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J. Cell Biol.,
Volume 140, Number 3, February 9, 1998 485-498
* Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710; and Cysteine-rich domains (Cys-domains) are
~50-amino acid-long protein domains that complex
two zinc ions and include a consensus sequence with six
cysteine and two histidine residues. In vitro studies
have shown that Cys-domains from several protein kinase C (PKC) isoforms and a number of other signaling
proteins bind lipid membranes in the presence of diacylglycerol or phorbol ester. Here we examine the second
messenger functions of diacylglycerol in living cells by
monitoring the membrane translocation of the green
fluorescent protein (GFP)-tagged first Cys-domain of
PKC- Department of
Biochemistry, University of Lausanne, 1066 Epalinges, Switzerland
(Cys1-GFP). Strikingly, stimulation of G-protein or tyrosine kinase-coupled receptors induced a
transient translocation of cytosolic Cys1-GFP to the
plasma membrane. The plasma membrane translocation was mimicked by addition of the diacylglycerol analogue DiC8 or the phorbol ester, phorbol myristate acetate (PMA). Photobleaching recovery studies showed
that PMA nearly immobilized Cys1-GFP in the membrane, whereas DiC8 left Cys1-GFP diffusible within
the membrane. Addition of a smaller and more hydrophilic phorbol ester, phorbol dibuterate (PDBu), localized Cys1-GFP preferentially to the plasma and nuclear membranes. This selective membrane localization was lost in the presence of arachidonic acid. GFP-tagged Cys1Cys2-domains and full-length PKC- also
translocated from the cytosol to the plasma membrane
in response to receptor or PMA stimuli, whereas significant plasma membrane translocation of Cys2-GFP
was only observed in response to PMA addition. These
studies introduce GFP-tagged Cys-domains as fluorescent diacylglycerol indicators and show that in living
cells the individual Cys-domains can trigger a diacylglycerol or phorbol ester-mediated translocation of
proteins to selective lipid membranes.
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