Definition

Protein phosphorylation is a post-translational modification of proteins in which a serine, a threonine or a tyrosine residue is phosphorylated by a protein kinase with the addition of a covalently bound phosphate group. There are several protein phosphorylation related peptides that belongs to regulatory domain of kinases used for studying the physiological role of enzymes.

Discovery

In 1906, Phoebus A. Levene at the Rockefeller Institute identified phosphate in the protein Vitellin ¹. In 1954, phosphorylation of a protein was observed when a liver enzyme catalyzed the phosphorylation of casein and became known as a protein kinase ².  A year later, the role of phosphorylation became more interesting as Fischer and Krebs, and Wosilait and Sutherland ³, showed that an enzyme involved in glycogen metabolism was regulated by the addition or removal of a phosphate, suggesting that reversible phosphorylation could control enzyme activity. A peptide fragment of the thyroid hormone (TR)-binding protein (TRBP), the LXXLL motif, phosphorylated on Ser884 important for receptor selectivity and LXXLL-binding affinity correlates with TRBP transcriptional activity ⁴. Aquaporin - 2 (254 - 267), pSER256, human, a peptide fragment of the human aquaporin-2 (AQP2) phosphorylated at Ser256, plays a key role in vasopressin signaling in the renal-collecting duct ⁵.

Structural Characteristics

Phosphorylation refers to the addition of a phosphate to one of the amino acid side chains of a protein. Phosphates are negatively charged so that their addition to a protein leads to conformational change of the protein. Many bacterial species express 'eukaryotic-like' Ser/Thr or Tyr protein kinases and phosphatases that are candidate mediators of developmental changes and host/pathogen interactions. Studies reveal that universal mechanisms of regulation and substrate recognition govern the functions of prokaryotic and eukaryotic Ser/Thr protein kinases (STPKs). Several structures also support novel mechanisms of regulation, including dimerization of STPKs, metal-ion binding and substrate mimicry ⁶. There are several small peptides are involved in regulation of protein phosphorylation. Sequence of [pSer884] Thyroid Hormone Binding Protein (TRBP), biotin labeled is BIOTIN-KDVTLT(pSer)PLLVNLLQSDIS ⁴. Aquaporin - 2 (254 - 267), pSER256, human sequence is RQ-pS-VELHSPQSLPR. These peptides are used for thyroid hormone and vasopressin-dependent trafficking studies respectively ⁵.

Mode of Action

The transfer of phosphates onto proteins is catalyzed by a variety of protein kinases in the cell. The hydroxyl groups (-OH) of serine, threonine, tyrosine or histidine amino acid side chains are commonly used for addition of phosphate group. A second class of enzymes, phosphatases, are responsible for the reverse reaction, in which phosphates are removed from a protein. Some of these enzymes are extremely specific, potentially phosphorylating or dephosphorylating only a few target proteins, while others are able to act broadly on many proteins. The examples of known targets of phosphorylation include most protein components of the cell, including enzymes, structural proteins, cell receptors, ion channels and signaling molecules. If a protein is controlled by its phosphorylation state, its activity at any one time will be directly dependent on the activity of the kinases and phosphatases that act on it. It is quite common for a phosphate group to be added or removed from a protein continually, a cycle that allows a protein to switch rapidly from one state to another ⁷. [pSer884] Thyroid Hormone Binding Protein (TRBP) peptide interacts with nuclear receptors. Ligand-dependent interaction of nuclear receptors and co-activators is a critical step in nuclear receptor-mediated transcriptional regulation. Serine884 of TRBP LXXLL motif is a key residue for receptor selectivity ⁴. Aquaporins form pores in the membranes of cells and selectively pass water molecules through the membrane, and prevent the crossing of sodium, potassium and other ions and small molecules. Human aquaporin-2 (AQP2) peptide AQP2 residues including S256 and S261, are altered in response to vasopressin. In the presence of vasopressin, AQP2 monophosphorylated at Ser256 and diphosphorylated AQP2 (pSer256/261) increase in abundance, whereas AQP2 monophosphorylated at Ser261 decreases, suggesting that both sites are involved in vasopressin-dependent AQP2 trafficking ⁵.

Functions

Regulate cell processes, through the use of phosphorylation cycles and cascades, the cell is able to regulate a diverse set of processes, including cellular movement, reproduction and metabolism. It is the simplicity, reversibility and flexibility of phosphorylation that explains why it has been adopted as the most general control mechanism of the cell.

Signaling networks, the study of phosphorylated proteins combined with mass spectrometry have been used to identify and quantify dynamic changes in phosphorylated proteins over time. Systematic analysis of complex phosphorylation networks has helped in understanding of cellular processes⁸.

 PKC, C2 Domain (49 - 53), This peptide belongs to the C2 regulatory domain of e-protein kinase C, also known as e-PKC/V1. This peptide, used in studying the physiological role of isozymes, is most likely the PKC-selective activator and is found to be cardioprotective ⁹.

Regulatory functions, several enzymes and receptors are switched "on" or "off" by phosphorylation and dephosphorylation. Reversible phosphorylation results in a conformational change in the structure of enzymes and receptors, causing them to become activated or deactivated. Many phosphorylation related peptides act as a modulator of enzyme function ¹⁰.

References

1.     Levene PA, Alsberg CL (1906). The cleavage products of vitellin. JBC., 2(1):127–133.

2.     Pawson T (1994). Introduction: Protein Kinases. Faseb J., 8:1112-1113.

3.     Fischer EH, Krebs EG  (1955). Conversion of phosphorylase b to phosphorylase a in muscle extracts. JBC., 216: 121-132.

4.     Ko L, Cardona GR, Iwasaki T, Bramlett KS, Burris TP, Chin WW (2002). Ser-884 adjacent to the LXXLL motif of coactivator TRBP defines selectivity for ERs and TRs. Mol. Endocrinol., 16:128-132.

5.     Hoffert JD, Pisitkun T, Wang G, Shen RF, Knepper MA (2006). Quantitative phosphoproteomics of vasopressin-sensitive renal cells: regulation of aquaporin-2 phosphorylation at two sites. PNAS., 103(18):7159-7164.

6.     Greenstein AE, Grundner C, Echols N, Gay LM, Lombana TN, Miecskowski CA, Pullen KE, Sung PY, Alber T (2005). Structure/Function Studies of Ser/Thr and Tyr Protein Phosphorylation in Mycobacterium tuberculosis. J Mol Microbiol Biotechno., 9(3-4):167-181.

7.     Sefton BM, Hunter T (1998). Protein Phosphorylation. San Diego: Academic Press.

8.     Olsen JV, Blagoev B, Gnad F, Macek B, Kumar C, Mortensen P, Mann M (2006). Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. Cell, 127(3):635–648.

9.     Brandman R, Disatnik MH, Churchill E, Mochly-Rosen D (2007). Peptides derived from the C2 domain of protein kinase C epsilon (epsilon PKC) modulate epsilon PKC activity and identify potential protein-protein interaction surfaces. J. Biol. Chem., 282(6):4113-4123.

10.  Stock JB, Ninfa AJ, Stock AM (1989). Protein phosphorylation and regulation of adaptive responses in bacteria. Microbiol. Rev., 53(4):450–490.