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Originally published In Press as doi:10.1074/jbc.M002964200 on May 30, 2000

J. Biol. Chem., Vol. 275, Issue 32, 24601-24607, August 11, 2000
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Protein Kinase C µ Is Regulated by the Multifunctional Chaperon Protein p32*

Peter StorzDagger §, Angelika HausserDagger , Gisela LinkDagger , Jürgen Dedio, Berhane Ghebrehiwet||, Klaus PfizenmaierDagger , and Franz-Josef JohannesDagger **DaggerDagger

From the Dagger  Institute of Cell Biology and Immunology, University of Stuttgart, Allmandring 31, 70569 Stuttgart, the ** Fraunhofer Institute for Interfacial Engineering, Nobelstraße 12, 70569 Stuttgart, the  Institute für Physiological Chemistry and Pathobiochemistry, University of Mainz, Duesbergweg 6, 55099 Mainz, Germany, and the || Department of Medicine and Pathology, State University of New York, Stony Brook, New York 11794

Received for publication, April 7, 2000, and in revised form, May 24, 2000

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We identified the multifunctional chaperon protein p32 as a protein kinase C (PKC)-binding protein interacting with PKCalpha , PKCzeta , PKCdelta , and PKCµ. We have analyzed the interaction of PKCµ with p32 in detail, and we show here in vivo association of PKCµ, as revealed from yeast two-hybrid analysis, precipitation assays using glutathione S-transferase fusion proteins, and reciprocal coimmunoprecipitation. In SKW 6.4 cells, PKCµ is constitutively associated with p32 at mitochondrial membranes, evident from colocalization with cytochrome c. p32 interacts with PKCµ in a compartment-specific manner, as it can be coimmunoprecipitated mainly from the particulate and not from the soluble fraction, despite the presence of p32 in both fractions. Although p32 binds to the kinase domain of PKCµ, it does not serve as a substrate. Interestingly, PKCµ-p32 immunocomplexes precipitated from the particulate fraction of two distinct cell lines, SKW 6.4 and 293T, show no detectable substrate phosphorylation. In support of a kinase regulatory function of p32, addition of p32 to in vitro kinase assays blocked, in a dose-dependent manner, aldolase but not autophosphorylation of PKCµ, suggesting a steric hindrance of substrate within the kinase domain. Together, these findings identify p32 as a novel, compartment-specific regulator of PKCµ kinase activity.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The protein kinases C (PKC)1 comprise a family of intracellular serine/threonine-specific kinases, which are implicated in signal transduction of a wide range of biological responses including changes in cell morphology, proliferation, and differentiation (1-3). The currently defined 11 members of the PKC family can be grouped into the three major classes of Ca2+-dependent classical PKCs, Ca2+-independent, novel PKCs, and Ca2+- and lipid-independent atypical PKCs as well as PKCµ and its mouse homologue PKD (4, 5), which do not conform to either one of these major classes and may thus define a new subgroup (6). PKCµ/PKD differ from the three major groups of PKC isozymes by the presence of an amino-terminal hydrophobic domain, an acidic domain (7), a pleckstrin homology domain within the regulatory region (8), and lack of a typical pseudosubstrate site. PKCµ is ubiquitously expressed, and evidence for the involvement of PKCµ in diverse cellular functions stems from reports showing enhancement of constitutive transport processes in PKCµ-overexpressing epithelial cells (9), G protein-mediated regulation of Golgi organization (10), and involvement in protection from apoptosis (11). Interestingly, PKCµ shows particularly high expression in thymus and hematopoietic cells suggesting a potential role in immune functions (12). In accordance with this is the finding that, upon B cell receptor stimulation, PKCµ is recruited together with the tyrosine kinase Syk and phospholipase Cgamma to the B cell receptor complex and negatively regulates phospholipase Cgamma activity (13).

In addition to lipid second messengers as regulators of PKC translocation and activation, there is increasing evidence for a role of regulatory proteins in controlling kinase activity and/or intracellular location of various PKC members. Thus, the identification of receptors of activated protein kinase C (14) as well as the binding of more general and of specific modulators such as 14-3-3 (15-17), PAR4, LIP (18, 19), and ZIP (20), respectively, points to a complex regulation of PKC-dependent intracellular pathways. Whereas the latter selectively bind to the C1 regulatory domain of the atypical PKClambda and zeta  and regulate kinase activity in a lipid messenger-independent manner (18), protein interacting with protein kinase Cs 1 was identified as a PKCalpha kinase domain-binding protein (21). By analogy, because of the ubiquitous expression of PKCµ and its apparent involvement in diverse cellular responses, the existence of cell type- and/or organelle-specific regulators of PKCµ can be postulated. Indeed, 14-3-3 proteins as well as phosphatidylinositol 4-kinases were recently identified to be associated specifically with the C1 region of PKCµ (17, 22).

To define other interacting proteins and to investigate their role in modulating kinase activity, we have used different PKCµ domains in various screening assays for PKCµ-binding proteins. The pleckstrin homology domain and the kinase domain of PKCµ were used in a yeast two-hybrid screen in order to identify new PKCµ-binding proteins. With the kinase domain as a bait, a novel PKC-binding protein was detected. We identified the multifunctional chaperon protein p32, previously described as a receptor of complement component C1q (23), the kininogen-binding protein p33 (24), and splicing factor associated protein p32 (25) as a general PKC interactor, and we describe in detail its interaction with PKCµ and the functional consequences on kinase activity.

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Yeast Two-hybrid Screening-- To introduce BamHI restriction sites, the kinase domain of PKCµ covering amino acids 570-911 was amplified using the following primers: 5'-ATCCTCATAGGATCCAAATCACTA-3' and 5'-ATCTCCTAGGATCCGTCAAAAC-3'. The amplified cDNA fragment was cloned either in pAS1 for yeast two-hybrid screening or in pGEX-3X (GST-µKin) to express glutathione S-transferase (GST) fusion proteins. The yeast strain Y190 was transformed with pAS1/PKCµ according to standard procedures (26). Expression of the fusion protein was verified by Western blot analysis of yeast lysates using a PKCµ-specific antibody. A pACT lambda  bacteriophage library of human-activated B-lymphocytes was converted in vitro to plasmids (27) and used to transform the pAS1/PKCµ-expressing Y190 yeast strain according to standard conditions (26). Clones were selected on the respective medium lacking tryptophan, leucine, and histidine containing 50 mM 3-amino-1,2,4-triazole (Sigma). Upon day 4 grown colonies were analyzed by lacZ staining. Blue colonies were streaked again and confirmed by lacZ staining. pACT plasmids were recovered by bacterial transformation of yeast isolated plasmids and subjected to dideoxy sequencing of both strands.

Recombinant PKCµ, Plasmid Constructs, and Cell Lines-- The production and purification of PKCµ from Sf158 insect cells overexpressing PKCµ has been described (28). To produce GST-p32 fusion proteins the cDNA fragment was amplified from the pACT-p32 plasmid using primers to introduce a BamHI site 5' of the ATG and cloned in frame in pGEX-3X. The fusion proteins for precipitation analysis were prepared according to standard procedures. The construction of the GFP-p32 construct (29) and the c-Myc-tagged PKCµ expression plasmid has been described previously (22, 28). The human SKW 6.4 B cell line (ATCC) was cultured in RPMI medium supplemented with 5% fetal calf serum. 293T cells were obtained from ATCC.

Antibodies and Reagents-- A rabbit antibody and a monoclonal antibody directed against gC1qR/p32 were used (29, 30). PKCµ, PKCzeta , PKCdelta , and JNK were detected with rabbit antibodies (Santa Cruz Biotechnology and Roche Molecular Biochemicals), PKCalpha and cytochrome c with monoclonal antibodies (Santa Cruz Biotechnology; PharMingen), and GST with a goat antibody (Amersham Pharmacia Biotech). Secondary alkaline phosphatase-conjugated goat anti-mouse IgG + IgM, goat anti-rabbit IgG antibodies, Cy3-conjugated goat anti-mouse and Cy2-conjugated goat anti-mouse antibodies were purchased from Dianova. Protease and phosphatase inhibitors were from Biomol. Phorbol ester (phorbol 12,13-dibutyrate, PdBu) and phosphatidylserine were purchased from Sigma.

Immunoprecipitation/GST Fusion Protein Precipitation Assays-- SKW 6.4 and Sf158 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 EDTA, pH 7.4, 1 mM NaF, 1 mM NaPP, 1 mM sodium orthovanadate, 1 mM sodium molybdate, 1 mM p-nitrophenyl phosphate, 1 µg/ml leupeptin, 1 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride). After 60 min cell lysis the lysates were centrifuged (10,000 × g, 15 min, 4 °C), and immunoprecipitation was performed as described (31). GST fusion protein pull-down assays were performed by incubation of 1 ml of lysate (representing 500,000 Sf158 cells or 50 × 106 SKW 6.4 cells) with the indicated amounts of GST fusion proteins coupled to glutathione-Sepharose for 60 min at 4 °C. Immunocomplexes or GST complexes were washed three times and applied to SDS-PAGE followed by transfer to nitrocellulose membrane. Western blot detection of PKCµ or p32 was performed according to standard procedures.

In Vitro Kinase Assays and Cellular Fractionation-- 80 ng of purified recombinant PKCµ from Sf158 cells were preincubated as indicated with different amounts of GST or GST-p32 in phosphorylation buffer (50 mM Tris, pH 7.5, 10 mM MgCl2, 2 mM dithiothreitol) for 10 min at room temperature. Kinase reaction in the absence or presence of phosphatidylserine/PdBu micelles, with or without 5 µg of aldolase as substrate, was started by addition of 4 µCi of [gamma -32P]ATP (Amersham Pharmacia Biotech) in 10 µl of kinase buffer, and incubation was carried out for 15 min at 37 °C. The reaction was stopped by adding 5× concentrated sample buffer, subsequently fractionated on 12% SDS-PAGE, transferred to a nitrocellulose membrane, and exposed upon autoradiography. Autoradiographs were analyzed by quantitative PhosphorImager analysis (Molecular Dynamics). After immunoprecipitation PKCµ substrate and autophosphorylation were determined in in vitro kinase assays as described (17). For cellular fractionation 4 × 108 SKW 6.4 and 3 × 108 293T cells were resuspended in lysis buffer containing no detergent and homogenized by applying 15 strokes with a "very tight-fitting" 5-ml Dounce homogenizer (Braun, Melsungen, Germany). Cellular debris was removed by centrifugation (800 × g, 5 min). The remaining lysate was centrifuged at 100,000 × g. The supernatant containing the cytosolic fraction was defined as the soluble fraction. The pellet was dissolved in lysis buffer containing 1% Triton X-100 and defined as the non-soluble fraction.

Confocal Immunofluorescence Analyses-- Cells were washed twice with PBS and fixed in 3.5% paraformaldehyde (in PBS) for 15 min at 37 °C. Permeabilization and blocking of the cells proceeded through incubation with 0.05% Tween 20 and 5% normal goat serum in PBS for 30 min. The cells were rinsed 3 times with PBS and then incubated with primary antibodies (0.05% Tween 20 and 5% normal goat serum in PBS). For immunofluorescence detection of the indicated proteins (see Figs. 3 and 4), cells were simultaneously incubated for 2 h with two different antibodies used in the following concentrations: PKCµ antibody at 4 µg/ml, anti-p32 or anti-coatomer CM1-A10 monoclonal antibodies at 3 µg/ml, p24 antibody at 1 µ g/ml, and anti-cytochrome C monoclonal antibody at 1 µg/ml. Incubation with Cy3- and Cy2-conjugated secondary antibodies (2 µg/ml) was performed for 1 h. Following staining, the cells were rinsed four times with PBS and mounted in mounting medium from Sigma (PBS/glycerol). In control stainings, no cross-reactivity of the anti-mouse and anti-rabbit antibodies was observed (data not shown). A Leica confocal laser-scanning microscope was used for colocalization studies. Simultaneous excitation of fluorescent dyes was achieved by an argon/krypton laser. The following adjustments were made: 1) excitation filter, short pass KP590; 2) beam splitter module, neutral beam splitter; 3) channel 1 Cy3 emission, barrier filter long pass OG590; and channel 2 Cy2 emission, barrier filter band pass BP535. Images were acquired through a plan 100 or 63× (1.3 oil immersion) objective. The cells were sliced into horizontal optical sections at an interval of 1 µm.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

p32 Binds to the Kinase Domain of PKCµ-- The cDNA fragment encoding for the PKCµ kinase domain was amplified by polymerase chain reaction, cloned in frame into pAS1 to be expressed as a fusion protein with the DNA binding domain of Gal4, and used in a two-hybrid screen in yeast (see scheme in Fig. 1A). After primary transfection of yeast and verification of PKCµ/Gal4 fusion protein expression by immunoblot using a PKCµ kinase domain-specific antibody (data not shown), yeast cells were secondarily transfected with pACT vector containing a human B cell library expressing fusion proteins with the Gal4 DNA activation domain. Transfectants were selected for growth on aminotriazole-containing minimal medium according to standard procedures (27, 32). Upon beta -galactosidase staining nine blue colonies growing at 50 mM 3-amino-1,2,4-triazole were identified. Plasmids were retrieved and subjected to sequence analysis of both strands using pACT-based primers. With the exception of one clone (Clone 138) all retrieved pACT plasmids displayed nonsense sequences resulting in premature translation termination and did not reveal Gal4 activation domain fusion proteins of significant length. Fig. 1B shows growth of Clone 138 on YPEG medium and the respective selection medium. The pACT plasmid was subjected to complete sequence analysis revealing a 1.5-kilobase pair cDNA insert. Upon data base searching, the sequence showed identity to a previously published cDNA coding for the glycoprotein p32/gC1q-R which binds to the globular head of C1q (23). In addition to the complete coding region of p32, 21 base pairs of the 5'-untranslated region were included in the pACT cDNA insert resulting in an in frame addition of seven amino acids between activation domain of Gal4 and p32.


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  		Fig. 1.   The kinase domain of PKCµ associates with p32. A, schematic drawing of PKCµ functional domains used for cloning of the kinase domain in the yeast expression vector (pAS1µKin). The fusion protein of GAL4 binding domain and PKCµ kinase domain was expressed in Y190 yeast strain, transfected with a B cell library containing GAL4 activating domain fusion proteins (see "Materials and Methods"). B, growth of yeast strains on rich media (YPEG, left panel) and selection media (HTL- + 50 mM aminotriazole, right panel). Y190 served as a negative control and Y190 pSE1111/pSE1112 as a positive control. Clone 138 encoding p32 was identified in the two-hybrid screen described here.

Biochemical Analysis of p32 Association with PKCµ and Other PKC Isotypes-- For an independent experimental verification of PKCµ-p32 interaction, precipitation assays with GST fusion proteins were performed. Therefore, the coding region of p32 was polymerase chain reaction-amplified, cloned in frame into pGEX-3X, and expressed as bacterial fusion protein with glutathione S-transferase. Purified GST-p32, immobilized on glutathione-Sepharose beads, was used to precipitate PKCµ from whole cell extracts of PKCµ-expressing Sf158 cells (Fig. 2A, top left panel). PKCµ could be specifically detected by Western blot analysis in GST-p32 precipitates using as little as 1 µg of fusion protein, whereas no signal was revealed in control precipitates using up to 16 µg of GST protein (Fig. 2A, left panels). GST 14-3-3tau , which efficiently associates with the PKCµ regulatory domain (17), served as a positive control (Fig. 2A, left panel, right lane). The respective amounts of fusion proteins used in precipitation assays were visualized by immunoblotting using an anti-GST antibody (Fig. 2A, bottom left panels). The same result was obtained using purified recombinant PKCµ (Fig. 2A, top right panel), indicating direct molecular interaction of PKCµ with p32. The reciprocal precipitation was carried out with extracts from the B cell line SKW 6.4, which expresses p32 in significant amounts (see Figs. 2C and 4) and a purified GST fusion protein of the PKCµ kinase domain (GST-µKin). Precipitates were analyzed by immunoblotting using a p32-specific rabbit antibody (Fig. 2B, top panel) or a GST-specific antibody to estimate GST loads (Fig. 2B, bottom panels). To demonstrate the specificity of the association, excessive amounts of GST protein (10-fold over GST-µKin) served as a negative control, which resulted in only a very weak staining (Fig. 2B, right lanes). Thus, the data presented here provide clear evidence of specific association of p32 with the kinase domain of PKCµ.


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  		Fig. 2.   PKCµ associates with p32 in vitro. A, Sf158 cell extracts expressing PKCµ were incubated with the indicated amounts of GST-p32, GST as a negative control, or GST-14-3-3tau as a positive control (left panels). In vitro binding of purified PKCµ to p32 is shown (right panels). 80 ng of purified PKCµ were precipitated with GST-p32 or GST proteins. PKCµ/GST-fusion protein complexes were harvested by incubation with glutathione-Sepharose beads and subjected to Western blot analysis using a PKCµ-specific antibody (top panels) as described under "Materials and Methods." GST (26 kDa) or GST-p32 fusion proteins (50 kDa) were detected using a goat anti-GST antibody (bottom panels). B, precipitation analysis of SKW 6.4 cells. 5 × 106 SKW 6.4 cells were lysed and incubated with 1 µg of GST-µKin or 10 µg of GST bound to glutathione-Sepharose beads as a negative control. Bound proteins were separated by 12% SDS-PAGE and subjected to immunoblot analysis using a p32 antibody (top panel) or a goat anti-GST antibody (bottom panel). C, coimmunoprecipitation of PKCµ with p32. 5 × 107 of SKW 6.4 cells were subjected to PKCµ or p32 immunoprecipitation using rabbit antibodies. Immunocomplexes were subjected to SDS-PAGE preceded by Western blot analysis using PKCµ-specific (top left panel) or p32-specific antibodies (bottom left panel). The experiments were performed three times with similar results.

In parallel to the GST-p32 precipitation assays (Fig. 2, A and B), association between PKCµ and p32 was independently demonstrated by reciprocal coimmunoprecipitation analysis using p32- and PKCµ-specific antisera (Fig. 2C). The somewhat weaker signal of p32 observed in PKCµ immunoprecipitates might be due to a steric hindrance by the PKCµ antibody, which is directed against carboxyl-terminal epitopes and thus could be in proximity to the p32 binding region. Phorbol ester treatment of cells or addition of phosphatidylserine/phorbol ester micelles to in vitro pull-down assays did not enhance p32 binding to PKCµ (data not shown), suggesting a constitutive, lipid-independent association of p32 in SKW 6.4 cells.

In order to assess the selectivity of p32 interaction with PKCµ, other PKC isotypes were analyzed by pull-down assays and coimmunoprecipitation analyses using three different recombinant PKC isotypes representing the three major PKC subgroups. By using GST-p32, in addition to PKCµ, a specific binding of PKCalpha , PKCzeta , and PKCdelta was noted (Fig. 3A). As a control, precipitation analysis of the c-Jun amino-terminal kinase (JNK) from lysates of 293T cells was performed. As shown in Fig. 3A (bottom panel), no binding of JNK could be detected. These results indicate a PKC-selective association of p32. Coimmunoprecipitation analyses using PKC-specific antibodies further confirmed interaction of p32 with members of the different PKC subgroups (Fig. 3B).


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  		Fig. 3.   p32 associates selectively with PKC isotypes. A, lysates from PKC-expressing Sf158 cells were used as a source for the indicated PKC isotypes. A lysate from 293T cells was used as a source for JNK. The amount of expressed PKC and JNK in cell lysates was estimated by Western blot analysis (left lanes) and the 10-fold amount was used for either GST or GST-p32 pull-down experiments detecting the indicated PKC isotypes by Western blot analysis. B, the indicated PKC isotypes were immunoprecipitated (IP) from lysates of 50 × 106 293 cells and either detected using isotype-specific antibodies (top panels) or a p32-specific antiserum (bottom panel). As a control total cell lysates (TCL) were compared for the presence of the indicated PKC isotypes and p32.

p32 Colocalizes with PKCµ in Mitochondria in SKW 6.4 Cells-- The association of PKCµ and p32 was further analyzed by confocal laser scanning microscopy. The literature on the cellular distribution of p32 is controversial, reporting p32 either localized at the cell membrane (33), intracellularly (34), or at mitochondria (29), which may reflect cell-specific differences. Therefore, we investigated the intracellular localization of p32 in the SKW 6.4 B cell line. As shown in Fig. 4, in these cells p32 is localized predominantly at intracellular compartments (Fig. 4, top row, left panel). Costaining with antibodies against cytochrome c (Fig. 4, top row, middle panel) resulted in a nearly identical staining pattern, which was confirmed by overlay analysis indicated by the blue color shown in the top right panel. In SKW 6.4 cells, PKCµ shows a broad speckled distribution throughout extranuclear regions of the cell (Fig. 4, middle row, left panel), with a clear enrichment in p32 positive, compartmentalized structures (Fig. 4, middle row, middle panel). Overlay of PKCµ- and p32-specific staining verifies a partial colocalization of both proteins (Fig. 4, middle row, right panel). A double staining with PKCµ (Fig. 4, bottom row, left panel) and cytochrome c (Fig. 4, bottom row, middle panel)-specific antibodies confirmed that PKCµ is partially located at mitochondria in SKW 6.4 cells (shown in blue at Fig. 4 in the bottom row, right panel). In 293T cells (Fig. 5, upper panel) and in SKW 6.4 cells, only a weak colocalization signal with p24 was revealed (Fig. 5, bottom panels), which is in accordance with an enrichment of PKCµ at mitochondria in the latter cell line. The data presented here thus indicate a cell type-specific compartmentalization/enrichment of PKCµ either at mitochondria, in the B cell line SKW 6.4 (Fig. 4), or at Golgi structures in 293T cells (Fig. 5).


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  		Fig. 4.   PKCµ associates with p32 in mitochondria. SKW 6.4 cells were coimmunostained with antibodies against PKCµ, p32, and cytochrome c. Cells proceeded to confocal laser scan analysis as described under "Materials and Methods." p32 (red) colocalizes with cytochrome c (green) as indicated by the blue color in the overlay (top row, right panel). PKCµ (red) colocalizes partially with p32 (green) shown by the overlay (middle row, right panel). PKCµ (red) associates partially with cytochrome C (green) as shown by the blue color (bottom row, right panel).


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  		Fig. 5.   PKCµ localizes at the Golgi compartment in 293T and not in SKW 6.4. Endogenously expressed PKCµ (green) in 293T cells colocalizes with coatomer/Golgi (CM1-A10 monoclonal antibody; red)-specific structure as indicated by the blue color (top row, right panel). PKCµ (green) does weakly colocalize with the Golgi-specific marker p24 (red) in SKW 6.4 cells (bottom row, right panel). Staining experiments were performed three times with similar results.

p32 Affects PKCµ Kinase Activity-- Since in in vitro studies p32 specifically binds to PKCµ and appears to be constitutively associated with the kinase in the B cell line SKW 6.4, we investigated whether it affects PKCµ kinase activity in vitro. Incubation of PKCµ with GST-p32 led to a slight enhancement of autophosphorylation (Fig. 6). We analyzed further whether substrate phosphorylation is affected by GST-p32 binding to PKCµ also. As shown in Fig. 6A, phosphorylation of the well known in vitro substrate aldolase (7, 17) was significantly inhibited over a 10-fold range (0.1-1 µg of p32). PKCµ-mediated aldolase phosphorylation was not affected in the presence of 1 µg of GST protein (Fig. 5B, right lane), indicating that inhibition of aldolase phosphorylation was not due to unspecific effects of the GST moiety. Quantitative analysis revealed, in the presence of 1 µg of GST-p32 an approximately 70% inhibition of aldolase phosphorylation (Fig. 7A, right panel). These data suggest that p32 binding to the kinase domain restricts, probably by steric hindrance, the substrate access to PKCµ.


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  		Fig. 6.   p32 inhibits PKCµ substrate phosphorylation. A, purified PKCµ was used for in vitro aldolase phosphorylation in the presence of the indicated amounts of GST-p32. The samples were fractionated by 12% SDS-PAGE, and the gel was dried and proceeded to quantitative phosphorimaging analysis upon autoradiography (right panel). B, inhibition of PKCµ aldolase phosphorylation of immunoprecipitates from 293T cells. Immunoprecipitates from 293T cells displaying high aldolase activity were incubated in vitro with the indicated amounts of GST-p32 or GST. The samples were fractionated by 12% SDS-PAGE and transferred to nitrocellulose, and the relative PKCµ loads were verified by Western blot analysis (data not shown). Aldolase phosphorylation was quantitatively evaluated by phosphorimaging analysis and is shown in a dose-response curve (right panel). C, in vitro inhibition of PKCµ kinase activity by p32 is independent of phorbol ester. Aldolase phosphorylation of purified PKCµ was performed in the presence of the indicated amounts of p32 and PdBu- containing micelles. Phosphorylation experiments were performed three times with similar results.


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  		Fig. 7.   Compartment-specific inhibition of PKCµ substrate phosphorylation. PKCµ was immunoprecipitated (IP) from soluble and non-soluble fractionated 293T and SKW 6.4 cells using equal amounts of cell lysates and subjected to in vitro auto- and substrate phosphorylation (B). PKCµ and p32 expression was monitored in PKCµ immunoprecipitates by Western blot analysis (A). Data from one of three experiments performed with similar results are shown. p, particulate fraction; s, soluble fraction.

A dose-dependent inhibition of substrate phosphorylation could be further demonstrated with in vitro kinase assays of immunoprecipitates (Fig. 6B). Addition of increasing amounts of p32 to PKCµ immunoprecipitates from 293T cells significantly inhibited aldolase phosphorylation, whereas autophosphorylation remained largely unaffected. In vitro inhibition of PKCµ substrate phosphorylation by p32 was not influenced by a concomitant phorbol ester activation (Fig. 6C) pointing to distinct regulation mechanisms.

p32 Selectively Associates with and Inhibits Aldolase Phosphorylation of PKCµ Located in the Particulate Fraction-- In SKW 6.4 cells, endogenous PKCµ has been localized predominantly to particulate structures, yet a weak cytosolic staining suggested a broader distribution to other compartments as well (see Fig. 4). Likewise, in several cell types, PKCµ also appeared to be enriched in particulate structures (9), but a partial cytosolic location has also been reported (36) and can be observed in the cell lines analyzed here (Figs. 4 and 5). In order to investigate whether or not the p32-PKCµ interaction and regulation of kinase activity is restricted to specific intracellular compartments, cell fractionation experiments were performed with 293T cells and SKW 6.4 cells. The results obtained support a compartment-specific regulation of PKCµ kinase activity by p32. Both cell lines express similar levels of endogenous p32 (Fig. 7A, bottom panel). PKCµ immunoprecipitates from soluble and particulate fractions of both SKW 6.4 and 293T cells were analyzed using PKCµ-specific antibodies. As shown in Fig. 7A, approximately equal amounts of PKCµ were present in either the soluble or the particulate fraction, yet p32 could be predominantly detected in PKCµ precipitates from the particulate fraction (Fig. 7A, middle panel), although, under the experimental conditions applied here, both fractions contain comparable amounts of p32 (Fig. 7A, bottom panel).

PKCµ immunoprecipitates were subjected to in vitro autophosphorylation and substrate phosphorylation. As shown in Fig. 7B, aldolase phosphorylation by PKCµ immunoprecipitates from the soluble fraction could be readily discerned, whereas PKCµ isolated from the particulate, p32-positive fraction did not show any detectable aldolase phosphorylation. Together with the data from in vitro kinase assays with purified PKCµ and p32-GST fusion proteins (Fig. 6), these findings indicate a p32-dependent regulation of compartmentalized PKCµ kinase activity and suggest a new mechanism of regulation of kinase activity via kinase domain interacting proteins, identifying an as yet unrecognized functional role of p32 in this process.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we identified by yeast two-hybrid screening a novel PKCµ-interacting protein, the previously described protein p32 (Fig. 1), which has been associated with multiple, chaperon-like functions. p32 may serve as a compartment-specific regulator of PKCµ kinase activity. Cellular colocalization of PKCµ and p32 at mitochondria was shown in the B cell line SKW 6.4 by confocal immunofluorescence microscopy (Fig. 4). Functional interaction of both proteins was shown by precipitation analysis with GST fusion proteins as well as by coimmunoprecipitation indicating a constitutive association of p32 with PKCµ (Fig. 2). As p32 causes inhibition of PKCµ substrate phosphorylation (Figs. 6 and 7), we propose a novel model of a chaperon-mediated control of PKCµ activation, in which PKCµ function is restricted to defined cellular compartments by the multifunctional protein p32. Accordingly, PKCµ kinase regulation by p32 may not only serve as a paradigm to explain a differential, cell-, and/or compartment-specific activation of ubiquitously expressed kinases by virtue of a cell-specific intracellular location/function of regulatory molecules but also provides new insight into regulation of kinase activity toward specific substrates by kinase domain interacting proteins (Figs. 6 and 7). These data show that PKCµ is, in addition to its regulation by lipids and 14-3-3 proteins (17), controlled by a p32-dependent mechanism that probably controls substrate access by steric hindrance.

So far, the biological role of p32 appeared rather unclear due to the diverse functions reported; p32 has been originally identified as a cell surface protein binding to the globular "heads" of the complement factor C1q (23). It also has been described as a cell surface kininogen-binding protein (24). In addition, several independent reports have described p32 as an intracellular protein (34, 37), which colocalizes in the endothelial cell line EA.hy926 with a mitochondrial marker protein (29). p32 has been shown to be important for the maintenance of mitochondrial oxidative phosphorylation (38). Mitochondrial functions of p32 are further indicated by the identification of a yeast homologue of p32, called Mam33p, that has been localized to the inner mitochondrial membrane (39). Other reports confirmed that p32 is located at mitochondria, but in addition a nuclear localization was found and a function of p32 as part of an import machinery was postulated (40). Moreover, p32 was described as part of the RNA splicing complex SF2 in HeLa cells (25). p32 has further been shown to associate with many viral proteins including HIV-1 Tat (41) and Rev (42) as well as with EBNA-1 of Epstein-Barr virus (43). The latter p32 functions are all considered to modulate transcription factor activity. The participation in different biological processes like mitochondrial functions, transcription- and splicing factor modulation, and potential role in complement cascade or blood coagulation (44) suggest a typical chaperon function of p32.

In this paper, we describe a novel aspect of p32 biology with a functional role as an inhibitor of kinase activity. The presented data show that p32 binds to the kinase domain of PKCµ and, without being a substrate, inhibits phosphorylation of aldolase, yet maintains or even enhances the level of autophosphorylation. As different phosphorylation sites trigger the activation state of PKC isoforms (45), similar mechanisms are conceivable for PKCµ. For the p32-mediated regulation of PKCµ activity, several possibilities may be considered. First, p32 may interfere with substrate phosphorylation by steric hindrance. Second, p32 binding to PKCµ could induce a conformational change such that endogenous autophosphorylation sites are preferentially used over cellular substrates. Third, the phosphorylation sites critical for kinase activation are blocked by p32, disabling substrate phosphorylation, yet leaving autophosphorylation at other serine residues of PKCµ unaffected. Several phosphorylation sites important for PKCµ activation have now been mapped within the catalytic domain (47), which is in accordance with the latter model of p32 interference with PKCµ function. Together, our data presented here thus indicate that, besides regulation of PKCµ kinase activity via the C1 domain either by activating lipid second messengers and phorbol ester (1, 3) or inactivating 14-3-3 proteins (17), other domains are involved in modulating PKCµ activity also. Since in contrast to 14-3-3, p32 does not affect lipid-induced PKCµ autophosphorylation (Fig. 6), we propose that PKCµ activity is controlled by at least two independent mechanisms. Moreover, our finding that p32 binds to different PKC isoforms points to a more general p32-based mechanism of controlling PKC kinase activity.

The differential cellular localization of p32 in different cell types (29, 40) may contribute to the compartment-specific functional role of various PKC isotypes, including PKCµ. As shown here, in the SKW 6.4 cell line, p32 largely colocalized with cytochrome c, indicative of a mitochondrial localization (Fig. 4). In full accordance with the in vitro binding studies, PKCµ partially colocalized with p32 at mitochondria, as revealed from confocal microscopy (Fig. 4) and cell fractionation studies (Fig. 7). Therefore, we propose that p32 is part of an intracellular receptor that retains PKCµ at intracellular compartments such as mitochondria and serves as a regulator of its kinase activity.

    ACKNOWLEDGEMENTS

We thank Stephen Elledge, Houston, TX, for the generous gift of the human B cell library and the two-hybrid reagents. We thank Felix Wieland (Heidelberg, Germany) and Kai Sohn (Stuttgart, Germany) for providing us with the p24 antibody. We also thank Heike Döppler for expert technical assistance in preparing the confocal images.

    FOOTNOTES

* This work was supported by the Deutsche Forschungsgemeinschaft Grant Jo227/4-3.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.

§ Current address: Dept. of Pathology, Beth Israel Deaconess Medical Center, 99 Brookline Ave., Research North RN-216, Boston, MA 02215.

Dagger Dagger To whom correspondence should be addressed. E-mail: FJJ@IGB.Fhg.dl.

Published, JBC Papers in Press, May 30, 2000, DOI 10.1074/jbc.M002964200

    ABBREVIATIONS

The abbreviations used are: PKC, protein kinase C; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PdBu, phorbol 12,13-dibutyrate; JNK, c-Jun amino-terminal kinase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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