Differential Downstream Functions of Protein Kinase Cη and -θ in EL4 Mouse Thymoma Cells*

Sensitive EL4 mouse thymoma cells (s-EL4) respond to phorbol esters with growth inhibition, adherence to substrate, and production of cytokines including interleukin 2. Since these cells express several of the phorbol ester-sensitive protein kinase C (PKC) isozymes, the function of each isozyme remains unclear. Previous studies demonstrated that s-EL4 cells expressed substantially more PKCη and PKCθ than did EL4 cells resistant to phorbol esters (r-EL4). To examine potential roles for PKCη and PKCθ in EL4 cells, wild type and constitutively active versions of the isozymes were transiently expressed using a Sindbis virus system. Expression of constitutively active PKCη, but not PKCθ, in s- and r-EL4 cells altered cell morphology and cytoskeletal structure in a manner similar to that of phorbol ester treatment, suggesting a role for PKCη in cytoskeletal organization. Prolonged treatment of s-EL4 cells with phorbol esters results in inhibition of cell cycling along with a decreased expression of most of the PKC isozymes, including PKCθ. Introduction of virally expressed PKCθ, but not PKCη, overcame the inhibitory effects of the prolonged phorbol ester treatment on cell cycle progression, suggesting a possible involvement of PKCθ in cell cycle regulation. These results support differential functions for PKCη and PKCθ in T cell activation.

Protein kinase C (PKC), 1 a family of phospholipid-dependent serine/threonine-specific kinases, has been implicated in numerous signaling pathways in lymphocytes and other cells (reviewed in Refs. [1][2][3]. At least 12 isozymes are recognized as members of the PKC family, and although all of these proteins share some structural similarities and rely on phospholipids for activation, many differences exist among them (reviewed in Refs. [2][3][4]. The isozymes exhibit diverse tissue distribution, subcellular localization, and requirements for diacylglycerol (DAG) and Ca 2ϩ as cofactors (reviewed in Refs. [2][3][4]. The conventional isozymes, PKC␣, -␤I, -␤II, and -␥, require both Ca 2ϩ and DAG; novel isozymes PKC␦, -⑀, -, and -are Ca 2ϩindependent; atypical isozymes PKC and -are Ca 2ϩ -independent and DAG-or phorbol ester-resistant. These differences argue for distinct functions of the isozymes. Altered expression or activity of an individual PKC isozyme can lead to specific changes in biological function. In the human Jurkat T lymphocyte line, antisense constructs (5), PKC downregulation (6), and co-expression with AP1 and nuclear factor of activated T cell transcription element reporter constructs (7) implicate PKC␣ in interleukin 2 (IL2) production. Microinjection of isozyme-specific antibodies has implicated a PKC␤ isozyme in down-regulation of elevated intracellular Ca 2ϩ in these cells (8). Ca 2ϩ -independent PKC isozymes also have been implicated in activation of Jurkat cells. Genot et al. (7) showed that PKC⑀, but not PKC␣, could induce expression of an NFkB reporter construct as well as expression of AP1 and nuclear factor of activated T cell transcription element reporter constructs, which PKC␣ also induced. However, different PKC isozymes have been implicated in some of these functions in other lymphocyte systems. Reasons for conflicting results may include cell-specific differences, redundancy in isozyme function, or incomplete inhibition, down-regulation, or activation of specific isozymes with the various reagents or methods used.
To investigate roles for individual PKC isozymes in various T cell functions, we have compared phorbol ester-sensitive (s) and -resistant (r) lines of EL4 mouse thymoma cells. s-EL4 cells, unlike r-EL4 cells, produce cytokines, adhere to plastic substrates, and become growth-inhibited when stimulated with phorbol esters (9,10). An explanation for these differences may be a divergence in the PKC expression profile of the two cell lines. Northern and Western analysis revealed that s-EL4 and r-EL4 cell lines expressed comparable amounts of PKC␣, -␤, and -␦ but that the r-EL4 cells produced substantially less PKC⑀ (11), PKC, and PKC (12). Long term phorbol ester treatment of s-EL4 cells resulted in the down-regulation of all of the PKC isozymes examined except for PKC, which exhibited a 5-fold increase in expression in comparison with control cells (12). These observations suggest that PKC⑀, PKC, and/or PKC may contribute to the phorbol ester-induced responses in s-EL4 cells. In support of a role for PKC, Baier et al. (13) noted that the overexpression of PKC in s-EL4 cells resulted in an increase in transcription of an IL2 reporter construct when cells were treated with phorbol ester. That group also showed that expression of a constitutively active PKC construct activated an AP1 reporter construct and that expression of a dom-inant negative PKC construct blocked it (14). Consistent with a role for this isozyme in T cell activation, Monks et al. (15) observed that antigen stimulation of T cell clones led to the selective activation and translocation of PKC concomitant with proliferation of the T cells, and similar induction of IL2and c-jun reporter constructs with expression of PKC was reported recently in Jurkat cells (16).
To elucidate further potential functions of PKC and the uniquely up-regulated PKC in EL4 cell activation, a virusbased transient expression system was used to introduce these PKC isozymes into the cells. Expression of constitutively active PKC in EL4 cells resulted in dramatic changes in the cell morphology as well as cytoskeletal organization that were similar to those observed in s-EL4 cells stimulated with phorbol ester. In contrast, expression of constitutively active PKC, but not PKC, counteracted the inhibitory effects of prolonged phorbol ester treatment on cell cycle progression. Taken together, these results suggest that PKC and PKC play distinct roles in cellular signaling, with PKC involved in cytoskeletal organization and PKC implicated in cell cycle progression.

EXPERIMENTAL PROCEDURES
Materials-DMEM, RPMI 1640, phosphate-buffered saline (PBS), trypsin/EDTA, and other tissue culture materials were purchased from Mediatech Inc. (Herndon, VA). Heat-inactivated fetal bovine serum was obtained from either Sigma or Summit Biotechnology (Fort Collins, CO). Penicillin/streptomycin was acquired from Life Technologies, Inc. Phorbol 12,13-dibutyrate (PDB) was obtained from Sigma. Restriction enzymes and modifying enzymes were purchased from New England Biolabs (Beverly, MA) and Life Technologies, Inc., and used essentially according to manufacturers' specifications. Rabbit polyclonal antibodies directed against the carboxyl termini of PKC isozymes were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal antibodies against Sindbis virus envelope glycoproteins were a generous gift from Dr. C. Rice (Washington University, St. Louis, MO) (17). Murine PKC and PKC cDNA were generous gifts from Dr. H. Mischak (Institute fur Klinische Molekularbiologie and Tumorgenetik, Munich, Germany). Horseradish peroxidase-conjugated secondary antibodies were purchased from Bio-Rad and Sigma. The enhanced chemiluminescence detection system (ECL) was purchased from Amersham Pharmacia Biotech.
Cell Culture-Baby hamster kidney-21 clone 13 cells (BHK) were obtained from American Type Culture Collection (ATCC, Rockville, MD) and maintained in DMEM supplemented with 10% fetal bovine serum, 10 g/ml penicillin, 10 g/ml streptomycin, and 2 mM glutamine and used between passages 6 and 14 after acquisition. s-and r-EL4 cells also were obtained from ATCC and maintained in RPMI 1640 supplemented with 10% fetal bovine serum, 10 g/ml penicillin, 10 g/ml streptomycin, and 2 mM glutamine. L929 fibroblasts were obtained from ATCC and were maintained in DMEM supplemented with 10% fetal bovine serum, 10 g/ml penicillin, and 10 g/ml streptomycin and were used between passages 8 and 17.
Generation of Sindbis Recombinants-The generation of double subgenomic Sindbis recombinants (dsSIN) capable of expressing either PKC or PKC was accomplished by excising cDNAs encoding PKC and PKC from plasmid vectors kindly provided by H. Mishack and then subcloning these cDNAs into the Sindbis plasmid pTE2JC1 (18). Fidelity of cloning was examined by analysis of restriction enzyme digestion. Those pTE2JC1 plasmids with the appropriate PKC or PKC inserts were amplified in Escherichia coli. pTE2JC1:CAT (chloramphenicol acetyltransferase) was described previously (18). Purified pTE2JC1:CAT, pTE2JC1:PKC, and pTE2JC1:PKC, linearized with the XhoI restriction enzyme, were employed as templates for in vitro transcription using SP6 RNA polymerase as described previously (18). 4 ϫ 10 6 BHK cells (10 7 /ml) were transfected with 5-10 g of the RNA transcripts by square pulse electroporation using a BTX820 square pulse generator (BTX Inc., San Diego) at 680 V for 99 s (5 pulses with 1-s interval between pulses). Approximately 24 h post-transfection, the medium was collected and assayed for infectious virus titer in L929 cells. The resulting recombinant viruses were called dsSIN:CAT, dsS-IN:PKC, and dsSIN:PKC for their ability to express CAT, PKC, and PKC, respectively.
The constitutively active clones of PKC and PKC were made by site-directed mutagenesis of their pseudosubstrate regions. Alanine 161 in PKC was replaced by a glutamate using oligonucleotides (5Ј-GC-CAAAGGGAGATGCGAAG-3Ј and 5Ј-CTTCGCATCTCCCTTTGGC-3Ј), and alanine 148 in PKC was replaced by glutamate using oligonucleotides (5Ј-GCCGAGGAGAGATCAAACA-3Ј and 5Ј-TGTTTGATCTCTC-CTCGGC-3Ј). The mutant clones of PKC and PKC were inserted into the pTE2JC1 plasmid, and recombinant viruses capable of expressing constitutively active PKC (dsSIN:PKC CA ) and constitutively active PKC (dsSIN:PKC CA ) were generated as described above. The kinasedead PKC mutant (dsSIN:PKC KD ) was generated by replacing lysine 384 with an alanine in the background of the A161E mutant. The oligonucleotides used for this mutation were 5Ј-ACGCCGTGGCCGT-GCTGAAGA-3Ј and 5Ј-TCAGCACGGCCACGGCGTAC-3Ј. Catalytic domains of PKC and PKC were amplified by polymerase chain reaction using 5Ј-CATATGTCTAGAACTCTCCTAGCAG-3Ј and 5Ј-CATATGTCTAGAATGCGCAGGACTTC-3Ј, respectively, as 5Ј primers and the SP6 primer in the vector as the 3Ј primer. Resulting DNA fragments were separated and cloned into pTE plasmid, and recombinant viruses capable of expressing the catalytic domains of PKC (dsSIN:PKC CD ) or PKC (dsSIN:PKC CD ) were generated as described above. PKC CD starts with engineered Met as an initiation codon and is followed by Asn-345 to the carboxyl terminus of the protein (residue 684). PKC CD starts with Met-355 as an initiation Met and continues to the end of the protein (residue 708). Appropriate protein expression was confirmed by detection of labeled polypeptides following in vitro transcription followed by translation in rabbit reticulocyte lysate and in the BHK cells by immunoblotting.
At least two clones were isolated from each independent plasmid construct. All experiments involving virus manipulation and handling were performed in a BL2 facility under the protocol approved by the Institutional Biosafety and Recombinant DNA Committee of the University of Virginia.
Infection of Cells with dsSIN Recombinants-Cells in monolayer or suspension culture were maintained in late logarithmic phase for infection with dsSIN recombinants. Medium was removed and viruses were added to a designated multiplicity of infection (m.o.i.) in PBS (with 2 mM CaCl 2 and 2 mM MgCl 2 ) or RPMI. 1 h later, the appropriate prewarmed medium was added to the cells which were then incubated at 37°C for the times indicated. For each experiment, two controls were used as follows: 1) addition of medium alone (mock infection), and 2) infection with dsSIN:CAT.
CAT Enzyme-linked Immunoadsorbent Assay-Cells infected with dsSIN:CAT were assayed for CAT activity after lysis in PBS containing 0.2% Tween 20 and 1% bovine serum albumin. The lysates were centrifuged, and the supernatants were subjected to a CAT enzyme-linked immunoadsorbent assay using the protocol provided by the manufacturer (5 Prime 3 3 Prime Inc., Boulder, CO) (21). All assays were done in duplicate and repeated at least three times.
F-actin Staining-Cells were plated onto glass coverslips and infected with the appropriate viruses as described above. At specific times post-infection, cells were fixed by incubating them in PBS containing 4% formaldehyde for 20 min and then washed with PBS. Permeabilization was accomplished by incubating the cells for 30 min in washing solution (PBS, 1% bovine serum, 0.025% Nonidet P-40 and 0.02% sodium azide). Cells were incubated for 30 min in washing solution containing 100 nM fluorescein isothiocyanate (FITC)-conjugated phalloidin (Sigma). Excess fluorescein was removed by washing the cells at least twice with the washing solution. The coverslips were mounted onto glass slides, and the cells were visualized by both phase contrast and fluorescent microscopy (at 515 nm) using a 40ϫ Planarphor objective lens.
Cell Cycle Analysis-Logarithmic phase s-EL4 cells were treated with 100 nM PDB or 0.01% ethanol vehicle and then incubated for 6, 12, or 18 h at 37°C. Cells were then permeabilized in an isotonic solution, and the DNA was stained using propidium iodide (50 g/ml in 0.3% Nonidet P-40, 100 g/ml boiled RNase A, and 0.1% sodium citrate). Stained cells were incubated at 4°C for at least 30 min. DNA content of the cells was examined using fluorescence activated cell sorting (FACS) at the FL2 channel wavelength on a FACScan instrument (Becton Dickinson), and results were analyzed by the CellQuest program (Becton Dickinson). i. was used to ensure that most of the cells were infected with multiple virus particles. Western analysis showed that both cell lines infected with dsSIN:PKC expressed significantly more PKC than did mock-or dsSIN:CAT-infected control cells (Fig. 1). Analysis of CAT activity after infection of s-EL4 and r-EL4 with dsSIN:CAT confirmed comparable expression in the two cell lines as did expression of viral envelope glycoproteins (17) (data not shown).

Expression of Viral PKC and PKC in EL4 Cells
In addition to enhanced expression of an approximately 80-kDa band in the dsSIN:PKC cells, prominent 45-50 kDa antibody-reactive bands were observed (Fig. 1). These smaller -reactive species may represent catalytic fragments. Overexpression of a similar 80-kDa immunoreactive band was detected in s-EL4 cells infected with dsSIN:PKC (Fig. 2B). When blots were overexposed, lower molecular weight bands were observed in cells overexpressing PKC as well. Similar results were observed in r-EL4 cells (data not shown).
To eliminate the need for stimulation of the cells with phorbol ester, recombinant Sindbis viruses capable of expressing constitutively active PKC (dsSIN:PKC CA ) and PKC (dsSIN: PKC CA ) were generated by introducing a point mutation in the pseudosubstrate region of each isozyme (7) as described under "Experimental Procedures." Overexpression of both PKC and constitutively active PKC was detected as early as 3 h post-infection in s-EL4 cells ( Fig. 2A), and the time course of expression in r-EL4 cells was nearly identical (data not shown). The degradation of constitutively active PKC protein was much faster when compared with that of PKC ( Fig. 2A). Similar results were observed for PKC (Fig. 2B).
Catalytic domain constructs of PKC (PKC CD ) and PKC (PKC CD ) also were expressed from infection of EL4 cells with Sindbis virus capable of expressing those active fragments. Good expression of 43-kDa PKC CD and 38-kDa PKC CD was achieved within 3.5 h of infection. The slight discrepancy between the calculated molecular masses of these fragments (39 and 40 kDa, respectively) and the size estimated from mobility in SDS-PAGE are consistent with the slight discrepancy in migration of the intact isozymes. These catalytic domain fragments, especially PKC CD , also underwent some degradation over the ensuing 7 h (Fig. 2, C and D). Similar results were obtained when these constructs were expressed in reticulocyte lysates in vitro as well as in BHK cells and in r-EL4 cells (data not shown).
Effect of PKC on the Cytoskeleton-To address whether PKC is involved in the regulation of cell morphology or adherence, logarithmic phase s-EL4 and r-EL4 cells were either mock-infected or infected with dsSIN:CAT, dsSIN:PKC CA, ds-SIN:PKC CD , dsSIN:PKC CA , or dsSIN:PKC CD for 5 h and then fixed onto glass coverslips using a solution of 4% formaldehyde in PBS. Fixed cells were permeabilized and stained with FITC-conjugated phalloidin that binds to F-actin. Inspection of EL4 cells by phase contrast and fluorescence microscopy revealed that phorbol ester stimulation or expression of constitutively active or catalytic domain PKC induced cytoskeletal changes. dsSIN:CAT-infected s-EL4 cells (Fig. 3, A and B), essentially identical to mock-infected cells (data not shown), were refractory indicating a rounded morphology. PDB treat- ment (Fig. 3, C and D) and overexpression of constitutively active PKC (Fig. 3, E and F) resulted in a flatter morphology, as shown by a decrease in refraction and the formation of ruffles at the membrane surface. Fluorescence microscopy confirmed that these new cellular structures contained a high concentration of F-actin (Fig. 3, D and F). These cytoskeletal changes were seen within 2 h of viral infection (data not shown). Infection with dsSIN:PKC CD caused a distinct morphological change in which the cells produced one or two long processes (Fig. 3, G and H) instead of large areas of membrane ruffling. In contrast, infection with dsSIN:PKC CD (Fig. 3, I and J), dsSIN:PKC CA or dsSIN:PKC KD (data not shown) caused minimal alteration in morphology from that of the CATexpressing cells.
PDB treatment of r-EL4 cells led to an increase in production of filipodia and reorganization of the cytoskeleton as demonstrated by the formation of new F-actin containing structures (Fig. 4, C and D). Infection of these cells with dsSIN:PKC CA (Fig. 4, E and F) caused the appearance of membrane ruffling somewhat like that observed in s-EL4 cells with PDB treatment or the expression of PKC CA. Expression of dsSIN: PKC CD in r-EL4 (Fig. 4, G and H) caused the appearance of one or two prominent actin-containing protrusions as it did in s-EL4 cells. However, expression of these active PKC constructs did not result in adhesion of the s-or r-EL4 cells to plastic (data not shown). Expression of constitutively active PKC (Fig. 4, I and J), the catalytic domain of PKC, or kinase dead PKC in r-EL4 cells did not alter morphology or induce cytoskeletal reorganization (data not shown). (10) with cell cycle blocks in G 1 and G 2 /M (22). This cell cycle inhibition may be due to the down-regulation of the majority of the PKC isozymes (12). Antigen-stimulated T cell clones show selective activation and translocation of PKC concomitant with the proliferation of those T cells (15), suggesting the involvement of PKC in the regulation of cell cycle progression. A potential connection between the activity of PKC and cell cycle progression in s-EL4 cells was examined by analysis of DNA content. Following treatment with 100 nM PDB or 0.01% ethanol vehicle control, s-EL4 cells were collected at 6, 12, and 18 h, permeabilized, and stained with propidium iodide. DNA histograms obtained by FACS analysis revealed a dramatic decrease in cell populations corresponding to S phase (the area between the diploid (2N) and tetraploid (4N) DNA peaks) as early as 6 h after treatment (Fig. 5). The proportion of cells in S phase was only 7% of the total population at 6 h after PDB treatment in comparison with 32% of the ethanol-treated control cells. [ 3 H]Thymidine incorporation experiments also showed greater than a 3-fold decrease in DNA synthesis after 6 h of PDB treatment and 1 h of pulse labeling (data not shown), consistent with earlier studies (10,22).

Involvement of PKC in Cell Cycle Progression-Prolonged treatment of EL4 cells with phorbol esters inhibits cell cycle progression
Potential roles for PKC and PKC in cell cycle progression were examined using a combination of prolonged PDB treatment, which down-regulates the majority of PKC isozyme activities but up-regulates PKC (12) plicity of 20 infectious particles per cell. Two h post-infection, cells were divided into two equal groups, and one group was treated with 100 nM PDB. After 6 h at 37°C, cellular DNA was stained with propidium iodide and then analyzed using FACS. Fig. 7 shows that expression of PKC can overcome the inhibi-tion of cell cycle progression induced by phorbol ester treatment. Cells infected with dsSIN:PKC or dsSIN:PKC and dsSIN:PKC progressed through the cell cycle in the presence of PDB, whereas cells infected with dsSIN:CAT or dsSIN:PKC did not. Similar results were obtained after 12 and 18 h of PDB treatment.
Expression of dsSIN:PKC CA also rescued PDB-treated cells from growth inhibition; however, expression of the catalytic domain of PKC in the cells did not (data not shown). Given the rapid degradation of the expressed catalytic fragment (Fig. 2), a potential function for the PKC catalytic domain in cell cycle progression cannot be ruled out. r-EL4 cells are not growthinhibited by phorbol ester treatment, and expression of PKC or -did not alter the cell cycle profiles (data not shown). DISCUSSION EL4 mouse thymoma cells have been used to help identify steps in phorbol ester-induced lymphocyte responses. s-EL4 cells respond with growth inhibition, adherence to plastic, and production of cytokines including IL2 (9 -14). A resistant line, r-EL4, lacks these responses (9,10), and overexpression of PKC isozymes or constitutively active constructs in sEL4 cells has supported a role for PKC in IL2 production (13,14). Previous studies revealed greatly diminished expression of PKCs -⑀, -, and -in the resistant cells (11,12). In attempt to determine whether PKCs -or -contribute to phorbol ester responses of EL4 thymoma cells, s-EL4 and r-EL4 cells were transiently infected with recombinant Sindbis viruses capable of expressing CAT (dsSIN:CAT), the wild type isozymes (dsSIN:PKC or -), constitutively active isozymes generated by mutation in the pseudosubstrate sites (dsSIN:PKC CA or -CA ), or catalytic domain constructs (dsSIN:PKC CD or -CD ). All recombinants were expressed in all of the cells as determined by Western blot analysis (Figs. 1, 2, and 6).
PKC immunoblots often revealed a doublet at approximately 80 kDa with an increase in dsSIN:PKC and dsSIN: PKC CA predominantly in the lower band. The upper band may represent a nonspecific cross-reacting protein or, given that PKCs are subject to phosphorylation at multiple sites (reviewed in Ref. 3), it may represent a more phosphorylated . Cells were permeabilized in isotonic solution, and their DNA was stained with propidium iodide. The DNA contents were examined by FACS analysis using the FL2 channel for at least 10,000 cells for each sample, and the data were analyzed using a CellQuest analysis program. The 2N peak represents diploid DNA content from cells that are in the G 1 cell cycle phase, and the 4N peak represents tetraploid DNA content from cells that are in G 2 /M. S phase cells are apparent from the signal between the 2N and the 4N peaks. A representative of five independent experiments is shown.
form of PKC, and increased expression of the smaller band in virus-infected cells may suggest incomplete phosphorylation of the virally expressed protein, perhaps because the high expression overwhelms available kinases.
In addition, expression of PKC-reactive bands at ϳ45-50 kDa was detected in the overexpressing cells. In the case of the dsSIN:PKC CA -infected cells, greatly increased expression of the ϳ50-kDa band was observed, and this band seemed to increase at the expense of the 80-kDa bands at long times (24 h) of infection (Fig. 2). Since the antibody is specific for a carboxyl-terminal peptide, it is likely that these bands represent degradation products containing catalytic domains of PKC.
Detection of a 50-kDa fragment is suggestive of accumulation of a catalytically active carboxyl-terminal fragment (PKM). PKCs of the classical type (␣, ␤, and ␥) are more susceptible to degradation by membrane-associated calpains when they are in their active conformation with the hinge region exposed (23,24). However, in EL4 cells the isozymes typically are further degraded (12). It would seem that PKC is not susceptible to this form of degradation since no degradation is observed with long term phorbol ester stimulation of s-EL4, and in fact, the cells up-regulate expression of this isozyme when other isozymes are degraded (12). It is possible that overexpressed PKC is present at sufficient concentration to experience some degradation via calpains. Another possibility is that a less phosphorylated form of the enzyme may be targeted for proteolysis. Finally, PKC␦ (25)(26)(27) and human PKC (28) are susceptible to caspase 3-mediated proteolysis at a DEVD site present in the V3 hinge region of those isozymes. Resultant generation of stable PKC␦ or PKC catalytic fragments in U937 myeloid leukemia cells has been implicated in apoptosis (26,28). PKC lacks a DEVD site in the V3 region so it is not clear whether this member of the Ca 2ϩ -independent PKCs also is susceptible to another form of proteolysis.
PKC immunoblots also showed lower molecular weight forms of overexpressed enzyme (Figs. 2 and 6), especially in the case of the constitutively active construct. Again, these are consistent with the size of catalytically active PKM. Human PKC is susceptible to caspase 3-mediated proteolysis at the V3 hinge region to generate a 42-kDa active fragment (28); however, murine PKC lacks the relevant DEVD site so it is not clear how the fragment is generated in the EL4 cells.
Expression of the constitutively active PKC caused a significant morphological change in both s-and r-EL4 cell lines. s-EL4 cells with dsSIN:PKC CA exhibited a flatter, less refractory appearance with membrane ruffling very similar to that of s-EL4 cells treated with 100 nM PDB for 30 min; and this change was accompanied, in both cases, by enhanced staining for F-actin in the new structures (Fig. 3). Demonstration of these changes as early as 2 h after infection helps argue for direct effects of PKC CA rather than effects mediated by other isozymes whose expression may have been altered. Further support for a specific effect of PKC expression was the failure of PKC (Fig. 3) or kinase-dead PKC (data not shown) to induce this morphological change. Expression of a catalytic domain construct of PKC in s-EL4 cells resulted in a distinct morphology with one or two long actin-containing processes (Fig. 3), suggesting that the intact version of the constitutively active PKC rather than the proteolyzed forms is required for the membrane ruffling. and allowed to incubate an additional 6 h at 37°C. Cells were permeabilized in isotonic solution, and their DNA was stained with propidium iodide. The DNA contents were examined by FACS analysis using the FL2 channel for at least 10,000 cells for each sample, and the data were analyzed using a CellQuest analysis program. A representative of three independent experiments is shown, and the mean Ϯ S.D. of % of cells in S phase is indicated in each panel.
In r-EL4 cells phorbol ester treatment caused production of numerous small filipodia rather than the large membrane ruffles observed in phorbol ester-treated s-EL4 cells (Fig. 4). Overexpression of PKC CA caused membrane ruffling with enhanced F-actin staining in the ruffles similar to that in s-EL4 cells (Fig. 4). This result is consistent with a requirement for PKC for these morphological changes since the control r-EL4 cells lack significant expression of PKC. Expression of the catalytic domain construct of PKC in r-EL4 cells resulted in the appearance of some longer processes as it did in s-EL4 cells. This different morphology of cells expressing catalytic domain PKC versus constitutively active PKC may reflect some difference in subcellular localization and/or substrate recognition of the two PKC constructs. Differences in subcellular localization might be expected since the catalytic domain constructs lack the membrane binding domains, and these domains have been observed to alter substrate specificity for PKC (29).
Alteration of cytoskeletal organization in response to PKC activation is not limited to EL4 cells but is a rather universal response in cells of different lineage and is characterized by changes in F-actin content and formation of new actin structures (30 -33). Cytoskeletal reorganization modulates many cellular functions such as adhesion (34), motility (35), and polarity (36,37). However, the roles of individual isozymes in regulation of the cytoskeleton are not well established. In several adherent cell lines including BHK, chicken embryo fibroblasts, and L929 fibroblasts, as well as in primary rat aortic smooth muscle cells, expression of constitutively active PKC but not constitutively active PKC resulted in fewer stress fibers and more extensions of plasma membrane as well as stronger staining of F-actin near the plasma membrane. 2 Goodnight et al. (38) also observed flattening of NIH3T3 fibroblasts and cytoplasmic blebbing upon phorbol ester treatment of NIH3T3 cells overexpressing PKC. Thus differences in expression of PKC correlate with morphological changes in a variety of cell types.
We have observed association of PKC with particulate fractions of s-EL4 cells in both control and phorbol ester-treated cells 3 as have Sansbury et al. (39). Basu (40) observed similar particulate localization in two breast cancer cell lines. Goodnight et al. (38) noted localization of PKC in a juxtanuclear area consistent with Golgi in untreated cells. Phorbol ester treatment resulted in translocation of a portion of PKC to the outer cell membrane as well as transient punctate nuclear staining potentially consistent with nuclear pores (38). Chida et al. (41) had observed expression of PKC in association with rough endoplasmic reticulum in keratinocytes and overexpressing COS cells, and Grief et al. (42) has reported nuclear staining of human keratinocytes with PKC antibody. Immunofluorescent localization of overexpressed PKC was not pursued here because of the inability to distinguish intact versus proteolyzed PKC with the available antibody.
Regulation of PKC appears to be quite distinct from that of other PKC isozymes. We had observed up-regulation of PKC in response to phorbol ester treatment of s-EL4 cells under conditions where other isozymes were significantly down-regulated (12), and Basu (40) observed similar up-regulation of PKC in breast cancer lines in correlation with protection of the cells against tumor necrosis factor-induced cytotoxicity. Although the functions of PKC in various cells are not yet well elucidated, the data available support distinct regulation of this isozyme and its unique involvement in cellular signaling. Evidence is consistent with a role in morphological changes that may contribute to a variety of functional alterations depending on the cell type.
Although expression of neither wild type nor constitutively active PKC affected morphology or cytoskeletal organization of EL4 cells (Figs. 3 and 4), overexpression of PKC, but not PKC, did have effects on cell cycle progression in EL4 cells (Fig. 7). Treatment of many hematopoietic cells including HL60 (43), s-EL4 (9,10,22), and T cells (44) with phorbol esters results in grown inhibition. A dramatic decrease in the number of s-EL4 cells entering S phase occurs after phorbol ester treatment (Fig. 5). This block was overcome by overexpression of PKC but not PKC (Fig. 7) implying a specific role for PKC in cell cycle progression. Sansbury et al. (39) overexpressed PKC in a different phorbol ester-resistant line of EL4 cells and observed a similar lack of effect on growth of the cells. The conclusion that PKC contributes to cell cycle progression is consistent with the report from Monks et al. (15) that T cell proliferation correlated with PKC translocation to the membrane. Phorbol ester treatment is expected to activate PKC in EL4 cells, but it is possible that other activated isozymes account for the growth inhibition and/or that down-regulation of PKC contributes to the cell cycle block. If PKC is sufficient for inducing cells to transit the cell cycle, one would have to conclude that it is not essential since r-EL4 cells lack significant PKC expression and continue to proliferate in the presence of phorbol ester. Indeed, expression of SIN:PKC in r-EL4 cells failed to exhibit an effect, probably because the cells continue to cycle in the presence of phorbol ester (data not shown). It is possible that other isozymes may substitute for a cell cycle progression function of PKC in those cells or that PKC is important for counteracting the effects of other PKC isozymes that r-EL4 cells also lack. Alternatively, cell cycle progression in r-EL4 cells may be completely independent of PKC activation.
Again, it is interesting that caspase 3-mediated proteolysis of PKC to generate an active catalytic fragment has been implicated in apoptosis in human U937 cells. Although the murine PKC lacks the DEVD site for this proteolysis, it is possible that the PKC fragment that is generated carries out a significant function distinct from that of the intact active isozyme. This, in fact, seems likely since the two forms of the enzyme would be expected to exhibit distinct subcellular localization and thus to encounter different substrates.
In summary, evidence has been presented that PKC and PKC mediate distinct downstream functions in EL4 cells with PKC contributing to actin cytoskeletal reorganization and morphological changes and PKC contributing to cell cycle progression as well as to IL2 production (13,14,16). More detailed analysis of the subcellular localization of these isozymes, their constitutively active forms, and potentially active fragments of the enzymes should help elucidate their respective functions as will identification of downstream substrates.