(Received for publication, April 28, 1997, and in revised form, June 18, 1997)
From the Molecular Mechanisms of Tumor Promotion Section,
Laboratory of Cellular Carcinogenesis and Tumor Promotion, NCI,
National Institutes of Health, Bethesda, Maryland 20892 and the
Department of Pharmacology, Uniformed Services University
of the Health Sciences, Bethesda, Maryland 20814
Emerging evidence suggests important differences
among protein kinase C (PKC) isozymes in terms of their regulation and
biological functions. PKC is regulated by multiple interdependent
mechanisms, including enzymatic activation, translocation of the enzyme
in response to activation, phosphorylation, and proteolysis. As part of
our ongoing studies to define the factors contributing to the specificity of PKC isozymes, we prepared chimeras between the catalytic
and regulatory domains of PKC, -
, and -
. These chimeras, which
preserve the overall structure of the native PKC enzymes, were stably
expressed in NIH 3T3 fibroblasts. Their intracellular distribution was
similar to that of the endogenous enzymes, and they responded with
translocation upon treatment with phorbol 12-myristate 13-acetate
(PMA). We found that the potency of PMA for translocation of the
PKC
/x chimeras from the soluble fraction was influenced by the
catalytic domain. The ED50 for translocation of
PKC
/
was 26 nM, in marked contrast to the
ED50 of 0.9 nM in the case of the PKC
/
chimera. In addition to this increase in potency, the site of
translocation was also changed; the PKC
/
chimera translocated
mainly into the cytoskeletal fraction. PKCx/
chimeras displayed twin
isoforms with different mobilities on Western blots. PMA treatment
increased the proportion of the higher mobility isoform. The two
PKCx/
isoforms differed in their localization; moreover, their
localization pattern depended on the regulatory domain. Our results
emphasize the complex contributions of the regulatory and catalytic
domains to the overall behavior of PKC.
Protein kinase C (PKC)1
is a major family of serine/threonine kinases that plays a crucial role
in cell signal transduction, regulating cell growth and differentiation
(1). Emerging evidence suggests important differences among PKC
isozymes both in their regulation and in their biological roles. Thus,
in K-562 erythroleukemia cells, PKC was implicated in mediating
PMA-induced cytostasis, whereas PKC
II was involved in proliferation
(2). In RBL-2H3 basophilic leukemia cells, the PKC
and -
isoforms
preferentially inhibited phospholipase C activity (3), whereas the
PKC
and -
isoforms linked the mast cell high affinity receptor
for IgE to the expression of c-fos and c-jun (4).
In PKC
knockout mice, signaling through the antigen
receptor-dependent signaling pathway was markedly impaired
(5). Not only may some PKC isoforms be active whereas others not for a
given response, but the actions of different isoforms may even be
antagonistic. In NIH 3T3 cells, for example, PKC
arrested cell
growth, whereas PKC
stimulated it (6, 7). As part of our ongoing
studies to explore the basis of specificity of PKC isozymes, we have
prepared chimeras between the regulatory and the catalytic domains of
PKC
, -
, and -
and investigated their behavior in intact
cells.
Protein kinase C consists of an N-terminal regulatory domain and a
C-terminal catalytic domain. The catalytic domain acts as a
serine/threonine-specific protein kinase, and the regulatory domain is
thought to inhibit this catalytic activity through a so-called
pseudosubstrate region near its N terminus. Immediately C-terminal to
this pseudosubstrate region is a pair of highly conserved zinc finger
structures termed the C1 domains that are the sites of phorbol ester
binding on the molecule and contribute to the association with anionic
phospholipid (8, 9). In the classic isozymes, ,
I,
II, and
, a second domain in the regulatory region, the C2 domain, bestows
Ca2+ dependence. The novel isozymes,
,
,
, and
, lack this region and correspondingly lack Ca dependence, although
they have a modified C2 homolog N-terminal to the C1 domain (10). The
individual C1 domains of PKC bind phorbol esters with similar affinity
to the intact PKC. X-ray crystallography of the PKC
C1b domain
revealed that the phorbol ester inserts into a hydrophilic cleft in an otherwise hydrophobic surface, promoting interaction of the C1 domain
with the membrane (11). It thus functions as a hydrophobic switch.
In the intact unstimulated cell, PKC is largely cytosolic with some proportion depending on the isoform and the cell type present in the membrane and cytoskeletal fractions (12, 13). Phorbol ester addition leads to translocation of PKC from the cytosol to the membranes, presumably reflecting enhanced membrane affinity of the C1-phorbol ester complex. This translocation provides one measure of the response of specific PKC isoforms in the context of the intact cell. Emerging understanding suggests that translocation should not only depend on the strength of the association between the C1 domain and the membrane but should also be coupled to other factors contributing to the membrane association and the energetics of conformational changes in the enzyme upon activation. Receptors for activated protein kinase C, the binding proteins for the regulatory domain of PKC, have been described (14), which stabilize the activated conformation of the enzyme (15). Specific substrates likewise can drive association, as elegantly shown by Jaken and co-workers (16). The state of phosphorylation of PKC is another important regulator that influences both activity and localization (17, 18). Finally, the ability of the pseudosubstrate domain to interact with the catalytic domain is central to its function.
We report here that the catalytic domain of PKC influences both the potency of phorbol ester for PKC translocation and the compartment to which PKC is translocated in response to phorbol ester.
Protein kinase C chimeras were
generated by swapping the regulatory and the catalytic domains of
PKC, PKC
, and PKC
. The regulatory domain of PKC
was
amplified by polymerase chain reaction (PCR) employing high fidelity
thermostable vent DNA polymerase using the following primers:
5
-CTCGAGATGGTAGTGTTCAATGGCCTTCTTA-3
and
5
-GCTGCCTTTGACTAGTACCTTGAT-3
. To amplify the catalytic
domain of PKC
, the primers we used were:
5
-GTACTAGTCAAAGGCAGCTTTGGCAA-3
and
5
-GACGCGTTCAGGGCATCAGGTCTTCACCAAA-3
. The regulatory and catalytic domains of PKC
were amplified by utilizing the primers below, respectively: 5
-GGGCTCGAGATGGCACCCTTCCTTCGCATTT-3
,
5
-GCTGCCTTTGACTAGTACTTTT-3
, 5
-GTACTAGTCAAAGGCAGCTTTGGCAA-3
, and
5
-CCACGCGTAATGTCCAGGAATTGCTCAAACTT-3
. To amplify the
regulatory and catalytic domains of PKC
, we employed the following
primers, respectively: 5
-CGCTCGAGATGGCTGACGTCTTCC-3
, 5
-GCTGCCTTTGACTAGTACCATGAGGAA-3
,
5
-GTACTAGTCAAAGGCAGCTTTGGCAA-3
, and
5
-CGACGCGTTACCGCGCTCTGCAGGATGG-3
. To reduce the chance of introducing mutations, we not only used high fidelity enzymes but we
also kept the number of PCR cycles low (8 cycles). To facilitate subsequent cloning steps, into the inner PCR primers we introduced a
unique restriction site (SpeI). After 8 cycles of polymerase chain reaction, we added an adenine overhang to the constructs with
Taq polymerase at 72 °C after removing the primers, and
we then ligated them into the pGEM-T vector. From this point on we employed only classical cloning techniques to further reduce the possibility of mutations in our constructs. Using the pGEM-T vector as
a shuttle vector we amplified the different PKC domains separately by
transforming them into bacteria; we then subcloned the catalytic domains into the vectors containing the regulatory domains using SpeI and MluI restriction enzymes. An important
advantage of this approach is that we could reconstruct the wild type
PKC
, -
, and -
isoforms using the same inserts as for the
chimeras, providing us with wild type controls constructed the same way
as the chimeras. Next we performed site-directed mutagenesis to mutate
the SpeI site back to the original sequence using the
following mutagenesis primers: for chimeras with PKC
regulatory
domain, 5
-TCCTCATGGTGCTGGGCAAAGGCAGC-3
; for chimeras with PKC
regulatory domain, 5
-CCAAAAAGTACTTGGCAAAGGCAGC-3
; and for those with
PKC
regulatory domain, 5
-CTTCATCAAGGTGTTAGGCAAAGGCAGC-3
. We used
the SpeI site for the selection. The chimeras along with the
wild type PKC isozymes were then subcloned into an epitope-tagging mammalian expression vector described in detail by Olah et
al. (19). The XhoI and MluI sites ensure
unidirectionality, and the vector attaches a C-terminal 12-amino acid
tag to the end of the proteins, originally derived from the C terminus
sequence of PKC
. Finally, our constructs were sequenced by Paragon
Biotech Inc. (Baltimore, MD) to assure that no mutations had been
introduced. The chimeras were designated as PKCx/y, where x and y refer
to the regulatory and the catalytic domains, respectively. Thus, PKC
/
, for example, refers to the chimera between the
regulatory domain and the
catalytic domain.
NIH 3T3 cells were grown in Dulbecco's modified Eagle's medium supplemented with 4500 mg/liter glucose, 4 mM L-glutamine, 50 units/ml penicillin, 50 µg/ml streptomycin (Advanced Biotechnologies Inc., Columbia, MD) and 10% fetal calf serum (Life Technologies, Inc.). The cells were transfected with either the empty vector or the different PKC expression vectors using Lipofectamine (Life Technologies, Inc.) following the procedure recommended by the manufacturer. The transfected cells were subsequently grown in selection medium containing 750 µg/ml G418 (Life Technologies, Inc.). After 12-18 days in selection medium, single colonies were picked and subsequently screened for the presence of different PKC chimeras by Western blot analysis. Where indicated, cells were treated with different concentrations (0.01 nM-10 µM) of PMA (LC Laboratories, Woburn, MA) for 1 h, 3 h, and 6 h at 37 °C; dimethyl sulfoxide was added to the control cells. Analyses were routinely carried out on pools of transfected cells, but all results were confirmed on individual clones.
Cell Lysis, Subcellular Fractionation, and Western Blot AnalysisThe cells were harvested into 20 mM Tris-Cl
(pH 7.4) containing 5 mM EGTA, 1 mM
4-(2-aminoethyl)benzenesulfonyl fluoride, and 20 µM
leupeptin and lysed by sonication. The cytosolic fraction represents
the supernatant following centrifugation at 100,000 × g for 1 h at 4 °C. The Triton X-100 soluble
particulate fraction was prepared by a 2-h extraction of the pellet
with the same buffer containing 1% Triton X-100 and a subsequent
centrifugation for 1 h at 100,000 × g. The
remaining pellet is the Triton X-100 insoluble fraction. The samples
were subjected to SDS-polyacrylamide gel electrophoresis according to
Laemmli (20) and transferred to nitrocellulose membranes. The protein
content of individual samples was determined (12) by staining the
Western blots with 0.1% Ponceau S solution in 5% acetic acid (Sigma).
The protein staining was found to be linear up to 30 µg of
protein/lane. The Ponceau S staining was removed by several washes with
phosphate-buffered saline (pH 7.4); the membranes were blocked with 5%
milk in phosphate-buffered saline and subsequently immunostained with
polyclonal antibodies generated against a polypeptide corresponding to
amino acids 726-737 of PKC (Life Technologies, Inc.). In some cases
the chimeras containing the
and the
catalytic domains were
detected with the corresponding anti-catalytic domain antibodies from
Upstate Biotechnology (Lake Placid, NY) and Research and Diagnostic
Antibodies (Berkeley, CA), respectively. Secondary antibodies were goat
anti-rabbit IgG coupled to horseradish peroxidase (Bio-Rad), and the
immunoreactive bands were visualized by the ECL Western blotting
detection kit purchased from Amersham Corp. The densitometric analysis
of the immunoblots and the normalization to the protein content of each individual lane were performed as described (12).
Protein kinase C activity was
assayed by measuring the incorporation of 32P from
[-32P]ATP (Amersham Corp.) into substrates (as
described previously (21)) in the presence of 100 µg/ml
phosphatidylserine and 1 µM PMA. Cell lysates were
partially purified on a HiTrap Q column (Pharmacia Biotech Inc.,
Uppsala, Sweden) and 10 µl of the partially purified cell lysates
were incubated in assay buffer containing 20 mM HEPES, pH
7.5, 10 mM MgCl2, 0.5 mM
CaCl2, 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 20 µM leupeptin, 10 µg/ml aprotinin (all
purchased from Sigma), 50 µM ATP, 1 µCi of
[
-32P]ATP, and 50 µM/assay myelin basic
protein (from bovine brain) (Sigma) as substrate at 30 °C for 10 min. The reaction was stopped by adding trichloroacetic acid at 10%
final concentration. After centrifugation for 5 min at 15 000 × g a 25-µl aliquot of the supernatant was spotted onto
phosphocellulose disks (Life Technologies, Inc.). The disks were washed
three times in 0.5% phosphoric acid and three times in distilled
water. The bound radioactivity was measured by liquid scintillation
counting. The kinase assay was linear with time over this incubation
period, and at 50 µM substrate was linear with the amount
of protein over the range of cell lysates used in the assays.
[3H]PDBu binding was measured by using the polyethylene glycol precipitation assay (21). Briefly, cell lysates (40-60 µg of protein/assay) were incubated with 20 nM [3H]PDBu in the presence of 100 µg/ml phosphatidylserine. Nonspecific binding, determined in the presence of 30 µM nonradioactive PDBu, was subtracted to give specific binding. Data presented represent triplicate determinations in each experiment.
We have constructed protein kinase C, -
, and -
chimeras
to study the relative contributions of the regulatory and the catalytic subunits of these isozymes to their behavior.
We have determined that these PKC chimeras can be stably
expressed in NIH 3T3 cells, bind
[3H]PDBu, and exhibit cofactor-dependent
kinase activity as do the wild type PKC isozymes (Table I, Fig.
1). Using a previously described tagging
system (19) we could readily distinguish the endogenous and
overexpressed isozymes of PKC/x and PKC
/x (sizes are determined
by the regulatory domain). In the case of PKC
/
and PKC
/
we
confirmed our findings by using anti-PKC
and anti-PKC
anti-catalytic antibodies. Because the levels of these overexpressed enzymes as well as the levels of the overexpressed PKC
/
were much
higher than the endogenous PKC
, interference by the endogenous PKC
was not a problem. The antibodies recognized the two previously described PKC
-specific bands at 90 and 93 Kd (7) of the overexpressed PKC
. PKC
/
and PKC
/
chimeras also
showed double bands on Western blots, suggesting that the
posttranslational phosphorylation of the PKC
catalytic domain was
similar to that of the wild type. In contrast, the PKC
/
and
PKC
/
chimeras show a single band suggesting that the
posttranslational modification of PKC
occurs only on the catalytic
domain.
|
To explore the role of the catalytic domains in phorbol ester response,
we determined the translocation and subcellular localization patterns
of the different chimeras after PMA treatment (Fig.
2). Based on the kinetics of
translocation of the endogenous PKC, -
, and -
isoforms in
these cells (12) (Table II), we examined the effects on the chimeras of treatment with varying doses of PMA for
1, 3, and 6 h. The translocation dose-response curves were stable
after 1 h of exposure to PMA, and at this time point down-regulation was not yet detectable (data not shown). Fig. 2
illustrates one representative dose-response experiment using pooled
transfected cells (at least three experiments were performed at each
time point). The tagged overexpressed wild type PKC isozymes (PKC
/
, PKC
/
, and PKC
/
) translocated with time courses
and dose-response curves similar to those of the endogenous enzymes reported earlier (12), arguing against artifacts caused either by the
overexpression or by the
-epitope tag (Table II). Experiments were
also performed with at least one clone of each chimera, yielding similar results to the pooled cultures.
|
The subcellular distribution of PKC chimeras in the untreated cells is
summarized in Table III. The distribution
of the overexpressed wild type enzymes was similar to that reported
previously for the endogenous enzymes (12) except that 10% of
PKC/
was found in the insoluble fraction. The control of
distribution in the case of PKC
/x (PKC
regulatory domain)
chimeras was dominated by the
regulatory domain. A role for the
catalytic domain was evident in the case of PKC
/
chimera;
moreover, the catalytic domain of PKC
seemed to have some effect in
bringing PKC
/
and PKC
/
into the insoluble fraction (PKC
/
was also higher there as expected). The PKC
/
distribution
differed from that of both parent isozymes with a much higher
proportion in the Triton X-100 insoluble fraction. The
cofactor-dependent stimulation of kinase activity was
somewhat lower for PKC
/
than that of the other chimeras (see Fig.
1); on the other hand, the PKC
/
chimera showed good
[3H]PDBu binding activity (see Table I). How these
differences may be related to the higher portion in the Triton X-100
insoluble fraction remains to be determined.
|
In the case of the PKC regulatory chimeras, the catalytic domain
markedly influenced the apparent affinity of PMA for the enzyme. PMA
was less potent in translocating the wild type PKC
/
than the
PKC
/
and
/
chimeras. The dose-response curves for the
decrease in PKC in the soluble fraction were quantitated and fitted to
the Hill equation (Fig. 3) In the case of
wild type PKC
/
, the ED50 for translocation was
26 ± 1 nM (n = five experiments) (similar to that reported earlier for the endogenous PKC
(12)) (Table II). It decreased to 9.1 ± 0.3 nM in the case
of the PKC
/
chimera (n = five experiments) and
was yet an order of magnitude more sensitive in the case of the
PKC
/
chimera (ED50, 0.91 ± 0.05 nM
(n = five experiments)). For the chimeras involving the
and
regulatory domains the ED50 values for
PMA-induced translocation were similar independent of the specific
regulatory or catalytic domain (PKC
/
, 9.3 ± 0.1 nM; PKC
/
, 12.4 ± 0.3 nM;
PKC
/
, 8 ± 1 nM; PKC
/
, 12 ± 1 nM; PKC
/
, 7.4 ± 0.2 nM; PKC
/
,
7.0 ± 0.4 nM; n = three experiments
for all values) (Fig. 4).
Not only did the PKC/
chimera have a dose-response curve that was
shifted to the left, but the destination of translocation changed;
i.e. the chimera translocated mostly to the Triton X-100 insoluble fraction. This shift in distribution was parallel to a
changing proportion of the PKC
/
chimera in the lower band as
compared with the upper band (easiest to observe in the total PKC
fraction). The lower band of PKC
/
was present just in the Triton
X-100 insoluble fraction, whereas the upper band was
predominantly in the cytosolic and particulate fractions (Fig.
2).
The PKC/
and PKC
/
chimeras also revealed a PMA dependent
shift in the proportions of the two bands with an increase in the lower
band at higher PMA concentrations. The subcellular distribution of the
two bands depended on the identity of the regulatory domain, whereas
the presence of the two bands depended on the
catalytic domain.
Compared with PKC
/
, PKC
/
and PKC
/
showed a reduced proportion of the upper band present in the Triton X-100 insoluble fraction. Unlike PKC
/
or PKC
/
, PKC
/
maintained large
amounts of the higher mobility isoform (lower band) in the cytosolic
and membrane fractions.
We determined the PMA dependence of the increase in the lower band for
the PKCx/ chimeras. The ED50 values were similar to those observed for the decrease in the soluble fraction (1.5 versus 0.91 nM for PKC
/
, 11.1 versus 8 nM for PKC
/
, and 9.1 versus 7.0 nM for PKC
/
). We conclude that
the two processes occur in parallel.
A major objective is to dissect the factors regulating the flow of
information through the families of PKC isoforms present in specific
cell types. For therapeutic intervention, isoform selective ligands
would greatly enhance specificity. Unfortunately it is becoming clear
that current in vitro binding assays to recombinant PKC
isozymes neglect major contributions to selectivity in the intact
cells. Thus, phorbol esters have a 4-fold weaker affinity for PKC
compared with PKC
in vitro (21), whereas PMA is 160-fold more potent for translocation of PKC
than of PKC
in mouse
keratinocytes (13). Selectivity depends, moreover, on the specific cell
type. In NIH 3T3 cells, the selectivity of PMA for PKC
compared with PKC
is only 3.5-fold versus the 160-fold in the
keratinocytes (12, 13). Our current results demonstrate that the
factors controlling the phorbol ester interactions depend on the
catalytic domain of PKC as well as on the phorbol ester binding C1
domains. Identification of the specific mechanisms by which the
catalytic domain contributes to the translocation remains to be
determined.
One possible mechanism by which the catalytic domain could influence
protein kinase C unfolding, and indirectly phorbol ester binding, would
be through the strength of the interaction between the pseudosubstrate
region and the catalytic site. Cantley and co-workers (22) have
examined in depth the substrate selectivities of the PKC isozymes. The
pseudosubstrate peptide shows similar Km values
for the
and
catalytic activities (
was not reported) (22).
Also, we had observed similar relative activities of the PKC
and
PKC
pseudosubstrates for PKC
(21). Although the regulation of PKC
isozymes by second messengers and membrane components has been
extensively studied (1, 23), the mechanisms by which PKCs can
separately modulate signals from distinct receptor pathways remain
under active investigation. In cells stimulated with hormones or
phorbol esters, most of the cellular PKC translocates to new
subcellular sites, including the plasma membrane (24), cytoskeleton
(15), nucleus (15, 25), and elsewhere (15). Furthermore, within the
same cell various isozymes may each be localized to different
subcellular sites after cell stimulation (25, 26). Translocation of
protein kinases to new sites necessarily alters their access to
substrates (15).
Increasing evidence implicates both the regulatory and the catalytic
domains in modulating PKC translocation. It has been previously
proposed that membrane binding of PKC in vivo reflects the
binding of the activated enzyme to the anchored receptors for activated
protein kinase C (27). This occurs via the regulatory domain of PKC
(14), stabilizing the active conformation of the enzyme (15). Protein
kinase C was also reported to bind to actin through a binding site
located within the regulatory domain (28). Conversely, there is strong
evidence for the role of the catalytic domain in isozyme-specific
localization. Although PKC
I and -
II differ only at the C
terminus, they localize differently (29), strongly arguing that unique
C-terminal sequences may target these isoforms to different subcellular
locations (30). Also, using PKC
and -
II chimeras, a region within
the catalytic domain of
II PKC was shown to be responsible for its
isotype-specific translocation to the nucleus (31). Targeting may also
occur by binding to cellular proteins that function as substrates.
Examples include myristoylated alanine-rich C kinase substrate,
-adducin, and kinesin light chain (29, 32). Furthermore, the
constitutive membrane association of the truncated PKC regulatory
domain, in contrast to the cytosolic localization of the holoenzyme,
argues for a role of the catalytic domain (33). Finally,
immunohistochemical studies reveal a role for the catalytic domain in
the pattern of localization and translocation of PKC
and PKC
chimeras.2 Complementing
these other studies, the findings described here show that the
catalytic domain can control the potency of PMA for driving
translocation.
Not surprisingly, phosphorylation has emerged as an important mechanism
of PKC regulation (17). Phosphorylation provides negative charges on
the so-called activation loop of PKC that is necessary for enzymatic
activity (34, 35). This transphosphorylation is followed by two
autophosphorylation steps. Both occur on the C terminus of the enzyme,
further stabilizing the catalytically active conformation and also
making the enzyme soluble (18). Our studies support a role for
phosphorylation in determining the localization of PKC, with PMA
changing the proportion of the higher mobility isoform of PKCx/
chimeras in the Triton X-100 insoluble fraction. At the same time the
localization patterns of these chimeras depend on the regulatory
domain. The fact that the phosphorylation state of PKCs as well as PKC
chimeras can change after PMA treatment emphasizes that protein
phosphatases may play a cell-specific role in targeting different PKCs
during translocation.
PMA translocates chimeras that have the same regulatory domains but
different catalytic domains with potencies that differ by an order of
magnitude. Phorbol esters bind to the C1 domains of PKC providing a
hydrophobic cap over a hydrophilic cleft (11). At the same time their
side chains contribute to stabilizing the enzyme at the membrane. Our
results suggest that binding of PMA reveals other site(s) that
facilitate(s) translocation (shift in ED50) and that
play(s) a role in targeting PKC to separate subcellular sites
(translocation of PKCx/ chimeras to the insoluble fraction).