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

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

E Oancea et al. J Cell Biol. 1998.

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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Figures

Figure 6

Figure 6

Plasma membrane translocation of Cys1-domains in the presence of ceramide and free fatty acids. (A) Cys1–GFP translocated to the plasma membrane in response to the addition of phorbol ester and diacylglycerol analogues, but not in response to the addition of ceramide or free fatty acids. The ability of DiC8 (100 μg/ml), PMA (1 μM), ceramide-C8 (10 μM), oleic acid (100 μM), and arachidonic acid (100 μM) to induce plasma membrane translocation was determined by recording confocal fluorescence images immediately before and 4 min after stimulation. The relative increase in plasma membrane fluorescence intensity was determined as described in Fig. 2_B_. Only DAG and PMA were able to induce a significant relative increase in the plasma membrane fluorescence of Cys1–GFP. (B) Arachidonic acid prevented the DiC8-mediated translocation of Cys1–GFP to the plasma membrane. The same concentrations of the analogues were used as in A. Ceramide or free fatty acids were added to RBL cells expressing Cys1–GFP 5 min before the addition of 100 μg/ml of DiC8. The relative increase in the plasma membrane fluorescence intensity was again calculated as described in Fig. 2_B_. Arachidonic acid significantly decreased the DiC8-induced plasma membrane localization of Cys1–GFP. (C) Fluorescence images of RBL cells treated with arachidonic acid and diacylglycerol. A series of images of RBL cells expressing Cys1–GFP were recorded before stimulation (left), 5 min after addition of arachidonic acid (100 μM; middle), and 5 min after adding DiC8 (100 μg/ml; right) to the arachidonic acid treated cells. The distribution of Cys1– GFP was markedly punctuate after arachidonic acid addition. The subsequent addition of DiC8 induced only minimal plasma membrane translocation but instead enhanced the particulate staining in the cytosol. (D) Diffusion analysis of cytosolic Cys1– GFP by photobleaching recovery experiments. A short laser pulse (8 ms) and sequential imaging were used for the analysis of cytosolic Cys–GFP diffusion (0.033 s between images). Two- dimensional Gaussian fits of the bleach profiles were used for the analysis (Subramanian and Meyer, 1997). The relative increase in the square radius is graphed as a function of time. As for the one-dimensional analysis, the diffusion coefficient is proportional to the slope of this curve.

Figure 2

Figure 2

Comparison of the time course of plasma membrane translocation of Cys1–GFP in response to activation of IgE or PAF receptors. (A) Sequential images of RBL cells expressing Cys1–GFP taken immediately before and 40, 80, and 200 s after cross-linking of the IgE receptors with 20 μg/ml DNP-BSA. The images shown were not corrected for photobleaching. (B) For each cell in a given image, a line intensity profile across the cell was obtained. Typical intensity profiles are shown at each of the four time points. (C) Schematic representation of the method used to calculate a relative increase in plasma membrane staining. A relative increase in plasma membrane localization was calculated from the plasma membrane [I mb] and the average cytosolic fluorescence intensity [I cyt], respectively. (D) The plasma membrane translocation was represented as a relative increase in plasma membrane localization [_R_] and plotted as a function of time. The antigen or the PAF ligand were added at t = 0 s. The resulting curves represent the time course of plasma membrane translocation of Cys1–GFP in response to IgE receptor cross-linking and PAF receptor activation, respectively.

Figure 1

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.

Figure 3

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.

Figure 4

Figure 4

Comparison of the apparent lateral membrane diffusion coefficient and apparent plasma membrane dissociation time of Cys1–GFP in response to the addition of PMA, PC-PLC, or DiC8. Fluorescence recovery after photobleaching was used to determine the diffusion coefficient and dissociation time of Cys1–GFP bound to the plasma membrane after either PMA, PC-PLC, or DiC8 addition. A small region of the plasma membrane was photobleached using a short laser pulse (8 ms), and sequential images were recorded every 330 ms for PC-PLC and DiC8 addition and every 1.5 s for PMA addition. (A) Example of four images of a cell expressing Cys1–GFP and stimulated with PC-PLC. The images shown were recorded immediately before and 0.33, 2, and 6 s after the photobleaching pulse. The plasma membrane bleach spot is indicated by the arrow. (B) Comparison of the recovery in fluorescence intensity of Cys1–GFP at the center of the bleach spot. (C) In each series of images, the one-dimensional fluorescence intensity profiles along the plasma membrane were measured as a function of time and each profile was fit by a Gaussian function. (D) Calculated relative increase in the square radius of each Gaussian profile as a function of time for three typical cells. Data for cells stimulated with PMA, PC-PLC, and DiC8 are shown. The apparent lateral plasma membrane diffusion coefficients are proportional to the slope of each linear fit (dy/dt = 4 × D). (E) Calculated membrane dissociation time courses for Cys1–GFP localized to the plasma membrane by PMA, PC-PLC or DiC8 addition.

Figure 5

Figure 5

Addition of PDBu identifies the nuclear membrane as a selective target for Cys1–GFP translocation. (A) Series of three confocal fluorescence images of RBL cells expressing Cys1–GFP and stimulated by addition of PDBu (1 μM). The image on the left was recorded before PDBu addition, the middle image 1 min after PDBu addition, and the image on the right 10 min after PDBu addition. PDBu induced an initial localization of the Cys1–GFP to the nuclear membrane (middle). This nuclear localization became weaker in time, possibly due to the translocation of Cys1–GFP to the plasma membrane (right). Images were corrected for photobleaching. (B) PDBu concentration dependence of the plasma and nuclear membrane translocation of Cys1–GFP. Maximum translocation is reached at ∼40 nM PDBu for the plasma membrane and at 300 nM for the nuclear membrane. (C) Time course of translocation of Cys1–GFP to plasma and nuclear membrane in response to PDBu (1 μM). The relative increase in plasma membrane fluorescence reached a plateau after ∼60–120 s, whereas the relative increase in nuclear membrane fluorescence reached a maximum during a similar time period but then slowly decreased over time. (D–F) Analysis of plasma and nuclear membrane photobleaching recovery experiments. In D, the raw fluorescence recovery traces are shown. Interestingly, the recovery was faster for the nuclear membrane than for the plasma membrane. E shows a graph of the square radius of the bleach profile as a function of time for nuclear and plasma membrane. The apparent lateral diffusion coefficient of the Cys1–GFP in the two membranes was similar in both membranes. F shows the calculated time course of dissociation of Cys1–GFP away from the nuclear versus plasma membrane. The significantly faster apparent dissociation time from the nuclear membrane suggests that Cys1-domains have a lower affinity for the nuclear membrane compared with the plasma membrane.

Figure 7

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.

Figure 8

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.

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