Real time visualization of protein kinase activity in living cells

Ren Hwa Yeh, Xiongwei Yan, Michael Cammer, Anne R. Bresnick, David S. Lawrence

Research output: Contribution to journalArticle

59 Scopus citations

Abstract

A library of fluorescently labeled protein kinase C (PKC) peptide substrates was prepared to identify a phosphorylation-induced reporter of protein kinase activity. The lead PKC substrate displays a 2.5-fold change in fluorescence intensity upon phosphorylation. PKC activity is readily sampled in cell lysates containing the activated PKCs. Immunodepletion of conventional PKCs from the cell lysate eliminates the fluorescence response, suggesting that this peptide substrate is selectively phosphorylated by PKCα, β, and γ. Finally, living cells microinjected with the peptide substrate exhibit a 2-fold increase in fluorescence intensity upon exposure to a PKC activator. These results suggest that peptide-based protein kinase biosensors may be useful in monitoring the temporal and spatial dynamics of PKC activity in living cells.

Original languageEnglish (US)
Pages (from-to)11527-11532
Number of pages6
JournalJournal of Biological Chemistry
Volume277
Issue number13
DOIs
StatePublished - Mar 29 2002

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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