GREEN FLUORESCENT PROTEIN (GFP)-TAGGED CYSTEINE-RICH DOMAINS FROM PROTEIN-KINASE-C AS FLUORESCENT INDICATORS FOR DIACYLGLYCEROL SIGNALING IN LIVING CELLS

Cysteine-rich domains (Cys-domains) are similar to 50-amino acid-long protein domains that complex two zinc ions and include a consensus seq uence with six cysteine and two histidine residues. In vitro studies h ave shown that Cys-domains from several protein kinase C (PKC) isoform s and a number of other signaling proteins bind lipid membranes in the presence of diacylglycerol or phorbol ester, Here we examine the seco nd messenger functions of diacylglycerol in living cells by monitoring the membrane translocation of the green fluorescent protein (GFP)-tag ged first Cys-domain of PKC-gamma (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 Cys 1-GFP in the membrane, whereas DiC8 left Cys1-GFP diffusible within th e membrane, Addition of a smaller and more hydrophilic phorbol ester, phorbol dibuterate (PDBu), localized Cys1-GFP preferentially to the pl asma and nuclear membranes, This selective membrane localization was l ost in the presence of arachidonic acid. GFP-tagged Cys1Cys2-domains a nd full-length PKC-gamma also translocated from the cytosol to the pla sma membrane in response to receptor or PMA stimuli, whereas significa nt plasma membrane translocation of Cys2-GFP was only observed in resp onse to PMA addition. These studies introduce GFP-tagged Cys-domains a s fluorescent diacylglycerol indicators and show that in living cells the individual Cys-domains can trigger a diacylglycerol or phorbol est er-mediated translocation of proteins to selective lipid membranes.