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Published online 21 October 2002. doi:10.1083/jcb.200203048
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© The Rockefeller University Press, 0021-9525/2002/10/291 $5.00
The Journal of Cell Biology, Volume 159, Number 2, 291-302

Article

 Ca2+-controlled competitive diacylglycerol binding of protein kinase C isoenzymes in living cells

Johannes C. Lenz1, H. Peter Reusch2, Nadine Albrecht1, Günter Schultz1 and Michael Schaefer1

1 Institut für Pharmakologie, Freie Universität Berlin, 14195 Berlin, Germany
2 Institut für Klinische Pharmakologie und Toxikologie, Freie Universität Berlin, 14195 Berlin, Germany

Address correspondence to Michael Schaefer, Institut für Pharmakologie, Freie Universität Berlin, Thielallee 67-73, 14195 Berlin, Germany. Tel.: 49-30-8445-1863. Fax: 49-30-8445-1818. E-mail: schae{at}zedat.fu-berlin.de

The cellular decoding of receptor-induced signaling is based in part on the spatiotemporal activation pattern of PKC isoforms. Because classical and novel PKC isoforms contain diacylglycerol (DAG)-binding C1 domains, they may compete for DAG binding. We reasoned that a Ca2+-induced membrane association of classical PKCs may accelerate the DAG binding and thereby prevent translocation of novel PKCs. Simultaneous imaging of fluorescent PKC fusion proteins revealed that during receptor stimulation, PKC{alpha} accumulated in the plasma membrane with a diffusion-limited kinetic, whereas translocation of PKC{varepsilon} was delayed and attenuated. In BAPTA-loaded cells, however, a selective translocation of PKC{varepsilon}, but not of coexpressed PKC{alpha}, was evident. A membrane-permeable DAG analogue displayed a higher binding affinity for PKC{varepsilon} than for PKC{alpha}. Subsequent photolysis of caged Ca2+ immediately recruited PKC{alpha} to the membrane, and DAG-bound PKC{varepsilon} was displaced. At low expression levels of PKC{varepsilon}, PKC{alpha} concentration dependently prevented the PKC{varepsilon} translocation with half-maximal effects at equimolar coexpression. Furthermore, translocation of endogenous PKCs in vascular smooth muscle cells corroborated the model that a competition between PKC isoforms for DAG binding occurs at native expression levels. We conclude that Ca2+-controlled competitive DAG binding contributes to the selective recruitment of PKC isoforms after receptor activation.

Key Words: protein kinase C; diglycerides; calcium signaling; signal transduction; protein transport


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