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Green fluorescent protein (GFP)-tagged cysteine-rich domains from protein kinase C as fluorescent indicators for diacylglycerol signaling in living cells.

Oancea E, Teruel MN, Quest AF, Meyer T - J. Cell Biol. (1998)

Bottom Line: This selective membrane localization was lost in the presence of arachidonic acid.GFP-tagged Cys1Cys2-domains and full-length PKC-gamma 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.

ABSTRACT
Cysteine-rich domains (Cys-domains) are approximately 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-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 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-gamma 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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Translocation of Cys2–GFP, Cys1Cys2–GFP, and full-length PKC-γ–GFP to the plasma membrane in response to receptor activation. (A) Schematic representation of the GFP-tagged  constructs used in these experiments: Cys2–GFP, Cys1Cys2– GFP, and full-length PKC-γ–GFP. The proteins were expressed  in RBL cells by microporation of in vitro transcribed RNA. (B)  SDS-PAGE of [35S]Met-labeled proteins of in vitro transcribed  GFP, Cys2–GFP, Cys1Cys2–GFP and PKC-γ–GFP. C–E represent series of three images of cells expressing the Cys2–GFP,  Cys1Cys2–GFP and PKC-γ–GFP fusion proteins, respectively.  The images were taken immediately before (left), 90 s after (middle), and 5 min after (right) stimulation with 100 nM PAF. Images  were recorded at low laser intensity and were not corrected for  photobleaching. A different plasma membrane translocation  characteristic was observed for the three fusion proteins. Only a  small fraction of the Cys2–GFP (D) translocated to the plasma  membrane in response to receptor activation, while cytosolic  Cys1Cys2–GFP (E) translocated more readily to the plasma  membrane. Both, Cys2–GFP and Cys1Cys2–GFP, had also a typically higher concentration of the protein localized to the nucleus  that was not significantly affected by receptor activation. (F) Full-length PKC-γ–GFP (E) showed significant nuclear exclusion in  resting cells and a maximal transient localization to the plasma  membrane in response to PAF receptor activation.
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Figure 7: Translocation of Cys2–GFP, Cys1Cys2–GFP, and full-length PKC-γ–GFP to the plasma membrane in response to receptor activation. (A) Schematic representation of the GFP-tagged constructs used in these experiments: Cys2–GFP, Cys1Cys2– GFP, and full-length PKC-γ–GFP. The proteins were expressed in RBL cells by microporation of in vitro transcribed RNA. (B) SDS-PAGE of [35S]Met-labeled proteins of in vitro transcribed GFP, Cys2–GFP, Cys1Cys2–GFP and PKC-γ–GFP. C–E represent series of three images of cells expressing the Cys2–GFP, Cys1Cys2–GFP and PKC-γ–GFP fusion proteins, respectively. The images were taken immediately before (left), 90 s after (middle), and 5 min after (right) stimulation with 100 nM PAF. Images were recorded at low laser intensity and were not corrected for photobleaching. A different plasma membrane translocation characteristic was observed for the three fusion proteins. Only a small fraction of the Cys2–GFP (D) translocated to the plasma membrane in response to receptor activation, while cytosolic Cys1Cys2–GFP (E) translocated more readily to the plasma membrane. Both, Cys2–GFP and Cys1Cys2–GFP, had also a typically higher concentration of the protein localized to the nucleus that was not significantly affected by receptor activation. (F) Full-length PKC-γ–GFP (E) showed significant nuclear exclusion in resting cells and a maximal transient localization to the plasma membrane in response to PAF receptor activation.

Mentions: In vitro studies have shown that the second Cys-domain of PKC-γ, the Cys1Cys2 tandem domains and the full-length PKC are also phorbol ester sensitive (Quest et al., 1994). Therefore, we tested whether these constructs can also be used as GFP-tagged probes to study diacylglycerol-mediated signal transduction (Fig. 7, A and B). As for the Cys1– GFP, the GFP-fusion constructs were expressed in RBL cells by RNA transfection (Fig. 7, C to E, left images). The expressed Cys2–GFP and Cys1Cys2–GFP were uniformly expressed in the cytosol and were in most cells enriched in the nucleoplasm (Fig. 7, C and D, left images). The expressed GFP-tagged full-length PKC-γ was largely cytosolically localized (Fig. 7 E, left image).


Green fluorescent protein (GFP)-tagged cysteine-rich domains from protein kinase C as fluorescent indicators for diacylglycerol signaling in living cells.

Oancea E, Teruel MN, Quest AF, Meyer T - J. Cell Biol. (1998)

Translocation of Cys2–GFP, Cys1Cys2–GFP, and full-length PKC-γ–GFP to the plasma membrane in response to receptor activation. (A) Schematic representation of the GFP-tagged  constructs used in these experiments: Cys2–GFP, Cys1Cys2– GFP, and full-length PKC-γ–GFP. The proteins were expressed  in RBL cells by microporation of in vitro transcribed RNA. (B)  SDS-PAGE of [35S]Met-labeled proteins of in vitro transcribed  GFP, Cys2–GFP, Cys1Cys2–GFP and PKC-γ–GFP. C–E represent series of three images of cells expressing the Cys2–GFP,  Cys1Cys2–GFP and PKC-γ–GFP fusion proteins, respectively.  The images were taken immediately before (left), 90 s after (middle), and 5 min after (right) stimulation with 100 nM PAF. Images  were recorded at low laser intensity and were not corrected for  photobleaching. A different plasma membrane translocation  characteristic was observed for the three fusion proteins. Only a  small fraction of the Cys2–GFP (D) translocated to the plasma  membrane in response to receptor activation, while cytosolic  Cys1Cys2–GFP (E) translocated more readily to the plasma  membrane. Both, Cys2–GFP and Cys1Cys2–GFP, had also a typically higher concentration of the protein localized to the nucleus  that was not significantly affected by receptor activation. (F) Full-length PKC-γ–GFP (E) showed significant nuclear exclusion in  resting cells and a maximal transient localization to the plasma  membrane in response to PAF receptor activation.
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Figure 7: Translocation of Cys2–GFP, Cys1Cys2–GFP, and full-length PKC-γ–GFP to the plasma membrane in response to receptor activation. (A) Schematic representation of the GFP-tagged constructs used in these experiments: Cys2–GFP, Cys1Cys2– GFP, and full-length PKC-γ–GFP. The proteins were expressed in RBL cells by microporation of in vitro transcribed RNA. (B) SDS-PAGE of [35S]Met-labeled proteins of in vitro transcribed GFP, Cys2–GFP, Cys1Cys2–GFP and PKC-γ–GFP. C–E represent series of three images of cells expressing the Cys2–GFP, Cys1Cys2–GFP and PKC-γ–GFP fusion proteins, respectively. The images were taken immediately before (left), 90 s after (middle), and 5 min after (right) stimulation with 100 nM PAF. Images were recorded at low laser intensity and were not corrected for photobleaching. A different plasma membrane translocation characteristic was observed for the three fusion proteins. Only a small fraction of the Cys2–GFP (D) translocated to the plasma membrane in response to receptor activation, while cytosolic Cys1Cys2–GFP (E) translocated more readily to the plasma membrane. Both, Cys2–GFP and Cys1Cys2–GFP, had also a typically higher concentration of the protein localized to the nucleus that was not significantly affected by receptor activation. (F) Full-length PKC-γ–GFP (E) showed significant nuclear exclusion in resting cells and a maximal transient localization to the plasma membrane in response to PAF receptor activation.
Mentions: In vitro studies have shown that the second Cys-domain of PKC-γ, the Cys1Cys2 tandem domains and the full-length PKC are also phorbol ester sensitive (Quest et al., 1994). Therefore, we tested whether these constructs can also be used as GFP-tagged probes to study diacylglycerol-mediated signal transduction (Fig. 7, A and B). As for the Cys1– GFP, the GFP-fusion constructs were expressed in RBL cells by RNA transfection (Fig. 7, C to E, left images). The expressed Cys2–GFP and Cys1Cys2–GFP were uniformly expressed in the cytosol and were in most cells enriched in the nucleoplasm (Fig. 7, C and D, left images). The expressed GFP-tagged full-length PKC-γ was largely cytosolically localized (Fig. 7 E, left image).

Bottom Line: This selective membrane localization was lost in the presence of arachidonic acid.GFP-tagged Cys1Cys2-domains and full-length PKC-gamma 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.

ABSTRACT
Cysteine-rich domains (Cys-domains) are approximately 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-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 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-gamma 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.

Show MeSH