TY - JOUR
T1 - Fluorometric detection of protein kinase Cα activity based on phosphorylation-induced dissociation of a polyion complex
AU - Toita, Riki
AU - Mori, Takeshi
AU - Naritomi, Yuki
AU - Kang, Jeong Hun
AU - Shiosaki, Shujiro
AU - Niidome, Takuro
AU - Katayama, Yoshiki
N1 - Funding Information:
This work was financially supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan . R.T. appreciates a Japan Society for the Promotion of Science (JSPS) Fellowship from MEXT.
PY - 2012/5/15
Y1 - 2012/5/15
N2 - Here, we report the fluorometric detection of protein kinase Cα (PKCα) activity in a cancerous cell lysate using a polyion complex (PIC) composed of a quencher (BHQ3)-modified chondroitin sulfate [CS(X)] and a dendrimer modified with a cationic peptide substrate (FKKQGSFAKKK-NH 2) and a near infrared (NIR) fluorophore (Cy5.5) (polymer 1). When polymer 1 formed the PIC with CS(X) through electrostatic interactions, the NIR fluorescence was quenched effectively via Förster resonance energy transfer (FRET) between Cy5.5 and BHQ3. However, this quenched fluorescence was recovered when the pendant peptides in polymer 1 were phosphorylated with PKCα due to the dissociation of the PIC. When PKCα was added to the PIC dispersion, a significant increase in fluorescence intensity was observed, whereas its fluorescence increase was inhibited with a PKCα inhibitor in a concentration-dependent manner. Furthermore, our PIC was robust enough to measure PKCα activity in a cancerous cellular lysate without purification. The PKCα-responsive PIC offers a simple, rapid, sensitive, and robust approach to detect PKCα activity in crude cellular lysates that would be suitable for drug screening formats and cancer diagnosis using crude cellular lysates.
AB - Here, we report the fluorometric detection of protein kinase Cα (PKCα) activity in a cancerous cell lysate using a polyion complex (PIC) composed of a quencher (BHQ3)-modified chondroitin sulfate [CS(X)] and a dendrimer modified with a cationic peptide substrate (FKKQGSFAKKK-NH 2) and a near infrared (NIR) fluorophore (Cy5.5) (polymer 1). When polymer 1 formed the PIC with CS(X) through electrostatic interactions, the NIR fluorescence was quenched effectively via Förster resonance energy transfer (FRET) between Cy5.5 and BHQ3. However, this quenched fluorescence was recovered when the pendant peptides in polymer 1 were phosphorylated with PKCα due to the dissociation of the PIC. When PKCα was added to the PIC dispersion, a significant increase in fluorescence intensity was observed, whereas its fluorescence increase was inhibited with a PKCα inhibitor in a concentration-dependent manner. Furthermore, our PIC was robust enough to measure PKCα activity in a cancerous cellular lysate without purification. The PKCα-responsive PIC offers a simple, rapid, sensitive, and robust approach to detect PKCα activity in crude cellular lysates that would be suitable for drug screening formats and cancer diagnosis using crude cellular lysates.
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U2 - 10.1016/j.ab.2012.01.036
DO - 10.1016/j.ab.2012.01.036
M3 - Article
C2 - 22342947
AN - SCOPUS:84859042294
SN - 0003-2697
VL - 424
SP - 130
EP - 136
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 2
ER -
