycolysis. Substrate-level phosphorylation is a type of Chemical reaction that results in the formation of Adenosine triphosphate (ATP by the direct transfer of a Phosphate See also Gluconeogenesis, which carries out a process wherein glucose is synthesized rather than catabolized ATP is synthesized at the expense of solar energy by photophosphorylation in the chloroplasts of plant cells. The production of ATP using the energy of sunlight is called photophosphorylation. Chloroplasts are Organelles found in Plant cells and eukaryotic Algae that conduct Photosynthesis.

Phosphorylation of sugars is often the first stage of their catabolism. Sugar is a class of edible Crystalline substances mainly Sucrose, Lactose, and Fructose. For the related metabolic process see Anabolism. Catabolism is the set of Metabolic pathways which break down molecules into It allows cells to accumulate sugars because the phosphate group prevents the molecules from diffusing back across their transporter.

External links

• Mammalian Phosphorylation Resource, which integrates information on available phospho-specific antibodies
• deltaMasses detection and localization of phosphorylations after mass spectrometry
• Functional analyses for site-specific phosphorylation of a target protein in cells (A Protocol)

References