title: Fluorometric detection of protein kinase Cα activity based on phosphorylation-induced dissociation of a polyion complex.
abstract: 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.
authors.affiliation: Graduate School of Systems Life Sciences, Kyushu University, Nishi-ku, Fukuoka, Japan.
authors.forename: Riki
authors.initials: R
authors.lastname: Toita
authors.forename: Takeshi
authors.initials: T
authors.lastname: Mori
authors.forename: Yuki
authors.initials: Y
authors.lastname: Naritomi
authors.forename: Jeong-Hun
authors.initials: JH
authors.lastname: Kang
authors.forename: Shujiro
authors.initials: S
authors.lastname: Shiosaki
authors.forename: Takuro
authors.initials: T
authors.lastname: Niidome
authors.forename: Yoshiki
authors.initials: Y
authors.lastname: Katayama
mesh_terms: D000595:Amino Acid Sequence; D015374:Biosensing Techniques / Q000379:methods*; D002232:Carbocyanines; D002457:Cell Extracts / Q000737:chemistry; D045744:Cell Line, Tumor; D002809:Chondroitin Sulfates / Q000737:chemistry*; D050091:Dendrimers / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes; D005470:Fluorometry; D006801:Humans; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis*; D010766:Phosphorylation; D051571:Protein Kinase C-alpha / Q000032:analysis*; D019265:Spectroscopy, Near-Infrared; D055672:Static Electricity
chemical_list: C098793:CY5.5 cyanine dye; D002232:Carbocyanines; D002457:Cell Extracts; D050091:Dendrimers; D005456:Fluorescent Dyes; D009842:Oligopeptides; C104700:PAMAM Starburst; D002809:Chondroitin Sulfates; D051571:Protein Kinase C-alpha