From the Biochemistry Department, Institute of Medical Biology, University of Tromsø, 9037 Tromsø, Norway
Received for publication, November 15, 2000, and in revised form, December 12, 2000
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ABSTRACT |
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The atypical protein kinase C (PKC) isoenzymes,
The protein kinase C
(PKC)1 family of
lipid-dependent serine/threonine kinases plays pivotal
roles in a wide variety of cellular processes (reviewed in Refs. 1-3).
Based on sequence homology, domain organization, and biochemical
properties, 10 different isoforms are grouped into three classes
denoted classical, novel, and atypical PKCs. Interestingly, in addition to cytoplasmic proteins nuclear proteins
also have been reported to act as substrates for aPKCs (31-33). The
RNA-binding protein nucleolin is phosphorylated by Specific targeting of signaling protein kinases to subcellular
compartments offers an important level of regulation in which the
accessibility of their specific activators and substrates can be
spatiotemporally limited. The presence of conserved nuclear localization signals (NLSs) allows rapid import to the cell nucleus via
the formation of trimeric NLS-importin Here we have studied the subcellular localization of Cell Cultures--
HeLa cells (ATCC CCL2) were grown in Eagle's
minimum essential medium supplemented with 10% fetal calf serum,
nonessential amino acids, 2 mM L-glutamine,
penicillin (100 units/ml), and streptomycin (100 µg/ml) (Life
Technologies, Inc). HEK293 cells were maintained in Dulbecco's
modified Eagle's medium supplemented with 10% fetal calf serum, and
the antibiotics described above. Subconfluent HeLa and HEK293 cells
were transfected using the calcium-phosphate coprecipitation method.
For the nuclear export experiments, leptomycin B, kindly provided by
Dr. M. Yoshida, Tokyo, was added to the medium to a final concentration
of 2 ng/ml.
Plasmid Constructions and Site-directed Mutagenesis--
The
murine Subcellular Localization Analyses and
Immunocytochemistry--
For the subcellular localization studies of
the different GFP fusion proteins, HeLa cells were seeded in 6-well
dishes at a density of 5 × 104 cells per well 24 h before transfection. The cells were transfected with 1 µg of
expression vectors for the different GFP fusions. The subcellular
localizations of the GFP fusion proteins in living cells were
visualized by fluorescence microscopy using a Leitz DMIRB invert
microscope equipped for fluorescence and with a Leica DC100 digital
camera. For DAPI staining the cells were fixed and permeabilized in 4%
paraformaldehyde, 0.01% Triton X-100 for 10 min at 4 °C, and the
DNA was stained with 1 µg/ml DAPI (Sigma) for 5 min at room
temperature. HA- Preparation of Cytosolic/Nuclear Extracts and Immune Complex
Kinase Assays--
The activities of the different GFP- Western Blot Analyses--
HeLa cells were seeded at 4 × 105 per 100-mm dish the day before transfection. The cells
were transfected with 10 µg of the different GFP- Different Subcellular Localization of Wild-type
To determine the subcellular localization of
Phosphorylation of a conserved threonine residue within the activation
loop of all PKCs is crucial for subsequent autophosphorylation and
activation of the enzyme (54). Substitution of this threonine in The Zinc Finger Domain of
The sequence identities between the zinc finger domain of
The zinc finger domain of the aPKCs does not contain classical
monopartite or bipartite NLSs. However, Leptomycin B Treatment Induces Nuclear Accumulation of
Characterization of an NES within Intramolecular Interactions between the N-terminal Pseudosubstrate
Sequence and the Catalytic Domain Inhibit Nuclear Localization of
Nuclear Import of Nuclear localization of classical and novel PKCs as well as aPKCs
has been observed previously (8, 35, 58-64). However, to our
knowledge, this is the first report where functional NLS and NES
sequences are identified within any PKC. We find that Our results suggest that the core of the NLS of Based on sequence analyses it has been suggested that both the
classical and the aPKCs may contain a bipartite NLS (35). For We have found that in contrast to wild-type As mentioned in the Introduction, nuclear localization of both We find that In a recent study Sanchez et al. (57) reported that /
- and
PKC, play important roles in cellular signaling
pathways regulating proliferation, differentiation, and cell survival.
By using green fluorescent protein (GFP) fusion proteins, we found that
wild-type
PKC localized predominantly to the cytoplasm, whereas both
a kinase-defective mutant and an activation loop mutant accumulated in
the nucleus. We have mapped a functional nuclear localization signal
(NLS) to the N-terminal part of the zinc finger domain of
PKC.
Leptomycin B treatment induced rapid nuclear accumulation of GFP-
as
well as endogenous
PKC suggesting the existence of a
CRM1-dependent nuclear export signal (NES). Consequently,
we identified a functional leucine-rich NES in the linker region between the zinc finger and the catalytic domain of
PKC. The presence of both the NLS and NES enables a continuous shuttling of
PKC between the cytoplasm and nucleus. Our results suggest that the
exposure of the NLS in both
- and
PKC is regulated by
intramolecular interactions between the N-terminal part, including the
pseudosubstrate sequence, and the catalytic domain. Thus, either
deletion of the N-terminal region, including the pseudosubstrate sequence, or a point mutation in this sequence leads to nuclear accumulation of
PKC. The ability of the two atypical PKC isoforms to
enter the nucleus in HeLa cells upon leptomycin B treatment differs
substantially. Although
PKC is able to enter the nucleus very
rapidly,
PKC is much less efficiently imported into the nucleus.
This difference can be explained by the different relative strengths of
the NLS and NES in
PKC compared with
PKC.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
PKC and
/
PKC
constitute the atypical PKCs (aPKCs). In contrast to the classical and
novel PKCs that contain two repeated diacylglycerol (DAG)-binding zinc
finger domains within their regulatory domains, the aPKCs have only a
single zinc finger domain that is unable to interact with DAG or
phorbol esters (4, 5). Consequently, they do not require DAG for their
activation. Atypical PKCs have instead been shown to be regulated
in vitro and in vivo by other lipid products such
as ceramide (6, 7) and phosphatidylinositol 3,4,5-trisphosphate, a
product of phosphatidylinositol 3-kinase (PI 3-kinase) (8-10). Consistently, aPKCs are strongly implicated as downstream effectors of
PI 3-kinase (8, 10-14). Recently, evidence has accumulated that imply
important roles for aPKCs in processes as diverse as proliferation (15,
16), differentiation (17-19), cell polarity (reviewed in Ref. 20),
insulin-mediated up-regulation of general protein synthesis (21),
glucose transport (22-24), up-regulation of
2 integrin
gene expression (25), and cell survival (26-30).
PKC in response
to nerve growth factor (NGF) treatment of PC12 cells (33).
Heterogeneous ribonucleoprotein-A1, another RNA-binding protein
involved in splicing and mRNA transport, is also a substrate of
PKC (31). Both nucleolin and heterogeneous
ribonucleoprotein-A1 shuttle between the cytoplasm and the nucleus. The
ubiquitously expressed transcription factor Sp1 is able to form a
complex with
PKC. In fact,
PKC phosphorylates Sp1 within the
DNA-binding domain and stimulates Sp1-mediated transactivation of the
vascular permeability factor/vascular endothelial growth factor
promoter (32). Nuclear localization of both
- and
PKC has been
demonstrated. NGF stimulation of PC12 cells led to rapid and transient
translocation of
PKC from the cytoplasm to the nucleus (33-35). In
resting HepG2 cells ectopically expressed
PKC was found both in the
cytoplasm and in the nucleus (8). Upon stimulation with either
platelet-derived growth factor or epidermal growth factor, the nuclear
pool of
PKC translocated in a wortmannin-sensitive manner to the
cytoplasm and to more compact structures within the nucleus.
-importin
complexes (36-40). During the last few years, short leucine-rich nuclear export
signals (NESs) have been identified within a variety of proteins like
human immunodeficiency virus-1 Rev (41), PKI (42), mitogen-activated
protein kinase/extracellular signal-regulated kinase kinase (43),
mitogen-activated protein kinase-activated protein kinase-2
(MAPKAP-kinase 2) (44), cyclin B (45), and phospholipase C-
1 (46).
NES-dependent nuclear export is inhibited by leptomycin B
that interferes with the binding of NES to CRM1/exportin 1 (47-51).
- and
PKC in
living cells using green fluorescent protein (GFP) fusion proteins. We
find that a kinase-defective mutant of
PKC accumulates in the cell
nucleus, whereas the wild-type kinase is mainly cytosolic. Inhibition
of CRM1-dependent nuclear export using leptomycin B leads
to rapid nuclear accumulation of both GFP-
and endogenous
PKC. By
deletion studies and site-directed mutagenesis, we identified both a
functional NLS and an NES in
PKC. These signals endow
PKC with
the ability to shuttle continuously between the cytoplasm and the
nucleus. Our results are compatible with the notion that the exposure
of the NLS in both
PKC and
PKC may be regulated by intramolecular
interactions between the N-terminal region and the catalytic domain of
the kinases. Also, we find that
PKC is much less efficiently
imported into the nucleus than
PKC in HeLa cells upon blockade of
nuclear export by leptomycin B treatment. This is most likely due to
differences in the relative strengths of the NES and NLS in the two
atypical PKCs.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
PKC cDNA was amplified from a mouse brain cDNA
library (Marathon Ready, CLONTECH) by PCR using
ExTaq polymerase (Takara Biomedicals). The PCR product was made blunt
and subcloned into the SmaI site of pUC18. Inspection of the
cDNA sequences show that both the murine
PKC and the human
PKC cDNAs actually contain an in frame ATG codon nine codons
upstream of the proposed start codon (5, 52). The Xenopus
PKC also contains this start codon eight amino acids upstream of the
second ATG codon. Since this is the first in frame ATG and the amino
acid sequences of this N-terminal extension also are conserved between
the species, we suggest that this most 5' ATG is the start codon of
/
PKC. Consequently, the numbering system used in this paper is
based on this. To generate pHA-
, pUC18-
PKC was digested with
EcoRI and XbaI, and the fragment containing the
coding region for full-length
PKC (amino acids 1-595 in our
numbering system) was inserted into the corresponding sites of
pcDNA3-HA. pcDNA3-HA was a kind gift from Dr. Jorge Moscat and
contains an influenza hemagglutinin (HA) epitope tag inserted into the
HindIII-EcoRI sites of pcDNA3 (Invitrogen).
pGFP-
was made from pHA-
by inserting the
EcoRI-XbaI (blunted) fragment encoding
PKC
into the EcoRI-SmaI sites of pEGFP-C1 vector
(CLONTECH). pGFP-
was made from pHA-
(kindly provided by dr. Jorge Moscat) by inserting an
EcoRI-XbaI (blunted) fragment encoding rat
PKC
into the EcoRI-SmaI sites of pEGFP-C1. The
expressions plasmids for HA-
K282W, GFP-
K282W, GFP-
T411A, GFP-
T411E, GFP-
K282W-R150E/R151E,
GFP-
(141-162)R150E/R151E, GFP-
F253A/L255A, GFP-
A129E,
GFP-
K281W, and GFP-
F252A/L254A were made by site-directed
mutagenesis according to the instruction manual for the Quick-Change
Site-directed Mutagenesis Kit (Stratagene) using the
PKCK282W,
PKCT411A,
PKCT411E,
PKCR150E/R151E,
PKCF253A/L255A,
PKCA129E,
PKCK281W, and
PKCF252A/L254A mutagenesis primers (Table I). The GFP-
and GFP-
deletion mutants were made using the following strategy. Different
parts of the
- or
PKC cDNAs were amplified by PCR using
primers that contained recognition sequences for specific restriction
enzymes. The PCR products were purified and digested with restriction
enzymes and inserted into the corresponding sites of pEGFP-C1. All
constructs were verified by sequencing. All GFP-
/-
constructs are
named according to the included parts of either
PKC or
PKC with
the amino acid positions shown in parentheses. To study the
localization of the regulatory domain of
PKC, two different
constructs were made encoding fusion proteins where GFP was either
fused to the N-terminal end or the C-terminal end. pUC18-reg
was
generated by PCR using pUC18-
PKC as template and the
PKCR.5pr and
256.3nt primers (Table I). The PCR product was made blunt and
subcloned into the SmaI site of pUC18. pUC18-reg
was
digested with NheI (blunted) and EcoRI, and the
reg
fragment was inserted into the ApaI (blunted) and
EcoRI sites of pEGFP-N1 (CLONTECH). To
make pGFP-
(1-256), preg
-GFP was cut with EcoRI and
BamHI, and the reg
fragment was cloned into the
corresponding sites of pEGFP-C1. pGFP-
(256-595) was made from a PCR
product generated by using pUC18-
PKC as template and the primers
PKCcat.5pr and
PKC.3nt. The PCR product was cut with
XbaI (blunted) and EcoRI and inserted into
SmaI-EcoRI digested pEGFP-C1. pGFP-
(1-139)
was constructed by inserting an EcoRI-MscI
fragment from pGFP-
(1-256) into the
EcoRI-SmaI sites of pEGFP-C1. pGFP-
(141-595)
was made by PCR using the
PKC141.5pr and the
PKC.3nt primers. The
product was cut with XbaI (blunted) and EcoRI and
cloned into the EcoRI-SmaI sites of pEGFP-C1.
pGFP-
(194-256) was constructed from a PCR product amplified using
the
194.5pr and
256.3nt primers, digested with EcoRI
and NheI (blunted), and inserted into the
EcoRI-SmaI sites of pEGFP-C1. To make
pGFP-
(141-194), the
141.5pr and
194.3nt primers were used,
and the PCR product was cut with EcoRI and BamHI
and inserted into the corresponding sites of pEGFP-C1. Exactly the same
strategy was used to make pGFP-
(1-194) and pGFP-
,(163-194)
using the primers
PKCR.5pr and
PKC194.3nt for pGFP-
(1-194)
and
PKC163.5pr and
PKC194.3nt for pGFP-
(163-194) (Table I).
pGFP-
(141-163) was made from pGFP-
(141-194) by inserting an
EcoRI-Eco0109I (blunted) fragment into
EcoRI-SmaI-digested pEGFP-C1. To make
pGFP-
(132-182) a cDNA fragment encoding the zinc finger domain
of
PKC was amplified from mouse brain cDNA library using the
m
PKC132.5pr and m
PKC182.3nt primers (Table I). The PCR product
was cut with BamHI and EcoRI before being
inserted into the corresponding sites of pEGFP-C1. To make
pGFP-
(37-88) and pGFP-
(37-155), containing one or both of the
zinc finger domains from murine
PKC fused to GFP, the same strategy
as for construction of pGFP-
(132-182) was used except that the
PKC37.5pr and
PKC88.3nt primers were used for pGFP-
(37-88)
and the
PKC37.5pr and
PKC155.3nt primers were used for
pGFP-
(37-155). pGFP-
(255-592) and pGFP-
(130-592) were made
from PCR products amplified using pHA-
PKC as template and the
cat.5pr and
PKC.3nt primers for pGFP-
(254-592) and primers
130.5pr and
PKC.3nt for pGFP-
(130-592). The PCR products were blunted, cut with EcoRI, and cloned into the
EcoRI-SmaI sites of pEGFP-C1. pGFP-
(1-255)
was made from a PCR product generated using the
PKC.5pr and
PKC255.3nt primers that was cut with EcoRI and
BamHI and inserted into the corresponding sites within
pEGFP-C1. pGFP-
(1-182) and pGFP-
(130-255) were generated
following exactly the same strategy as for pGFP-
(1-255) using the
PKC5.pr and
PKC182.3nt primers for pGFP
(1-182) and the
primers
PKC130.5pr and
PKC255.3nt for pGFP-
(130-255).
Sequences of oligonucleotides used as PCR primers for plasmid
constructions and site-directed mutagenesis
and HA-
K282W were detected using a monoclonal
anti-HA antibody (12CA5, Roche Molecular Biochemicals). Subconfluent
HEK293 cells in 24-well culture dishes were cotransfected with 0.4 µg
of the different GFP-
/-
-expressing plasmids and 0.4 µg of
vectors expressing either HA-
or HA-
K282W. Twenty four h later
the cells were fixed by adding freshly made paraformaldehyde directly
to medium to a final concentration of 4%. The cells were permeabilized
for 10 min on ice using methanol pre-chilled at
20 °C. The
aldehyde groups were quenched by incubating the cells with 10 mM glycine, pH 8.5, for 5 min at room temperature. The fixed cells were incubated with 3% pre-immune goat serum in
phosphate-buffered saline for 1 h at room temperature before
incubation with the anti-HA antibody diluted 1:500 in blocking solution
for 1 h at room temperature or overnight at 4 °C. The
immunostaining was developed using an Alexa 594-conjugated goat
anti-mouse IgG secondary antibody (Molecular Probes) diluted 1:500 in
blocking solution. Endogenous
PKC in HeLa cells was detected by
staining with an anti-
PKC antibody (clone 41, Transduction
Laboratories) at a dilution of 1:200.
/-
mutants and HA-
/-
were measured in total cellular extracts by
immune complex kinase assays using histone H1 as substrate.
Subconfluent cultures of HeLa cells in 100-mm diameter Petri dishes
were transfected with 10 µg of vectors expressing the different
GFP-
/-
mutants or HA-
/-
. The cells were harvested by
trypsinization 24 h post-transfection, and nuclear extracts were
prepared as described (53). All buffers contained 1 tablet per 10 ml of
Complete Mini, EDTA-free protease inhibitor mixture (Roche Molecular
Biochemicals), 1 mM sodium vanadate, and 10 mM
-glycerophosphate. Following isolation of the nuclei by
centrifugation, the supernatant containing the cytosolic extract was
removed. Finally, the cytosolic and nuclear extracts were mixed to give
total cellular extracts. Approximately equal amounts of the different
GFP- or HA-tagged proteins were immunoprecipitated from total cell
extracts as follows. Total cellular extracts from 100-mm culture dishes
were preincubated with unsaturated 50% gel-slurry solution of protein
A-Sepharose CL-4B beads (Amersham Pharmacia Biotech) for 20 min at
4 °C and then incubated for 2 h at 4 °C with either 1 µg
of anti-HA antibody (12CA5, Roche Molecular Biochemicals) or 2 µg of
polyclonal anti-GFP antibody (Molecular Probes) in a total volume of
400 µl in HA lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 1 mM EGTA, 1%
Triton X-100). Fifteen µl of bovine serum albumin-saturated 50%
gel-slurry was added to the samples before continuing the incubation
for 1 h at 4 °C. The samples were washed five times in HA lysis
buffer and once in
/
-kinase buffer (35 mM Tris-HCl,
pH 7.5, 10 mM MgCl2, 0.5 mM EGTA,
0.1 mM CaCl2). The complexes were resuspended
in 15 µl of kinase buffer containing 3 µg of histone H1
(Calbiochem), 60 µM ATP, and 2 µCi of
[
-32P]ATP and incubated at 30 °C for 20 min. The
kinase reactions were terminated by adding 3.8 µl of 5×
SDS-polyacrylamide gel electrophoresis load buffer, and the samples
were boiled immediately for 5 min. The samples were run on a 10%
polyacrylamide gel and electrotransferred to a nitrocellulose membrane
(Hybond ECL, Amersham Pharmacia Biotech). The phosphorylated proteins
were detected and quantitated using a PhosphorImager (Molecular
Dynamics). The amount of immunoprecipitated GFP-
/-
and HA-
was
determined by probing the membrane with the specific anti-
PKC
antibody or an anti-
PKC antibody (Upstate Biotechnology) that
recognizes both
PKC and
PKC. The chemiluminescence signals from
the blots were detected using a LumiImager F1 (Roche Molecular
Biochemicals) and quantitated as Boehringer light units using
the LumiAnalyst 3.0 software. The relative activities of GFP-
and
HA-
were determined as PhosphorImager units of phosphorylated
substrate divided on the Boehringer light units representing the amount
of kinase used.
/-
expression
vectors. Twenty four h post-transfection the cells were scraped
directly in 100 µl of 2× SDS-polyacrylamide gel electrophoresis gel
load buffer, boiled for 5 min, and sonicated briefly. The samples were
run on 10% SDS-polyacrylamide gels and blotted onto Hybond
nitrocellulose membranes. The membranes were blocked in 5% nonfat dry
milk in TBST (10 mM Tris-HCl, pH 8.0, 150 mM
NaCl, 0.1% Tween 20) for 1 h at room temperature and then incubated with the primary antibody diluted in TBST for 1 h at room temperature or overnight at 4 °C. The following primary
antibodies were used: anti-GFP, polyclonal (diluted 1:2000,
CLONTECH), anti-
PKC (0.1 µg/ml, clone 41, Transduction Laboratories), anti-
PKC (0.5 µg/ml, Upstate
Biotechnology, Inc.), and anti-HA (1 µg/ml, 12CA5, Roche Molecular
Biochemicals). The membranes were washed 6 times in TBST and incubated
with horseradish peroxidase-conjugated anti-rabbit IgG or anti-mouse
IgG secondary antibodies (0.2 µg/ml, Transduction Laboratories) for
1 h at room temperature. The washing step described above was
repeated, and the membranes were developed using the ECL system
following the instructions of the manufacturer (Amersham Pharmacia Biotech).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
PKC and Two
Mutants with Single Mutations in the Catalytic Domain--
Vectors for
expression of murine
PKC with either an HA epitope tag or enhanced
green fluorescent protein (GFP) fused to its N terminus were
constructed. The expressed proteins were denoted HA-
and GFP-
,
respectively. A point mutation was introduced into the ATP-binding site
to generate a kinase-defective mutant of
PKC,
K282W. To test if
the relatively large GFP moiety affected the kinase activity of
PKC,
immune complex kinase assays were performed to compare the ability of
HA-
and GFP-
to phosphorylate histone H1. As shown in Fig.
1, HA-
and GFP-
immunoprecipitated from transiently transfected HeLa cells showed similar activities with
GFP-
even being a bit more active than HA-
. The GFP fusion to the
ATP-binding site mutant (GFP-
K282W) showed no activity as expected.
This strongly suggests that the fusion of GFP to the N-terminal of
PKC has no significant effect on kinase activity.
View larger version (25K):
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Fig. 1.
The kinase activity of
PKC is not affected by the GFP fusion partner.
Subconfluent HeLa cells in 100-mm dishes were transfected with 10 µg
of either pEGFP-C1 vector or expression constructs for GFP-
,
GFP-
K282W, or HA-
. Twenty four h after transfection, whole cell
extracts were made by mixing nuclear and cytosolic extracts as
described under "Materials and Methods." GFP, GFP-
,
GFP-
K282W, or HA-
were immunoprecipitated (IP) using
an anti-GFP antibody or an anti-HA antibody, and their kinase
activities were determined using histone H1 as substrate. Protein
loading was determined by immunoblotting (WB) with an
antibody against
PKC. The relative activities represent the amount
of substrate phosphorylated and quantitated using a PhosphorImager
divided on the amount of kinase protein used in the assays quantitated
by means of a LumiImager.
PKC in living cells,
HEK293 and HeLa cells transiently transfected with vectors expressing
either GFP-
, GFP-
K282W, or GFP alone were analyzed by
fluorescence microscopy. GFP-
was mainly localized to the cytoplasm
in both cell lines although a fraction of the protein was detected in
the nucleus (Fig. 2A).
Surprisingly, the GFP-
K282W mutant was localized to the cell nucleus
in both cell types. The GFP protein alone was distributed diffusely
throughout both the cytoplasm and the nucleus. To see if HA-tagged
or
K282W displayed the same subcellular localization as their GFP
counterparts, HEK293 cells were cotransfected with GFP-
and
HA-
K282W or GFP-
K282W and HA-
. The subcellular localization of
HA-
and HA-
K282W was determined in fixed cells by
immunocytochemistry using an anti-HA monoclonal antibody. As shown in
Fig. 2B, HA-
was localized predominantly to the
cytoplasm, whereas HA-
K282W accumulated in the nucleus. The nuclear
localization of GFP-
K282W was also verified by confocal laser
microscopy of transiently transfected NIH 3T3 cells (data not shown).
Western blot analyses of whole cell extracts and immunostaining with a
monoclonal antibody recognizing specifically the C-terminal catalytic
domain of
PKC showed that the nuclear staining was due to
full-length protein and not caused by a proteolytic fragment containing
GFP and only part of
PKC (data not shown).
View larger version (51K):
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Fig. 2.
Different subcellular localization of
wild-type PKC and two mutants with no or
reduced activity. A, subcellular distribution of
GFP-
and kinase-defective GFP-
K282W in HeLa and HEK293 cells.
Subconfluent cultures of HeLa (upper panel) or HEK293
(lower panel) cells seeded in 6-well dishes were transfected
with 1 µg of vectors expressing either GFP alone, GFP-
, or
GFP-
K282W. The subcellular localization in living cells was
visualized by fluorescence microscopy. HEK293 cells expressing
GFP-
K282W were stained with DAPI to visualize the nuclei. The nuclei
of HeLa and HEK293 cells expressing GFP-
K282W are indicated by
arrows. B, similar subcellular distribution of
HA-tagged (red) and GFP-tagged versions of wild-type and
kinase-defective
PKC. HEK293 cells were seeded in 24-well dishes and
cotransfected with expression vectors (0.4 µg) for HA-
and
GFP-
K282W (upper panel) or HA-
K282W and GFP-
(lower panel). Twenty four h after transfection the cells
were fixed and stained with an anti-HA antibody and analyzed by
fluorescent microscopy. Arrows indicate the nuclei of
coexpressing cells. C, kinase activity of different GFP-
mutants. Whole cell extracts from transiently transfected HeLa cells
expressing either GFP alone, GFP-
, GFP-
K282W, GFP-
T411E, or
GFP-
T411A were subjected to immunoprecipitation (IP)
using an anti-GFP antibody. The kinase activities were determined using
histone H1 as substrate. Autophosphorylation of immunoprecipitated
proteins are indicated (32P-GFP-
).
The loading of immunoprecipitated proteins was visualized by
immunoblotting with an antibody cross-reacting with both
- and
PKC. D, subcellular localization of GFP-
T411E and
GFP-
T411A in transiently transfected HeLa cells.
PKC
(Thr-410) with an acidic amino acid created a constitutively activated
kinase, whereas replacing it with alanine severely reduced the
catalytic activity (13, 55). To test if mutation of the corresponding
Thr-411 site in
PKC affected the subcellular localization of the
kinase, GFP fusion proteins containing alanine (GFP-
T411A) or
glutamate (GFP-
T411E) substitutions at this site were expressed in
HeLa cells. The catalytic activity of the T411A mutant was significantly reduced compared with wild-type
PKC and the T411E mutant (Fig. 2C). Interestingly, similar to the
kinase-defective ATP-binding site mutant, GFP-
T411A was mainly
nuclear in living HeLa cells, whereas GFP-
T411E displayed a
predominantly cytosolic localization just as the wild-type
PKC (Fig.
2D). Taken together, these results indicate that the
ATP-binding site and the T411A mutations may somehow affect the overall
conformation of the protein so that signals governing subcellular
localization are exposed differently in these mutants compared with the
wild-type enzyme. However, as demonstrated below the observed nuclear
accumulation is not correlated to the activity status of the kinase.
PKC Contains a Nuclear Localization
Signal--
Proteins larger than about 40-60 kDa cannot enter into
the nucleus through the nuclear pore complex by passive diffusion (38, 39). Since both HA-
K282W (67 kDa) and GFP-
K282W (92 kDa) are too
large to diffuse into the nucleus, it seemed logical to assume that
PKC could contain a functional nuclear localization signal (NLS) or
be transported via interaction with a partner protein containing an
NLS. Thus, to map the region(s) of
PKC required for nuclear
localization, deletion mutants were made in the context of GFP fusion
proteins (Fig. 3A). Plasmids
expressing the different deletions were transfected into HeLa cells,
and the expression of GFP fusion proteins with correct sizes was
verified by Western blot analyses using an anti-GFP antibody (Fig.
3B). Fluorescence microscopy of living cells revealed that
GFP-
(256-595) corresponding to the catalytic domain of
PKC was
mainly localized diffusely in the cytoplasm (Fig. 3C). Due
to a low level of expression of this construct in HeLa cells,
GFP-
(256-595) was also transiently expressed in HEK293 cells.
Compared with the distribution in HeLa cells, GFP-
(256-595) was
even more excluded from the nucleus in HEK293 cells. In contrast, a
fusion protein containing the regulatory domain, GFP-
(1-256) was
primarily localized to the cell nucleus. We next analyzed which part of
the regulatory domain that was responsible for the observed nuclear
accumulation (Fig. 3C). GFP-
(1-139), containing the
first 139 amino acids of
PKC including the pseudosubstrate sequence,
was diffusely localized throughout the cell. The molecular mass
of this construct is ~42 kDa so the protein will probably enter the
nucleus by passive diffusion (38, 39). In contrast, a GFP fusion
protein corresponding to the zinc finger domain of
PKC,
GFP-
(141-194), was exclusively localized to the nucleus. This
fusion protein further accumulated in structures corresponding to the
nucleoli. These observations clearly suggest that the zinc finger
domain contains a functional NLS. Surprisingly, GFP-
(194-256),
containing the variable linker region between the zinc finger domain
and the ATP-binding site, was excluded from the nucleus. According to
the theoretical size of this fusion protein (about 35 kDa) one would
expect that it could enter the nucleus by diffusion. Therefore, this
fusion protein may be sequestered in the cytoplasm by an anchoring
protein, or it may be actively exported from the nucleus.
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Fig. 3.
Expression and subcellular localization of
GFP fusion proteins containing different parts of the
PKC protein. A, schematic
representation of different GFP-
constructs. The numbers
in parentheses refer to amino acid positions defining the
parts of the
PKC protein included in the fusions. B,
immunoblotting of GFP-
deletion mutants. HEK293 cells were
transfected with either pEGFP-C1 or 10 µg of expression vectors for
either GFP-
, GFP
(256-595), GFP
(1-256), GFP
(1-139),
GFP
(141-194), or GFP
(194-256). The cells were harvested 24 h post-transfection, and total cellular proteins were separated in 10%
SDS-polyacrylamide gels. The proteins were electrotransferred to a
membrane that was subsequently probed with an anti-GFP antibody.
C, subcellular localization of the indicated GFP
deletion
mutants.
PKC and
those of classical and novel PKCs vary from 35 to 48%, whereas there
is 74% identity between the zinc finger domains of
PKC and
PKC
(Fig. 4A). We therefore asked
whether the zinc finger domain of
PKC, like that of
PKC, was able
to direct a GFP fusion protein to the nucleus. To this end, HeLa cells
were transfected with a construct expressing a GFP fusion protein
containing the complete zinc finger domain of murine
PKC,
GFP-
(130-182). Indistinguishable from the results with the
corresponding
PKC construct, this fusion protein was exclusively
nuclear demonstrating that the zinc finger domain of
PKC also
contains an NLS (Fig. 4C). However, GFP fusion proteins
containing either the first or both zinc finger domains of the
classical isoform
PKC, GFP-
(37-88), and GFP-
(37-155) did not
accumulate in the cell nuclei but rather in punctate structures
in the cytoplasm (Fig. 4C). Thus, the ability to translocate
to the nucleus is not a conserved feature of PKC zinc finger
domains.
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Fig. 4.
The zinc finger region of atypical
PKCs contain a nuclear localization signal. A,
alignments of amino acid sequences of the zinc finger domains of murine
PKC (SwissProt accession number Q62074), rat
PKC (P09217), PKC3
from C. elegans (GenBankTM accession number
AF025666), murine
PKC (P28867), and murine
PKC (P20444). The
sequences of both zinc finger domains of
PKC are shown with
PKC-1
as the most N-terminal zinc finger. Asterisks above the
alignment denote the basic residues in the putative core NLS. The
location of the two Arg residues mutated to Glu is indicated
(EE). Conserved positions are indicated below the
alignment (b, basic; h, hydrophobic; and
a, aromatic). B, a three-dimensional structure
model of the zinc finger domain of murine
PKC is shown to the
right with the Arg-150 and Arg-151 residues indicated. The
side chains of other Arg and Lys residues are also shown
(blue). The model was obtained from the Swiss Model
Repository. The x-ray structure model of the second zinc finger of
PKC with the phorbol ester
12-O-tetradecanoylphorbol-13-acetate (in
space-filling mode) bound and the two zinc atoms is shown to the
left (77). C, subcellular localization of GFP
fusions containing zinc finger regions of
PKC and
PKC. Constructs
encoding GFP-
(130-182) containing the zinc finger region of murine
PKC, GFP-
(37-88) containing the first zinc finger of
PKC, and
GFP-
(37-155) containing both zinc fingers of
PKC were expressed
in HeLa cells. D, mutation of Arg-150 and Arg-151 within the
zinc finger of
PKC prevents nuclear accumulation of kinase-defective
PKC. The subcellular localization of GFP-
K282W and
GFP-
K282W-R150E/R151E in which Arg-150 and Arg-151 are
replaced by glutamate residues was analyzed in HeLa cells.
E, GFP-
(141-162) containing the first 22 amino acids of
the
PKC zinc finger accumulates in the nucleus, whereas the NLS
mutant GFP-
(141)-R150E/R151E is diffusely localized
throughout the cell.
PKC,
PKC, and PKC3 from
Caenorhabditis elegans contain a cluster of four basic amino acids in the N-terminal part of the zinc finger
(KRF/LNRR in Fig. 4A). This motif is
not found in the classical and novel PKCs as exemplified by the second
zinc finger of
PKC and both zinc fingers of
PKC in Fig.
4A. In a three-dimensional structure model of the zinc
finger domain of
PKC, constructed based on the solved structure of
the corresponding zinc finger of
PKC, these basic residues
are exposed on the surface. Particularly, Arg-150 and Arg-151 are
ideally positioned to interact with either lipid cofactors or other
proteins (Fig. 4B). To see if introduction of two acidic
amino acids in this sequence could interfere with the nuclear
localization of kinase-defective
PKC, Arg-150 and Arg-151 were
substituted with glutamate residues giving
GFP-
K282W-R150E/R151E. Interestingly, these mutations
abolished the nuclear accumulation of kinase-defective GFP-
K282W.
GFP-
K282W-R150E/R151E was either diffusely distributed
throughout the cells or totally excluded from the nucleus (Fig.
4D). Thus, Arg-150 and Arg-151 seem to be critically
involved in nuclear localization of
PKC. To see if the N-terminal
half of the
PKC zinc finger, including the critical arginine
residues, was sufficient for mediating nuclear translocation,
GFP-
(141-162) encoding a GFP fusion protein containing the first 22 amino acids of the zinc finger was made. GFP-
(141-162) accumulated
in the nuclei of transiently transfected HeLa cells (Fig.
4E). In contrast to the GFP fusion protein containing the complete zinc finger, GFP-
(141-162) did not accumulate in the nucleoli. Next, we mutated Arg-150 and Arg-151 in the context of the
GFP-
(141-162) protein. GFP-
(141-162)R150E/R151E
displayed the same diffuse subcellular distribution as GFP itself (Fig. 4E). Thus, our results strongly suggest that
PKC contains
a functional NLS within the first 22 amino acids of the zinc finger
domain and that Arg-150 and Arg-151 are critical residues within this NLS.
PKC--
To determine whether the predominantly cytoplasmic
localization of wild-type
PKC is caused by the presence of a
leucine-rich NES, GFP-
-transfected HeLa cells were treated with
leptomycin B (LMB). Interestingly, treatment with LMB for 2 h
induced nuclear accumulation of the fusion protein (Fig.
5A). To determine whether LMB
could induce nuclear accumulation of endogenous
PKC, HeLa cells were
either left untreated or treated with LMB for 2 h. The cells were
fixed and the subcellular localization of endogenous
PKC was
determined by immunocytochemistry using a specific anti-
PKC antibody. In untreated cells
PKC was diffusely localized mainly in
the cytoplasm with only a fraction of the protein in the nucleus (Fig.
5B, left panel). Importantly, LMB treatment induced
redistribution of endogenous
PKC from the cytoplasm to the nucleus
(Fig. 5B, right panel). The nuclear accumulation of
endogenous
PKC in response to LMB was rapid, being significant after
15 min and almost completed after 30 min (Fig. 5C). These
results demonstrate that
PKC is exported from the nucleus by a
mechanism either involving a functional cis-acting,
LMB-sensitive NES or via an NES-containing interaction partner.
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Fig. 5.
LMB induces nuclear accumulation of
GFP- and endogenous
PKC. A, HeLa cells were transfected
with expression vector for GFP-
as described in the legend to Fig.
3A. Twenty four h after transfection, cells were either left
untreated or treated with 2 ng/ml LMB for 2 h, and the subcellular
localization of GFP-
was determined. B, HeLa cells were
cultured in Eagle's minimum essential medium supplemented with 10%
fetal calf serum and either left untreated or treated with LMB (2 ng/ml) for 2 h. The cells were fixed, permeabilized, and stained
with an anti-
PKC antibody that shows no cross-reactivity against
PKC. C, an experiment performed similarly as in
B except that LMB treatment was for 0-, 15- and 30 min,
respectively.
PKC--
Since
GFP-
(194-256), despite of its small size, was completely excluded
from the nucleus (see Fig. 3C), we speculated that this part
of
PKC, corresponding to the linker region between the zinc finger
domain and the ATP-binding site, could be involved in active export
from the nucleus. Interestingly, we identified a region (amino acids
248-255) that displayed significant similarity to previously
identified NES sequences (Fig.
6A). To determine whether this
region mediates nuclear export of
PKC, we generated a mutant of
PKC in which two presumably critical hydrophobic amino acids,
Phe-253 and Leu-255, are replaced by alanine residues. Similar
mutations within NES sequences in other proteins abolish the function
of the NES (41-43, 45, 46). In contrast to the wild-type kinase,
GFP-
F253A/L255A accumulated in the nucleus (Fig. 6B).
This strongly suggests that cytoplasmic localization of
PKC is
conferred by an NES in the linker region between the regulatory and the
catalytic domains.
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Fig. 6.
Characterization of an NES within the linker
region of PKC. A, alignments
of NES sequences from mitogen-activated protein kinase/extracellular
signal-regulated kinase kinase (43), PKI (42), Rev (41)(18), PLC
1
(46), and the atypical PKCs. Important hydrophobic residues are
boxed. B, mutation of critical hydrophobic
residues within the NES (Phe-253 and Leu-255) blocks nuclear export of
PKC. HeLa cells were transiently transfected with expression vectors
for GFP-
or for GFP-
F253A/L255A, and the subcellular localization
was determined 24 h after transfection.
PKC--
To begin unraveling whether intramolecular interactions
between the N-terminal part and the catalytic domain might mask the NLS
in
PKC, a GFP-
fusion protein in which the first 140 amino acids
were deleted was transiently expressed in HeLa cells. Contrary to the
full-length, wild-type kinase, GFP-
(141-595) localized exclusively
to the nucleus (Fig. 7). Since this
fusion protein lacks the autoinhibitory pseudosubstrate sequence, the
kinase activity of GFP-
(141-595) was increased compared with the
wild-type enzymes (data not shown). To determine whether disruption of
the intramolecular interaction between the pseudosubstrate sequence and
the catalytic domain led to nuclear localization, a point mutant of
GFP-
in which Ala-129 within the pseudosubstrate sequence was
replaced by glutamate, GFP-
A129E, was generated and transiently expressed in HeLa cells. Such a mutant has previously been demonstrated to be constitutively activated presumably due to the lack of
interaction of the pseudosubstrate sequence with the substrate
interaction site in the catalytic domain (56). Consistent with the
notion of a conformational change exposing the NLS, GFP-
A129E
displayed nuclear accumulation (Fig. 7). Taken together, these results
and those presented earlier indicate that it is not the activity status of the kinase as such that determines the subcellular localization. Instead, intramolecular interactions between the catalytic domain and
the pseudosubstrate sequence inhibit the nuclear localization of
PKC
by inducing a conformation where the NLS is masked.
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Fig. 7.
Intramolecular interactions between the
N-terminal pseudosubstrate sequence and the catalytic domain inhibit
nuclear localization of PKC. HeLa cells
were transfected with the indicated GFP-
fusion constructs, and the
subcellular localization was determined 24 h following
transfection.
PKC Is Much Less Efficient Than That of
PKC
in LMB-treated HeLa Cells--
The aPKC subtypes,
PKC and
PKC,
have the same structural organization and display considerable sequence
homology especially within their catalytic domains. To establish if
PKC was similarly distributed within the cell as
PKC, we made a
vector expressing GFP fused to full-length
PKC, pGFP-
. GFP-
was exclusively localized to the cytoplasm in untreated cells.
Surprisingly, GFP-
did not accumulate in the nucleus upon LMB
treatment for 2 h but was distributed diffusely all over the cell
(Fig. 8A). However, after long
term treatment with LMB (16 h), GFP-
accumulated in the nuclei of the transfected cells (data not shown). Since we did not have antibodies available that will only recognize
PKC specifically, without detecting
PKC, we were not able to analyze the subcellular distribution of endogenous
PKC by immunocytochemistry. To test whether kinase-defective
PKC localized differently from wild-type
PKC, GFP-
K281W mutated in the ATP-binding site was expressed in
HeLa cells. Contrary to kinase-defective
PKC, GFP-
K281W was predominantly localized in the cytoplasm in a manner similar to wild-type GFP-
(Fig. 8C). Kinase assays performed
following immunoprecipitation of GFP-
or GFP-
K281W from extracts
of transiently transfected HeLa cells showed that GFP-
was active,
whereas GFP-
K281W had no intrinsic kinase activity (Fig.
8C). Similar to GFP-
, GFP-
was as active as an HA
epitope-tagged
PKC (data not shown). Next, we mutated the putative
NES sequence in GFP-
generating GFP-
F252A/L254A. Compared with
wild-type GFP-
, this construct distributed more diffusely all over
the cells in transiently transfected HeLa cells but in contrast to
GFP-
F253A/L255A, GFP-
F252A/L254A did not accumulate in the
nucleus (data not shown). Taken together these results indicate that
the nuclear import of
PKC is much less efficient than that of
PKC. To sort out if this is due to intrinsic differences in the
relative strength/exposure of the NLS and NES in the two kinases,
several GFP-
deletion mutants were made, and their subcellular
distribution was compared with the distribution of corresponding
GFP-
mutants. Very interestingly, GFP-
(130-592) that lacks the
first 129 amino acids of
PKC, including the pseudosubstrate sequence, displayed the complete opposite localization compared with
the corresponding GFP-
(141-595) being entirely excluded from the
nucleus (Fig. 8D). However, LMB treatment induced rapid nuclear accumulation of this construct. Fig.
9 gives an overview of the subcellular
localization of various GFP-
and -
mutants before and after LMB
treatment. Our observations indicate that in
PKC the NES is a
stronger signal than the NLS, and nuclear localization is only observed
when the NES motif is removed or functionally inhibited. In contrast,
in
PKC the NLS is more potent than the NES when both signals are
exposed, and the nuclear import of
PKC is much more efficient than
that of
PKC.
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Fig. 8.
GFP- does not
accumulate in the nucleus following a 2-h treatment with LMB.
A, HeLa cells were seeded in 6-well dishes, and subconfluent
cells were transfected with 1 µg of a GFP construct containing
wild-type rat
PKC. Twenty four h later the subcellular distribution
of GFP-
was analyzed by fluorescence microscopy in cells which were
either left untreated or treated with LMB (2 ng/ml) for 2 h.
B, a kinase-defective mutant of
PKC does not accumulate
in the nucleus upon expression in HeLa cells. GFP-
and GFP-
K281W,
which contain an inactivating mutation in the ATP-binding site, were
expressed in HeLa cells. The subcellular localization was determined
24 h post-transfection. C, kinase activity of GFP-
and GFP-
K281W. HeLa cells were seeded in 100-mm dishes the day
before transfection, and subconfluent cultures were transfected with 10 µg of either an expression vector for GFP-
or an expression vector
for GFP-
K281W. Cells were harvested 24 h after transfection,
and the kinase activities of GFP-
and GFP-
K281W were assayed.
Autophosphorylation of immunoprecipitated (IP) GFP-
is
indicated. A Western blot (WB) of the immunoprecipitated
proteins used in the kinase assays is also shown. D,
deletion of the N-terminal regulatory domain containing the
pseudosubstrate sequence of
PKC does not cause nuclear accumulation
in the absence of LMB (2 ng/ml for 2 h).
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Fig. 9.
Summary of subcellular localizations of GFP
fusion proteins containing full-length or different parts of
- or
PKC before and after
LMB treatment. HeLa cells in 6-well dishes were transfected with 1 µg of expression vector for each indicated GFP fusion protein and
left untreated or treated with LMB (2 ng/ml) for 2 h at 24 h
post-transfection before being analyzed by fluorescence microscopy. The
locations of the NLS and NES motifs are shown (open diamond
and triangle, respectively). C, cytoplasm;
N, nucleus; C+N, both cytoplasm and nucleus;
nd, not determined.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
PKC shuttles
very rapidly and continuously between the nucleus and the cytoplasm.
This rapid nucleocytoplasmic shuttling occurs both in noncycling
serum-starved cells and in cycling cells proliferating in serum.
PKC consists of the
hexapeptide KRFNRR located in the
N-terminal part of the zinc finger domain (amino acids 146-151). This
basic cluster is conserved in aPKCs from different species as well as
in C. elegans PKC3 (KRLNRR) but not
in classical and novel PKCs (Fig. 4A). An exception is
provided by murine
PKC which contains a Gly residue instead of an
Arg (KRFNGR), whereas the rat sequence contains the Arg. GFP fusion
proteins containing the zinc finger region of either
PKC or
PKC
(both rat and murine) localize exclusively to the nucleus. In contrast,
GFP fusion constructs expressing either one or both zinc fingers of
classical
PKC are excluded from the nucleus and rather distributed
into punctate structures in the cytoplasm. A recent report (65)
demonstrated that GFP fusion constructs that expressed only one or both
of the zinc fingers of
PKC localized to the cytoplasm of rat
basophilic leukemia cells. Upon treatment with various stimuli
including phorbol esters, the zinc finger region of
PKC translocated
to the plasma membrane. Thus, although evident for the aPKCs, nuclear
localization is not a conserved feature of PKC zinc fingers as such.
PKC
this NLS would encompass two basic amino acids in the pseudosubstrate
sequence (Arg-133 and Lys-134) and Arg-150 and Arg-151 in the motif
identified by us (KRFNRR) in the zinc finger domain. Two
such basic clusters within a bipartite NLS are interdependent on each
other to mediate nuclear localization (36). The GFP construct
containing the zinc finger region lacks the upstream basic cluster in
this suggested bipartite NLS. Since this construct is exclusively
localized to the cell nucleus, we do not think that a bipartite NLS is
involved in nuclear translocation of
PKC. The KRFNRR motif, although
shorter, is most similar to a type of monopartite NLSs enriched in
arginine residues identified in the Tat and Rev proteins of human
immunodeficiency virus-1 and the Rex protein of human T-cell leukemia
virus type 1. These proteins have been demonstrated to be imported into
the nucleus by importin
in an importin
-independent manner (66,
67).
PKC that mainly
localized to the cytoplasm, two different point mutations in the
catalytic domain led to nuclear accumulation of full-length
PKC.
Nuclear accumulation also occurred with deletion mutants lacking either
the catalytic domain or the 140 N-terminal amino acids including the
pseudosubstrate sequence. Importantly, the A129E point mutation in the
pseudosubstrate sequence, which disrupts the interaction between this
autoinhibitory sequence and the substrate interaction site in the
catalytic domain, also led to nuclear accumulation of full-length
PKC fused to GFP. The deletion mutant is exclusively nuclear,
whereas the point mutant is found also in the cytoplasm. This
difference in the extent of relocalization relative to the wild-type
enzyme is consistent with previous findings showing that regions of the
regulatory domain of PKCs outside the pseudosubstrate sequence
contribute to autoinhibition (68). We therefore suggest that
intramolecular interactions between the catalytic domain and the
N-terminal part of the protein regulate the conformation of the protein
in such a way that the accessibility of the NLS, the NES, or both
signaling sequences is affected. Such a model of regulation has been
suggested for the serine/threonine kinase MAPKAP kinase-2 (44).
According to this model, an NLS within MAPKAP kinase-2 is exposed both
in the inactive and active enzyme. In the inactive enzyme an NES motif
is masked due to intramolecular interaction between an autoinhibitory
region and the catalytic domain. Consequently, inactive MAPKAP kinase-2
is localized to the nucleus. However, upon phosphorylation and
activation by p38, the intramolecular interaction is relieved leading
to unmasking of the NES. When both the NLS and NES are exposed, the
protein is exported from the nucleus more efficiently than it is
imported (44). For
PKC we suggest that it is primarily the exposure of the NLS that is regulated through intramolecular interactions between the catalytic domain and the N-terminal region. An interaction between the pseudosubstrate sequence in the N-terminal parts of the PKC
enzymes and the substrate interaction site in the catalytic domain is
well documented (69). To understand fully the intramolecular interactions regulating activity and subcellular localization, it will
be necessary to determine the three-dimensional structure of both
wild-type and mutant
PKC.
PKC
and
PKC has been reported (8, 33, 35). Recently, it was shown that
translocation of
PKC to the nucleus following NGF stimulation of
PC12 cells probably depends on nuclear PI 3-kinase activity (34).
Interestingly, evidence for the existence of nuclear PI 3-kinase
activity has been provided (70-72). It has earlier been proposed that
conventional PKC isoforms may continuously shuttle in and out of the
nucleus and become "trapped" in the nucleus by an increase in the
nuclear level of diacylglycerol (73). In line with this hypothesis,
Neri et al. (34) suggest that
PKC is similarly trapped
following an increase in nuclear phosphatidylinositol
3,4,5-trisphosphate. Due to their large size a functional NLS is
required for nuclear import of aPKCs. We find that the zinc finger
domain contains a functional, although atypical, basic NLS. This signal
functions independently of a structurally intact zinc finger in the
context of a GFP fusion. However, we cannot rule out the possibility
that in the context of the full-length protein an intact zinc finger is
required for nuclear accumulation. This is particularly the case since
the two Arg residues we mutated to Glu resulting in loss of nuclear
import may also be involved in the binding of phosphatidylinositol
3,4,5-trisphosphate. Thus, a conformational change may expose the NLS
which then enables nuclear import. Subsequently, the protein may become
trapped in the nucleus due to binding of nuclear phosphatidylinositol
3,4,5-trisphosphate by the zinc finger.
PKC is much more inefficiently imported into the
nucleus than
PKC upon inhibition of nuclear export. This is in
apparent conflict with the work showing rapid nuclear translocation of
PKC following NGF treatment of PC12 cells. However, it is possible
that nuclear translocation of
PKC is more tightly regulated than
that of
PKC perhaps via posttranslational modifications induced by
specific stimuli. Another possibility is that both Neri et
al. (34) and Wooten et al. (35) are actually looking more at
PKC than
PKC since the antibodies they used actually recognize both isoforms of aPKCs. We find that PC12 cells express
PKC using a specific monoclonal antibody recognizing only
PKC. However, a similar antibody recognizing only
PKC is not available.
PKC
colocalized with a putative anchoring protein called p62 into punctate, vesicle-like structures in the cytoplasm corresponding to late endosomes. The concept of p62 serving as an anchoring protein, or
perhaps more precisely a scaffolding protein, for aPKCs is very
interesting since p62 also seems to be involved in recruiting other
proteins into complexes harboring aPKCs (74-76). We have found that,
when overexpressed, p62 is able to redistribute kinase-defective
PKC
from the nucleus to the cytoplasm and that this ability is dependent on
a direct interaction between these two
proteins.2 Thus, it is clear
that in addition to regulation of subcellular localization by
conformational changes affecting NLS and NES function, the localization
of aPKCs is also being regulated by proteins with scaffolding functions
such as p62.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to M. Yoshida for the
generous gift of leptomycin B and to J. Moscat for pcDNA3-HA and
pHA-. We thank T. Lamark for helpful discussions.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from the Norwegian Cancer Society, the Norwegian Research Council, the Aakre Foundation, and the Blix Foundation (to T. J.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Fellow of the Norwegian Cancer Society.
§ To whom correspondence should be addressed: Dept. of Biochemistry, Institute of Medical Biology, University of Tromsø, 9037 Tromsø, Norway. Tel.: 47-776-44720; Fax: 47-776-45350; E-mail: terjej@fagmed.uit.no.
Published, JBC Papers in Press, December 13, 2000, DOI 10.1074/jbc.M010356200
2 M. Perander, G. Bjørkøy, T. Lamark, and T. Johansen, manuscript in preparation.
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ABBREVIATIONS |
---|
The abbreviations used are: PKC, protein kinase C; aPKC, atypical protein kinase C; GFP, green fluorescent protein; LMB, leptomycin B; NES, nuclear export signal; NLS, nuclear localization signal; PCR, polymerase chain reaction; DAG, diacylglycerol; PI 3-kinase, phosphatidylinositol 3-kinase; NGF, nerve growth factor; DAPI, 4,6-diamidino-2-phenylindole; HA, hemagglutinin; nt, nucleotide; MAPKAP-kinase 2, mitogen-activated protein kinase-activated protein kinase-2.
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