Real time visualization of protein kinase activity in living cells.
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 PKCalpha, beta, and gamma. 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.
Yeh
Ren-Hwa
RH
Department of Biochemistry, The Albert Einstein College of Medicine of Yeshiva University, Bronx, New York 10461-1602, USA.
Yan
Xiongwei
X
Cammer
Michael
M
Bresnick
Anne R
AR
Lawrence
David S
DS
D000595:Amino Acid Sequence; D005456:Fluorescent Dyes; D006367:HeLa Cells; D006801:Humans; D011493:Protein Kinase C / Q000378:metabolism*
D005456:Fluorescent Dyes; D011493:Protein Kinase C