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

Translocation of Cys1–GFP in response to the addition  of PMA or DiC8. Cells expressing Cys1–GFP were stimulated  with either 1 μM PMA (A) or 100 μg/ml DiC8 (B). The left panels show DIC images of the cells before stimulation. The middle  and right panels show fluorescent confocal fluorescence images  recorded immediately before and 5 min after stimulation, respectively. Addition of PMA or DiC8 induced the translocation of most  internal Cys1–GFP to the plasma membrane. The right images  were corrected by an average photobleaching rate. (C) A less significant translocation was observed when cells expressing the proline mutant of Cys1 (mCys1–GFP) were stimulated with 1 μM  PMA. D and E show the concentration dependence of the translocation of Cys1–GFP to the plasma membrane in response to  the addition of different concentrations of PMA (D) and DiC8  (E). F and G show the time course of translocation of the Cys1– GFP probe upon addition of PMA (1 μM) or DiC8 (100 μg/ml).  The translocation is shown as a relative increase in the plasma  membrane fluorescence (R) as a function of time after PMA or  DiC8 addition.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2140171&req=5

Figure 3: Translocation of Cys1–GFP in response to the addition of PMA or DiC8. Cells expressing Cys1–GFP were stimulated with either 1 μM PMA (A) or 100 μg/ml DiC8 (B). The left panels show DIC images of the cells before stimulation. The middle and right panels show fluorescent confocal fluorescence images recorded immediately before and 5 min after stimulation, respectively. Addition of PMA or DiC8 induced the translocation of most internal Cys1–GFP to the plasma membrane. The right images were corrected by an average photobleaching rate. (C) A less significant translocation was observed when cells expressing the proline mutant of Cys1 (mCys1–GFP) were stimulated with 1 μM PMA. D and E show the concentration dependence of the translocation of Cys1–GFP to the plasma membrane in response to the addition of different concentrations of PMA (D) and DiC8 (E). F and G show the time course of translocation of the Cys1– GFP probe upon addition of PMA (1 μM) or DiC8 (100 μg/ml). The translocation is shown as a relative increase in the plasma membrane fluorescence (R) as a function of time after PMA or DiC8 addition.

Mentions: As discussed in the introduction, PMA can potently activate cPKCs by directly binding to their Cys-domains. Therefore, we tested the effect of PMA on expressed Cys1–GFP. In response to extracellular addition of PMA, Cys1–GFP translocated from the cytosol to the plasma membrane (Fig. 3 A). The left panel in this figure shows a differential interference contrast (DIC) image of a group of RBL cells, the middle panel shows a confocal fluorescence image of the initially homogenous distribution of Cys1–GFP and the right panel shows the plasma membrane distribution of Cys1–GFP 5 min after PMA addition. As a control for the specificity of PMA-induced plasma membrane translocation, the addition of the bioinactive 4α isomer of PMA, instead of the bioactive 4β isomer, did not translocate Cys1–GFP to the plasma membrane (data not shown).


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 Cys1–GFP in response to the addition  of PMA or DiC8. Cells expressing Cys1–GFP were stimulated  with either 1 μM PMA (A) or 100 μg/ml DiC8 (B). The left panels show DIC images of the cells before stimulation. The middle  and right panels show fluorescent confocal fluorescence images  recorded immediately before and 5 min after stimulation, respectively. Addition of PMA or DiC8 induced the translocation of most  internal Cys1–GFP to the plasma membrane. The right images  were corrected by an average photobleaching rate. (C) A less significant translocation was observed when cells expressing the proline mutant of Cys1 (mCys1–GFP) were stimulated with 1 μM  PMA. D and E show the concentration dependence of the translocation of Cys1–GFP to the plasma membrane in response to  the addition of different concentrations of PMA (D) and DiC8  (E). F and G show the time course of translocation of the Cys1– GFP probe upon addition of PMA (1 μM) or DiC8 (100 μg/ml).  The translocation is shown as a relative increase in the plasma  membrane fluorescence (R) as a function of time after PMA or  DiC8 addition.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Translocation of Cys1–GFP in response to the addition of PMA or DiC8. Cells expressing Cys1–GFP were stimulated with either 1 μM PMA (A) or 100 μg/ml DiC8 (B). The left panels show DIC images of the cells before stimulation. The middle and right panels show fluorescent confocal fluorescence images recorded immediately before and 5 min after stimulation, respectively. Addition of PMA or DiC8 induced the translocation of most internal Cys1–GFP to the plasma membrane. The right images were corrected by an average photobleaching rate. (C) A less significant translocation was observed when cells expressing the proline mutant of Cys1 (mCys1–GFP) were stimulated with 1 μM PMA. D and E show the concentration dependence of the translocation of Cys1–GFP to the plasma membrane in response to the addition of different concentrations of PMA (D) and DiC8 (E). F and G show the time course of translocation of the Cys1– GFP probe upon addition of PMA (1 μM) or DiC8 (100 μg/ml). The translocation is shown as a relative increase in the plasma membrane fluorescence (R) as a function of time after PMA or DiC8 addition.
Mentions: As discussed in the introduction, PMA can potently activate cPKCs by directly binding to their Cys-domains. Therefore, we tested the effect of PMA on expressed Cys1–GFP. In response to extracellular addition of PMA, Cys1–GFP translocated from the cytosol to the plasma membrane (Fig. 3 A). The left panel in this figure shows a differential interference contrast (DIC) image of a group of RBL cells, the middle panel shows a confocal fluorescence image of the initially homogenous distribution of Cys1–GFP and the right panel shows the plasma membrane distribution of Cys1–GFP 5 min after PMA addition. As a control for the specificity of PMA-induced plasma membrane translocation, the addition of the bioinactive 4α isomer of PMA, instead of the bioactive 4β isomer, did not translocate Cys1–GFP to the plasma membrane (data not shown).

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