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

Research output: Contribution to journalArticle

6 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)130-136
Number of pages7
JournalAnalytical Biochemistry
Volume424
Issue number2
DOIs
Publication statusPublished - May 15 2012

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Phosphorylation
Protein Kinase C
Fluorescence
Polymers
Infrared radiation
Dendrimers
Peptides
Preclinical Drug Evaluations
Fluorophores
Protein C Inhibitor
Chondroitin Sulfates
Energy Transfer
Protein Kinase Inhibitors
Coulomb interactions
Static Electricity
Energy transfer
Purification
Screening
Substrates
Pharmaceutical Preparations

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Biophysics
  • Molecular Biology
  • Cell Biology

Cite this

Fluorometric detection of protein kinase Cα activity based on phosphorylation-induced dissociation of a polyion complex. / Toita, Riki; Mori, Takeshi; Naritomi, Yuki; Kang, Jeong Hun; Shiosaki, Shujiro; Niidome, Takuro; Katayama, Yoshiki.

In: Analytical Biochemistry, Vol. 424, No. 2, 15.05.2012, p. 130-136.

Research output: Contribution to journalArticle

Toita, Riki ; Mori, Takeshi ; Naritomi, Yuki ; Kang, Jeong Hun ; Shiosaki, Shujiro ; Niidome, Takuro ; Katayama, Yoshiki. / Fluorometric detection of protein kinase Cα activity based on phosphorylation-induced dissociation of a polyion complex. In: Analytical Biochemistry. 2012 ; Vol. 424, No. 2. pp. 130-136.
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