Ratiometric real-time measurement of protein kinase activity with fluorophore labeled polyion complexes

【Introduction】 The dysfunction of protein kinase (PK) activity results in various human diseases. Thus, a measurement method for PKs is not only valuable for understanding the molecular mechanism of complicated cellular... [ view full abstract ]

【Introduction】
The dysfunction of protein kinase (PK) activity results in various human diseases. Thus, a measurement method for PKs is not only valuable for understanding the molecular mechanism of complicated cellular events but also for diagnosis and drug discovery. Recently, fluorescence probes which get turn on during PK-mediated phosphorylation of substrate peptides are one of the attractive methods due to sharp contrast in simple optical readout systems. However, it is still difficult to realize quantitative real-time analysis and monitoring of PK activity in living cells. Therefore, we demonstrate a novel quantitative real-time detection system of PK activity. Our system is based on a polyion complex (PIC) nanoparticle embedded with a PK-responsive fluorescence signal and a second constant internal standard signal.
【Methods】
5-(and 6) -carboxytetramethyl-rhodamine (TAMRA) was modified to poly-L-aspartic acid (pAsp-TAMRA) as anionic polymer. As for cationic polymer, Cy5 and protein kinase Cα (PKCα) substrate peptide were modified to Dextran (Dex-Cy5-pep). After PICs formation of these polymers through electrostatic interaction, various concentrations of the PKCα were added and changes in the TAMRA and Cy5 fluorescence intensities were monitored.
【Results and Discussion】
Once pAsp-TAMRA and Dex-Cy5-pep formed the PICs, TAMRA fluorescence was selectively quenched and Cy5 fluorescence tended to be constant. This uneven quenching behavior of the two signals was controlled by adjusting each fluorophore content in each polymer. High content of TAMRA in pAsp-TAMRA (2.1 mol%) induced concentration quenching of TAMRA, while low content of Cy5 in Dex-Cy5-pep (0.4 mol%) did not show the quenching. In the monitoring of each fluorescence intensities with phosphorylation reaction of PICs, the recovery of TAMRA fluorescence intensity was observed with increasing reaction time and PKCα concentration. On the other hand, Cy5 fluorescence intensity was still constant. This result indicated that the electrostatic interaction between two types of polymers were weaken by phosphorylation and PICs were dissociated, which resulted in TAMRA fluorescence recovery from quenching. In this research, we realized this probe enables quantitative and real-time detection of PKCα activity. In near future, we will apply this PIC type probe to the measurement of PK activity in living cells.