Kinase sensors

Some cellular assays also present the ability to measure kinase activation using recombinantly expressed intracellular sensor molecules that fluoresce in response to phosphorylation. These sensors are reviewed by Lawrence et al. (2007). The most interesting versions use yellow and blue fluorescent proteins to create a FRET through phosphorylation-induced folding with an intramolecular SH2 domain. SH2 domains are known to bind tightly to phosphotyrosine molecules and have been successfully shown to serve as phosphotyrosine detectors. In this type of assay, imaging is not necessary because the total fluorescence can be measured in a population of cells using a standard plate reader. 

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Regulatory Hydrogenase Histidine Kinase Sensor Response Regulator... 

This system is employed by prokaryotic organisms, and homologous pathways have recently been identified in eukaryotes. The prototypical two-component pathway consists of two proteins A protein histidine kinase (sensor kinase) and a response regulator. Histidine kinases are very distinct from the superfamily of conventional protein serine/threonine and tyrosine kinases. The histidine kinases auto-phosphorylate on histidine residues and are involved in the phosphorylation of aspartate amino acids and their targets. 

Many strategies using fluorescence have been employed to detect kinase activity. Generally, these kinase sensors manifest increased fluorescence emission upon phosphorylation, while both dual fluorophore (fluorescence resonance energy transfer, FRET, Box 1.1) sensors and single fluorophore-containing sensors have been developed. 

Figure 1.4 Sox-containing kinase activity sensors, (a) Schematic of Sox-based peptide sensors of kinase activity and (b) p-turn-focused kinase sensors inciuding C-terminai recognition eiements.

Hardie DG, Hawley SA, Scott JW (2006) AMP-activated protein kinase - development of the energy sensor concept. J Physiol 574 7-15... 

Since the first report of cameleon, sensors based on the same approach have been designed to detect small molecules (including cAMP [162, 163], cGMP [164], Ins(l,4,5)P3 [165, 166], lipids [167, 168], and sugars [169]) and the activity of a wide variety of kinases [122, 168, 170, 171]. 

Sharma, V., Wang, Q. and Lawrence, D. S. (2008). Peptide-based fluorescent sensors of protein kinase activity Design and applications. Biochim Biophys. Acta. 1784, 94—99. 

Bearson BL and Bearson SM. 2008. The role of die QseC quorum-sensing sensor kinase in colonization and norepinephrine-enhanced motility of Salmonella enterica serovar Typhimurium. Microb Pathogenesis... 

Thus, lipoproteins could be injected over the surface of a lipid covered SPR sensor in a detergent free buffer solution and showed spontaneous insertion into the artificial membrane.171 Again two hydro-phobic modifications are necessary for stable insertion into the lipid layer, whereas lipoproteins with a farnesyl group only dissociate significantly faster out of the membrane. Therefore the isoprenylation of a protein is sufficient to allow interaction with membraneous structures, while trapping of the molecule at a particular location requires a second hydrophobic anchor. Interaction between the Ras protein and its effector Raf-kinase depends on complex formation of Ras with GTP (instead of the Ras GDP complex, present in the resting cell). If a synthetically modified Ras protein with a palmi-... 

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