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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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Phorbol ester sensitivity of Cys2–GFP, Cys1Cys2–GFP  and full-length PKC-γ–GFP. (A) In vitro binding of Cys2–GFP,  Cys1Cys2–GFP, and PKC-γ–GFP to lipid vesicles in the presence  of phorbol ester. In vitro translated 35S labeled fusion proteins  were used. The amplitude of each bar represents the percentage  of total counts retrieved in the vesicle fraction. The amplitude of  each bar represents an average of two samples from the same experiment with the number of counts in the vesicle fraction expressed as a percentage of total counts added (% bound). Two  separate experiments with phosphatidylserine vesicles and one  experiment with a phosphatidylserine/phosphatidylcholine mixture (1:4 ratio of lipids) gave similar results. (B–D) Series of three  images of cells expressing the Cys2–GFP, Cys1Cys2–GFP, and  PKC-γ–GFP fusion proteins respectively. The left panels show  differential interference contrast images of the cells before stimulation. The middle and right panels show fluorescent confocal fluorescence images recorded immediately before and 5 min after  stimulation with 1 μM PMA. All three fusion proteins show maximal translocation of the fusion proteins from cytosol to the  plasma membrane in the presence of PMA. For Cys2–GFP and  Cys1Cys2–GFP (B and C), the nuclear localized fusion proteins  did not significantly redistribute after PMA addition. Images  were not corrected for photobleaching.
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Figure 8: Phorbol ester sensitivity of Cys2–GFP, Cys1Cys2–GFP and full-length PKC-γ–GFP. (A) In vitro binding of Cys2–GFP, Cys1Cys2–GFP, and PKC-γ–GFP to lipid vesicles in the presence of phorbol ester. In vitro translated 35S labeled fusion proteins were used. The amplitude of each bar represents the percentage of total counts retrieved in the vesicle fraction. The amplitude of each bar represents an average of two samples from the same experiment with the number of counts in the vesicle fraction expressed as a percentage of total counts added (% bound). Two separate experiments with phosphatidylserine vesicles and one experiment with a phosphatidylserine/phosphatidylcholine mixture (1:4 ratio of lipids) gave similar results. (B–D) Series of three images of cells expressing the Cys2–GFP, Cys1Cys2–GFP, and PKC-γ–GFP fusion proteins respectively. The left panels show differential interference contrast images of the cells before stimulation. The middle and right panels show fluorescent confocal fluorescence images recorded immediately before and 5 min after stimulation with 1 μM PMA. All three fusion proteins show maximal translocation of the fusion proteins from cytosol to the plasma membrane in the presence of PMA. For Cys2–GFP and Cys1Cys2–GFP (B and C), the nuclear localized fusion proteins did not significantly redistribute after PMA addition. Images were not corrected for photobleaching.

Mentions: Nevertheless, in vitro translated Cys2–GFP, Cys1Cys2– GFP and PKC-γ–GFP all bound lipid vesicles in a phorbol ester-dependent manner (Fig. 8 A). Furthermore, all three constructs showed marked plasma membrane translocation in response to PMA (Fig. 8, B–D). Only the initially nuclear prelocalized Cys2–GFP and Cys1Cys2–GFP molecules was not significantly affected by the addition of PMA. In contrast, addition of PDBu induced a translocation of Cys2–GFP and Cys1Cys2–GFP to the plasma as well as nuclear membrane (data not shown, similar observations were made for Cys1–GFP in Fig. 6). No significant nuclear membrane localization of PKC-γ–GFP was observed after addition of PDBu, possibly because the full-length PKC-γ was largely nuclear excluded. While these measurements give additional insights into the function of the two Cys-domains in the context of the plasma membrane translocation of PKC-γ holoenzyme, they also suggest that the Cys1–GFP probe is better suited as a fluorescent indicator for studying diacylglycerol signaling.


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)

Phorbol ester sensitivity of Cys2–GFP, Cys1Cys2–GFP  and full-length PKC-γ–GFP. (A) In vitro binding of Cys2–GFP,  Cys1Cys2–GFP, and PKC-γ–GFP to lipid vesicles in the presence  of phorbol ester. In vitro translated 35S labeled fusion proteins  were used. The amplitude of each bar represents the percentage  of total counts retrieved in the vesicle fraction. The amplitude of  each bar represents an average of two samples from the same experiment with the number of counts in the vesicle fraction expressed as a percentage of total counts added (% bound). Two  separate experiments with phosphatidylserine vesicles and one  experiment with a phosphatidylserine/phosphatidylcholine mixture (1:4 ratio of lipids) gave similar results. (B–D) Series of three  images of cells expressing the Cys2–GFP, Cys1Cys2–GFP, and  PKC-γ–GFP fusion proteins respectively. The left panels show  differential interference contrast images of the cells before stimulation. The middle and right panels show fluorescent confocal fluorescence images recorded immediately before and 5 min after  stimulation with 1 μM PMA. All three fusion proteins show maximal translocation of the fusion proteins from cytosol to the  plasma membrane in the presence of PMA. For Cys2–GFP and  Cys1Cys2–GFP (B and C), the nuclear localized fusion proteins  did not significantly redistribute after PMA addition. Images  were not corrected for photobleaching.
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Figure 8: Phorbol ester sensitivity of Cys2–GFP, Cys1Cys2–GFP and full-length PKC-γ–GFP. (A) In vitro binding of Cys2–GFP, Cys1Cys2–GFP, and PKC-γ–GFP to lipid vesicles in the presence of phorbol ester. In vitro translated 35S labeled fusion proteins were used. The amplitude of each bar represents the percentage of total counts retrieved in the vesicle fraction. The amplitude of each bar represents an average of two samples from the same experiment with the number of counts in the vesicle fraction expressed as a percentage of total counts added (% bound). Two separate experiments with phosphatidylserine vesicles and one experiment with a phosphatidylserine/phosphatidylcholine mixture (1:4 ratio of lipids) gave similar results. (B–D) Series of three images of cells expressing the Cys2–GFP, Cys1Cys2–GFP, and PKC-γ–GFP fusion proteins respectively. The left panels show differential interference contrast images of the cells before stimulation. The middle and right panels show fluorescent confocal fluorescence images recorded immediately before and 5 min after stimulation with 1 μM PMA. All three fusion proteins show maximal translocation of the fusion proteins from cytosol to the plasma membrane in the presence of PMA. For Cys2–GFP and Cys1Cys2–GFP (B and C), the nuclear localized fusion proteins did not significantly redistribute after PMA addition. Images were not corrected for photobleaching.
Mentions: Nevertheless, in vitro translated Cys2–GFP, Cys1Cys2– GFP and PKC-γ–GFP all bound lipid vesicles in a phorbol ester-dependent manner (Fig. 8 A). Furthermore, all three constructs showed marked plasma membrane translocation in response to PMA (Fig. 8, B–D). Only the initially nuclear prelocalized Cys2–GFP and Cys1Cys2–GFP molecules was not significantly affected by the addition of PMA. In contrast, addition of PDBu induced a translocation of Cys2–GFP and Cys1Cys2–GFP to the plasma as well as nuclear membrane (data not shown, similar observations were made for Cys1–GFP in Fig. 6). No significant nuclear membrane localization of PKC-γ–GFP was observed after addition of PDBu, possibly because the full-length PKC-γ was largely nuclear excluded. While these measurements give additional insights into the function of the two Cys-domains in the context of the plasma membrane translocation of PKC-γ holoenzyme, they also suggest that the Cys1–GFP probe is better suited as a fluorescent indicator for studying diacylglycerol signaling.

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
Related in: MedlinePlus