Limits...
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.

Show MeSH

Related in: MedlinePlus

Receptor-mediated plasma membrane translocation of  the GFP-tagged first Cys-domain from PKC-γ. (A) Schematic representation of the domain composition of conventional PKC and  of the Cys1–GFP fusion construct used in the experiments. The  Cys1–GFP construct consists of the first cysteine-rich domains of  PKC-γ tagged with GFP at its COOH-terminal end. The protein  was expressed in adherent RBL cells by microporation of in vitro  transcribed RNA. After 3–12 h, cells were imaged using confocal  fluorescence microscopy. (B) SDS-PAGE of expressed protein  after in vitro translation of DNA encoding GFP, Cys1–GFP, and  a Cys1GFP with a Pro 46 to Ala mutation (mCys1–GFP). (C)  Binding of [35S]Met-labeled fusion protein to lipid vesicles in the  presence or absence of the phorbol ester PDBu. The same labeled proteins as in B were used for the liposome binding assay.  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. (D) Series  of three images of cells expressing RNA transfected Cys1–GFP.  The images were taken immediately before and 90 s and 5 min after cross-linking of the IgE receptors by addition of 20 μg/ml  DNP-BSA. Images were corrected by an average photobleaching  rate. (E) The same translocation of the Cys1–GFP probe was observed in cells with stable transfected PAF receptors and activated with PAF (100 nM). The images shown were recorded immediately before, 60 s and 5 min after stimulation (images were  not corrected for photobleaching). (F and G) No significant enhancement of the plasma membrane fluorescence was observed  when cells expressing mCys1–GFP (F) or GFP alone (G) were  stimulated with 100 nM PAF. The three images shown were recorded at the same time points before and after stimulation as  those in D.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2140171&req=5

Figure 1: Receptor-mediated plasma membrane translocation of the GFP-tagged first Cys-domain from PKC-γ. (A) Schematic representation of the domain composition of conventional PKC and of the Cys1–GFP fusion construct used in the experiments. The Cys1–GFP construct consists of the first cysteine-rich domains of PKC-γ tagged with GFP at its COOH-terminal end. The protein was expressed in adherent RBL cells by microporation of in vitro transcribed RNA. After 3–12 h, cells were imaged using confocal fluorescence microscopy. (B) SDS-PAGE of expressed protein after in vitro translation of DNA encoding GFP, Cys1–GFP, and a Cys1GFP with a Pro 46 to Ala mutation (mCys1–GFP). (C) Binding of [35S]Met-labeled fusion protein to lipid vesicles in the presence or absence of the phorbol ester PDBu. The same labeled proteins as in B were used for the liposome binding assay. 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. (D) Series of three images of cells expressing RNA transfected Cys1–GFP. The images were taken immediately before and 90 s and 5 min after cross-linking of the IgE receptors by addition of 20 μg/ml DNP-BSA. Images were corrected by an average photobleaching rate. (E) The same translocation of the Cys1–GFP probe was observed in cells with stable transfected PAF receptors and activated with PAF (100 nM). The images shown were recorded immediately before, 60 s and 5 min after stimulation (images were not corrected for photobleaching). (F and G) No significant enhancement of the plasma membrane fluorescence was observed when cells expressing mCys1–GFP (F) or GFP alone (G) were stimulated with 100 nM PAF. The three images shown were recorded at the same time points before and after stimulation as those in D.

Mentions: The conventional isoforms of PKC: PKC-α, PKC-β1, PKC-β2, and PKC-γ (cPKCs), each contain two Cys-domains in their regulatory region (Fig. 1 A). For PKC-γ, previous in vitro studies have shown that fusion constructs of glutathione S transferase (GST) with either the first or second Cys-domain bind to lipid vesicles in the presence of phorbol ester (i.e., Quest and Bell, 1994). To investigate the functions of diacylglycerol and phorbol ester in intact cells, we tagged the first Cys-domain of PKC-γ with GFP. In vitro translation showed that the protein encoded by the construct (Cys1–GFP) has the expected molecular mass (Fig. 1 B) and can bind to lipid vesicles in the presence of phorbol ester (Fig. 1 C). When a conserved proline residue within the Cys-domain (Pro 46) was replaced by a glycine residue (mCys1–GFP), the Cys-domain showed a markedly reduced ability to bind lipid vesicles in the presence of phorbol ester (Fig. 1 C). Such an important function of the conserved proline residue in phorbol ester binding has been predicted by a sequence comparison of all Cys-domains that bind phorbol ester in vitro (Kazanietz et al., 1994).


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)

Receptor-mediated plasma membrane translocation of  the GFP-tagged first Cys-domain from PKC-γ. (A) Schematic representation of the domain composition of conventional PKC and  of the Cys1–GFP fusion construct used in the experiments. The  Cys1–GFP construct consists of the first cysteine-rich domains of  PKC-γ tagged with GFP at its COOH-terminal end. The protein  was expressed in adherent RBL cells by microporation of in vitro  transcribed RNA. After 3–12 h, cells were imaged using confocal  fluorescence microscopy. (B) SDS-PAGE of expressed protein  after in vitro translation of DNA encoding GFP, Cys1–GFP, and  a Cys1GFP with a Pro 46 to Ala mutation (mCys1–GFP). (C)  Binding of [35S]Met-labeled fusion protein to lipid vesicles in the  presence or absence of the phorbol ester PDBu. The same labeled proteins as in B were used for the liposome binding assay.  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. (D) Series  of three images of cells expressing RNA transfected Cys1–GFP.  The images were taken immediately before and 90 s and 5 min after cross-linking of the IgE receptors by addition of 20 μg/ml  DNP-BSA. Images were corrected by an average photobleaching  rate. (E) The same translocation of the Cys1–GFP probe was observed in cells with stable transfected PAF receptors and activated with PAF (100 nM). The images shown were recorded immediately before, 60 s and 5 min after stimulation (images were  not corrected for photobleaching). (F and G) No significant enhancement of the plasma membrane fluorescence was observed  when cells expressing mCys1–GFP (F) or GFP alone (G) were  stimulated with 100 nM PAF. The three images shown were recorded at the same time points before and after stimulation as  those in D.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2140171&req=5

Figure 1: Receptor-mediated plasma membrane translocation of the GFP-tagged first Cys-domain from PKC-γ. (A) Schematic representation of the domain composition of conventional PKC and of the Cys1–GFP fusion construct used in the experiments. The Cys1–GFP construct consists of the first cysteine-rich domains of PKC-γ tagged with GFP at its COOH-terminal end. The protein was expressed in adherent RBL cells by microporation of in vitro transcribed RNA. After 3–12 h, cells were imaged using confocal fluorescence microscopy. (B) SDS-PAGE of expressed protein after in vitro translation of DNA encoding GFP, Cys1–GFP, and a Cys1GFP with a Pro 46 to Ala mutation (mCys1–GFP). (C) Binding of [35S]Met-labeled fusion protein to lipid vesicles in the presence or absence of the phorbol ester PDBu. The same labeled proteins as in B were used for the liposome binding assay. 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. (D) Series of three images of cells expressing RNA transfected Cys1–GFP. The images were taken immediately before and 90 s and 5 min after cross-linking of the IgE receptors by addition of 20 μg/ml DNP-BSA. Images were corrected by an average photobleaching rate. (E) The same translocation of the Cys1–GFP probe was observed in cells with stable transfected PAF receptors and activated with PAF (100 nM). The images shown were recorded immediately before, 60 s and 5 min after stimulation (images were not corrected for photobleaching). (F and G) No significant enhancement of the plasma membrane fluorescence was observed when cells expressing mCys1–GFP (F) or GFP alone (G) were stimulated with 100 nM PAF. The three images shown were recorded at the same time points before and after stimulation as those in D.
Mentions: The conventional isoforms of PKC: PKC-α, PKC-β1, PKC-β2, and PKC-γ (cPKCs), each contain two Cys-domains in their regulatory region (Fig. 1 A). For PKC-γ, previous in vitro studies have shown that fusion constructs of glutathione S transferase (GST) with either the first or second Cys-domain bind to lipid vesicles in the presence of phorbol ester (i.e., Quest and Bell, 1994). To investigate the functions of diacylglycerol and phorbol ester in intact cells, we tagged the first Cys-domain of PKC-γ with GFP. In vitro translation showed that the protein encoded by the construct (Cys1–GFP) has the expected molecular mass (Fig. 1 B) and can bind to lipid vesicles in the presence of phorbol ester (Fig. 1 C). When a conserved proline residue within the Cys-domain (Pro 46) was replaced by a glycine residue (mCys1–GFP), the Cys-domain showed a markedly reduced ability to bind lipid vesicles in the presence of phorbol ester (Fig. 1 C). Such an important function of the conserved proline residue in phorbol ester binding has been predicted by a sequence comparison of all Cys-domains that bind phorbol ester in vitro (Kazanietz et al., 1994).

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