Protein Kinase C µ Is Negatively Regulated by 14-3-3 Signal Transduction Proteins*

Angelika HausserDagger , Peter StorzDagger , Gisela LinkDagger , Hartmut StollDagger , Yun-Cai Liu§, Amnon Altman§, Klaus PfizenmaierDagger , and Franz-Josef JohannesDagger

From the Dagger  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

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 14-3-3tau isoform has been shown to associate with protein kinase C theta  and to negatively regulate interleukin-2 secretion. Here we present data that 14-3-3tau 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 14-3-3tau 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 14-3-3tau binding region is mapped within the regulatory C1 domain. Binding of 14-3-3tau 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, 14-3-3tau is not a substrate of PKCµ. In contrast 14-3-3tau strongly down-regulates PKCµ kinase activity in vitro. Moreover, overexpression of 14-3-3tau significantly reduced phorbol ester induced activation of PKCµ kinase activity in intact cells. We therefore conclude that 14-3-3tau is a negative regulator of PKCµ in T cells.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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, PKCtheta , belonging to the novel PKC subfamily (4), is suggested from recent studies: PKCtheta is translocated upon antigen-specific stimulation to the membrane interface between T cells and antigen presenting cells, implicating a physical interaction of PKCtheta either directly with the T cell receptor complex or other T cell receptor proximal signaling molecules (5).

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 14-3-3tau 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 14-3-3tau 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 14-3-3tau binding to PKCtheta negatively affects the stimulation of the interleukin-2 promotor and prevents PKCtheta translocation to the membrane (18), supporting a role of 14-3-3 proteins in the regulation of PKC activation in T cells.

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 14-3-3tau proteins. The 14-3-3tau binding site within PKCµ could be located to the C1 regulatory region. 14-3-3tau interacts preferentially with the activated, phosphorylated PKCµ and down-regulates kinase activity, suggesting that 14-3-3tau is a regulator of PKCµ functions in T cells.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Recombinant PKCµ, Plasmid Constructs, and Cell Lines-- The production of Sf158 insect cells overexpressing PKCµ (25) and the construction of 14-3-3tau and zeta  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 PKCµDelta 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). PKCµDelta 1-340 was constructed by cutting pCDNA3/PKCµDelta 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.

Immunoprecipitation by Antibodies and GST-14-3-3tau 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 14-3-3tau 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.

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 14-3-3tau 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 14-3-3tau precipitation of PKCµ.

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 [gamma -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 14-3-3tau or GST added.

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 [gamma -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 14-3-3tau 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. 14-3-3tau binding to activated PKCµ was analyzed essentially as described (29). Detection of bound 14-3-3tau was carried out by a 2-h incubation with 10 µg/ml GST 14-3-3tau fusion protein and visualized using an alkaline phosphatase-coupled anti-GST secondary antibody.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

14-3-3tau Specifically Associates with PKCµ in Vitro-- 14-3-3tau has been recently reported to associate with PKCtheta , which is highly expressed in T cells (4, 5). To test whether 14-3-3tau would also interact with another T cell expressed isoform, PKCµ, we analyzed recombinant PKCµ for potential 14-3-3tau association. GST 14-3-3tau fusion proteins were used to precipitate PKCµ expressed in Sf158 cells. As shown in Fig. 1A, in GST pulldown assays a 14-3-3tau dose-dependent binding of PKCµ can be detected by immunoblot analysis (upper panel), showing best detection using 4 µg of 14-3-3tau GST fusion protein. 14-3-3tau 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 14-3-3tau GST protein, as shown by comparison with PKCµ immunoprecipitation by PKCµ-specific polyclonal antibodies (Fig. 1A, left lane), even when the GST 14-3-3tau 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 14-3-3tau 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 14-3-3tau fusion protein was used to precipitate PKCµ from lysates of 60 × 106 Jurkat-TAg cells. Detection was carried out as described for A. C, 14-3-3tau is coprecipitated with PKCµ in 293 and COS cells. 293 cells (left panels) were cotransfected with PKCµ and 14-3-3tau 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 14-3-3tau (lower panels). Detection of 14-3-3tau was performed with a 14-3-3tau mAb and an alkaline phosphatase-based detection system. Stable PKCµ overexpressing COS transfectants (right panels) were transfected with the indicated amounts of a 14-3-3tau expression plasmid or vector alone. PKCµ was immunoprecipitated and analyzed for the presence of 14-3-3tau as for 293 cells.

Next, the association of 14-3-3 proteins with endogenous PKCµ was investigated. 14-3-3tau 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, 14-3-3tau and 14-3-3zeta (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).

14-3-3tau 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 14-3-3tau association (data not shown); cotransfection of PKCµ and 14-3-3tau 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 14-3-3tau 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, 14-3-3tau can also be coprecipitated with PKCµ upon transient overexpression using 10 µg of the respective 14-3-3tau expression construct (Fig. 1C, lower right panel). As the subtype-specific anti-14-3-3tau mAb is directed against an epitope within the potential binding site of target proteins,2 the reciprocal immunoprecipitation experiment was precluded.

14-3-3tau 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 14-3-3tau , we transiently overexpressed in 293 cells an amino-terminal PKCµ deletion mutant and a mutant lacking in addition the C1 binding region. The mutants PKCµDelta 1-79 and PKCµDelta 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). 14-3-3tau 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 14-3-3tau GST precipitates from 293 cells expressing wild type PKCµ and the PKCµDelta 1-79 mutant. Although expressed at high level (Fig. 2B), the PKCµDelta 1-340 mutant was not detectable in 14-3-3tau GST precipitates (Fig. 2C). Similar data were obtained by overexpressing the PKCµ kinase domain (data not shown). These findings indicate a binding of 14-3-3tau approximately within the region between amino acid 80-340 containing the complete C1 regulatory domain of PKCµ.


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Fig. 2.   14-3-3tau 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 14-3-3tau binding domain in PKCµ. A 14-3-3tau 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.

14-3-3tau 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 14-3-3tau 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 14-3-3tau binding capacity. As shown in Fig. 2A, all mutants were equally well expressed in 293 cells. In 14-3-3tau 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 14-3-3tau 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.

14-3-3tau Associates with Phosphorylated PKCµ-- As shown for the association of 14-3-3tau 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 14-3-3tau GST fusion proteins. Indeed, PKCµ could be more efficiently precipitated with 14-3-3tau 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 14-3-3tau 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 14-3-3tau 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 14-3-3tau with in vivo activated PKCµ was further demonstrated by Far Western analysis, where binding of 14-3-3tau to PKCµ was probed with 14-3-3tau 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 14-3-3tau binding to activated PKCµ. A, phorbol ester stimulation enhances PKCµ-14-3-3tau 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 14-3-3tau -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 14-3-3tau -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 14-3-3tau GST overnight with an anti-GST antibody (left panel) or by anti-PKCµ mAb (right panel) as described under "Experimental Procedures."

Binding of 14-3-3tau 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 14-3-3tau GST fusion proteins. As shown in Fig. 4, upon overexpression of the PKCµK612W mutant, no detectable autophosphorylation and subsequently no precipitation by 14-3-3tau 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 14-3-3tau GST proteins (Fig. 4, upper panel). These data provide further evidence that 14-3-3tau association requires autophosphorylation of PKCµ. In an independent approach to scrutinize phosphorylation dependence of 14-3-3tau 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.   14-3-3tau binding to PKCµ is dependent on PKCµ kinase activity. The PKCµ kinase dead mutant PKCµK612W, a pleckstrin homology domain deletion mutant (PKCµDelta 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 14-3-3tau 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.   14-3-3tau 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 14-3-3tau GST fusion protein (bottom panel). 14-3-3tau GST precipitates were subjected to SDS-PAGE and PKCµ was detected by immunoblotting with a PKCµ-specific rabbit antiserum.

14-3-3tau 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 14-3-3tau 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 14-3-3tau 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 14-3-3tau GST fusion protein (Fig. 6, top panel). PKCµ autophosphorylation was substantially inhibited already at a concentration of 1 µM 14-3-3tau GST, and a complete inhibition was noted at approximately 20 µM of 14-3-3tau 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 14-3-3tau 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 14-3-3tau 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 14-3-3tau , no bands were detectable in autoradiographs of SDS gels, the data further show that 14-3-3tau is not phosphorylated by PKCµ in vitro (Fig. 6, top panel).


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Fig. 6.   14-3-3tau 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 14-3-3tau 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.

Next we analyzed whether 14-3-3tau affects PKCµ kinase activity in intact cells (Fig. 7). Jurkat-TAg cells were transfected with control vectors or a 14-3-3tau 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 14-3-3tau in both assays, PKCµ autophosphorylation as well as aldolase phosphorylation (Fig. 7, upper panels).


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Fig. 7.   14-3-3tau overexpression inhibits PKCµ kinase activity in vivo. A, inhibition of PKCµ kinase activity in vivo. Jurkat-TAg cells were transfected with a 14-3-3tau 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 14-3-3tau 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

In this study, we identify PKCµ as a novel 14-3-3tau interacting protein and show that PKCµ kinase activity is negatively regulated by 14-3-3tau . The specificity of PKCµ/14-3-3tau 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 14-3-3tau binding, (ii) a requirement of autophosphorylation for efficient 14-3-3tau binding, and (iii) a highly effective down-regulation of kinase activity upon 14-3-3 binding in cell free assays and intact cells.

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, 14-3-3tau has been described to inhibit PKCtheta regulated interleukin-2 expression in T cells by preventing its translocation to the membrane (18). Together with other studies, in which binding of 14-3-3tau 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.

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 PKClambda and zeta  (33, 34). The fact that regulatory lipids and proteins can bind within the same region necessitated precise identification of the binding site of 14-3-3tau in PKCµ.

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 PKCepsilon and PKCgamma (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 14-3-3beta , 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 14-3-3tau 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, 14-3-3tau binding to PKCµ, whereas the simultaneous mutation of both motifs nearly completely abrogated 14-3-3tau binding (Fig. 2C). These findings suggest that PKCµ uses a similar serine-based motif for 14-3-3tau 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 14-3-3tau binding to PKCµ is dramatically enhanced upon phorbol ester stimulation of PKCµ autophosphorylation. Similar data have been reported for the interaction of 14-3-3tau 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 14-3-3tau 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 14-3-3tau acts as an allosteric inhibitor of already activated PKCµ rather than a competitor of activating lipid messengers. Binding of 14-3-3tau to PKCµ appears of functional significance as shown by a highly efficient in vitro inhibition of PKCµ by micromolar concentrations of 14-3-3tau (Fig. 6) and a significant reduction of PKCµ activity in vivo upon moderate overexpression of 14-3-3tau in T cells (Fig. 7).

In conclusion, we propose that 14-3-3tau 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 14-3-3tau , which subsequently down-regulates PKCµ kinase activity. As 14-3-3tau 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.

    FOOTNOTES

* 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.

    ABBREVIATIONS

The abbreviations used are: PKC, protein kinase C; GST, glutathione S-transferase; Pdbu, phorbol 12,13-dibutyrate; PAGE, polyacrylamide gel electrophoresis; mAb, monoclonal antibody.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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