From the
We have used immunocytochemical analyses to characterize the
subcellular distribution of protein kinase C (PKC)-
The PKC(
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-
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-
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-
Untreated
PKC-
Immunofluorescent localization of PKC-
Immunofluorescent staining of control
3T3-MTH-
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-
Several of the PKC isoforms appear
to target components of the cytoskeleton after activation: PKC-
Our immunofluorescence studies on
PKC-
Other studies have shown phorbol
ester-induced translocation of PKC-
Localization of PKC-
PKC-
PKC-
PKC-
Three isozymes,
PKC-
We compared the effects of TPA and a synthetic
diacylglycerol analog, 1,2-dioctanoylglycerol (DiC
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-
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.
We thank Dr. Denise R. Cooper for the anti PKC-
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
, -
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.
)
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).
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.
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) .
, 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.
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 10
cells/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.
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-
Fig. 2
compares the cellular localization
of PKC-II with Actin
Microfilaments
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-
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- and PKC-
Associate with the Endoplasmic
Reticulum and Cell Junctures
,-
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.
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.
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-
Immunocytochemical examination of the
subcellular localization of PKC-, -
, and -
Are Associated with the
Golgi Apparatus
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.
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.
, 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.
,
-
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) .
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).
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.
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.
, -
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) .
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.
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.
, -
, 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) .
), to
determine if another, perhaps more physiological, activator of PKC
could induce the association of PKC-
II with actin and PKC-
,
-
, and -
with Golgi. DiC
induced 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 DiC
induction of PKC translocation was transient
(as expected; Ref. 3); thus, by 4 h the staining patterns of the cells
resembled untreated cells.
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.
Table: Summary of immunocytochemical localization
of different PKC
isozymes
, 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.
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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.