1823
Journal of Cell Science 111, 1823-1830 (1998)
Printed in Great Britain © The Company of Biologists Limited 1998
JCS1547
Nuclear import of protein kinase C occurs by a mechanism distinct from the
mechanism used by proteins with a classical nuclear localization signal
Dirk Schmalz, Ferdinand Hucho and Klaus Buchner*
Institut für Biochemie der Freien Universität Berlin, Arbeitsgruppe Neurochemie, Thielallee 63, 14195 Berlin, Germany
*Author for correspondence (e-mail: [email protected])
Accepted 14 April 1998
SUMMARY
Protein kinase C does not have any known nuclear
localization signal but, nevertheless, is redistributed from the
cytoplasm to the nucleus upon various stimuli. In NIH 3T3
fibroblasts stimulation with phorbol ester leads to a
translocation of protein kinase C α to the plasma membrane
and into the cell nucleus. We compared the mechanism of
protein kinase C α’s transport into the nucleus with the
transport mechanism of a protein with a classical nuclear
localization signal at several steps. To this end, we comicroinjected fluorescently labeled bovine serum albumin to
which a nuclear localization signal peptide was coupled,
together with substances interfering with conventional
nuclear protein import. Thereafter, the distribution of both
the nuclear localization signal-bearing reporter protein and
protein kinase C α was analyzed in the same cells. We can
show that, in contrast to the nuclear localization signaldependent transport, the phorbol ester-induced transport of
protein kinase C α is not affected by microinjection of
antibodies
against
the
nuclear
import
factor
p97/importin/karyopherin β or microinjection of nonhydrolyzable GTP-analogs. This suggests that nuclear
import of protein kinase C α is independent of p97/
importin/karyopherin β and independent of GTP. At the
nuclear pore there are differences between the mechanisms
too, since nuclear transport of protein kinase C α cannot be
inhibited by wheat germ agglutinin or an antibody against
nuclear pore complex proteins. Together these findings
demonstrate that the nuclear import of protein kinase C α
occurs by a mechanism distinct from the one used by classical
nuclear localization signal-bearing proteins at several stages.
INTRODUCTION
translocates through the NPC in a GTP-dependent manner with
the help of two small proteins, the GTPase Ran/TC4 (Melchior
et al., 1993; Moore and Blobel, 1993) and the protein
p10/pp15/NTF-2 (Moore and Blobel, 1994; Paschal and
Gerace, 1995). Inside the nucleus the GTP form of Ran binds
to importin β and thereby displaces importin α as well as the
NLS-bearing protein (Rexach and Blobel, 1995; Görlich et al.,
1996). For a detailed review of the NLS-dependent nuclear
transport see Görlich and Mattaj (1996) and Nigg (1997).
In addition to the classical NLS-dependent protein import,
alternative ‘roads’ have been described (Pollard et al., 1996;
Sweitzer and Hanover, 1996; Michael et al., 1997) which differ
in several features, thus leading to an increasingly complex
picture of nuclear transport. The NLS-dependent transport (often
investigated using the SV 40 large-T-antigen-NLS chemically
crosslinked to a reporter protein like BSA-TRITC) can be
blocked by wheat germ agglutinin (WGA) (Adam et al., 1990)
and mAb 414, an antibody against the nuclear pore complex
protein p62 (Davis and Blobel, 1986; Marshallsay and
Lührmann, 1994). Furthermore the NLS-dependent transport is
dependent on GTP (Moore and Blobel, 1993; Görlich et al.,
1996) and independent of the cytoskeleton (Schmalz et al.,
1996). In another pathway, the shuttling hnRNP A1 protein is
directed to the nucleus by a 38 amino acid domain called the
Compartmentalization of the eukaryotic cell raises the question
of how both molecules and signals are transported across
cellular membranes. In the case of the cell nucleus, proteins
have to be transported in both directions through the nuclear
pores which span the two membranes constituting the nuclear
envelope. In recent years considerable progress has been made
in understanding nuclear import of proteins bearing a
‘classical’ nuclear localization signal (NLS) which consist of
one or two clusters of basic amino acids (Dingwall and Laskey,
1991). A variety of factors required for this multistep process
have been identified.
The present understanding is that the first step of nuclear
import of a protein bearing a classical NLS is the recognition
of the NLS by an NLS receptor in the cytoplasm. This NLSreceptor is called importin α or karyopherin α (Görlich et al.,
1994; Moroianu et al., 1995) (for additional synonyms and
names of yeast homologues see Nigg, 1997).
The cargo-NLS receptor complex then interacts with
importin/karyopherin β (Görlich et al., 1995; Radu et al., 1995)
via the the importin-β-binding domain localized on importin α
(Görlich et al., 1996; Weis et al., 1996). The trimeric complex
subsequently docks onto the nuclear pore complex (NPC) and
Key words: Protein kinase C, Nuclear transport, Cell nucleus,
Phorbol ester
1824 D. Schmalz, F. Hucho and K. Buchner
M9-region (Pollard et al., 1996). In this case the NLS-receptor
is a protein called transportin, which is distantly related to
importin β. The transportin-dependent mechanism is sensitive to
WGA, energy-depletion and non-hydrolyzable GTP-analogs
(Pollard et al., 1996). Furthermore, both ways appeared to be
calcium independent. In a third mechanism, proposed by
Sweitzer and Hanover (1996), protein import depends on
calcium, calmodulin and ATP, but not on GTP. Very recently,
Michael et al. (1997) reported that shuttling of the hnRNP K
protein depends on a new type of shuttling domain (KNS,
hnRNP K nuclear shuttling domain). Nuclear import and nuclear
export of hnRNP K are independent of both GTP-hydrolysis and
any cytosolic factors (Michael et al., 1997). The picture becomes
even more complicated if one includes the findings about the
nuclear import of other cargo such as snRNPs: Michaud and
Goldfarb (1992) found that the import of several, but not all, U
snRNPs is sensitive to WGA. These investigators also found a
considerable transport of U3 snRNP even under conditions
which abolished NLS-BSA-TRITC transport by mAb 414.
Protein kinases may function as vehicles to transport
information. Changes in a kinase’s subcellular localization may
be seen as information transduction. The protein kinase C
family, presently made up of eleven known isoforms which
differ in their mechanism of activation (for review see Dekker
and Parker, 1994; Nishizuka, 1995), is one of the kinase
families involved in signal transduction. The presence of PKC
isoforms has been described for many cellular compartments,
and there are many examples of translocation to other
compartments, including the cell nucleus, upon various stimuli
(e.g. Leach et al., 1989; Goodnight et al., 1994; Disatnik et al.,
1994). This reinforces the fact that PKC isoforms play an
important role in signal transduction toward and within the cell
nucleus (for review see Buchner, 1995). Besides a stimulusdependent translocation of PKC into the nucleus, a constitutive
localization of PKC in this compartment has been described
(e.g. Beckmann et al., 1994; Disatnik et al., 1994).
No isoform of PKC contains any sequence resembling a
known NLS, so the means of nuclear transport of PKC remains
to be elucidated. To investigate PKC’s nuclear import we chose
a system which allows a rapid and strong induction of nuclear
uptake: the translocation of PKCα from the cytosol to the nucleus
occurring in NIH 3T3 fibroblasts after stimulation with the
phorbol ester PMA (Leach et al., 1989). There seem to be
remarkable differences between the transport mechanisms of
PKCα and an NLS-bearing reporter protein, since we find the
nuclear import of PKCα in these cells dependent on the
cytoskeleton’s integrity, whereas we don’t find any influence of
the cytoskeleton on the NLS-dependent nuclear transport
(Schmalz et al., 1996). In our present study we compared the
NLS-dependent and the kinase’s pathways in more detail. To this
end we co-microinjected the reporter protein NLS-BSA-TRITC
with various additives known to affect the nuclear uptake of the
reporter protein and then looked for any alterations of the kinase’s
translocation in injected cells after stimulation with phorbol ester.
We can show that in contrast to the NLS-dependent
transport, nuclear import of PKCα is not blocked by an
antibody against importin/karyopherin β and is not inhibited
by the nuclear pore interacting compounds WGA and mAb
414. Furthermore, nuclear uptake of PKCα appears to be
independent of GTP, thus indicating that RanTC4 most
probably is not involved in this process. These striking
differences strongly suggest that PKCα may be transported
into the nucleus by a mechanism distinct from the NLSdependent nuclear transport.
MATERIALS AND METHODS
Tissue culture and drug treatment
The NIH 3T3 fibroblasts were a kind gift from Katrin Saar (Institut
für Humangenetik, Berlin). Cells were maintained in Dulbecco’s
modified Eagle’s medium (Cytogen), containing sodium pyruvate, 1.0
g/l glucose, 100 U/ml penicillin, 100 µg/ml streptomycin and 10%
fetal calf serum (Gibco) in a humidified atmosphere of 5% carbon
dioxide at 37°C. Cells were passaged every three days using
trypsin/EDTA (Gibco). They were incubated in serum-free medium
for approximately 4-6 hours prior to use in indirect
immunofluorescence or microinjection.
Exposure to PMA (phorbol 12-myristate 13-acetate, 160 nM for ten
minutes), colchicine (10 mM, one hour) or taxol (10 µM, one hour)
was carried out in serum-free medium (PMA and colchicine were
purchased from Sigma; taxol was purchased from RBI).
To induce energy depletion, cells seeded on multitest glass-slides
were incubated at 37°C for 30 minutes in phosphate buffered saline
(PBS, 137 mM sodium chloride, 2.7 mM potassium chloride, 8 mM
disodium
hydrogenphosphate
and
1.5
mM
potassium
dihydrogenphosphate) containing 10 mM sodium azide (to block the
respiratory chain) and 5 mM deoxyglucose (an inhibitor of glycolysis).
Following this, the cells were stimulated with 160 nM PMA for 10
minutes. To test the reversibility of the azide/deoxyglucose treatment,
the drug-containing solution was removed after 40 minutes and
replaced by serum-free medium. Cells were allowed to recover for 2
hours before they were stimulated with PMA.
Immunocytochemistry
To perform indirect immunocytochemistry, cells were seeded on
multitest glass slides. Cells were fixed with 3% paraformaldehyde in
PBS for 15 minutes and permeabilized with 80% methanol in PBS at
−20°C for twenty minutes. Blocking was performed using 3% bovine
serum albumin in PBS for 15 minutes. Antibodies were diluted in PBS
with 3% BSA at the following dilutions: anti-PKC-α (Upstate
Biotechnology Incorporated, Lake Placid) at 1:100 for 45 minutes, or
anti-β-tubulin (Boehringer) at 1:250 for 45 minutes. Goat anti-mouse
Fc-Cy2 (Dianova, Hamburg) was used as the secondary antibody at a
dilution of 1:400. Preparations were mounted in Fluoromount G (Serva)
and analyzed with a Leica TCS 4D confocal laser scanning microscope.
Preparation of NLS-TRITC-BSA fluorescent conjugate
Synthetic peptides containing the SV 40 large-T-antigen wild-type
nuclear localization signal (NLS; sequence: CGTGPKKKRKVGG)
were obtained from Bachem (Heidelberg) and as a kind gift from
Victor Tsetlin of the Shemyakin-Ovchinnikov Institute of Bioorganic
Chemistry in Moscow, Russia. Conjugation with TRITC-BSA
(Sigma) was performed as described (Adam et al., 1990). In short,
peptides were resuspended in 50 mM Hepes, pH 7.0, reduced with 50
mM dithiothreitol and desalted on a Sephadex G-10 column. TRITCBSA was activated with a 20-fold molar excess of the sulfo-SMCC
(sulfosuccinimydyl
4-(N-maleimidomethyl)
cyclohexane-1carboxylate) crosslinking reagent (Pierce). Following the activation,
a 50-fold molar excess of the reduced peptides was added and the
mixture was incubated at 4°C in darkness overnight. The NLSTRITC-BSA conjugates were desalted on a Sephadex G-25 column,
dialyzed against transport buffer and the protein concentration was
adjusted to 1 mg/ml using the method of Bradford (1976).
Microinjection
Microinjection was used to investigate the accumulation of fluorescent
conjugates under various conditions in intact cells. Cells were grown
Nuclear import of protein kinase C 1825
on gridded coverslips (‘CELLocates’, Eppendorf, Germany) and coinjected with NLS-TRITC-BSA or TRITC-BSA (Sigma) and the
additive indicated using a manual system with Femtotips (Eppendorf,
Germany). The injection pressure was 250 hPa, the injection time was
700 milliseconds. Approximately 10−15 to 10−14 liters of the protein
solution (1-10% of total cellular volume) were injected into each cell.
For injection a solution containing 1 mg/ml NLS-BSA-TRITC in
transport buffer (110 mM potassium acetate, 20 mM Hepes, pH 7.3, 5
mM sodium acetate, 2 mM magnesium acetate, 1 mM EGTA, 2 mM
dithiothreitol) was blended with either a solution containing 20 mg/ml
WGA in a ratio of 1:4, or a solution containing 250 mM GMP-PNP
in a ratio of 1:5 and centrifuged at 13,000 rpm in an Eppendorf
centrifuge for 15 minutes prior to injection. To monitor the inhibitory
effect of the monoclonal antibody against p97 (Alexis, San Diego), the
antibody solution was dialyzed against transport buffer and blended in
a ratio of 1:1 with the NLS-BSA-TRITC solution. mAb 414 (1 mg/ml;
Babco, Lakeside) was not dialyzed but blended with NLS-BSATRITC in a ratio of 1 plus 10 and used directly for microinjection.
For a typical experiment, cells were allowed to sit for 20 minutes
at 5% carbon dioxide and 37°C in a humidified atmosphere after
injection. When stimulation with phorbol ester was carried out, PMA
was added to a final concentration of 160 nM 20 minutes after
injection and the preparation was allowed to stand for an additional
10 minutes. Thereafter, cells were fixed and indirect
immunocytochemistry to detect the subcellular distribution of PKC
was carried out as described above.
if the cytoskeleton is important in an earlier step of activation,
prior to the translocation step. To address this question we tried
to directly compare the behaviour of both NLS-BSA-TRITC
and PKCα in one cell using a monoclonal antibody against
p97/importin/karyopherin β. This antibody is described as
blocking nuclear uptake of NLS-BSA-TRITC in permeabilized
cells (Chi et al., 1995). Since this mAb was raised against a
protein from bovine erythrocytes, we first checked its
capability to also recognize its cognate in murine NIH 3T3
RESULTS
Participation of the cytoskeleton in PKCα nuclear
uptake
In an earlier study (Schmalz et al., 1996) we found that in NIH
3T3 fibroblasts PKCα transport is dependent on intact
cytoskeleton. Since the necessity of intact microtubules for the
PKC-transport raises the question of whether or not some kind
of vesicular transport might be involved, we examined the
effect of the microtubule-stabilizing drug taxol on PKCα
nuclear transport. Taxol is known to decrease the level of
vesicular transport (Hamm-Alvarez et al., 1994).
We first checked the effects of both colchicine and taxol on
the tubulin-cytoskeleton of 3T3 fibroblasts. The concentration
of colchicine that we used results in an almost complete
destruction of the microtubules. Compared to the effects of
colchicine, the effects of taxol appeared to be less drastic.
Nevertheless, morphological changes which have been
described elsewhere (Schiff and Horwitz, 1980), such as
rounding up of many cells and bundling of microtubules, could
be observed (data not shown). Treatment of control cells with
PMA induced nuclear accumulation of PKCα (Fig. 1A,B).
Inducing the kinase’s transport by treatment with PMA after
incubation with colchicine or taxol, we found that the transport
is heavily reduced after pretreatment with colchicine (Fig.
1C,D) but unaffected in the case of taxol (Fig. 1E,F). This also
indicates that the inhibitory effect of microtubule-disrupting
agents is not due to blockage of vesicular transport.
Involvement of importins/karyopherins α/β and
GTP/Ran in nuclear transport of PKCα
The striking differences concerning the importance of the
cytoskeleton’s integrity in nuclear accumulation of NLS-BSATRITC and PKCα raise the question of whether PKCα is
transported into the nucleus via a novel pathway, independent
of the well-characterized importin/Ran GTP-dependent way, or
Fig. 1. Influence of the cytoskeleton on PKCα’s nuclear
accumulation. NIH 3T3 fibroblasts were either untreated (A,B) or
treated with 10 mM colchicine (C,D) or 10 mM taxol (E,F) for 60
minutes before stimulation with 160 nM PMA (10 minutes), fixed,
processed for immunocytochemistry and analyzed with confocal
laser scanning microscopy as described in Materials and Methods.
(A,C,E) Cells show normal, cytosolic distribution of PKCα before
stimulation with PMA. (B) The kinase’s translocation into the
nucleus upon stimulation with PMA for 10 minutes in control cells
without pretreatment is visible. This translocation is inhibited by
pretreatment with 10 µM colchicine (C,D), whereas pretreatment
with 10 µM taxol has no significant influence on PKCα’s nuclear
accumulation (E,F). Bar, 10 µm.
1826 D. Schmalz, F. Hucho and K. Buchner
cells by western blotting. In fact, at a dilution of 1:1,000, the
mAb recognized a single protein of an apparent molecular
mass of 90 kDa (data not shown).
If, as in the preparations shown in Fig. 2A and B, only the
reporter protein NLS-BSA-TRITC is injected, the cells show the
expected nuclear accumulation of the fluorescent protein (Fig.
2A). Fig. 2B reveals that the normal PKCα distribution is not
affected, even if NLS-BSA-TRITC is accumulated in the nucleus
(Fig. 2A), indicating that microinjection itself does not alter the
subcellular distribution of PKCα in NIH 3T3 fibroblasts.
Co-injection of mAb p97 and NLS-BSA-TRITC totally
abolished NLS-dependent protein import in resting (Fig. 2C), or
PMA-stimulated (Fig. 2E) cells. The distribution and overall
staining of NLS-BSA-TRITC was not significantly different for
non-stimulated and PMA-stimulated cells (Fig. 2C,E). In contrast
to the bright nuclear signals in the control situation (Fig. 2A), we
observed a punctate pattern in the cytoplasm of generally very
weak intensity, in some cases making it difficult to obtain clear
pictures. Apparently, NLS-BSA-TRITC, if not transported into
the nucleus, accumulates in cytoplasmic organelles and is rapidly
degraded. The PKCα distribution is normal in control cells (Fig.
2D), but treatment with PMA results in an accumulation of the
kinase in the nucleus, despite the presence of mAbp97 inside the
cells (Fig. 2F). These data suggest that PKCα transport is
independent of p97/importin/karyopherin β.
To investigate the GTP-requirement of the nuclear uptake of
PKCα we used the two non-hydrolyzable GTP-analogs
5′guanylylimidodiphosphate (GMP-PNP) (Fig. 3.1) and GTPγ-S (Fig. 3.2). After co-microinjection of GMP-PNP and NLSBSA-TRITC we found PKCα in the cytoplasm in resting (Fig.
3.1B), and a nuclear accumulation in PMA-stimulated cells
(3.1D). In contrast to this, we found a complete inhibition of
the nuclear uptake of NLS-BSA-TRITC in both resting and
PMA-stimulated cells (Fig. 3.1A,C). The same experiment was
carried out using GTP-γ-S as non-hydrolyzable-GTP-analog.
The result was the same as that with GMP-PNP: total inhibition
of NLS-dependent transport, but no interference with PKCα
transport (Fig. 3.2), showing that the nuclear import of PKCα
is independent of GTP.
Energy requirement of PKCα transport
Since our experiments shown in Fig. 3 ruled out GTP’s
involvement in PKCα transport, only two explanations for the
observed nuclear uptake remain possible: (a) passive
diffusion, or (b) active transport via an ATP-dependent
alternative pathway. In order to exclude the first possibility we
examined the kinase transport under energy-depletion
conditions. In contrast to control cells (Fig. 4A,B), cells which
were depleted of cellular energy by incubation for 30 minutes
in PBS containing 5 mM deoxyglucose and 10 mM sodium
azide (Fig. 4C,D), show some morphological changes,
probably due to the drug treatment, and are unable to transport
the kinase into the nucleus after stimulation with phorbol ester.
However, after removal of azide and deoxyglucose and
recovery in medium for 2 hours, PMA induced nuclear
translocation of PKCα again (Fig. 4E,F), indicating that the
blockage of nuclear transport is indeed due to energy depletion
and not to general cell damage.
PKCα behavior at the nuclear pore
Many studies have used wheat germ agglutinin (WGA) to
Fig. 2. Nuclear translocation of PKCα is not inhibited by an antip97/karyopherin/importin β antibody. The TRITC-fluorescence
(A,C,E) and the CY-2-fluorescence representing PKCα’s subcellular
distribution (B,D,F) were monitored by confocal laser scanning
microscopy. (A,B) Control: microinjection of NLS-BSA-TRITC
alone results in a nuclear accumulation of this reporter protein (A),
but leaves the cytosolic distribution of PKCα unaffected (B). Cells
were co-microinjected with NLS-BSA-TRITC and an antibody
against p97/importin/karyopherin β (C-F) and then treated with PMA
(E,F), or left untreated (C,D). The nuclear PKCα localization after
PMA stimulation despite microinjection of anti-p97 antibody (F)
indicates that PKCα transport into the nucleus is independent of
p97/importin/karyopherin β. Bar, 10 µm.
block the function of the nuclear pores in import of NLSBSA-TRITC in permeabilized cells (e.g. Adam et al., 1990;
Moore and Blobel, 1992) or the import of snRNPs in
microinjected oocytes (e.g. Michaud and Goldfarb, 1992). We
therefore co-microinjected NLS-BSA-TRITC and WGA
(ratio 1:5) into NIH 3T3 fibroblasts and analyzed the
subcellular distribution of both the reporter protein and PKCα
after cells were treated with PMA. In Fig. 5C it can be seen
that in the presence of WGA and PMA NLS-BSA-TRITC is
not transported into the nucleus but rather accumulated, and
Nuclear import of protein kinase C 1827
most likely degraded, in cytoplasmic vesicles. In contrast to
this, Fig. 5D clearly shows the nuclear accumulation of
PKCα after treatment with phorbol ester, despite the presence
of WGA. Fig. 5A and B, respectively, show that in non-PMAtreated cells the inhibitory effect of WGA on nuclear
transport of NLS-BSA-TRITC is also present, and that the
PKCα distribution is cytoplasmic, as it normally is for
unstimulated cells.
Since WGA seems to block only the function of nuclear
pores in the protein import process, still allowing some import
of several snRNPs, we checked the effect of a second poreinteracting compound, the mAb 414 (Davis and Blobel, 1986).
This mAb is known to bind to the nuclear pore complex
proteins p62 and to other nuclear pore complex proteins
containing the FXFXG-motif and to abolish nuclear import in
most cases, but does not completely block the import of U3
snRNP (Marshallsay and Lührmann, 1994; Michaud and
Goldfarb, 1992). We co-microinjected NLS-BSA-TRITC and
mAb 414, and, in analogy to experiments described above,
found a total inhibition of NLS-BSA-TRITC transport in both
stimulated and unstimulated cells (Fig. 6A,C). In contrast to
this, the same cells show nuclear accumulation of PKCα after
treatment with phorbol ester (Fig. 6D). Again, the cytoplasmic
distribution of PKCα without stimulation is unaffected by the
microinjection-procedure (Fig. 6B).
1) GMP-PNP
DISCUSSION
Taken together, our data suggest that nuclear transport of
PKCα in NIH 3T3 fibroblasts depends on metabolic energy
and occurs via a pathway distinct from the one used by
classical NLS-bearing proteins at several stages. In contrast to
this pathway, nuclear import of PKCα appears to be
independent of p97/importin/karyopherin β and independent of
GTP. Also at the nuclear pore there are differences between the
mechanisms, since nuclear transport of PKCα cannot be
inhibited by WGA or mAb 414. Another striking difference
between the NLS-dependent transport and the PKCα-pathway
is the latter’s dependency on intact cytoskeleton.
Role of the cytoskeleton
Studying the taxol-effect on nuclear transport of PKCα, we
expand our previous findings (Schmalz et al., 1996). The
inhibition of transport by destroying the cytoskeleton might
indicate that vesicular transport is involved. Since taxol is
described only as reducing, but not entirely abolishing,
vesicular transport (Hamm-Alvarez et al., 1994), this
possibility still cannot be entirely ruled out, but has become
much less probable in the light of our observation that taxol
does not inhibit nuclear import of PKCα. Since there are
numerous reports of interactions between cytoskeletal
2) GTP-γ-S
Fig. 3. Non-hydrolyzable GTP-analogs do not inhibit PKCα’s nuclear transport. Cells were co-microinjected with either GMP-PNP and NLSBSA-TRITC (part 1), or GTP-γ-S and NLS-BSA-TRITC (part 2). Cells were left without stimulation with PMA (A,B) or were stimulated with
160 nM PMA for 10 minutes (C,D). The nuclear accumulation of PKCα after PMA stimulation despite microinjection of GMP-PNP (1D) or
GTP-γ-S (2D), respectively, reveals the independence of PKCα transport from GTP. Bars, 10 µm.
1828 D. Schmalz, F. Hucho and K. Buchner
Fig. 5. WGA does not block nuclear import of PKCα. Comicroinjection of WGA and NLS-BSA-TRITC prevents the nuclear
uptake of NLS-BSA-TRITC (A), again leaving PKCα’s distribution
unaltered (B). After stimulation with PMA the nuclear uptake of
NLS-BSA-TRITC is inhibited by WGA (C), but, in contrast to this,
PKCα is accumulated in the nucleus despite the presence of WGA
(D). Bar, 10 µm.
Fig. 4. PKCα’s nuclear transport requires energy. Confocal
micrographs showing the subcellular distribution of PKCα. Nuclear
import of PKCα after stimulation with phorbol ester, which is clearly
visible in control preparations (A,B), does not take place if cells were
depleted of energy by incubation with sodium azide and deoxyglucose
(C,D). Following 2 hours recovery in glucose-containing medium after
removal of azide and deoxyglucose, cells showed a PMA-induced
nuclear uptake of PKCα (E,F), indicating that the blockage of import
was not due to general cell damage. Bar, 10 µm.
components and PKC-isoforms (for review see Jaken, 1992),
it appears more likely that the cytoskeleton’s integrity plays a
role in the steps leading to the interaction with a hypothetical
PKC-binding and -transport protein (see below) rather than in
the following translocation step.
Dependence of metabolic energy
Depletion of metabolic energy by using sodium azide and
deoxyglucose abolished nuclear uptake of PKCα, which has a
molecular mass of 80 kDa. This observation is in accordance
with the general view that proteins with a molecular mass larger
than 40-50 kDa cannot enter the cell nucleus by diffusion through
the nuclear pore complex (Görlich and Mattaj, 1996; Nigg,
1997). However, some small proteins, such as histone H1, appear
to also be imported into the nucleus in an energy-dependent
manner (Breeuwer and Goldfarb, 1990). In this study it was
shown that treatment with carbonyl cyanide ptrifluoromethyloxyphenylhydrazone (FCCP, an inhibitor of
mitochondrial electron transport) and deoxyglucose blocked
nuclear import of histone H1. Comparable to our observations
on the blockage of PKC’s nuclear import, this effect could be
reversed by removal of the drugs (Breeuwer and Goldfarb, 1990).
In the case of PKCα it may be possible, although not very
probable, that, instead of (or in addition to) an energydependent process at the nuclear pore, an energy-dependent
step occurs during the release from a cytoskeletal anchor.
Role of cytosolic import factors
To compare the role of cytosolic import factors in the transport
pathways of NLS-BSA-TRITC and PKCα in intact cells we
microinjected an antibody against p97/importin/karyopherin β
known to block nuclear transport in permeabilized cells (Chi
et al., 1995). To study the transport behaviour of PKCα we
could not use a permeabilized-cell assay, which has been used
in most studies of nuclear protein import, because we found
that the standard permeabilizing procedure itself leads to a
change in the intracellular distribution of PKCα. In contrast to
this, microinjection leaves the kinase’s subcellular distribution
unaffected (e.g. Fig. 2B).
Although the antibody against p97 and the mAb against
PKCα were both mouse IgGs, we see clear differences in the
Nuclear import of protein kinase C 1829
nuclear immunoreactivity between PMA-treated and untreated
cells. Since, to current knowledge, p97/importin/karyopherin β
remains at the nuclear envelope during all steps of nuclear
import (Nigg, 1997) and anti-p97 yields a cytoplasmic and
nuclear envelope-staining in immunocytochemistry (Chi et al.,
1995), we can be quite sure that our data reflect the intra-nuclear
PKCα distribution, barely masked by p97 immunoreactivity.
Similar considerations are valid for mAb 414.
There is a growing family of importin/karyopherin βhomologs: ‘transportin’ (Pollard et al., 1996); ‘karyopherin β3’
(Yaseen and Blobel, 1997). This family’s growth supports the
hypothesis that importin/karyopherin β-homologs might be the
archetypical import factors and that importin/karyopherin α
plays a role as an adaptor for a certain kind of NLS (which is
surely not present in PKC). Because of the diversity among the
importin/karyopherin βs, our finding that anti-p97 does not
inhibit the PKCα transport does not entirely rule out an
involvement of an as yet unidentified cytosolic import factor
related to or similar to importin/karyopherin β.
Involvement of Ran/TC4
Our data strongly suggest that transport of PKCα is independent
of GTP and that the GTPase Ran/TC4 is most likely not involved.
Clearly Ran/TC4 is involved in the NLS-dependent transport
pathway, as well as in the NES-mediated nuclear export (NES,
nuclear export signal) (Richards et al., 1997) and much effort has
been spent investigating the function of Ran/TC4 in nuclear
Fig. 6. mAb 414 does not inhibit PKCα’s nuclear transport. Comicroinjection of mAb 414, a monoclonal antibody against nuclear
pore complex proteins, and BSA-NLS-TRITC results in an inhibition
of the reporter protein’s nuclear uptake both in unstimulated (A), and
in PMA-stimulated cells (C). Also, in this case the PKCα distribution
is cytosolic in unstimulated cells (B), but there is a strong nuclear
accumulation of the kinase after treatment with PMA (D). Bar, 10 µm.
transport in some detail (Melchior et al., 1993; Moore and Blobel,
1993; Görlich et al., 1996). But beside these findings there are
also reports of GTP-independent transport pathways. Sweitzer
and Hanover (1996) discovered a calcium- and ATP-dependent
import pathway of NLS-BSA-TRITC in HeLa cells. This
pathway is described as activated after release of intracellular
calcium in a calmodulin-dependent manner (Sweitzer and
Hanover, 1996). However, in contrast to PKCα import, this GTPindependent pathway was blocked by WGA, so that it appears
unlikely that the mechanism of PKC nuclear import is identical
to this calcium-activated mechanism. In addition to that, the
transport of sn RNPs in HeLa cells seems to be at least less
sensitive to non-hydrolyzable GTP-analogs (Palacios et al.,
1996), or even insensitive to this kind of inhibitors (Marshallsay
et al., 1996) when compared to the transport of NLS-BSATRITC. Recently, Michael et al. (1997) described the import of
hnRNP K in HeLa cells as being independent of GTP. Together,
these findings exemplify that GTP-hydrolysis is not a general
feature of transport processes across the nuclear envelope.
Apparently, the mechanism of PKC import belongs to the
growing group of GTP-independent transport mechanisms.
Behavior of PKC at the nuclear pore
To investigate the behavior of PKCα at the nuclear pore we
used two components known to inhibit the transport of NLSBSA-TRITC at the pore level: WGA and mAb 414, a
monoclonal antibody directed against the nucleoporins
containing the FXFXG motif (Davis and Blobel, 1986). mAb
414 is described as blocking nuclear transport as well as or
even better than WGA, but since there are reports that the
transport of U3 snRNP in Xenopus oocytes is not abolished
(although reduced) (Michaud and Goldfarb, 1992), it cannot be
concluded that mAb 414 blocks the pore completely. Within
this framework, our finding that PKCα-transport cannot be
blocked by mAb 414 does not exclude transport through the
nuclear pore. But, obviously, the transport pathway used by the
kinase is, also at this point, mechanistically different from the
pathway used by classical NLS-bearing proteins. This
interpretation leaves open the question whether the means of
transport are different, or if functionally different nuclear
pores, used by different classes of import cargo, exist.
A possible model
We found that transport of PKCα differs from transport of
NLS-BSA-TRITC at several steps, but the question remains as
to which ‘road’ the kinase takes into the nucleus. The simplest
model is that PKCα has a unknown, certainly non-canonical
NLS which is hidden in inactive PKC. Activation of PKC is
accompanied by a change in conformation (Newton, 1995)
leading to the exposure of regions in the protein which are not
accessable in the inactive state. An unknown nuclear import
factor then may be able to bind to the NLS exposed after
activation. Based on our data on the role of the cytoskeleton in
the nuclear translocation of PKCα we find it attractive to
speculate that the interaction of a putative import factor and
PKCα in the cytoplasm is dependent on the cytoskeleton’s
integrity.
The nuclear import factor transporting PKC may be
considered as a special form of a binding protein for activated
PKC that is responsible for targeting the kinase toward the cell
nucleus. Several PKC-binding proteins have already been
1830 D. Schmalz, F. Hucho and K. Buchner
identified (Mochly-Rosen et al., 1991; Ron et al., 1994; Hyatt et
al., 1994; Staudinger et al., 1995) which may function as
targeting factors. However, none of these so far has been
implicated in PKCα’s nuclear translocation. Once in the nucleus,
PKC-binding proteins may help to keep the enzyme there.
Indeed, we found nuclear proteins that bind activated PKCα in
a blot overlay assay (U. Rosenberger et al., unpublished).
To identify the nuclear import factor/PKC-binding protein a
functional assay using permeabilized cells is needed which
would allow adding back cytosolic components. Presently, we
are trying to establish such an assay in our lab.
This work was supported by the Deutsche Forschungsgemeinschaft
(Sfb 515) and the Fond der Chemischen Industrie. Excellent technical
assistance from Doris Krück is gratefully acknowledged.
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