From the Institute of Cell Biology and Immunology,
University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany and
the § Division of Cell Biology and Immunbiology, La Jolla
Institute for Allergy and Immunology,
San Diego, California 92121
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ABSTRACT |
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Recent studies have documented direct interaction
between 14-3-3 proteins and key molecules in signal transduction
pathways like Ras, Cbl, and protein kinases. In T cells, the 14-3-3 Members of the protein kinase C
(PKC)1 family of
intracellular serine kinases play critical roles in the regulation of a
variety of intracellular signaling processes. Much attention has been focused on the role of PKCs in T cell signaling (for review see Refs.
1-3). Phorbol ester responsive PKCs in general have long been
associated with T cell activation and a prominent role of one
particular subtype, PKC During T cell signaling events evidence of an involvement of members of
the 14-3-3 proteins, an abundant group of acidic proteins originally
found in brain extracts (6-8), has been obtained. For example, it has
been demonstrated that the 14-3-3 We have recently described a novel PKC isotype termed PKCµ (19),
which, although ubiquitously expressed, shows particularly high
expression in thymus and hematopoetic cells (20). PKCµ displays, in
addition to the conserved kinase and regulatory domains in common to
all PKC isoforms, structural features like a hydrophobic amino-terminal
domain, an acidic regulatory domain (21), and a pleckstrin homology
domain (22). First evidence for involvement of PKCµ in diverse
cellular functions stems from reports showing enhancement of
constitutive transport processes in PKCµ overexpressing epithelial
cells (23) and PKCµ activation during antigen receptor-mediated signaling in B cells (24).
In the present study, we demonstrate by binding studies and pulldown
assays as well as by transient expression in the T cell line Jurkat
that PKCµ specifically associates in vitro and in vivo with 14-3-3 Recombinant PKCµ, Plasmid Constructs, and Cell Lines--
The
production of Sf158 insect cells overexpressing PKCµ (25) and the
construction of 14-3-3 Immunoprecipitation by Antibodies and GST-14-3-3 Transfections--
7.5 × 106 Jurkat-TAg cells
were seeded per 60-mm-diameter dish in 5 ml of RPMI supplemented with
10% fetal calf serum and transfected with 5 µg of DNA and 20 µl of
Superfect reagent (Qiagen) according to the manufacturer's protocol.
Cells were harvested and analyzed 48 h upon transfection by
immunoprecipitation analysis as described above. In the case of
14-3-3 In Vitro Kinase Assays--
Jurkat-TAg cells were stimulated
with phorbol 12,13-dibutyrate (PdBu, 100 nM) for the
indicated times, lysates were prepared, and PKCµ was
immunoprecipitated. PKCµ autophosphorylation was determined in an
in vitro kinase assay as described previously (28). In
brief, the immunoprecipitates were washed twice in lysis buffer and
once in phosphorylation buffer (50 mM Tris, pH 7.5, 10 mM MgCl2, 2 mM dithiothreitol). The
immune complexes were mixed with 10 µl of phosphorylation buffer
containing 0.2 µl of [ Phosphatase Treatment and Far Western Blot Analysis--
PKCµ
was immunoprecipitated from 5 × 106 Sf158 cells using
4 µl of a rabbit antiserum raised against a carboxyl-terminal
epitope. Protein G-Sepharose bound immune complexes were in
vitro phosphorylated as described above and washed twice to remove
nonincorporated [ 14-3-3
Next, the association of 14-3-3 proteins with endogenous PKCµ was
investigated. 14-3-3
14-3-3 14-3-3
14-3-3 14-3-3
Binding of 14-3-3 14-3-3
Next we analyzed whether 14-3-3 In this study, we identify PKCµ as a novel 14-3-3 14-3-3 binding to several signal transducers (7-10) including PKC
isotypes (18, 41, 42) has been reported, but controversial data exist
as to the functional role of these interactions (7, 8, 41). Of
relevance to the findings reported here, 14-3-3 Activation of conventional and novel PKC isotypes typically occurs by
binding of second messengers like diacylglycerol or phorbol ester to
the C1 region (28, 30-32). The C1 region further serves as a binding
region for regulatory proteins, as has been shown for the atypical
PKC Phosphoserine binding motifs for 14-3-3 proteins like
RSXpSXP and
RXXpSXP have been identified by extensive screening using peptid libraries (36). These motifs are present and
functional in several already known 14-3-3 binding proteins including
PKC In conclusion, we propose that 14-3-3
isoform has been shown to associate with protein kinase C
and to
negatively regulate interleukin-2 secretion. Here we present data that
14-3-3
interacts with protein kinase C µ (PKCµ), a subtype that
differs from other PKC members in structure and activation mechanisms. Specific interaction of PKCµ and 14-3-3
can be shown in the T cell
line Jurkat by immunocoprecipitiation and by pulldown assays of either
endogenous or overexpressed proteins using PKCµ-specific antibodies
and GST-14-3-3 fusion proteins, respectively. Using PKCµ deletion
mutants, the 14-3-3
binding region is mapped within the regulatory
C1 domain. Binding of 14-3-3
to PKCµ is significantly enhanced
upon phorbol ester stimulation of PKCµ kinase activity in Jurkat
cells and occurs via a Cbl-like serine containing consensus motif.
However, 14-3-3
is not a substrate of PKCµ. In contrast 14-3-3
strongly down-regulates PKCµ kinase activity in vitro. Moreover, overexpression of 14-3-3
significantly reduced phorbol ester induced activation of PKCµ kinase activity in intact cells. We
therefore conclude that 14-3-3
is a negative regulator of PKCµ in
T cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, belonging to the novel PKC subfamily (4),
is suggested from recent studies: PKC
is translocated upon
antigen-specific stimulation to the membrane interface between T cells
and antigen presenting cells, implicating a physical interaction of
PKC
either directly with the T cell receptor complex or other T cell
receptor proximal signaling molecules (5).
isotype interacts with the
catalytic subunit of the phosphoinositide 3-kinase (9) and the Cbl
protooncogene (10), affecting Ras-dependent T cell
receptor-mediated signaling leading to NF-AT activation (11). Besides T
cell-specific functions 14-3-3 proteins have been shown to be involved
in mitogenic pathways of other cells as well, affecting regulation of
the Raf kinase (7, 12), cell cycle (13), and anti-apoptotic pathways
(14-16). The mechanism, by which 14-3-3 influences Raf is still
unresolved, as recent data suggest that activation of Raf by 14-3-3 may
in fact be due to stabilization of an activation complex rather than a
direct stimulation of Raf activity (17). A stabilizing role in the formation of signaling complexes can be deduced from the capacity of
14-3-3
isoform to form dimers in vitro (10). The
recruitment of signal transducers like Cbl (10) and phosphoinositide
3-kinase (9) in T cells further supports a potential role of 14-3-3 dimers in the assembly and/or regulation of signaling complexes. Evidence for an active regulatory function of 14-3-3 proteins stems
from the finding that 14-3-3
binding to PKC
negatively affects
the stimulation of the interleukin-2 promotor and prevents PKC
translocation to the membrane (18), supporting a role of 14-3-3 proteins in the regulation of PKC activation in T cells.
proteins. The 14-3-3
binding site within
PKCµ could be located to the C1 regulatory region. 14-3-3
interacts preferentially with the activated, phosphorylated PKCµ and
down-regulates kinase activity, suggesting that 14-3-3
is a
regulator of PKCµ functions in T cells.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
glutathione S-transferase (GST) fusion proteins has been described previously (9). The human T
lymphoma cell line Jurkat-TAg (26) was maintained in RPMI 1640 medium
supplemented with 10% fetal calf serum. GST fusion proteins were
isolated according to the manufacturer's instructions (Amersham
Pharmacia Biotech). In brief fusion proteins were bound to
glutathione-Sepharose and quantitated upon Coomassie staining by
densitometric scanning, calibrated against an albumin standard. PKCµ
deletion mutant PKCµ
1-79 was constructed by digesting pBpl4 (19) with ApaI and NsiI. Overhanging 5' and 3' ends
were filled with the Klenow enzyme, and the 2.9 kilobase PKCµ
fragment was isolated and ligated in EcoRV-digested
pCDNA3 (Invitrogen). PKCµ
1-340 was constructed by
cutting pCDNA3/PKCµ
1-79 with HindIII,
isolating a 800-base pair HindIII fragment followed by
religating the vector/PKCµ portion. Additionally these mutants were
cloned in other expression vectors and verified by transient expression
(27). PKCµ point mutations (serine to alanine exchange) were created
using a polymerase chain reaction approach according to the
manufacturer's instructions (Quickchange site-directed mutagenesis,
Stratagene) and were verified by dideoxy sequencing of both strands.
COS transfectants stably overexpressing PKCµ were generated by
transfecting COS cells with PKCµ wild type cloned in the expression
vector pCDNA3 followed selection of transfectants in neomycin (400 µg/ml) containing media for a period of 20 days. Single colonies were
analyzed for PKCµ overexpression by Western blot analysis.
Precipitation
of PKCµ--
Sf158 or Jurkat-TAg cells were lysed at 4 °C in
lysis buffer (20 mM Tris, pH 7.4, 2 mM
MgCl2, 1% Triton X-100, 1 mM sodium orthovanadate, 150 mM NaCl, 10 µg/ml leupeptin, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM NaF, 1 mM nitrophenylphosphate) using a sonifier. After
centrifugation of cell debris (15 min, 15 000 rpm, type 5403, Eppendorf) GST precipitation was done by incubation with the indicated
amounts of GST fusion proteins coupled to glutathione-Sepharose in 1-ml
lysate portions (500 000 Sf 158 cells or 60 × 106
Jurkat-TAg cells) for 90 min at 4 °C. For immunoprecipitation of
PKCµ from Jurkat-TAg cells, a PKCµ antiserum was used as described earlier (28). Immunocomplexes were harvested by incubation with protein
G-Sepharose (Pharmacia, 30 µl/2 × 107 cell
equivalents) for 30 min at 4 °C. Immunocomplexes or GST complexes
were washed three times in lysis buffer and applied to SDS-PAGE
following transfer to a nitrocellulose membrane. Western blot detection
of PKCµ or 14-3-3
was performed according to standard conditions
using monoclonal antibodies as described earlier (18, 28). GST was
detected using an anti-GST mAb (Santa Cruz). Visualization for all
Western blots shown was performed using an alkaline phosphatase-based detection system according to standard conditions.
overexpression experiments, PKCµ was immunoprecipitated and
in vitro autophosphorylated as described below.
Exponentially growing 293 cells, 40-80% confluent, were transfected
with the indicated plasmids using 2 µg of DNA and 10 µl of
Superfect reagent for each well of a 6-well plate or 10 µg of DNA and
60 µl of Superfect reagent for a 100-mm plate. Extracts from one well
were used for each immunoprecipitation and GST 14-3-3
precipitation
of PKCµ.
-32P]ATP (Amersham Pharmacia
Biotech) and incubated for 10 min at 37 °C. The reaction was stopped
by adding 5× SDS-PAGE sample buffer, fractionated by SDS-PAGE followed
by transferring to a nitrocellulose membrane, and visualized by
phosphoimaging (Molecular Dynamics). For the in vitro
inhibition assays, 80 ng of purified PKCµ enzyme from Sf158 cells
(25) was used with the indicated amounts of GST 14-3-3
or GST added.
-32P]ATP. Bound PKCµ was eluted in
a final volume of 100 µl upon adding 50 µl of immunizing peptide (1 mg/ml) by incubating 30 min at 4 °C. PKCµ was incubated with
Phosphatase 2A (0.4 units) for the indicated times. Equal aliquots were
subjected either to GST 14-3-3
precipitation followed PKCµ
immunodetection or to direct immunoblot analysis to compare
precipitation efficacies. For Far Western analysis, PKCµ from 80 × 106 Jurkat TAg cells was immunoprecipitated as
described. Aliquots of immunoprecipitates were subjected to SDS-PAGE
and transferred to a polyvinylidene difluoride membrane. PKCµ
detection was carried out using a PKCµ mAb. 14-3-3
binding to
activated PKCµ was analyzed essentially as described (29). Detection
of bound 14-3-3
was carried out by a 2-h incubation with 10 µg/ml
GST 14-3-3
fusion protein and visualized using an alkaline
phosphatase-coupled anti-GST secondary antibody.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Specifically Associates with PKCµ in
Vitro--
14-3-3
has been recently reported to associate with
PKC
, which is highly expressed in T cells (4, 5). To test whether 14-3-3
would also interact with another T cell expressed isoform, PKCµ, we analyzed recombinant PKCµ for potential 14-3-3
association. GST 14-3-3
fusion proteins were used to precipitate
PKCµ expressed in Sf158 cells. As shown in Fig.
1A, in GST pulldown assays a 14-3-3
dose-dependent binding of PKCµ can be detected
by immunoblot analysis (upper panel), showing best detection
using 4 µg of 14-3-3
GST fusion protein. 14-3-3
binding to
PKCµ is specific because no binding to the respective amount of GST
proteins was detectable. Only a fraction of total recombinant PKCµ
was precipitated with 14-3-3
GST protein, as shown by comparison
with PKCµ immunoprecipitation by PKCµ-specific polyclonal
antibodies (Fig. 1A, left lane), even when the
GST 14-3-3
concentration was increased to 20 µg (data not
shown).
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Fig. 1.
14-3-3 interacts with
PKCµ. A, PKCµ expressed in
Sf158 cells was precipitated with the indicated amounts of 14-3-3
GST bacterial fusion protein and the respective GST protein as a
control. PKCµ (left lane) was immunoprecipitated
(IP) under similar conditions (500,000 Sf158 cells) as a
positive control using a polyclonal rabbit antibody specific for
PKCµ. Bound PKCµ was detected by immunoblot analysis using a PKCµ
mAb and an alkaline phosphatase-coupled secondary antibody. GST was
visualized using an anti-GST mAb as described under "Experimental
Procedures." B, PKCµ can be specifically precipitated by
14-3-3 in Jurkat-TAg cells. 10 µg of GST 14-3-3
fusion protein was
used to precipitate PKCµ from lysates of 60 × 106
Jurkat-TAg cells. Detection was carried out as described for
A. C, 14-3-3
is coprecipitated with PKCµ in
293 and COS cells. 293 cells (left panels) were
cotransfected with PKCµ and 14-3-3
expression vectors as described
under "Experimental Procedures." 40 h after transfection
PKCµ immunoprecipitates were analyzed by Western blot for the
presence of PKCµ (upper panels) and 14-3-3
(lower
panels). Detection of 14-3-3
was performed with a 14-3-3
mAb
and an alkaline phosphatase-based detection system. Stable PKCµ
overexpressing COS transfectants (right panels) were
transfected with the indicated amounts of a 14-3-3
expression
plasmid or vector alone. PKCµ was immunoprecipitated and analyzed for
the presence of 14-3-3
as for 293 cells.
GST fusion proteins were used to precipitate PKCµ from extracts of Jurkat-TAg cells. As shown in Fig.
1B, endogenous PKCµ could be specifically precipitated
from lysates of Jurkat-TAg cells. Both 14-3-3 isoforms, 14-3-3
and
14-3-3
(30), were equally suited to precipitate PKCµ. The
respective controls, glutathione S-transferase, and as a
control for nonspecific binding, the pleckstrin homology domain of
PKCµ expressed as a GST fusion protein did not detectably precipitate
PKCµ in pulldown assays (Fig. 1B, left lanes).
association with PKCµ was also shown in coprecipitation
experiments using PKCµ-specific antibodies. As in 293 cells endogenous PKCµ levels are too low to detect 14-3-3
association (data not shown); cotransfection of PKCµ and 14-3-3
was performed in 293 cells. Additionally, 14-3-3 was transiently overexpressed in
stable COS-PKCµ transfectants, and PKCµ was immunoprecipitated from
lysates of double transfectants. In both cases, different amounts of
14-3-3 DNA were used for transfection to ensure optimum expression. As
shown in Fig. 1C (left panels), in cotransfected 293 cells 14-3-3
can be readily detected in PKCµ
immunoprecipitates upon appropriate expression of both cDNAs
(PKCµ/14-3-3 DNA ratio 1:10). Likewise, in stably PKCµ expressing
COS transfectants, 14-3-3
can also be coprecipitated with PKCµ
upon transient overexpression using 10 µg of the respective 14-3-3
expression construct (Fig. 1C, lower right
panel). As the subtype-specific anti-14-3-3
mAb is directed
against an epitope within the potential binding site of target
proteins,2 the reciprocal
immunoprecipitation experiment was precluded.
Binds to a Serine-dependent Motif within the
C1 Region of PKCµ--
The cysteine fingers in the C1 region of PKCs
have been previously reported to be the binding site for second
messengers as well as for regulatory proteins affecting protein kinase
activity (31-34). Fig. 2A
displays the location of these domains in PKCµ. In an attempt to
identify potential binding sites of 14-3-3
, we transiently
overexpressed in 293 cells an amino-terminal PKCµ deletion mutant and
a mutant lacking in addition the C1 binding region. The mutants
PKCµ
1-79 and PKCµ
1-340 constructed
by deletion analysis initiating translation at Met-80 or Met-341 (see
"Experimental Procedures") were used. Transfection of these mutants
in 293 cells resulted in the expression of approximately 100- and
70-kDa variants of PKCµ as shown by immunoprecipitation (Fig.
2B). 14-3-3
GST fusion proteins were used to precipitate PKCµ, and the mutants from lysates of 293 cells were transfected with
the respective expression constructs. As shown in Fig. 2C, PKCµ could be readily detected in 14-3-3
GST precipitates from 293 cells expressing wild type PKCµ and the PKCµ
1-79 mutant. Although expressed at high level (Fig. 2B), the
PKCµ
1-340 mutant was not detectable in 14-3-3
GST
precipitates (Fig. 2C). Similar data were obtained by
overexpressing the PKCµ kinase domain (data not shown). These
findings indicate a binding of 14-3-3
approximately within the
region between amino acid 80-340 containing the complete C1 regulatory
domain of PKCµ.
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Fig. 2.
14-3-3 associates
with the C1 domain of PKCµ. A,
schematic outline of the structural domains of PKCµ wild type and
mutants generated for identification of the 14-3-3 binding site.
B, expression of PKCµ mutants. 293 cells were transfected
with the indicated PKCµ mutants cloned in the pCDNA3 expression
vector. Truncated and wild type PKCµ proteins were immunoprecipitated
using an antiserum directed against a carboxyl-terminal epitope. PKCµ
was visualized by immunostaining using a PKCµ mAb. C,
determination of the 14-3-3
binding domain in PKCµ. A 14-3-3
GST fusion protein was used to precipitate wild type and the mutated
PKCµ proteins upon transient overexpression in 293 cells. Detection
was by Western blotting with a PKCµ-specific mAb. D,
in vitro autophosphorylation activity of the
PKCµDM mutant. The indicated PKCµ constructs were
transfected in 293 cells, and PKCµ was immunoprecipitated and
in vitro autophosphorylated. Shown are autoradigraphs
(lower panel) and Western blot detection of PKCµ
(upper panel). The blots were scanned to determine relative
phosphorylation efficacy.
binding has been reported to involve a serine consensus motif
like RSXSXP (35, 36) or
RX1-2SX2-3S (37).
Therefore, we searched for potential serines matching the predicted
consensus sequences within the C1 region of PKCµ. Two serine regions,
serine 205/208 (RRLSNVSLT) and serine 219/223
(IRTSSAELST; Fig. 2A), show some similarity to the predicted 14-3-3
binding consensus sequences. Of
interest, these regions also exert homology to the predicted consensus
sequence of PKCµ substrates (38), therefore potentially representing
an autophosphorylation site (see below). The indicated serine pairs
were mutated to alanine (PKCµS205A,S208A and
PKCµS219A,S223A) and expressed in 293 cells (Fig. 2,
A and B). The sets of mutants were further
combined in another expression plasmid carrying the double mutant
(PKCµDM: S205A,S208A/S219A,S223A; Fig. 2A)
and, upon transient expression in 293 cells, analyzed for 14-3-3
binding capacity. As shown in Fig. 2A, all mutants
were equally well expressed in 293 cells. In 14-3-3
GST
precipitates, both the PKCµS205A,S208A mutant and the
PKCµS219A,S223A mutant, were still detectable, but in
contrast, the mutant lacking both serine motifs, PKCµDM, could hardly be detected in 14-3-3
GST precipitates (Fig.
2C). This suggests that both serine motifs, Ser-205/208 and
Ser-219/223, are involved in PKCµ binding. To investigate potential
autophosphorylation of serine 205/208 or serine 219/223, the mutants
were expressed in 293 cells, and in vitro
autophosphorylation assays were performed. As shown in Fig.
2D, the double mutant showed significant reduction in
autophosphorylation (30%) compared with PKCµ wild type, whereas both
PKCµS205A,S208A and PKCµS219A,S223A mutants
display only weak reduction in PKCµ autophosphorylation (data not
shown). PKCµ contains approximately 10 phosphorylation
sites.3 Thus likely mutation
of one site is probably below the detection level. Together with the
data of the 14-3-3 pulldown assays, these findings, point to serine
205/208 and serine 219/223 as functional important phosphorylation
sites in PKCµ.
Associates with Phosphorylated PKCµ--
As shown for
the association of 14-3-3
with Cbl, serine phosphorylation of Cbl is
essential (37). We therefore tested whether activated PKCµ, which has
been shown to be exclusively phosphorylated on serine residues (28),
displays enhanced binding of 14-3-3
GST fusion proteins. Indeed,
PKCµ could be more efficiently precipitated with 14-3-3
GST fusion
proteins upon phorbol ester stimulation of Jurkat-TAg cells (Fig.
3A). Upon stimulation of cells
with phorbol ester for 5 and 10 min, respectively, an approximately 4- and 10-fold enhancement of 14-3-3
binding to PKCµ was observed (Fig. 3A). Control immunoprecipitation of PKCµ performed
in parallel from aliquots (20 × 106 cells) of the
culture verified approximately equal amounts of PKCµ in each group
(Fig. 3A, lower panel). Activation of PKCµ by
phorbol ester treatment of cells was assessed by in vitro
autophosphorylation of immunoprecipitates (Fig. 3A,
middle panel). This revealed in accordance with earlier
findings (20) a moderate stimulation of kinase activity by phorbol
ester, which is also evident from a shift toward slower migrating bands
(Fig. 3A, middle and lower panels).
Enhanced binding of 14-3-3
to phosphorylated PKCµ explains its
relatively weak binding to PKCµ isolated from untreated Sf158 cells
(Fig. 1A) that displays only a low basal PKCµ activity. Association of 14-3-3
with in vivo activated PKCµ was
further demonstrated by Far Western analysis, where binding of
14-3-3
to PKCµ was probed with 14-3-3
GST fusion proteins and
subsequent detection by anti-GST antibodies. Although upon cellular
stimulation by PdBu PKCµ was present in equal amounts in
immunoprecipitates (Fig. 3B, right panel),
detection of PKCµ with the 14-3-3 probe was only possible upon
preactivation of PKCµ (Fig. 3B, left
panel).
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Fig. 3.
Enhanced 14-3-3
binding to activated PKCµ.
A, phorbol ester stimulation enhances PKCµ-14-3-3
interaction. Jurkat-TAg cells were stimulated with phorbol ester (100 nM) for the time indicated, and lysates were prepared.
PKCµ was either immunoprecipitated by a polyclonal PKCµ antiserum
or precipitated by 14-3-3
-GST fusion protein. GST proteins served as
negative control. To verify in vivo activation by PdBu,
aliquots of the lysates were immunoprecipitated by anti-PKCµ and
subjected to in vitro autophosphorylation and
autoradiography (middle panel). PKCµ was visualized by
Western blot analysis (lower panel). B, Far
Western analysis of 14-3-3
-PKCµ interaction. Jurkat-TAg cells were
stimulated with 100 nM PdBu, and PKCµ was
immunoprecipitated and subjected to SDS-PAGE as described. Detection
was carried out upon preincubating blots with 14-3-3
GST overnight
with an anti-GST antibody (left panel) or by anti-PKCµ mAb
(right panel) as described under "Experimental
Procedures."
to PKCµ is dependent on endogenous kinase
activity. A kinase dead PKCµ mutant, PKCµK612W (27, 39)
displaying no detectable autophosphorylation (Fig.
4, upper panel) was tested for
potential precipitation by 14-3-3
GST fusion proteins. As shown in
Fig. 4, upon overexpression of the PKCµK612W mutant, no
detectable autophosphorylation and subsequently no precipitation by
14-3-3
was detectable. In contrast, PKCµ wild type and a
pleckstrin homology domain deletion mutant, which has been previously
shown to exert constitutive kinase activity (40), were shown to be efficiently precipitated by 14-3-3
GST proteins (Fig. 4, upper panel). These data provide further evidence that 14-3-3
association requires autophosphorylation of PKCµ. In an independent
approach to scrutinize phosphorylation dependence of 14-3-3
binding,
PKCµ immunoprecipitates from Sf158 cell were in vitro
autophosphorylated and subsequently treated with phosphatase 2A.
Concommitant with a time-dependent dephosphorylation of
PKCµ, a strong reduction in the amount of 14-3-3-precipitable kinase
was noted (Fig. 5, top and
bottom panel). Western blot analysis ensured that
phosphatase treatment did not affect PKCµ protein levels (Fig. 5,
middle panel).
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Fig. 4.
14-3-3 binding to
PKCµ is dependent on PKCµ
kinase activity. The PKCµ kinase dead mutant
PKCµK612W, a pleckstrin homology domain deletion mutant
(PKCµ
PH), PKCµ wild type (PKCµWT), and
the respective vector control were transfected in 293 cells and
immunoprecipitated (IP) with a PKCµ-specific antiserum
(right lanes) or precipitated with 14-3-3
GST fusion
proteins. Both PKCµ precipitates were subjected to in
vitro autophosphorylation and exposed to autoradiography upon
SDS-PAGE (upper panel) and Western blot analysis
(lower panel).
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Fig. 5.
14-3-3 binding to
PKCµ is phosphatase-sensitive. PKCµ
immunocomplexes from Sf158 cells were in vitro
autophosphorylated. PKCµ was eluted from the protein G beads by
incubating with immunizing peptide and subjected to phosphatase 2A
treatment for the indicated times. Aliquots were removed, subjected to
direct SDS-PAGE and immunoblot analysis (middle panel)
followed by autoradiography (top panel) or precipitated
using an 14-3-3
GST fusion protein (bottom panel).
14-3-3
GST precipitates were subjected to SDS-PAGE and PKCµ was
detected by immunoblotting with a PKCµ-specific rabbit
antiserum.
Inhibits PKCµ Kinase Activity in Vitro and in
Vivo--
14-3-3 binding to phosphorylated target proteins has been
shown to modify cellular responses. For example the 14-3-3-mediated sequestration of the proapoptotic factor Bad, upon its serine phosphorylation by AKT/PKB, destroys the Bad-Bcl-2 complex and thus
modifies the apoptotic response of affected cells (14-16). As
14-3-3
binds to serine phosphorylated PKCµ (Fig. 4), a similar sequestration mechanism could occur. As a consequence, a reduction of
PKCµ kinase activity would be conceivable. Therefore, we tested whether the presence of 14-3-3
interferes with PKCµ kinase
activity. Purified PKCµ from Sf158 cells (25) was subjected to
in vitro kinase assays in the presence of various amounts of
14-3-3
GST fusion protein (Fig. 6,
top panel). PKCµ autophosphorylation was substantially
inhibited already at a concentration of 1 µM 14-3-3
GST, and a complete inhibition was noted at approximately 20 µM of 14-3-3
GST (Fig. 6, top panel). The
GST control protein did not affect PKCµ autophosphorylation up to a
concentration of 20 µM (Fig. 6, top panel).
Autophosphorylation was also not affected in the presence of the same
molar concentrations of a typical substrate-like syntide 2 (Ref. 25 and
data not shown). These findings point to a specific inactivation of
PKCµ kinase upon 14-3-3
binding, which was corroborated by
analysis of substrate phosphorylation. Similar as shown for the
autophosphorylation, a quantitative inhibition of PKCµ substrate
phosphorylation was obtained in the presence of 20 µM
14-3-3
GST. A quantitative analysis of inhibition of PKCµ
autophosphorylation activity revealed an IC50 of
approximately 4 µM (Fig. 6, bottom panel) for
autophosphorylation and substrate phosphorylation alike. Phosphopeptide
analysis of purified recombinant PKCµ revealed 10 distinct peptides
indicating phosphorylation sites.3 Therefore in the
experiment shown in Fig. 6, inhibition of autophosphorylation activity
largely reflects other than the 14-3-3 binding sites. Moreover, because
at the position of GST 14-3-3
, no bands were detectable in
autoradiographs of SDS gels, the data further show that 14-3-3
is
not phosphorylated by PKCµ in vitro (Fig. 6, top panel).
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Fig. 6.
14-3-3 binding
inhibits PKCµ kinase activity in
vitro. Autophosphorylation and in vitro
phosphorylation of the PKCµ substrate aldolase of purified
recombinant PKCµ was measured in the presence of the indicated
amounts of 14-3-3
GST fusion protein or GST. Inhibition of kinase
activity has been quantified by phosphoimage scanning and is shown as a
14-3-3 dose response curve in the lower panel. Data from one
of four experiments performed with similar results are shown.
affects PKCµ kinase activity in
intact cells (Fig. 7). Jurkat-TAg cells
were transfected with control vectors or a 14-3-3
expression
construct (18), and PKCµ kinase activity was measured in
immunoprecipitates by in vitro autophosphorylation and
substrate phosphorylation. A 40% reduction of PKCµ kinase activity
was revealed upon transfection of 14-3-3
in both assays, PKCµ
autophosphorylation as well as aldolase phosphorylation (Fig. 7,
upper panels).
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Fig. 7.
14-3-3
overexpression inhibits PKCµ kinase
activity in vivo. A, inhibition of
PKCµ kinase activity in vivo. Jurkat-TAg cells were
transfected with a 14-3-3
pEFNeo expression vector or vector alone.
40 h after transfection cells were stimulated for 10 min with PdBu
following PKCµ immunoprecipitation. Immunoprecipitates were aliquoted
and either in vitro autophosphorylated or used to
phosphorylate the substrate aldolase. Shown are autoradiographs
(upper panels) upon overnight exposition. PKCµ and
14-3-3
expression was determined by Western blot analysis
(lower panels). Shown is a representative experiment of
three with similar inhibition of relative PKCµ activation (0.6 ± 0.14).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
interacting
protein and show that PKCµ kinase activity is negatively regulated by
14-3-3
. The specificity of PKCµ/14-3-3
interaction and its relevance is evident from (i) identification of the binding site in the
C1 regulatory domain of PKCµ containing serine motifs for autophosphorylation and 14-3-3
binding, (ii) a requirement of autophosphorylation for efficient 14-3-3
binding, and (iii) a highly
effective down-regulation of kinase activity upon 14-3-3 binding in
cell free assays and intact cells.
has been described
to inhibit PKC
regulated interleukin-2 expression in T cells by
preventing its translocation to the membrane (18). Together with other
studies, in which binding of 14-3-3
to the phosphoinositide 3-kinase
(9) and to dictyostelium myosin II heavy chain kinase (41) was also
found to cause inhibition of the respective enzymatic activities, a
more general function of 14-3-3 as a negative regulator of signal
transduction pathways can be assumed.
and
(33, 34). The fact that regulatory lipids and proteins
can bind within the same region necessitated precise identification of
the binding site of 14-3-3
in PKCµ.
and PKC
(36). In contrast, a novel motif has been identified
in Cbl (37), displaying
RX1-2SX2-3S, which
differs basically from the above motif by absence of prolins. A motif
similar to the latter containing one serine
(RSLS359VE), mediating the binding to 14-3-3
,
has been identified in the phosphatase protein-tyrosine phosphatase 1 (43). Two consensus sequences matching the Cbl-derived consensus motif
were found to comprise two spatially related potential 14-3-3
binding regions within the PKCµ C1 regulatory domain, located between
amino acids 80-340 (Fig. 2A). The mutational analyses
performed here provide direct evidence for the involvement of both the
serine 205/208 (RRLSNVSLT) and serine 219/223
(IRTSSAELST) motif in 14-3-3 binding, as mutation
of only one motif retained, in each case, 14-3-3
binding to PKCµ,
whereas the simultaneous mutation of both motifs nearly completely
abrogated 14-3-3
binding (Fig. 2C). These findings
suggest that PKCµ uses a similar serine-based motif for 14-3-3
binding as Cbl (37). Of note, we obtained evidence that both of these
serine motifs (Ser-219/223) serve as autophosphorylation sites of
PKCµ, which is in accordance with a requirement of phosphoserines for
14-3-3 binding. This is underlined by the finding that 14-3-3
binding to PKCµ is dramatically enhanced upon phorbol ester
stimulation of PKCµ autophosphorylation. Similar data have been
reported for the interaction of 14-3-3
and Cbl, which also requires
serine phosphorylation of Cbl for efficient 14-3-3 binding (37). It is
further of interest to note that the two 14-3-3
binding motifs are
located within the 80-amino acid spacer (19) between the two zinc
fingers of PKCµ. Thus, the 14-3-3 binding site is spatially separated
from the lipid messenger/phorbolester binding site located within the
cysteine-rich zinc fingers (31, 32). Both the distinct sites used for
lipid and 14-3-3 binding and the prerequisite of lipid
messenger-dependent autophosphorylation for efficient
14-3-3 binding clearly favor a model of a sequential action of these
two PKCµ regulators. We propose that 14-3-3
acts as an allosteric
inhibitor of already activated PKCµ rather than a competitor of
activating lipid messengers. Binding of 14-3-3
to PKCµ appears of
functional significance as shown by a highly efficient in
vitro inhibition of PKCµ by micromolar concentrations of
14-3-3
(Fig. 6) and a significant reduction of PKCµ activity
in vivo upon moderate overexpression of 14-3-3
in T cells
(Fig. 7).
plays a role as a negative
feedback regulator of PKCµ, ensuring a tight control of kinase
activity. Upon binding of activating second messengers to the zinc
fingers, PKCµ undergoes autophosphorylation and exerts enhanced
kinase activity toward appropriate substrates. Serine phosphorylation
of defined regions of the regulatory domain of PKCµ in turn creates a
high affinity binding site for 14-3-3
, which subsequently
down-regulates PKCµ kinase activity. As 14-3-3
is a T
cell-specific isoform of this family of adapter/regulator proteins and
PKCµ is not only highly expressed in T cells but also participates in
T cell antigen receptor-mediated signal
events,4 the biological
significance of the PKCµ-14-3-3 interaction becomes apparent.
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FOOTNOTES |
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* This work was supported by Deutsche Forschungsgemeinschaft Grant Jo227/4-2.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: Inst. of Cell Biology and Immunology, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany. Tel.: 49-711-685-6995; Fax: 49-711-685-7484; E-mail: Franz-Josef.Johannes{at}po.uni-stuttgart.de.
2 Y. C. Liu, unpublished observations.
3 F. J. Johannes and T. Herget, unpublished observations.
4 F. J. Johannes, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: PKC, protein kinase C; GST, glutathione S-transferase; Pdbu, phorbol 12,13-dibutyrate; PAGE, polyacrylamide gel electrophoresis; mAb, monoclonal antibody.
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REFERENCES |
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