Lund University, Department of Laboratory Medicine, Molecular Medicine, Malmö University Hospital, S-205 02 Malmö, Sweden
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Abstract |
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To investigate the role of protein kinase C
(PKC) isoforms in regulation of neurite outgrowth,
PKC,
II,
, and
fused to enhanced green fluorescent protein (EGFP) were transiently overexpressed in
neuroblastoma cells. Overexpression of PKC
-EGFP
induced cell processes whereas the other isoforms did
not. The effect of PKC
-EGFP was not suppressed by
the PKC inhibitor GF109203X. Instead, process formation was more pronounced when the regulatory domain
was introduced. Overexpression of various fragments
from PKC
regulatory domain revealed that a region
encompassing the pseudosubstrate, the two C1 domains, and parts of the V3 region were necessary and
sufficient for induction of processes. By deleting the
second C1 domain from this construct, a dominant-negative protein was generated which suppressed processes induced by full-length PKC
and neurites induced during retinoic acid- and growth factor-induced
differentiation. As with neurites in differentiated neuroblastoma cells, processes induced by the PKC
-
PSC1V3 protein contained
-tubulin, neurofilament-160, and F-actin, but the PKC
-PSC1V3-induced
processes lacked the synaptic markers synaptophysin
and neuropeptide Y. These data suggest that PKC
,
through its regulatory domain, can induce immature neurite-like processes via a mechanism that appears to
be of importance for neurite outgrowth during neuronal differentiation.
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Introduction |
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THE regulation of neurite outgrowth during neuronal
differentiation is complex and likely to involve multiple signal transduction components. One group of
enzymes that has been suggested to be involved in this
process is the protein kinase C (PKC)1 isoform family.
Several PKC isoforms have been shown to be present in
growing axons both in vivo and in vitro (Ide, 1996). There is also experimental evidence for a function of PKC in the
regulation of neurite outgrowth in neuronal differentiation model systems, such as PC12 cells (Hundle et al.,
1995
, 1997
) and neuroblastoma cells (Parrow et al., 1992
,
1995
; Fagerström et al., 1996
).
PKC comprises a family of serine/threonine protein kinases, consisting of at least 11 different isoforms, divided
into subgroups depending on structural similarities and requirement for activators. The classical PKCs (,
I,
II,
and
) are Ca2+-dependent and activated by diacylglycerol
and phorbol esters. Novel PKCs (
,
,
, and
) are activated by diacylglycerol and phorbol esters, but are Ca2+-independent. The atypical PKC isoforms
and
/
are insensitive to diacylglycerol and phorbol ester and are also
Ca2+-independent. Finally, PKCµ is structurally unique,
but is activated by phorbol esters (Nishizuka, 1992
; Newton, 1995
; Liu, 1996
).
The PKC molecule consists of one NH2-terminal regulatory domain (RD) and one COOH-terminal catalytic domain. In the resting state the enzyme is kept inactive by a
pseudosubstrate motif in the RD bound to the catalytic
site. To become active this locked conformation has to be
changed and this is assumed to be caused by the binding of
activators to the RD (Newton, 1997). The RD from classical and novel PKC isoforms contains two classes of domains, C1 and C2, which are targets for PKC activators.
Diacylglycerol and phorbol ester bind C1 domains and in
classical isoforms the C2 domain binds Ca2+. On the other
hand, the C2 domain in novel isoforms does not bind Ca2+,
putatively explaining the Ca2+ independence of these isoforms (Stabel and Parker, 1991
; Nishizuka, 1992
; Ponting
and Parker, 1996
).
Besides being the target for PKC activators, the RD also
has been shown to be responsible for protein-protein interaction, which may direct each isoform to unique intracellular sites. RACKs (receptors for activated C-kinase)
constitute one class of PKC-binding proteins that interact
with activated PKC and this binding is to a large extent
mediated via the C2 domain (Mochly-Rosen and Gordon,
1998). RACKs have been identified for PKC
(Ron et al., 1994
) and
(Csukai et al., 1997
), and overexpression of either the entire C2 domain or peptides derived thereof has
been shown to block isoform-specific translocation and/or
activation of individual isoforms (Ron et al., 1995
; Johnson
et al., 1996
; Hundle et al., 1997
). There are also several reports demonstrating protein interaction sites in the region
comprising the two C1 domains (Prekeris et al., 1996
;
Matto-Yelin et al., 1997
; Yao et al., 1997
), suggesting that
depending on the interaction partner, different PKC domains may be of importance. Since there is little evidence
for a substrate of PKC that is preferentially phosphorylated by one or several isoforms, but not by others, at least
some of the isoform-specific effects observed have been
attributed to the fact that different isoforms will localize to
different intracellular sites. Due to these assumptions, and
since RDs in several cases determine interaction partners
and subcellular localization sites, RDs have been considered to be acting isoform specifically in a dominant-negative manner (Jaken, 1996
). Studies have shown specific effects of overexpression of RDs from individual isoforms
(Liao et al., 1994
; Cai et al., 1997
). However, there are also
reports demonstrating that the effect of the full-length
PKC can be mimicked by parts of, or the entire, RD indicating that some PKC effects may actually be mediated via
this domain (Lehel et al., 1995a
; Singer et al., 1996
). Furthermore, studies with chimeras consisting of PKC molecules with the regulatory and catalytic domain derived
from different isoforms have shown that isoform specificity may be mediated via either domain (Acs et al., 1997
;
Wang et al., 1998
). Thus, to understand the molecular
mechanisms for a PKC effect there is a need to identify
which isoforms exert the effect and which domain(s) is/are
involved in mediating it.
Neuroblastoma cell lines have been used extensively as
in vitro model systems to study mechanisms regulating
neuronal differentiation. In this study, two neuroblastoma
cell lines, SH-SY5Y and SK-N-BE(2), were used. Both of
these cell lines can be induced to differentiate with a plethora of factors (Påhlman et al., 1981; Melino et al., 1993
;
Lavenius et al., 1994
, 1995
; Rossino et al., 1995
). In several of these differentiation protocols there is evidence for the
involvement of PKC. In particular, neurite outgrowth appears to involve PKC, and a number of PKC isoforms are
present in growth cones of the differentiating cells (Parrow et al., 1995
; Fagerström et al., 1996
). Experiments with
high phorbol ester concentrations, which cause selective
down regulation of PKC isoforms, have suggested that
novel isoforms may be of importance in neurite outgrowth (Fagerström et al., 1996
).
The aim of this study was to investigate whether increased levels of a particular PKC isoform would be sufficient to induce growth of neurites in neuroblastoma cells.
To accomplish this, cDNA coding for the classical and
novel PKC isoforms that are consistently expressed in
neuroblastoma cell lines and tumor specimens, PKC,
II,
, and
(Zeidman et al., 1999
), was introduced into an expression vector and human neuroblastoma cells were
transfected with these plasmids. To identify cells overexpressing the proteins, the COOH-terminal ends of the isoforms were fused to enhanced green fluorescent protein
(EGFP). The results demonstrate that PKC
is the only
isoform that can induce processes in neuroblastoma cells
and that this effect is independent of the catalytic activity of the enzyme. By making a series of constructs expressing
isolated domains of PKC
, this study demonstrates that
the effect is mediated by a region from PKC
encompassing the pseudosubstrate, the two C1 domains, and parts of
the V3 region. The data also indicate that this effect is of
importance for neurite outgrowth during neuronal differentiation.
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Materials and Methods |
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Plasmids
cDNA coding for full-length human PKC isoforms ,
II,
, and
; the RD
from PKC
,
II,
,
,
, and
; or smaller fragments of PKC
were generated by PCR with introduction of appropriate restriction enzyme sites in
the primers. The DNA fragments were introduced into the pEGFP-N1
vector (Clontech Laboratories, Inc.), thereby fusing the PKC cDNA with
EGFP cDNA. The schematic structures of the protein products coded for
by the different expression vectors are shown in Fig. 1 A. Templates for
the PCR reactions were for PKC
,
, and
cDNA from SH-SY5Y cells;
for PKC
II ATCC plasmid 80047 (Hocevar et al., 1993
); for PKC
ATCC
plasmid 80049 (Aris et al., 1993
); and for PKC
cDNA generated from
human placenta mRNA (Clontech Laboratories, Inc.). The PKC
plasmids
PSC1aV3E and
PSC1bV3E (
PSC1V3E with DNA coding for either the second or the first C1 domain deleted) were generated with primers designed to amplify the entire
PSC1V3E plasmid, excluding the DNA
coding for the domain that should be deleted. An MluI site was introduced in each primer, the PCR product was cleaved with MluI, and ligated. Table I lists the primers used to generate the PKC fragments. All
PCR reactions were performed with Pfu polymerase (Stratagene) to minimize introduction of mutations and all PCR-generated fragments used in
this study were sequenced. The generation of the protein products of anticipated sizes were confirmed by transfecting the expression vectors into
COS cells with the calcium phosphate method (Sambrook et al., 1989
) and
subjecting the cell lysate to Western blot analysis (Fig. 1, B-D). In addition, the NheI/SalI fragments from
FL and
FL (full-length PKC) were
inserted into the CMS-EGFP vector (Clontech Laboratories, Inc.) to obtain expression of PKC and EGFP as two separate proteins.
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Western Blot Analysis
COS cells were transfected with different expression vectors, washed with
PBS, and lysed in buffer (10 mM Tris, pH 7.2, 160 mM NaCl, 1% Triton
X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulphate, 1 mM
EGTA, 1 mM EDTA, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM
phenylmethylsulfonyl fluoride). Lysates were centrifuged for 10 min at
15,000 g and 25 µg of protein was electrophoretically separated on an SDS
polyacrylamide gel and thereafter transferred to Hybond-C extra nitrocellulose filter (Nycomed-Amersham, Inc.). EGFP- or PKC-immunoreactivity was analyzed with antibodies directed against green fluorescent protein (GFP; Clontech Laboratories, Inc.) or PKC,
II,
, or
(Santa
Cruz), and detected with an HRP-labeled secondary antibody using the SuperSignal system (Pierce Chemical Co.) as substrate. The chemiluminescence was detected with a CCD camera (Fuji Photo Film Co.).
Cell Culture
Human neuroblastoma SH-SY5Y, SH-SY5Y/TrkA, and SK-N-BE(2) cells were maintained in MEM supplemented with 10% FBS, 100 IU/ml penicillin, and 100 µg/ml streptomycin (GIBCO BRL). For transfection experiments, SH-SY5Y and SH-SY5Y/TrkA cells were trypsinized and seeded at a density of 350,000 cells/35-mm cell culture dish on glass coverslips in serum free medium. After 20 min the medium was changed to medium containing serum and antibiotics, and incubated for 24 h before start of the transfections. SK-N-BE(2) cells were seeded on glass coverslips in regular growth medium (300,000 cells per dish) and transfections were initiated 24 h after seeding. In experiments where cells were treated with 12- O-tetradecanoylphorbol-13-acetate (TPA; Sigma Chemical Co.) for 4 d, or with growth factors for 40 h, the density at cell seeding was 250,000 cells/35-mm dish.
SH-SY5Y cells were transfected using 3.5 µl Lipofectin (GIBCO BRL) and 1.8 µg of DNA/ml serum free medium and SK-N-BE(2) cells were transfected with 4 µl Lipofectamine (GIBCO BRL) and 2 µg DNA, essentially according the supplier's protocol.
For differentiation studies, SH-SY5Y/TrkA cells were treated for 40 h with 100 ng/ml NGF (Promega Corp.), and SK-N-BE(2) cells with 10 µM retinoic acid (RA; Sigma Chemical Co.) or 25 ng/ml ciliary neurotrophic factor (CNTF; Promega Corp.).
Morphology Studies
16 h after the end of transfections (unless otherwise stated) cells were fixed in 4% paraformaldehyde in PBS for 4 min, mounted on microscopy slides using a PVA-DABCO solution (9.6% polyvinyl alcohol, 24% glycerol, and 2.5% 1,4-diazabicyclo[2.2.2]octane in 67 mM Tris-HCl, pH 8.0), and used for morphological studies. Digital images were captured with a Sony DKC 5000 camera system. The transfected cells were considered to have long processes if the length of the process exceeded that of two cell bodies. At least 200 transfected cells per experiment were counted.
Confocal Microscopy
Cells were transfected, fixed, and mounted as for morphology studies.
Cells expressing various PKC-EGFP constructs and Texas red-phalloidin-stained F-actin were examined using a Bio-Rad MRC 1024 confocal
system fitted with a Nikon Diaphot 300 microscope using a Nikon plan-apo 60 × 1.2 NA water immersion lens.
Immunofluorescence and Staining of F-Actin
Cells grown on glass coverslips were fixed with 4% paraformaldehyde as
above. For detection of -tubulin, synaptophysin, and neuropeptide Y
(NPY), cells were permeabilized and blocked with 1% BSA/0.02% saponin in PBS. The primary antibody (monoclonal mouse anti-
-tubulin
[Sigma Chemical Co.] diluted to 1:2,000; monoclonal mouse antisynaptophysin [clone SY38, DAKOPATTS] diluted to 1:10; or polyclonal rabbit
anti-NPY [Biogenesis] diluted to 1:40, respectively) was incubated for 1 h
in blocking/permeabilization solution. The secondary antibody (donkey
anti-mouse IgG-TRITC [Jackson ImmunoResearch Laboratories, Inc.]
diluted to 1:100 for
-tubulin and 1:20 for synaptophysin detection; or
donkey anti-rabbit IgG-TRITC [Jackson ImmunoResearch Laboratories,
Inc.] diluted to 1:300 for NPY staining) was incubated for 1 h in blocking
solution. Extensive washing with PBS and blocking/permeabilization solution was done between all steps. For detection of neurofilament-160 (NF-160), cells were blocked for 30 min with 3% BSA in PBS and incubation
with monoclonal mouse anti-NF-160 (Sigma Chemical Co.) diluted to
1:50 was performed for 3 h. Secondary antibody donkey anti-mouse IgG-TRITC (Jackson ImmunoResearch Laboratories, Inc.) was diluted to
1:300 and incubated for 1 h after extensive washing with PBS. For staining
of F-actin, cells were fixed with 4% paraformaldehyde. Cells were treated
for 5 min with 0.1% Triton X-100 in PBS and incubated for 10 min with 2 µg/ml TRITC-conjugated phalloidin (Sigma Chemical Co.) in PBS. For
confocal studies, fixed cells were blocked and permeabilized with 5% donkey serum and 0.3% Triton X-100 in TBS, and stained for 20 min with
Texas red-conjugated phalloidin (Molecular Probes, Inc.; 25 µl/ml blocking/permeabilization solution). Coverslips were mounted on object slides
with 20 µl PVA-DABCO.
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Results |
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PKC Induces Processes in Neuroblastoma Cells
To investigate whether increased levels of a specific PKC
isoform are sufficient to induce neurites, expression vectors coding for four different PKC isoforms were transfected into neuroblastoma cells. The classical and novel
isoforms consistently expressed in neuroblastoma cells,
PKC, PKC
II, PKC
, and PKC
(Zeidman et al., 1999
),
were selected for this approach. The cDNAs coding for
these isoforms were fused to cDNA coding for EGFP,
generating a PKC-EGFP fusion protein when expressed.
To confirm the generation of fusion proteins, COS cells
were transiently transfected with these plasmids, and cell
lysates were subjected to Western blot analysis using isoform-specific antibodies (Fig. 1 B), which demonstrated the formation of proteins of the anticipated sizes.
SH-SY5Y and SK-N-BE(2) neuroblastoma cells were
transfected with the vectors and the morphology of transfected cells was visualized with fluorescence microscopy
(Fig. 2 A). When EGFP alone was expressed in SH-SY5Y
and SK-N-BE(2) cells, the fluorescence was distributed throughout the cell. FLE and
IIFLE (full-length PKC
bound to EGFP) were mainly localized in the cytoplasm
and were absent from the nucleus.
FLE localized
throughout the entire cell, whereas
FLE localized mainly
to the cell periphery and, in some cells, to perinuclear
structures (Fig. 2 A). All fusion proteins gave rise to fluorescence of similar intensity in the transfected cells, indicating that there were no major differences in the expression levels of fusion proteins in individual cells.
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The morphological effects of the overexpression of PKC
isoforms were quantified by counting the number of transfected cells with cell processes longer than the length of
two cell bodies. In SK-N-BE(2) cells, overexpression of
FLE induced long processes in 41% of the transfected
cells, a substantially higher number than cells expressing
EGFP only, where 6% of transfected cells had long processes. This effect was specific for PKC
, as overexpression of neither
FLE,
IIFLE, nor
FLE resulted in an increased number of cells with long processes (Fig. 2 B). A
similar, but less pronounced pattern was observed in SH-SY5Y cells where overexpression of
FLE lead to 23%
transfectants with long processes compared with 12% for
cells expressing EGFP only. As in the case of the SK-N-BE(2) cells, overexpression of other PKC isoforms did
not induce processes (Fig. 2 C). To exclude a potential role
of EGFP in the PKC
effect, cDNA for PKC
and
were
transferred from
FL and
FL, respectively, to the CMS-
EGFP vector as a control. In these constructs PKC and
EGFP are expressed as separate proteins. SK-N-BE(2)
cells were transfected with these vectors, and 5% of PKC
and 31% of PKC
overexpressing cells had processes. This
demonstrates that the process induction of PKC
-EGFP is
not dependent on EGFP.
To investigate whether the changes in cell morphology
provoked by overexpression of PKC-EGFP can be blocked
by inhibition of PKC, the transfectants were treated with
GF109203X (Fig. 2 D). This inhibitor did not cause a decrease in the percentage of transfected cells with long processes. The concentration used (2 µM) is in the range that
inhibits the catalytic activity of classical and novel PKC
isoforms in vitro (Martiny-Baron et al., 1993
) and blocks
TPA-induced expression of fos and jun genes in neuroblastoma cells (Ding et al., 1998
). Thus, the induction of processes by PKC
appears to be independent of the catalytic activity of the enzyme.
The Regulatory Domain of PKC Is Sufficient to
Induce Processes
The fact that overexpression of full-length PKC induced
processes in the presence of GF109203X suggested an independence of the kinase activity. To analyze whether the
PKC RD is sufficient for the effect, vectors coding for the
RDs of PKC
,
,
, and
fused to EGFP, were created.
The RDs of the remaining novel isoforms PKC
and
PKC
, which are not expressed in neuroblastoma cells,
were also included as a comparison (Fig. 1 A). All constructs were sequenced and found free of mutations. The
constructs were expressed in COS cells (Fig. 1 C) where
Western blot analysis confirmed formation of proteins of
the anticipated sizes.
SH-SY5Y and SK-N-BE(2) cells were transfected with
the vectors and all fusion proteins gave rise to fluorescence of similar intensity in transfected cells (Fig. 3), with
the exception of RDE.
RDE caused a weaker fluorescence suggesting lower levels of this protein. As in the case
for full-length PKC
and PKC
II, their corresponding
RD-EGFP fusion proteins localized mainly outside the
nucleus with a tendency to perinuclear enrichment (Fig. 3 A). Neither of these RDs induced a major increase in the
number of cells with processes (Fig. 3, B and C). In
contrast, transfection with the
RDE,
RDE, and
RDE
constructs led to a drastic change in cell morphology,
most prominent in
RDE transfectants. Overexpression of
these proteins gave rise to 19% (
RDE), 32% (
RDE),
and 25% (
RDE) SH-SY5Y cells with long processes. The
corresponding numbers for SK-N-BE(2) cells were 55%
(
RDE), 56% (
RDE), and 46% (
RDE). The fusion proteins seemed to be localized mainly to perinuclear structures and the cell periphery.
RDE seemed to localize to all parts of the cells and long processes were induced in
12% of the transfected SH-SY5Y cells and 20% of the SK-N-BE(2) cells.
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The Pseudosubstrate, C1 Domains, and Parts of the
V3 Domain from PKC Are Required for
Process Induction
To clarify which parts of the RD that are essential for the
induction of processes, a series of constructs coding for different parts of RDE was created (Fig. 1 A). The constructs were sequenced and transfected into COS cells,
where proteins of expected sizes were detected in cell lysates with Western blot analysis using a GFP antibody
(Fig. 1 D). The PKC
subdomains were expressed in SH-SY5Y (Fig. 4, A and B) and SK-N-BE(2) cells (Fig. 4 C), and proteins gave rise to bright fluorescence of similar intensity suggesting no major difference in intracellular concentration.
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All fusion proteins containing the two C1 domains
(RDE,
C2PSC1E,
PSC1E,
PSC1V3E,
C1V3E, and
C1E; see Fig. 1 A for structural description) were not detected in the nucleus, and displayed a tendency to enrich in
perinuclear structures (Fig. 4 A). Some fusion proteins,
particularly
RDE and
PSC1V3E, also seemed to localize
to the plasma membrane. C2-containing proteins without the C1 domains (
C2E and
C2PSE; see Fig. 1 A) localized throughout the cell, and the smaller proteins (
PSE
and
PSC1aE) were primarily present in the nucleus.
When cell morphology was examined, it was evident
that the fragment from PKC containing the pseudosubstrate, the C1 domains, and the V3 region (
PSC1V3E)
was necessary and sufficient to induce processes (Fig. 4,
A-C). 48% of the SH-SY5Y cells expressing this protein exhibited long processes. In SK-N-BE(2), the corresponding number was 59%. When the pseudosubstrate
(
C1V3E) or the V3 (
PSC1E) was removed from the
PSC1V3 fragment, no substantial induction of processes
could be observed in either cell line. It is notable that in
SH-SY5Y cells more
PSC1V3- than
RDE-expressing
cells had processes (48% versus 36%), suggesting that removal of the C2 domain enhances the process-inducing capacity (Fig. 4 B). It was also evident that the other constructs did not have a major effect on process induction.
Intracellular Distribution of PKC
Subdomain Fragments
Fluorescence microscopy suggested that the PKC fragments localized to different intracellular sites. To investigate a possible correlation between the localization and
process-inducing ability of the fragments, transfected SH-SY5Y cells were analyzed with confocal microscopy (Fig.
5). Full-length PKC
fused to EGFP localized uniformly
outside the nucleus. The smallest fragment that induced processes,
PSC1V3E, displayed a distinct plasma membrane localization. Removal of the pseudosubstrate led to
the complete loss of plasma membrane localization, as
C1V3E could only be seen in the perinuclear area of the
cell. This suggests that the pseudosubstrate might be necessary for targeting of PKC
to the plasma membrane
(Fig. 5). Removal of the hinge region from the PSC1V3
fragment generating
PSC1E, which is incapable of inducing processes, did not cause a loss of plasma membrane localization (Fig. 5). In conclusion, these data suggest that
localization to the plasma membrane, for which the pseudosubstrate and the C1 domains are required, is necessary,
but not sufficient for the process induction. The V3 region
needs to be present for optimal function of the fragment.
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Inhibition of Process Outgrowth by the Use of
Inhibitory PKC Constructs
The previous results demonstrate that PKC through the
PSC1V3 fragment has the capacity to induce processes in
neuroblastoma cells. To address the question of whether
this capacity is a part of the molecular events driving neurite outgrowth in neuroblastoma cells differentiating in response to growth factors and RA, an attempt was made to
find an
PSC1V3E-derived construct that could inhibit the
process formation, putatively by acting in a dominant-negative manner. The two constructs that were most similar to
PSC1V3E, i.e.,
PSC1E and
C1V3E, and did not display
a process-inducing capacity, were initially evaluated for
this purpose. Neither construct had a major effect on neurite outgrowth in RA-differentiated SK-N-BE(2) cells
(data not shown). Thereafter, cDNA coding for either the first (C1a) or the second (C1b) C1 domain was deleted
in the
PSC1V3E construct, forming
PSC1bV3E and
PSC1aV3E, respectively (Fig. 1, A and D). SK-N-BE(2)
cells were transfected with these vectors, and vector coding for EGFP only (Fig. 6 A). Neither protein induced processes in untreated cells, demonstrating that both C1
domains are required for this effect. In fact, there was a
slight suppression of the number of cells with processes in
PSC1aV3E-expressing cells (Fig. 6 A). After treatment
with RA, 57% of EGFP-expressing cells and 52% of
PSC1bV3E-transfected cells had neurites. In contrast,
only 18% of
PSC1aV3E-expressing cells had processes, demonstrating a neurite suppressing effect of this protein.
Treatment with CNTF gave results that followed the same
pattern as in RA, albeit with generally fewer neurite extending cells (Fig. 6 A).
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The constructs were also evaluated for NGF-driven neurite outgrowth of SH-SY5Y cells stably expressing the
high affinity NGF receptor, TrkA (Fig. 6 B). Also in this
differentiation protocol, expression of PSC1aV3E, but
not
PSC1bV3E, caused a substantial decrease in the
number of neurite-bearing cells, both in control and
NGF-exposed cells. These results demonstrate that the
protein lacking the second C1 domain (
PSC1aV3) inhibits neurite outgrowth in several neuronal differentiation
protocols, whereas the protein with the first C1 domain
deleted (
PSC1bV3E) has no such effect.
To test whether the C1-deleted constructs have similar
effects on processes induced by overexpression of PKC
or
PSC1V3E,
FLE and
PSC1V3E were cotransfected
with
PSC1aV3E or
PSC1bV3E at a 1:3 ratio into SK-N-BE(2) cells (Fig. 6, C and D). Cotransfection with
PSC1bV3E gave rise to fewer cells with processes than
when either
FLE or
PSC1V3E alone was transfected,
but substantially more process-bearing cells than when
PSC1bV3E alone was transfected into the cells. It is likely
that the lower number of cells with processes in this
cotransfection protocol could be due to a significant proportion of cells expressing only
PSC1bV3E, cells that will fluoresce, but will not have processes. On the other
hand, cotransfection with
PSC1aV3E gave a lower number of cells with processes than did cotransfection with
PSC1bV3E. Thus, the
PSC1V3 fragment with the second C1 domain deleted (
PSC1aV3E) acts in a dominant-negative manner both suppressing processes induced by
overexpression of PKC
and inhibiting neurite outgrowth
in several neuronal differentiation protocols. This suggests
that the effect of the PSC1V3 region from PKC
may be a
common mechanism for these processes.
Characteristics of PSC1V3E-induced Processes
All PKC-derived, process-inducing constructs caused
similar morphological changes of transfected cells. The
outgrowth of processes was accompanied by a shrinkage
of the cytoplasm and a rounding up of the cell body, which
was most apparent in SK-N-BE(2) cells. Untreated SH-SY5Y cells generally have smaller cell bodies than SK-N-BE(2) cells, but a tendency towards rounding up of the cell body was observed in the SH-SY5Y cells, also. The
overall morphology of the processes differed slightly between the two cell lines. In SH-SY5Y cells, generally one
process per cell was observed, but this process frequently
carried several branches of various lengths (Figs. 2 A,
; 3 A,
; and 4 A, PSC1V3), but in some cells two or more
processes extending from the same cell were seen (Fig. 3
A,
and
). The SK-N-BE(2) cells generally had more
than one process per cell, and these processes were frequently branched.
To address whether the PSC1V3E-induced processes
have characteristics associated with neurites, expression of
cytoskeletal components and synaptic markers were analyzed. The
PSC1V3E-induced processes in SH-SY5Y
cells were compared with neurites obtained after 4 d of
treatment with 16 nM TPA, a protocol that causes SH-SY5Y cells to differentiate neuronally (Fig. 7). The experiments show that both
PSC1V3E-induced processes and
the neurites of differentiated SH-SY5Y cells were composed of
-tubulin (Fig. 7, A-D) and NF-160 (Fig. 7, E-H).
The cells were also stained for F-actin (Fig. 7, I-L), which
besides staining of the main branches of the processes, also
revealed an intense staining either at the tip of the processes (Fig. 7 L) or at sites where the processes have sharp
bends (not shown). These actin-rich structures resemble the growth cones in TPA-differentiated cells (Fig. 7 J),
suggesting that
PSC1V3E-induced processes express
growth cones. Staining for the presence of synaptic proteins NPY (Fig. 7, M-P) and synaptophysin (Fig. 7, Q-T)
in TPA differentiated SH-SY5Y cells (Fig. 7, M, N, Q, and
R) was positive, while the processes of cells transfected with
PSC1V3E were negative (Fig. 7, O, P, S, and T).
This shows that
PSC1V3E-induced processes are neurite-like, but lack important properties of functional neurites.
Furthermore, no overall increase in the expression of NPY
or synaptophysin could be detected in the
PSC1V3E-
transfected cells, suggesting that this PKC
fragment does
not induce complete differentiation of neuroblastoma
cells. The characteristics of processes induced by
FLE
were similar to
PSC1V3E-induced processes (not shown).
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Colocalization of PKC-EGFP and F-Actin
An interesting issue is why overexpression of the RDs of
both PKC and PKC
(
RDE and
RDE) induced processes, whereas for full-length isoforms the same effect
only was obtained with PKC
(
FLE) and not with PKC
(
FLE). A unique feature of PKC
, compared with other
isoforms, is the presence of an actin-binding site between
the C1 domains (Prekeris et al., 1996
). Binding to F-actin via this site in vitro has been shown to maintain PKC
in
an open conformation (Prekeris et al., 1998
), which may
result in exposure of structures in the RD essential for the
process-inducing capacity of this isoform. If this interaction is important for the process induction of
FLE, it
would be expected to detect colocalization of F-actin and
FLE. F-actin in
FLE-transfected SH-SY5Y cells was
stained with Texas red-conjugated phalloidin and the
colocalization of F-actin and
FLE was analyzed with confocal microscopy. Several processes were analyzed and it
was evident that the proteins were colocalized in some
parts of the processes (Fig. 8). This result thus indicates
that an interaction between
FLE and F-actin may take
place in the processes.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
This study was designed to examine the role of PKC isoforms in neurite outgrowth regulation and identify structures in the PKC molecule of importance for its function in
this process. For this purpose, we used neuroblastoma cell
lines which have been extensively used to study neuronal
differentiation. Of the classical and novel PKC isoforms
that are consistently expressed in neuroblastoma cells
(PKC,
II,
and
; Zeidman et al., 1999
), only overexpression of PKC
induced processes in these cells. PKC
has been suggested to be of importance for neurite outgrowth in PC12 cells where overexpression of PKC
, but
not PKC
, potentiated NGF-induced neurite outgrowth
(Hundle et al., 1995
). The effect of PKC
in PC12 cells was
suppressed by PKC inhibitors, which contrasts the results
in the present study which demonstrates that the effect of
PKC
was independent of its kinase activity. Furthermore,
in PC12 cells, overexpression of PKC
did not by itself induce processes. It is thus likely that PKC
may regulate
neurite outgrowth by a number of mechanisms. In neuroblastoma cells, several PKC isoforms are enriched in
growth cones, but studies with phorbol ester treatment, which downregulates the classical isoforms, have suggested a role for PKC
or another novel isoform in supporting the growth cone (Fagerström et al., 1996
). These
facts, together with the results from the present study,
highlight PKC
as one PKC isoform of importance in neurite outgrowth regulation.
In this study, the PKC isoforms were fused to EGFP to
visualize transfected cells and to facilitate an examination
of the intracellular distribution of the expressed proteins.
EGFP, in its native fluorescent form, is a highly condensed
molecule (Ormö et al., 1996). Approaches to fuse PKC
isoforms with GFP variants have been successfully used to
follow the translocation of PKC
II (Feng et al., 1998
),
PKC
(Sakai et al., 1997
), PKC
(Ohmori et al., 1998
; Shirai et al., 1998
), and PKC
(Shirai et al., 1998
). When examined, this fusion has been shown not to influence the catalytic activity of the enzyme. GFP variants have also
been fused to smaller proteins or isolated domains, such as
histone 2B (Kanda et al., 1998
), pleckstrin homology domains (Stauffer et al., 1998
), and PKC C1 domains (Oancea et al., 1998
) without any obvious loss of function. Furthermore, as shown in this study, regardless if the position
of EGFP was at the COOH terminus of intact PKC
or if
it was placed COOH-terminally of the RD in constructs where the catalytic domain was deleted, processes were induced in neuroblastoma cells. This suggests that the effect
on the process induction is independent of the position of
EGFP. Processes were also induced when PKC
, without
being fused to EGFP, was overexpressed. Several subdomains of PKC
that were fused to EGFP did not induce processes at all, further indicating that the effects observed in this study are not mediated by EGFP.
As previously mentioned, the effect of PKC was independent of enzymatic activity and of the presence of the
catalytic domain, since expression of the RD was sufficient
to induce processes. In fact, the RD could induce processes more potently than the full-length PKC
, suggesting
that the catalytic domain may inhibit this function of the
RD. The RD from PKC
and
also induced processes in
the transfected cells, despite the inability of full-length PKC
to do so. Cells transfected with
RDE displayed less
fluorescence than the other RD transfectants, probably indicating a lower level of expression of fusion protein in
these transfectants. It is possible that the RD from PKC
would have had the same effect if the protein levels in
each individual cell had been higher. These results may
suggest that the novel isoforms PKC
and PKC
, and perhaps PKC
, could have the capacity to induce processes under proper conditions. An interesting feature possibly
explaining the selective effect of full-length PKC
, is the
actin binding site which is present only in this isoform
(Prekeris et al., 1996
). When PKC
binds actin it is maintained in an open active confirmation exposing the catalytic domains and the RDs (Prekeris et al., 1998
), which
thereby can exert its activity. There was a colocalization of
FLE and F-actin in processes, a finding which may indicate that this interaction might be important for the selective effect of PKC
, although further experimentation is
necessary to establish this interaction as crucial for process induction.
The finding that the PKC effect is insensitive to PKC
inhibitors and could be mimicked by the RD is somewhat
surprising. Since RDs of PKC isoforms have been suggested to act in a dominant-negative manner, the effects
obtained in this study may be due to a dominant-negative effect of PKC
and its RD on another endogenous PKC
isoform. If this were the case, it would be expected to see
an induction of processes upon inhibition of this isoform
with PKC inhibitors. However, treatment of the neuroblastoma cells with GF109203X did not cause an elevated
number of processes. It could be argued that this lack of
process induction is due to the fact that GF109203X also
inhibits other kinases that are critical for the induction of
processes. If so, it would be expected that GF109203X
should suppress the processes also in PKC
-overexpressing cells, since the kinase of importance for processes also
would be inhibited under these conditions. Furthermore, if
the effects of the PKC
constructs are dominant-negative,
the suppression by
PSC1aV3E of PKC
-induced processes, RA-, and NGF-induced neurites implies that this
construct would act in a dominant-negative manner towards a dominant-negative effect in the first case, whereas
in the latter protocols it would simply act in a dominant-negative way. Therefore, we think that the most plausible
explanation for the effects observed in this paper is that
PKC
RD induces processes through a mechanism that
does not involve dominant-negative effects.
There are other reports where parts of, or the entire
PKC RD exert the same effects as the complete enzyme.
PKC was shown to activate phospholipase D in a PKC
activator-dependent, but PKC activity-independent fashion, and phospholipase D was activated by PKC
regulatory, but not catalytic domain in vitro (Singer et al., 1996
).
Another example is the inhibition of Golgi-specific sulfation of glycosaminoglycan chains in cells overexpressing PKC
, which can be mimicked by overexpressing the
PKC
C1 domains only (Lehel et al., 1995a
).
When examining the role of the different domains of
PKC RD in process induction, it was evident that a fragment centering on the two C1 domains was sufficient and
necessary for this effect. Interestingly, the C2 domain,
which is of importance for RACK binding (Csukai et al.,
1997
), was not of importance for the process-inducing capacity. In fact, expression in SH-SY5Y cells suggested that removal of the C2 domain from the RD, generating
PSC1V3, slightly increased the ability to induce processes.
There are several examples demonstrating that protein interaction with the RD is mediated via the C1 domains. Beside the previously mentioned actin binding site in PKC
located between the C1 domains (Prekeris et al., 1996
), a
homologue of 14-3-3 has been shown to bind the Dictyostelium myosin II heavy chain-specific PKC through the
PKC C1 domain (Matto-Yelin et al., 1997
). In addition,
binding of the pleckstrin homology domain from the tyrosine kinase Btk was shown to be dependent on the pseudosubstrate and the C1 domain from PKC
(Yao et al., 1997
). Using an overlay assay, it was shown that the second C1 domain from PKC
bound several proteins from
Xenopus laevis oocyte cytosol extracts (Pawelczyk et al.,
1998
). Taken together, these results indicate an important
role for the C1 domains in PKC protein interactions. Thus,
it is conceivable that the effects observed in this study are
due to the C1 domains interacting with other proteins,
thereby eliciting the observed morphological changes. However, there was also a dependence on the pseudosubstrate and parts of the V3 domain for the induction of processes. These structures have been shown to be of importance for localization of PKC
C1 domains to the plasma
membrane in NIH3T3 fibroblasts (Lehel et al., 1995b
).
In line with that report, the process-inducing fragment, PSC1V3, localized almost exclusively to the plasma membrane, but this localization was lost when the pseudosubstrate was removed. This was accompanied with a loss of
process-inducing capacity, which suggests that a plasma
membrane localization is necessary for this effect. However, a plasma membrane localization per se of the C1 domains is not sufficient, since the PSC1 fragment to a large
extent appeared to be present at the plasma membrane
without inducing processes.
Removal of the second, but not of the first, C1 domain
generated a fragment that suppressed neurite outgrowth
during RA-, CNTF-, and NGF-driven neuronal differentiation. Since this same fragment also acted in a dominant-negative manner towards processes induced by PKC
overexpression, these results suggest that the observed effects of PKC
is not only observed upon overexpression of
the protein, but may indeed be of importance for neurite outgrowth that accompanies neuronal differentiation.
However, given the abundance of proteins with C1 domains (Hurley et al., 1997
), it cannot be excluded that during neuronal differentiation effects reported in this study
are mediated via other C1 containing proteins. The results
obtained with the C1-deleted constructs also illustrate the
different properties of the two C1 domains that have been described (Szallasi et al., 1996
; Hunn and Quest, 1997
;
Bögi et al., 1998
).
From the present results, it is not possible to draw definite conclusions regarding the mechanisms whereby PKC
constructs elicit processes. To exclude the possibility that
the increase in process-bearing cells is not due to a selection of cells with process-inducing capacity, the number
of SH-SY5Y cells expressing EGFP or
RDE following
transfection were counted. There was a lower percentage
of
RDE-expressing cells (4.1 ± 0.5% of EGFP- versus
2.8 ± 0.6%
RDE-expressing cells), but this difference is
too low to account for the increase in process-bearing cells (
5% in EGFP- to 32% in
RDE-expressing cells). Furthermore, the few processes that could be observed in
EGFP-expressing cells were much shorter than processes
in cells transfected with PKC
constructs. This was also
true for EGFP-expressing cells that were kept in culture
for up to 4 d. This suggests that transfection with PKC
constructs does not result in an enhancement of a basal rate of process generation, but rather induces some events
that eventually lead to the generation of neurite-like processes. This process generation may be mediated via cytoskeletal mechanisms, effects on the interaction of the
cell with the substratum, or some other mechanism. It
does not seem to involve altered expression of differentiation-coupled genes, since no increase in expression of
NPY or synaptophysin, in the cell bodies or the processes, could be observed in
PSC1V3E-overexpressing cells.
These proteins are elevated upon neuronal differentiation
of neuroblastoma cells. Thus, it is likely that PKC
overexpression induces processes in undifferentiated cells and
does not elicit a complete neuronal differentiation program. Both
-tubulin and NF-160 were present at apparently similar levels in the processes in
PSC1V3E-overexpressing cells and in neurites in neuronally differentiated
neuroblastoma cells, indicating that the processes induced
by the PKC
fragment to some extent display neuronal
features. Such a dissociation between the physical induction of neurites and the accompanying increase in neuronal differentiation markers generally present in neurites
has also been observed after overexpression of a constitutively active phosphatidylinositol 3 kinase in PC12 cells
(Kobayashi et al., 1997
).
In conclusion, this study demonstrates that PKC, but
not PKC
,
II, or
, induces neurite-like processes in neuroblastoma cells and this effect can be ascribed to a region
encompassing the pseudosubstrate, the two C1 domains,
and parts of the V3 domain. Identification of a dominant-negative construct derived from this region indicates that
this effect of PKC
is of relevance for neurite outgrowth
during neuronal differentiation.
![]() |
Footnotes |
---|
Address correspondence to Christer Larsson, Lund University, Department of Laboratory Medicine, Molecular Medicine, Entrance 78, 3rd floor, Malmö University Hospital, S-205 02 Malmö, Sweden. Tel.: 46-40-337-404. Fax: 46-40-337-322. E-mail: christer.larsson{at}molmed.mas.lu.se
Received for publication 22 December 1998 and in revised form 25 March 1999.
Financial support was obtained from The Swedish Society for Medical
Research, The Swedish Cancer Society, The Children's Cancer Foundation of Sweden, HKH Kronprinsessan Lovisas förening för barnasjukvård,
Malmö University Hospital Research Funds, and Magnus Bergvall, Crafoord, Ollie and Elof Ericsson, Hans von Kantzow, Gunnar, Arvid, and
Elisabeth Nilsson, and John and Augusta Persson Foundations.
We thank Linda Pettersson and Stefan Seth for technical assistance.
![]() |
Abbreviations used in this paper |
---|
CNTF, ciliary neurotrophic factor; EGFP, enhanced green fluorescent protein; FL, full-length PKC; FLE, full-length PKC bound to EGFP; GFP, green fluorescent protein; NF-160, neurofilament-160; NPY, neuropeptide Y; PKC, protein kinase C; RA, retinoic acid; RACK, receptor for activated C-kinase; RD, regulatory domain; TPA, 12-O-tetradecanoylphorbol-13-acetate.
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