Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
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Originally published online as doi:10.1189/jlb.0206109 on October 31, 2006

Published online before print October 31, 2006
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(Journal of Leukocyte Biology. 2007;81:509-519.)
© 2007 by Society for Leukocyte Biology

Protein kinase C activity is required for cytotoxic T cell lytic granule exocytosis, but the {theta} isoform does not play a preferential role

Michael J. Grybko, Arun T. Pores-Fernando, Georjeana A. Wurth and Adam Zweifach1

Department of Physiology and Biophysics, University of Colorado Health Sciences Center, Aurora, Colorado, USA

1 Correspondence at current address: Department of Molecular and Cell Biology, University of Connecticut at Storrs, 91 North Eagleville Road, Storrs, CT 06269-3125. E-mail: adam.zweifach{at}uconn.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CTLs kill virus-infected, tumor, and transplanted targets via secretion of lytic agents including perforin and granzymes. Knowledge of the signals controlling this important process remains vague. We have tested the idea that protein kinase C (PKC){theta}, a member of the novel PKC (nPKC) family, which has been shown to play a preferential role in critical Th cell functions, plays a similar, preferential role in CTL lytic granule exocytosis using T acute lymphoblastic leukemia-104 (TALL-104) human leukemic CTLs as a model. We provide evidence consistent with the idea that PKC activity is important for the degranulation step of lytic granule exocytosis, as opposed to upstream events. In contrast with previous work, our results with pharmacological agents suggest that conventional PKCs (cPKCs) and nPKCs may participate. Our results suggest that stimulation with soluble agents that bypass the TCR and trigger granule exocytosis activates PKC{alpha} and PKC{theta}, which can both accumulate at the site of contact with a target cell, although PKC{theta} did so more often. Finally, using a novel assay that detects granule exocytosis specifically in transfected, viable cells, we find that overexpression of constitutively active mutants of PKC{alpha} or PKC{theta} can synergize with increases in intracellular [Ca2+] to promote granule exocytosis. Taken together, our results lend support for the idea that PKC{theta} does not play a preferential role in CTL granule exocytosis.

Key Words: cytotoxicity • PKC • flow cytometry


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
One of the key mechanisms CTLs use to kill target cells is exocytosis of lytic granules, specialized secretory lysosomes that contain cell-killing agents such as perforin and granzymes (reviewed in refs. [1 2 3 ]). Knowledge of the signaling pathways controlling this important process remains fairly rudimentary. In recent years, protein kinase C (PKC){theta}, a member of the novel PKC (nPKC) family, which also includes the {delta}, {epsilon}, {eta}, and µ isoforms, has been shown to play preferential roles in important Th cell functions such as IL-2 gene expression, JNK activation [4 , 5 ], and Fas ligand (FasL) expression [6 , 7 ]. nPKCs lack the Ca2+-binding C2 domains of conventional PKCs (cPKCs: {alpha}, ßI, ßII, and {gamma} isoforms) and are thought to be activated physiologically by diacylglycerol, for which phorbol myristate acetate (PMA) serves as a potent substitute (reviewed in ref. [8 ]).

It has been known for many years that TCR-stimulated lytic granule exocytosis is PKC-dependent [9 10 11 ] and that treatment of CTLs with phorbol esters such as can synergize with increases in intracellular-free [Ca2+] ([Ca2+]i) to promote granule exocytosis [11 , 12 ]. Recent work has shown that PKC{theta} localizes to the immunological synapse in CTLs contacting relevant targets [13 14 15 ], as it has been shown to do in Th cells contacting APCs [16 , 17 ]. Taken together, these results naturally lead to the attractive idea that PKC{theta} is also likely to play a critical role in granule exocytosis. However, there are two problems with this idea. First, it is now clear that there are non-PKC targets of PMA, including Ras guanine nucleotide-releasing proteins (RasGRPs), which are thought to be involved in activation of ERKs [18 ], which are now known to be important for granule exocytosis [18 19 20 ]. Thus, the ability of PMA to synergize with [Ca2+]i increases in promoting granule exocytosis and does not necessarily prove that PKC participates. Second, TCR-stimulated granule exocytosis is a complex process that involves multiple steps upstream of degranulation, including formation of the immunological synapse, reorientation of lytic granules, and the cell’s microtubule organizing center (MTOC) and Golgi apparatus to face the target [21 22 23 ]. Involvement of PKC in TCR-stimulated granule exocytosis therefore does not unambiguously establish a role for PKC in the actual degranulation step of the lytic interaction.

Even less evidence supports the view that PKC{theta} plays a preferential role in granule-dependent killing by CTLs; in fact, what little evidence is available argues against the idea. Pardo et al. [24 ] reported that inhibitors of cPKCs but not nPKCs inhibited TCR-stimulated, granule-dependent cytotoxicity and showed that overexpression of mutant, constitutively active PKC{alpha}, but not constitutively active PKC{theta}, could synergize with ionomycin to drive granule exocytosis. Furthermore, Puente et al. [25 ] provided evidence that PMA fails to activate PKC{theta} in cloned, murine CTLs, as assessed using in vitro kinase assays or by measuring binding of PKC{theta} to membranes. This argues that PKC{theta} cannot mediate the effects of PMA in promoting granule exocytosis. Recent further work by this group [26 ] has shown that ERK activation in response to solid-phase CD3 is dependent on nPKC activity, as it is not inhibited by cPKC blockers, and granule exocytosis stimulated by solid-phase CD3 or target cells is sensitive to cPKC blockers. It is important that normal granule exocytosis was observed in CTLs derived from PKC{theta} knockout mice, although there was clear up-regulation of other PKC isoforms.

We have investigated the involvement of PKC in lytic granule exocytosis in T acute lymphoblastic leukemia (TALL)-104 human leukemic CTLs [27 ], which we have used as a model of CTL function and signaling in a number of studies [20 , 28 29 30 ]. We find that TALL-104 cells express multiple PKC isoforms, including at least one cPKC (PKC{alpha}) and one nPKC (PKC{theta}). We provide evidence consistent with the idea that PKC activity plays a key role in the degranulation step of lytic granule exocytosis, as opposed to upstream steps. In contrast with previous work [24 ], our results with pharmacological inhibitors of PKCs are consistent with the idea that cPKCs and nPKCs may participate. Our results suggest that stimulation with soluble agents that bypass the TCR and trigger granule exocytosis activates PKC{alpha} and PKC{theta}—results that contrast with those of Puente et al. [18 ]. We analyzed the distribution of PKC{alpha} and PKC{theta} in conjugates with target cells and found that both could, in some cases, be found enriched at the immunological synapse, in striking contrast to what has been reported in Th cells, where localization at the synapse is exclusive to PKC{theta} [31 ]. Finally, using a novel assay that detects granule exocytosis specifically in transfected, viable cells, we found that overexpression of constitutively active mutants of PKC{alpha} or PKC{theta} can synergize with [Ca2+]i increases to promote granule exocytosis. Taken at face value, our results suggest that PKC{alpha} may be more effective than PKC{theta}. These results are thus on the one hand consistent with the results of Pardo et al. [24 ], in that they suggest that PKC{alpha} may in fact play a preferential role but conversely, differ from their results in that they indicate that PKC{theta} can have a functional effect, albeit at higher expression levels relative to the endogenous protein. Taken together, our results add support for the idea that PKC{theta} is unlikely to play the kind of preferential role in CTL granule exocytosis that it does in Th cell functions.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals and reagents
Salts for physiological solutions were from Sigma-Aldrich (St. Louis, MO). FCS was from Atlas Biologicals (Ft. Collins, CO). Dynabeads M450 antihuman pan T cell beads (Dynal Invitrogen, Brown Deer, WI) were used for solid-phase anti-CD3 stimulation. Thapsigargin (TG) and PMA were from Alexis Biochemicals (San Diego, CA). PE-labeled anti-CD107a mAb (Clone H4A3) and isoform-specific PKC mAb were purchased from BD Biosciences (San Diego, CA). The CD107a antibody was concentrated before use as described previously [30 ]. The antiphospho(ser) PKC antibody [raised against the sequence (R/K)X(phosph-S)(Hyd)(R/K)] was from Cell Signaling Technologies (Beverley, MA). Secondary antibodies were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). Protease inhibitor and phosphatase inhibitor cocktails were from Calbiochem/EMD Biosciences (San Diego CA).

Cells and solutions
All cell types used were obtained from American Type Culture Collection (Manassas, VA). TALL-104 cells were grown in Iscove’s medium supplemented with 10% FCS and 100 IU IL-2 in a humidifier incubator at 37°C in 10% CO2. Jurkat cells and Raji B cells were grown in RPMI + 10% FCS. Ringer’s solution contained (in mM) 145 NaCl, 4.5 KCl, 1 MgCl2, 2 CaCl2, 5 HEPES, and 10 glucose (pH 7.4 with NaOH).

Western blotting
Whole cell lysates, prepared from 2.5–5 x 106 TALL-104 cells or Jurkat cells per experimental condition, were run on gels and transferred to nitrocellulose membranes as described previously [20 ]. Bradford assays were used to determine protein concentration in supernatants so that equal amounts of protein could be loaded. When blots were quantitated, Image J (http://rsb.info.nih.gov/ij/) was used to select regions of interest, and mean intensity was computed after subtraction of the mean intensity of a background region of identical size located between lanes. Regions used were strips that comprised the entire molecular weight range of the gel and were slightly narrower than the spacing between lanes.

N{alpha}-Benzyloxycarbonyl-L-lysine thiobenzyl ester (BLT)-esterase assays
Granyzme B released from TALL-104 cells was assayed by measuring hydrolysis of BLT [32 ], as described previously [20 , 29 ]. The percentage of BLT-esterase activity released spontaneously was measured from unstimulated CTLs, which were treated identically to stimulated cells and incubated in normal Ringer’s. Release was calculated as absorbancesupernatant/absorbancepellet + absorbancesupernatant. Spontaneous release was subtracted from percentage release obtained upon stimulation to obtain percentage-specific stimulated release.

Immunocytochemistry and flow cytometry
For immunocytochemistry experiments, Caltag’s Fix/Perm kit was used according to the manufacturer’s protocol (Caltag Laboratories/Invitrogen, Burlingame, CA). In the experiments (Fig. 3) , a FITC-labeled secondary antibody was used following the primary antibodies. For the experiments (see Figs. 4 and 6 ), Zenon reagents (Molecular Probes/Invitrogen, Eugene OR) were used to pre-label primary antibodies according to the manufacturer’s instructions. Following staining, cells were then allowed to settle on coverslips, and Prolong antifade reagent (a hardening mounting medium from Molecular Probes/Invitrogen) was applied.


Figure 3
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  		Figure 3. Stimulation induces rapid translocation of PKC{alpha} and PKC{theta} to the plasma membrane in TALL-104 cells. (A) Confocal micrographs showing the subcellular localization of PKC{alpha} and PKC{theta} immunoreactivity in unstimulated cells (upper) and in cells stimulated with TG + PMA for 30 min (lower). (B) Live-cell time-lapse imaging of PKC-GFP fusion proteins. (1) Images of the subcellular localization of PKC{alpha} before and at various times after stimulation with TG + PMA (upper) and of PKC{theta} before and after stimulation with PMA (lower). Images were acquired at the times (relative to stimulation) indicated. (2) Plots of the normalized ratio of membrane to cytosolic PKC for PKC{alpha} (upper) and PKC{theta} (lower). Different symbols correspond to different cells analyzed.

 

Figure 4
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  		Figure 4. PKC{alpha} and PKC{theta} can accumulate at the site of contact with a target cell. TALL-104 cells were allowed to contact Raji B lymphoma cells, which were coated with a CD3/CD19 bsAB, then fixed and permeabilized, and stained with PKC isoform-specific antibodies, which were prelabeled with isotype-specific Fab fragments. (A) Phase contrast image showing two TALL cells in contact with a larger Raji in the center. (B) Immunolocalization of PKC{theta}. (C) Immunolocalization of PKC{alpha}. Original scale bar is 7 µM.

 

Figure 6
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  		Figure 6. Expression of constitutively active mutant PKC{alpha} and PKC{theta} GFP fusion proteins in TALL-104 cells. (A) Plot of PE fluorescence versus GFP fluorescence for cells transfected with GFP (i, upper) or mutant PKC{alpha}-GFP (ii, lower). (B) Plot of PE fluorescence versus GFP fluorescence for cells transfected with GFP (i, upper) or mutant PKC{theta}-GFP (ii, lower). (A and B) Contour plots drawn at the 2% probability level; and outliers are shown. (C) Representative micrographs showing cells transfected with mutant PKC{alpha}-GFP. (D) Representative micrographs of cells transfected with mutant PKC{theta}-GFP. In both cases, fluorescence was clearly associated with the plasma membrane. Images were collected at the same gain settings and then autoscaled.

 
For flow cytometry to detect lysosome-associated membrane protein (LAMP CD107A) externalization, cells were first washed with normal Ringer’s and then stimulated as indicated in the conditions described. The anti-LAMP antibody was concentrated 20-fold from the stock provided by the manufacturer using Millipore Amicon columns with a 5000 m.w. cutoff (Millipore, Bedford, MA) and then diluted 1:50 before use. Cells were suspended in PBS, supplemented with 0.2% paraformaldehyde after processing. Flow cytometry was done on a Coulter Cytomics FC500 (Beckman Coulter, Miami, FL), except the experiments to determine levels of expression of PKC mutants; these were performed on a FACSCalibur (BD Biosciences, San Jose, CA). FlowJo software (TreeStar, Ashland, OR) was used to analyze data off-line. For multicolor experiments, data were acquired without hardware compensation. Unstained and single-color controls were run, and data were compensated off-line using FlowJo.

Fluorescence imaging
Image acquisition was performed on three systems. For live cell imaging of PKC-GFP fusion protein dynamics and for investigation of the effects of rottlerin on PKC isoform localization, a system built around a Nikon TE200 inverted microscope (Nikon, Melville, NY), which has been described previously [28 ], was used. Slidebook software (Intelligent Imaging Innovations, Denver, CO) was used to control the hardware. Images in Fig. 3 were acquired with a Zeiss LSM 510 laser-scanning confocal microscope at the University of Colorado Health Sciences Center (UCHSC) Light Microscopy Facility (Aurora) using a 63x Apochromat oil immersion objective and optimized pinhole settings. Images in Fig. 4 were acquired with a Leica SP2 laser-scanning confocal microscope at the Flow Cytometry and Confocal Microscopy Facility at the University of Connecticut (Storrs), using a 100x objective and pinhole settings yielding a depth of focus of 2 Airy units (see Fig. 4 ).

cDNA constructs and transfections
Mouse PKC{theta} and mutant PKC{theta} A148E, both in the pEF expression vector [5 ], were a generous gift of Dr. Gottfried Baier (University of Innsbruck, Austria). Rabbit PKC{alpha} and PKC{alpha}R22A/A25E, in the SR{alpha} expression vector [33 ], were given to us by Dr. Mary Reyland (UCHSC). Coding sequences were transferred into plasmid vector-expressing enhanced GFP (pEGFP)-N1 (Clontech, Mountain View, CA) by using standard PCR methods to add unique restriction sites (KpnI and BamHI for PKC{theta} constructs and KpnI and EcoRI for PKC{alpha} constructs). PCR products were then ligated into the multiple cloning site of pEGFP-N1. TALL-104 cells were transfected using an Amaxa Nucleofector (Amaxa Biosystems, Gaithersburg, MD) using program T-20 and solution V, and experiments were performed 5–7 h post-transfection. In our hands, expression of GFP or GFP fusion proteins can be detected ~2 h after nucleofection, and expression is essentially maximal 5–7 h postnucleofection. At longer times, expression levels are comparable, but cell viability is decreased. As assessed using LAMP assays, cells respond normally 2 h after nucleofection.

Statistics
Statistical analysis of results was performed with Instat (Graphpad Software, San Diego, CA). One-way paired ANOVA was used with Dutton’s post-test to compare experimental to control datasets.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
TALL-104 cells express multiple PKC isoforms
We examined the expression of PKC isoforms in TALL-104 human leukemic CTLs using Western blotting (Fig. 1 ). We probed lysates prepared from human brain, Jurkat human leukemic Th cells, and TALL-104 cells. With the antibodies we had, we were able to confirm the presence of PKC{alpha} and PKC{theta} in TALL-104 cells at levels that were comparable with those of the control lysates. PKCß and PKC{epsilon} were detected, although apparently at substantially lower levels relative to the controls. The antibodies we had to PKC{delta} and PKC{eta} failed to detect bands of the appropriate size in human brain lysates, which we used as a positive control (rat brain is listed as the appropriate positive control by the supplier, but we were concerned about species specificity.) Thus, although we cannot reach a conclusion about the relative expression of these PKC isoforms, it is clear that TALL-104 CTLs express at least one representative of the cPKCs and the nPKCs at levels comparable with cells or tissues that are known sources.


Figure 1
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  		Figure 1. TALL-104 cells express multiple PKC isoforms. Western blotting was used to probe the expression of different PKC isoforms in lysates prepared from human brain, Jurkat human leukemic Th cells, and TALL-104 CTLs. Protein (10 µg) was loaded for each sample, and blots were probed with isoform-specific mAb.

 
Probing the involvement of PKCs in TCR-dependent and TCR-independent granule exocytosis with pharmacological tools
We used pharmacological agents as an initial means of testing the involvement of PKCs in granule exocytosis (Fig. 2 ). We tested the effects of Ro31-8220 (Ro, 4 µM; a general PKC inhibitor), Go6976 (Go, 4 µM, an inhibitor of classical PKC isoforms), and rottlerin (20 µM, a drug that has been used as an nPKC inhibitor [6 , 34 ]) on exocytosis stimulated by solid-phase anti-CD3 or by 1 µM Thapsigargin (TG) in combination with 50 nM PMA. TG is a drug that blocks smooth endoplasmic reticulum Ca2+-adenosinetriphosphatase [35 ], depleting stores and activating Ca2+ influx in CTLs [36 ]. We used BLT-esterase assays [32 ] to measure exocytosis. Cells were preincubated in the presence of drugs for 5 min, and drugs were present during stimulation. Spontaneous release, assessed by incubating cells in the presence of the drug without stimulating them, was measured in each experiment and subtracted from release caused by stimulation to obtain the specific granule exocytosis. Results are presented in Figure 2A . The general PKC inhibitor Ro essentially completely blocked exocytosis stimulated by both means. Go inhibited granule exocytosis stimulated by beads or by TG + PMA by ~50%. Like Ro, rottlerin also almost completely blocked exocytosis stimulated by anti-CD3 beads and by TG + PMA.


Figure 2
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  		Figure 2. PKC inhibitors indicate that PKC is involved in lytic granule exocytosis but do not identify PKC{theta} as the isoform involved. (A) BLT-esterase assays were performed to assess the effects of PKC inhibitors on lytic granule exocytosis stimulated by anti-CD3 beads (i) or by TG + PMA (ii). (B) A phospho(ser)-PKC substrate antibody was used to probe lysates from TALL-104 cells, from unstimulated cells (Ctrl), from cells stimulated with TG + PMA (STIM), and from stimulated cells treated with Ro31-8220 (Ro), Go6976 (Go), or rottlerin (Rott). (i) A representative blot; (ii) quantitation from three such experiments. Data are normalized to the levels measured in unstimulated cells. The regions of interest used to quantitate gels comprised regions that were slightly narrower than the space between lanes and extended the length of the entire lane. Asterisks indicate results that differ at the P < 0.05 level.

 
As a means of testing whether the drugs we used in the experiments described above have the expected pharmacological effects on PKC activation, we used a commercially available antibody against the sequence (R/K)X(phospho-S)(Hyd)(R/K) [37 ] to probe using Western blotting patterns of PKC activation under the conditions described above in Figure 2A 2ii . This antibody is expected to detect proteins that have been phosphorylated by PKC, although it is not clear whether there would be expected to be any selectivity for substrates preferred by different PKC classes. A representative blot is shown in Figure 2B 2i , and quantitation of data from three experiments (including the one shown in Fig. 2B 2i ) is shown in Figure 2B 2ii . Note that in experiments such as this, we do not know the identity of any of the bands detected and thus, decided it was most appropriate to analyze entire lanes rather than specific bands. Consistent with the idea that the antibody detects phosphorylated PKC substrates, we found that stimulation with TG + PMA caused an approximate fourfold increase in immunoreactivity, which was almost blocked entirely by the general PKC inhibitor Ro. It is surprising that neither Go nor rottlerin had a statistically significant inhibitory effect on the increase in immunoreactivity caused by stimulation with TG + PMA, despite the fact that there were bands or smears of immunoreactivity that appeared to be reduced by treatment with Go, particularly, in the 50- to 100- and 25- to 35-kilodalton ranges. The failure of rottlerin to affect phosphorylation of PKC substrates is particularly striking, as this drug inhibited granule exocytosis completely, while Go had only a partial effect.

We also examined using immunocytochemistry whether rottlerin prevented the translocation of PKC{theta} and PKC{alpha} to the plasma membrane (data not shown), reasoning that selective prevention of PKC{theta} translocation might provide some alternative evidence for inhibitory activity of rottlerin. We found that rottlerin altered the subcellular localization of both PKC{alpha} and PKC{theta} in unstimulated cells, creating a more punctate distribution than was observed in control cells. Furthermore, there was no apparent association of either PKC isoform with the plasma in rottlerin-treated cells following stimulation with TG + PMA.

PKC{alpha} and PKC{theta} translocate to the plasma membrane rapidly upon stimulation
As described above, TALL-104 cells express PKC{alpha} and PKC{theta} at high levels. We used microscopy to examine changes in the subcellular localization of these PKC isoforms as a result of stimulation (Fig. 3 ), reasoning that the kinetics of translocation or the location to which the PKC isoform moved might provide information about which isoform participates in granule exocytosis. In a first set of experiments, we used antibodies against PKC{alpha} and PKC{theta} to assess their localization in unstimulated cells and in cells stimulated with TG and PMA for 30 min (Fig. 3A) . In unstimulated cells, both isoforms were present throughout the cytoplasm but were largely excluded from the nucleus. In contrast, in cells treated with TG and PMA, there was a clear association of both isoforms with the plasma membrane and a concomitant decrease in the amount in the cytoplasm.

To further investigate translocation of PKC isoforms to the plasma membrane, we monitored the dynamics of PKC{alpha} and PKC{theta} in live cells (Fig. 3B) . We transfected cells with vectors encoding wild-type PKC{alpha} or PKC{theta} fused to GFP and performed time-lapse imaging. As was the case with the immunocytochemical experiments described above, PKCs were localized primarily in the cytoplasm in unstimulated cells. Stimulation (with PMA alone in the case of PKC{theta} or with TG+PMA for PKC{alpha}) caused rapid translocation of both PKC isoforms to the plasma membrane. Translocation was, in many cases, complete within ~6 min of stimulation for both isoforms.

PKC{alpha} and PKC{theta} can localize to the immunological synapse
In Th cells, PKC{theta} has been shown to be the only isoform that localizes to the site of contact with an APC [16 ]. PKC{theta} has also been shown to localize to the contact site with a target cell in CTLs [13 14 15 ]. However, whether PKC{theta} is the only isoform to do so in CTLs has, to our knowledge, not been examined. We used immunocytochemistry to probe the localization of PKC{alpha} and PKC{theta} in TALL-104 cells contacting Raji B lymphocytes, which were coated with a CD3-containing bispecific antibody (bsAB) to trigger TCR-mediated killing (Fig. 4 ). We have shown previously that bsAB trigger reorientation and target-directed exocytosis of lytic granules in TALL-104 cells [28 , 29 ]. We used commercial, isotype-specific FAb to pre-label the isoform-specific anti-PKC antibodies, enabling us to localize both PKC isoforms simultaneously. We found that in ~20% of cells in which PKC{theta} was enriched at the contact site, PKC{alpha} was too. In the other cases, only PKC{theta} was enriched. We did not observe cases in which PKC{alpha} was enriched at the contact, and PKC{theta} was not. These results indicate that localization to the immunological synapse is not exclusive to PKC{theta} in TALL-104 cells, as PKC{alpha} can, in some cases, be found there as well.

Constitutively active mutants of PKC{alpha} or PKC{theta} can substitute for PMA in promoting lytic granule exocytosis
As a final means of probing the involvement of PKCs in lytic granule exocytosis, we examined whether expression of constitutively active, mutant PKCs [5 , 33 ] can substitute for PMA in promoting granule exocytosis. To do this, we exploited a relatively new assay of lytic granule exocytosis, which detects transfer of LAMP-1 from lytic granules, where it normally resides to the plasma membrane as a consequence of exocytosis [38 39 40 41 ]. In this method, as we apply it, cells are stimulated in the presence of an anti-LAMP mAb, which labels any LAMP exposed to the extracellular solution via exocytosis [30 ]. We have shown previously that LAMP-1 externalization can be used to monitor lytic granule exocytosis in TALL-104 cells using confocal microscopy or flow cytometry and suspected that using flow cytometry, this method could be modified to monitor specifically the exocytic responses of viable, transfected cells. Furthermore, we reasoned that this method might make it possible to determine the effects of specific levels of expression—something that is not possible using existing techniques and might be important in revealing effects that are only apparent in cells with high levels of expression.

To test the utility of this method (Fig. 5 ), we transfected cells with GFP and monitored LAMP-1 staining using flow cytometry under three conditions: no stimulation, stimulation with TG alone, and stimulation with TG + PMA. In all cases, the duration of incubation was 50 min, the same as for the BLT-esterase assays shown in Figure 2A . The distribution of GFP fluorescence intensity for cells selected for viability based on forward- and side-scatter (FSC and SSC, respectively) is shown (see Fig. 5A ). The bars marked in Figure 5A indicate GFP-negative cells (1) and cells with increasing levels of expression (2–5). These bars denote gates that were then used to assess LAMP staining of cells with different levels of GFP expression. Figure 5B shows histograms of anti-LAMP-1 staining intensity for cells from the different gating regions shown in Figure 5A , arranged in columns corresponding to increasing GFP fluorescence intensity from left to right. For GFP-negative cells (Column 1), stimulation with TG and PMA (bottom row) caused a large increase in anti-LAMP-1 fluorescence compared with unstimulated cells (top row), consistent with our previous results. There was no effect of increasing levels of GFP expression on the exocytic responses of stimulated cells (compare histograms in Columns 1–5 of the bottom row). Stimulation with TG alone (middle row) caused a smaller increase in anti-LAMP-1 fluorescence than stimulation with TG + PMA, consistent with the idea that PMA is required for robust granule exocytosis. Again, as GFP fluorescence increased, there was essentially no effect on anti-LAMP-1 staining under any of the stimulation conditions (compare histograms in the middle row of Columns 1–5). Figure 5C quantitates the results of this experiment, along with two others, plotting LAMP-1 staining intensity versus GFP intensity. Unstimulated cells are represented by circles, cells stimulated with TG are represented by squares, and cells stimulated with TG + PMA are represented by triangles. These results indicate that the exocytic responses of GFP-expressing cells can be monitored using LAMP-1 exposure, that responses of cells with different levels of expression can be examined specifically, and that expressing GFP alone does not alter responses.


Figure 5
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  		Figure 5. LAMP externalization can be used to monitor the exocytic responses of transfected cells. (A) Histogram of GFP fluorescence for cells transiently transfected with GFP. The bars indicate gating regions that were used to analyze LAMP fluorescence in the panels below. (B) Histograms of anti-LAMP fluorescence for cells, from the experiment shown in A, left unstimulated (top row), stimulated with TG alone (middle row), or stimulated with TG + PMA (bottom row). Histograms are arranged in columns that correspond to the gating regions shown in A. (C) Quantitation of results from three such experiments, including the one shown in A and B. The geometric mean (g.m.) of LAMP staining intensity is plotted versus the g.m. of GFP fluorescence intensity. •, Results from unstimulated cells; {blacksquare}, cells stimulated with TG alone; {blacktriangleup}, cells stimulated with TG + PMA. Error bars are standard deviations.

 
As a prerequisite to ultimately testing functional effects of constitutively active mutant PKCs, we sought to examine the levels of expression that we could attain and to determine whether PKC mutants expressed fused to GFP were associated with the membrane (Fig. 6 ). We reasoned that membrane localization would be consistent with the idea that they have catalytic activity. To examine expression levels, we transfected cells with GFP alone or with mutant PKC-GFP fusion proteins and then fixed them and stained them with PKC isoform-specific mAb, which we prelabeled with PE-conjugated, high-affinity, isotype-specific FAb fragments. Figure 6A shows results obtained for cells transfected with mutant PKC{alpha}, and Figure 6B shows results from cells tranfected with mutant PKC{theta}. In both cases, there was no increase in the PE fluorescence of cells transfected with GFP alone, but cells transfected with mutant PKC-GFP fusion proteins showed clear increases in PE fluorescence. Thus, cells transfected with mutant PKC-GFP fusion proteins, which express higher levels of GFP, express more PKC, as expected. These data can, after subtraction of nonspecific staining (determined from cells stained with Fab only, not shown), be normalized to the endogenous levels of expression, allowing the relative overexpression of the PKC mutants to be estimated. At the lowest level of mutant PKC expression at which significant functional effects are seen (corresponding to Gating Region 4 in Fig. 7 ), cells express approximately fourfold more PKC{alpha} and ~25-fold more PKC{theta} than untransfected or GFP-transfected controls. At the highest levels of expression (comparable with Gating Region 5 in Fig. 7 ), we estimate that PKC mutant-transfected cells express approximately tenfold more PKC{alpha} or ~70-fold more PKC{theta}. Of course, at any given level of GFP expression, the total amount of either mutant expressed is similar. Note that one major caveat with regard to these results is that it is not clear how the catalytic activity of the mutants compares with the catalytic activity of the stimulated wild-type PKCs or to each other.


Figure 7
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  		Figure 7. Constitutively active, mutant PKC{alpha} or PKC{theta} can synergize with an increase in [Ca2+]i to promote lytic granule exocytosis. (A and D) Histograms from one representative experiment each of GFP fluorescence for cells transfected with mutant PKC{alpha}-GFP (A) or mutant PKC{theta}-GFP. The bars represent gates that were used to analyze LAMP-PE fluorescence in subsequent panels. PKC alpha A/E, mutant PKC{alpha}R22A/A25E; PKC theta A/E, mutant PKC{theta}A148E. (B and E) Representative histograms of anti-LAMP fluorescence for cells transfected with mutant PKC{alpha}-GFP (B) or PKC{theta}-GFP (E) from the experiments shown above. Histograms are arranged in columns numbered corresponding to the bars representing gating regions in the histograms above. Unstimulated cells are shown in the top rows, cells stimulated with TG in the middle rows, and cells stimulating the TG + PMA in the bottom rows. (C and F) Plots of the g.m. of anti-LAMP fluorescence versus the g.m. of GFP fluorescence for mutant PKC{alpha}-GFP (C) and mutant PKC{theta}-GFP (F). •, Results from unstimulated cells; {blacksquare}, cells stimulated with TG alone; {blacktriangleup}, cells stimulated with TG + PMA. *, Values that differ at the P < 0.05 level from GFP-negative controls. Error bars are standard deviations. Compare this with Figure 5C .

 
Figure 6C and 6D , shows confocal micrographs of cells transfected with mutant PKC-GFP fusion proteins. For both mutants, GFP fluorescence was apparently associated with the plasma membrane, as would be expected if the mutant PKC were active. Note that in the case of PKC{theta}, there was also clear, punctuate, intracellular localization. The nature of these structures is unclear.

To examine the ability of constitutively active, mutant PKCs to support lytic granule exocytosis, we transfected cells with mutant PKC{alpha}-GFP or mutant PKC{theta}-GFP and examined responses in unstimulated cells and in cells stimulated with TG or with TG + PMA (Fig. 7 ). Figure 7A and 7D , shows typical profiles of GFP intensities for mutant PKC{alpha}-GFP- and PKC{theta}-GFP-expressing cells, respectively. Figure 7B and 7E , shows histograms of anti-LAMP staining intensity for cells transfected with mutant PKC{alpha}-GFP and PKC{theta}-GFP, arranged in columns corresponding to the gating regions shown by bars in Figure 7A or 7D . As was the case for GFP alone, there was no increase in anti-LAMP staining with higher expression levels of either mutant in unstimulated cells (Fig. 7B and 7E , top rows). In contrast, an enhancement of LAMP-1 staining was observed in cells stimulated with TG, which expressed either PKC mutant at high levels (Fig. 7B and 7E , middle rows, Columns 4 and 5 on the right). In cells stimulated with TG and PMA, an enhancement of exocytosis was also detected at the highest expression level. To analyze these results further, we computed the g.m. of LAMP-PE fluorescence and plotted it versus the g.m. of GFP fluorescence. (Analyzing the data this way offers one advantage over simply analyzing the percentage of cells that respond: It takes into account any changes in the amplitude of responses.) Pooled results from three experiments for each mutant are quantitated in Figure 7C and 7F . Circles represent unstimulated cells, squares represent cells stimulated with TG, and triangles represent cells stimulated with TG and PMA. The asterisks indicate levels of LAMP-1 staining that differ at the P < 0.05 level from GFP-negative controls. These results indicate that expression of constitutively active mutants of PKC{alpha} or PKC{theta} can substitute for PMA in synergizing with TG to promote lytic granule exocytosis. PKC{alpha} is apparently more effective than PKC{theta}. To confirm that these results are not due to toxicity caused by expression of the mutant PKCs, we nucleofected cells with GFP, mutant PKC{alpha}-GFP, or mutant PKC{theta}-GFP, waited 7 h, and then stimulated them for 50 min with TG in normal extracellular solution. We then assessed cell viability using propidium iodide (PI) staining, after gating cells on FSC and SSC, as we did for measurement of exocytosis (data not shown). The majority of cells was PI-negative, regardless of whether they expressed GFP or mutant PKCs fused to GFP. There was no increase in the percentage of PI-positive cells at higher levels of expression, where effects of expressing mutant PKCs were observed.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our results provide strong support for the idea that activation of PKC is critical for the actual exocytosis of lytic granules, as opposed to upstream steps involved in the lytic interaction, as we find that the general PKC inhibitor Ro blocks granule exocytosis stimulated by solid-phase anti-CD3 and by TG + PMA (Fig. 2) . The latter result is especially important, as stimulation with TG + PMA is unlikely to trigger formation of an immunological synapse or reorientation of lytic granules, the Golgi apparatus or the MTOC. Note that our results do not rule out a role for PKC in those upstream processes, and Nesic et al. [42 ] have previously provided evidence that PKC is also important for them.

Additional experimental confirmation of an important role of PKC in mediating the effects of PMA in lytic granule exocytosis comes from the experiments shown in Figure 7 , demonstrating that mutant, constitutively active PKC{alpha} or PKC{theta} can substitute for PMA in promoting lytic granule exocytosis when [Ca2+]i is elevated with TG. It is important that these results do not agree completely with the one previous paper that has examined this issue in CTLs. Pardo et al. [24 ] reported that expression of mutant, constitutively active PKC{alpha}, but not PKC{theta}, could synergize with ionomycin to promote lytic granule exocytosis, and overexpression of constitutively active PKC{theta} promoted FasL production. One possible explanation for this discrepancy is that PKC{theta} may, in their experiments, have been expressed that were sufficient at fairly low levels to support FasL production, but not lytic granule exocytosis. The method they used—they contransfected cells with PKC mutants and truncated CD2 and then used magnetic isolation to select the transfected cells—does not allow the level of expression of the mutant PKC to be assessed. Our results, obtained, it must be noted, in a leukemic cell line, demonstrate that at similarly high absolute levels of expression, either mutant is able to produce functional effects. Relative to endogenous levels, however, more PKC{theta} was required, consistent with the idea that PKC{alpha} may be more effective. However, there are a number of potential caveats to the interpretation of these results. First, it is not clear that both mutants have similar catalytic activity. Second, it appears as though some PKC{theta} but not PKC{alpha} is associated with intracellular structures rather than the plasma membrane. Our data indicate that stimulation causes both PKC isoforms to translocate primarily to the plasma membrane. Note that the ability of mutant PKCs to substitute for PMA in driving granule exocytosis (ref. [24 ] and the present study) indicates that non-PKC targets of PMA such as RasGRP, are not critical for the degranulation step of lytic granule exocytosis or that high levels of PKC activity can substitute for their effects.

The results shown in Figure 3 provide clear evidence that stimulation triggers translocation of PKC{alpha} and PKC{theta} to the plasma membrane. In the case of PKC{theta}, translocation could be triggered by PMA alone, without the need of an increase in [Ca2+]i, as expected on the basis of the Ca2+ insensitivity of nPKCs. Regulation of PKCs is complex, but it is thought that membrane binding is sufficient to activate PKC, provided that three critical sites are phosphorylated. It is currently thought that these sites are phosphorylated in mature PKCs, which reside in the cytosol and are capable of translocation to the plasma membrane [8 ]. Assuming that this is correct, our results indicate that PMA activates PKC{theta} and thus, stands in contrast to the results of Puente et al. [18 ], who used cell fractionation to isolate plasma membranes and then probed them for PKC{theta} using Western blotting and also performed kinase assays with immunoprecipitated PKC{theta} isolated from the fractions. They found that neither soluble CD3 nor PMA triggered binding of PKC{theta} to the membrane or increased its activity in kinase assays, but solid-phase anti-CD3 antibodies did. We cannot account for the discrepancy between their results and ours, except to note that kinase assays with PKC can be technically difficult as a result of the need to provide exogenous lipids [43 ]. Puente et al. [24 ] do not mention having done so. Note that taken together with our previous work [30 ], our results indicate that translocation of PKC{alpha} and PKC{theta} precedes lytic granule exocytosis, which occurs with a lag of ~5 min following stimulation with TG and PMA.

The results of Figure 2B are also consistent with the idea that stimulation with TG + PMA activates PKC in CTLs, as enhanced immunoreactivity of an antibody expected to react with the phosphorylated PKC substrates was observed in a Ro-dependent manner in cells stimulated with TG + PMA. However, neither Go nor rottlerin, both of which blocked granule exocytosis, significantly inhibited substrate phosphorylation as assessed with this method, a result that is particularly surprising in the case of rottlerin, which blocked exocytosis completely. One possible explanation for this result is that the antibody does not recognize substrates that are phosphorylated by nPKCs, as the sequence against which the antibody was raised is the cPKC consensus substrate phosphorylation site [37 ]. However, Thuille et al. [44 ] showed that this antibody (Gottfried Baier, personal communication) detects increases in substrate phosphorylation when Jurkat T cells are transfected with constitutively active, catalytically competent, mutant PKC{theta}. There is in fact currently controversy in the literature about the effects of rottlerin, which was identified originally specifically as a PKC{delta} inhibitor [34 ] rather than a nPKC inhibitor, as the title of the paper might suggest. Davies et al. [45 ] assessed the specificity of a number of protein kinase inhibitors and reported that rottlerin did not block PKC{delta} or PKC{alpha} but did inhibit other kinases, such as p38-regulated/activated kinase and MAPK-activated protein kinase 2. Recent work suggests that rottlerin inhibits mitochondrial energy production in pancreatic acinar cells and that this accounts for rottlerin’s inhibitory effects on secretion [46 ]. The best evidence that rottlerin blocks PKC{theta} was provided by Villalba et al. [6 ], who showed that rottlerin inhibited recombinant PKC{theta} assayed in vitro. However, inspection of their data indicates that half-maximal inhibition occurred at a rottlerin concentration of ~100 µM, far higher than we used or others typically use. Thus, although rottlerin blocked granule exocytosis completely (Fig. 2) , we do not feel it is appropriate to make strong conclusions based on the effect. Rottlerin could also inhibit granule exocytosis by blocking one of rottlerin’s alternate targets or by depleting cellular energy stores. The incomplete block of exocytosis by Go provides the strongest pharmacological evidence we have that supports the involvement of a nPKC such as PKC{theta}.

In Th cells, demonstrating a selective role for PKC{theta} in several key functions appears to have been straightforward. Werlen et al. [4 ] found that constitutively active PKC{theta} but not PKC{alpha} or PKC{epsilon} could drive JNK activation and transcription of IL-2 reporter constructs, but all three mutants could support ERK activation. In their study, Go was used to assess the contribution of cPKCs and was shown to have minimal effect on NF-AT activation triggered by PMA and constitutively active mutant calcineurin. Ghaffari-Tabrizi et al. [5 ] showed that mutant PKC{theta} but not PKC{alpha} could support JNK activation and IL-2 promoter activation, while dominant-negative mutants of PKC{theta} or PKC{alpha} could suppress JNK activation. Villalba et al. [6 ] showed that constitutively active PKC{theta} could preferentially drive FasL production and reported that rottlerin, which as described above and used as a specific PKC{theta} inhibitor, inhibited FasL production. Villunger et al. [7 ] also showed that constitutively active PKC{theta} could preferentially drive FasL production and apoptotic cell death. These results, taken together with the fact that only PKC{theta} translocates to the immunological synapse [31 ], make a compelling case that PKC{theta} has a preferential role.

The situation in CTLs has received far less attention. Ours is only the third study of which we are aware that performs experiments explicitly testing the possibility that there is a preferential role for PKC{theta} in lytic granule exocytosis (the other two, as described above, were by Pardo et al. [24 ] and Puente et al [26 ]). None has yielded results that support the idea, despite the fact that methods and reagents that were used were similar to those used successfully in demonstrating a preferential role for PKC{theta} in Th cell functions. At this point, it appears likely that PKC{theta} does not have a preferential role in CTL lytic granule exocytosis and that instead, multiple PKC isoforms can contribute.


   ACKNOWLEDGEMENTS
 
This work was supported by National Institutes of Health Grant R01 AI054839 to A. Z. The authors acknowledge the help of Dr. Karen Helm and the University of Colorado Cancer Center Flow Cytometry Core, Mr. Steven Fadul and the UCHSC Light Microscopy Core Facility, and Dr. Carol A. Norris and the University of Connecticut Flow Cytometry and Confocal Microscopy Facility. We also thank Dr. Gottfried Baier of the University of Innsbruck for providing mutant and wild-type PKC{theta} and for his personal communication regarding the source of the antibody used in Thuille et al. [44 ] and Dr. Mary Reyland of UCHSC for providing mutant and wild-type PKC{alpha}.

Received February 21, 2006; revised September 7, 2006; accepted October 11, 2006.


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 DISCUSSION
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