is expressed in mast cells and is functionally involved in Fc
receptor I signaling
* Experimental Immunology Branch, National Cancer Institute, and
Section on Chemical Immunology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland
Correspondence: Stephen Shaw. National Cancer Institute, 9000 Rockville Pike, Bldg. 10/4B36, Bethesda, MD 20892. E-mail: sshaw{at}nih.gov
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, a member of the PKC family with
demonstrated function in a limited range of cell types. We found that
PKC is expressed in bone marrow-derived mast cells and in the
RBL-2H3 mast cell line. PKC underwent translocation to the membrane
in response to Fc
receptor I (FcR I) activation. Receptor
activation induced phosphorylation of PKC . The tyrosine
phosphorylation of PKC is delayed relative to PKC 
and coincides
temporally with PKC association with c-src family members Lyn and
Src. Studies of RBL-2H3 cells transduced with PKC constructs
indicated a role for PKC in receptor-induced activation of
extracellular regulated kinases, interleukin-3 gene transcription, and
degranulation in response to antigen stimulation. These studies extend
the known functions of PKC to another important immune cell type and
indicate the concurrent participation of multiple PKCs in the FcR
I-mediated response of mast cells.
Key Words: mast cells • kinases • protein • signal transduction • Fc receptors
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, which is a member of the novel PKC subfamily.
Compared with the other PKCs, its expression is much more tissue
restricted, with demonstrated expression only in skeletal muscle,
endothelial cells, megakaryoblasts, and T lymphocytes [1
,
2
]. PKC plays important roles in T-cell receptor
(TCR) signal transduction in mature T cells [3
]. In PKC
knockout mice, peripheral-blood T lymphocytes have a major defect
in their proliferative response to TCR stimulation. The underlying
mechanisms include defects in activator protein-1 and nuclear
factor-
B transcription factor activation, resulting in a dramatic
decrease in interleukin (IL)-2 production and high-affinity IL-2
receptor expression after stimulation. Although the full signaling
pathway involving PKC in T cells is not fully defined, a number of
important pieces are understood. Upon T-cell contact with
antigen-presenting cells, PKC is recruited to the contact area,
along with other signaling molecules [4
,
5
]. PKC has been shown to associate with Fyn, Vav,
and Lck in T lymphocytes, and hence it might be involved in the
regulation of those proteins that are important in T-cell signal
transduction [6
7
8
]. PKC is tyrosine phosphorylated
in T cells by Lck after TCR stimulation, and the tyrosine
phosphorylation is important for its signaling ability
[8
].
Although PKC is special in terms of its role in TCR signal
transduction, its regulation shares common features with other PKCs.
PKC activity is under autoinhibition in resting state, due to the
binding of its pseudosubstrate with its kinase domain. Stimulation of
many receptors can produce second messengers including the lipid
diacylglycerol. PKC can bind with the lipid on the membrane via its C1
domain, resulting in its translocation from cytosol to membrane.
Partial penetration of the C1 domain into the lipid bilayer of the
membrane leads to dissociation of pseudosubstrate from the kinase
domain, and the kinase domain becomes available to phosphorylate
substrate [9
]. Besides the allosteric activation
described above, PKC kinase activity is also regulated by
phosphorylation of three conserved serine-threonine residues in its
kinase domain. Without phosphorylation at those sites, PKC has little
or no kinase activity [10
]. Regulation of
phosphorylation at these residues may differ depending on cell type and
PKC isoform. Tyrosine phosphorylation has also been shown for some
PKCs. For example, PKC has been found to be tyrosine phosphorylated
in mast cells after Fc receptor (FcR) I stimulation, and this
phosphorylation increases its kinase activity and changes its substrate
specificity [11
].
Although mast cells are part of the innate immune system, they can be
effectors for the adaptive immune response via their FcR binding of
immunoglobulin (Ig). Stimulation of mast cells via antigen
cross-linking of IgE bound to their FcR I results in their
activation and degranulation. RBL-2H3 is a rat basophilic leukemia cell
line that expresses FcR I and is widely used to study the molecular
basis of mast cell activation. Signal transduction via FcR I shares
many features with signal transduction via the T-cell antigen receptor.
When bound with IgE and cross-linked by antigen, the receptor cluster
gives rise to proximity between a receptor-associated Src family kinase
(Lyn) and its substrate on a neighboring receptor [12
],
namely tyrosine residues in the immunoreceptor tyrosine-based
activation motif on the FcR I ß and 
chains. The
phosphorylation of the tyrosine-based activation motif creates a novel
binding site for subsequent recruitment and activation of the kinase
Syk [13
], which activates downstream targets, causing
secretion of inflammatory mediators by exocytosis, synthesis and
secretion of cytokines, and other biochemical responses
[14
].PKC has been shown to be an important player in
FcR I phosphorylation and Syk activation as well as in other
signaling events [15
16
17
18
19
20
21
]. But as in other cell systems,
it is still a challenge to understand the mechanism of signal
transduction involving PKCs and to differentiate the role of different
isoforms of PKC.
We explored the possibility that PKC might be expressed in mast
cells. The results reported here demonstrated both expression of PKC
in mast cells and its function in FcR I-mediated activation of
mast cells. Although there are strong parallels between the behaviors
of PKC and PKC in this system, differences in phosphorylation
kinetics and in association with src kinases also suggest somewhat
different roles for these closely related isoforms. Because
phosphorylation of PKCs and their translocation are critical to PKC
activation, these aspects of PKC have been explored in the RBL-2H3
model system.
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and were purchased
from Transduction Laboratories, Lexington, KY. 4G10 antiphosphotyrosine
mAb was from Upstate Biotechnology Inc., Lake Placid, NY. Src and Lyn
polyclonal antibodies (Abs) were from Santa Cruz Biotechnologies, Santa
Cruz, CA. Phosphospecific mitogen-activated protein (MAP) kinase
(including extracellular regulated kinase [ERK], p38, and jun
N-terminal kinase [JNK]) Abs were purchased from New England Biolabs,
Beverly, MA. dinitrophenyl (DNP)-specific mouse IgE (mIgE) was
previously described [22
]. All figures represent results
of at least two representative experiments.
Cell culture and stimulation
Bone marrow-derived mast cells were cultured essentially as
previously described [23
]. Briefly, bone marrow was
extracted from femurs and tibias of 6- to 8-week-old mice and cultured
in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS) and
20 ng/mL of IL-3 for 4–5 weeks. Determination of FcR I expression
and toluidine blue staining of metachromatic granules were used to
analyze the mast cell population. After 4–5 weeks, cultures were
>95% mast cells. Mice were caged and used in accordance with the
National Institutes of Health regulations and animal study proposal
A98-04-03. The RBL-2H3 cell line was maintained in minimum essential
medium supplemented with 10% FCS. For antigen stimulation, cells were
plated in six-well plates 1 day before stimulation. Four hours before
stimulation, cells were changed to fresh medium containing 1 µg/mL of
mIgE specific for DNP. Immediately before the antigen was added, the
cells were washed twice with serum-free medium. For stimulation, cells
were incubated in serum-free medium containing antigen (DNP-human serum
albumin, concentration indicated in each experiment) at 37°C for the
indicated times. The reaction was stopped by removing the medium,
washing with ice-cold phosphate-buffered saline once, and adding 1 mL
of ice-cold lysis buffer (150 mM NaCl, 50mM Tris-HCl [pH 7.4], 1%
Triton x-100, 2 mM sodium orthovanadate, 5 mM sodium pyrophosphate,
protease inhibitor cocktail (Boehringer Mannheim, Indianapolis, IN)].
The plates were placed on ice for 30 min, and the lysate was recovered.
Lysates were centrifuged at 11,000 g for 20 min, and
supernatants were recovered for Western blots or immunoprecipitation.
Constructs and Expression
PKC complementary DNA (cDNA) was cloned from Jurkat T cells
by reverse transcriptase-polymerase chain reaction (PCR) and subcloned
into the Semliki Forest virus vector (pSFV). PKC mutants were made
generally as previously described [24
]. Briefly, the
constitutively active PKC mutant was made by mutating the Arg-145
and Arg-146 in the pseudosubstrate motif to Ile and Trp, respectively,
by site-directed mutagenesis. Those two positively charged residues are
important for the binding of the pseudosubstrate motif with the kinase
domain. Mutation of those two residues results in a kinase whose
pseudosubstrate motif cannot bind the kinase domain and thus does not
require lipid activators to phosphorylate its substrate or to
translocate to the plasma membrane. The inactive mutant of PKC was
made by changing Lys-409 in the ATP-binding site of the kinase domain
to a Trp. In other isoforms, the mutant competes with the native form
for substrates but cannot phosphorylate them. Fidelity of the
constructs was determined by direct sequencing. All PKC constructs
generated were tagged either with green fluorescent protein (GFP) or an
influenza hemagglutinin peptide at the C terminus. Expression of the
constructs was with a modified protocol using the Semliki Forest virus
expression system from Life Technologies, Inc., Rockville, MD, as
previously described [25
]. Levels of cellular expression
of PKC after transfection into RBL-2H3 were consistent among these
constructs as determined by blotting the cell lysate with hemagglutinin
peptide antibody in repeated experiments (e.g., Fig. 7
below).
 View larger version (19K): [in a new window] |
Figure 7. PKC enhances ERK1-ERK2 activation induced by FcR I
cross-linking. (A) PKC (hemagglutinin-tagged) wild-type (WT),
constitutively active mutant which has point mutations in the
pseudosubstrate site (M1), an inactive mutant which has a point
mutation in its ATP-binding site (M2), or a control GFP construct was
transduced into RBL-2H3 cells using the pSFV expression system. The
cells were stimulated or not with 1 ng/mL of antigen for 10 min. The
cell lysate were blotted with phospho-ERK-specific Ab and ERK Ab. The
cell lysate was also blotted with hemagglutinin tag mAb as a control
for equivalent expression level of PKC . The result showed that
constitutively active PKC enhances ERK activation induced by
antigen stimulation. (B) Constitutively active PKC and a control
GFP construct were transduced into RBL-2H3 cells using the pSFV
expression system. The cells were stimulated with different
concentrations of antigen. The cell lysate was blotted with
phospho-ERK-specific Ab. The ERK2 bands were quantified with a
densitometer, and the relative value was drawn in the bar graph as
pairs representing active PKC and control GFP construct-expressing
cells at the indicated antigen concentrations.
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.
PCR and sequencing
To confirm the expression of PKC in mast cells, we used a
PCR-based strategy. Because rat PKC has not been cloned, the design
of the PCR primers for PKC was based on mouse and human PKC
cDNA sequences. We compared mouse and human PKC cDNA sequences and
found that two fragments of human PKC cDNA sequence (GenBank no.
L07032) were completely conserved in mouse PKC . We assumed that the
corresponding sequence would also be conserved in rat PKC . The
homologous sequence encompasses base pairs 327–415 (88 nucleotides)
and 1184–1213 (29 nucleotides). We generated two pairs of primers
based on the two homologous sites: two 5' primers (AGAGCTGAAACCTCAAG
and CTGGAAATGAGTGACAC) in the first fragment and one 3' primer
(GAAGACCTTGCCAAAAC) in the second fragment. Human PKC and rat PKC
cDNAs were used as positive and negative control templates for the
PCR. PCR was performed using the above primers and a previously
described [26
] cDNA library from RBL-2H3 cells as
template. The resulting PCR fragments were cloned into the TOPO TA
cloning vector from Invitrogen, (Carlsbad, CA) and sequenced.
RNA protection assay
Total RNA was extracted from RBL-2H3 cells by using Tri Reagent
from Molecular Research Center, Inc., Cincinnati, OH. A Multi-probes
RNA protection assay kit was purchased from PharMingen, San Diego, CA,
and the included instructions were followed.
Phosphopeptide analysis
RBL-2H3 cells (6 x 106/dish) were plated
into 10-cm-diameter culture dishes 1 day before experiments. Cells were
washed once with phosphate-free medium and were incubated for 1 h
in the same medium. Three milliliters of phosphate-free medium
containing 5% dialyzed FCS, 2 mCi [32P]–orthophosphate,
and 1 µg/mL of mIgE were then added to each dish. The cells were
further incubated for 4 h at 37°C. Subsequently the cells were
washed once with serum-free medium and were stimulated or not with 300
ng/mL of antigen for 5 min followed by lysis with 1 mL of lysis buffer.
Precleared lysates were reacted with mAb to PKC , and recovered
protein was resolved by SDS-4–20% PAGE. After autoradiography the
detected band was excised, and protein was extracted from the gel. The
recovered protein was subsequently digested with trypsin, and generated
peptides were resolved by thin-layer chromatography.
Degranulation assay
Degranulation in RBL-2H3 cells infected with Semliki Forest
virus encoding different constructs of PKC and RacV12, was determined
as previously described [20
]. Briefly, confluent cells
(2 x 106/well) in a six-well plate were infected and
preloaded for 4 h with DNP-specific IgE (1 µg/mL) in minimum
essential medium. Sensitized cells were then washed twice, stimulated
with 10 ng/mL of DNP-human serum albumin for 20 min in Tyrode’s buffer
[20
] at 37°C, and placed on ice. Supernatants were
quickly collected and kept on ice. For determination of hexosaminidase
activity in supernatants, aliquots were incubated in the presence of 1
mM 4-p-nitrophenyl N-acetyl-ß-glucosaminide in
0.1 M sodium citrate (pH 4.5) for 20 min at 37°C. Reaction was
stopped by addition of carbonate/bicarbonate buffer, and
p-nitrophenol was detected by absorbance at 402 nm. Total
hexosaminidase content was determined from cells solubilized with 0.5%
Triton X-100 in Tyrode’s buffer. The extent of degranulation was
calculated by dividing absorbance in the supernatants by the sum of
absorbance in the supernatants and in solubilized cells. Relative
values obtained were normalized to degranulation in GFP-transduced
cells.
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is expressed in RBL-2H3 cells is quite restricted in tissue
specificity, we considered the possibility that PKC might
contribute to signal transduction in other hematopoietic cells besides
T cells. Given the strong similarities in signal production by the TCR
in T cells and the FcR I in mast cells, we hypothesized that PKC
might contribute to FcR I signaling in mast cells. We began testing
this hypothesis by Western blot and PCR analysis in RBL-2H3 cells,
which are a rat basophilic leukemia cell line commonly used to model
signaling in mast cells. As shown in Figure 1A
, Western blot analysis using a well-characterized mAb specific
for PKC demonstrated the presence of PKC protein in the lysate
of RBL-2H3 cells at the same molecular weight as the PKC in the
positive control lysate from Jurkat cells. As expected, lysate from COS
cells, which are known not to express PKC , showed no band at the
expected molecular weight for PKC . To rule out the possibility that
the mAb was cross-reacting with PKC , an immunoprecipitation was
done using PKC mAbs, and samples were blotted with either PKC
or PKC mAbs. The result showed that no PKC was brought down by
PKC mAbs (Fig. 1B) .
 View larger version (36K): [in a new window] |
Figure 1. PKC is detected in RBL-2H3 cells by Western blot and reverse
transcriptase-PCR. (A) Lysates were prepared from Jurkat, RBL-2H3, and
COS cells. Samples were resolved in 8% SDS–PAGE, transferred to
nitrocellulose followed by immunoblot analysis with mAb 27 specific for
PKC , and detected by enhanced chemiluminescence. (B) RBL-2H3 cell
lysate was immunoprecipitated by using PKC mAb, resolved on SDS
gel, and blotted with either PKC mAb or PKC mAb. (C) Using two
pairs of oligonucleotide primers (two 5' primers with one 3' primer, as
described in Materials and Methods) designed from the human and mouse
sequences, PCR was performed on a cDNA library from RBL cells, together
with positive and negative control templates. The sizes of the
amplified products were consistent with the expected lengths.
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mAb, a PCR was designed to
confirm the expression of PKC mRNA in RBL-2H3 cells. The primers
were designed as described in Material and Methods, based on the
sequence of human and mouse PKC . Amplification with those primers
gave the expected DNA fragment when a rat RBL-2H3 cell cDNA library or
human PKC cDNA was used as template (Fig. 1C)
. The same primers
showed no amplification product when mouse PKC cDNA was used as
template. The PCR fragment was cloned into a TA vector and sequenced.
Nucleotide sequence analysis demonstrated high sequence identity to
mouse and human PKC (92% and 85% respectively), but only 58%
identity with rat PKC , indicating that it is the rat homolog of PKC
. Thus, RBL-2H3 expresses PKC mRNA and protein.
PKC in RBL-2H3 cells translocates in response to antigen
stimulation of the FcR I.
Translocation to the plasma membrane is an important part of both
the localization and activation of PKC; experimentally, this is often
determined by comparing the recovery of PKC from the cytosolic fraction
and particulate fraction. Therefore, we analyzed whether PKC
translocates in response to antigen stimulation via FcR I. The cells
were precoated with 1 µg/mL of mIgE specific for DNP for 4 h and
stimulated with DNP-human serum albumin for the indicated time. The
cells were lysed by sonication, and the lysate was fractionated as
described in Materials and Methods. The lysate from each fraction was
blotted with PKC mAb. As early as 1 min after antigen exposure,
there was a dramatic reduction of PKC in the cytosolic fraction and
corresponding increases in both of the particulate fractions
(Fig. 2A
). Of the two particulate fractions, there was a higher level of
basal recovery of PKC and in the ultraspeed pellet, in which
plasma membrane was enriched. Even at low antigen concentrations (1
ng/mL) there was an increase in PKC recovery in both particulate
fractions. The translocation became much more dramatic at higher
antigen concentrations. Compared with PKC , PKC had a slightly
lower basal level in the particulate fraction, and PKC
translocation needed a somewhat higher antigen concentration to reach
saturation level (Fig. 2B)
.
 View larger version (36K): [in a new window] |
Figure 2. PKC translocates from the cytosol to particulate fractions in
response to FcR I stimulation. (A) RBL-2H3 cells were stimulated or
not with 300 ng/mL of antigen for the indicated times. The cells were
then sonicated and fractionated as described in Materials and Methods.
After stimulation, PKC in both high-speed and ultraspeed pellets
increased, with a concomitant decrease in the cytosol fraction. (B)
RBL-2H3 cells were stimulated with different concentrations of antigen
for 5 min and fractionated, and PKC translocation was analyzed by
Western blot in comparison with PKC . Recovery of the relevant PKC
isoform in each fraction is expressed as a percentage of the recovery
of that isoform in the same fraction at the 100-ng/mL condition.
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translocation to the plasma
membrane after antigen stimulation (Fig. 3
). RBL-2H3 cells were transduced with PKC -GFP. Four hours after
transduction, the cells were stimulated or not with 300 ng/mL of
antigen for 5 min followed by fixation with 4% paraformaldehyde. In
the absence of stimulation, PKC showed a diffuse cytosolic
distribution; it was enriched in the vicinity of the nucleus but was
excluded from the nucleus. After stimulation, the intensity of the PKC
at the cell periphery increased dramatically, and its intensity in
the cytosol decreased; as a result, the cytosol-nuclear boundary was
much less visible. The result indicated that PKC in RBL-2H3 cells
responded to FcR I stimulation, which is consistent with the
biochemical evidence of translocation.
 View larger version (75K): [in a new window] |
Figure 3. PKC translocation observed by microscopy. PKC -GFP was
transduced into RBL-2H3 cells. The cells were stimulated or not with
100 ng/mL of antigen for 5 min, fixed, and visualized with a Zeiss
Axioplan 2 microscope (Carl Zeiss, Inc., Thornwood, NY).
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after antigen stimulation. undergoes phosphorylation during FcR I response both to
confirm its involvement in FcR I signaling and as a step in
understanding the mechanism of PKC signaling in RBL-2H3 cells.
RBL-2H3 cells were labeled with [32P]orthophosphate as
described in Materials and Methods and stimulated or not with 300 ng/mL
of antigen for 5 min. The cell lysates were immunoprecipitated with PKC
mAb; the recovered protein was resolved by SDS-PAGE and
autoradiographed. The result shows that PKC has some basal level of
phosphorylation. Phosphorylation increased dramatically after 5 min of
antigen stimulation (Fig. 4A
). The band was excised and digested with trypsin, and the
peptides were resolved by thin-layer chromatography; phosphorylation of
two peptides increased dramatically after antigen stimulation,
indicating at least two major phosphorylation sites in PKC that
were induced by antigen stimulation (Fig. 4B)
.
 View larger version (35K): [in a new window] |
Figure 4. PKC phosphorylation analysis using 32P in vivo
labeling. RBL-2H3 cells were 32P labeled, stimulated or not
with 300 ng/mL of antigen for 5 min, and lysed. (A) The lysate was
immunoprecipitated with PKC mAb or a control normal mouse IgG
(NmIgG). The precipitate was resolved on SDS gel and autoradiographed.
The results show increased phosphorylation of PKC after
stimulation. (B) The bands corresponding to phosphorylated PKC in
the above gels were subjected to phosphopeptide mapping as described in
Materials and Methods.
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and has been reported for PKC [8
]. To
determine whether PKC is tyrosine phosphorylated after antigen
stimulation, RBL-2H3 cells were stimulated with antigen and lysed with
Triton X-100 lysis buffer. The lysate was immunoprecipitated with PKC
mAb, and resolved proteins were blotted with antiphosphotyrosine
mAb 4G10 and compared with PKC . The results demonstrated that PKC
undergoes tyrosine phosphorylation after antigen stimulation
(Fig. 5A
). As previously described [11
], tyrosine
phosphorylation of PKC typically peaks at 1 min following antigen
stimulation and declines thereafter. In contrast, minimal tyrosine
phosphorylation of PKC was observed at 1 min after stimulation, and
peak phosphorylation typically was observed 10–20 min later. The delay
in tyrosine phosphorylation of PKC (relative to PKC) suggested
additional upstream signaling components regulating the phosphorylation
of PKC .
 View larger version (31K): [in a new window] |
Figure 5. Tyrosine phosphorylation of PKC in RBL-2H3 cells and
bone marrow-derived mast cells (BMMC). RBL-2H3 cells or BMMC were
stimulated or not with 300 ng/mL of antigen for the indicated times.
PKC in the cell lysate was immunoprecipitated with PKC mAb,
resolved by SDS gel, and blotted with anti phosphotyrosine mAb 4G10. As
a comparison, PKC tyrosine phosphorylation was analyzed in the same
way. After stripping, the membranes were blotted again with PKC or
PKC mAb to assure equal loading. Result shown is representative of
four independent experiments. (A) Kinetics of PKC tyrosine
phosphorylation in comparison with PKC . (B) BMMC lysate, together
with COS cell lysate as negative control, was blotted with PKC mAb
to show that PKC is also expressed in BMMC. (C) PKC in the BMMC
lysate, stimulated or not with antigen for 5 min, was
immunoprecipitated and blotted with 4G10 antiphosphotyrosine antibody
or PKC mAb after stripping.
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expression in RBL-2H3 is just an aberrant special case. To address
this issue, we looked at expression of PKC in primary cultured mast
cells. By Western blot, we confirmed that mast cells express PKC
and, in a more important observation, that the PKC in primary
cultured mast cells was also tyrosine phosphorylated in response to
antigen stimulation (Fig. 5B
and 5C) .
Antigen-induced association of Src and Lyn with PKC
We hypothesized that PKC might become associated with other
signaling components as it participates in signal transduction. In the
Western blots examining PKC tyrosine phosphorylation, we observed
two other tyrosine-phosphorylated molecules in the PKC
immunoprecipitate. The molecular weight of those molecules was
consistent with that of members of the Src kinase family. The membrane
was stripped and reblotted with antibodies specific for two Src family
members expressed in RBL-2H3 cells—Src and Lyn. Reblotting confirmed
that Src and Lyn coprecipitated with PKC . Src and Lyn had marginal
basal binding with PKC that increased dramatically after 9 and 27
min of antigen stimulation. The strong binding of Src and Lyn with PKC
coincided with the strong tyrosine phosphorylation of PKC
(Fig. 6
).
 View larger version (74K): [in a new window] |
Figure 6. Antigen-induced association of Src and Lyn with PKC . RBL-2H3 cells
were stimulated or not with 300 ng/mL of antigen. The cells were lysed
and PKC in the lysate was immunoprecipitated with PKC mAb. In
the upper panel, an antiphosphotyrosine blot revealed not only
tyrosine-phosphorylated PKC but also other bands whose molecular
weights suggested Src family kinases. The lower panels show results of
sequential stripping and reblotting with Lyn and then Src Abs to detect
coprecipitates and with PKC mAb to confirm equal loading of PKC
in each lane.
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enhances ERK1/ERK2 activation induced by FcR I
cross-linking. [27
, 28
]. To determine
whether PKC might be involved in ERK activation in RBL-2H3 cells,
PKC wild type and mutant constructs (constitutive active and
inactive mutants) were transduced into RBL-2H3 cells using the pSFV
expression system. Since ERK activation is regulated by phosphorylation
of critical sites, we analyzed its activation by Western blot analysis
using Abs specific for phosphorylation of ERK on its activation loop.
RBL-2H3 cells were stimulated or not with the indicated concentration
of antigen for 10 min, and the lysates were analyzed by Western blot.
In the absence of antigen stimulation, there was little stimulation of
ERK phosphorylation by any of the constructs. In contrast, following
antigen stimulation, the constitutively active PKC -expressing cells
had a much stronger ERK activation than control GFP
construct-expressing cells (Fig. 7A
). Control Western blots for ERK showed equivalent loading of ERKs
in each lane and for PKC showed equivalent expression of the
different constructs. These results demonstrate that PKC could
contribute to the activation of ERK. Note that if endogenous PKC in
RBL cells is optimal for the response, protein derived from the
transduced constructs will not have an observable effect. Therefore,
others [27
, 28
] and we have used
constitutively active constructs in order to favor their contribution
over the endogenous protein. The fact that the transduced
constitutively active PKC contributed principally following
antigen-stimulation suggested that its role in the process
fundamentally resembles that of the endogenous protein. Augmentation of
ERK activation was observed at each of the antigen concentrations
tested (Fig. 7B)
; however, the relative contribution from the
transduced constitutively active PKC became progressively less at
higher antigen concentrations. Inactive PKC -transduced cells had a
lower ERK activation than that of controls (Fig. 7A)
, indicating that
in this response the inactive form acted as a dominant negative; this
result was likewise consistent with a contribution of PKC in the
physiologic response.
PKC augments IL-3 mRNA in response to FcR I cross-linking.
Since cytokine synthesis and secretion are an important response
of mast cells to antigen stimulation, we investigated the effects of
transduced PKC constructs on the level of IL-3 mRNA expression in
response to antigen stimulation. Cells were transduced with relevant
constructs and stimulated or not with indicated concentrations of
antigen for 2 h, and total RNA was extracted from the cells and
analyzed by RNA protection assay for the amount of relevant cytokine
mRNA. As shown in Figure 8
, constitutively active PKC enhanced IL-3 gene transcription by
30%, while inactive PKC acted as a dominant negative and inhibited
IL-3 gene transcription by about 50%. The effect was specific for IL-3
since tumor necrosis factor (TNF)-
RNA was marginally reduced rather
than augmented by constitutive active PKC . In these studies, as in
many others, the inhibitory effects of dominant-negative constructs may
be promiscuous with respect to isotype inhibition, and therefore they
will need confirmation by other approaches.
 View larger version (22K): [in a new window] |
Figure 8. PKC augments IL-3 mRNA in response to FcR I cross-linking. PKC
-constitutive active mutant (M1), inactive mutant (M2), or the
control GFP construct was transduced into RBL-2H3 cells using the pSFV
expression system. The cells were stimulated or not with 10 ng/mL of
antigen for 2 h. Total RNA was extracted from the cells, and the
level of cytokine mRNA was detected by RNA protection assays. After
quantitation with a densitometer, the relative cytokine expression was
expressed as a percentage of the amount present in the cell transfected
with the control GFP construct (represented by the horizontal line at
100). Results shown are the mean plus or minus standard error of the
mean for four independent experiments. The changes in IL-3 were both
statistically significant (P < 0.01, two-tailed
t-test) while the decrease in TNF- was of borderline
statistical significance (P = 0.06).
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increases degranulation of RBL cells in response to antigen
stimulation.. Degranulation was assessed by measuring the
release of hexosaminidase from RBL-2H3 cells after antigen stimulation.
As shown in Figure 9
, cells expressing either constitutively active or wild-type PKC
showed higher degranulation than GFP-expressing control cells. The
increases were approximately half of that observed with the positive
control construct known to augment degranulation [30
], a
constitutively active Rac construct (RacV12). In contrast, RBL-2H3
cells transduced with the inactive form PKC have roughly the same
extent of degranulation as GFP-transduced control cells. Thus in
contrast to the cytokine induction, the inactive form does not function
as a dominant negative in degranulation.
 View larger version (21K): [in a new window] |
Figure 9. Expression of PKC in RBL-2H3 cells increases its degranulation in
response to antigen stimulation. PKC wild-type (WT), constitutively
active mutant (M1), inactive mutant (M2), or constitutively active Rac
(RacV12) were transduced into RBL-2H3 cells. Degranulation in response
to antigen stimulation was measured and compared with degranulation in
GFP transduced control cells, as described in Materials and Methods.
The horizontal line on the graph represents the degranulation in GFP
control cells, which is normalized as 1. The result shown is an average
of four independent experiments. Statistical analysis was done by
Student’s t-test, which showed that WT, M1, and
Rac-transduced cells have significantly higher degranulation compared
with GFP-transduced cells (P < 0.01).
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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is unusually tissue restricted, with
demonstrated expression in a limited range of cells including T cells,
endothelial cells, and skeletal muscle cells. There are only two cell
types in which it has been shown to play a functional role—T
lymphocytes and endothelial cells [3
, 24
].
In T lymphocytes, PKC undergoes specific localization to the site
of T cell contact with antigen-presenting cells [4
,
5
] and plays a critical role in stimulation of IL-2
production by mature T lymphocytes [3
]. In the present
study we showed that PKC was expressed in another important immune
cell, mast cells, and that PKC was functionally involved in a
central signaling pathway for that cell, namely stimulation via FcR
I.
Translocation to the membrane and its own phosphorylation are cardinal
features of PKC activation [9
, 10
]. By both
of these criteria, PKC in mast cells underwent activation during
FcR I-mediated stimulation. First, using biochemical and microscopic
approaches, we found that PKC translocated in response to antigen
stimulation. Of particular note, translocation did not require
stimulation with high antigen concentrations but rather resembled
PKC in its dose response to even low antigen concentrations.
Membrane translocation is a virtually absolute requirement for PKC
activation, since diacylglycerol-facilitated insertion into the lipid
bilayer is critical to the enzyme’s release from autoinhibition.
Furthermore, that localization contributes to the substrate selectivity
for membrane-proximal proteins [31
, 32
].
Phosphorylation of conserved serine or threonine residues in the
catalytic domain is another mode by which PKC kinase activity is
regulated. Our in vivo-labeling experiment revealed increased
phosphorylation of PKC after antigen stimulation in mast cells.
Phosphopeptide mapping indicated that there are at least two major
phosphorylation sites. This observed phosphorylation could be either
tyrosine phosphorylation or serine-threonine phosphorylation or both.
For several reasons, we infer that the majority of this phosphorylation
detected by radiolabeling is on serine-threonine. First, the dominant
phosphorylation on other PKC isoforms is at three classic sites of
serine-threonine in the catalytic domain [33
]. Second,
the peptides showing the strongest induced phosphorylation also had
substantial basal phosphorylation; since we know from our tyrosine
phosphorylation studies that there is no basal tyrosine phosphorylation
for PKC , it is likely that what we had seen was phosphorylation at
serine-threonine residues in the catalytic domain of PKC .
Serine-threonine phosphorylation can also happen in the regulatory
domain of PKC after the enzyme is activated [34
], but it
generally happens in a low stoichiometry and has not yet been well
characterized. Thus, translocation and phosphorylation indicate
activation of PKC in response to FcR I signaling, which implies
a functional role of PKC in FcR I signal transduction.
Tyrosine phosphorylation can also regulate PKC function. Many of the
members of PKC family have been found to undergo tyrosine
phosphorylation in response either to receptor stimulation or
H2O2 stimulation in a variety of cells
[35
, 36
]. Tyrosine phosphorylation of PKC
can modify its kinase activity and substrate specificity, or regulate
its association with other signal-transducing molecules. Tyrosine
phosphorylation has been demonstrated for one site on PKC during
T-cell activation [8
]. Since PKC is the most closely
related isoform and PKC undergoes tyrosine phosphorylation in this
same model system, tyrosine phosphorylation of PKC is also a
relevant precedent. PKC can undergo phosphorylation at multiple
sites in both the regulatory and catalytic regions [37
,
38
]. As shown in the foregoing experiments, PKC
underwent tyrosine phosphorylation in mast cell in response to FcR I
engagement. Sequential steps in phosphorylation cascades could give
rise to characteristic differences in kinetics of phosphorylation of
the different component proteins, as has been observed in this model
system [39
]. Our findings demonstrated that PKC
tyrosine phosphorylation peaks within 1 min after stimulation, but PKC
tyrosine phosphorylation happens minutes later. This difference
contrasts with the rapid translocation of both isoforms (within 1 min).
It implies that despite their high sequence similarity, PKC and PKC
are differentially regulated by tyrosine phosphorylation. Although
it has been shown that tyrosine phosphorylation of PKC modifies its
kinase activity, we did not see significant change of PKC in vitro
kinase activity after stimulation (data not shown). The role of
tyrosine phosphorylation of PKC in mast cells may be similar to
what has been shown for PKC in T cells, where tyrosine
phosphorylation of PKC did not change its in vitro kinase activity
but did cause functional defects in proliferation-induction
[8
].
In T lymphocytes, PKC has been shown to be constitutively
associated with Lck, with no change upon stimulation
[8
]. In mast cells, PKC has been shown to be
associated with both Src and Lyn; the former is constitutive, while the
latter is induced by receptor ligation [40
]. Our
findings demonstrate that PKC association with Src kinases in mast
cells has its own unique character. PKC has little or no
association with either Src or Lyn when the cell is not stimulated. But
the association was induced by antigen stimulation in both cases.
Association of PKC with Src kinases coincides with PKC tyrosine
phosphorylation but is delayed relative to peak phosphorylation of the
src kinases [41
], suggesting that tyrosine
phosphorylation of PKC regulates the association. The association
of PKC with Lyn has been shown to be mediated by phosphorylated
Tyr-52 in PKC and the SH2 domain in Lyn [40
]. PKC
might use the same mechanism in association with Src kinases.
If PKC is involved in signal transduction of FcR I in mast
cells, a change in PKC kinase activity should have some functional
effect on the biological response of the cell to receptor engagement.
There is convincing evidence that PKC contributes to MAP kinase
activation, although the mechanism is still not entirely clear. For
example, when constitutively active PKC , ß, , , 
, and
were transfected into COS cells, they all induced ERK activation
[27
]. In a similar finding in T cells, constitutively
active PKC , , and all activated ERK kinase
[28
]. In addition, PKC may have a singular capacity
to activate JNK kinase in synergy with calcineurin [28
];
however, the significance of this finding is unclear since JNK
activation is normal in PKC knockout mice [3
].
Our data demonstrated that constitutively active PKC enhances ERK
activation induced by antigen stimulation, suggesting that PKC
contributes to receptor-induced ERK activation. In contrast, the
dominant negative PKC inhibited ERK activation, which is also
consistent with that role. We also evaluated JNK and p38 MAP kinase
activation in constitutively active PKC -transduced mast cells and
found no change (data not shown); the fact that constitutively active
PKC does not cause JNK or p38 MAP kinase activation is confirmation
of the specificity of the enhanced receptor-induced ERK activation by
PKC . We adopted the strategy of using a constitutively active PKC
construct based on the work of others [27
,
28
, 42
]; the objective was to be able to
observe functional activities of the transduced isoform above the
presumably optimal concentration of the endogenous protein. The
dependence of enhanced ERK phosphorylation-activation on FcR I
stimulation by the constitutively active PKC provided reassurance
that the observed activity is still driven by the normal
receptor-mediated signal. Cytokine synthesis and secretion are
important elements of the biological responses of mast cells after
stimulation by antigen. Furthermore, MAP kinases have been shown to be
involved in the transcriptional regulation of cytokine gene expression
[43
44
45
]. We found that enhanced production of IL-3 mRNA
in constitutively active PKC -expressing mast cells after FcR I
stimulation correlates well with the results described above for ERK
activation, namely enhanced FcR I-stimulated ERK activation in
constitutively active PKC -transduced cells. This effect is specific
since it is not observed for other genes such as TNF-.
PKC also facilitated degranulation of RBL-2H3 cells in response to
antigen stimulation, albeit not as strongly as Rac. This finding is
consistent with the hypothesis that the endogenous PKC is involved
in degranulation. It is interesting that the inactive mutant of PKC
, which inhibited ERK activation and IL-3 transcription, did not
have a similar dominant negative effect on degranulation. Conversely,
the wild-type PKC , which showed no effect on ERK activation and
IL-3 transcription, appeared to be at least as effective as the
constitutively active mutant in enhancing degranulation. The
differences in the effects of the PKC mutant on different aspects of
RBL-2H3 response to antigen stimulation indicate that PKC might be
involved in more than one pathway in FcR I signaling, and probably
PKC functions in a different manner in different signaling
pathways.
The two most closely related novel PKC isoforms, PKC and PKC ,
both participate in the response of mast cells to antigen via their
FcR I receptor; however, they might participate in different ways,
as evidenced by differing patterns of tyrosine phosphorylation and
regulated association with Src kinases. The transduced PKC in mast
cells shows a functional effect on ERK activation, IL-3 production, and
degranulation induced by FcR I stimulation. Thus, the expression and
functional importance of PKC are not restricted to T cells but also
include mast cells.
Received October 13, 2000; revised December 13, 2000; accepted December 15, 2000.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
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