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J. Biol. Chem., Vol. 277, Issue 8, 6490-6496, February 22, 2002

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Protein Kinase C (PKC)-mediated PKCµ Activation Modulates ERK and JNK Signal Pathways^*

Ilona Brändlin, Susanne Hübner, Tim Eiseler, Marina Martinez-Moya, Andreas Horschinek, Angelika Hausser, Gisela Link, Steffen Rupp, Peter Storz^§, Klaus Pfizenmaier, and Franz-Josef Johannes^¶

From the Fraunhofer Institute for Interfacial Engineering, Nobelstraße 12, the  Institute of Cell Biology and Immunology, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany, and the ^§ Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215

Received for publication, June 29, 2001, and in revised form, December 4, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Protein kinase C (PKC), a family of lipid-activated serine kinases, is involved in multiple functions in the regulation of growth control. The PKC-related isoform PKCµ/PKD has been implicated in mitogenic signal cascades because of the activation of p42/p44 MAPK leading to Elk1-mediated gene transcription, and PKCµ/PKD has been shown to be activated via a PKC-dependent pathway. By using confocal analyses, we demonstrate here that PKCµ partially colocalizes with PKC in different cell types. Colocalization depends on the presence of the PKCµ pleckstrin homology domain. Coexpression of constitutively active PKC with PKCµ leads to a significant enhancement of the PKCµ substrate phosphorylation capacity as a result of an increased phosphorylation of the activation loop Ser^738/742 of PKCµ, whereas Ser⁹¹⁰ autophosphorylation remains unaffected. In vitro phosphorylation experiments show that PKC directly phosphorylates PKCµ on activation loop serines. Consequently, the p42 MAPK cascade is triggered leading to an increase in reporter gene activity driven by a serum-responsive element in HEK293 cells. At the same time, PKC-mediated JNK activation is reduced, providing evidence for a mutual regulation of PKCµ/PKC affecting different arms of the p38/ERK/JNK pathways. Our data provide evidence for the sequential involvement of selective PKC isoforms in kinase cascades and identify the relevant domains in PKCµ for interaction with and activation by PKC as pleckstrin homology domain and activation loop.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Activation of protein kinases through growth factor receptors is achieved through a complex network of intracellular signal processes involving second messengers, protein-protein interaction, and the phosphorylation/dephosphorylation of interacting proteins. Protein kinases of the C family (PKC)¹ have been shown to be involved in the signal transduction of a wide range of biological responses, including changes in cell morphology, proliferation, and differentiation (1, 2). PKCs comprise a family of intracellular serine/threonine-specific kinases; depending on the isoform, their activation is typically initiated by Ca²⁺, lipid second messengers, and/or protein activators. In addition, subsequent multiple phosphorylation steps caused by upstream kinases contribute to a fully active state of PKCs (3).

Consisting of PKCµ (4) and its mouse homologue termed PKD (5), PKC (6), and PKD2 (7), the subgroup of PKC-related kinases shares common structures such as amino-terminal cysteine fingers that define the structural basis for lipid-mediated PKC activation. PKCµ/PKD differs from the other PKC isozymes by the presence of an acidic domain (8), a PH domain within the regulatory region (9), and the lack of a typical pseudosubstrate site. PKCµ/PKD is ubiquitously expressed and involved in diverse cellular functions such as transport processes and the G protein-mediated regulation of Golgi organization (10, 11) as well as anti-apoptotic functions (12).

PKCµ/PKD activation occurs through several mediators via a PKC-dependent pathway (13). Activation of PKCµ by the growth factor PDGF via a phospholipase C-mediated pathway (14) points to a role in the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) cascade. Subsequently, activation of the MAPK by PKCµ was demonstrated (15). PKD has been shown to be constitutively associated with PKC via its PH domain, leading to enhanced -peptide phosphorylation of PKD immunoprecipitates from cells coexpressing PKC (16). Activation of PKD results in phosphorylation of five serine residues (17). In contrast to other PKCs, phosphorylation of the carboxyl-terminal serine is defined as an autophosphorylation event and is not performed by upstream kinases (18). Two other phosphorylation sites, serines 738 and 742, have been identified (19). In addition to lipid second messenger-induced activation, PKCµ kinase activity is regulated by PKCµ-binding proteins p32 and 14-3-3, which apparently affect substrate access and down-regulate kinase activity (20, 21).

Because PKC has been shown to be selectively associated with PKD, PKC can be considered as a potential upstream kinase of PKD/PKCµ triggering PKCµ-dependent downstream pathways. This is suggested by recent data showing enhancement of PKD kinase activity upon transient coexpression with constitutively active PKC (22). We have carried out this study to analyze the activation of PKCµ by PKC in detail and to identify upstream activators and potential downstream pathways. These data show intracellular colocalization of PKCµ with PKC as well as PKC-dependent PKCµ activation via phosphorylation of PKCµSer^738/742. PKC-mediated activation of PKCµ leads to the inhibition of PKC-triggered JNK activity and to increased PKCµ-dependent MAPK activity, providing evidence for a mutual regulation of these PKC members affecting different arms of the p38/ERK/JNK pathways.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cloning of Expression Constructs-- The construction of the PKCµ expression vectors has been described previously (23). The PKC-GFP expression vector was constructed by amplifying the PKC coding region from cDNA of JurkatTAg cells, using the primers 5'-CCCAAGCTTATGTCGTCTGGCACCATGAAGTTC-3' introducing a HindIII site upstream of the ATG and 5'-CGGAATTCCTTGGTTGCAATTCTGGAGACAC-3,' creating an EcoRI site at amino acid 682 of PKC. After digestion, the fragment was cloned in pEGF-N3 (CLONTECH) to express a carboxyl-terminal GFP fusion protein PKC-GFP. This construct codes for a 107-kDa fusion protein that can be detected either by a GFP or by the carboxyl-terminal PKC antibody. To construct untagged PKC, the PCR fragment was generated with a 3'-primer 5'-GGAATTCCTATGGTTGCAATTCTGGAGACAC-3', which created an EcoRI site downstream from the stop codon and cloned in pcDNA3 using the same restriction sites. The PKC_A/E and PKC_K/R expression constructs were a gift of Gottfried Baier, Innsbruck, Austria. PKC_A/E carries a mutation in the pseudosubstrate site at alanine 161, creating a lipid-independent constitutively active enzyme, and PKC_K/R is mutated in the ATP-binding site at lysine 384 (24). The construction of the PKCµ-GFP expression constructs has been described by Hausser et al.² Recombinant expression and purification of PKCµ in Sf158 cells has been described previously (25). Hemagglutinin-tagged PKCµ_K612W was cloned in pFAST-Bac (Invitrogen) allowing infection of insect cells. Recombinant protein was purified using an nickel-nitrilotriacetic acid affinity column.

Antibodies and Reagents-- Rabbit antibodies (Santa Cruz Biotechnology) were used for PKCµ and PKC detection. Phosphospecific anti-rabbit antibodies directed against phosphoserine 744/748 and 916 of PKD (Ser^738/742 and Ser⁹¹⁰ in PKCµ) were purchased from Cell Signaling. Western blot detection was carried out using an alkaline phosphatase-based detection system (Dianova). The cell lines used (HEK293 and MCF7) were obtained from ATCC.

Transfection of HEK293 and MCF7 Cells-- 600,000 HEK293 cells were seeded in 6-cm plates 24 h before transfection. Cells were transfected using 5 µg of DNA with 30 µl of Superfect (Qiagen) and incubated for 2 h. The transfection mixtures were replaced by fresh media, and cells were lysed 48 h after transfection (see below). JNK assays were performed as described (26). Serum-responsive element-driven luciferase activity was monitored using a 4xSRE-Tk-luc reporter gene construct. Normalization for transfection efficiencies was performed by coexpression with a -galactosidase reporter gene construct (pCH110) as described (15). Experiments were carried out in triplicate and repeated eight times.

Immunofluorescence Analysis-- PKCµ-GFP, PKCµ_PH-GFP, and PKC were transfected in HEK293 and MCF-7 cells. For MCF7 cells the FuGENE 6 transfection reagent (Roche Molecular Biochemicals) was used according to the manufacturer's instructions. 24 h after transfection cells were washed twice with PBS and fixed in 4% paraformaldehyde (in PBS) for 15 min at 37 °C. Permeabilization and blocking of the cells were performed by incubation with 0.05% Tween 20 and 5% fetal calf serum in PBS for 30 min. The cells were rinsed 3 times with PBS and then incubated with the primary antibody PKC (0.2 µg/ml, Santa Cruz Biotechnology) for 2 h (0.05% Tween 20 and 5% fetal calf serum in PBS). For immunofluorescence detection of PKC, cells were incubated with a secondary anti-rabbit antibody (Alexa-546 conjugated, 1:1000, MobiTec) for 2 h. After staining, the cells were rinsed four times with PBS and prepared for microscopic analysis. Images were acquired using a confocal laser scanning microscope (TCS SP2, Leica) equipped with a x100/1.4 HCX PlanAPO oil-immersion objective. GFP was excited with an argon laser (488 nm line), whereas Alexa-546 was excited using a helium-neon laser (543 nm line). Each image represents a two-dimensional parallel projection of sections in the z series taken at 0.8-0.9-µm intervals across the cell.

Immunoprecipitation Assays and in Vitro Kinase Assays-- Cells were lysed at 4 °C in lysis buffer (20 mM Tris/HCl, pH 7.4, 1% Triton X-100, 150 mM NaCl, 5 mM MgCl₂, 1 µg/ml leupeptin, 1 µg/ml aprotinin, 1 mM phenylmethanesulfonyl fluoride, 2 mM sodium orthovanadate, 1 mM sodium fluoride). In some assays 1 mM microcystin and 300 nM okadaic acid were added. After 30 min of incubation on ice, the lysates were centrifuged (10,000 × g, 15 min, 4 °C), and immunoprecipitation was performed as described (27). In brief 1 ml of lysate was incubated for 1.5 h at 4 °C with 5 µl of the antibody. Immunocomplexes were harvested by adding 30 µl of protein G-Sepharose, incubated for 1 h, and washed twice with lysis buffer and one time with PBS. The samples were fractionated by SDS-PAGE followed by transfer to a nitrocellulose membrane. For in vitro kinase assays the immunoprecipitates were washed additionally in 1 ml of phosphorylation buffer (50 mM Tris, pH 7.5, 10 mM MgCl₂, 2 mM dithiothreitol). Kinase reaction with or without 5 µg of aldolase/syntide 2 as a substrate was started by adding of 4 µCi of [-³²P]ATP (Amersham Biosciences) in 20 µl of kinase buffer and incubated for 15 min at 37 °C. For in vitro phosphorylation of recombinant PKCµ_WT and PKCµ_K612W by recombinant PKC (Calbiochem), 110 ng of each protein were used in a volume of 40 µl of phosphorylation buffer. Enzyme assays were carried out in the presence of lipid micelles as described (25). In vitro activation of autophosphorylation and substrate phosphorylation of 5 µg of myelin basic protein (Sigma) and 5 µg of syntide 2 (Sigma) by recombinant PKC in the presence or absence of lipid micelles was tested (data not shown). Recombinant PDK1 was a gift of Alex Toker, Boston. Phosphorylation reactions were stopped by adding 5× concentrated sample buffer, subsequently fractionated on SDS-PAGE, transferred to a nitrocellulose membrane, and exposed to autoradiography. Syntide 2 phosphorylation was monitored by autoradiography after fractionation on 20% SDS-PAGE followed by drying of the gel. Autoradiographs were analyzed by using PhosphorImaging analysis (Molecular Dynamics).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

PKCµ Colocalizes with PKC-- PKD has been shown to be associated with PKC using biochemical techniques as demonstrated by in vitro pull-down assays using GST fusion proteins and coimmunoprecipitations (16). PKCµ, the human homologue of PKD, has been reported to be localized at the Golgi compartment, in particular in cells of epithelial origin (10), whereas PKC has been described to be predominantly localized on the rough endoplasmic reticulum in COS cells and in keratinocytes (28). Based on the biochemical data, we questioned whether both enzymes colocalize in HEK293 cells. Immunofluorescence analyses were performed after the transient coexpression of a PKCµ-GFP fusion protein with PKC wild type (PKC_WT), a constitutively active mutant (PKC_A/E), and kinase-dead mutant (PKC_K/R). As shown in Fig. 1, PKCµ was localized in perinuclear structures (Fig. 1, left-hand panels). Immunostaining of PKC_WT revealed similar perinuclear staining structures (Fig. 1, upper row, center). An overlay of both pictures demonstrates partial colocalization, indicated by the yellow color (Fig. 1, upper row, right-hand panel). The relative intensity of red and green fluorescence along the indicated white lines is displayed (Fig. 1, right profiling histograph). An equal distribution of maximum fluorescence intensities provides further support for a colocalization of both enzymes (Fig. 1, right profiling histograph). Similarly to PKC wild type, expression of constitutively active PKC_A/E as well as kinase-dead PKC_K/R resulted in the same staining pattern, i.e. displaying partial colocalization with PKCµ. Because PKC has been reported to be associated with PKCµ via its PH domain (16), a PKCµ_PH-GFP construct was tested for colocalization with PKC. In contrast to wild type PKCµ, colocalization with PKC was not detected under the experimental conditions employed here (Fig. 1, 4th row). These results clearly indicate that the intracellular colocalization of PKCµ and PKC depends on the presence of the PKCµ PH domain. Colocalization of PKCµ with PKC was further demonstrated in the mammary epithelial cell line MCF7. Similarly as shown for HEK293 cells, colocalization could be shown by coexpression of PKC wild type as well as after expressing constitutively active PKC_A/E or kinase-dead PKC_K/R (Fig. 1, lower panels).

View larger version (50K): [in this window] [in a new window]   Fig. 1.   Subcellular colocalization of PKCµ-GFP and PKC. PKCµ-GFP, PKCµPH-GFP, and the PKC constructs indicated were transiently coexpressed in HEK293 (upper panels) and MCF7 cells (lower panels). Intracellular colocalization was analyzed using confocal immunofluorescence microscopy. 24 h after transfection, cells were fixed and stained for PKC with an anti-PKC rabbit antiserum followed by incubation with Alexa-546-labeled anti-rabbit antibody. PKCµ-GFP (green) and PKC (red) stains were combined (right panels). The overlay is indicated by the yellow color. Relative red and green fluorescence intensities of selected areas indicated by the white line are displayed in profiling histographs at the right.

PKC Is an Upstream Kinase of PKCµ-- PKCµ/PKD activation has been reported to be mediated by distinct cellular stimuli-like growth factors, neuropeptides, immunoreceptor agonists, or G protein-coupled receptors via a PKC-dependent pathway (13, 29). PKC colocalizes with PKCµ (Fig. 1) and has been shown to interact physically via the PH domain and activate PKD (17). Therefore, PKC may act as a direct upstream kinase of PKCµ/PKD, potentially phosphorylating activation-relevant serine residues (17), in a similar way that PDK1 has been reported to activate several other PKC isotypes (3, 30). This prompted us to analyze in detail the activation of human PKCµ by PKC. PKCµ and PKC_WT, constitutively active PKC_A/E, and kinase-dead PKC_K/R were coexpressed in HEK293 cells, immunoprecipitated, and used for in vitro kinase assays. As shown in Fig. 2 (upper right panel), it was possible to demonstrate the autophosphorylation of constitutively active PKC_A/E and of wild type PKC. As expected, kinase activity is absent in PKC_K/R immunoprecipitates. Furthermore, PKCµ immunoprecipitates of cells cotransfected either with constitutively active PKC_A/E, kinase-dead PKC_K/R, or wild type PKC showed a significant increase in autophosphorylation (Fig. 2, upper panel). As a negative control, kinase-dead PKCµ_K612W was cotransfected with the indicated PKC mutants not resulting in any autophosphorylation activity. Protein loads of all immunoprecipitates used were confirmed by Western blot analysis (Fig. 2, middle panels).

View larger version (45K): [in this window] [in a new window]   Fig. 2.   PKC activates PKCµ. The indicated expression constructs were transfected in HEK293 cells. PKCµ (left panels) or PKC (right panels) was immunoprecipitated and subjected to in vitro kinase assay. Autophosphorylation (upper panel) and aldolase substrate phosphorylation (lower panel) were measured after SDS-PAGE fractionation and transferred to a membrane by overnight autoradiography. In this experiment a carboxyl-terminal PKCWT-GFP expression construct was used migrating at 107 kDa. Transgene expression was monitored using Western blot analysis of immunoprecipitates (middle panels). Autoradiographs from aldolase phosphorylation were quantified by PhosphorImaging analysis. IVK, in vitro kinase assay.

In contrast to the enhancement of PKCµ autophosphorylation, on coexpression of PKC mutants only the cotransfection of PKCµ with the constitutively active PKC_A/E showed a significant increase in PKCµ substrate phosphorylation (Fig. 2). Coexpression of PKC_A/E with PKCµ leads to a 6-fold enhancement of aldolase phosphorylation (Fig. 2, lower diagram), which is considered to be a specific PKCµ substrate in vitro (Fig. 2, lower panels). Immunoprecipitates of PKC mutants used here do not show detectable aldolase phosphorylation (Fig. 2, lower right panel).

Various PKCµ phosphorylation site-specific antibodies were used to analyze PKCµ activation by PKC in more detail. Ser⁹¹⁰ (equivalent to Ser⁹¹⁶ in PKD) and Ser^738/742 (equivalent to Ser^744/748 in PKD) were shown to be phosphorylated during PKD activation (18, 31). PKCµ and the indicated PKC mutants were coexpressed in HEK293 cells, and the phosphorylation of the respective serine residues in PKCµ was demonstrated by Western blot analysis. As expected, enhanced phosphorylation of Ser⁹¹⁰ and Ser^738/742 could be demonstrated on phorbol ester stimulation (Fig. 3). Expression of constitutively active PKCµ_PH and PKCµ_AD leads to the enhancement of auto- and substrate phosphorylation (15) (and data not shown) and could consequently be detected using Ser(P)⁹¹⁰ and Ser(P)^738/742 antibodies. Kinase-dead PKCµ_K612W was detected only weakly by Ser(P)^738/742 and Ser(P)⁹¹⁰ antibodies (Fig. 3A, lower panels), probably resulting from transphosphorylation by endogenous PKCµ. Coexpression of PKCµ with PKC_A/E leads to higher Ser^738/742 phosphorylation compared with Ser⁹¹⁰, whereas the Ser(P)^738/742 signal was strongly reduced on expression of kinase-dead PKC_K/R. The Ser(P)⁹¹⁰ signal was not differentially affected by coexpression of the various PKC mutants, as expected from the data shown in Fig. 2.

View larger version (32K): [in this window] [in a new window]   Fig. 3.   A, PKC selectively phosphorylates Ser738/742 of PKCµ. The indicated constructs were transiently expressed in HEK293 cells. Transgene expression of PKCµ was monitored by Western blot analysis using a PKCµ rabbit antiserum (upper panel). Selective PKCµ phosphorylation was shown by Western blot analysis using phosphospecific antibodies recognizing either Ser(P)910 (middle panel) or Ser(P)738/742 (lower panel). As a positive control, PKCµ-transfected cells were stimulated with phorbol ester. B, specificity of PKCµ phosphoserine antibodies. Ser(P)910 and Ser(P)738/742 antibodies do not cross-react with other PKC isotypes. Extracts of Sf9 cells expressing PKC and immunoprecipitates of PKC and PKC were analyzed by Western blot using isotype-specific antibodies and Ser(P)910 and Ser(P)738/742 antibodies as indicated.

Coexpression of PKC_WT and PKCµ led to a slight increase in Ser(P)^738/742 phosphorylation. Coexpression of kinase-dead PKCµ_K612W with PKC_A/E leads to phosphorylation of Ser^738/742 indicative of an upstream function of PKC (Fig. 3). These findings clearly indicate that the coexpression of PKC with PKCµ leads to phosphorylation of Ser^738/742, which subsequently triggers the substrate phosphorylation of PKCµ independently of the autophosphorylation of Ser⁹¹⁰. Ser⁹¹⁰ can therefore be confirmed as an autophosphorylation site, whereas Ser^738/742 is phosphorylated by an upstream kinase, triggering PKCµ substrate phosphorylation capacity. Ser(P)^738/742 and Ser(P)⁹¹⁰ antibodies used in this study are specific for the detection of activated forms of PKCµ. They do not show a cross-reaction with the PKC isotypes PKC and PKC. For PKC a very weak cross-reaction was observed (Fig. 3B).

PKC Directly Activates PKCµ-- As PKCµ activation by PKC occurs upon coexpression of both enzymes in living cells, it does not necessarily indicate direct activation. Independent kinases like PDK1 mediating activation loop phosphorylation of PKCs (3, 32) could not be excluded. To demonstrate direct activation of PKCµ by PKC, purified recombinant expressed kinases were used to show PKCµ phosphorylation on activation loop Ser^738/742 (Fig. 4, lower panels). As shown in Fig. 2 this transphosphorylation leads to enhanced in vitro substrate phosphorylation that can be measured also with the PKCµ substrate syntide 2 (25). PKCµ_WT can be directly phosphorylated in vitro by recombinant PKC leading to enhanced transphosphorylation and substrate (syntide 2) phosphorylation (Fig. 4, upper panels). In addition kinase-dead recombinant PKCµ_K612W was expressed in insect cells, purified, and included in the in vitro kinase assay. This mutant does not show any intrinsic autophosphorylation (Fig. 4, upper panel) enabling the measurement of direct transphosphorylation in vitro. Upon incubation of PKCµ_K612W with PKC, a significant transphosphorylation signal was observed. In addition, although to a lower degree, detection by Ser(P)^738/742-specific antibodies but no enhancement of syntide 2 phosphorylation could be demonstrated. Furthermore PDK1 was tested for direct phosphorylation of PKCµ_K612W too. As shown in Fig. 4 (right lanes), neither enhancement of transphosphorylation nor enhancement in detection efficiency through activation loop-specific Ser(P)^738/742 antibodies could be shown. The respective controls PKCµ_K612W and PDK1 did not show any syntide 2 phosphorylation, whereas PKC and PKCµ_WT alone show a weak syntide 2 phosphorylation.

View larger version (59K): [in this window] [in a new window]   Fig. 4.   In vitro activation of PKCµ by PKC. Recombinant PKCµ, PKCµK612W, PKC, and PDK1 were incubated for in vitro phosphorylation as indicated. Reactions were subdivided to monitor autophosphorylation (upper panel) followed Western blot detection of PKCµ (lower panel) and PKC/PDK1 (not shown). Furthermore aliquots of the kinase reaction were used to monitor syntide 2 substrate phosphorylation and activation loop phosphorylation using Ser(P)738/742-specific antibodies (middle panels).

These data clearly demonstrate that PKC acts as a direct PKCµ kinase leading to activation loop phosphorylation and enhanced in vitro substrate phosphorylation.

PKC Enhances PKCµ-mediated Activation of the p42/44 MAPK Cascade-- We have shown that constitutively active PKCµ_PH activates the p42/44 MAPK cascade triggering SRE-mediated gene activation (15). These data, as well as earlier studies (33) correlating high PKCµ expression with proliferative effects in mouse keratinocytes, indicated a PKCµ function in mitogenic pathways. Coexpression of PKC_A/E with PKCµ results in PKCµ activation by phosphorylation of Ser^738/742_, leading to the enhanced substrate phosphorylation capacity of PKCµ in cell-free kinase assays (see above). To verify that the PKC-mediated transactivation of PKCµ is also effective in intact cells and translates into an enhancement of cellular responses such as MAPK-dependent gene expression, SRE-driven reporter gene assays were performed as a parameter of MAPK activation. The serum-responsive element in the c-fos promoter is necessary and sufficient for growth factor-mediated transcription. A reporter gene construct containing a basal thymidine kinase promoter followed by four SRE recognition sites and a luciferase gene was used (15).

As a positive control, PKCµ-expressing cells were stimulated with phorbol ester, which led to a 5-fold increase in SRE-driven luciferase activity compared with nonstimulated vector-transfected cells (Fig. 5A). Expression of PKC_WT, constitutively active PKC_A/E or kinase-dead PKC_K/R alone did not stimulate SRE-driven luciferase activity in HEK293 cells.

View larger version (28K): [in this window] [in a new window]   Fig. 5.   Coexpression of PKC and PKCµ enhances SRE-driven luciferase activity of p42/p44 MAPK. A, HEK293 cells were cotransfected with the indicated expression constructs and with a 4xSRE-Tk-luciferase reporter construct and pCH110 for normalization. 24 h after transfection cells were shifted for 15 h to a serum-free medium, harvested, and proceeded for the measurement of luciferase activity (see "Materials and Methods"). Luciferase activity was normalized against transfection rates by measuring -galactosidase activity. B, activation of p42/p44-MAPK was measured after expression of the indicated constructs by Western blot analysis using either p42/p44 antibodies (upper panel) or phospho-p42/p44 specific antibodies (lower panel).

After coexpression of PKCµ with PKC_WT an approximately 9-fold and with PKC_A/E a 5-fold enhancement of SRE-driven luciferase activity was demonstrated (Fig. 5A). Similar results were obtained in NIH3T3 cells (data not shown). As expected from the in vitro phosphorylation studies (Fig. 2), the coexpression of kinase-dead PKC_K/R and PKCµ also enhanced SRE-driven luciferase activity, albeit to a much lower extent as compared with functional PKC.

To demonstrate further PKC/PKCµ-mediated MAPK stimulation, activation of p42/44 was measured using phosphorylation site-specific antibodies. As shown in Fig. 5B, the phorbol ester stimulation of HEK293 cells transiently expressing PKCµ led to a significant enhancement of p42/p44 phosphorylation in a similar way as described previously (15). Coexpression of PKCµ with PKC_A/E led to an increase of p42 but not of p44 phosphorylation (Fig. 5B), verifying PKCµ dependence of this effect. As a control, expression levels of p42 and p44 were monitored by Western blot (Fig. 5B, upper panel). These data clearly indicate that physiological relevant functions of PKCµ like the activation of the MAPK cascade are mediated through PKC activation.

Coexpression of PKCµ and PKC Affects PKC-mediated JNK Activation-- PKC coexpression with PKCµ enhances SRE-driven luciferase activity indicating a physiological role of PKC-mediated activation of PKCµ. Under the experimental conditions applied here, JNK was found not to be affected by overexpression of PKCµ (15). To date no data on PKC regulation of p42 and JNK in HEK293 cells were available, but in this study we were able to demonstrate the direct influence of PKC overexpression on JNK. As shown above in Fig. 5A, PKC expression failed to activate SRE-driven reporter genes, indicating that PKC does not directly activate the p42 MAPK cascade in this cell type. However, PKC was found to stimulate JNK activation, as shown by the 5-fold increased phosphorylation of the amino-terminal GST-c-Jun fusion protein by endogenous JNK immunoprecipitated from PKC_A/E-overexpressing cells. As a positive control, tumor necrosis factor-stimulated HEK293 cells were used, resulting in an approximately 4-fold enhancement of GST-c-Jun phosphorylation. PKCµ_WT served as a negative control as well as constitutively active PKCµ_PH, which did not or only weakly activated JNK (Fig. 6). Interestingly, on coexpression of PKCµ with constitutively active PKC_A/E, a reduction of PKC-mediated JNK activation could be observed (Fig. 6), indicating a negative feedback regulation of PKCµ on PKC kinase activity. These findings are corroborated by expression of the kinase-dead mutant PKCµ_K612W leading to a 4-fold increase JNK activity. Expression levels of all constructs used were verified by Western blot analysis (upper panels).

View larger version (48K): [in this window] [in a new window]   Fig. 6.   Coexpression of PKCµ with PKC inhibits PKC-mediated JNK activity. The indicated constructs were transiently coexpressed in HEK293 cells. 24 h after transfection cells were serum-starved overnight, and JNK immunoprecipitates were prepared to phosphorylate GST-c-Jun fusion proteins. Relative enhancement of JNK activation was estimated by quantitative PhosphorImaging analysis of the autoradiograph (lower panel). Transgene expression is shown by Western blot analysis. The amounts of GST-c-Jun fusion protein used are visualized by Ponceau staining (lower panel). TNF, tumor necrosis factor.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

It has been shown previously that PKD is activated via a PKC-dependent pathway (13). Demonstrating the physical association of PKD with PKC (16), it is conceivable that PKC serves as an upstream kinase phosphorylating PKD. In accordance with this view we show by confocal analysis that the human isozyme PKCµ colocalizes partially with PKC, a prerequisite for direct functional interaction. We could show that colocalization occurs in perinuclear compartments. The coexpression of constitutively active PKC with PKCµ leads to a phosphorylation enhancement of Ser^738/742 in PKCµ, previously thought to act as autophosphorylation sites. The data presented here now suggest that these serines, which are located in the activation loop of PKCµ, are transphosphorylation sites targeted by an upstream activating kinase. In colocalization studies (Fig. 1), the previous finding of PKC/PKCµ interaction via the PKCµ PH domain (16) and the PKC dependence of PKCµSer^738/742 phosphorylation (Figs. 3 and 4) demonstrates PKC as the direct upstream kinase of PKCµ. The discovery that activation loop phosphorylation is essential for substrate phosphorylation further explains earlier observations of the regulation of PKCµ kinase activity.

PDK1 has been shown to act as an upstream kinase for several PKC isotypes (3, 32) (e.g. leading to activation loop phosphorylation of Thr⁵⁰⁰ of PKCII) but does not phosphorylate the PKCµ activation loop (Fig. 4). The PDK1 phosphorylation sites in the activation loop of several kinases contain threonines, whereas in the homologous position of PKCµ a serine is located. Furthermore, the primary amino acid sequence surrounding Ser^738/742 diverges significantly from known PDK1 sites. According to the primary structure of the activation loop of PKCµ and our data (Fig. 4), PDK1 can be excluded as a direct PKCµ kinase.

We have recently shown that the chaperon-like protein p32 binds to the PKCµ kinase domain and significantly reduces compartment-specific PKCµ substrate phosphorylation but interferes only weakly with the autophosphorylation capacity of PKCµ (21). According to the data presented here, we conclude that p32 binds to the kinase domain and interferes by steric hindrance with the PKCµ activation loop, thereby inhibiting substrate phosphorylation.

The PH domain of PKD/PKCµ has been implicated in the negative regulation of kinase activity (15, 34). Therefore, on binding PKC to the PKCµ PH domain, a negative block could be released, probably leading to conformational changes and enhanced autophosphorylation but not affecting substrate phosphorylation (Fig. 2). According to this model, kinase-dead PKC_K/R activates PKCµ autophosphorylation by conformational changes but does not affect substrate phosphorylation, which in turn needs to be triggered independently by PKC kinase activity. Within this activation model, the PH domain as well as PH domain-mediated interaction serve as an initial step unfolding PKCµ and thus enabling autophosphorylation of Ser⁹¹⁰. In addition unfolding may further contribute to complete activation by unknown endogenous factors explaining the PKCµ/PKC_K/R-mediated SRE-driven luciferase activity (Fig. 5A).

Although the molecular mode of PKCµ activation is currently not clear, we were able to show that PKC serves as a direct upstream activator for PKCµ-triggered cellular processes (15). The coexpression PKC with PKCµ leads to the enhancement of SRE-driven luciferase activity (Fig. 5A). Although PKC acts as a PKCµ activator, it is interesting to note that, by coexpression of both kinases, PKCµ-mediated effects are enhanced, but simultaneously PKC-triggered cellular processes activating JNK are reduced. The differential triggering of JNK and p42/p44 MAPK cascades by PKCs have been reported previously (35). Furthermore, it has been shown that PKCµ negatively interferes with the EGF receptor-mediated JNK activation in HEK293 cells (36). In this study the authors suggest that PKCµ phosphorylation of Thr^654,669 of the EGF receptor negatively affects JNK-activity, whereas EGF-triggered MAPK activity remains unaffected. PKCµ/PKD is activated by PDGF via a PKC-mediated pathway (29) and not by EGF (36). Because PDGF induces suppression of EGF-mediated JNK activation, a PDGF-triggered pathway leading via PKC to PKCµ activation can be postulated. This subsequently results in EGF receptor phosphorylation and the down-regulation of JNK, whereas mitogenic pathways remain unaffected. Our results point to the existence of a complex network of interfering kinase cascades triggered by PKCs and PKCµ.

	ACKNOWLEDGEMENTS

We thank Gottfried Baier, University of Innsbruck, for the PKC constitutively active and the kinase-dead mutants. We are grateful for the help of Eva Behrle and Annett Burzlaff in performing immunofluorescence analysis.

	FOOTNOTES

^* This work was supported by Grant 03121805 from the Bundesministerium für Bildung und Forschung and the Deutsche Forschungsgemeinschaft, SFB 495/B5.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: Fraunhofer Institute for Interfacial Engineering, Nobelstraße 12, 70569 Stuttgart, Germany. Tel.: 49-711-970-4244; Fax: 49-711-970-4200; E-mail: FJJ@IGB.FhG.de.

Published, JBC Papers in Press, December 6, 2001, DOI 10.1074/jbc.M106083200

² Hausser, A., Link, G., Bamberg, L., Burzlaff, A., Lutz, S., Pfizenmaier, K., and Johannes, F. J. (2002) J. Cell Biol. in press.

	ABBREVIATIONS

The abbreviations used are: PKC, protein kinase C; SRE, serum-responsive element; PKCµ, protein kinase C µ; PDK1, phosphoinositide-dependent kinase, PKD, protein kinase D; MAPK, mitogen-activated protein kinase; JNK, c-Jun amino-terminal kinase; EGF, epidermal growth factor, PDGF, platelet-derived growth factor; PH, pleckstrin homology; GFP, green fluorescence protein; ERK, extracellular signal-regulated kinase; GST, glutathione S-transferase; PBS, phosphate-buffered saline.

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