Regulation of Protein Kinase C
by the B-cell Antigen
Receptor*
Sharon A.
Matthews
,
Rashmi
Dayalu
,
Lucas J.
Thompson
, and
Andrew M.
Scharenberg
§
From the
Department of Pediatrics and Immunology,
University of Washington and Children's Hospital and Regional
Medical Center, Seattle, Washington 98195
Received for publication, November 5, 2002, and in revised form, December 13, 2002
 |
ABSTRACT |
Diacylglycerol-dependent signaling
plays an important role in signal transduction through T- and
B-lymphocyte antigen receptors. Recently, a novel serine-threonine
kinase of the protein kinase C (PKC) family has been described and
designated as PKC
. PKC
has two putative diacylglycerol binding C1
domains, suggesting that it may participate in a novel
diacylglycerol-mediated signaling pathway. Here we show that both
endogenous and recombinant PKC
are trans-located to the plasma
membrane and activated by the diacylglycerol mimic phorbol
12-myristate 13-acetate. Mutational analysis demonstrates that PKC
activation is dependent on trans-phosphorylation of two conserved
activation loop serine residues. We also find that PKC
is an
important physiologic target of the B-cell receptor (BCR), because
PKC
is found to be abundantly expressed in chicken and human B-cell
lines and, in addition, exhibits robust activation after BCR
engagement. Genetic and pharmacologic analyses of BCR-mediated PKC
activation indicate that it requires intact phospholipase C
and PKC
signaling pathways. Furthermore, in co-transfection assays, PKC
can
be trans-phosphorylated by the novel PKC isozymes PKC
, PKC
,
or PKC
but not the classical PKC enzyme, PKC
.
Taken together, these results suggest that PKC
is an important
component of signaling pathways downstream from novel PKC enzymes
after B-cell receptor engagement.
 |
INTRODUCTION |
One of the earliest detectable events following engagement of
lymphocyte antigen and Fc receptors is activation of the phospholipase C isozyme
(PLC
)1
(reviewed in Refs. 1-5). Activated PLC
acts to hydrolyze the membrane lipid phosphatidylinositol 4,5-bisphosphate, resulting in the generation of the second messengers diacylglycerol (DAG) and
inositol 3,4,5-trisphosphate. Soluble inositol 3,4,5-trisphosphate diffuses through the cytoplasm to bind to and gate inositol
3,4,5-trisphosphate receptor ion channels expressed on intracellular
calcium store membranes, thereby initiating a general increase in
cytosolic Ca2+, which is a critical component of antigen
and Fc receptor cell activation signals (reviewed in Refs. 6-9 and by
others). In contrast, DAG remains associated with cellular membranes
and serves as an essential cofactor in the assembly of a functional
"signalsome" in the subplasmalemmal region beneath engaged
receptors. Whereas a large body of evidence from studies of PLC
and
PKC signaling indicates that the DAG-dependent component of
antigen and Fc receptor signals influence diverse aspects of immune
cell biology (2, 10-16), understanding the molecular mechanisms
through which DAG acts requires a detailed knowledge of the direct
targets of DAG and how they are influenced by the production of DAG
following receptor engagement.
A novel serine-threonine kinase with two potential DAG-binding C1
domains has recently been cloned and designated PKC
, but its
activation mechanism and the identity of cell surface receptors that
utilize its signaling capacity remain uncharacterized. As our initial
analyses indicated that PKC
is abundantly expressed in human
B-cells, we investigated whether PKC
was involved in signals
mediated by the B-cell antigen receptor (BCR). Utilizing a combination
of biochemical, genetic, and pharmacologic approaches, here we show
that PKC
is a downstream effector for BCR-mediated DAG production
and that its activation mechanism probably involves DAG-mediated
membrane trans-location followed by trans-phosphorylation of two
conserved residues within its "activation loop" by novel PKC enzymes.
 |
MATERIALS AND METHODS |
Reagents--
Constitutively active PKC mutants were obtained
from David Rawlings (Department of Pediatrics, University of
Washington) and Peter Parker (Protein Phosphorylation Laboratory,
Cancer Research UK). An M2 FLAG monoclonal antibody covalently coupled
to agarose beads was from Sigma. A polyclonal antibody recognizing the
carboxyl-terminal 16 residues of human and chicken PKC
(HFIMAPNPDDMEEDP) was generated by standard immunological techniques
and affinity-purified against the immunizing peptide. Monoclonal PKC
antibodies and a V5 epitope antibody were from Transduction
Laboratories and Invitrogen, respectively. A polyclonal antibody that
specifically recognizes a phosphorylated serine 735 residue in the
activation loop of PKC
was obtained from Doreen Cantrell (Lymphocyte
Activation Laboratory, Cancer Research UK). This antibody is directed
against a phosphorylated epitope that is conserved in all three members
of the PKD kinase family. F(ab')2 fragments of anti-human
IgM were from Jackson Laboratories. A monoclonal-stimulating antibody
recognizing the chicken BCR (M4) was purified from hybridoma
supernatant using standard procedures. The classical/novel PKC
inhibitor Ro-31-8025 was from Calbiochem. All of the other reagents
were from standard suppliers or as indicated in the text.
Cell Culture and Transient Transfections--
Human Raji and
Ramos and mouse A20 B-cell lines were maintained in RPMI 1640 medium
supplemented with 10% fetal bovine serum, 10 units/ml
penicillin/streptomycin, 2 mM glutamine, and 50 µM 2-
-mercaptoethanol. Chicken DT40 cells were
cultured in RPMI 1640 medium in the presence of 10% fetal bovine
serum, 1% chicken serum, 10 units/ml penicillin/streptomycin, and 2 mM glutamine. HEK 293 endothelial cells expressing the TET
Repressor protein were maintained in DMEM supplemented with 10%
fetal bovine serum, 10 units/ml penicillin/streptomycin, 2 mM glutamine, and 5 µg/ml blasticidin.
Transient transfection of HEK 293 cells was carried out using a Beckman
Gene-Pulser electroporation apparatus. 1 × 107
cells/0.5 ml serum-free media were pulsed in 0.4-cm cuvettes with 10 µg of plasmid DNA at 330 volts and 1000 microfarads before diluting with 10 ml of complete medium. Cells were allowed to recover
overnight before experimental use. For transfection of A20 B-cells, the
electroporation conditions used were 250 volts and 950 microfarads.
cDNA Cloning and Mutagenesis--
The PKC
coding sequence
was PCR-amplified from a human brain cDNA library
(Clontech) using
5'-ACGTGCGGCCGCTGTCTGCAAATAATTCCCCTCCATCAGCCCAG-3' forward and
5'-ACGTTCTAGATTAAGGATCTTCTTCCATATCATCTGGATTAGG-3' reverse primers. The PCR fragment was subcloned
NotI/XbaI (sites are underlined) into
a modified pcDNA4/TO doxycycline-inducible mammalian expression
vector. This modified vector contains an in-frame FLAG epitope coding
sequence, resulting in the expression of an amino-terminally tagged
FLAG-PKC
protein. A similar method was used to construct the
pcDNA4/TO FLAG-PKD vector. To generate the pcDNA4/TO GFP-PKC
construct, the coding sequence for GFP was PCR-amplified with
5'-HindIII and 3'-NotI restriction sites. This
PCR fragment was then cloned into the pcDNA4/TO vector, and the
PKC
coding sequence was cloned in-frame COOH-terminal to the GFP
sequence using NotI and XbaI restriction sites.
PKC
, PKC
, and PKC
mutants were cloned into a modified
pcDNA5/TO vector with an in-frame amino-terminal V5 epitope tag.
Site-specific mutations within the catalytic domain of PKC
,
resulting in single or double amino acid substitutions, were generated
by overlap PCR using wild-type PKC
as the template. Mutants were
generated using the above primers together with internal forward and
reverse primers complementary to each other and containing specific
nucleotide substitutions as required. Primers (forward sequence only
shown) containing the desired mutation(s) (underlined) were
as follows: PKC
-K605N,
5'-GGGAGGGATGTGGCTATTAACGTAATTGATAAGATGAG-3'; PKC
-S731A/S735A,
5'-CATTGGTGAAAAGGCATTCAGGAGAGCTGTGGTAGGAACTCCAGC-3'; and
PKC
-S731E/S735E,
5'- CATTGGTGAAAAGGAATTCAGGAGAGAGGTGGTAGGAACTCCAGC-3'.
Following the second PCR reaction, the amplified cDNAs were
subcloned (NotI/XbaI) into the modified
pcDNA4/TO expression vector. Constructs were sequenced using an
Applied Biosystems automated DNA sequencer before they were used in
transient expression experiments. Protein expression was induced by
treating cells with 5 µg/ml doxycycline for 24 h.
Cell Lysis and Immunoprecipitation--
Cells were lysed in a
buffer containing 50 mM Tris-HCl, pH 7.4, 2 mM
EGTA, 2 mM EDTA, 1 mM dithiothreitol, protease
inhibitors, 1 mM AEBSF, and 1% Triton X-100. Exogenously
expressed PKC
was immunoprecipitated with either a FLAG monoclonal
antibody or with an affinity-purified PKC
antibody recovered with
protein G-Sepharose beads and resuspended in 2× SDS-PAGE reducing
sample buffer (1 M Tris-HCl, pH 6.8, 0.1 mM
Na3VO4, 6% SDS, 0.5 M EDTA, 4%
2-
mercaptoethanol, 10% glycerol, 0.01% bromphenol blue).
Immunoprecipitates were separated by 8% SDS-PAGE, transferred to
polyvinylidene difluoride membrane, and Western blotted with
appropriate antibodies.
Cell Fractionation--
DT40 B-cells were washed in ice-cold
phosphate-buffered saline and resuspended in 1 ml of ice-cold
fractionation buffer (10 mM Tris, pH 7.4, 2 mM
EDTA, 2 mM EGTA, 1 mM dithiothreitol, protease inhibitors, 1 mM AEBSF). Cells were lysed by
homogenization, and unbroken cells/nuclear debris were removed by
centrifugation at 800 × g for 10 min at 4 °C. The
supernatant was subjected to high speed ultracentrifugation at
100,000 × g for 30 min at 4 °C, resulting in a
soluble cytosolic fraction and an insoluble membrane pellet. The
membrane pellet was solubilized in 1 ml of fractionation buffer containing 1% Triton-X for 20 min at 4 °C before insoluble material was removed by centrifugation at 20,000 × g for 10 min
at 4 °C. PKC
was then immunoprecipitated from both cytosolic and
membrane fractions and analyzed by Western blotting.
In Vitro Kinase Assays--
Immunocomplexes were washed twice in
lysis buffer (described above) and once in kinase buffer (30 mM Tris-HCl, pH 7.4, 10 mM MgCl2).
PKC
autophosphorylation was determined by incubating immunocomplexes
with 20 µl of kinase buffer containing 100 µM [
-32P]ATP at 30 °C for 10 min. Reactions were
terminated by the addition of 2× SDS-PAGE sample buffer, and the
samples were analyzed by 8% SDS-PAGE and autoradiography.
Microscopy--
For immunofluorescent localization of endogenous
PKC
, DT40 B-cells were resuspended in phosphate-buffered saline and
allowed to attach to polylysine-coated glass bottom dishes (MatTek
Inc.). The cells were then left untreated or treated with 50 ng/ml PMA for 10 min before fixing with 4% paraformaldehyde for 15 min at room
temperature. The cells were then permeabilized with a 0.5% saponin
buffer and sequential incubation with primary (anti-PKC
, 1 µg/ml)
and secondary antibodies (anti-rabbit Alexa Fluor 488, 1:3000
dilution) for 20 min. After each step, the cells were washed three times in phosphate-buffered saline containing 1% bovine serum
albumin. For GFP visualization, A20 B-cells transiently expressing
GFP-PKC
were plated on polylysine-coated glass bottom dishes
in phosphate-buffered saline and allowed to adhere before stimulation
with 50 ng/ml PMA. The cells were excited with a 495-nm wavelength
light, and emitted light was imaged using an IMAGO CCD camera set for a
2-s exposure using a Zeiss microscope and TillVision software. All of
the experiments presented a representative of two to three independent experiments.
 |
RESULTS |
Although the PKC
gene and transcripts have been described
previously (17), there is no present literature regarding its regulation or receptor systems that utilize PKC
as a signaling mechanism. However, PKC
has two putative C1 domains. These domains (see schematic in Fig. 1A) of
~50 residues are thought to bind the lipid second messenger
diacylglycerol, suggesting that PKC
might participate in a
diacylglycerol-mediated signaling pathway. As the tumor-promoting
phorbol esters serve as pharmacological substitutes for DAG and mimic
many aspects of the biological activity of DAG, we used one of them,
PMA, to evaluate the potential involvement of PKC
in DAG signaling
by imaging the subcellular localization of PKC
in cells that had
been left untreated or treated with PMA (Fig. 1B). As can be
seen, both endogenous PKC
(imaged in fixed and antibody-stained
cells, top panels) and GFP-tagged PKC
(imaged in live
cells, bottom panels) are substantially redistributed from
the cytosol to the plasma membrane in response to PMA treatment, consistent with DAG serving as a membrane recruitment signal for PKC
.

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Fig. 1.
Activation of PKC by
the DAG mimic PMA. A, schematic of PKC showing position
of C1 domains, the single central pleckstrin homology (PH)
domain, and the location of ATP binding site and putative activation
loop serines. B, membrane trans-location of endogenous and
GFP-tagged PKC by PMA. Top panels, trans-location of
endogenous PKC . DT40 B-cells were left untreated or treated with PMA
for 10 min, fixed, stained with an anti-PKC antibody, and imaged.
Bottom panels, GFP-tagged PKC was expressed in A20
B-cells, and live cells were imaged during PMA stimulation. Images
shown are before and 8 min after the addition of PMA. The homogenous
staining pattern obtained upon imaging the GFP-tagged PKC in live
cells suggests that the punctate staining pattern observed for
endogenous PKC (both before and after PMA treatment) is an artifact
of the fixation process. C, PMA induces activation and
serine 735 phosphorylation of PKC . Left panel, expression
of FLAG-tagged PKC . A FLAG-PKCn expression construct was transfected
into HEK 293 cells under control of a doxycycline-responsive promoter.
Cells were lysed, immunoprecipitated with the indicated antibody, and
analyzed by anti-FLAG immunoblotting. Right panel,
anti-pS735 antibody recognizes activated FLAG-PKD and FLAG-PKC . HEK
293 cells expressing either FLAG-PKD or FLAG-PKC were lysed,
immunoprecipitated with anti-FLAG antibody, and analyzed by in
vitro kinase assay, anti-pS735 immunoblotting, and anti-FLAG
immunoblotting. D, PMA induces the activation of endogenous
PKC in chicken and human B-cell lines. The indicated cell lines were
treated or not treated with PMA, lysed, immunoprecipitated with
anti-PKC , and analyzed by anti-pS735 and PKC
immunoblotting.
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Recruitment to the plasma membrane often serves as a means for
activation of protein kinases, and PMA has previously been shown to
induce both membrane recruitment and activation of PKD1, one of the
closest homologues of PKC
(18, 19). To understand how membrane
recruitment affects PKC
function, we produced a FLAG-tagged PKC
construct as a backbone for mutational analysis of PKC
activation
and confirmed its expression after transfection of HEK 293 cells (Fig.
1C, left panel). The treatment of cells expressing FLAG-PKC
with PMA induced easily detectable enzymatic activation as measured by an in vitro kinase assay (Fig.
1C, right panel, top blot). For many
serine-threonine kinases, phosphorylation within the activation loop
serves as a marker for enzymatic activation. The putative activation
loop residues of PKC
are serines 731 and 735. Therefore, we utilized
an antibody targeted specifically at phosphoserine 735 and its
surrounding region to analyze activation loop phosphorylation of
FLAG-PKC
. Anti-pS735 immunoreactivity strongly correlated with the
activation state of FLAG-PKC
, suggesting a role for phosphorylation
of this residue during PKC
activation (Fig. 1C,
right panel, middle blot). We further utilized
anti-pS735 to examine at the activation of endogenous PKC
. As
our initial analyses (data not shown) had indicated the presence of
abundant PKC
in several B-cell lines, we evaluated whether PMA
treatment could activate endogenous PKC
in chicken DT40 B-cells and
the Raji and Ramos human B-cell lines (Fig. 1D). As can be
seen, PMA treatment strongly induced anti-pS735 immunoreactivity of
anti-PKC
immunoprecipitates, indicating that endogenous PKC
is
activated by PMA in the same manner as the recombinant FLAG-PKC
.
To further investigate the role of pS735 phosphorylation in PKC
activation, we constructed a kinase-deficient mutant of PKC
on the
FLAG-PKC
backbone via the mutation of a conserved lysine residue
within the putative ATP-binding cassette of the kinase domain
(FLAG-PKC
-KN). This mutant had no detectable kinase activity as
assessed by an in vitro kinase assay (Fig.
2A, top panel). However, a comparison of the anti-pS735 immunoreactivity induced by PMA
treatment of wild-type FLAG-PKC
with that of the FLAG-PKC
-KN mutant demonstrated essentially intact phosphorylation of this site
(Fig. 2A), indicating that this site is trans-phosphorylated by an upstream kinase in intact cells. In some proteins whose function
is modulated by phosphorylation at serine or threonine residues, the
replacement of the regulatory serine or threonine residues with
negatively charged glutamate or aspartate residues induces the protein
to act as if it is constitutively phosphorylated at the mutated sites.
Conversely, the replacement with alanine produces a protein whose
function can no longer be modulated by phosphorylation. Therefore, we
further analyzed the activation mechanism PKC
by producing mutants
on the FLAG-PKC
backbone with potentially activating mutations at
positions 731 and 735 (serine to glutamate, S731E/S735E) or
deactivating mutations (serine to alanine at the same positions,
S731A/S735A) and analyzing their responses to PMA treatment (Fig.
2B). Although the S731A/S735A mutant is no longer activated
by PMA, the S731E/S735E mutant shows high constitutive activity that is
PMA-independent, indicating that phosphorylation at serines 731 and 735 is both necessary and sufficient for PKC
activation. When viewed in
conjunction with the redistribution to the plasma membrane induced by
PMA treatment, a compelling model for PKC
activation can be
constructed in which its activation occurs as the result of its
membrane trans-location and subsequent trans-phosphorylation by an
upstream PMA-regulated protein kinase on serine 731/735.

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Fig. 2.
Activation of PKC is
via trans-phosphorylation of serines 731 and 735. A, PMA
induces trans-phosphorylation of serine 735 of PKC . HEK 293 cells
were transiently transfected with pcDNA4/TO vectors driving the
expression of WT or PKC -KN mutant. The cells were left untreated or
treated with PMA, and expressed proteins were analyzed by anti-FLAG
immunoprecipitation followed by either in vitro kinases
assays (measuring PKC autophosphorylation) or by anti-pS735 and
anti-FLAG immunoblotting. Note that the anti-pS735 immunoreactivity
together with the lack of kinase activity of the PKC -KN mutant
demonstrates that a significant fraction of anti-pS735 immunoreactivity
of stimulated PKC is because of a trans-phosphorylation event.
B, phosphorylation of activation loop serines is necessary
and sufficient for PKC activation. HEK 293 cells were transiently
transfected with pcDNA4/TO vectors driving the expression of the
indicated constructs, treated with doxycycline to induce protein
expression, and treated or not treated with PMA. Cells were lysed, and
expressed proteins were analyzed by anti-FLAG immunoprecipitation
followed by in vitro kinase assay and anti-FLAG
immunoblotting. SS/EE, S731E/S735E;
SS/AA, S731A/S735A.
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Based on its abundance in B-cells, its activation by the DAG mimic PMA
and the well established role of PLC
in B-cell antigen receptor
signal transduction, we next investigated whether PKC
was activated
after B-cell antigen receptor engagement. The engagement of the BCR on
either chicken DT40 B-cells (Fig.
3A) or human Raji and Ramos
human B-cells (Fig. 3B) induced strong and rapid activation of PKC
as assessed by the induction of anti-pS735 immunoreactivity. To address the question of where activated PKC
is localized within the cell, fractionation experiments were preformed in both PMA and
BCR-stimulated B cells. As illustrated in Fig. 3C, PKC
trans-locates from the cytosol to the membrane fraction in response to
PMA treatment (Fig. 3C, lower panels), consistent
with the observation that PMA induces the trans-location of GFP-PKC
from the cytosol to the plasma membrane (see Fig. 1B). In
addition, pS735 immunoblotting reveals that activated PKC
is
restricted to the membrane fraction of PMA-treated B-cells (Fig.
3C, upper panels). In contrast, a portion of
PKC
trans-locates to the membrane fraction of BCR-stimulated B-cells, and activated PKC
is detectable in both the cytosolic and
membrane fractions (Fig. 3C). Kinetic analysis indicates
that PKC
rapidly redistributes from the cytosol to the membrane
compartment of B-cells in response to BCR ligation (within <30 s) and
that activated PKC
is found in cytosolic and membrane compartments both at early (<30 s) and late (
10 min) time points (data not shown).

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Fig. 3.
B-cell receptor engagement induces activation
of endogenous PKC in chicken and human B-cell
lines. A, chicken DT40 B-cells were treated or not treated
with anti-chicken IgM, lysed at the indicated times, immunoprecipitated
with anti-PKC , and analyzed by anti-pS735 and PKC immunoblotting.
B, human Raji and Ramos B-cell lines were treated or not
treated with F(ab')2 fragments of anti-human IgG, lysed at
the indicated times, immunoprecipitated with anti-PKC , and analyzed
by anti-pS735 and PKC immunoblotting. C, chicken DT40
B-cells were left untreated ( ) or were treated with either
anti-chicken IgM (BCR) or with 50 ng/ml PMA for 3 min as indicated.
Cytosolic and membrane fractions were prepared as described under
"Experimental Procedures," and PKC activity was analyzed by
anti-pS735 and PKC immunoblotting.
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That the activation of PKC
by BCR ligation is entirely dependent on
PLC
activation was demonstrated through the use of DT40 B-cell lines
engineered to be deficient in individual components of the signaling
cascade, leading to PLC
activation (Fig.
4A). Lyn-deficient DT40 cells
have intact but delayed PLC
activation (20). They also exhibit
relatively intact but delayed activation of PKC
(peak activation
occurs at >10 min as opposed to ~1 min in wild-type DT40 cells).
This closely tracks the published time course of PLC
activation as
measured by inositol phosphate turnover (20). In contrast, DT40 cell
lines deficient in BLNK, BTK, and PLC
2, each of
which have completely abrogated PLC
activation (reviewed in Ref.
21), show completely abrogated PKC
activation. Note that in addition
PMA-mediated PKC
activation is intact in all of the DT40 cell lines
tested, eliminating the possibility that direct effects of the
deficiency of these proteins might be affecting PKC
activation.
Consistent with these results (Fig. 4B), the treatment of
cells with the putative DAG antagonist calphostin C also abrogated
BCR-mediated PKC
activation in chicken and human B-cells.

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Fig. 4.
BCR-activation of PKC
is dependent on PLC and DAG.
A, all panels, wild-type and mutant DT40 B-cell
lines lacking the indicated signaling molecules were analyzed for
BCR-mediated activation of PKC . Cells were left unstimulated ( ) or
were stimulated with either 50 ng/ml PMA for 10 min (P) or
with 10 µg/ml anti-chicken IgM (B) for the indicated
times. Cells were lysed, and the endogenous PKC was
immunoprecipitated (IP). Samples were analyzed by SDS-PAGE
and Western blotting using the indicated antibodies. B,
wild-type DT40 B-cells were left untreated or were treated with 3.5 µM of the competitive DAG antagonist calphostin C
(Cal.C) prior to PMA (P) or BCR stimulation
(times are indicated) and analysis of PKC activity as in A. DMSO, Me2SO.
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The above results demonstrate that PLC
is a probable source of DAG
for BCR-mediated PKC
activation. Because DAG would plausibly serve
to membrane target and activate both classical and novel PKC
enzymes in the same general microdomain area(s) as PKC
would be
localized, we investigated whether either of these classes of
enzymes might serve as an upstream activating kinase for PKC
. Consistent with this possibility, the treatment of B-cells with the
classical/novel PKC inhibitor Ro-31-8025 completely blocked BCR-mediated PKC
activation (Fig.
5A). Whereas this inhibitor is
thought to be relatively specific for the classical and novel classes
of PKC enzymes relative to other serine/threonine kinases (including
PKD1, the closest homologue of PKC
(22)), the use of inhibitors is
always open to questions regarding specificity within the cellular
environment. Therefore, to further evaluate the role of
PKC-dependent trans-phosphorylation as an activation mechanism for PKC
, we tested the ability of activated mutants of
various PKC subtypes to induce PKC
phosphorylation (and thus activation) in a heterologous expression assay. The expression of
constitutively activated mutants of novel PKC isozymes (
,
, and
) produced robust constitutive activation of PKC
in the absence
of PMA stimulation (Fig. 5B). In contrast, the co-expression of kinase-deficient or wild-type PKC
or PKC
had little or no effect on basal or PMA-induced activation of PKC
. Interestingly, the
expression of a constitutively activated classical PKC enzyme, PKC
,
produced no detectable change in either constitutive or PMA-induced
PKC
activation (Fig. 5C), suggesting that PKC
is a
poor substrate for PKC
and potentially other classical PKC isoforms.

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Fig. 5.
Novel PKC isoforms control the
phosphorylation and thus activity of PKC in
intact cells. A, the classical/novel PKC-specific inhibitor
Ro-31-8425 blocks PKC activation. Wild-type DT40 B-cells were left
untreated or were treated with 5 µM of the
classical/novel PKC inhibitor Ro-31-8425 prior to PMA (P) or
BCR (for the times indicated) stimulation. Cells were lysed, and
endogenous PKC was immunoprecipitated (IP) and analyzed
by Western blotting with the indicated antibodies. DMSO,
Me2SO. B, top and middle
panels, HEK 293 cells were co-transfected with pcDNA4/TO
FLAG-PKC and either a control vector or different novel PKC
expression constructs as indicated. KD, kinase-deficient;
DA, dominant active; wt, wild-type. Cells were
treated or not treated with PMA and lysed, and endogenous PKC was
immunoprecipitated and analyzed by Western blotting with the indicated
antibodies. Bottom panels, the expression of the various
novel PKC enzymes was confirmed by Western blotting with anti-PKC
antibodies or antibody against a V5 epitope tag. C,
top and middle panels, HEK 293 cells were
co-transfected with pcDNA4/TO FLAG-PKC and a control vector, a
dominant active PKC construct, or a dominant active PKC
expression construct as indicated. Cells were treated or not treated
with PMA and lysed, and endogenous PKC was immunoprecipitated and
analyzed by Western blotting with the indicated antibodies.
Bottom panel, the expression of the PKC DA mutant protein
was confirmed by Western blotting with an anti-PKC antibody.
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 |
DISCUSSION |
We have analyzed the activation mechanism of the novel
serine-threonine kinase PKC
and show that PKC
is activated by PMA and BCR-mediated DAG production via the trans-phosphorylation of two
serine residues (Ser 731 and Ser 735) within its activation loop. The
ability of activated mutants of novel PKC isozymes but not the
classical PKC enzyme, PKC
, to induce constitutive PKC
activation
suggests that this trans-phosphorylation event may be mediated
primarily by novel PKC enzymes. As PKC
exhibits robust activation in
response to BCR engagement, our results suggest that PKC
is an
important downstream target of activated novel PKC enzymes during BCR signaling.
The closest homologues of PKC
are PKD1/PKCµ and PKD2, and together
these three kinases form a distinct protein kinase subfamily. They
share a predicted tertiary structure that includes two C1 domains
contained in their amino-terminal halves, a single central pH domain,
and closely homologous kinase domains in their COOH-terminal halves.
Consistent with their structural similarity to PKC
, PKD1 and PKD2
(similar to PKC
) appear to act downstream from both DAG and protein
kinase C enzymes. Although the data in this paper suggest that PKC
appears to relatively specifically targeted by novel PKC isoforms, PKD1
and PKD2 are activated by both classical and novel PKCs (19, 23-25).
From the standpoint of their catalytic domains, these three kinases are
only distantly related to the ACG kinases (consisting of the PKA, PKC,
and PKG protein kinase families). Instead, their kinase domains exhibit
the closest sequence similarity to those of calcium-regulated kinases
(25). Consistent with this finding, small peptides phosphorylated by
PKD1 in vitro do not appear to significantly overlap with
those phosphorylated by classical/novel PKC enzymes (23, 26),
suggesting that PKC
/PKD1/PKD2 substrates represent distinct
signaling pathways downstream from DAG and PKCs.
Whether PKD1, PKD2, and PKC
share similar substrate ranges or
downstream biological effector functions remains to be demonstrated. However, their position downstream from novel PKCs suggests that one or
more of them is involved in linking novel PKC activation with effector
responses downstream from the BCR in B-cells. In this regard, a recent
report (14) has implicated the novel PKC isoform PKC
in controlling
the mechanisms of anergy and tolerance in B-cells. Because PKD1
and PKC
are both expressed in B-cells and appear to be targets of
novel PKC enzymes, either one or both could plausibly function as a
link between PKC
(and possibly other novel PKC enzymes) and
downstream effectors and mechanisms involved in the creation of B-cell
energy and tolerance. Determining whether PKC
and/or its homologues
operate in this pathway or an alternative signaling pathway will
depend on the future development of genetic or pharmacologic tools for
the manipulation of their signaling function.
 |
ACKNOWLEDGEMENT |
We gratefully acknowledge Tomohiro Kurosaki
for the Lyn, PLC
2, BTK, and BLNK-deficient DT40 B-cell lines.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grant GM45901 (to A. M. S).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed: Dept. of Pediatrics,
University of Washington and Children's Hospital and Regional Medical
Center, 1959 N. E. Pacific Ave., Seattle, WA 98195. Tel.: 206-221-6446; Fax: 206-221-5469; E-mail:
andrewms@u.washington.edu.
Published, JBC Papers in Press, December 27, 2002, DOI 10.1074/jbc.M211295200
 |
ABBREVIATIONS |
The abbreviations used are:
PLC
, phospholipase C isozyme
;
PMA, phorbol 12-myristate 13-acetate;
PKC, protein kinase C;
PKD, protein kinase D;
BCR, B-cell receptor;
HEK, human embryonic kidney;
GFP, green fluorescent protein;
AEBSF, 4-(2'-aminoethyl)-benzenesulfonyl fluoride hydrochloride.
 |
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