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

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

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][2][3]. Phorbol ester responsive PKCs in general have long been associated with T cell activation and a prominent role of one particular subtype, PKC, 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).
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 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.
We have recently described a novel PKC isotype termed PKC (19), which, although ubiquitously expressed, shows particularly high expression in thymus and hematopoetic cells (20). PKC 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 PKC in diverse cellular functions stems from reports showing enhancement of constitutive transport processes in PKC overexpressing epithelial cells (23) and PKC 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 PKC specifically associates in vitro and in vivo with 14-3-3 proteins. The 14-3-3 binding site within PKC could be located to the C1 regulatory region. 14-3-3 interacts preferentially with the activated, phosphorylated PKC and down-regulates kinase activity, suggesting that 14-3-3 is a regulator of PKC functions in T cells.

EXPERIMENTAL PROCEDURES
Recombinant PKC, Plasmid Constructs, and Cell Lines-The production of Sf158 insect cells overexpressing PKC (25) and the construction of 14-3-3 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 supple-mented 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. PKC deletion mutant PKC ⌬1-79 was constructed by digesting pBpl4 (19) with ApaI and NsiI. Overhanging 5Ј and 3Ј ends were filled with the Klenow enzyme, and the 2.9 kilobase PKC fragment was isolated and ligated in EcoRVdigested pCDNA3 (Invitrogen). PKC ⌬1-340 was constructed by cutting pCDNA3/PKC ⌬1-79 with HindIII, isolating a 800-base pair HindIII fragment followed by religating the vector/PKC portion. Additionally these mutants were cloned in other expression vectors and verified by transient expression (27). PKC 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 PKC were generated by transfecting COS cells with PKC wild type cloned in the expression vector pCDNA3 followed selection of transfectants in neomycin (400 g/ml) containing media for a period of 20 days. Single colonies were analyzed for PKC overexpression by Western blot analysis.
Immunoprecipitation by Antibodies and GST-14-3-3 Precipitation of PKC-Sf158 or Jurkat-TAg cells were lysed at 4°C in lysis buffer (20 mM Tris, pH 7.4, 2 mM MgCl 2 , 1% Triton X-100, 1 mM sodium orthovanadate, 150 mM NaCl, 10 g/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 ϫ 10 6 Jurkat-TAg cells) for 90 min at 4°C. For immunoprecipitation of PKC from Jurkat-TAg cells, a PKC antiserum was used as described earlier (28). Immunocomplexes were harvested by incubation with protein G-Sepharose (Pharmacia, 30 l/2 ϫ 10 7 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 PKC 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. Transfections-7.5 ϫ 10 6 Jurkat-TAg cells were seeded per 60-mmdiameter dish in 5 ml of RPMI supplemented with 10% fetal calf serum and transfected with 5 g of DNA and 20 l 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 overexpression experiments, PKC was immunoprecipitated and in vitro autophosphorylated as described below. Exponentially growing 293 cells, 40 -80% confluent, were transfected with the indicated plasmids using 2 g of DNA and 10 l of Superfect reagent for each well of a 6-well plate or 10 g of DNA and 60 l of Superfect reagent for a 100-mm plate. Extracts from one well were used for each immunoprecipitation and GST 14-3-3 precipitation of PKC.
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 PKC was immunoprecipitated. PKC 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 MgCl 2 , 2 mM dithiothreitol). The immune complexes were mixed with 10 l of phosphorylation buffer containing 0.2 l of [␥-32 P]ATP (Amersham Pharmacia Biotech) and incubated for 10 min at 37°C. The reaction was stopped by adding 5ϫ 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 PKC enzyme from Sf158 cells (25) was used with the indicated amounts of GST 14-3-3 or GST added.
Phosphatase Treatment and Far Western Blot Analysis-PKC was immunoprecipitated from 5 ϫ 10 6 Sf158 cells using 4 l 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 [␥-32 P]ATP. Bound PKC was eluted in a final volume of 100 l upon adding 50 l of immunizing peptide (1 mg/ml) by incubating 30 min at 4°C. PKC was incubated with Phosphatase 2A (0.4 units) for the indicated times. Equal aliquots were subjected either to GST 14-3-3 precipitation followed PKC immunodetection or to direct immunoblot analysis to compare precipitation efficacies. For Far Western analysis, PKC from 80 ϫ 10 6 Jurkat TAg cells was immunoprecipitated as described. Aliquots of immunoprecipitates were subjected to SDS-PAGE and transferred to a polyvinylidene difluoride membrane. PKC detection was carried out using a PKC mAb. 14-3-3 binding to activated PKC was analyzed essentially as described (29). Detection of bound 14-3-3 was carried out by a 2-h incubation with 10 g/ml GST 14-3-3 fusion protein and visualized using an alkaline phosphatase-coupled anti-GST secondary antibody.

14-3-3 Specifically Associates with PKC 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, PKC, we analyzed recombinant PKC for potential 14-3-3 association. GST 14-3-3 fusion proteins were used to precipitate PKC expressed in Sf158 cells. As shown in Fig. 1A, in GST pulldown assays a 14-3-3 dose-dependent binding of PKC can be detected by immunoblot analysis (upper panel), showing best detection using 4 g of 14-3-3 GST fusion protein. 14-3-3 binding to PKC is specific because no binding to the respective amount of GST proteins was detectable. Only a fraction of total recombinant PKC was precipitated with 14-3-3 GST protein, as shown by comparison with PKC immunoprecipitation by PKC-specific polyclonal antibodies (Fig. 1A, left lane), even when the GST 14-3-3 concentration was increased to 20 g (data not shown).
Next, the association of 14-3-3 proteins with endogenous PKC was investigated. 14-3-3 GST fusion proteins were used to precipitate PKC from extracts of Jurkat-TAg cells. As shown in Fig. 1B, endogenous PKC 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 PKC. The respective controls, glutathione S-transferase, and as a control for nonspecific binding, the pleckstrin homology domain of PKC expressed as a GST fusion protein did not detectably precipitate PKC in pulldown assays (Fig. 1B, left lanes).
14-3-3 association with PKC was also shown in coprecipitation experiments using PKC-specific antibodies. As in 293 cells endogenous PKC levels are too low to detect 14-3-3 association (data not shown); cotransfection of PKC and 14-3-3 was performed in 293 cells. Additionally, 14-3-3 was transiently overexpressed in stable COS-PKC transfectants, and PKC 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 PKC immunoprecipitates upon appropriate expression of both cDNAs (PKC/14-3-3 DNA ratio 1:10). Likewise, in stably PKC expressing COS transfectants, 14-3-3 can also be coprecipitated with PKC upon transient overexpression using 10 g 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.
14-3-3 Binds to a Serine-dependent Motif within the C1 Region of PKC-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 PKC. In an attempt to identify potential binding sites of 14-3-3, we transiently overexpressed in 293 cells an amino-terminal PKC deletion mutant and a mutant lacking in addition the C1 binding region. The mutants PKC ⌬1-79 and PKC ⌬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 PKC as shown by immunoprecipitation (Fig. 2B). 14-3-3 GST fusion proteins were used to precipitate PKC, and the mutants from lysates of 293 cells were transfected with the respective expression constructs. As shown in Fig. 2C, PKC could be readily detected in 14-3-3 GST precipitates from 293 cells expressing wild type PKC and the PKC ⌬1-79 mutant. Although expressed at high level (Fig. 2B), the PKC ⌬1-340 mutant was not detectable in 14-3-3 GST precipitates (Fig.  2C). Similar data were obtained by overexpressing the PKC 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 PKC.
14-3-3 binding has been reported to involve a serine consensus motif like RSXSXP (35,36) or RX 1-2 SX 2-3 S (37). Therefore, we searched for potential serines matching the predicted consensus sequences within the C1 region of PKC. 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 PKC substrates (38), therefore potentially representing an autophosphorylation site (see below). The indicated serine pairs were mutated to alanine (PKC S205A,S208A and PKC S219A,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 (PKC DM : 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 PKC S205A,S208A mutant and the PKC S219A,S223A mutant, were still detectable, but in contrast, the mutant lacking both serine motifs, PKC DM , 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 PKC 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 PKC wild type, whereas both PKC S205A,S208A and PKC S219A,S223A mutants display only weak reduction in PKC autophosphorylation (data not shown). PKC 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 PKC.
14-3-3 Associates with Phosphorylated PKC-As shown for the association of 14-3-3 with Cbl, serine phosphorylation of Cbl is essential (37). We therefore tested whether activated PKC, which has been shown to be exclusively phosphorylated on serine residues (28), displays enhanced binding of 14-3-3 GST fusion proteins. Indeed, PKC 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 PKC was observed (Fig. 3A). Control immunoprecipitation of PKC performed in parallel from aliquots (20 ϫ 10 6 cells) of the culture verified approximately equal amounts of PKC in each group (Fig. 3A, lower panel). Activation of PKC 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 PKC explains its relatively weak binding to PKC isolated from untreated Sf158 cells (Fig. 1A) that displays only a low basal PKC activity. Association of 14-3-3 with in vivo activated PKC was further demonstrated by Far 3 F. J. Johannes and T. Herget, unpublished observations.

FIG. 1. 14-3-3 interacts with PKC.
A, PKC 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. PKC (left lane) was immunoprecipitated (IP) under similar conditions (500,000 Sf158 cells) as a positive control using a polyclonal rabbit antibody specific for PKC. Bound PKC was detected by immunoblot analysis using a PKC mAb and an alkaline phosphatase-coupled secondary antibody. GST was visualized using an anti-GST mAb as described under "Experimental Procedures." B, PKC can be specifically precipitated by 14-3-3 in Jurkat-TAg cells. 10 g of GST 14-3-3 fusion protein was used to precipitate PKC from lysates of 60 ϫ 10 6 Jurkat-TAg cells. Detection was carried out as described for A. C, 14-3-3 is coprecipitated with PKC in 293 and COS cells. 293 cells (left panels) were cotransfected with PKC and 14-3-3 expression vectors as described under "Experimental Procedures." 40 h after transfection PKC immunoprecipitates were analyzed by Western blot for the presence of PKC (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 PKC overexpressing COS transfectants (right panels) were transfected with the indicated amounts of a 14-3-3 expression plasmid or vector alone. PKC was immunoprecipitated and analyzed for the presence of 14-3-3 as for 293 cells.
Western analysis, where binding of 14-3-3 to PKC was probed with 14-3-3 GST fusion proteins and subsequent detection by anti-GST antibodies. Although upon cellular stimulation by PdBu PKC was present in equal amounts in immunoprecipitates (Fig. 3B, right panel), detection of PKC with the 14-3-3 probe was only possible upon preactivation of PKC (Fig. 3B, left panel).
Binding of 14-3-3 to PKC is dependent on endogenous kinase activity. A kinase dead PKC mutant, PKC K612W (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 PKC K612W mutant, no detectable autophosphorylation and subsequently no precipitation by 14-3-3 was detectable. In contrast, PKC 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 PKC. In an independent approach to scrutinize phosphorylation dependence of 14-3-3 binding, PKC immunoprecipitates from Sf158 cell were in vitro autophosphorylated and subsequently treated with phosphatase 2A. Concommitant with a time-dependent dephosphorylation of PKC, a strong reduction in the amount of 14-3-3precipitable kinase was noted (Fig. 5, top and bottom panel). Western blot analysis ensured that phosphatase treatment did not affect PKC protein levels (Fig. 5, middle panel).

14-3-3 binding to phosphorylated target proteins has been
shown to modify cellular responses. For example the 14-3-3mediated 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 PKC (Fig. 4), a similar sequestration mechanism could occur. As a consequence, a reduction of PKC kinase activity would be conceivable. Therefore, we tested whether the presence of 14-3-3 interferes with PKC kinase activity. Purified PKC 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). PKC autophosphorylation was substantially inhibited already at a concentration of 1 M 14-3-3 GST, and a complete inhibition was noted at approximately 20 M of 14-3-3 GST (Fig. 6, top panel). The GST control protein did not affect PKC autophosphorylation up to a concentration of 20 M (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 PKC kinase upon 14-3-3 binding, which was corroborated by analysis of substrate phosphorylation. Similar as shown for the autophosphorylation, a quantitative inhibition of PKC substrate phosphorylation was obtained in the presence of 20 M 14-3-3 GST. A quantitative analysis of inhibition of PKC autophosphorylation activity revealed an IC 50 of approximately 4 M (Fig. 6, bottom panel) for autophosphorylation and substrate phosphorylation alike. Phosphopeptide analysis of purified recombinant PKC 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 PKC in vitro (Fig. 6, top panel).
Next we analyzed whether 14-3-3 affects PKC kinase activity in intact cells (Fig. 7). Jurkat-TAg cells were transfected FIG. 5. 14-3-3 binding to PKC is phosphatase-sensitive. PKC immunocomplexes from Sf158 cells were in vitro autophosphorylated. PKC 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 PKC was detected by immunoblotting with a PKC-specific rabbit antiserum.
with control vectors or a 14-3-3 expression construct (18), and PKC kinase activity was measured in immunoprecipitates by in vitro autophosphorylation and substrate phosphorylation. A 40% reduction of PKC kinase activity was revealed upon transfection of 14-3-3 in both assays, PKC autophosphorylation as well as aldolase phosphorylation (Fig. 7, upper panels). DISCUSSION In this study, we identify PKC as a novel 14-3-3 interacting protein and show that PKC kinase activity is negatively regulated by 14-3-3. The specificity of PKC/14-3-3 interaction and its relevance is evident from (i) identification of the binding site in the C1 regulatory domain of PKC 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.
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 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.
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 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 PKC.
Phosphoserine binding motifs for 14-3-3 proteins like RSX-pSXP 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⑀ and PKC␥ (36). In contrast, a novel motif has been identified in Cbl (37), displaying RX 1-2 SX 2-3 S, which differs basically from the above motif by absence of prolins. A motif similar to the latter containing one serine (RSLS 359 VE), 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 PKC 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 PKC, whereas the simultaneous mutation of both motifs nearly completely abrogated 14-3-3 binding (Fig. 2C). These findings suggest that PKC 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 PKC, 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 PKC is dramatically enhanced upon phorbol ester stimulation of PKC 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 PKC. Thus, the 14-3-3 binding site is spatially separated from the lipid FIG. 6. 14-3-3 binding inhibits PKC kinase activity in vitro. Autophosphorylation and in vitro phosphorylation of the PKC substrate aldolase of purified recombinant PKC 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.
FIG. 7. 14-3-3 overexpression inhibits PKC kinase activity in vivo. A, inhibition of PKC 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 PKC immunoprecipitation. Immunoprecipitates were aliquoted and either in vitro autophosphorylated or used to phosphorylate the substrate aldolase. Shown are autoradiographs (upper panels) upon overnight exposition. PKC 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 PKC activation (0.6 Ϯ 0.14). 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 messengerdependent autophosphorylation for efficient 14-3-3 binding clearly favor a model of a sequential action of these two PKC regulators. We propose that 14-3-3 acts as an allosteric inhibitor of already activated PKC rather than a competitor of activating lipid messengers. Binding of 14-3-3 to PKC appears of functional significance as shown by a highly efficient in vitro inhibition of PKC by micromolar concentrations of 14-3-3 (Fig. 6) and a significant reduction of PKC activity in vivo upon moderate overexpression of 14-3-3 in T cells (Fig. 7).
In conclusion, we propose that 14-3-3 plays a role as a negative feedback regulator of PKC, ensuring a tight control of kinase activity. Upon binding of activating second messengers to the zinc fingers, PKC undergoes autophosphorylation and exerts enhanced kinase activity toward appropriate substrates. Serine phosphorylation of defined regions of the regulatory domain of PKC in turn creates a high affinity binding site for 14-3-3, which subsequently down-regulates PKC kinase activity. As 14-3-3 is a T cell-specific isoform of this family of adapter/regulator proteins and PKC 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 PKC-14-3-3 interaction becomes apparent.