(Received for publication, November 7, 1994; and in revised form, June 20, 1995)
From the
Protein kinase C (PKC) has been found to have unique
properties among the PKC isozymes in terms of its membrane association,
oncogenic potential, and substrate specificity. Recently we have
demonstrated that PKC
localizes to the Golgi network via its zinc
finger domain and that both the holoenzyme and its zinc finger region
modulate Golgi function. To further characterize the relationship
between the domain organization and the subcellular localization of
PKC
, a series of NIH 3T3 cell lines were created, each
overexpressing a different truncated version of PKC
. The
overexpressed proteins each were designed to contain an
-epitope
tag peptide at the COOH terminus to allow ready detection with an
antibody specific for the tag. The subcellular localization of the
recombinant proteins was analyzed by in vivo phorbol ester
binding, immunocytochemistry, and cell fractionation followed by
immunoblotting. Results revealed several regions of PKC
that
contain putative subcellular localization signals. The presence either
of the hinge region or of a 33-amino-acid region including the
pseudosubstrate sequence in the recombinant proteins resulted in
association with the plasma membrane and cytoskeletal components. The
catalytic domain was found predominantly in the cytosolic fraction. The
accessibility and thus the dominance of these localization signals is
likely to be affected by the overall conformation of the recombinant
proteins. Regions with putative proteolytic degradation sites also were
identified. The susceptibility of the overexpressed proteins to
proteolytic degradation was dependent on the protein conformation.
Based on these observations, a model depicting the interaction and
hierarchy of the suspected localization signals and proteolytic
degradation sites is presented.
Protein kinase C (PKC) ()is a family of more than 10
isozymes of serine/threonine protein kinases that are central to many
signal transduction pathways(1, 2, 3) .
Although these isozymes share a similar structural domain organization,
differences in their substrate specificity, cofactor requirements,
tissue and cellular distribution, and subcellular localization suggest
that each of the different PKC isozymes plays a specific and distinct
regulatory role(s) in cellular signal transduction. However, the
elucidation of PKC isozyme-specific functions has been hindered by the
presence of multiple isozymes in the majority of tissues and cell lines
studied, as well as by the almost complete lack of isozyme-specific in vivo activator or inhibitor molecules(4) . To date
the most successful approaches to reveal PKC isozyme-specific phenomena
have been either to overexpress a single isozyme in a given cell
type(5, 6, 7) or to carry out antisense
treatment of cells to specifically decrease the level of just one
isotype(8, 9) .
Among the PKC isozymes, only
PKC has been reported to exhibit full oncogenic
potential(7, 10) . PKC
also has been implicated
in the regulation of other biological processes, including antiviral
resistance(11) , hormone secretion(12) , and regulation
of transporters (13, 14) . PKC
displays unusual
membrane association (13, 15) and a restricted in
vitro substrate specificity(16) . Recently, we reported
that PKC
exhibited a unique association with Golgi membranes via
its zinc finger domain and, in accordance with this subcellular
localization, specifically modulated Golgi function(14) . These
results suggested that the zinc finger domain of PKC
might act as
a specific localization signal.
Subcellular localization could be a key component in determining the functional and regulatory specificity of a given PKC isozyme. The PKC isozymes have been reported to localize to various subcellular compartments(17, 18, 19, 20, 21, 22, 23) , other proteins that appear to serve as anchoring factors for PKC have been cloned(24) , and regions with potential subcellular localization signals have been postulated for some of the isoforms(14, 19) . However, the precise determinants of subcellular localization and their potential importance in defining the function of a given PKC molecule have not yet been fully characterized for any of the isozymes.
A general model for the regulation of PKC activity often includes the translocation (tight association) of PKC to membranes in response to activator binding, accompanied by a conformational change within the lipid environment of the membrane that results in a catalytically active PKC(1) . This is followed by rapid inactivation, apparently due to a decrease in activator binding and dissociation of PKC from the membrane complex. However, if PKC remains tightly associated with the membrane for longer time periods, as in the case of activation by phorbol 12-myristate 13-acetate (PMA), the activated PKC becomes susceptible to down-regulation through proteolytic degradation(26, 27) . Although translocation is a rapid, dramatic subcellular redistribution of PKC, almost nothing is known about the molecular mechanism(s) and in vivo determinants of this process(2, 25) . Likewise, little is known of the events involved in the down-regulation and proteolytic degradation of PKC(27) . Whereas one protease cleavage site has been mapped to the border of the hinge region and catalytic domain of the cPKC isozymes(26) , the protease(s) involved in vivo is not clear(27) . Further, it is likely that other protease-sensitive sites also are involved in the progressive degradation of PKC.
PKC, a novel PKC isozyme, is
one of the PKC isotypes reported to undergo translocation and to be
susceptible to proteolytic degradation (13, 15, 28, 29) . To explore the
effects of various domains of PKC
on its conformation, subcellular
localization, activator-induced translocation, and proteolytic
stability, we have utilized stably transfected NIH 3T3 cell lines that
overexpress different domain fragments of PKC
. An epitope tag was
added to the COOH termini of the mutant proteins to allow uniform
detection by an antibody recognizing the tag peptide(30) . We
have used these overexpressed recombinant polypeptides to identify
regions of the molecule possibly involved in determining its
subcellular distribution and translocation. In addition, we have
investigated the proteolytic susceptibility of these recombinant
proteins and have found several protease-sensitive regions.
Figure 1:
Domain organization of PKC and its
various recombinant
-epitope-tagged derivatives. The numbers
denote the NH
- and COOH-terminal amino acids of the various
constructs. PS and ATP correspond to the
pseudosubstrate region and the ATP-binding site, respectively, whereas
represents the 12-amino-acid
-epitope tag. With mouse
PKC
cDNA as template, PCR fragments were generated for the various
recombinant proteins and cloned into the
-epitope tagging vector,
p
MTH, as described under ``Experimental
Procedures.''
Figure 2:
Characterization of the recombinant
protein overexpressor cell lines by immunoblotting. Overexpressor cells
were lysed at 80% confluency in SDS-PAGE sample buffer, 10 µg of
protein/lane was applied to SDS-PAGE, and immunoblot analysis was
carried out with the anti -epitope tag antibody (see
``Experimental Procedures''). Molecular mass markers are
shown at the left.
Figure 3:
Analysis of the subcellular distribution
of the various PKC recombinant fragments by cell fractionation and
immunoblotting. Confluent cells were deprived of serum and induced in
the presence of 20 µM zinc acetate for 16 h, disrupted by
Dounce homogenization, and fractionated by centrifugation into nuclear,
particulate, and cytosolic fractions as described under
``Experimental Procedures.'' The nuclear and particulate
fractions were further separated by Triton X-100 detergent extraction,
resulting in detergent-soluble (nuclear and particulate) fractions, and
detergent-insoluble (nuclear scaffold and cytoskeleton) fractions. 10
µg of protein/lane was applied to SDS-PAGE, and the recombinant
proteins in each fraction were detected by immunoblotting. The arrows indicate the major subcellular localization differences
between the
2 and
3 recombinant proteins and the
5 and
6 proteins, respectively.
Figure 4:
Immunocytochemical analysis of NIH 3T3
cell lines overexpressing the various -epitope-tagged recombinant
proteins, with the empty p
MTH vector as a control. The cells were
fixed with paraformaldehyde, solubilized by exposure to Triton X-100
detergent, and incubated with the anti-
-tag antibody as described
under ``Experimental Procedures.'' Immunofluorescent staining
was employed using Cy3-conjugated anti-rabbit antibody. A,
1; B,
2; C,
3; D,
4; E,
5; F,
6; G,
7; H,
holo PKC
overexpressor cells. I presents cells
transfected with the empty p
MTH vector. The large arrows point to the plasma membrane, whereas the small arrows designate the Golgi. Note the strictly perinuclear decoration of
cells overexpressing the
3 and
6 proteins. The bar represents 20 µm.
The short term, PMA-induced subcellular redistribution of the
-epitope-tagged mutant proteins also was investigated by
immunocytochemistry. Most of the mutant proteins (including the
1
and
5 proteins, data not shown) were completely translocated to
the plasma membrane within 10 min of treatment of the cells with PMA (Fig. 5). The subcellular distribution of the
4 catalytic
domain fragment, which does not contain the phorbol ester-binding
domain, was not affected by PMA treatment, whereas the
3 zinc
finger domain fragment was observed to translocate at a much slower
rate (data not shown).
Figure 5:
PMA-induced subcellular redistribution of
the PKC recombinant proteins. The overexpressor cells were exposed
to PMA for 10 min and then fixed and stained as described in the legend
to Fig. 4. A and B, holo PKC
; C and D,
2; E and F,
6; G and H,
7 overexpressor cells. A, C, E, and G, untreated cells. B, D, F, and H, cells after PMA treatment. The large arrows indicate the plasma membrane, whereas the small arrows designate the Golgi. The bar represents
20 µm.
Figure 6:
Analysis of the proteolytic degradation of
the various overexpressed PKC mutant proteins. The overproducer
cells were grown in DMEM supplemented with 10% fetal calf serum and 75
µM zinc acetate and were maintained for an additional
3-4 days in the same composition medium after reaching confluency
to allow accumulation of the indicated recombinant proteins to as high
a level as possible. The cells then were harvested in hot SDS sample
buffer to prevent further degradation, and 50 µg of protein/lane
was applied to SDS-PAGE and analyzed by immunoblotting as described
under ``Experimental Procedures.'' The nonspecific
immunoreactivity in the 90-kDa range in lanes from
1 to
4 is
most likely due to sample overloading. Molecular mass markers are shown
at the left.
Recently we showed that the zinc finger region of PKC
localizes exclusively to the Golgi and suggested that this domain may
function as a localization signal for the holoenzyme(14) . In
the present study we have employed NIH 3T3 cell lines stably
overexpressing various
-epitope-tagged domain fragments of
PKC
to better characterize the role(s) that the protein domains of
PKC
may play in determining subcellular localization and
structural stability. The subcellular distribution of the mutant
proteins was analyzed by in vivo [
H]PDBu
binding assay, immunocytochemistry, and cell fractionation followed by
immunoblotting. Our results indicate that in addition to the zinc
finger domain, at least three other regions of PKC
may function as
subcellular localization signals. The pseudosubstrate and hinge
regions, which are part of the
2 and
7 mutant proteins,
respectively, but which are absent from the
3 protein, facilitated
plasma membrane and cytoskeletal association ( Fig. 3and 4).
Further, the catalytic domain seems to contain determinants for
cytosolic localization, because only those mutant proteins that contain
this domain exhibited cytosolic distribution. Alternatively, the
diffuse distribution conferred by the presence of the catalytic domain
might be interpreted to indicate a lack of localization signal in this
region. To answer this question would require further experiments.
Because activator-induced conformational changes and membrane association appear to be involved with the proteolytic down-regulation of PKC, the susceptibility of the recombinant proteins to proteolysis also was assessed (Fig. 6). It is of interest that although PMA induced proteolytic degradation, the degradation products themselves were detected only in confluent, resting cells. Whether proteolysis under the two conditions operates via the same mechanism and generates the same fragments requires further investigation. Our current hypothesis is that they do, only the faster rate of PMA-induced degradation does not allow the accumulation and thus the detection of the intermediate products.
The degradation products of holo-PKC
and of the
2 and
5 mutant proteins allowed us to identify
three protease-sensitive regions of PKC
: between the
pseudosubstrate region and zinc finger domain, at the border of the
hinge region and catalytic domain, and within the catalytic domain,
probably close to the ATP-binding site. Proteolytic cleavage between
the hinge region and the catalytic domain is thought to be an initial
step in the degradation of PKC. Cleavage at this site has been reported
for a number of PKC isozymes, including PKC
, and is carried out in vivo by unknown proteases (27) or in vitro by incubation with trypsin or
calpain(26, 28, 29) . An additional cleavage
at or near the ATP-binding site conceivably could serve to rapidly
inactivate the unregulated catalytic domain. The presence of another
protease-sensitive site between the pseudosubstrate region and the zinc
finger domain has been observed in vitro(35) . The
results presented in Fig. 6indicate that this cleavage site
also plays a role in the degradation of PKC
in vivo as
well.
Some of the findings presented in this communication cannot be
accounted for simply by the presence or absence of individual
localization signals or protease-sensitive sites. For example, the
6 protein is unstable, whereas the larger
5 and the smaller
7 proteins are more stable. Further, the
6 protein localized
exclusively to the Golgi, whereas the larger
5 and the smaller
7 proteins exhibited a broader subcellular distribution. To help
clarify the results reported and discussed here, we have summarized the
observations in the schematic model presented in Fig. 7.
Although additional information probably is required for a complete
understanding of the structural interrelationships of PKC
, this
schematic representation of intracellular domain-domain interactions is
in agreement with the results obtained and also takes into account
features of previous models depicting the structure-function
relationship of PKC(25, 36) . According to this model,
in the resting state the holo PKC
protein is folded into a compact
structure via two strong interdomain interactions: between the
pseudosubstrate region and the substrate recognition pocket of the
catalytic domain and between the hinge region and the catalytic domain (Fig. 7A). The former interaction has been postulated
in earlier models ( (25) and references therein), whereas the
latter may be identical with the activated C kinase receptor-binding
site pseudo activated C kinase receptor interaction suggested by Ron et al.(24) . Further, in our representation the plasma
membrane localization signal may be identical with the activated C
kinase receptor-binding portion of the hinge region(24) . In
this folded, compact conformation neither the ``dominant''
localization signals (pseudosubstrate region, zinc finger domain,
localization signal in the hinge region; see also below) nor the
protease-sensitive sites are primarily exposed on the surface. Thus, in
this state the protein is proteolytically stable and found diffusely
distributed primarily in the cytosol due to the presence of putative
cytoplasmic localization signal(s) within the catalytic domain (Fig. 3; (28) ). Deletion of the NH
-terminal
133 amino acids of the V
region, which results in
the
5 recombinant protein, had no effect on the localization or
stability of the molecule. The corresponding NH
-terminal
region was also found to be dispensible in determining the cofactor
dependence of the closely related nPKC
isozyme(25) ,
although its possible function in Ca
binding was
suggested by sequence comparison(37) .
Figure 7:
A schematic model for PKC depicting
domain-domain interactions, putative localization signals, and
proteolytic degradation sites. The empty and gray boxes represent the pseudosubstrate and hinge regions, respectively. The stipled two-tooth comb-like structures represent the zinc
finger region, and the small black dots represent
protease-sensitive sites. A, the completely folded structure
of holo PKC
in its inactive state. Two strong interdomain
interactions are suggested: between the pseudosubstrate region and
catalytic domain and between the hinge region and the catalytic domain.
Note that the dominant localization signals (pseudosubstrate region,
zinc finger domain, hinge region), as well as the protease-sensitive
sites, are buried within the molecule. B, represents the
structure of the
6 recombinant protein. The lack of the
pseudosubstrate region results in partial unfolding, which gives the
zinc finger domain dominance in determining the subcellular
localization and also exposes a protease-sensitive site. The plasma
membrane localization signal present in the hinge region remains
inaccessible. C, activation induces unfolding of the holo
PKC
protein. Binding of an activating ligand (large black
oval) to the zinc finger region induces this major conformational
change due to disruption of the two interdomain interactions. The
``sticky'' surfaces of the pseudosubstrate and hinge regions
then can function as localization signals for tight association with
membranes. The three putative proteolytic degradation sites also would
be exposed in this membrane-associated, activated conformational
state.
In contrast, deletion
through the pseudosubstrate region, represented by the 6 mutant
protein, apparently induces major conformational changes by disrupting
one of the crucial interdomain interactions (Fig. 7B).
In this state the protein would be in a partially unfolded conformation
with at least some of the protease-sensitive sites now accessible. This
would be consistent with the low level of the
6 protein found in
the cell. The plasma membrane localization signal of the hinge region,
which would be exposed on the surface of the smaller
7 protein, is
still buried in this case. On the other hand, the zinc finger region
would be readily accessible in this conformation of the
6 protein
to dominantly exert its Golgi localization effect over the cytosolic
distribution provided by the catalytic domain.
This model is in
agreement with the recent hypothesis that cofactor-induced activation
of PKC is accompanied by the dissociation of the pseudosubstrate region
from the substrate-binding domain, along with association of the now
exposed NH-terminal region with acidic phospholipids of
membranes(25, 38) . Further, our results suggest that
PMA treatment also disrupts the other intramolecular interaction
between the hinge region and the catalytic domain (Fig. 7C). The observation that PMA induces the
translocation of the strictly Golgi-localized
6 protein to the
plasma membrane (Fig. 5), apparently by allowing the
localization signal of the hinge region to appear on the surface, is in
accordance with this hypothesis. According to this model the
activator-induced conformational changes allow all three dominant
localization signals to become exposed, thus providing sufficient
information for the activated molecule to redistribute to its required
subcellular localization. The exposed pseudosubstrate and hinge
regions, as a function of their cytoskeletal binding capacities (Fig. 3), also might play a role in the subcellular
redistribution process itself for PKC
. The identification of
interactions between cytoskeletal proteins and the pseudosubstrate
region of PKC supports this hypothesis(36) .
Consistent with this model is the suggestion that proteolytic cleavage at the site between the pseudosubstrate region and the zinc finger domain, as demonstrated in Fig. 6, may be one of the first steps in the down-regulation of activated PKC. This would serve to lock the protein into an open, proteolytically susceptible conformation and also would remove one of the postulated localization signals. Further studies are required to define the physiological relevance of this cleavage site.
The results presented also establish an apparent order of dominance
among the localization signals of PKC. In the absence of
activation by PMA, the zinc finger region shows clear dominance over
the cytoplasmic localization signal(s) of the catalytic domain in the
6 protein, whereas it can contribute equally with the
pseudosubstrate and hinge regions in defining the localization of the
2 and
7 proteins, respectively ( Fig. 3and Fig. 4). With PMA treatment, however, the pseudosubstrate and
hinge regions are clearly dominant in directing the localization of the
PKC
mutant proteins over both the zinc finger and
catalytic domains. Because in the
2 and
7 recombinant
proteins both the pseudosubstrate and hinge regions would be exposed on
the surface of the molecule even prior to PMA binding, it seems
unlikely that their contribution would increase with PMA treatment.
Rather, a decreased ability of the zinc finger region to act as a Golgi
localization signal upon PMA binding would explain the complete Golgi
to plasma membrane translocations of the
2,
6, and
7
proteins (Fig. 5). (
)
These findings raise several
questions. Is the hypothesis that the zinc finger region can serve as a
localization signal applicable to other PKC isozymes? What happens to
the degradation products of PKC? Because, in contrast to the
catalytic domain fragment, most of the mutant proteins representing the
regulatory domains of PKC
proved to be fairly stable, it is
conceivable that they might accumulate to relatively high levels as
degradation products of endogenous PKC
. Do these proteolytic
fragments have any biological function? Our observation that the
Golgi-localized zinc finger domain affects Golgi function suggests that
proteolytically generated fragments of PKC may play a role in cellular
regulation(13) . Additional studies utilizing PKC mutant
proteins as described are required to answer these important
considerations.