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J. Biol. Chem., Vol. 279, Issue 34, 35702-35708, August 20, 2004

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Protein Kinase C Translocates to the Perinuclear Region to Activate Phospholipase D1^*

Tianhui Hu and John H. Exton

From the Howard Hughes Medical Institute and Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37212

Received for publication, March 2, 2004 , and in revised form, June 7, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The inhibition of phorbol ester activation of phospholipase D1 (PLD1) by protein kinase C (PKC) inhibitors has been considered proof of phosphorylation-dependent activation of PLD1 by PKC. We studied the effect of the PKC inhibitors Ro-31-8220 and bisindolylmaleimide I on PLD1 activation and found that they inhibited the activation by interfering with PKC binding to PLD1. Further studies showed that only unphosphorylated PKC could bind to and activate PLD1 and that both inhibitors induced phosphorylation of PKC. The phosphorylation status of either PLD1 or PKC per se did not affect PLD1 activation in vitro. Immunofluorescence studies showed that PLD1 remained in the perinuclear region after phorbol ester treatment, whereas PKC translocated from cytosol to both plasma membrane and perinuclear regions. Both Ro-31-8220 and bisindolylmaleimide I blocked the translocation of PKC to the perinuclear region but not to the plasma membrane. Studies with okadaic acid suggested that phosphorylation regulated the relocation of PKC from the plasma membrane to the perinuclear region. It is proposed that localization and interaction of PKC with PLD1 in the perinuclear region is required for PLD1 activation and that PKC inhibitors inhibit this through phosphorylation of PKC, which blocks its translocation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Phospholipase D (PLD)¹ is a ubiquitous enzyme that hydrolyzes phosphatidylcholine to phosphatidic acid and choline and is involved in many important intracellular processes such as vesicle trafficking in Golgi, exocytosis, endocytosis, cytoskeletal reorganization, respiratory burst, and protein expression (1). Two isoforms of mammalian PLD have been cloned. PLD1 can be regulated by many factors such as protein kinase C (PKC) and members of the Rho and Arf families of small G proteins (2–5), whereas PLD2 exhibits a high basal activity and shows little or no response to PKC, Rho, or Arf in vitro (6–8). PKC is considered a major regulator of PLD1, and its role has been explored extensively. The PKC phosphorylation and interaction sites on PLD1 have been widely studied (9, 10). There are also several reports indicating that the PLD interaction sites on PKC may exist in both the N terminus (11, 12) and the C terminus (13). Previous studies have shown that phosphorylation is not required for the in vitro activation of PLD by PKC (3, 13–15). However, the role of phosphorylation in the regulation of PLD in vivo remains uncertain.

Our recent work has provided in vivo evidence that PKC activates PLD1 through a protein-protein interaction and that phosphorylation of PLD1 results in inactivation (13). However, other groups have provided evidence that phosphorylation of PLD1 is needed for its activation by PKC. The effect of PKC inhibitors such as Ro-31-8220 on PLD1 activation has been considered one of the major pieces of evidence for a role of phosphorylation (for references, see Ref. 1). Ro-31-8220 (RO), an ATP analog and a potent PKC kinase inhibitor (16, 17), markedly inhibits PLD1 activation induced by 4-phorbol 12-myristate 13 acetate (PMA) in vivo, consistent with the view that PKC activates PLD1 by phosphorylation. However, the mechanism by which RO inhibits PLD1 activity has not been studied. As a potent inhibitor of PKC, RO could also change PKC autophosphorylation and affect its cellular localization. It could also block the interaction of PKC with PLD1.

PLD1 exhibits variable patterns of subcellular membrane localization depending on the cell type (18). In mammalian cells, PLD1 is enriched in the perinuclear region, which may include the Golgi apparatus (18, 19). Some reports indicate that PLD1 also localizes to secretory granules, late endosomes, and lysosomes (18, 20–22). Cell fractionation studies show that PLD1 activity is restricted to caveolin-enriched membranes in some cell lines (23–25). There is other evidence that PLD1 might localize at the plasma membrane (26, 27). It is usually considered that after short term stimulation, PLD1 stays at its perinuclear location and does not undergo translocation. However, there is a report showing that upon longer time stimulation with antigen, PLD1 can translocate to the plasma membrane in RBL-2H3 cells (27). A recent report has also shown that PLD1 translocates from perinuclear endosomes and Golgi to the plasma membrane 2 h after PMA stimulation of COS-7 cells (20).

It has been established that PKC is predominantly cytosolic but translocates to the membrane fraction after PMA stimulation (28). However, the exact subcellular location of PKC after stimulation varies dependent on the cell line and stimulus. Some studies indicate that PKC translocates from cytosol to plasma membrane after stimulation (29, 30) and concentrates in cell-cell contact areas (31), whereas other groups provide evidence that PKC translocates to the nucleus after PMA stimulation (32). Other reports indicate that PKC may translocate to both the plasma membrane and perinuclear structures that may be the endoplasmic reticulum (33, 34) or recycling endosomes (35). There is evidence that PKC translocation is closely correlated with its phosphorylation status. For example, it has been shown that dephosphorylated PKC relocates from the plasma membrane to the perinuclear region (36). Another report indicates that a site-specific phosphorylation of PKC induces resistance to translocation and down-regulation (37).

In this study, the effect of RO on PLD1 was studied, and the results showed that RO inhibits PLD1 activation by interfering with the association of PKC with PLD. In addition, cell imaging provides evidence that PKC and PLD1 colocalize in response to PMA.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials—PMA, bovine serum albumin, Nonidet P-40, phosphatidylinositol 4,5-bisphosphate, protein phosphatase 2A (PP2A), microcystin, 2% gelatin solution, and horseradish peroxidase-conjugated secondary antibody were from Sigma. Dipalmitoylphosphatidylcholine, phosphatidylethanolamine, and phosphatidylbutanol (PtdBut) standard were from Avanti Polar Lipids Corp. L--Dipalmitoyl[2-palmitoyl-9,10-³H(N)]-phosphatidylcholine and [³H]myristic acid were from PerkinElmer Life Sciences. Protein G-agarose beads, Dulbecco's modified Eagle's medium, penicillin, streptomycin, fetal bovine serum, Tris-glycine SDS-polyacrylamide gels, pcDNA3.1(+), pcDNA3.1(His A,B,C) vectors, and anti-Xpress monoclonal antibody were from Invitrogen. The transfection reagent FuGENE 6 and the protease inhibitor mixture were from Roche Applied Science. COS-7 cells were from American Type Culture Collection. Anti-PKC monoclonal antibody was from BD Transduction Laboratories. Anti-phosphothreonine polyclonal antibody was from Zymed Laboratories Inc.. Plasmid and PCR product purification kits were from Qiagen. Anti-rabbit IgG, horseradish peroxidase, ECL reagent, and film were from Amersham Biosciences. The pEGFP-C2 vector was from Clontech. The anti-rhodamine red anti-mouse IgG and the SlowFade light antifade kit were from Molecular Probes.

Plasmid Construction—The rat PLD1 was cloned into pcDNA3.1 (His) vector with the N-terminal Xpress tag. The rat PKC was subcloned at the EcoRI site into pcDNA3.1 (+) vector and pEGFP-C2 vector. All of the constructs were sequenced to verify the coding regions and were well expressed in COS-7 cells.

Cell Culture and Transfection—COS-7 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 100 units/ml penicillin, 100 µg/ml streptomycin, and 10% fetal bovine serum in 5% CO₂. Six-well plates were seeded with 2 x 10⁵ cells/well, and 10-cm dishes were seeded with 8 x 10⁵ cells 24 h before transfection with FuGENE 6 according to the manufacturer's instructions.

In Vivo PLD Assay—After 5 h of transfection, the cells in six-well plates were serum-starved overnight (0.5% fetal bovine serum in Dulbecco's modified Eagle's medium) in the presence of 1 µCi/ml [³H]myristic acid. PLD activity was assayed by incubating the cells with 0.3% 1-butanol for 20 min before 30 min of PMA (100 nM) treatment and measuring the formation of [³H]PtdBut as a percentage of total labeled lipids as described before (38).

Subcellular Fractionation—After transfection and starvation overnight, 10-cm dishes of COS-7 cells were washed once with ice-cold phosphate-buffered saline (PBS) and then harvested using lysis buffer (25 mM Hepes, pH 7.2, 10% glycerol, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, and protease inhibitor mixture). After 10 s of sonication two times, the cell lysate was first centrifuged at 500 x g for 10 min to remove unbroken cells. The supernatant was then spun at 120,000 x g for 45 min at 4 °C to separate the cytosolic and crude membrane fractions.

In Vitro PLD Assay—For in vitro assay, COS-7 cells were transfected with PLD1 or PKC. The supernatant containing overexpressed PKC was used as the PKC fraction, and the crude membranes containing PLD1 were resuspended in lysis buffer and used as PLD1 fraction. The PLD1 activity was measured by the formation of [³H]PtdBut in vitro as described (4). Briefly, phospholipid vesicles generated from phosphatidylethanolamine/phosphatidylinositol 4,5-bisphosphate/phosphatidylcholine (16:1.4:1) containing [palmitoyl-³H]phosphatidylcholine (0.5 µCi/reaction) were used with 1-butanol (0.6%) as substrate. The reaction mixtures were incubated at 37 °C for 30 min and stopped with chloroform/methanol/HCl (50:98:2). The lipids were extracted from the organic phase and resolved by thin layer chromatography. Bands comigrating with a PtdBut standard were quantitated by liquid scintillation counting.

Immunoprecipitation and Western Blotting—COS-7 cells cultured in 10-cm plates were transfected and starved overnight as described above. The cells were washed once with ice-cold PBS and harvested using immunoprecipitation buffer containing 25 mM Hepes, pH 7.2, 10% glycerol, 1 mM EDTA, 1 mM EGTA, 50 mM KCl, 10 mM NaF, 10 mM Na₄P₂O₇, 1.2 mM Na₃VO₄, 1% Nonidet P-40, and protease mixture. The cell suspension was sonicated for 10 s and then spun at 120,000 x g for 45 min to pellet the detergent insoluble fraction. The supernatant was then precleared by mixing it with 1 µg of affinity-purified mouse IgG and 20 µl of a 1:1 slurry of protein G beads for 1 h at 4 °C. The mixture was then spun, and the supernatant was incubated with 2 µl of anti-Xpress antibody and 20 µl of protein G beads overnight. The immunoprecipitates were washed four times with the immunoprecipitation buffer and then resuspended in SDS sample buffer. The samples were analyzed by SDS-PAGE on 8% gels and transferred to polyvinylidene difluoride membranes (Immobilon-P; Millipore). The blots were then blocked with 1% BSA and incubated with primary antibody followed by incubation with horseradish peroxidase-conjugated secondary antibody. The bands were detected using ECL.

Dephosphorylation—PLD1 and PKC were immunoprecipitated with anti-Xpress antibody and anti-PKC antibody, respectively, and washed twice with the immunoprecipitation buffer to remove proteins nonspecifically bound to the protein G beads and then washed an additional two times with dephosphorylation buffer (25 mM Hepes, pH 7.2, 50 mM KCl, 10% glycerol, 1 mM dithiothreitol, and protease inhibitor mixture) to remove the detergent in the immunoprecipitation buffer. The resulting products on the protein G-agarose beads were then suspended in the reaction buffer and treated either with no addition or with the addition of PP2A or with the PP2A preinactivated with 1 µM microsystin, a potent inhibitor of protein phosphatase types 1 and 2A (39). After 30 min of incubation at room temperature, the reaction was stopped by the addition of SDS sample buffer and analyzed by Western blotting.

Immunofluorescence Microscopy—COS-7 cells were grown on 2% gelatin-coated coverslips. 5 h after transfection with EGFP-PKC or Xpress-PLD1 or both, the cells were starved in Dulbecco's modified Eagle's medium for an additional 20 h. After treatment with PMA, the cells were washed with ice-cold PBS and fixed in 3.7% formaldehyde for 30 min and permeabilized with 0.1% Triton X-100 in PBS for 5 min at room temperature. The fixed cells were washed three times with PBS, blocked with 10% horse serum and 1% BSA in PBS for 1 h, and then incubated with anti-Xpress antibody (1:1000) in PBS solution with 3% BSA for another 1 h. After being washed three times, the cells were stained with rhodamine red anti-mouse IgG (1:1000) in PBS solution with 3% BSA for 1 h. After washing three times, the cells were equilibrated and mounted on slides using the SlowFade light Antifade kit (Molecular Probes) and examined using an Axiovert S100 Microscope connected with an AxioCam HRM high resolution CCD camera system (Carl Zeiss). The images were captured using Axiovision 3.1 software (Carl Zeiss) and processed using Photoshop 6.0 (Adobe).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The PKC Inhibitor Ro-31-8220 Inhibits PMA-stimulated PLD1 Activity by Interference with PLD1 and PKC Association—The effects of different concentrations of RO on the time course of PtdBut accumulation in COS-7 cells stimulated with PMA are shown in Fig. 1A. The results indicate that RO almost completely inhibits PMA-stimulated PLD1 activity at a concentration of 1 µM. Because binding is essential for PKC to activate PLD1 (13), we tested the effects of RO on PMA-stimulated PLD1 and PKC binding using coimmunoprecipitation. Fig. 1B shows that PMA increased the binding between PLD1 and PKC and that RO blocked this binding at the concentration of 1 µM.² Cell fractionation showed that only the membrane-associated fraction of PKC bound to PLD1 (data not shown). The results indicate that RO can inhibit PMA-stimulated PLD1 activity by interference with PLD1 and PKC binding.

View larger version (28K): [in this window] [in a new window]   FIG. 1. The effects of RO on PMA-stimulated PLD1 activity and PLD1 binding with PKC in COS-7 cells. COS-7 cells were cotransfected with PLD1 and PKC. 5 h after transfection, the cells were starved for 24 h. A, COS-7 cells were pretreated with different concentrations of RO for 30 min. PMA (100 nM) was added at 2, 5, and 15 min before another 2-min incubation with 1-butanol (0.3%), and a PLD assay was carried out. In Me2SO samples, the cells were pretreated with RO (5) µM for 30 min, and Me2SO (0.1% v/v) was then added at 2, 5, and 15 min. B, COS-7 cells were pretreated with RO (1 µM) for 30 min. PMA (100 nM) was incubated with the cells for 15 min. The cells were lysed and immunoprecipitated using anti-Xpress antibody and Western blotted using both anti-Xpress and anti-PKC antibodies. A lysate from PKC-overexpressing cells was used as PKC standard (right panel). C, COS-7 cells were pretreated with RO (1 µM) for 30 min. PMA (100 nM) was then incubated with the cells for 15 min. The cells were lysed and immunoprecipitated using anti-PKC antibody and then analyzed by 6% SDS-PAGE, followed by Western blotting using anti-PKC antibody (upper panel) or anti-Thr(P) polyclonal antibody (lower panel). D, COS-7 cells were treated with PMA (100 nM), RO (1 µM), or Bis-I (1 µM) for 15 min before being lysed and fractionated into cytosol and membrane fractions as described under "Experimental Procedures." The fractions were analyzed by 8% SDS-PAGE and Western blotted with anti-PKC antibodies. Ct, control; C, cytosol; M, membrane. The data are representative of at least four experiments.

RO Blocks PMA-stimulated PLD1 and PKC Binding by Phosphorylation of PKC—The finding that RO blocked PLD1 and PKC binding was unexpected. As an ATP analog, RO may also have effects on PKC autophosphorylation. PKC exhibits different phosphorylation states with varying effects on activity (40). We therefore determined the influence of the phosphorylation status of PKC on its binding to PLD1. The experiment employed a gel system that resolved phosphorylated PKC from the nonphosphorylated form. The results show that, compared with the PKC standard that showed both phosphorylated and nonphosphorylated forms, as verified by blotting with anti-Thr(P) antibodies (not shown), only the less phosphorylated, fast moving band of PKC bound to PLD1 (Fig. 1B). The effect of RO on PKC autophosphorylation was also tested, and the results are shown in Fig. 1C. Both the band shift and the Thr phosphorylation results show that RO induced autophosphorylation of PKC. The data also show that RO did not modify the phosphorylation of PKC induced by PMA. Fig. 1D shows that RO induced the membrane translocation of PKC.

To gain support for the view that the effect of RO on PLD1 activity and PLD1 and PKC binding is due to its effect on PKC and not to some nonspecific effect, Bis-I, another PKC kinase inhibitor with a similar structure to RO (17) was tested. Fig. 2A shows that Bis-I completely inhibited PMA-stimulated PLD1 activity at a concentration of 1 µM and also blocked the PMA-stimulated PLD1 and PKC binding (Fig. 2B). In agreement with the results of Fig. 1B, only the less phosphorylated form of PKC bound to PLD1 (Fig. 2B). Like RO, Bis-I increased the autophosphorylation of PKC and caused minimal inhibition of the effect of PMA (Fig. 2C). Bis-I also induced the membrane translocation of PKC (Fig. 1D).

View larger version (28K): [in this window] [in a new window]   FIG. 2. The effects of Bis-I on PKC and PLD1 binding and PKC phosphorylation. COS-7 cells were cotransfected with PLD1 and PKC. 5 h after transfection, they were starved for another 24 h. A, COS-7 cells were pretreated with Bis-I (1 µM) for 30 min, and PMA (100 nM) was added at 2, 5, and 15 min before another 2-min incubation with 1-butanol (0.3%) and assay for PLD activity. B, COS-7 cells were treated with Bis-I (1 µM) for 30 min. PMA (100 nM) was incubated with cells for 15 min. The cells were lysed and immunoprecipitated using anti-Xpress antibody and Western blotted using both anti-Xpress and anti-PKC antibodies. A lysate from cells overexpressing PKC was used as a standard (right panel). C, COS-7 cells were treated with Bis-I (1 µM) for 30 min before incubation with PMA (100 nM) for 15 min. The cells were lysed and immunoprecipitated using anti-PKC antibody and Western blotted using anti-Thr(P) antibody. The data are representative of at least three separate experiments.

View larger version (26K): [in this window] [in a new window]   FIG. 3. The effects of phosphorylation status of both PLD1 and PKC on PLD1 activity assayed in vitro. A, membranes from PLD1-transfected cells and cytosol from cells transfected with PKC were obtained for in vitro assay. Dephosphorylation was carried out as described under "Experimental Procedures." Samples treated with microcystin (1 µM) to inactivate PP2A were used as control. PLD/DP means only PLD1 was dephosphorylated; PKC/DP means that only PKC was dephosphorylated; Both/DP means that both PLD1 and PKC were dephosphorylated. B, to test whether PP2A and microcystin functioned properly, COS-7 cells were lysed and immunoprecipitated using anti-PKC antibody. After washing four times, protein G-agarose beads were dephosphorylated with PP2A or microcystin-pretreated PP2A (PP2A*). The data are representative of at least three separate experiments.

It is concluded from these experiments that the effect of phosphorylation of PKC on its interaction with PLD1 observed in vivo is not due to the phosphorylation per se but is dependent on other cellular components.

PKC Translocates to Both the Plasma Membrane and Perinuclear Region after PMA Stimulation—It is known that PKC and PLD1 have different cellular locations in unstimulated cells in vivo and that activation of PKC is associated with membrane translocation (for references see Refs. 1 and 35). To test the possibility that PKC might translocate to the site of PLD1, N-terminal GFP-tagged PKC and Xpress-tagged PLD1 were coexpressed in COS-7 cells. Their cellular localizations before and after PMA stimulation are shown in Fig. 4. The figure shows that, before PMA stimulation, PKC was present in the cytosol, whereas PLD1 was located in punctate perinuclear structures. After PMA stimulation, PKC translocated to both the plasma membrane and the perinuclear region, whereas PLD1 stayed condensed at this region. Merging of the images shows that PMA induced marked perinuclear colocalization of PKC and PLD1. Additional studies with the nuclear marker Hoechst 33342 confirmed their perinuclear location (data not shown).

View larger version (20K): [in this window] [in a new window]   FIG. 4. Colocalization of PLD1 and PKC in COS-7 cells. COS-7 cells were cotransfected with Xpress PLD1 and GFP-PKC and starved 5 h later. After 24 h of starvation, the cells were treated with PMA (300 nM) for 15 min and stained as described under "Experimental Procedures." The data are representative of at least five separate experiments. Bar, 15 µm.

View larger version (20K): [in this window] [in a new window]   FIG. 5. The effect of RO and Bis-I on PMA stimulated PKC translocation. COS-7 cells were transfected with GFP-PKC and starved 5 h later. After 24 h of starvation, the cells were pretreated with RO (500 nM) or Bis-I (500 nM) for 30 min and then treated with PMA (300 nM) for 15 min. After washing and fixation, the cells were stained as described under "Experimental Procedures." The data are representative of at least five separate experiments. Bar, 15 µm.

View larger version (15K): [in this window] [in a new window]   FIG. 6. The effect of okadaic acid on PLD1 activity and PKC translocation in COS-7 cells. COS-7 cells were cotransfected with Xpress PLD1 and PKC. 5 h after transfection, the cells were starved for 24 h. A, COS-7 cells were pretreated with different concentrations of OA for 1 h and then incubated with 1-butanol (0.3%) for another 10 min. PMA (5 nM) was added, and the cells were incubated for 5 min before performing an in vivo PLD assay. B, to test whether OA functioned correctly, the OA (500 nM) sample used for PLD assay was also Western blotted using anti-Thr(P) antibody. C, COS-7 cells were transfected with GFP-PKC and starved 5 h later. After 24 h starvation, the cells were treated with OA (500 nM) for 1 h. After PMA (50 nM) stimulation for 15 min, the cells were washed and fixed for microscope observation. The data are representative of at least three separate experiments. Bar, 15 µm.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

A surprising result was the stimulation of the phosphorylation of PKC exerted by RO (Fig. 1C) and Bis-I (Fig. 2C) in vivo. Because it is very unlikely that these inhibitors, which are ATP analogs and act by competing with ATP, would directly stimulate the phosphorylation of PKC, it seems that another protein kinase could be involved, as discussed below. Alternatively, the inhibitors may act by blocking the dephosphorylation of PKC, which has been observed in some cell lines (36, 41). Another surprising result was that RO and Bis-I induced the translocation of PKC to the membrane fraction (Fig. 1D). However, because of the opposite effects of the inhibitors and PMA on the activation of PLD1, it seems unlikely they both translocated PKC to the same membrane(s). In fact, Fig. 5 shows that the inhibitors did not induce the same intracellular translocations of PKC as those seen with PMA.

A key issue to be resolved in the present study is why PMA, which induced phosphorylation of PKC, caused activation of PLD1 and promoted the association of PKC with PLD1 i.e. changes opposite to those observed when PKC phosphorylation was increased by RO or Bis-I (Figs. 1 and 2). One obvious explanation is that the protein kinase (or protein phosphatase) involved in the phosphorylation induced by the inhibitors differs from PKC. This could result in the phosphorylation of different residues. As described above, the intracellular localization of PKC seen with PMA differed from that seen with the inhibitors. Although there could be many reasons for this, it could reflect a difference in PKC phosphorylation (36, 37, 41, 44). However, we cannot provide definitive proof of this hypothesis because the kinase (phosphatase) involved in the inhibitor effects has not been identified.

The translocation experiments provide an explanation of how PMA might induce the activation of PLD1 by a protein-protein interaction. They confirm many observations that PLD1 is located predominantly, but not exclusively, in the perinuclear region (7, 19, 27, 42, 43) but, more importantly, illustrate that PMA causes the translocation of PKC to the perinuclear region as well as to the plasma membrane (Fig. 4 and Ref. 35) and that RO and Bis-I block the localization of PKC to the perinuclear region (Fig. 5). Because our present and previous data (13) indicate that the association of PKC with PLD is required for PMA activation of PLD1, their colocalization in the perinuclear region would be expected to lead to PLD1 activation. Likewise, the inhibition of this colocalization by RO and Bis-I could explain why these inhibitors block the activation.

The results with RO and Bis-I support previous findings that the phosphorylation status of PKC is closely related with its cellular localization (36, 37, 41, 44), although some of these studies did not define the membrane fraction(s) involved. Our results also show that RO and Bis-I phosphorylate PKC, and it is hypothesized that this makes it unable to translocate to perinuclear region. Phorbol esters presumably activate PKC at the plasma membrane. Thus it is likely that PKC initially translocates to the plasma membrane and subsequently relocates to the perinuclear region (36). There is evidence that this relocation is due to its dephosphorylation by PP2A and can be blocked by RO (36). This provides support for the idea that RO and Bis-I block the PKC relocation to the perinuclear region by increasing its phosphorylation, thus inhibiting its binding to and activation of PLD1. The inhibitors would be expected to block the phosphorylation of PKC induced by PMA. However, it is evident from Figs. 1C and 2C that they induce phosphorylation of PKC per se and have little or no effect on the phosphorylation induced by PMA. As discussed above, it is likely that the inhibitors act through another protein kinase or a protein phosphatase.

To further prove the relationship between PKC phosphorylation and its localization, OA, a PP2A inhibitor, was used to study its effect on PKC translocation and activation of PLD1. The results of Fig. 6 showed that OA partially inhibited PKC relocation to the perinuclear region and thus inhibited the activation of PLD1. The results with RO, Bis-I, and OA suggest PMA induced PKC translocation to the plasma membrane and then relocation to the perinuclear region to activate PLD1.

In our study PKC was tagged with GFP to track its translocation in COS-7 cells. Although GFP has been widely used in many previous reports to study the localization and translocation of PKC, we considered the possible effect of GFP on PKC localization. The GFP results showed that when GFP alone was expressed it localized inside nuclei (data not shown). When the translocation of GFP-PKC was analyzed using cell fractionation, the results showed that GFP-PKC had the same translocation ability as nontagged PKC (data not shown). We also compared the PLD activity increase induced by nontagged PKC or GFP-tagged PKC and found no difference (data not shown). Therefore we conclude that GFP tagging does not affect the ability of PKC to translocate or activate PLD.

In summary, the present findings present a novel mechanism by which PKC could activate PLD1 in vivo. They also illustrate that only the nonphosphorylated form of PKC can interact with PLD1 and that PKC and PLD1 can colocalize in the perinuclear region following PMA stimulation. They also reveal some unexpected findings with respect to two widely used PKC inhibitors, namely that they induce phosphorylation of PKC in vivo and block its interaction with PLD1. We propose this as a mechanism by which they inhibit PMA activation of PLD1.

	FOOTNOTES

 
^* This work was partly supported by the Vanderbilt Ingram Cancer Center. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Rm. 831 Light Hall, Vanderbilt University, Nashville, TN 37232. Tel.: 615-322-6494; Fax: 615-322-4381; E-mail: john.exton{at}vanderbilt.edu.

¹ The abbreviations used are: PLD, phospholipase D; PKC, protein kinase C; RO, Ro-31-8220; Bis-I, bisindolylmaleimide I; PMA, 4-phorbol 12-myristate 13 acetate; PP2A, protein phosphatase 2A; PtdBut, phosphatidylbutanol; OA, okadaic acid; PBS, phosphate-buffered saline; BSA, bovine serum albumin; GFP, green fluorescent protein.

² The fraction of PKC binding to PLD1 before and after PMA stimulation was quantitated by densitometry, and the results showed that approximately 0.2% of total PKC bound to PLD1 before PMA stimulation, and 2.7% of total PKC bound to PLD1 after PMA stimulation (data not shown).

³ It should be noticed that in the PP2A-treated samples the PKC bands are stronger compared with the relevant other lanes (Fig. 3B). One possibility is that dephosphorylated PKC is more easily extracted from the beads in the SDS sample buffer. Another possibility is that the dephosphorylated form may be less susceptible to proteolysis.

	ACKNOWLEDGMENTS

 
We thank Judy Nixon for preparation of the manuscript.

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