Protein Kinase Cα Translocates to the Perinuclear Region to Activate Phospholipase D1*

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 PKCα. 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 PKCα binding to PLD1. Further studies showed that only unphosphorylated PKCα could bind to and activate PLD1 and that both inhibitors induced phosphorylation of PKCα. The phosphorylation status of either PLD1 or PKCα per se did not affect PLD1 activation in vitro. Immunofluorescence studies showed that PLD1 remained in the perinuclear region after phorbol ester treatment, whereas PKCα translocated from cytosol to both plasma membrane and perinuclear regions. Both Ro-31-8220 and bisindolylmaleimide I blocked the translocation of PKCα to the perinuclear region but not to the plasma membrane. Studies with okadaic acid suggested that phosphorylation regulated the relocation of PKCα from the plasma membrane to the perinuclear region. It is proposed that localization and interaction of PKCα with PLD1 in the perinuclear region is required for PLD1 activation and that PKC inhibitors inhibit this through phosphorylation of PKCα, which blocks its translocation.

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.
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)(3)(4)(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)(14)(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 12myristate 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)(24)(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.
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 (ϩ) 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 g/ml streptomycin, and 10% fetal bovine serum in 5% CO 2 . Six-well plates were seeded with 2 ϫ 10 5 cells/well, and 10-cm dishes were seeded with 8 ϫ 10 5 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 Ci/ml [ 3 H]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 [ 3 H]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 ϫ g for 10 min to remove unbroken cells. The supernatant was then spun at 120,000 ϫ 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 [ 3 H]PtdBut in vitro as described (4). Briefly, phospholipid vesicles generated from phosphatidylethanolamine/phosphatidylinositol 4,5-bisphosphate/phosphatidylcholine (16:1.4:1) containing [palmitoyl-3 H]phosphatidylcholine (0.5 Ci/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 Na 4 P 2 O 7 , 1.2 mM Na 3 VO 4 , 1% Nonidet P-40, and protease mixture. The cell suspension was sonicated for 10 s and then spun at 120,000 ϫ g for 45 min to pellet the detergent insoluble fraction. The supernatant was then precleared by mixing it with 1 g of affinity-purified mouse IgG and 20 l 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 l of anti-Xpress antibody and 20 l 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 M 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
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 M. 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 M. 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-stimu-

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 M) 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. lated PLD1 activity by interference with PLD1 and PKC␣ binding.
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 M 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).
The Phosphorylation Status of Either PLD1 or PKC␣ Does Not per Se Affect PMA-stimulated PLD1 Activity in Vitro-The preceding results showed that phosphorylated PKC␣ was unable to bind to PLD1 and that RO and Bis-I inhibited PLD1 and PKC␣ binding by phosphorylating PKC␣ and making it unable to bind to PLD1. To see whether the phosphorylation status of either PLD1 or PKC␣ would affect in vitro PLD1 activity, the Ser/Thr phosphatase PP2A was used to treat membranes containing PLD1 and cytosol containing PKC␣ for 30 min before conducting an in vitro PLD assay. Pretreatment of PP2A with microcystin, a potent PP2A inhibitor (39)  was analyzed, and the results are shown in Fig. 3B. The results show that PP2A did dephosphorylate PKC␣ and that microcystin blocked this effect. 3 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).
RO or Bis-I Blocks PMA-stimulated Perinuclear Translocation of PKC␣-To test whether RO or Bis-I could affect PKC␣ translocation, GFP-tagged PKC␣ was expressed in COS-7 cells. The cells were then pretreated with RO or Bis-I (500 nM) before PMA stimulation. The results shown in Fig. 5 indicated that neither RO nor Bis-I blocked PKC␣ translocation from the cytosol to the plasma membrane. However, both inhibitors prevented PKC␣ from translocating to the perinuclear region. The inhibition of PKC␣ perinuclear translocation could explain why RO and Bis-I block the binding between PLD1 and PKC␣.
Effects of Okadaic Acid on PMA Activation of PLD1 and Translocation of PKC␣ to the Perinuclear Region-A previous report has indicated that PKC␣ perinuclear translocation may be due to its dephosphorylation following its translocation from the cytosol to the plasma membrane (36). To explore the possible role of dephosphorylation, okadaic acid (OA), a PP2A inhibitor was used to treat the cells, and its effects on PLD1 activity and PKC␣ translocation were observed. Fig. 6A shows that OA partially inhibited 5 nM PMA-stimulated PLD1 activity in a concentrationdependent manner. Concentrations of OA higher than 500 nM were not tested because the cells showed loss of viability. The effects of OA (500 nM) on PMA-stimulated PKC␣ phosphorylation were also tested, and Fig. 6B shows the expected increase. Fig.  6C also shows that OA partially blocked PKC␣ translocation to the perinuclear region. These results support the view that the translocation of PKC␣ from cytosol to perinuclear region involves its dephosphorylation. DISCUSSION The present study arose from an effort to find if the inhibitory effect of RO on PLD1 activity involved an inhibition of phosphorylation or not. Our results show that RO inhibits PLD1 activity by blocking the PMA-stimulated association of PKC␣ with PLD1 (Fig. 1). Previous studies have shown that PKC␣ auto-phosphorylation is closely related to its activity and that the enzyme exists in different phosphorylation states (36,37,40,41). Our study found that only the unphosphorylated form of PKC␣ can bind to PLD1. This finding provides a clue as to why RO inhibits the binding of PKC␣ to PLD1. Fig. 1D shows that treatment with RO causes phosphorylation of PKC␣, and this is associated with its inability to bind to PLD1. Similar results were obtained with Bis-I, another kinase inhibitor of PKC␣ (Fig. 2). These results support the view that PKC␣ can activate PLD1 by a protein-protein interaction but that only unphosphorylated PKC␣ can bind to PLD1.
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 proteinprotein 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.