 |
INTRODUCTION |
Signal transduction networks comprise parallel pathways that
achieve integrated responses at multiple levels including second messenger accumulation and activation of kinases and transcription factors. Two major pathways involve nonreceptor protein-tyrosine kinases (1) and the protein kinase C
(PKC)1 family of
serine/threonine kinases (2). Direct interaction between the 
(3)
and 
(4) members of the PKC family and particular protein-tyrosine
kinase signaling proteins have recently been described. These reports
are of interest because precedents for direct integration of
serine/threonine and tyrosine kinase pathways by protein-protein
interactions are rare, with perhaps the most prominent being
identification of ras as a mediator between receptor
tyrosine kinases and the MAPK pathway of serine/threonine kinases
(5).
T-cell receptor (TCR) stimulation leads to hydrolysis of
phosphatidylinositol-4,5-bisphosphate to produce diacylglycerol (DAG) and inositol-3,4,5-trisphosphate (6). These result, respectively, in
direct activation of PKC and in increased intracellular calcium, which
can then influence PKC activation among its other effects. Use of
activators and inhibitors has established that PKC activation in
T-cells is necessary but not sufficient for proliferation and production of characteristic cytokines (7). Furthermore, in the murine
thymoma line EL-4, transiently overexpressing 
PKC, the PKC activator
phorbol-12-myristate-13-acetate (PMA) increased transcription of a
reporter gene regulated by the IL-2 promoter (8). Although T-cells
express 10 of the 11 PKC isozymes (9), PKC is of special interest
because it is the most nearly T-cell-restricted isozyme in its
expression (10). In addition, activation of T-cells results in PKC
translocation to the zone of TCR clustering present at the contact
between the T-cell and an antigen-presenting cell (APC) (11).
PKC-mediated events can operate in a manner largely independent of
other signaling pathways (12). A prominent parallel pathway following
stimulation of the TCR involves activation of a tyrosine kinase
signaling cascade (6). Among the protein-tyrosine kinases that have
been described as reversibly associated with the TCR complex are src
family members p59fyn and p56lck and syk family member ZAP-70 (6).
p59fyn is notable for its low fractional stoichiometry of association
with the TCR CD3 complex, which suggests a transient modulatory role,
and for its associations with a wide range of other proteins implicated
in T-cell signaling including ZAP-70 (13), c-cbl (14), fas (15), shc
(16), IL-7 receptor (17), CD43 (18), 
-tubulin (19),
inositol-3,4,5-trisphosphate kinase (20) and the
inositol-3,4,5-trisphosphate receptor (21). These interactions,
mediated primarily through SH2 and SH3 domains, can be short lived. In
the case of ZAP-70, for example, the association with p59fyn in a
T-cell hybridoma was found as early as 10 s after activation,
peaked at 5 min, and was gone by 10 min (13). Although 10 min is much
longer than the interaction between a typical enzyme and substrate, it
is short compared with the time course for expression of mitogenic
cytokines, implying that a variety of other interactions are also
likely to be involved in the regulation of cytokine secretion.
T-cells express one of the two splicing variants of p59fyn, the other
being found primarily in brain (22). The enzyme is posttranslationally
modified by N-terminal myristylation or palmitoylation, which is
thought to facilitate membrane localization (23). Although clearly
implicated in T-cell signaling by biochemical and genetic experiments
using transgenic mice (24), the precise physiological functions of
p59fyn remain unclear. It is believed to be involved in TCR-induced
calcium release from intracellular stores mediated by the
inositol-3,4,5-trisphosphate receptor (25), a process thought to be
regulated in part by PKC as well. In other hematopoietic cells, p59fyn
is associated with Btk (26) and with WASp (27); mutations in each have
been implicated in human immune disorders.
Recent progress in the understanding of PKC function has focused on the
fact that individual isozymes translocate from one cell compartment to
other subcellular sites following physiological stimulation, often
manifested as a shift in distribution from the soluble fraction to the
particulate fraction. This phenomenon is believed to reflect a
conformational change in PKC leading to specific interaction with
particular anchoring proteins, designated receptors for activated C
kinase (RACKs) (28). Restricting localization to defined sites within
the cell represents a general strategy (29) that serves to limit the
substrates exposed to the catalytic site of each PKC isozyme, providing
physiological specificity for a family of enzymes that can
phosphorylate a wide range of proteins in vitro. Two such
anchoring proteins have been described in detail, RACK1, specific for
PKC (30), and RACK2, specific for PKC (31). Each is found in the
particulate fraction and shows saturable binding to its cognate PKC
following stimulation. In addition to promoting localization, anchoring
proteins can also potentiate PKC catalytic activity, presumably by
stabilizing the active conformation (29). Both RACK1 and RACK2 have
sequence homology to the WD-40 family of proteins previously implicated in signaling (32), and in both cases, the interaction is centered on
the regulatory domain of PKC, which has the most extensive sequence
variation within the PKC family. Notably, neither RACK serves as a
substrate for PKC itself (30, 31). However, other PKC-binding proteins,
several of which are implicated in regulating membrane-cytoskeleton
interactions, have been shown to be substrates (33). Of particular
relevance to the present work, the pleckstrin homology domains of Tec
family protein-tyrosine kinases Btk and Emt have been suggested as
possible anchors (3). The more distantly related atypical class PKC
has been shown to both bind and phosphorylate ZIP, a protein thought to
provide a scaffold-linking PKC to cytokine receptor tyrosine kinases
(4).
Given the pharmacologically established significance of PKC in general
for the T-cell response (2) and the intracellular localization of
PKC in close proximity to the T-cell receptor following activation
(11), specific functions for endogenous anchoring proteins of PKC
are likely. Support for this view comes from disruption of
activation-induced translocation and anchoring of PKC in Jurkat
T-cells by overexpression of either the human immunodeficiency virus
protein Nef (34) or the signaling protein 14-3-3-
(35). In the
latter case, PKC-dependent activation of the IL-2 promoter
was shown to be inhibited. Recent work using electroporated antibodies
to disrupt PKC function in peripheral blood lymphocytes has further
implicated PKC in early responses to T-cell activation, specifically
up-regulation of the IL-2 receptor (36). In the present work,
identification of p59fyn as an endogenous anchoring protein for PKC
has provided a basis for experiments indicating a role for this
interaction in the regulation of IL-4 in nontransformed T-cell lines,
using both electroporated antibodies and a small organic compound as
pharmacological probes.
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EXPERIMENTAL PROCEDURES |
Reagents--
Phorbol-12-myristate-13-acetate (PMA) was from LC
Laboratories. Phosphatidylserine (PS) and diacylglycerol (DAG) were
from Avanti. Ionomycin and tetanus toxoid were from Calbiochem. Lectin from Phaseolus vulgaris phytohemagglutinin, piceatannol,
enolase, and histone type IIIs were from Sigma. TER14687
((±)-2-(N,N-dimethylaminomethyl)-1indanone HCl)
was obtained from Sigma Aldrich (catalog number S793558). OKT3 antibody
to CD3 (ATCC) was purified from mouse ascites on a protein A sorbent
kit (Pierce).
Recombinant Proteins--
Human PKC was PCR-amplified from
Jurkat cell cDNA, cloned into the baculovirus transfer vector
pBlueBacHisB (Invitrogen), and confirmed by sequencing. The
recombinant N-terminal His-tagged PKC was isolated from Sf-9 insect
cells on a nickel chelation resin (Qiagen) according to the
manufacturer's protocols. The resulting protein was 85% pure by
silver-stained SDS-PAGE gel and was catalytically active for
autophosphorylation and using histone IIIs as substrate.
PKC-V1 domain (amino acids 1-140) and the V1 domain of 
PKC
(amino acids 1-140) were PCR-amplified from Jurkat cell cDNA and
expressed in XL1-Blue strain of Escherichia coli
(Stratagene) using pQE-30 vector (Qiagen) with an N-terminal His tag
for nickel chelation purification and modified to encode a C-terminal
c-myc epitope for immunological detection.
Human p59fyn cDNA was PCR-amplified from human T-cell cDNA,
cloned in-frame into pMAL-c2 expression vector to produce a fusion protein with maltose-binding protein (MBP), and the sequence was verified using an ABI373 sequencer (Applied Biosystems). Protein was
purified on an amylose affinity column following the manufacturer's protocols (New England Biolabs). The resulting MBP-p59fyn fusion protein was 90% pure and was catalytically active as measured by
autophosphorylation and by using enolase as substrate. Similarly, p59fyn cDNA was cloned into the pGEX-3X expression vector (Amersham Pharmacia Biotech) to generate GST fusion proteins and purified using
the manufacturer's protocols. The resulting GST-p59fyn fusion protein
was 90% pure and was also catalytically active.
In Vitro Kinase Assays--
Recombinant MBP-p59fyn or MBP (2.5 µg) was incubated with or without substrates, 2.5 µg of PKC-V1,
and/or 2 µg of acid-denatured enolase, in 50 µl of assay buffer (10 mM MnCl2, 40 mM Hepes buffer, pH
7.6, and 5 µCi of [
-32P]ATP) for 15 min at room
temperature (37). Reactions were stopped by adding 12.5 µl of 5×
SDS-PAGE sample buffer and boiling for 5 min. Samples were resolved by
SDS-PAGE, transferred onto nitrocellulose, and exposed to x-ray film
using an intensifying screen at 
80 °C. Parallel manipulations were
used for PKC autophosphorylation or phosphorylation of histone using
50 ng of PKC in the absence or presence of 40 µg/ml histone type
IIIs (Sigma) with or without activating lipids (DAG and
phosphatidylserine, 0.8 and 50 µg/ml, respectively) and 10 µg of
GST-p59fyn, all in 20 mM MgCl2, 20 mM Tris-HCl, pH 7.5, 12 mM 2-mercaptoethanol,
20 µM ATP, and 5 µCi [-32P]ATP.
Affinity Sorbent Binding--
100 µg of recombinant His-tagged
PKC-V1 was immobilized on 0.5 ml of agarose-nickel resin (Qiagen)
and incubated for 30 min at room temperature with 0.5% Triton
X-100-soluble Jurkat cell extract, prepared in solubilization buffer
(150 mM NaCl, 20 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1 mM EGTA, 0.2 mM sodium
orthovanadate, 0.2 mM phenylmethylsulfonyl fluoride, and 20 µg/ml each of leupeptin, aprotinin, and soybean trypsin inhibitor).
The resin was washed three times with 50 mM Tris-HCl, pH
7.5, 12 mM 2-mercaptoethanol, 0.2 M NaCl, 0.1%
polyethylene glycol, and eluted with standard SDS-PAGE sample buffer:
1% SDS, 1% 2-mercaptoethanol, 30 mM Tris-HCl, pH 6.8, 10% glycerol, 0.05% bromphenol blue. The sample was directly loaded
on a 10% SDS-PAGE gel followed by Western blotting; after transfer to
nitrocellulose membrane, the resolved and immobilized proteins were
probed with anti-phosphotyrosine antibodies (Transduction Labs, 1:1000)
or anti-p59fyn antibodies (Transduction Labs, 1:500) overnight at
4 °C. The membranes were washed three times with PBS/Tween (140 mM NaCl, 8 mM Na2PO4,
1.5 mM KH2PO4, 3 mM
KCl, pH 7.0, 0.05% Tween 20) then incubated with anti-mouse IgG
horseradish peroxidase-conjugated antibodies (Amersham Pharmacia
Biotech) diluted 1:1000 in PBS/Tween + 2% BSA for 1 h at room
temperature. After washing, the membranes were subjected to a
chemiluminescent reaction utilizing luminol (Sigma) as a substrate,
followed by immediate exposure to autoradiography film (Eastman Kodak
Co.).
Microplate Binding Assay--
Polystyrene 96-well plates
(Corning) were coated with indicated amounts of purified MBP-p59fyn or
MBP in PBS for 1 h at 37 °C. The plates were washed with TBST
buffer (20 mM Tris, pH 7.4, 150 mM NaCl, and
0.05% Tween 20), blocked with 1% BSA in PBS at room temperature for
1 h, and washed again with TBST. Recombinant PKC or V1
fragments of PKC or PKC were added in a buffer containing 10 mM MnCl2, 150 mM NaCl, 40 mM Hepes, pH 7.6, 1 mM ATP, and 0.1% BSA and
incubated 30 min with shaking at room temperature. The plates were then
washed 5 times with TBST buffer followed by incubation with
peroxidase-conjugated anti-PKC antibody in 1% BSA/PBS (Transduction Labs) for 1 h at room temperature. The wells were washed 5 times with TBST buffer and developed using the 3,3',5,5'-tetramethylbenzidine peroxidase substrate system according to the manufacturer's directions (Kirkegaard and Perry Laboratories), followed by reading OD at 650 nm.
Background binding of PKC constructs to MBP alone, <0.1 OD, was
subtracted to yield the specific binding to MBP-p59fyn. No signal was
seen in the absence of PKC.
Yeast Two-hybrid Analysis--
The Saccharomyces
cerevisiae strain L40 (MATa trp1 leu2 his3
LYS2::lexA HIS3
URA3::lexA-lacZ) and vectors
were provided by S. Hollenberg (38). PKC and its V1 domain (amino
acids 1-140) and the V1 domain of PKC (amino acids 1-140) were
PCR-amplified from the corresponding plasmid cDNA and cloned
in-frame downstream of LexA DNA binding polypeptide in BTM116 vector to
form the bait construct. DNA fragments encoding full-length and
truncated variants of p59fyn were PCR-amplified from human T-cell
cDNA and cloned in-frame downstream of VP16 transcription
activation domain in pVP16 vector to form the prey construct. The
full-length human p59fyn in which Lys-296 was mutated to Ala (K296A)
was PCR-amplified from the wild type p59fyn cDNA. Full-length,
regulatory (amino acids 1-248), and kinase (amino acids 249-509)
domains of human p56lck were PCR-amplified from corresponding cDNA
(39) and also cloned into pVP16 vector. Human (CD4+ T-cells, purified
from the blood of normal donors) or murine (HT2 cell line) T-cell
cDNA libraries were similarly prepared as prey, with
>105 independent clones in each.
Interaction analysis between fusion bait and prey proteins in yeast was
performed by first introducing the LexA fusion bait plasmids into L40
yeast using the standard lithium acetate method (40), testing for
background -galactosidase activity, and then transforming the
resultant bait yeast strain with the prey to be tested. The
transformants were plated to synthetic UTL medium lacking leucine and
tryptophane to maintain selection for the prey and bait plasmids,
respectively, and to synthetic THULL medium lacking histidine, leucine,
tryptophane, uracil, and lysine to assay for protein-protein
interaction. Colonies from the UTL plates were picked and plated on
THULL medium grids to retest the interaction observed on primary THULL
medium plates. About 25 independent transformants were analyzed for
each bait-prey pair.
Yeast grown on THULL grids were assayed for -galactosidase activity
by a qualitative filter lift assay (40). For quantitative analysis, at
least six independent transformants were pooled to generate mixed
liquid cultures, which were grown in UTL medium overnight, then
inoculated into fresh UTL or THULL media at 1:10 dilution and grown
overnight at 30 °C. A600 was taken, and
100-500 µl of the culture was used for the assay, which was
performed as described (40). Relative -galactosidase activity was
expressed in Miller units as A420 × 100)/A600/min of incubation/(ml of yeast culture), with the very low background (no prey) subtracted. Absolute units varied with age of the yeast cultures and have not been normalized; each figure panel is from a single experiment.
T-cell Lines--
Human peripheral blood lymphocytes isolated
from normal donors were used to generate CD4(+) T-cell lines by
standard methods, including TT7.5 and T17.5, on which most of the
studies were conducted. Briefly, peripheral blood lymphocytes were
stimulated with immobilized anti-CD3 (OKT3 from ATCC) on tissue culture
plates in the presence of 20 ng/ml IL-4 and 10 µg/ml anti-IL-12
antibody (R&D Systems). Cells were cultured for 3-5 days, then
expanded in media containing 10 ng/ml IL-2 and 10 ng/ml IL-4 (R&D
Systems). The lines were maintained by anti-CD3 stimulation every 21 days, followed by expansion with IL-2 + IL-4. Cells were maintained in
modified Yssel's media (Iscove's modified media supplemented with 2%
heat-inactivated human AB serum, 2.5 mg/ml AlbuMAX II, 1×
insulin-transferrin-selenium supplement, and 1% penicillin,
streptomycin (all from Life Technologies, Inc.)) in the presence of 10 ng/ml IL-2 and 10 ng/ml IL-4 (R&D Systems). Jurkat cells (ATCC) were
maintained in RPMI 1640 (Life Technologies, Inc.), supplemented with
10% fetal bovine serum (HyClone Laboratories) and 2.5%
penicillin/streptomycin (Life Technologies, Inc.).
Cells were generally cultured overnight in modified Yssel's media
without IL-2 and IL-4 before stimulation. Typically, 5 × 107 cells were washed with PBS, pelleted, and resuspended
in 2 × 5 ml warmed modified Yssel's media. Direct PKC
stimulation was achieved by treatment with 10 nM PMA and
the calcium ionophore ionomycin at 25 µg/ml or by a combination of 20 nM PMA plus 1 µg/ml phytohemagglutinin for 10-15 min at
37 °C followed by washing in 10 ml of PBS. Alternatively, cells were
stimulated for 1 h using OKT3 (1 µg/ml), an antibody to the CD3
complex of the TCR.
Immunoprecipitation Analyses--
Cells were lysed by
resolubilizing cell pellets in solubilization buffer + 1% Triton
X-100, followed by a 10-min incubation in an ice bath. Lysates were
clarified by centrifugation at 12,000 × g for 10 min
at 4 °C. Triton-soluble protein was quantified by BCA protein assay
(Pierce). A minimum of 350 µg of lysate protein was used for each
immunoprecipitation, to which was added 2.5-5 µg of monoclonal
antibody to PKC (Transduction Laboratories), polyclonal antibody to
p59fyn (Upstate Biotechnology), or control antibodies. After incubation
overnight (16 h) at 4 °C with end-over-end rotation on a rotating
platform, protein PLUS G/A-agarose (Oncogene) was added, and the
mixture was incubated for an additional 20 min at 4 °C with
rotation. The agarose was then washed four times with
immunoprecipitation buffer.
SDS-PAGE sample buffer was added to the washed immune complexes and
boiled for 5 min. Samples were resolved on 8 or 10% SDS-PAGE gels and
transferred to nitrocellulose. The membranes were incubated in blocking
buffer (5% nonfat dry milk in PBS) for 1 h at room temperature,
followed by overnight incubation at 4 °C with primary antibodies,
anti-PKC monoclonal antibodies (Transduction Laboratories), or
anti-p59fyn polyclonal antibodies (Santa Cruz Biotechnology) diluted
1:500 in 1% BSA in PBS. Membranes were washed 3 times for 10 min in
PBS, 0.05% Tween 20. Secondary antibody conjugated to peroxidase was
added at a concentration of 1:1,000 in 1% BSA in PBS for 1 h at
room temperature. The membranes were washed as above and developed for
electrochemiluminescence detection as described by the manufacturer
(Amersham Pharmacia Biotech).
Protein Expression Analyses--
T-cells grown and stimulated as
described above were incubated for 24 h in culture media,
following which secreted cytokines were assayed using enzyme-linked
immunosorbent assay reagents from R&D Systems or
BIOSOURCE, following the manufacturer's protocols for detection of bound biotinylated antibodies with
peroxidase-streptavidin (Pierce) using 3,3',5,5'-tetramethylbenzidine
substrate (Kirkegaard and Perry). Experimentation focused on cells that
express IL-4 as well as -IFN but not IL-2. Expression of surface
antigens was measured 3-24 h after stimulation using fluorescently
labeled antibodies (Pharmingen) and a FACSCalibur flow cytometer with CellQuest software (Becton Dickinson). Cells, ~5 × 105/assay, were suspended in PBS + 1% FCS, 0.1% sodium
azide and incubated with antibody at room temperature for 30 min. Cells were washed three times and read immediately or stored in 0.5% formaldehyde in the dark at 4 °C for less than 5 days. An aliquot of
TER14687 from frozen Me2SO stocks was added to the cells
during stimulation. Antibodies to PKC and p59fyn (Santa Cruz
Biotechnology) or to choline acetyl transferase (CAT) (Promega), as a
control, were electroporated into the cells. A Bio-Rad Gene Pulser II
with Capacitance Extender II was used at 350 V and 250 microfarads. 1 × 107 cells were suspended in a sterile cuvette in
ice-cold PBS with antibody for 10 min, then electroshocked and rested
at room temperature for 15 min before stimulation. Under these
conditions, >50% of cells were able to take up antibody from the
media, without substantial cytotoxicity.
| |
RESULTS |
In Vitro Binding--
The first variable region of novel class PKC
isozymes had previously been shown to contain anchoring protein binding
sites (41). Accordingly, initial experiments were designed using that domain as a biochemical bait to identify PKC-binding proteins. Sorbents were prepared from PKC-V1, comprising residues 1-140. Adsorbed and eluted proteins from Jurkat lymphoma cell extracts were
characterized using antibodies to phosphotyrosine (Tyr(P)) and to known
T-cell-signaling proteins. These results led to the preliminary
identification of p59fyn as the most prominent adsorbed Tyr(P)-containing protein (Fig. 1). The
other bands in lane 3 stained with anti-Tyr(P) antibody are
attributable to leached-off protein contaminants present in the PKC
preparation used to make the sorbent (see lane 2) or to
p59fyn degradation products (see lane 4). In control
experiments using sorbents constructed from the V1 regions of PKC
and PKC, no band at the molecular weight of p59fyn was observed (not
shown).
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Fig. 1.
Binding of p59fyn to
PKC-V1 affinity sorbent. Recombinant PKC-V1
was immobilized via a histidine tag on a nickel-agarose resin and
incubated with Jurkat cell extracts. Lane 1, total Jurkat
phosphotyrosine (Tyr(P))-containing proteins; lane
2, background Tyr(P) immunoreactivity present in the PKC-V1
preparation before the addition of Jurkat cell extracts; lanes
3 and 4, proteins eluted from the resin with SDS-PAGE
sample buffer following washing. Proteins were visualized by Western
blotting; after separation on a 10% gel, the proteins were transferred
to nitrocellulose and probed with anti-Tyr(P) antibody (lanes
1-3) or anti-p59fyn antibody (lane 4).
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To further establish that PKC can bind to p59fyn, purified
recombinant constructs were used in a 96-well microplate binding assay
(Fig. 2). A solid phase capture format
was used, with p59fyn immobilized as a fusion protein with MBP. Based
on the concentration needed for half-maximal binding under these
conditions, the affinity of the interaction was estimated to be 5 × 10-9 M-1. Similar results were
obtained using GST-p59fyn immobilized on nitrocellulose. An important
feature of these results is that the binding of the PKC-V1 region
was approximately equivalent to that seen with the full-length
recombinant PKC, whereas the binding of the corresponding V1 region
of the closely related novel class PKC isozyme was substantially
weaker (Fig. 2B). Binding was quite reproducible in
immediate replicates, with interassay coefficient of variation below
10% and background values well below the specific binding. The only
significant source of variability was dependence on the PKC-activating
phospholipids, DAG and phosphatidylserine, possibly reflecting
variation among the different batches of recombinant proteins in
posttranslational modifications, e.g. phosphorylation or
minor proteolysis, or in carryover of lipid activators from the
baculovirus production system.
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Fig. 2.
Binding of recombinant
PKC to p59fyn in a microplate assay.
A, titration of MBP-p59fyn immobilized on a microplate is
shown for the indicated amounts of PKC in solution. B,
the V1 variable domains of PKC and PKC were compared for ability
to bind MBP-p59fyn. Detection of bound protein was accomplished using
an enzyme-conjugated antibody. The average of duplicate determinations
for a representative replicate of at least four different protein
preparations is plotted, with background signal (~0.1 optical
density) found with MBP alone subtracted.
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As described previously for other PKC isozymes in the presence of their
anchoring proteins (28), the presence of p59fyn potentiated the kinase
activity of PKC, increasing maximal phosphorylation of histone IIIs
in an in vitro kinase assay (Fig.
3 lanes 7 and 8),
with a more modest effect on autophosphorylation (lanes 3 and 4). The figure shows representative results from three
independent preparations of PKC. Increased phosphorylation in
lanes 7 and 8 at the molecular weight of PKC
is attributed to histone dimers.
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Fig. 3.
Potentiation of PKC
kinase activity by p59fyn. PKC autophosphorylation (lanes
1-4) and phosphorylation of histone IIIs (lanes 5-8)
were assayed by incorporation of radiolabeled phosphate in the absence
or the presence of GST-p59fyn, and in the absence or presence of
DAG/phosphatidylserine (PS), as indicated. Equal amount of
material was loaded in each SDS gel lane.
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Association in T cells--
Following preliminary studies in
Jurkat lymphoma cells, the binding of the two proteins was examined
in nontransformed T-cells. As shown in Fig.
4A, an antibody against PKC
co-precipitated p59fyn from a T-cell supernatant. Conversely, shown in
Fig. 4B, an antibody to p59fyn co-precipitated PKC. Cell
activation is known to result in segregation of some TCR-associated
proteins into detergent-insoluble microdomains (29); p59fyn in
particular was difficult to solubilize quantitatively, and therefore,
no attempt was made to determine the stoichiometry of association in
resting cells compared with activated cells. The specificity of the
interaction between PKC and p59fyn in T-cells was established through a series of negative results. The interaction of PKC with
RACK1, originally described in cardiac myocytes (30), was confirmed in
Jurkat cells by immunoprecipitation studies. By this technique, PKC
did not bind to p59fyn, and PKC did not bind to RACK1. Furthermore,
neither inositol-3,4,5-trisphosphate kinase nor p56lck were
immunoprecipitated with PKC-specific antibody under conditions where
p59fyn was detected.
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Fig. 4.
Co-immunoprecipitation from T-cells of
PKC and p59fyn. A, TT7.5 T-cell
extracts immunoprecipitated (IP) with anti-PKC;
B, T17.5 T-cells immunoprecipitated with anti-p59fyn.
Proteins were separated by SDS-PAGE, transferred to nitrocellulose, and
probed with anti-p59fyn (A) or anti-PKC (B),
with the Mr region of interest on the blots
illustrated. Lane 1, controls; for A, protein
G-agarose with no cell extract added, and for B, excess
p59fyn peptide (residues 35-51) was added to block the precipitating
antibody. Lane 2, co-immunoprecipitate of unstimulated cell
extracts. Lane 3, co-immunoprecipitate of cells stimulated
with PMA + phytohemagglutinin (A) or PMA alone
(B). Equal aliquots of cell extract were loaded in each
lane.
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p59fyn Kinase Activity--
When tested in vitro,
PKC-V1 was a substrate for recombinant p59fyn fused to MBP (Fig.
5). Controls showed that the MBP portion was irrelevant to the activity (lanes 5-8). Six tyrosines
are present in PKC-V1, and one of these, Tyr-53, is conserved in PKC-V1, which is also a substrate for MBP-p59fyn in
vitro, although to a lesser extent than PKC-V1 (lanes
2 and 4). PKC did not compete as strongly with the
known p59fyn substrate, enolase, as did PKC-V1 (lanes 1 and 3 versus 9). Consistent with a role for
phosphorylation in stabilizing the interaction, inclusion of ATP in the
microplate binding assay improved the signal to noise ratio (not
shown). Further, p59fyn catalytic activity was required for binding in
the yeast two-hybrid constructs described below.
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Fig. 5.
In vitro phosphorylation of
PKC-V1 by p59fyn. Kinase reaction products
were separated by SDS-PAGE (14% gel) and autoradiographed. Recombinant
PKC-V1 and PKC-V1, both, functioned as substrates of MBP-p59fyn,
competitive with enolase, but to a greater extent in the case of
PKC-V1 (lanes 1-4). MBP alone had no kinase activity on
any of the components (lanes 5-8), whereas the MBP-p59fyn
fusion was competent to phosphorylate enolase (lane 9)
and itself (lane 10).
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Yeast Two-hybrid Analysis--
Additional evidence for a direct
interaction, consistent with the immunoprecipitation results and
in vitro recombinant protein binding data, was obtained
using the yeast two-hybrid system. Fragments of PKC fused to lexA
formed a DNA binding bait, and fragments of p59fyn fused to VP16 formed
an RNA polymerase-activating prey, with -galactosidase as the
reporter gene turned on by bait binding to prey. Because the
combination of full-length PKC and p59fyn was found to be toxic to
the yeast, the experiments focused on the PKC-V1 region. The other
major variable portion of the PKC regulatory domain, V3 comprising
residues 282-385, produced a high background signal as bait even in
the absence of a prey and was not analyzed further.
Fig. 6 presents a summary of PKC-V1
binding to p59fyn. The strongest binding was to the kinase domain,
although some binding to the regulatory domain was observed. When
transfected into the same parental bait construct yeast strain,
approximately equal amounts of the fusion proteins were produced for
full-length p59fyn and for both the kinase and regulatory
domains, as measured by Western blotting with anti-p59fyn
antibodies. Thus, the reporter gene signal is attributable to
variations in the affinity of the interacting bait and prey
proteins.
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Fig. 6.
Binding of PKC-V1 to
p59fyn in yeast two-hybrid system. A, binding of
PKC-V1 bait to FL (full-length), K (kinase
domain), R (regulatory domain) of p59fyn (open
bars) compared with p56lck (hatched bars) assayed by
quantitative -galactosidase liquid culture assays and reported in
Miller units. The solid bar represents binding to K296A
("kinase-dead") mutant of p59fyn. B, binding of
PKC-V1 bait to full-length p59fyn prey in the absence and presence
of 60 µM TER14687, compared with control bait (Tal1) and
prey (E2A).
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Specificity of the interaction was explored in two respects. First, the
V1 region of PKC, which is the isozyme most closely related to
PKC by sequence, showed no binding to p59fyn or its major fragments.
Second, p56lck, which is closely related to p59fyn by sequence (39) and
is also critical for the TCR-mediated signal transduction, showed
reduced binding. The quantitative comparison of PKC-V1 binding to
p59fyn and p56lck is shown in Fig. 6A. Unlike p59fyn, for
which the kinase domain was as effective as the intact protein, the two
major domains of p56lck were not nearly as effective as the whole
protein. Furthermore, when p59fyn kinase and regulatory domain prey
constructs were mixed into a murine T-cell cDNA library and
screened using PKC-V1 as bait, the positive clones recovered were
predominantly from p59fyn, consistent with the degree to which they had
been spiked into the library. Likewise, screening a human cDNA
library yielded several p59fyn clones but no p56lck clones.
Also shown in Fig. 6A is evidence that the catalytic
function of p59fyn is necessary for binding to PKC-V1; the K296A
p59fyn mutant, which has no kinase activity (42), did not bind to
PKC-V1. Both wild type full-length p59fyn and the kinase domain
constructs were catalytically active in the yeast cells as determined
by anti-Tyr(P) Western blot analysis of total yeast proteins. In contrast, yeast cell extracts containing PKC-V1 bait alone or in
combination with the K296A mutant had no detectable Tyr(P) by the same analysis.
The yeast two-hybrid system was further used to characterize TER14687,
(±)-2-(N,N-dimethylaminomethyl)-1-indanone HCl,
a compound identified by preliminary screening of an empirically
diverse set of compounds (43) for activity in a Jurkat cell IL-2
production assay (35). In the yeast reporter assay, the compound was
found to block the association between PKC-V1 and p59fyn (Fig.
6B). Because this compound undergoes a spontaneous
elimination at neutral pH to form a more reactive compound, the active
species probably results from reaction with a media component; the
final active structure has not yet been identified. Whatever the
ultimate structure of TER14687, however, its effects are apparently
specific to the interaction of interest because it had no effect on a
Tal1/E2A two-hybrid construct. In this control, an active transcription factor is reconstituted by noncovalent association of domains from two
separate transcription factors. Moreover, when added to media at the 60 µM concentration used in the two-hybrid experiments, TER14687 did not affect growth of normal yeast cells.
Physiological Role--
Prior work had shown that disrupting
PKC translocation by overexpressing a binding protein suppressed
IL-2 production in Jurkats (35) and that PKC was one of the first
PKC isozymes to be activated following T-cell stimulation (36). Under
the conditions used here, PMA stimulation caused all isozymes studied to translocate from the soluble to particulate fraction in both Jurkat
lymphoma cells and normal T-cell lines. These include closely related
PKC and PKC as well as the more distantly related PKC. However, PKC was the only isozyme that translocated following physiological stimulation using the OKT3 antibody to CD3 (Fig. 7A). PKC translocation in
nontransformed T-cells following OKT3 activation was blocked by
TER14687 (Fig. 7B). No effect of the compound on other
isozymes was observed in Jurkats or normal T-cells. Thus, a compound
that had been found to specifically inhibit PKC binding to p59fyn
was also found to cause a selective effect on PKC translocation.
TER14687 therefore provides a useful tool for exploring the
physiolgical consequences of PKC translocation.
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Fig. 7.
Modulation of isozyme translocation.
A, translocation from soluble cytoplasmic (S) to
particulate (P) fraction for PKC and PKC following
treatment of nontransformed T-cells with the PKC agonist PMA or with
the OKT3 antibody to CD3. B, translocation of PKC induced
by OKT3 following a 15-min preincubation with 10 µM
TER14687. Proteins were visualized by Western blotting as in Fig.
1.
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OKT3 stimulation of normal human T-cells in the presence of 5 µM TER14687 resulted in substantial suppression of the
up-regulation of surface antigens normally associated with T-cell
activation. The results shown in Fig.
8A for CD69 are representative
of effects observed in parallel experiments examining CD25 and CD40L.
Expression of the cytokines IL-4 and -IFN was also measured for
several cell lines that express these cytokines but not IL-2 upon
stimulation. Culture supernatants collected 24 h after OKT3
stimulation were analyzed by enzyme-linked immunosorbent assay assays;
tritiated thymidine uptake was used to assess the proliferation
response. The representative example in Fig. 8 (panels B-D)
shows that TER14687 caused a more pronounced suppression of IL-4 as
compared with -IFN. Considering all experiments together, there was
a trend to reduced proliferation at higher doses of TER14687, possibly as a secondary consequence of the reduction in secretion of IL-4, which
acts as an autocrine growth factor. Reductions in -IFN were
correlated with reduced proliferation, whereas the more extreme reductions of IL-4 secretion were independent of effects on
proliferation.
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Fig. 8.
TER14687 effects on protein expression.
A, effect of 5 µM TER14687 on expression of
CD69 T-cell surface antigen induced by overnight stimulation with OKT3
antibody to the CD3 complex, measured by FACS cytometry. The effect of
varying doses of TER14687 following 24-h OKT3 stimulation is shown for
proliferation (panel B) and secretion of IL-4 (panel
C) and -IFN (panel D). The absolute levels of
-IFN are 10-fold higher than IL-4.
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Specificity of the TER14687 results was enhanced by several negative
results. At 5 µM in the cell media, TER14687 had no
effect on calcium flux, an early sequel to OKT3 activation that was
blocked by 25 µM piceatannol, a tyrosine kinase
inhibitor. Cytotoxic effects of TER14687 were modest, below 5 µM; the higher tolerance of yeast cells to TER14687 may
be because of active export pumps, which are known to be readily
inducible in yeast (44). At 50 µM in vitro,
the compound did not inhibit the kinase activity of either PKC or p59fyn.
Additional indications that the cytokine effects arose from the direct
interaction between PKC and p59fyn were provided by exploratory
experiments using antibodies to either PKC or p59fyn as
pharmacological probes, introduced into the cells by electroporation. For these experiments, the cells were stimulated for 30 min using PMA
and ionomycin, conditions that produced results comparable with OKT3
activation with regard to PKC translocation and cytokine secretion.
Suppression of IL-4, with little effect on -IFN, was seen with
antibodies to either protein but not with a control antibody against
choline acetyl transferase (CAT) (Fig.
9). As shown in the figure, these results
were significantly more variable in replicate experiments than was the
case with TER14687, possibly because of variable degradation of the
antibody. Consistent with a progressive degradation of the antibody, it
was found that doubling the length of time the cells were stimulated
eliminated the IL-4 suppression effect of the antibodies electroporated
into the cells before stimulation. OKT3 activation, which required 60 min of stimulation to increase cytokine secretion, was thus not
feasible using antibodies as pharmacological probes.
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Fig. 9.
Electroporated antibody effects on cytokine
expression. Antibodies were introduced into T-cells under
electroporation conditions in which >50% of the cells took up
antibody with minimal loss in cell viability. The effects of antibodies
to PKC and to p59fyn on IL-4 expression induced by PMA + ionomycin
are compared with a control anti-CAT antibody. Filled bars
are with antibody; open bars are electroporated mock
experiment without antibody; all are normalized against cells that were
not electroporated.
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DISCUSSION |
The key experimental result that has emerged from the present
study is that a specific protein-tyrosine kinase, p59fyn, interacts with a specific PKC isozyme, PKC, in a manner that goes well beyond
the typical interaction of an enzyme with its substrate. Rather, the
interaction is quite analogous to that previously described between
other PKC isozymes and their anchoring RACKs (28). This result,
together with other recent evidence expanding the range of direct PKC
interactions with proteins involved in signaling pathways (3, 4), thus
has ramifications beyond T-cell biology.
Signal transduction networks are inherently nonlinear, replete with
feedback loops, and convergent in several aspects. This complexity
makes definitive proof for the role of any single component difficult
to achieve and even more difficult for the interaction of two
components. Accordingly, the credibility of p59fyn as an anchoring
protein for PKC is fortified by being the common conclusion from
three independent lines of experimentation. First, the two proteins
interact in vitro, as measured by using affinity sorbents on
cell extracts (Fig. 1) as well as by direct binding of recombinant proteins (Fig. 2). Furthermore, the presence of p59fyn potentiates the
kinase activity of PKC on another substrate (Fig. 3), a property shared with other RACKs.
Second, the two proteins are associated in vivo, as detected
by co-immunoprecipitation from cell extracts (Fig. 4). The use of
nontransformed human T-cell lines, rather than immortalized tumor lines
or genetically engineered overexpressing lines, enhances the
credibility of this result. One possible divergence from the previous
PKC/RACK literature concerns the dependence of the interaction on
activating phospholipids. Lipid-derived second messengers, normally
produced intracellularly upon cell activation, induce a change in
conformation that both activates catalytic activity and enables RACK
binding (2). The activation dependence observed for PKC binding to
p59fyn was variable in the T-cells examined. The known segregation of
TCR-associated proteins into detergent-insoluble domains following
activation (29) precludes accurate quantitation of the interaction
between PKC and p59fyn in Triton extracts, however. The issue could
not be resolved from in vitro dependence on DAG for binding
because removing cytokines from the cell media overnight consistently
reduced the amount of particulate fraction PKC to some degree,
suggesting that the cytokines needed to promote growth of the cells
also caused a variable level of activation.
Third, the yeast two-hybrid system established that the interaction
encompasses an extended region of p59fyn, with strong binding via the
kinase domain of p59fyn (Fig. 6). This result is consistent with
evidence that PKC is a substrate for tyrosine phosphorylation by
p59fyn (Fig. 5). PKC obtained from T-cells by immunoprecipitation
also contained phosphotyrosine, as determined by Western blot analysis
using Tyr(P)-specific antibody. The stable nature of the binding
revealed by co-immunoprecipitation is unusual for enzyme interactions
with substrates, although there is precedence for catalytic activity
increasing stability of a binding event. Specifically, experiments with
a kinase-dead mutant showed that p59fyn kinase activity is required for
p59fyn binding to ZAP-70 (13), analogous to the present result showing
that p59fyn must be catalytically active to interact with PKC-V1 in
the yeast two-hybrid format (Fig. 6A). Importantly, both
two-hybrid and immunoprecipitation experiments showed strong
specificity for the interaction compared with closely related proteins
for both binding partners (PKC in place of PKC, and p56lck in
place of p59fyn). These results do not preclude the possibility that
additional cognate proteins may exist to which PKC binds, however.
It would be surprising if there were no physiological effects
attributable to the interaction between these two proteins, each of
which has individually been implicated in T-cell signaling. TER14687,
which blocks their association in the yeast two-hybrid system (Fig.
6B) and specifically prevents normal translocation of PKC
in T-cells (Fig. 7) does indeed show specific suppressive effects on
production of cytokines (Fig. 8B) as well as surface antigens characteristic of activated T-cells (Fig. 8A).
Furthermore, a differential effect on expression of IL-4 compared with
-IFN was observed, extending prior work in Jurkat cells showing that overexpressed 14-3-3- protein is both a translocation inhibitor and
IL-2 suppressor (35). The active species formed in cells from TER14687
is not known. The consistent findings using antibodies to either PKC
or p59fyn as pharmacological probes (Fig. 9) strengthens the validity
of the results, extending prior antibody electroporation work (36).
Because the expression of -IFN was 10-fold higher than IL-4 in the
cell lines studied, the specificity may reflect a quantitative effect
rather than a qualitative difference in the regulation of the two
cytokines. Whatever the precise consequences are for the signal
network, the available pharmacological data suggest that compounds
disrupting the interaction of PKC and p59fyn could have therapeutic
utility for suppression of IL-4, a key cytokine implicated in allergy
(45).
PKC and the Tyrosine
Kinase p59fyn*
TOP
ABSTRACT
INTRODUCTION
To whom correspondence should be addressed. Tel.: 415-567-6815;
Fax: 415-567-6819; E-mail: kauvarLM{at}earthlink.net.
