©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Immunocytochemical Localization of Eight Protein Kinase C Isozymes Overexpressed in NIH 3T3 Fibroblasts
ISOFORM-SPECIFIC ASSOCIATION WITH MICROFILAMENTS, GOLGI, ENDOPLASMIC RETICULUM, AND NUCLEAR AND CELL MEMBRANES (*)

JoAnne Goodnight (1), Harald Mischak (2), Walter Kolch (2), J. Frederic Mushinski (1)(§)

From the (1) Molecular Genetics Section, Laboratory of Genetics, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892-4255 and the (2) Institute for Clinical Molecular Biology and Tumor Genetics, GSF-Forschungszentrum Hämatologikum Marchioninistrasse 25, D-81377 Munich, Federal Republic of Germany

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have used immunocytochemical analyses to characterize the subcellular distribution of protein kinase C (PKC)-, -I, -II, -, -, -, -, and - in NIH 3T3 fibroblasts that overexpress these different PKC isozymes. Immunofluorescence studies and Western blotting with antibodies specific for individual isoforms revealed that before activation the majority of the PKCs are not membrane-bound and are diffusely distributed throughout the cytoplasm. In addition, a fraction of PKC- and - appears membrane-bound and concentrated in the Golgi apparatus. Activation of each isozyme's kinase activity (with the exception of PKC-) by treatment of these cells with the phorbol ester 12- O-tetradecanoylphorbol-13-acetate results in isozyme-specific alterations of cell morphology, as well as in a rapid, selective redistribution of the different PKC isozymes to distinct subcellular structures. Within minutes after 12- O-tetradecanoylphorbol-13-acetate treatment, PKC- and - concentrate at cell margins. In addition, PKC- accumulates in the endoplasmic reticulum, PKC-II associates with actin-rich microfilaments of the cytoskeleton, PKC- accumulates in Golgi organelles, and PKC- associates with nuclear membranes. Our results demonstrate that each activated PKC isozyme specifically associates with a particular cellular structure, presumably containing the substrate for that isozyme. These findings support the hypothesis that PKC substrate specificity in vivo is mediated, at least in part, by the restricted subcellular locale for each PKC isozyme and its target protein.


INTRODUCTION

The PKC()isozymes comprise a family of at least 11 different serine/threonine kinases that have been implicated in a variety of cellular responses including proliferation, differentiation, gene expression, membrane transport, and secretion of hormones and neurotransmitters (1, 2, 3) . The PKC family can be classified into three major groups, namely, classical (cPKC-, -I, -II, and -; Ref. 4), novel (nPKC-, -, -, -, and -µ; Refs. 5-8), and atypical (aPKC- and -, which is the human counterpart of mouse PKC-; Refs. 9-11).

Classical PKCs and novel PKCs translocate from the cytosol to membranous sites upon activation with diacylglycerol (1) . TPA and certain other phorbol esters mimic diacylglycerol in that they can bind and activate all PKCs, except the atypical PKCs, by substituting for endogenous diacylglycerol (12, 13) . It is widely recognized that phorbol esters such as TPA are promoters for skin tumors, and they have pleiotropic and often tissue-specific effects that include alterations in cell morphology (14) , redistribution of the actin cytoskeleton (15) , and loss of fibronectin from the cell surface (16) , as well as the regulation of cellular responses such as proliferation, differentiation, gene expression, and membrane transport (reviewed in Refs. 1-3).

Although individual isozymes demonstrate only subtle differences in enzymatic properties, ligand binding and substrate specificity in vitro (17, 18) , the isoforms exhibit different tissue- and cell-type-specific expression patterns in vivo (1, 19, 20, 21, 22, 23) , suggesting unique, specific functions for each PKC isoform. Determination of specific biological roles for each isozyme and whether these roles are the same in all cell types has been complicated by the large size of this family and the fact that most cell types express only a few of the isozymes. Therefore, only a few biological functions have been defined for particular isoforms. For example, PKC- and -, but not PKC-, -, or -, mediate differentiation of 32D cells (a mouse myeloid cell line) into mature macrophages (24) . Overexpression of PKC- in NIH/3T3 cells leads to oncogenic transformation (25, 26) , whereas PKC- inhibits proliferation (25, 27) . These findings led us and others (28) to hypothesize that the wide spectrum of functions ascribed to PKC in vivo could be achieved from a family of functionally similar enzymes if there were isozyme-specific differences in subcellular localization.

Earlier studies (19, 20) showed that PKCs could be induced to associate with areas of cell-cell contact and cytoskeletal elements, respectively, in different cell types. More recently, isotype-specific antisera were used to show that, in activated mouse T lymphocytes, PKC-II interacts with cytoskeletal elements like ankyrin and spectrin (29) and that each of six PKC isozymes in rat cardiac myocytes had a distinct subcellular localization before and after treatment with TPA or norepinephrine (30) .

We determined to test the theory that even in the same cell different PKC isozymes translocate to different organelles upon activation by phorbol esters. We used isozyme-specific antiserums to study the localization of the different PKC isozymes in clones of NIH 3T3 fibroblasts that overexpressed each of the individual isozymes. The NIH 3T3 system had the advantage of being well characterized by a variety of laboratories. It also enabled us to monitor TPA-induced translocation of all eight isozymes within the same cell line and allowed extensive negative controls, since our clone of wild-type NIH 3T3 cells appears to express only one isozyme, PKC-, in significant amounts (25, 31) . These results could be compared with the published results in cell types that express multiple PKCs to determine whether different cell types show the same relocalization patterns and what the effect of introduction of expression of normally silent PKC genes might have on fibroblast physiology. As predicted, a unique pattern of subcellular localization was found for each PKC isozyme in the overexpressing cells, suggesting that each isozyme associates with its own specific substrate at particular sites.


MATERIALS AND METHODS

Expression Vectors

The expression vectors that were used have been described previously (24, 25) . In brief, pMTH (kindly provided by Dr. G. Shen-Ong) contains the mouse metallothionine promoter and the neomycin resistance gene as a selectable marker (32) . pMTH-, -II, -, -, -, and - were constructed by inserting the corresponding blunt-ended cDNAs into the blunt-ended BamHI site of pMTH as described (24) . In addition, an expression vector for PKC-I was generously provided by Dr. I. B. Weinstein (33) , and rat PKC- cDNA, a kind gift from Dr. J. Knopf (4) , was similarly cloned into pMTH.

Generation of Overexpressing Cell Lines

Clones of NIH 3T3 cells that overexpress one of the different PKC isoforms were produced and characterized essentially as described elsewhere (25) , with the exception of 3T3-PKC-I overexpressers that were only recently generated. The PKC--overexpressing NIH 3T3 cells were a kind gift from Dr. J. Silvio Gutkind (34) . Stably transfected cell lines were selected with G418, and 12 resistant clones from each transfection were selected at random and screened for PKC protein expression by Western blot analyses. The clones that displayed the highest protein expression were used in the following experiments.

Protein Analysis and Antibodies

For analysis of protein expression of each of the PKC isozymes, cells were grown in DMEM containing 10% FCS and 4 mM glutamine until 75% confluent. The cells were washed twice in PBS, lysed and scraped in protein lysis buffer (20 mM Tris, pH 7.5, 2 mM EDTA, 2 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml leupeptin, 80 µg/ml aprotinin, 0.1% 2-mercaptoethanol, and 1.2% Triton X-100). The cell lysates were sonicated for 10 s on ice and boiled for 3 min in SDS-PAGE sample buffer (35) , and equivalent amounts of protein, as determined using a Bio-Rad protein detection kit, were separated on a 10% SDS-polyacrylamide gel and electrophoretically transferred to nitrocellulose (BA 85, Schleicher & Schuell). The membrane was incubated with blocking solution (5% dried milk powder in TBS), and the different PKC isoforms were immunodetected using the following isozyme-specific antibodies: mouse monoclonal anti-PKC- (Upstate Biotechnologies Inc.), rabbit anti-pan-PKC- antiserum (36, a generous gift of Dr. Denise Cooper), rabbit anti-PKC- antiserum (Research and Diagnostics Laboratories), rabbit anti-PKC- and rabbit anti-PKC- antisera (Life Technologies, Inc.), rabbit anti-PKC- antiserum (Boehringer Mannheim), and rabbit anti-PKC- antiserum (18) . The membranes were incubated for 2 h in the appropriate primary antibody diluted in blocking solution. Next, the membranes were washed three times in TBS containing 0.2% Tween-20 and then incubated in species-specific horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, Inc.). Immunoreactive bands were developed using a chemiluminescent substrate (Kirkegaard & Perry) and visualized by exposure to film.

Cytoplasmic and Membrane Protein Fractionations

NIH-3T3-PKC-overexpressing cell lines were grown in DMEM containing 10% FCS and 4 mM glutamine until 75% confluent. The cells were left untreated or treated with 100 nM TPA for 15 min, 60 min, or 4 h and subsequently washed twice with PBS at room temperature. To prepare cytoplasmic fractions, cells were lysed and scraped in 400 µl of protein lysis buffer (see above) containing no Triton X-100, sonicated for 10 s on ice, and centrifuged at 100,000 g for 60 min. The supernatant (cytoplasmic fraction) was saved and stored at 80 °C, and the pellet (membrane fraction) was resuspended in protein lysis buffer containing 1.2% Triton X-100 and sonicated for 10 s on ice. Western blot analyses were performed on these two fractions as described above.

Immunofluorescence

Clones of NIH 3T3 cells that overexpress one of the PKC isoforms were seeded in eight-well Lab-Tek Chamber Slides (Nunc) at a concentration of 5 10cells/well in DMEM supplemented with 10% FCS and 4 mM glutamine. For experiments involving phorbol ester treatment, cells were treated with 100 nM TPA for various times. Cells were then rinsed briefly in PBS, fixed in 100% methanol at 20 °C for 15 min, and again rinsed in PBS. Comparable results were obtained when the cells were fixed in 3% paraformaldehyde, except for PKC- overexpressers, in which nuclear envelope staining was lost with the latter fixation. Fixed cells were permeabilized in TBS containing 0.25% Triton X-100, and nonspecific reactive sites were blocked with TBS + 0.25% Triton X-100 containing 5% normal goat serum and 5% nonfat dry milk (immunofluorescence-blocking solution) for 30 min at room temperature. Cells were incubated for 2 h at room temperature with the appropriate primary PKC isoform-specific antibody diluted in (immunofluorescence-blocking solution. Between antibody incubations, the cells were washed five times in TBS containing 0.25% Triton X-100. Primary antibodies were detected by a species-specific secondary antibody. Secondary antibodies used were FITC-conjugated donkey anti-mouse IgG and FITC-conjugated donkey anti-rabbit IgG (both obtained from Jackson ImmunoResearch Laboratories, Inc.), and rhodamine-conjugated goat anti-mouse IgG and rhodamine-conjugated goat anti-rabbit IgG (both from Organon Teknika Corp.). The ER was identified by staining with a rabbit anti-ER antibody (37) , and the Golgi apparatus was identified by staining with a mouse anti-mannosidase II monoclonal antibody (37, both generously provided by Drs. Richard Klausner and Julie Donaldson, National Institutes of Health). Coverslips were mounted on the slides using Fluoromount (Kirkegaard & Perry Laboratories), and the cells were observed on a fluorescence microscope (Zeiss Axiophot) and photographed using Kodak P800/1600 ASA film and a 63 objective. For actin visualization, the cells were fixed in 3.7% (v/v) formaldehyde in PBS (pH 7.4) for 30 min at room temperature, rinsed three times in PBS, permeabilized in PBS/0.1% Triton X-100, and stained with 100 ng/ml rhodamine phalloidin (Molecular Probes Inc.) for 30 min at room temperature. The cells were washed three times with PBS and mounted with coverslips.


RESULTS

Morphological Changes Induced in PKC-overexpressing Clones of NIH 3T3 Cells

Before activation of their PKCs by TPA, all of the overexpressing clones showed no significant morphological differences from the wild-type cells. After TPA treatments, most of the cultures, which were still subconfluent, showed signs of stimulation, i.e. ruffling of the cell membranes seen in 3T3-MTH- (Figs. 3 and 7). Some of the overexpressers, however, underwent more dramatic changes in shape when their PKCs were activated. After 15 min of TPA treatment, PKC-II-overexpressing fibroblasts dramatically flattened out and displayed unusually pronounced ruffling of the edges of the plasma membrane. These edges are the sites to which a portion of activated PKC-II relocates ( cf. Fig. 2). In response to TPA treatment, PKC--overexpressers rapidly rounded up, and their cytoplasms projected blebs ( cf. Fig. 5 ). Upon TPA treatment, 3T3-MTH- cells underwent striking morphological alterations that were characterized by long cytoplasmic extensions ( cf. Figs. 3 and 6). These morphological changes, seen maximally at the 4-h time point, were accompanied by a severe retardation of proliferation, as has been reported previously (25, 27) .


Figure 2: Immunofluorescent co-localization of PKC-II and actin in 3T3-MTH-II cells. Cells that overexpress PKC-II were simultaneously double stained for PKC-II ( left panels) and F-actin ( right panels) after 100 nM TPA treatment for 0 min, 15 min, or 4 h as described under ``Materials and Methods.'' White bar indicates 10 µm.




Figure 5: Immunofluorescent localization of PKC- in 3T3-MTH- cells. A, 3T3-MTH- cells were treated with 100 nM TPA for 0 min, 15 min, 60 min, or 4 h and stained with PKC--specific antibody as described under ``Materials and Methods.'' B, immunofluorescent co-localization of PKC- and the Golgi apparatus (arrowheads). Cells that overexpress PKC- were either untreated ( left panels) or incubated for 15 min in the presence of 100 nM TPA ( right panels). The cells were subsequently fixed and simultaneously co-stained as described under ``Materials and Methods'' with PKC--specific antiserum ( upper panels) and rabbit anti-mannosidase II antibody ( lower panels). White bar indicates 10 µm.



PKC Antibody Specificity

We assessed the isozyme specificity of the PKC antibodies using Western blots of total cell extracts of wild-type NIH 3T3 cells and overexpressing clonal derivatives, which had been electrophoresed on 10% SDS-polyacrylamide gels. As shown in Fig. 1 A, parental NIH 3T3 cells and all overexpressers expressed high levels of PKC-, while other PKC isoforms were basically not detectable by antibody staining. As expected, NIH 3T3 cells that had been stably transfected with MTH-II, -, -, -, -, or - expression vectors expressed substantial amounts of the appropriate isoforms, while the presence of these isoforms in wild-type or other overexpressers was essentially undetectable. Equal loading of each cell lysate was verified by Coomassie-stained gels (data not shown). The PKC isozyme-specific antisera used in this report recognized a single band of the expected apparent mass, did not cross-react with other isozymes, and therefore made suitable probes for immunocytochemical localization of PKC isozymes in 3T3-PKC-overexpressing cells. 3T3-MTH-I overexpressers were compared to the other PKC-overexpressing cell lines by Western analysis using pan-PKC- antiserum or rabbit anti-PKC-I antiserum (Life Technologies, Inc.) at a later time; therefore, the results are not included in Fig. 1 A. As expected, the antiserum that is specific for PKC-I did not cross-react with PKC-II or any other isozymes and detected PKC-I protein as a single 82-kDa band only in the 3T3-I overexpressers (data not shown). Since this antibody worked poorly in our immunofluorescence studies, we used the aforementioned pan-PKC--specific antibody for the immunocytochemical analysis of the PKC-I and -II overexpressers.


Figure 1: Western blots. A, control NIH 3T3 cells and NIH 3T3 cells that overexpress PKC-, -II, -, -, -, -, and - (indicated at the top) were probed with isozyme-specific antibodies to PKC (indicated along the left margin). Total protein lysates (100 µg) were electrophoresed on 10% SDS-polyacrylamide gels and electroblotted onto nitrocellulose membranes. B, translocation of PKC isozymes at different times after treatment with 100 nM TPA in 3T3-PKC-overexpressing cell lines (indicated along the right margin). Cytosolic and membrane fractions were electrophoresed in 10% SDS-polyacrylamide gels, followed by Western blotting.The immunoreactive bands for PKC- (82 kDa), PKC-II (82 kDa), PKC- (82 kDa), PKC- (82 kDa), PKC- (95 kDa), PKC- (72 kDa), and PKC- (indicated by arrow, 82 kDa) were visualized as described under ``Materials and Methods.''



Translocation of PKC Isozymes over Time in Response to TPA Treatment

Previous reports have demonstrated that the activation of PKC by phorbol ester results in the rapid redistribution of the enzyme from a predominately cytosolic location in resting cells to a membrane-associated site during stimulation (38) . To study the kinetics of phorbol ester-induced activation of the PKC isozymes, we used the seven isozyme-specific antisera for Western blot analyses of cytosol (supernatant) and membrane-bound (pellet) fractions of cell lysates. These studies were performed on untreated PKC overexpressers and after 15 min, 1 h, and 4 h of treatment with 100 nM TPA. Fig. 1B illustrates that all the isozymes are present in abundant amounts in the cytosolic fractions of untreated cells, presumably indicating inactive PKC protein. In addition, significant amounts of PKC-I, -, -, and - are membrane-associated in untreated cells, presumably indicating activation of a portion of these isozymes. TPA-induced activation of the PKC isozymes resulted in translocation of each isozyme, with the notable exception of PKC- (39) , albeit to varying degrees. For example, complete translocation from the cytoplasm to membranes of PKC-, -II, -, -, and - was observed within 1 h. In contrast, TPA treatment of PKC-I and - overexpressers results in only partial translocation to the membrane fraction, even after 4 h. These data demonstrate that the PKC isoforms differ with respect to responsiveness to activation by TPA and suggest that their selective activation might reflect different subcellular localization that could be detected using immunofluorescence analysis. PKCs are thought to be down-regulated, i.e. completely lost to immunochemical detection, after prolonged, e.g. 18-h, activation, such as by high doses of TPA (40) . The Western blots in Fig. 1 B show that the yield of PKC- from membranes was lower than that of other isozymes, but neither its level in the membrane fraction nor that of any other isozyme decreased by the 4-h time point. Thus 4 h of TPA treatment did not down-regulate the overexpressed PKCs, and cells at time points between 15 min and 4 h of TPA treatment would be appropriate for examination by immunofluorescence microscopy.

Immunofluorescence Studies of Individual PKC Isozymes

The PKC-isozyme-specific antisera were next used to study the intracellular localization of each PKC isozyme. To eliminate nonspecific background staining, primary antibodies that had not been affinity-purified (anti-pan-PKC- that reacts with both PKC-I and -II, anti-PKC- and anti-PKC-) were absorbed with NIH 3T3 cell lysates that had been immobilized on glutaraldehyde-activated cartridges (FMC-Bioproducts). To ensure that the staining that we obtained by the PKC antisera was specific, many negative controls were included, but for the sake of brevity these results are not shown. Preincubating all the anti-peptide antibodies with the immunizing peptide abrogated immunofluorescence in every case (data not shown). Furthermore, only the anti-PKC- antiserum stained wild-type NIH 3T3 cells, as expected (data not shown). Identical immunofluorescence and staining patterns were also seen in NIH 3T3 cells that overexpress each of the PKC isozymes following stable transfection with Harvey-Sarcoma virus-based pLTR expression vectors that contain the same cDNAs that were cloned into the pMTH vector (25, 26) . To ensure that the immunofluorescence results were not due to clonal artifacts, we examined bulk cultures of each of the PKC-overexpressing cell lines and obtained staining patterns identical to those obtained from the cloned cell lines (data not shown).

Association of Activated PKC-II with Actin Microfilaments

Fig. 2 compares the cellular localization of PKC-II in cells that overexpress PKC-II (3T3-MTH-II) in the presence or absence of TPA. In untreated 3T3-MTH-II cells, PKC--specific antibody diffusely stains nearly the entire cytoplasm, concentrating in a broad area surrounding, yet sparing, the nucleus. After TPA treatment, the cells dramatically flatten and display pronounced ruffled edges of the plasma membrane. Furthermore, PKC-II redistributes to the plasma membrane with marked redistribution to membrane ruffles and filamentous structures that traverse the cytoplasm, resembling the pattern expected for actin-rich cytoskeletal components. To confirm the notion that activated PKC-II translocates to actin-rich microfilaments, 3T3-MTH-II cells were stained simultaneously with pan-PKC- antiserum and rhodamine phalloidin, a phallotoxin that stains filamentous (F-) actin (41) . All the right-hand panels of Fig. 2 show the characteristic filamentous pattern of F-actin detected by rhodamine-phalloidin staining of the same cells shown on the left. The two lower left panels illustrate the translocation of PKC-II following treatment with TPA for 15 min and 4 h. Activation of PKC-II resulted in partial redistribution to the plasma membrane and revealed intense staining of the actin-rich microfilament lattice. The patterns obtained by co-staining with anti-pan-PKC- and phalloidin were essentially identical in five additional, independent experiments.

PKC- and PKC- Associate with the Endoplasmic Reticulum and Cell Junctures

To ascertain whether other activated PKC isoforms also redistribute to actin-rich microfilaments in response to TPA, we stained NIH-3T3 cells that overexpress the other PKC isozymes with the corresponding isozyme-specific antisera to compare the cellular localization of PKC-,-I, -, -, -, -, and - in the presence and absence of TPA. Fig. 3 illustrates that the other PKC isozymes, including PKC-I, the isoform most closely related to PKC-II, did not assume an actin-like distribution following TPA treatment. With a few exceptions, untreated cells showed a diffuse distribution of each PKC throughout the cytoplasm, but after TPA treatment each isozyme displayed a unique redistribution pattern. After 15 min in 100 nM TPA, PKC- redistributes to the cell periphery of 3T3-MTH- cells and accumulates in cell margins, including cell-cell junctures. Another portion of activated PKC- concentrates in punctate regions in the cytoplasm, near the nucleus, a pattern similar to one expected for ER. The intense punctate staining was confirmed to be that of the ER using double labeling of the same cells with mouse anti-PKC- and rabbit anti-ER antisera (Fig. 4, upper and lower right panels). Colocalization of PKC- and ER proteins was also observed in untreated cells (Fig. 4, upper and lower left panels), suggesting that PKC- associates with resident ER membrane proteins even before activation. The association may be loose before activation, because PKC- does not appear to be membrane-bound until after TPA treatment (Fig. 1 B).


Figure 3: Immunofluorescent localization of PKC isoforms in TPA-activated and unactivated 3T3-PKC-overexpressing cells. NIH 3T3 cells that overexpress PKC-, -I, -, -, -, -, or - were untreated ( left panels) or treated ( right panels) for 15 min with 100 nM TPA. The cells were fixed and stained as described under ``Materials and Methods'' with the corresponding isoform-specific antibodies.




Figure 4: Immunofluorescent co-localization of PKC- and resident ER proteins. Cells that overexpress PKC- were either untreated ( left panels) or incubated for 15 min in the presence of 100 nM TPA ( right panels). The cells were subsequently fixed and simultaneously stained as described under ``Materials and Methods'' with mouse anti-PKC- antiserum ( upper panels) and rabbit anti-ER antibody ( lower panels). White bar indicates 10 µm.



TPA treatment of PKC-I-overexpressing cells induced translocation of PKC-I to ruffled membrane edges, similar to the redistribution induced in 3T3-PKC-II cells, but PKC-I did not redistribute into a pattern that resembled actin-containing microfilaments (Fig. 3). Furthermore, we observed more pronounced membrane ruffling in TPA-treated 3T3-PKC-I cells than that which was seen in 3T3-MTH-II cells. Staining of PKC-, -, and - overexpressers revealed an association of these isoforms with the Golgi apparatus, which will be discussed in detail later.

Untreated PKC- overexpressers revealed a punctate (ER-like) staining throughout the cytoplasm, while TPA treatment induced a substantial portion of PKC- to redistribute to the plasma cell membrane, concentrating particularly in areas of cell-cell contact rather than at the growing edges of the cells. Neither untreated or TPA-treated 3T3-MTH- cells showed convincing colocalization of PKC- and ER, but a loose association at zero time cannot be completely ruled out, owing to the widespread nature of the staining by both antisera (data not shown). Any association would have to be loose, because there is very little membrane-associated PKC- at zero time (Fig. 1 B). Another striking and unique characteristic of activated PKC- was an intense perinuclear localization, suggesting that a portion of activated PKC- targets some component of the nuclear envelope.

Immunofluorescent localization of PKC- in 3T3-MTH- cells revealed a diffuse cytoplasmic staining that did not change significantly after TPA treatment. This finding is not surprising, since this atypical PKC isoform is unable to bind phorbol ester, and it does not associate with membranes after TPA treatment (Fig. 1 B).

PKC-, -, and - Are Associated with the Golgi Apparatus

Immunocytochemical examination of the subcellular localization of PKC- indicated that this isoform is evenly distributed throughout the cytoplasm of untreated 3T3-MTH- cells and completely excluded from the nuclei (Fig. 5 A). In response to TPA, the cells dramatically and rapidly round up, the cytoplasm projects blebs, and the cells reveal intense staining of a compact juxtanuclear structure that resembles the Golgi apparatus (15 min). After longer TPA treatment (60 min and 4 h), a fraction of PKC- is found in clusters at the cell membrane, but the majority of this isoform remains associated with the compact Golgi-like structure. To confirm the concentration of activated PKC- in Golgi, we simultaneously stained cells with anti-PKC- and an antibody to the cis/medial Golgi marker, mannosidase II (Ref. 37, Fig. 5B). The upper left panel shows diffuse cytoplasmic staining of PKC- in untreated cells, detected by rabbit anti-PKC- and FITC-labeled goat anti-rabbit IgG, whereas the lower left panel shows the expected perinuclear localization of the mannosidase II-rich Golgi component detected by mouse anti-mannosidase II and rhodamine-labeled goat anti-mouse IgG. The two upper right panels depict the TPA-induced redistribution of PKC- to perinuclear Golgi-like structures (indicated by arrowheads), and the lower right panels show that the most brightly stained regions colocalize with mannosidase II protein, confirming their identity as Golgi components.

Immunofluorescent staining of control 3T3-MTH- cells revealed that PKC- is localized throughout the cytoplasm as well as concentrated in a perinuclear region resembling the Golgi (Fig. 6 A). Upon TPA treatment, 3T3-MTH- cells undergo striking morphological alterations that are characterized by the development of long cytoplasmic extensions. These morphological changes, seen maximally in the 4-h time point, are accompanied by a severe retardation of proliferation, as has been reported previously (25, 27) . Colocalization studies using anti-PKC- and anti-mannosidase II antisera revealed that in untreated cells PKC- colocalizes with the mannosidase II-rich Golgi component (Fig. 6 B). Interestingly, the Golgi appears at the base from which the cellular processes emanate. After TPA treatment, PKC- remains associated with the Golgi organelle, however, a portion of the staining by anti-PKC- and anti-mannosidase II relocates from its normal juxtanuclear position to the long processes. TPA treatment also induces a portion of PKC- to redistribute to the cell periphery as seen in the upper right panel of Fig. 6 B.


Figure 6: Immunofluorescent localization of PKC- in 3T3-MTH- cells. A, 3T3-MTH- cells were treated with 100 nM TPA for 0 min, 15 min, 60 min, or 4 h and stained with 3T3-adsorbed PKC--specific antibody as described under ``Materials and Methods.'' B, immunofluorescent co-localization of PKC- and the Golgi apparatus. Cells that overexpress PKC- were either untreated ( left panels) or incubated for 15 min in the presence of 100 nM TPA ( right panels). The cells were subsequently fixed and simultaneously co-stained as described under ``Materials and Methods'' with PKC--specific antiserum ( upper panels) and rabbit anti-mannosidase II antibody ( lower panels). White and black bars indicate 10 µm.



As shown in Fig. 7 ( A and B), PKC- resides chiefly in the Golgi apparatus in untreated PKC--overexpressing cells. Double labeling of 3T3-MTH- cells using anti-mannosidase II and anti-PKC- antisera demonstrated virtually identical staining for both proteins (Fig. 7 B), confirming that the perinuclear concentration of PKC- involved the Golgi. After TPA stimulation, we observed PKC- staining at the outer cellular membrane (Fig. 7 A, 15 min, 60 min, and 4 h) as well as transient punctate staining of the nuclear membrane, suggestive of nuclear pores (Fig. 7 A, 15 min and 4 h; Fig. 7 B, right panels). In addition, a portion of PKC- remained associated with the Golgi apparatus throughout the duration of the TPA treatment.


Figure 7: Immunofluorescent localization of PKC- in 3T3-MTH- cells. A, 3T3-MTH- cells were treated with 100 nM TPA for 0 min, 15 min, 60 min, or 4 h and stained with 3T3-absorbed PKC--specific antibody as described under ``Materials and Methods.'' B, co-localization of PKC- and the Golgi apparatus. Cells that overexpress PKC- were untreated ( left panels) or incubated with 100 nM TPA for 15 min ( right panels). The cells were subsequently fixed and simultaneously co-stained as described under ``Materials and Methods.'' The upper left and right panels show staining of PKC- in the Golgi apparatus, detected by rabbit anti-PKC- and FITC-labeled goat anti-rabbit IgG. The lower left and right panels show the localization of mannosidase II, detected by mouse anti-mannosidase II and rhodamine-labeled goat anti-mouse IgG. White and black bars indicate 10 µm.




DISCUSSION

The data presented in this paper demonstrate the differential localization of eight PKC members in NIH 3T3 cells that overexpress one PKC isoform. Our results, which are summarized in , clearly indicate that, with the exception of PKC-, all PKC isozymes translocate not only to the plasma membrane but also to different intracellular sites upon activation by TPA, presumably at the sites where the isozymes find their substrates. In most cases the associations are stable ones, with very little down-regulation from the membrane locations seen by 4 h.

Several of the PKC isoforms appear to target components of the cytoskeleton after activation: PKC-, -I, -II, -, and - become associated with cell margins and points of contact with other cells, and a portion of the PKC-II dramatically redistributes to actin-containing microfilaments in response to TPA. The TPA-induced translocation of PKC- to the periphery of the cell is in agreement with the results of experiments done in normal rat embryo fibroblast cells (19) and rat myocytes grown in serum (30) . What is more, several proteins within the focal contact matrix, such as -actinin, talin, and vinculin, have been shown to be substrates for PKC (42, 43) .

Our immunofluorescence studies on PKC- suggested that another portion of this particular isoform associates with the ER, even before activation (Fig. 4), although after TPA activation, the ER staining appears more concentrated and punctate (Fig. 3). Since Western blot studies (Fig. 1 B) showed no membrane-bound PKC- before activation by TPA, we must conclude that the apparent association of a small fraction of PKC- with ER in untreated cells is only very weak. While most proteins that are synthesized in the ER are transported to the Golgi apparatus for sorting and distribution to other destinations such as the cell surface, lysosome, or secretory vesicles, other proteins remain in the ER as permanent residents. This may be the case for activated PKC-, presumably indicating that it may have a functional role in this organelle. Some ER transmembrane proteins contain an ER-targeting motif (44, 45) that consists of Lys- X- X or Lys- X-Lys- X- X. Since the typical site of PKC phosphorylation consists of a serine or threonine in the vicinity of basic residues (46) , one might predict phosphorylation of ER membrane proteins by PKC-, which associates with the ER after activation by TPA (Fig. 4).

Other studies have shown phorbol ester-induced translocation of PKC- to the nuclear envelope of fibroblasts (47, 48, 49) and of PKC-, -I, -, -, and - in cardiac myocytes grown in defined medium (30) . We, however, did not observe nuclear localization of PKC- in TPA-treated NIH 3T3 cells (data not shown) or 3T3-MTH- cells (Fig. 4). We cannot completely explain this discrepancy, but it probably is due to the different cell lines that were used or the different culture conditions employed.

Localization of PKC-II differed from the other PKC isozymes in that TPA treatment induced redistribution of PKC-II to a concentration in microfilaments, on the cell membrane and at ruffled membrane edges. This is consistent with the findings of Gregorio et al. (29) and Disatnik et al. (30) that PKC-II associates with cytoskeletal elements in T lymphocytes and cardiac myocytes, respectively. In our studies, TPA-stimulated 3T3-PKC-I, and - cells also showed relocalization of their particular PKC to the ruffled cell periphery but not to actin-rich microfilaments. The molecular events responsible for membrane ruffling are not known, although potential mechanisms have been proposed that involve components of the cytoskeleton and the polymerization of actin at the inner surface of the plasma membrane. PKC has been implicated in rearrangement of microtubules, microfilaments, actin-binding proteins (such as vinculin, myosin heavy and light chains), and vimentin intermediate filaments in a variety of cell types: B16a melanoma cells (50) , vascular endothelial cells (51) , and neutrophils (52) ; however, it was not known which PKC isoform(s) are involved in these processes. Our studies suggest that PKC-II is particularly closely associated with the actin component of the cytoskeleton. This isoform may phosphorylate actin directly or one of its binding proteins, as suggested by the reports that PKC is capable of phosphorylating the cytoskeletal proteins talin (53) and vinculin (42) . Further studies are designed to investigate the nature of the PKC-II-cytoskeleton interaction in greater depth in vitro.

PKC-, -I, and - may also be involved with the cytoskeleton in the ruffling process, but they do not associate with F-actin. A predominant, specific substrate for PKC, MARCKS (myristoylated, alanine-rich protein kinase C substrate), has been shown to be an F-actin cross-linking protein (54) . Its cross-linking activity is inhibited by PKC-mediated phosphorylation, resulting in the displacement of MARCKS from F-actin, while subsequent dephosphorylation results in reassociation with F-actin. This phosphorylation-dependent association/disassociation is another possible mechanism through which one or more of these PKCs could regulate actin-membrane interaction (55) .

PKC-I and -II are the products of alternative splicing and differ only in their carboxyl termini (having 50 or 52 unique terminal amino acids, respectively) (56) . Therefore, our data and that of Disatnik et al. (30) , which show different relocalization sites for PKC-I and PKC-II, strongly suggest that the carboxyl terminus of these isozymes is critical in targeting their translocation to their proper substrates or sites of action. It is reasonable to predict that the carboxyl terminus may be responsible for the targeting of other isozymes as well. Experiments are under way to test this hypothesis.

PKC- and PKC- not only concentrate at cell-cell junctures after TPA treatment, they also relocate to the vicinity of the nucleus, albeit with important morphological differences. The anti-PKC- antibody staining of 3T3-MTH- cells revealed an intense staining of a perinuclear ring that resembles the nuclear envelope, while activated PKC- translocated to discrete structures resembling nuclear pores in its overexpresser. Disatnik et al. (30) also reported nuclear relocalization of PKC- after exposure of cardiac myocytes to serum or TPA, but they also showed nuclear relocalization of PKC-. Our interpretation of the perinuclear localization of activated PKC- was that it targeted Golgi structures (see below). Chida et al. (57) presented evidence that PKC- is associated with rough endoplasmic reticulum rather than with nuclei. We tested this notion by staining our PKC--overexpressing fibroblasts (and all the overexpressing clones; data not shown) with the ER-specific antiserum before and after TPA treatment, and PKC colocalized with ER convincingly only in TPA-treated 3T3-MTH- (Fig. 4). The PKC--specific antiserum staining patterns of 3T3-MTH- were also somewhat different from those seen in keratinocytes and PKC--overexpressing COS cells (57) . The latter resembled the patterns seen in our untreated 3T3-MTH- (Fig. 4). This difference must be one of many expected examples of cell-type differences in localization of individual PKC isoenzymes. Greif et al. (58) reported that PKC- was found within the nucleus of human keratinocytes. Chida et al. (57) did not agree with this finding, and we did not find PKC- within the nucleus of 3T3-MTH- either, but our cells showed speckled staining of the nuclear surface. Several studies have shown that the nuclear envelope can play a role in signal transduction pathways after activation of PKC (47, 48, 59, 60) , although the molecular mechanism by which PKC molecules transduce signals to the nucleus has not been determined.

Three isozymes, PKC-, -, and -, associate with the Golgi apparatus, but there are differences among these isozymes in the way this occurs. PKC- appears to colocalize with mannosidase II only after TPA stimulation, while PKC- and PKC- can be seen, at least in part, to have concentrated in the mannosidase II-rich Golgi even before TPA is introduced into the culture medium. These results are consistent with the data shown in Fig. 1 B, namely that a substantial amount of PKC- and -, but very little of PKC-, appears membrane-bound, i.e. activated (perhaps by serum components), even in the absence of TPA. TPA treatment causes the Golgi to become very compact in all three overexpressers, but there are morphological differences, namely the PKC--overexpressing cells round up and concentrate the PKC/Golgi at one pole of the nucleus, PKC--overexpressing cells slow their growth rate and translocate some of the PKC and Golgi proteins into the unusual cytoplasmic processes, and PKC- overexpressers flatten out and transiently lose the compact appearance of the Golgi. It is our working hypothesis that these three isozymes contribute in different ways to processing and transport of proteins through the Golgi network. Exactly what form these contributions might take is unknown, but recent work has shown that activated PKC (isoform unknown) plays a role in binding two protein components of intracellular vesicles, ADP-ribosylation factor, and -COP (110-kDa coat protein), to Golgi membranes (61) . Another possible role for PKC in Golgi-related activities relates to the unusual, long, cellular processes that appear when PKC- overexpressers are treated with TPA. These components may be similar to the long tubulovesicular processes that extend from the Golgi along microtubules, which appear when rat kidney cells are treated with brefeldin A to induce retrograde transport of Golgi proteins into the ER (62) .

We compared the effects of TPA and a synthetic diacylglycerol analog, 1,2-dioctanoylglycerol (DiC), to determine if another, perhaps more physiological, activator of PKC could induce the association of PKC-II with actin and PKC-, -, and - with Golgi. DiCinduced redistribution effects that were virtually identical to those induced by TPA for all of the PKC overexpressers (data not shown). The most obvious difference was that DiCinduction of PKC translocation was transient (as expected; Ref. 3); thus, by 4 h the staining patterns of the cells resembled untreated cells.

It is not yet clear how many of the specific sites for relocalization of individual isozymes identified in NIH 3T3 overexpressers will be found in all cells. It is reasonable to expect that there will be differences, since individual PKC isozymes serve biologically distinct functions in different cell types. Localization of PKC- seems different in 3T3 overexpressers (Golgi) and in keratinocytes and COS overexpressers (rough ER). However, it is interesting that our results in exogenously overexpressed PKCs are more similar than different from those of six naturally expressed isoenzymes in myocytes (30) reported while our manuscript was in preparation. These authors used immunofluorescence to localize PKC isoenzymes in cardiac myocytes before and after stimulation with serum, norepinephrine and TPA. Their studies and ours detected a PKC-II association with cytoskeleton, a PKC-, -I, -II, -, and - association with the cell periphery after activation, and PKC- and - association with perinuclear structures. The biggest difference between the two sets of results lies in the widespread finding of virtually all PKCs within or near the nucleus by Disatnik et al. (30) , and a much more limited nuclear association in our results. Part of this discrepancy may be due to differences in culture conditions, and part may be due to the difference in cell types studied.

Studies that explored the substrate specificity of individual PKC isozymes in vitro suggested that the different PKCs phosphorylated most of the substrates investigated with quite similar, although slightly different, efficiencies (18) . The diverse biological effects of overexpressed PKC isozymes in vivo (24, 25) strongly argue for specific functions for each of the PKC isozymes. The results presented here suggest that substrate choice in vivo could be determined by distinct compartmentalization of the different PKC isozymes. Thus, the PKC multigene family appears not to be merely a set of functionally redundant protein kinases; instead, each isoform may have a distinct physiological role and a specific substrate or subcellular site in which it works.

  
Table: Summary of immunocytochemical localization of different PKC isozymes



FOOTNOTES

*
This work was supported in part by Grant 10318 from the Deutsche Krebshilfe Doktor Mildred Scheel Stiftung. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Bldg. 37, Rm. 2B04, National Institutes of Health, 37 CONVENT DR. MSC 4255, BETHESDA, MD 20892-4255.

The abbreviations used are: PKC, protein kinase C; DiC, 1,2-dioctanoylglycerol; MARCKS, myristoylated alanine-rich protein kinase C substrate; TPA, 12- O-tetradecanoylphorbol-13-acetate; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; TBS, Tris-buffered saline; FITC, fluorescein isothiocyanate; ER, endoplasmic reticulum; PBS, phosphate-buffered saline.


ACKNOWLEDGEMENTS

We thank Dr. Denise R. Cooper for the anti PKC- antibody, Drs. J. Silvio Gutkind and Fabio Gusovsky for the PKC--overexpressing NIH 3T3 cell line, Dr. I. Bernard Weinstein for the PKC-I expression vector, and Drs. Julie Donaldson and Richard Klausner for the anti-ER and anti-mannosidase II antibodies as well as for help with the interpretation of the immunofluorescence data. We also thank Dr. Marcelo Kazanietz for helpful discussions and a critical reading of the manuscript.


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