Fluorometric detection of protein kinase Cα activity based on phosphorylation-induced dissociation of a polyion complex

Riki Toita, Takeshi Mori, Yuki Naritomi, Jeong Hun Kang, Shujiro Shiosaki, Takuro Niidome, Yoshiki Katayama

    研究成果: ジャーナルへの寄稿学術誌査読

    6 被引用数 (Scopus)

    抄録

    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.

    本文言語英語
    ページ(範囲)130-136
    ページ数7
    ジャーナルAnalytical Biochemistry
    424
    2
    DOI
    出版ステータス出版済み - 5月 15 2012

    !!!All Science Journal Classification (ASJC) codes

    • 生物理学
    • 生化学
    • 分子生物学
    • 細胞生物学

    フィンガープリント

    「Fluorometric detection of protein kinase Cα activity based on phosphorylation-induced dissociation of a polyion complex」の研究トピックを掘り下げます。これらがまとまってユニークなフィンガープリントを構成します。

    引用スタイル