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Protein kinase C targeting is regulated by temporal and spatial changes in intracellular free calcium concentration [Ca²⁺]_i

CHRISTIAN MAASCH, STEFAN WAGNER^*, CARSTEN LINDSCHAU, GABI ALEXANDER, KLAUS BUCHNER^*, MAIK GOLLASCH, FRIEDRICH C. LUFT and HERMANN HALLER¹

Medizinische Hochschule Hannover, Hannover, Germany;
^* Institute of Biochemistry, Free University, Berlin, Germany; and
Franz Volhard Clinic and the Max-Delbrück Center for Molecular Medicine, Medizinische Fakultät der Charité, Humboldt University of Berlin, Germany

¹Correspondence: Medizinische Hochschule Hannover, OE 6840, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany. E-mail: haller.hermann{at}mh-hannover.de

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Protein kinase C (PKC) isoforms exert specific intracellular functions, but the different isoforms display little substrate specificity in vitro. Selective PKC isoform targeting may be a mechanism to achieve specificity. We used a green fluorescent fusion protein (GFP) to test the hypothesis that local changes in [Ca²⁺]_i regulate translocation of PKC and that different modes of Ca²⁺ and Ca²⁺ release play a role in PKC targeting. We constructed deletion mutants of PKC to analyze the Ca²⁺-sensitive domains and their role in targeting. Confocal microscopy was used and [Ca²⁺]_i was measured by fluo-3. The fusion protein PKC-GFP was expressed in vascular smooth muscle cells and showed a cytosolic distribution similar to the wild-type PKC protein. The Ca²⁺ ionophore ionomycin induced a speckled cytosolic PKC-GFP distribution, followed by membrane translocation, while depolarization by KCl induced primarily membrane translocation. Selective voltage-operated Ca²⁺ channel opening led to a localized accumulation of PKC-GFP near the plasma membrane. Opening Ca²⁺ stores with InsP₃, thapsigargin, or ryanodine induced a specific PKC-GFP targeting to distinct intracellular areas. The G-protein-coupled receptor agonist thrombin induced a rapid translocation of the fusion protein to focal domains. The tyrosine kinase receptor agonist PDGF induced Ca²⁺ influx and led to a linear PKC-GFP membrane association. PKC-GFP deletion mutants demonstrated that the C2 domain, but not the catalytic subunit, is necessary for Ca²⁺-induced PKC targeting. Targeting was also abolished when the ATP binding site was deleted. We conclude that PKC can rapidly be translocated to distinct intracellular or membrane domains by local increases in [Ca²⁺]_i. The targeting mechanism is dependent on the C2 and ATP binding site of the enzyme. Localized [Ca²⁺]_i changes determine the spatial and temporal targeting of PKC.—Maasch, C., Wagner, S., Lindschau, C., Alexander, G., Buchner, K., Gollasch, M., Luft, F. C., Haller, H. Protein kinase C targeting is regulated by temporal and spatial changes in intracellular free calcium concentration [Ca²⁺]_i.

Key Words: isoforms • green fluorescent protein • vascular smooth muscle cells • receptor coupling • translocation • mutation

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
CA²⁺ and phospholipid-dependent protein kinase C (PKC) isoforms play an important role in intracellular signaling (1 , 2) . PKC isoforms are characterized by an NH₂-terminal regulatory domain containing binding sites for Ca²⁺, phosphatidylserine, and diacylglycerol (or tumor-promoting phorbol esters), a small central hinge region, and a COOH-terminal catalytic domain (3) . PKC is regulated by multiple interdependent mechanisms including enzymatic activation, translocation of the enzyme in response to activation, phosphorylation, and proteolysis. The PKC isoforms are divided into three subfamilies based on differences in the regulatory domain (4) . Since the isoforms are expressed on different genes, have a strictly regulated tissue expression, and display biochemical differences, they seem to exert different biological functions (5 6 7 8) . However, in vitro the different PKC isoforms demonstrate little substrate specificity. Therefore, other mechanisms must be responsible for their differential effects. One possibility is directed translocation of PKC isoforms to their respective targets (9) . Immunocytochemistry and confocal microscopy have been used to demonstrate that PKC can be translocated to the plasma membrane, cytoskeleton, perinuclear and nuclear area, and other intracellular targets (for a review, see refs 9 10 11 ). Targeting is important in specific substrate recognition and responses on hormonal stimulation (12 , 13) . One possible targeting mechanism for protein kinases and phosphatases is through association with anchoring proteins that tether the enzymes to subcellular structures and organelles. Several anchoring proteins for PKC have been described but their actual role in PKC isoform targeting remains unclear (11) .

Since PKC is translocated by Ca²⁺ and phospholipids, a local increase of these substances could also influence intracellular targeting (12 , 14) . In fact, several authors have shown that Ca²⁺ induces a shift of PKC to the cell membrane and to the nucleus (15 , 16) . Recently, a major role for local [Ca²⁺]_i changes in signaling was described. [Ca²⁺]_i increases beneath the cell membrane, so-called Ca²⁺-‘sparks’, have been reported (17 , 18) that exert specific local intracellular effects. Whether or not these local changes affect subcellular PKC isoform localization and targeting is unknown.

We tested the hypothesis that the local [Ca²⁺]_i changes regulate PKC translocation and that different modes of Ca²⁺ influx and release play a role in PKC targeting. We used a green fluorescent fusion protein (GFP) to directly analyze the intracellular movements of the enzyme. Depending on the construct, GFP permits enzymatic activity and allows the direct observation of PKC. Fusion to GFP for PKC and PKCßII have been used to analyze PKC translocation in living cells. We generated a PKC-GFP fusion protein and investigated the influence of [Ca²⁺]_i on the temporal and spatial PKC distribution in vascular smooth muscle cells (VSMC). We then generated different PKC mutants to identify the responsible domains.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
[-³²P]-ATP was purchased from Amersham Pharmacia Biotech (Freiburg, Germany). Monoclonal antibody against PKC was from Upstate Biotechnologies (Lake placid, N.Y.). Mammalian expression vector pDB 755 was obtained from Dr. Y. Nishizuka (Kobe University). Vectors pEGFP-N1, pEGFP-N2, pEGFP-C1, pGFP-C1, and the monoclonal antibody against GFP were from Clontech (Palo Alto, Calif.). TOPO pCR 2.1 was from Invitrogen (Groningen, Netherlands). Baculo transfer vector pAcGHLT-A was from PharMingen (San Diego, Calif.). Restriction enzymes were obtained from Promega (Madison, Wis.). 1,2-bis(2-Aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), and BAPTA-AM, thrombin, PDGF-AB, histone III, SL-myo-inositol 1,4,5-trisphosphate, and D-myo trisphosphate were from Sigma (Deisenhofen, Germany). Fluo-3-AM, and marina blue-conjugated antibodies were from Molecular Probes (Eugene, Oreg.). Antibody against ryr1/2 ryanodine receptor was from Dianova (Hamburg, Germany). Ionomycin, 1-oleoyl-2-acetyl-sn-glycerol, and BayK8644 were purchased from Calbiochem (Bad Soden, Germany). Purified Ryanodine R-100 was from Research Biochemicals Int. (Natick, Conn.). Chamber slides were from NUNC (Naperville, Ill.). All other chemicals were obtained from Merck (Darmstadt, Germany); U.S.E. Mutagenesis Kit was from Pharmacia (Freiburg, Germany).

VSMC culture
Rat aortic VSMC were cultured by procedures as published previously (8) . Briefly, the rats (12–14 wk) were killed instantly and their thoracic aortas were excised. After adherent fat and connective tissue were removed, the aortas were cut longitudinally and the endothelial cells were removed by gentle scraping with fine forceps. The aortas were then minced into small pieces and incubated at 37°C for 2 h in phosphate-buffered saline (PBS) without calcium, but with 1 mg/ml collagenase (type I, 150 IU/mg, Worthington Biochemical Corp. (Freehold, N.J.), 0.5 mg/ml elastase (type III, 40 IU/mg, Sigma), and 0.5 mg/ml trypsin inhibitor (Sigma). After 2 h, Dulbecco’s modified Eagle’s medium (DME/F-12) containing 10% fetal calf serum (FCS, Life Technologies, Inc., Eggenstein, Germany) was added to the suspension to inactivate enzymes. The cells were then centrifuged at 120 g for 10 min and the pellet was resuspended in M199 with 10% FCS. The cells were then seeded at a density of 3–5 x 10⁵/cm² and cultured at 37°C in 95% humidified air with 5% CO₂. Cells until up to passage 3 were used in all experiments. For ryanodine experiments, cells were cultured until passage 6. The phenotype of the cultured VSMC was determined by staining the cells for -actin and desmin. Antibodies for smooth muscle specific -actin and desmin were obtained from BioMakor (Rehovot, Israel) and Boehringer Mannheim (Roche, Mannheim, Germany).

Construction and expression of plasmids encoding PKC-GFP fusion protein A rat PKC cDNA in a pDB 755 vector was kindly provided by Dr. Y. Nishizuka (19) . The cDNA was subcloned into pEGFP-N2 for transfection and expression of the fusion protein. Several mutations and deletion constructs were made by polymerase chain reaction or restriction/ligation as described in more detail elsewhere (S. Wagner et al., unpublished results). Structures of the mutated proteins are displayed at the top of Fig. 10 .

View larger version (37K): [in this window] [in a new window]   Figure 10. Calcium dependency of PKC-GFP deletion mutants. Transfected cells are shown at rest (0 s) and after exposure to ionomycin (10-7 M) for 30 and 180 s. A) Deletion of the calcium binding C2-domain of PKC abolished the ionomycin-induced translocation of the fusion protein completely. B) Deletion of the catalytic domain of PKC resulted in a membrane association in resting cells. Exposure to ionomycin resulted in a translocation of PKC-GFP to the cytosol and formation of a speckled pattern. These spots accumulated over 180 s and were only reversible after >15 min. C) Deletion of the catalytic and the hinge region resulted in a mostly nuclear and little membrane localization of PKC-GFP in resting cells. Ionomycin induced after 30 s a nuclear export of PKC-GFP resulted in an accumulated pattern at the plasma membrane and the nuclear membrane (180 s). D) Expression of the C1-domain fused to GFP showed a pole-like perinuclear and little nuclear localization in resting cells. Ionomycin induced no translocation of PKC (C1)-GFP. E) Deletion of the regulatory domain of the enzyme resulted in a diffuse cytosolic and nuclear localization at resting state. Ionomycin-induced Ca2+ influx leaded to a more coarse distribution after a prolonged time period. F) Site-directed mutagenesis of the ATP binding site resulting in an inactive enzyme showed a membrane-association of the fusion protein in the resting state when expressed in VSMC and prevented the ionomycin-induced translocation.

View larger version (78K): [in this window] [in a new window]   Figure 10C.

View larger version (55K): [in this window] [in a new window]   Figure 10E.

Transfection of VSMC
Transient transfection into VSMC was carried out using SuperFect (Qiagen, Hilden, Germany). Cells were seeded on 4-well glass chamber slides (NUNC) for 1 day prior to transfection and washed twice with PBS without Ca²⁺/Mg²⁺. They were subsequently incubated with SuperFect, 0.5 µg plasmid-DNA/well in M199 culture medium (Seromed, Berlin, Germany) for 3 h at 37°C, and 5% CO₂ and washed twice with PBS without Ca²⁺/Mg²⁺. After the transfection, cells were cultured at 31.5°C to obtain optimal fluorescent intensity of GFP. The fluorescence of PKC-GFP was monitored 24 h after transfection.

PKC activity
PKC activity of cel lysates was measured using histone III pseudo substrate phosphorylation. Assay conditions were as follows: 20 mM Tris-HCl, pH 7.4, 10 µM MgCl₂, 1 mM CaCl₂, 10 µM ATP, 10–15 µCi/ml [g-³²P]-ATP, 40 µg/ml phosphatidyl serine/sn-dioctanoyl-glycerol vesicles, and 200 µg/ml histone IIIS as substrate in a final volume of 500 µl at 30°C for 10 min. The phosphorylated proteins were analyzed by sodium dodecyl sulfate (SDS) -gel electrophoresis, followed by autoradiography.

Confocal microscopy
Translocation of PKC-GFP was analyzed by confocal microscopy (MRC 1024; Bio-Rad, Munich, Germany) using both an argon/krypton and a UV laser at 363 nm/488 nm/568 nm excitation and 460 nm/515 nm/580 nm long-pass barrier filter sets, respectively. PKC-GFP fluorescence was measured using an argon/krypton laser at 488 nm excitation and a Nikon Flour 40 x (NA 1.15) water immersion lens. The transfected cells were washed twice with HEPES buffer (5 mM HEPES, pH 7.3, 135 mM NaCl, 5.4 mM KCl, 5 mM glucose) containing 1.5 mM CaCl₂. Afterward, the cells were incubated for at least 2–4 min in HEPES buffer with 1.5 mM CaCl₂ for adaptation to the medium. The cells expressing PKC-GFP (10–40% of total cell population) were observed by confocal microscopy. PKC-GFP signals were collected using the Bio-Rad time course software function with single line excitation (488 nm) in different, stimuli-dependent time intervals (1, 5,10, 15, 20 s) between images. Various drugs were applied during the scanning of PKC-GFP-transfected cells.

Measurement of [Ca²⁺]_i
[Ca²⁺]_i was measured using fluo-3 (Molecular Probes). Cells adherent to coverslips were washed three times with HEPES buffer with 1.5 mM CaCl₂ and incubated with 5 µM fluo-3-AM for 20 min at 37°C, 5% CO₂. Imaging was performed using a Bio-Rad MRC1024 confocal microscope system. Relative increase in [Ca²⁺]_i was assessed with fluo-3 using an excitation wavelength at 488 nm and an emission wavelength at 522/35 nm. Stimuli were added during scanning. Quantification of relative fluorescence increase was measured using the regions of interest (ROI) tool at the ‘time course’ software of the MRC1024 and displayed as arbitrary units subtracted by background ROI. For buffering of extracellular Ca²⁺, cells were loaded with fluo-3 as described before and washed three times with HEPES with 1 µM BAPTA. For buffering of [Ca²⁺]_i, cells were loaded with 5 µM BAPTA-AM after loading them with fluo-3.

[Ca²⁺]_i release by InsP₃
Cells were transfected as described and washed three times with HEPES buffer with 1.5 mM CaCl₂. Cells were then incubated with 30 µg/ml saponin for 1 min at 37°C, 5% CO₂ and three times for 2 min with calcium-free HEPES buffer; 1 µM inositol-triphosphate (InsP₃) was added during scanning by confocal microscopy.

Immunocytochemistry
Immunocytochemistry and confocal microscopy were performed as described recently (8) . Transfected VSMC were fixed with 4% paraformaldehyde and permeabilized with ice-cold 80% methanol. After incubation with 3% skimmed milk in phosphate buffer solution (SM/PBS) for 60 min, the preparation was incubated for 1 h at room temperature with the mouse anti-PKC-antibody. The preparation was washed three times with PBS and then exposed to the secondary antibody (marina blue-conjugated anti-mouse IgG at 1:100, 0.1% bovine serum albumin/PBS) for 60 min. For confocal microscopy, the preparation was mounted with Aqua Poly/Mount (Polysciences, Warrington, Pa.). A Bio-Rad MRC 1024 confocal imaging system (Bio-Rad Laboratories, Munich, Germany) with a UV laser at 363 nm excitation and an argon/krypton laser at 488 nm excitation was used. At least 25–30 cells from each of three independent experiments were examined under each experimental condition. Images were acquired in the normal scanning mode with a Kalman filter of 3.

Immunoblotting
Cultured VSMC were scraped off and treated with cold buffer (50 mM Tris-HCl pH 7,4, 2% SDS, 1 mM EDTA, 10 mM EGTA, 25 µM leupeptin, 1 mM PMSF). After estimating the protein content by UV measurement, 50 µg of protein was loaded onto each lane of a 12% SDS-polyacrylamide gel electrophoresis. After separation, the samples were electroblotted onto Immobilon-P membranes (Millipore, Eschborn, Germany). The membranes were successively incubated, first with blocking buffer containing 137 mM NaCl, 20 mM Tris-HCl pH 7.5, 10% (w/v) skimmed milk, 0.2% (v/v) Tween-20, and 0.02% NaN₃ for more than 120 min at room temperature. The next incubation was conducted with the specified antibody diluted in incubation buffer containing 137 mM NaCl, 20 mM Tris-HCl (pH 7.5) with 1% bovine serum albumin at room temperature. After two rinses and three washings for more than 5 min with blocking buffer without skimmed milk, a final incubation was carried out in with a peroxidase-labeled goat anti-mouse antibody (Pierce Chemicals, Oud-Beijerland, Netherlands) in incubation buffer for more than 2 h. After rinsing and washing as above, visualization was archived by chemiluminescence on X-ray films.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
We first characterized the expression and localization of PKC-GFP in rat VSMC by Western blot and confocal microscopy and compared the results with the wild-type PKC protein. Figure 1A shows the immunoblots for PKC (left panel) and PKC-GFP (right panel) from nontransfected control cells (lane 1) and cells transfected with the fusion protein PKC-GFP (lane 2). PKC was detected at 83 kDa. PKC-GFP-transfected cells expressed a single band at ~ 111 kDa for anti-PKC as well as for anti-GFP. Figure 1B shows fluorescent photomicrographs of a resting VSMC transfected with the PKC-GFP fusion protein. GFP-transfected VSMC were fixed, stained for PKC, and analyzed with confocal microscopy. In resting cells, PKC was localized by immunostaining in the perinuclear area (left). Perinuclear localization of the fusion protein was detected by direct observation of GFP fluorescence (middle). Colocalization of PKC-GFP fluorescence and anti-PKC immunofluorescence demonstrated that the fusion protein localizes to the same area as PKC (right). Last, Fig. 1C shows the total PKC activity in transfected and nontransfected control cells as assessed by substrate phosphorylation. The transfected cells showed a significantly increased PKC activity as compared to controls (P<0.05, n=4), indicating that the PKC-GFP fusion protein is enzymatically active.

View larger version (28K): [in this window] [in a new window]   Figure 1. Characterization of PKC-GFP fusion protein expressed in rat VSMC by Western blot analysis, confocal microscopy, and PKC activity. A) Immunoblots of cell lysate from nontransfected (lane 1) and PKC-GFP-transfected (lane 2) VSMC probed with a monoclonal antibody against PKC (left) and polyclonal anti-GFP (right). PKC-GFP-transfected cells expressed a band at approximately 111 kDa and 83 kDa for anti-PKC as well as a single band for anti-GFP. B) Colocalization (right) of PKC (left) and the PKC-GFP fusion protein (middle) demonstrating that GFP does not affect the cytosolic localization of the protein. C) PKC activity of GFP-transfected (left column) and nontransfected (right column) VSMC. Transfected cells show an increased level of enzymatic activity, indicating that the GFP part of the fusion protein does not affect the enzymatic activity.

We then analyzed the effects of a rapid, generalized increase in [Ca²⁺]_i using the ionophore ionomycin (10^-7 M) (Fig. 2 , representative of >50 experiments). In resting VSMC, PKC-GFP showed an intense perinuclear fluorescence. Within 10 s after ionomycin, the homogeneous fluorescence changed to a more speckled pattern in the cytosol. At 15 s, a PKC-GFP accumulation at the cell membrane was first visible, which increased rapidly (30 s) and remained unchanged for several minutes (Fig. 2A ). When the same experiments were carried out in the presence of the chelator BAPTA (10^-7 M), ionomycin had no effect on PKC-GFP distribution (Fig. 2A , lower right). We then analyzed the ionophore-induced increase in [Ca²⁺]_i and compared the result to the temporal course of PKC translocation (Fig. 2B ). Ionomycin (10^-7 M) led to a generalized increase in [Ca²⁺]_i within 3–6 s, followed by a more gradual decrease. After 35 s, [Ca²⁺]_i had returned to basal levels. Thus, the [Ca²⁺]_i rise preceded the changes in PKC-GFP redistribution by several seconds whereas PKC-GFP was still associated with the cell membrane after [Ca²⁺]_i had returned to basal levels.

View larger version (35K): [in this window] [in a new window]   Figure 2. Effects of ionomycin on translocation of PKC-GFP (A) and [Ca2+]i (B). A) Ionomycin (10-/7 M) induced a rapid intracellular redistribution from a homogeneous cytosolic to a speckled pattern in the cytosol. The cytosolic dots were more pronounced after 15–30 s. During the same time period, the first membrane association of PKC-GFP was visible. After 30 s, both cytosolic dots and membrane association remained unchanged for several minutes. After 40 min the translocation was reversible (data not shown). In the presence of the external Ca2+ chelator BAPTA, the ionomycin-induced translocation of PKC-GFP was completely abolished (lower right). B) Ionomycin induced a rapid increase in [Ca2+]i within 4–6 s, reaching its maximum after 15 s; it remained increased until 40 s. Extracellular BAPTA virtually abolished the [Ca2+] increase.

We next analyzed the effects of voltage-gated ion channel opening on PKC-GFP distribution. Depolarization of the cell membrane using KCl (40 mM) induced a slow translocation of the fusion protein to the plasma membrane (Fig. 3A , representative of > 30 experiments). Membrane association was first visible after 30 s and completed after 90 s; PKC-GFP remained associated with the plasma membrane for > 30 min. Figure 3B shows the temporal changes in [Ca²⁺]_i after depolarization. [Ca²⁺]_i increased rapidly and remained elevated for ~100 s. We next analyzed the effects of the L-type Ca²⁺ channel agonist BayK 8664 (10^-7 M) on the intracellular distribution of PKC-GFP (Fig. 4A ). BayK induced a rapid (<10 s) accumulation of fluorescence at distinct spots (size<1.5 µm) near the cell membrane (Fig. 4A , representative of >30 experiments). No continued PKC-GFP pattern (as after membrane depolarization) was observed. Within 2–3 min, the BayK-induced translocation to the cell membrane was reversible. The changes in [Ca²⁺]_i are shown in Fig. 4B . The increase in [Ca²⁺]_i was much smaller than the alterations observed after membrane depolarization.

View larger version (31K): [in this window] [in a new window]   Figure 3. Effects of plasma membrane depolarization by KCl on translocation of PKC-GFP (A) and [Ca2+]i (B). A) Depolarization of VSMC by 40 mM KCl results in a rapid translocation of cytosolic PKC-GFP to the plasma membrane. The first membrane association was visible after 20 s and further increased up to 60 s. The cytosolic PKC-GFP decreased concomitantly. The KCl response was slowly reversible after 30 min. In the presence of the external Ca2+ chelator BAPTA, the KCl-induced translocation of PKC-GFP was completely abolished (lower right). B) Depolarization of VSMC by 40 mM KCl induced a rapid increase in intracellular calcium within 5 s, reaching a maximum after 20 s and slowly decreasing over the next 150 s. Extracellular BAPTA reduced calcium influx by 75%.

View larger version (42K): [in this window] [in a new window]   Figure 4. Effects of BayK on translocation of PKC-GFP (A) and [Ca2+]i intracellular calcium concentration (B). A) BayK (10-7 M) induced a rapid translocation of PKC-GFP to focal spots at the plasma membrane within 10–20 s. These spots slowly disappeared over 90 s. The spot size (~0.8 µm) increased until 20 s and decreased from 25–250 s. The presence of BAPTA completely abolished the formation and appearance of spots. B) BayK induced a moderate increase in [Ca2+]i with a peak at 5 s, followed by a slightly elevated [Ca2+]i. With BAPTA no significant increase in [Ca2+]i could be observed.

We analyzed the effects of intracellular [Ca²⁺]_i release. Thapsigargin (10^-7 M) induced a slow (within 40–80 s) appearance of a speckled cytosolic PKC-GFP pattern with no apparent membrane association (Fig. 5A , representative of >30 experiments). No plasma membrane association of PKC-GFP was observed. Chelating of [Ca²⁺]_i by preincubation with BAPTA-AM prevented the thapsigargin-induced changes in PKC translocation (Fig. 5A , lower right) whereas chelating of extracellular Ca²⁺ ions with BAPTA had no effect (data not shown). The thapsigargin-induced changes in [Ca²⁺]_i were also slow and showed a close correlation with the initiation of PKC-GFP translocation (Fig. 5B , representative of >20 experiments). We then used ryanodine (10^-7 M) to release Ca²⁺ from intracellular stores. Since we have previously shown that the intracellular distribution of the ryanodine receptors change with cell differentiation and is dependent on the passage number, we analyzed freshly isolated VSMC (passage 0) and after passage 6 (Fig. 6 ). The right panels show the immunocytochemistry for the ryanodine receptors in the respective cells. In differentiated cells at passage 0, the ryanodine receptors were distributed in clusters along the cell membrane and little in the cytosol (Fig. 6A ). After exposure of the cells to ryanodine, PKC accumulated rapidly (<10 s) in focal spots near the plasma membrane in a pattern similar to the receptor distribution (Fig. 6A , left panels, representative of >20 experiments). In contrast, cells of passage 6 showed a more cytosolic pattern of receptor distribution, with a maximum in the perinuclear area (Fig. 6B , left panels). In these cells, ryanodine induced a rapid translocation of PKC-GFP in a similar distribution (Fig. 5B , representative of >20 experiments). The time course of PKC translocation was the same in cells from passages 0 and 6. We observed a similar PKC translocation with InsP₃-induced Ca²⁺ release (Fig. 7 ). Immunostaining for InsP₃ receptor revealed a close association of the receptor along the cell membrane and in the perinuclear area (right panel). Exposure of the cells to InsP₃ induced a rapid translocation of PKC-GFP to the plasma membrane and focal accumulation in the cytosol.

View larger version (29K): [in this window] [in a new window]   Figure 5. Effect of thapsigargin on translocation of PKC-GFP (A) and on [Ca2+]i (B). A) Thapsigargin induced a slow formation of a focal spots within the cytosol and in the perinuclear region (50 s). This process increased over 200 s and the spots slowly dissolved within 400 s Loading VSMC with the membrane-permeable Ca2+ chelator BAPTA-AM abolished completely the effect of thapsigargin on PKC-GFP, whereas extracellular by BAPTA had no influence (data not shown). B) Thapsigargin induced a slow increase in [Ca2+]i reaching its maximum after 50 s and returning to baseline after 150 s. BAPTA-AM reduced the increase in [Ca2+]i after thapsigargin stimulation below 10%. Extracellular BAPTA had no influence on the increase in [Ca2+]i after stimulation.

View larger version (49K): [in this window] [in a new window]   Figure 6. Effect of ryanodine on translocation of PKC-GFP in freshly isolated VSMC (A) and after passage 6 (B). A) Ryanodine (10-7 M) induced in freshly isolated cells a rapid translocation of PKC-GFP to specific spots near the plasma membrane (10 s), which had disappeared within 40–60 s. Immunostaining of ryanodine receptors (A, right panel) showed a clustered receptor localization near the plasma membrane and few receptors in the cytosol. B) In dedifferentiated VSMC (passage 6), ryanodine (10-7 M) induced a speckled cytosolic PKC-GFP pattern with no translocation toward the cell membrane. The time course of the ryanodine effect was rapid and comparable to the effects seen in panel A. Immunostaining of ryanodine receptors in dedifferentiated VSMC (B, right panel) showed a receptor localization in the cytosol and the perinuclear region.

View larger version (48K): [in this window] [in a new window]   Figure 7. Effect of InsP3 on translocation of PKC-GFP. InsP3 induced within 20 s a translocation of PKC-GFP toward the cell membrane and focal spots in the cytosol. This translocation was persistent and reversed only after 600 s. After immunostaining of InsP3 receptor I (7, right panel) a linear localization near the plasma membrane and a perinuclear association of the receptor were observed.

We then investigated the effects of receptor-mediated [Ca²⁺]_i transients on PKC-GFP translocation after stimulation with thrombin (0.1 U/ml; Fig. 8A ) and PDGF (1 ng/ml; Fig. 8B ); the figures are representative of at least 15 independent experiments. Thrombin induced a rapid (15 s) PKC-GFP translocation toward distinct areas near or at the plasma membrane. This focal accumulation slowly changed into a more linear pattern and was reversed after 30 min. The translocation of PKC-GFP to the focal areas by thrombin was completely abolished by blockade of [Ca²⁺]_i release by BAPTA-AM (Fig. 8A , left panel). In contrast, PDGF induced a distinctly continued association of PKC-GFP with the plasma membrane. The PDGF-induced translocation of PKC-GFP was slow and totally dependent on Ca²⁺ influx from the extracellular space (Fig. 8B , right panel).

View larger version (47K): [in this window] [in a new window]   Figure 8. Effect of thrombin (A) and PDGF-AB (B) on translocation of PKC-GFP. A) Thrombin (0.1 U/ml) induced a rapid translocation of PKC-GFP to spots near the plasma membrane at 30 s. These spots were persistent and the translocation was reversible after 2 min. Chelating of intracellular Ca2+ ions by loading cells with BAPTA-AM prior to stimulation abolished this effect completely, whereas extracellular BAPTA had no significant effect. B) PDGF-AB (1 ng/ml) induced a translocation of PKC-GFP from the cytosol to the plasma membrane within 60 s, which was reversible after 8–9 min. Stimulation of the cells in the presence of BAPTA abolished the translocation of PKC-GFP completely, whereas BAPTA-AM had no effect.

The panels in Fig. 9 show height coded projections of Ca²⁺-induced translocation patterns of PKC-GFP. Figure 9A shows PKC-GFP in a resting VSMC, with evenly distributed enzyme in the cytosol and steep gradients toward the nucleus and the plasma membrane. Figure 9B demonstrates the differential effect of Ca²⁺ influx on PKC-GFP translocation. Depending on the mode of Ca²⁺ influx, PKC-GFP was either translocated to areas in both the cytosol and plasma membrane (ionomycin, Fig. 9B ), mainly to the plasma membrane (membrane depolarization by KCl, Fig. 9D ), or to focal membrane areas by localized calcium channel opening (BayK 8664, Fig. 9C ). In contrast, Ca²⁺ release from intracellular stores (thapsigargin, Fig. 9E ; InsP₃, Fig. 9F ) targeted PKC-GFP to focal areas within the cytosol; these areas were ~0.5 µm wide. Last, it seems that physiological agonists that activate PKC target the enzyme to completely different areas within the cell. Thrombin (Fig. 9G ) induced the translocation of copious PKC-GFP to localized areas near the plasma membrane, whereas PDGF (Fig. 9F ) led to a PKC-GFP translocation in small amounts evenly distributed at the plasma membrane.

View larger version (61K): [in this window] [in a new window]   Figure 9. Height coded projection of PKC-GFP in VSMC at rest (A) and after exposure to ionomycin (10-7 M) for 10 s (B), the calcium channel opener BayK 8644 (10-7 M) for 20 s (C), KCL (40 mM) for 20 s, thapsigargin (10-7 M) (E), InsP3 (1 µM) (F), thrombin (0.1 U/ml) for 10 s (G), and PDGF (1 ng/ml) (H). Depending on the mode of [Ca2+] influx and/or release, PKC-GFP was targeted to focal or linear areas at the plasma membrane or accumulated in small spots in the cytosol.

View larger version (62K): [in this window] [in a new window]   Figure 9E.

After having shown that localized changes in [Ca²⁺]_i concentration target PKC-GFP and that the mode of Ca²⁺ entry or release has distinct effects on enzyme localization, we analyzed which domains in the PKC molecule mediate the Ca²⁺-induced translocation. Different deletion mutants of PKC-GFP were constructed and transfected into VSMC; the cells were then exposed to ionomycin (10^-7 M). The results of these experiments are shown in Fig. 10 . Deletion of the C2 domain had no effect of the PKC distribution in resting cells (Fig. 10A , left panel). The ionomycin-induced increase in [Ca²⁺]_i did not induce a translocation of PKC-GFP and had no effect on enzyme distribution. In contrast, deletion of the catalytic domain changed the enzyme distribution in the resting cells (Fig. 10B , left panel). Changes in [Ca²⁺]_i induced a translocation of PKC-GFP to focal areas in the cytosol similar to the pattern observed with the full-length fusion protein. Deletion of the catalytic and the hinge domain of PKC resulted in mostly nuclear and little membrane localization in resting cells. Ionomycin induced a nuclear export of PKC-GFP resulting in a accumulated pattern at the plasma membrane and a continued pattern at the nuclear membrane (Fig. 1C ). Expression of the C1-domain of PKC showed a pole-like perinuclear and little nuclear localization. Ionomycin induced no translocation of PKC-GFP (Fig. 10D ). Deletion of the regulatory domain of the enzyme changed the distribution in resting cells to a more diffuse pattern, even including the nucleus. Ionomycin induced a coarser pattern after a prolonged time period (Fig. 10E ). Last, site-directed mutagenesis of the ATP binding site resulting in an inactive enzyme showed a membrane association of the enzyme in the resting state and prevented ionomycin-induced translocation (Fig. 10F ).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
We used a GFP fusion protein to analyze the [Ca²⁺]_i-induced targeting of PKC in VSMC and tested the hypothesis that localized [Ca²⁺]_i changes determine the targeting of the enzyme to distinct areas in the cell. We demonstrated that depending on the mode of transmembranous calcium flux, PKC is translocated to different areas of the cell membrane and that [Ca²⁺]_i release from intracellular stores targets the enzyme to focal areas in the cytosol within 1 µm of the site of release. Our experiments indicate that the intracellular targeting of PKC may vary depending on the differentiation state of the cells. We then demonstrated that physiological agonists that activate PKC target the enzyme to completely different intracellular areas. Last, using PKC deletion mutants, we investigated which domains are responsible for the [Ca²⁺]_i-induced translocation and identified three domains that regulate PKC targeting in living cells.

The analysis and comparison of the temporal changes in [Ca²⁺]_i and translocation of PKC demonstrated that changes in [Ca²⁺]_i and the velocity of PKC translocation are closely correlated. Rapid changes in Ca²⁺ influx or release induce, after a short delay, a translocation of the enzyme within 10–20 s. In contrast, the relatively slow increase in [Ca²⁺]_i after thapsigargin resulted in a slower translocation of the enzyme. These findings suggest that the translocation of PKC is dependent on a threshold Ca²⁺ concentration and translocation occurs only when local concentrations of more than 200 nM have been reached (20) . The different time courses in PKC translocation was nicely demonstrated after stimulation of the cells with thrombin and PDGF, respectively. Both agonists induce activation and translocation of PKC (21) ; however, whereas thrombin led to an immediate translocation in our experiments, the effects of PDGF on PKC translocation were only visible after 60 s, which is compatible with a slow PDGF-induced calcium influx.

In contrast to the close relationship between translocation of PKC and the [Ca²⁺]_i increase, the duration of the PKC translocation was not related to the changes in [Ca²⁺]_i concentration. In all experimental protocols, focal accumulation of PKC persisted after the [Ca²⁺]_i concentration had returned to resting levels. These observations suggest that [Ca²⁺]_i plays an important role in initiation of translocation while other mechanisms are responsible for prolonged association. Using GFP fusion proteins, Oancea and co-workers have recently shown that lipid binding is responsible for the prolonged translocation of PKC in living cells (22 , 23) . The duration of PKC translocation in this late phase of the response is quite variable, and ranged from 30 s in the case of BayK 8664-induced [Ca²⁺] influx to more than 20 min after KCL-induced membrane depolarization. The extent (height) of the local [Ca²⁺]_i increase may be responsible for the activation of different phospholipases and generation of activating lipids (24 , 25) . Our findings contrast with the recent observations by Oancea and Meyer, who observed a rapid shuttling of PKC from the membrane after cessation of the [Ca²⁺]_i signal (23) . Different states of PKC membrane association may exist. The use of other PKC mutants to investigate the underlying molecular mechanisms will be of interest.

Our results demonstrate in living cells that the mode of [Ca²⁺]_i entry into the cytosol and localization of the [Ca²⁺]_i rise influence enzyme targeting. Originally it was believed that the increase in [Ca²⁺]_i leads to a general association of PKC with cellular membranes (26 , 27) . A translocation of PKC isoforms to the plasma membrane has been observed in living cells by using fluorescent probes (28 , 29) . Several authors have shown that PKC can be directed to different intracellular targets on hormonal stimulation. These intracellular targets include the nucleus, cytoskeleton, microtubules, and contractile fibers (9 , 30 31 32 33 34) . The targeting of the enzyme to these sites may be regulated by specific binding of PKC to so-called receptors of activated protein kinase C (RACKs). We now provide evidence that localized changes in [Ca²⁺]_i concentrations are important in targeting PKC to distinct areas in the cell. Transmembranous Ca²⁺ influx has previously been suggested to lead to localized PKC activation (2) . However, our data suggest that local changes in [Ca²⁺]_i induce targeting and enzyme accumulation; the underlying mechanisms are unclear. Possibly, the local increase in [Ca²⁺]_i increases the affinity of binding sites for PKC at plasma membranes. More likely is the recently described increased membrane interaction of the enzyme by small increases in [Ca²⁺]_i (24) . A further increase in [Ca²⁺]_i would then lead to enzyme activation in these areas. An analysis of our data shows that the areas of local PKC accumulation are 0.8–1.5 µm wide. This size links PKC accumulation to the recently described generation of Ca²⁺ sparks. These local Ca²⁺ transients are caused by the opening of one or the coordinated opening of a number of tightly clustered ryanodine-sensitive Ca²⁺ release (RyR) channels in the sarcoplasmic reticulum. Modulation of spark frequency seems to be able to influence cell function by activating cyclic nucleotides (35 , 36) . Our results suggest that calcium-dependent PKC isoforms may also be influenced by sparks.

Mochly-Rosen and co-workers have described so-called RACKs (receptors for activated protein kinase C) (37) . Other binding proteins, so-called STICKs (substrates that interact with protein kinase C), which bind the inactive form of the enzyme, have also been described (10) . Recently Mineo et al. have reported a PKC binding protein, called sdr (serum deprivation response), which targets PKC to distinct domains of the cell membrane, the caveolae (38) . These different PKC binding proteins could be involved in the compartmentalization of the PKC-GFP fusion protein observed after the different stimuli. So far, however, these binding proteins have been described with specificity only for different PKC isoforms (37) . In our experiments, the single isoform PKC, was differentially distributed after cell exposure to different agonists. This observation raises the possibility that Ca²⁺ is not only important to increase the avidity of PKC for phospholipids, but may also regulate the binding to a variety of PKC binding proteins in different cellular compartments. The sdr binding protein described by Mineo et al. (38) shows [Ca²⁺]_i-dependent binding for PKC. In their experiments, the presence or absence of [Ca²⁺] regulated the distribution of PKC (and other PKC isoforms) from caveolae. In contrast, phorbol ester did not influence the binding of PKC to this protein. This finding supports the assumption that the local fluctuations in [Ca²⁺]_i regulate the amount of PKC bound to specific intracellular receptors.

In summary, we used a GFP fusion protein for PKC to investigate the hypothesis that the local changes in [Ca²⁺]_i regulate translocation of PKC and that different modes of [Ca²⁺]_i influx and release play a role in targeting PKC to distinct areas in the cell. We demonstrated that localized intracellular changes in [Ca²⁺] determine the spatial and temporal targeting of PKC. In addition, we constructed deletion mutants of PKC and showed that the calcium-induced targeting is dependent on the C2 and ATP binding site of the enzyme. Our results suggest that changes in cytosolic calcium are a major regulator of PKC targeting and that this mechanism may be one explanation for isoform-specific substrate recognition and differential responses of PKC on hormonal stimulation.

	ACKNOWLEDGMENTS

 
This work was supported by a grant in aid from the Deutsche Forschungsgemeinschaft to H.H.

Received for publication April 21, 1999. Revision received March 7, 2000.

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