Protein phosphorylation/dephosphorylation plays a central role in cell signaling. Phosphopeptides have been proven to be critical components in designing assays for protein phosphorylation.

Engstrrom, in 1978 described protein phosphorylation plays a central role in neural and hormonal control of cellular activity. Zetterqvist et al., 1976 suggested multiple basic amino acids, particularly arginine, at the phosphorylation site are prerequisites for an appreciable rate of the phosphorylation by cyclic AMP-dependent protein kinase 1.  Arginine forms stable complexes with orthophosphate esters due to the combined influence of electrostatic interaction and hydrogen- bonding.  Indeed, two guanidine nuclei can simultaneously interact with a single phosphate entity. Such interactions could occur on the phosphorylated peptides, the newly phosphorylated serine residue forming an intra peptide interaction with one or more adjacent arginine residues, thus inducing a conformation change in the product. Such interactions would be much weaker with lysine and histidine 2

Structural Characteristics
Phosphorylation of serine-14 in glycogen phosphorylase a, the N-terminal portion of this protein is involved in a conformation change that brings the phosphorylated serine in close proximity to a number of arginine residues, including arginine- 16 3. BRCT tandem domains, found in many proteins involved in DNA damage checkpoint and DNA repair pathways, were recently shown to be phosphopeptide binding motifs. Using solution nuclear magnetic resonance (NMR) spectroscopy and mutational analysis, Botuyan MVE et al., in 2004 have characterized the interaction of BRCA1-BRCT domains with a phosphoserine-containing peptide derived from the DNA repair helicase BACH1. They showed that a phenylalanine in the +3 position from the phosphoserine of BACH1 is bound to a conserved hydrophobic pocket formed between the two BRCT domains and that recognition of the phosphate group is mediated by lysine and serine side chains from the amino-terminal BRCT domain. Mutations that prevent phosphopeptide binding abolish BRCA1 function in DNA damage-induced checkpoint control. NMR data also reveal a dynamic interaction between BRCA1-BRCT and BACH1, where the bound phosphopeptide exists as an equilibrium of two conformations and where BRCA1-BRCT undergoes a transition to a more rigid conformation upon peptide binding 4

Mode of Action
Cellular phosphorylation is a reversible, covalent modification of a protein or lipid that modify the activity of the phosphorylated molecule by inducing conformational changes within the molecule. This modification occurs either through the addition of phosphate groups via the transfer of the terminal phosphate from a phosphate donor, e.g. ATP to an amino acid residue and/or by their removal.  The reversible phosphorylation of proteins can result in the activation or termination of many important cellular events including cell signaling, growth, and differentiation. Intracellular phosphorylation by protein kinases, triggered in response to extracellular signals, provides a mechanism for the cell to switch on or off many diverse processes. These processes include metabolic pathways, kinase cascade activation, membrane transport, gene transcription, and motor mechanisms. Although the natural substrates of protein kinases and phosphatases are usually proteins (e.g. Myelin basic protein, GSK-3 fusion protein, and Bad fusion protein, etc.), peptides and phosphopeptides are increasingly used for analyzing protein kinases and phosphatases (instead of the natural protein substrates), particularly for high throughput screening of protein kinase and phosphatase inhibitors 5, 6.


Fluorescent labeled and biotinylated peptides and corresponding phosphopeptides are increasingly used for analyzing protein kinases and phosphatases (to replace the radioactive phosphopeptides and phosphoproteins 7

Fluorescent labeled peptides and phosphopeptides (substrates for protein phosphatases and reference standards/competing tracers for the kinase reactions) are critical components of many commercial assay kits for protein kinases and phosphatases.

ELISA-based kinase assays, can be used to selectively measure the activity of either PKC or PKA. The micro strips coated with an immobilized peptide based on a pseudo substrate sequence along with the   reagents used for both types of kinase reactions and subsequent ELISA 8.

Fluorescence polarization is one of the best methods suitable for high throughput analysis of protein kinases and phosphatases.


1.     Camussi G, Bussolino F, Salvidio G, Baglioni C (1987). Tumor necrosis factor/cachectin stimulates rat peritoneal macrophages and human endothelial cells to synthesize and release platelet activating factor. J. Exp. Med., 166:1390-1404.

2.     Cotton FA, Hazen EE, Legg MJ (1979). Staphylococcal nuclease: proposed mechanism of action based on structure of enzyme-thymidine 3',5'-bisphosphate-calcium ion complex at 1.5-A resolution. PNAS., 76(6):2551-2555.

3.     Fletterick RJ, Sprang S, Madsen NB (1979). Analysis of the surface topography of glycogen phosphorylase a: implications for metabolic interconversion and regulatory mechanisms. J. Biochem., 57(6):789-797.

4.     Botuyan MV, Nominé Y, Yu X, Juranic N, Macura S, Chen J, Mer G (2004). Structural Basis of BACH1 Phosphopeptide Recognition by BRCA1 Tandem BRCT Domains. Structure, 12(7):1137-1146.

5.     Kennelly PJ(2001). Protein phosphatases--a phylogenetic perspective. Chem Rev., 101:2291-2312.

6.     Hunter T (1995). Protein kinases and phosphatases: the yin and yang of protein phosphorylation and signaling. Cell, 80:225-236.

7.     Parker GJ, Law TL, Lenoch FJ, Bolger RE (2000). Development of high throughput screening assays using fluorescence polarization: nuclear receptor-ligand-binding and kinase/phosphatase assays. J Biomol Screen., 5(2):77-88.

8.     Versteeg HH, Nijhuis E, van den Brink GR, Evertzen M, Pynaert GN, van Deventer SJ, Coffer PJ, Peppelenbosch MP (2000). A new phosphospecific cell-based ELISA for p42/p44 mitogen-activated protein kinase (MAPK), p38 MAPK, protein kinase B and cAMP-response-element-binding protein. Biochem J., 350(3):717-722.


If you are unable to find your desired product please contact us for assistance or send an email to info@biosyn.com

Product Name Catalog # Unit Price/Unit 
2 CDC25C, CHK1 and CHK2 Substrate, phosphorylated
12874-01 1 mg $1,013 cart inquire
Angiotensin II Substrate
12860-01 1 mg $998 cart inquire
CK1 Peptide Substrate [pS7]
14745-01 1 mg $635 cart inquire
CREBtide [KRREILSRRPSYR], Phosphorylated, C - term
12869-01 1 mg $845 cart inquire
Insulin Receptor (1142 - 1153) pTyr1146
14748-01 1 mg $818 cart inquire
Insulin Receptor (1142 - 1153) pTyr(1146 ,1150, 1151)
TRDI - pY - ETD - pY - pY - RK
14747-01 1 mg $1,007 cart inquire
Keratin K18 - C
12872-01 1 mg $710 cart inquire
Platelet - Derived Growth Factor ß - Receptor (719 - 723)
12861-01 1 mg $845 cart inquire
SH2 Domain Ligand (2)
12865-01 1 mg $1,080 cart inquire
Threonine Phosphopeptide PKC Substrate 4 phosphorylated
KR - pT - IRR
14824-01 1 mg $594 cart inquire
Tyrosine Kinase Peptide 3 [RRLIEDAE - pY - AARG],
12853-01 1 mg $1,335 cart inquire

Biosynthesis Inc.