Regulation of ADAM12 Cell-surface Expression by Protein Kinase C ϵ*

The ADAM (a disintegrin and metalloprotease) family consists of multidomain cell-surface proteins that have a major impact on cell behavior. These transmembrane-anchored proteins are synthesized as proforms that have (from the N terminus): a prodomain; a metalloprotease-, disintegrin-like-, cysteine-rich, epidermal growth factor-like, and transmembrane domain; and a cytoplasmic tail. The 90-kDa mature form of human ADAM12 is generated in the trans-Golgi through cleavage of the prodomain by a furin-peptidase and is stored intracellularly until translocation to the cell surface as a constitutively active protein. However, little is known about the regulation of ADAM12 cell-surface translocation. Here, we used human RD rhabdomyosarcoma cells, which express ADAM12 at the cell surface, in a temporal pattern. We report that protein kinase C (PKC) ϵ induces ADAM12 translocation to the cell surface and that catalytic activity of PKCϵ is required for this translocation. The following results support this conclusion: 1) treatment of cells with 0.1 μm phorbol 12-myristate 13-acetate (PMA) enhanced ADAM12 cell-surface immunostaining, 2) ADAM12 and PKCϵ could be co-immunoprecipitated from membrane-enriched fractions of PMA-treated cells, 3) RD cells transfected with EGFP-tagged, myristoylated PKCϵ expressed more ADAM12 at the cell surface than did non-transfected cells, and 4) RD cells transfected with a kinase-inactive PKCϵ mutant did not exhibit ADAM12 cell-surface translocation upon PMA treatment. Finally, we demonstrate that the C1 and C2 domains of PKCϵ both contain a binding site for ADAM12. These studies show that PKCϵ plays a critical role in the regulation of ADAM12 cell-surface expression.

Important in vivo functions have been reported for several ADAMs (3). For example, the finding that ADAM9, -10, and -17 have, or mediate, ␣-secretase activity could be used to design new treatment strategies for Alzheimer's disease (7,14,16,17). Overexpression of ADAMs has been observed in many human cancers (18,19), suggesting that ADAMs could promote tumor growth and metastasis by modulating growth factor shedding and cell adhesion. A recent genome-wide scan and polymorphism analysis of a large group of patients identified ADAM33 on chromosome 20 as a putative asthma susceptibility gene (20). We have demonstrated that ADAM12-S, which is present in the serum of pregnant women but not in that of women who are not pregnant (21), can be used as a first-trimester maternal serum marker for Down syndrome (22). Gene-ablation experiments in mice revealed that ADAM17 (TACE)-deficient mice have severe perinatal and postnatal defects primarily related to eye, hair, and skin anomalies, including failure of eyelid fusion (10). In contrast, ADAM9-deficient mice have an apparently normal phenotype (10,14). ADAM12 deficiency confers increased perinatal mortality, although the reason for this is not yet well understood (23). Surviving ADAM12-null mice have defects in adipose tissue (23), and mice overexpressing ADAM12 under the muscle creatine kinase promoter exhibit increased adipogenesis (24), supporting the idea that ADAM12 is involved in mesenchymal cell differentiation.
ADAM12, originally named meltrin-␣ (25), has been implicated in muscle cell function in vivo and in vitro (25)(26)(27)(28)(29)(30). In the original study, expression of a truncated version of ADAM12, lacking the prodomain and metalloprotease domain, was found to stimulate muscle-cell fusion in cultured C2C12 cells, whereas full-length ADAM12 inhibited the fusion process (25). It was demonstrated later that ADAM12 overexpression in C2C12 cells induced a quiescence-like phenotype and that the cell-adhesion domains and cytoplasmic tail were required for mediating cell cycle arrest (31). In addition, it was demonstrated that the level of endogenously produced ADAM12 was higher in proliferating myoblasts and reserve cells than in well differentiated myotubes (25,27,31). We reported, using a different cell-differentiation system (3T3-L1 preadipocytes) (32), that ADAM12 was prominently expressed in proliferating preadipocytes, although the levels subsequently decreased during differentiation into fully mature adipocytes. The level of cell-surface ADAM12 seemed to be highest at the onset of differentiation. These and other studies have demonstrated that ADAM12 has a specific temporal expression pattern in several tissue compartments during development, regeneration, and in disease (12,18,19,(23)(24)(25)(26)(27)(28)(29)(30)33). However, little is known about the regulation of ADAM12 activation and translocation to the cell surface. Herein, we report that PKC⑀, a novel PKC isoform (34), induces the translocation of ADAM12 to the cell surface in a manner that is dependent on its catalytic activity.

EXPERIMENTAL PROCEDURES
Cell Lines and Antibodies-The following cell lines were obtained from American Type Culture Collection: human myoblastic rhabdomyosarcoma cells (RD), breast adenocarcinoma cells (MCF-7), African green monkey cells (COS-7), and Chinese hamster ovary cells (CHO-K1). For all cell lines, except CHO-K1 cells, we used Dulbecco's modified Eagle's medium supplemented with GlutaMAX I, 4500 mg/l glucose, 50 units/ml penicillin, 50 g/ml streptomycin, and 10% FBS (Invitrogen). CHO-K1 cells were grown in Dulbecco's modified Eagle's medium/ Ham's F12 medium with the same supplements. Differentiation of RD cells was induced in confluent cultures by substituting 10% FBS with 2% horse serum in otherwise complete growth medium. MCF-7 cells expressing full-length ADAM12 under a tetracycline-regulated system were generated according to the instructions provided by the manufacturer (BD Biosciences Clontech). In brief, ADAM12 full-length cDNA was subcloned into the SalI/EcoRV sites of a pTet-On regulatory plasmid. The resulting constructs were transfected into MCF-7 cells using electroporation in the presence of 100 g/ml hygromycin and 1 g/ml tetracycline, both obtained from Sigma-Aldrich (Vallensbaek, Denmark). The transfected MCF-7 cells were grown in medium (see above) containing 1 g/ml tetracycline. In some experiments, cells were treated for 15 min with 0.1 M phorbol 12-myristate 13-acetate (PMA; Calbiochem), 0.1 M calphostin C (Calbiochem), or no additive.
Transient Transfection Assays-Cells were transfected using Fu-Gene 6 transfection reagent (Roche Diagnostic), or LipofectAMINE 2000 (Invitrogen) in serum-free medium according to the manufacturer's instructions, and analyzed 1 or 2 days later. Full-length ADAM12 or vector control cDNA was transfected into CHO-K1 and COS-7 cells for use as positive or negative controls, respectively, in Western blot and immunostaining experiments. For positive controls in FACS experiments, CHO-K1 cells were transiently transfected with cDNA encoding full-length ADAM12 or ADAM12-⌬cyt, a membrane-inserted ADAM12 protein lacking the cytoplasmic tail (35).
Preparation of Cellular Extracts, Immunoprecipitation, and Western Blotting-RD cells, with or without PMA treatment, and CHO cells transfected with full-length ADAM12 were washed two times with ice-cold PBS and lysed in RIPA buffer (50 mM Tris-HCl, pH 7.4, 1% Triton X-100, 25 mM HEPES, 150 mM NaCl, 0.2% deoxycholate, 5 mM MgCl 2 , 1 mM Na 3 VO 4 , 1 mM NaF, and a protease inhibitor mixture (Complete EDTA-free protease inhibitor mixture tablets; Roche Molecular Biochemical)). The protein concentration of extracts was measured using the BCA protein assay kit according to the instructions from the manufacturer (Pierce Biotechnology), and samples were normalized for protein content. To detect desmin and actin expression, total RIPA extracts (50 g total protein/well) were separated by 4 -12% polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. For immunoprecipitation, protein extracts were pre-incubated for 1 h with 50 l of protein-G beads alone to reduce nonspecific protein binding. The lysates were further incubated overnight with 50 l of protein-G prebound with ADAM12 mAbs 6E6, 6C10, and 8F8. After incubation of protein extracts with protein-G beads/mAbs or control IgG, the beads were centrifuged and washed four times with RIPA buffer. Proteins bound to the beads were eluted in sample buffer and boiled for 5 min. After centrifugation, the supernatants were analyzed by 8, 14, or 15% polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The membranes were stained using ADAM12 polyclonal antisera (rb 122), PKC (␣, ␦, or ⑀), desmin, actin, GFP, or myc antibodies as the primary antibody, and horseradish peroxidase-conjugated anti-rabbit antibody or HRP-conjugated anti-mouse antibody, respectively, as the secondary antibody. The SuperSignal Western blotting detection system (Pierce) was used for visualization.
To enrich for membrane fractions, cells were washed twice with ice-cold PBS, then scraped into cold buffer A (16.8 mM HEPES pH 8.0, 2.0 mM MgCl 2 , 0.88 mM EDTA, 1 mM Na 3 VO 4 , 1 mM NaF, and the protease inhibitor mixture described above), homogenized by 30 strokes in a Dounce homogenizer on ice, and then centrifuged at 200 ϫ g for 10 min. Supernatants were centrifuged, the protein content was measured, and aliquots with equal amounts of protein were further centrifuged at 200,000 ϫ g for 30 min. The resulting membrane-enriched pellets were dissolved in RIPA buffer and used for immunoprecipitation or immunoblotting. An equal amount of 2ϫ sample buffer was added, and samples were analyzed by Western blotting for ADAM12 (rb 122), and PKC␣, ␦, and ⑀ content.
Flow Cytometry-ADAM12 mAbs (clones 6E6 and 4G2) were biotinylated using EZ-link N-hydroxysuccinimide-LC-LC-Biotin (Pierce) according to the manufacturer's instructions. Cells were detached with trypsin/EDTA and allowed to recover for 5 min at 37°C in growth medium supplemented with 10% FBS, then transferred to ice and washed twice in ice-cold washing buffer (PBS supplemented with 1% bovine serum albumin). Cells were incubated with biotinylated 6E6 and 4G2 mAbs or isotype control Ab. After 20 min at 4°C, cells were fixed in 4% paraformaldehyde for 15 min and washed twice with washing buffer. Cells were further incubated with streptavidin-phycoerythrin conjugate for 20 min at 4°C, followed by two washes. Cells were finally resuspended in 300 l of washing buffer for flow cytometric analyses performed according to standard settings on a FACStar PLUS flow cytometer with CELLQUEST software (both from BD Biosciences).
Cell Attachment Assays-Cell attachment assays were performed as described previously (18,40,41). In brief, MaxiSorp 96-well plates (Nalge Nunc International) were coated (overnight at 4°C) with 10 g/ml of the IgG fraction of polyclonal anti-ADAM12 Ab (rb134) and the corresponding preimmune IgG in 0.1 M NaHCO 3 buffer, pH 9.5. RD and MCF-7 cells were then treated with 0.1 M PMA and/or 0.1 M calphostin C for 30 min and examined for their ability to attach to the different substrates for 1 h in serum-free medium. The number of adherent cells were quantitated as described previously (40,41) Immunostaining and Imaging-Cultured cells were analyzed by immunoperoxidase or immunofluorescence staining using either adherent or suspended cells. All procedures were performed at room temperature. To reveal the presence of ADAM12 at the surface of adherent cells, cells were incubated with monoclonal antibodies (a mixture of 6E6, 6C10, and 8F8) to ADAM12 or control mouse IgG 1 for 1 h at 4°C, then fixed in 4% paraformaldehyde in PBS for 5 min. Bound antibody was visualized using the DakoChemMate detection kit, which is based on an indirect streptavidin-biotin technique using a biotinylated secondary antibody. To reveal the presence of intracellular ADAM12 or MyoD, cells were rinsed with PBS, fixed in 4% paraformaldehyde for 5 min at room temperature, rinsed in PBS, permeabilized with 0.1% Triton X-100 in PBS for 5 min, and incubated with polyclonal anti-ADAM12 antiserum (rb 122) or preimmune serum, a monoclonal antibody recognizing MyoD, or a control mouse IgG 1 for 1 h followed by visualization as described above. To stain the cell surface of suspended cells, cells were detached with trypsin/EDTA or enzyme-free cell dissociation buffer (both from Invitrogen), restored for 5 min at 37°C in growth medium supplemented with 10% FBS, transferred to ice, and incubated for 30 min with monoclonal antibodies (a mixture of 6E6, 6C10, and 8F8) or control mouse IgG 1 . The cells were rinsed once in PBS, fixed in 4% paraformaldehyde for 2 min, and immediately centrifuged onto glass slides in a Cytospin Microfuge (2 min; Shandon, Pittsburgh) and air-dried. Detection was performed using rabbit anti-mouse-TRITC.
To detect ADAM12 in focal adhesions and to visualize stress fibers, cells were allowed to attach and spread onto a tissue culture plastic substratum for 1 h followed by treatment with PMA for 30 min. Subsequently, cells were fixed with 4% paraformaldehyde in PBS for 5 min at 4°C. For double immunostaining, cells were incubated for 1 h with anti-vinculin monoclonal antibodies and with ADAM12 polyclonal antiserum (rb 122) or preimmune serum. Detection was performed using secondary antibodies: swine anti-rabbit-FITC and rabbit anti-mouse-TRITC. TRITC-phalloidin (Molecular Probes, Leiden, The Netherlands) was used to stain actin in stress fibers.
Cells were examined using an inverted Zeiss Axiovert microscope equipped with phase contrast optics and connected to a PentaMAX chilled, charge-coupled device camera. The images were processed using the Metamorph Software Program (Universal Imaging Corporation). For scoring of the number of cells expressing ADAM12 or PKCs, the cells were examined under the microscope using a 63ϫ/1.4 numerical aperture plan-APOCHROMAT oil immersion objective. Cells were scored blindly by two independent researchers, from ϳ10 randomly selected fields per experiments, and data shown are representative results from at least three independent experiments.

ADAM12 at the Cell Surface in Early Differentiating RD
Rhabdomyosarcoma Cells-We have previously demonstrated that the human RD embryonal rhabdomyosarcoma cell line expresses ADAM12 mRNA (28). As shown in Fig. 1A (lane 4), ADAM12 protein could be detected in growing RD cells by immunoprecipitation and Western blotting using specific antibodies. ADAM12 appeared as an M r 110,000 proform (the catalytically inactive zymogen) and an M r 90,000 mature form (the active form in which the prodomain is cleaved). An additional M r 70,000 band degradation product was often detected in these cells. No bands were observed when the specific ADAM12 mAbs were replaced with control IgG (Fig. 1A, lane  3). CHO-K1 cells transfected with full-length human ADAM12 were used as a positive control (Fig. 1A, lane 2) and transfection with an empty control vector provided a negative control (Fig. 1A, lane 1). We further compared the expression of ADAM12 in RD cells before confluence (day Ϫ2), at confluence (day 0), and after stimulating muscle cell differentiation for 2 days (day 2) by culturing in the presence of 2% horse serum. The total amount of ADAM12 was similar or slightly increased upon the onset of differentiation and, as expected, the level of desmin expression seemed to slightly increase (Fig. 1B). FACS analysis demonstrated that RD cells exhibited low but detectable amounts of ADAM12 protein at the cell surface (Fig. 1C).
Morphological analysis of RD cells demonstrated a heterogeneous population of cells. That is, subconfluent cultures grown in Dulbecco's modified Eagle's medium with 10% FBS contained round, polygonal, and spindle-shaped cells. Immunostaining revealed that more than half of these growing RD cells expressed ADAM12 and fewer than 25% expressed MyoD, an early marker of myogenic differentiation (Fig. 1, D and E). An average of 29% (based on three separate experiments) expressed neither ADAM12 nor MyoD (a), 48% expressed ADAM12 but not MyoD (b), no cells expressed MyoD alone (c), and 23% expressed both ADAM12 and MyoD (d). These results indicate that ADAM12 expression precedes the expression of MyoD during myogenic differentiation in RD cells.
Because ADAM12 may have an important role in ectodomain shedding and/or cell interactions when located at the cell surface in cultured cells, we examined the distribution of ADAM12 without (Fig. 1, F-H) and with cell permeabilization (Fig. 1, I-K). In subconfluent cultures, few cells exhibited ADAM12 immunostaining at the cell surface. ADAM12 immunostaining was particularly concentrated in areas of cell-cell contacts (Fig.  1F, double arrows) and along the entire cell surface (Fig. 1G, single arrows). In cultures grown in 2% horse serum for 4 days, elongated, myotube-like cells located on top of flattened polygonal cells were seen. These myotube-like cells exhibited distinct punctate cell-surface ADAM12 immunostaining (Fig. 1H, arrows). Immunostaining with antibodies against skeletal myosin (fast) was used to detect muscle differentiation (data not shown). To allow for detection of intracellularly located ADAM12, parallel cultures were permeabilized with Triton X-100 before immunostaining with ADAM12 antibodies (Fig. 1, I-K). The intensity of intracellular ADAM12 immunoreactivity seemed to be higher in the early subconfluent cultures (Fig. 1, I and J) than in the more differentiated cultures (Fig. 1K). The results described above indicate that ADAM12 is enriched at the cell surface of RD cells during early stages of myogenic differentiation and raises the question of what might regulate its translocation from intracellular stores to the cell surface.
PMA Treatment Increases ADAM12 Cell-surface Expression-It has previously been demonstrated that PKC␦ together with ADAM9 is involved in enhanced TPA-induced ectodomain shedding of heparin-binding epidermal growth factor (13). This led us to test whether PKC activation might influence the translocation of ADAM12 to the cell surface. When untreated RD cells were immunostained with antibodies against ADAM12, only a few cells exhibited cell surface immunofluorescence staining (Fig. 2, A and I). However, when the cells were treated with the general PKC activator PMA for 30 min before immunostaining, ϳ30% of the cells were ADAM12-positive at the cell surface, and the intensity of the immunostaining reaction was increased (Fig. 2, B and I). Likewise, ADAM12-expressing MCF-7 breast carcinoma cells (tet-off) treated with PMA exhibited a greater proportion of positive cells (Fig. 2F) than ADAM12-expressing MCF-7 cells that were not PMA-treated (Fig. 2E). MCF-7 cells that do not express ADAM12 (tet-on) did not exhibit ADAM12 immunostaining, with or without PMA treatment (Fig. 2, C and D). COS-7 cells transiently transfected with full-length ADAM12 exhibited the same pattern of increased ADAM12 cell-surface localization upon PMA treatment (Fig. 2, G and H). Pretreatment of the RD cells with calphostin C (an inhibitor of PKC) for 15 min before PMA treatment abolished the increase in ADAM12 cell-surface expression (Fig. 2I). Further evidence for increased ADAM12 cell-surface expression was obtained using a cell attachment assay (Fig. 2, J and K). 96-well plates were coated with anti-ADAM12 IgG (rb134) or the corresponding preimmune IgG. RD cells (Fig. 2J) or ADAM12-expressing MCF-7 cells (Fig. 2K), with or without prior PMA treatment, were added to the wells for 1 h, after which the number of adherent cells was quantified. Fig. 2J shows that the binding of PMA-treated RD cells to anti-ADAM12-IgG was significantly greater than the binding of non-treated RD cells. Enhanced binding was not detected in PMA-treated RD cells on plates coated with preimmune IgG (Fig. 2J). Likewise, PMA-treated ADAM12-expressing MCF-7  4). After immunoprecipitation, Western blotting was performed using rabbit ADAM12 antiserum (rb122). B, ADAM12 expression in RD cells was examined at different stages of growth and differentiation. At day Ϫ2, cells are growing. At day 0, when cells are reaching confluence, differentiation was induced by switching the medium to Dulbecco's modified Eagle's medium ϩ2% horse serum. Cells were harvested after 2 days in differentiation medium (day 2). Immunoprecipitation was performed with a mixture of ADAM12 monoclonal antibodies, and Western blotting was performed using an ADAM12 antiserum (rb122). Crude extracts were also analyzed by Western blotting using antibodies against desmin and actin as a loading control. C, FACS analysis using the ADAM12 monoclonal antibodies 6E6 (red line) and 4G2 (green line) reveals low levels of ADAM12 cell-surface expression in RD cells. The isotype control is shown as a gray peak. D and E, growing RD cells were stained with antibodies to ADAM12 (brownish cytoplasmic staining) and MyoD (brownish nuclear staining). Cells expressing neither ADAM12 nor MyoD were designated a; cells expressing ADAM12 but not MyoD were designated b, cells expressing MyoD but not ADAM12 were designated c, and cells expressing both ADAM12 and MyoD were designated d. A total of ϳ500 cells in three separate experiments were evaluated, and the percentage of a, b, c, and d cells was estimated. F-H, these images show ADAM12 cell surface immunostaining (no pre-treatment with Triton X-100). I-K, these images show ADAM12 cytoplasmic staining (after pretreatment with Triton X-100). Hematoxylin was used for counterstaining in (D, F-K). Double arrows point at cell-surface ADAM12 immunostaining in areas of cell-cell contact. Single arrows point to diffuse punctate cell-surface ADAM12 immunostaining. cells bound more efficiently to anti-ADAM12 IgG than untreated cells (Fig. 2K). A corresponding increase in MCF-7 cell binding was not seen when cells were plated on preimmune IgG. All cells tested bound equally well to fibronectin (data not shown). We further tested whether the increase in ADAM12 cell-surface expression could be confirmed by FACS analysis; however, no significant changes could be detected by this assay (data not shown).  H). In I, the number of ADAM12-positive cells was estimated in RD cell cultures in the presence and absence of PMA and/or calphostin C. Significantly more cells exhibited ADAM12 cell-surface immunostaining after PMA treatment (*, p Ͻ 0.0001) and this effect was inhibited by calphostin C (**, p Ͻ 0.006). Cell attachment assays were performed in which RD cells (J) and MCF-7 cells that do or do not express ADAM12 (K) were treated with PMA (or vehicle control) for 30 min and then allowed to attach to anti-ADAM12 IgG (rb134) (black column) or the corresponding preimmune IgG (white column). Significantly more PMA-treated RD cells (J) attached to anti-ADAM12 IgG (p Ͻ 0.0002) and significantly more ADAM12-expressing MCF-7 cells (K) attached to anti-ADAM12 IgG (p Ͻ 0.05) than to preimmune IgG. MCF-7 cells that do not express ADAM12 did not attach to anti-ADAM12 IgG, regardless of the presence or absence of PMA. L-O, the effect of PMA on the pattern of F-actin and vinculin staining in freshly adherent RD cells is shown. Without PMA treatment, RD cells contain an elaborate pattern of stress fibers (L) that becomes reorganized into a cortical network after a 30-min PMA treatment (M). In addition, the cells become slightly smaller and more rounded. Focal adhesion formation, as assessed by vinculin immunostaining, seemed slightly more distinct in cells without PMA treatment (N) than those with PMA treatment (O). Co-immunostaining of vinculin (P) and ADAM12 (Q) in RD cells without PMA treatment demonstrated that ADAM12 was found in focal adhesions in these adhering RD cells.
To test whether ADAM12 was located at cell surfaces facing the substratum and, if so, whether PMA would influence this distribution pattern, cells were first allowed to attach for 1 h to a plastic substrate or to fibronectin, then PMA (or vehicle control) was added for another 30 min. PMA treatment induced a change in RD cell shape; cells became smaller and slightly more rounded. Consistent with this finding, stress fibers (as assessed by F-actin staining) were significantly attenuated and actin became organized in a cortical network (Fig. 2, compare L and M). The degree of focal adhesion formation, as assessed by anti-vinculin immunostaining, was also attenuated (Fig. 2,  compare N and O). ADAM12 codistributed with most of these vinculin-positive focal adhesions at the periphery of the cells (Fig. 2, P and Q); notably, this pattern did not seem to change after PMA treatment (data not shown). Together, these results suggest that PMA enhances the transport of ADAM12 from an intracellular pool to the cell surface, and because calphostin C inhibits this effect, these processes seem to be regulated by PKC(s).
Role of PKC⑀ and Its Catalytic Activity in Translocation of ADAM12 to the Cell Surface-We next sought to determine which PKC isoform was involved in the PMA-induced translocation of ADAM12 to the cell surface of RD cells. Membrane fractions were isolated, extracted in RIPA buffer, and examined by Western blotting using isoform-specific anti-PKC antibodies (Fig. 3A). In untreated RD cell membranes, PKC␣ and ⑀ were either not detected, or were present in low levels in the membrane fractions, whereas PKC␦ was present at high levels.
PMA stimulation induced a rapid increase of both PKC␣ and PKC⑀ in the membrane fractions, whereas PKC␦ levels apparently were not affected. Pretreatment of cells with the PKC inhibitor calphostin C inhibited the PMA-induced PKC⑀ increase in the membrane fraction; however, no inhibition of PKC␣ was seen.
To determine which PKCs ADAM12 interacts with, we analyzed extracts of RD cell membrane fractions from PMAtreated cells by co-immunoprecipitation. A mixture of anti-ADAM12 mAbs or preimmune IgG was used for precipitation and antibodies to the PKC isoforms were used for detection in the presence or absence of PMA treatment (Fig. 3, B-D). ADAM12 and the PKC⑀ isoform were associated in the membrane-enriched fractions, and the association was significantly increased in these fractions after PMA treatment (Fig. 3B). Evidence for a complex between ADAM12 and PKC⑀ was confirmed by the reverse experiment, in which extracts of membrane-enriched fractions were immunoprecipitated with antibodies to PKC⑀ and Western blotted with antiserum to ADAM12 (Fig. 3C). In contrast, ADAM12 did not seem to be present in complexes with PKC␣ (data not shown) nor with PKC␦ in membrane-enriched fractions irrespective of treatment with PMA (Fig. 3, C and D). In each experiment, a positive control was included in which Western blotting of ADAM12 or PKC⑀/PKC␦ (from crude membrane fractions of PMA-treated cells) was performed in parallel (see first lane in Fig. 3, B-D, respectively). As a loading control, the immunoprecipitates were probed with the respective antibodies   FIG. 3.

PMA-induced interaction between endogenous PKC⑀ and ADAM12 in RD cell membrane fractions.
A, Western blot analysis was used to detect PKC isoforms ␣, ␦, and ⑀ in membrane fractions from cells pre-treated with PMA and/or calphostin C, as indicated. PKC⑀ became translocated to the membrane fractions upon PMA treatment, and this effect was inhibited by calphostin C. B-D, the first lanes show direct Western blot analyses of ADAM12, PKC⑀, and PKC␦ in crude membrane fractions (CM) from PMA-treated cells. The remaining lanes represent co-immunoprecipitation of extracts prepared from membrane fractions followed by Western blotting, as indicated. B, ADAM12 monoclonal antibodies or control IgG were used for immunoprecipitation followed by Western blotting with an antibody to PKC⑀ and PKC␦, and ADAM12 antiserum (rb122). C and D show the reverse experiments in which the PKC⑀ (C) or PKC␦ (D) antibody was used for immunoprecipitation followed by Western blotting with an ADAM12 antiserum (rb122).
(ADAM12, PKC⑀, or PKC␦) that were used for immunoprecipitation. In addition, in each experiment immunoprecipitation was performed with a control IgG to confirm the specificity of the interaction. These results demonstrated that the activated form of PKC⑀, but not PKC␣ or -␦, was associated with ADAM12 in membrane-enriched fractions of PMA-treated RD cells.
We next studied the distribution of ADAM12 in RD cells transfected with full-length EGFP-tagged PKC␣, -␦, and -⑀ constructs. The day after transfection, cells were treated with PMA for 30 min and adherent nonpermeabilized cells were analyzed by immunostaining for ADAM12 and EGFP fluorescence to detect the various PKC isoforms (Fig. 4). In untreated cells, diffuse cytoplasmic EGFP fluorescence was seen but no EGFP fluorescence was associated with the cell membrane (Fig. 4, A and C). No ADAM12 cell-surface immunostaining could be detected (Fig. 4, B and D). Upon treatment with PMA, transfected RD cells displayed cell-membrane EGFP fluorescence indicating the translocation of PKC␣ and -⑀ to the cell membrane (Fig. 4, E and G). Notably, among those cells transfected with PKC␣ or -⑀ (Fig. 4, E and G), only those transfected with the PKC⑀ construct (Fig. 4G) exhibited ADAM12 cellsurface immunostaining (Fig. 4H). As expected, some of the non-transfected RD cells exhibited ADAM12 at the cell surface upon PMA treatment (Fig. 4F). However, cell counts showed that up to 40% of the PKC⑀-transfected cells exhibited ADAM12 cell-surface immunostaining, compared with ϳ20% of total cells (Fig. 4I). The effect on ADAM12 localization upon transfection with the PKC␦ construct was difficult to evaluate as most of the transfected RD cells dramatically changed shape and became much smaller (data not shown).
To confirm the role of PKC⑀ in translocation of ADAM12 to the cell surface, RD cells were transfected with myristoylated PKC␣-EGFP (MyrPKC␣-E), myristoylated PKC⑀-EGFP (MyrPKC⑀-E), or myristoylated kinase-inactive PKC⑀ (MyrPKC⑀KD-E). One day after transfection non-permeabilized cells were stained with antibodies against ADAM12 and analyzed for EGFP-fluorescence. ADAM12 was found to be localized at the cell surface in a substantial portion of cells expressing myristoylated wild-type PKC⑀ (Fig. 5, C and D). In contrast, ADAM12 was not translocated to the cell surface in cells transfected with MyrPKC␣-E or MyrPKC⑀KD-E (Fig. 5, B and F). Quantitative analysis revealed that ϳ30% of Myr-PKC⑀-E transfected cells showed ADAM12 cell surface immunostaining, compared with 8% of cells transfected with MyrPKC⑀KD-E-and 3% of MyrPKC␣-E-transfected cells (Fig.  5G). These results indicate a PKC⑀ catalytic activity-dependent translocation of ADAM12 to the cell surface of RD cells.
To confirm that the catalytic activity of PKC⑀ was required for cell surface translocation of ADAM12, RD cells were transfected with GFP-tagged full-length PKC⑀ (PKC⑀) or a kinaseinactive PKC⑀ mutant (PKC⑀KD). The proportion of transfected cells that expressed ADAM12 at the cell surface in response to PMA was estimated. As shown in Fig. 6A, PMA treatment increased ADAM12 translocation in PKC⑀-transfected cells but failed to induce ADAM12 translocation in cells expressing the PKC⑀ kinase-inactive mutant. Cell counts show that ADAM12 membrane translocation was found in only 8% of PKC⑀KD transfected cells compared with 29% of total cells (Fig. 6B). These results provide additional evidence that the catalytic activity of PKC⑀ is indeed required for ADAM12 translocation to the cell membrane.
We next used Triton-permeabilization to visualize intracellular ADAM12 and PKC⑀ before and after treatment with PMA. As shown in Fig. 7, A and B, PKC⑀ and ADAM12 were both concentrated in a perinuclear compartment of untreated PKC⑀-transfected cells. A similar pattern of localization was observed for kinase-inactive PKC⑀ and ADAM12 in untreated PKC⑀KD-transfected cells (Fig. 7, E and F). PMA treatment induced a rapid translocation of both PKC⑀ and ADAM12 to the cell surface in PKC⑀-transfected cells, as revealed by GFP fluorescence and ADAM12 immunostaining (Fig. 7 C and D,  arrows). However, we found no PMA-induced translocation of ADAM12 to the cell surface in PKC⑀KD transfected cells (Fig.  7H) although PKC⑀KD readily translocated to the cell membrane (Fig. 7G, arrow). These results show that PKC⑀ and ADAM12 may associate in a perinuclear compartment but PKC⑀ catalytic activity is required for the translocation of ADAM12 to the cell surface.
PKC⑀ Interaction with ADAM12 Is Mediated through the Regulatory Domain of PKC⑀-The PKC protein is composed of two separate domains: the NH 2 -terminal regulatory and the COOH-terminal catalytic domains (Fig. 8A). To map the ADAM12 binding site in PKC⑀, transient transfections with EGFP-tagged full-length PKC⑀ (PKC⑀FL-E) or PKC⑀ regulatory (PKC⑀RD-E) or catalytic (PKC⑀CD-E) domains were performed. The day after transfection, cells were either left untreated or treated with PMA for 30 min. Co-immunoprecipitation experiments were performed using extracts of membrane-enriched fractions and a mix of anti-ADAM12 mAbs for immunoprecipitation followed by Western blotting with a GFP antibody. As demonstrated in Fig. 8B (lanes 1 and 2), fulllength PKC⑀ interacted with ADAM12 in the membrane-enriched fractions upon treatment with PMA. A 113-kDa band corresponding to the PKC⑀FL-E was detected in the precipitate (Fig. 8B, lane 2). When analyzing PMA-treated cells transfected with either the regulatory or the catalytic domain of PKC⑀ (Fig. 8B, lanes 3 and 4), it was observed that ADAM12 interacted only with the regulatory domain (Fig. 8B, lane 4). The band was found at ϳ70 kDa corresponding to the expected size of PKC⑀-RD-E. No interaction between ADAM12 and PKC⑀-CD-E (expected size, 68 kDa) was found after PMA treatment. To monitor transfection efficiency, cell lysates from the same transfected cells were analyzed by Western blotting using the GFP antibody (Fig. 8B, lanes 5-8). Finally, we tested the interaction between ADAM12 and the C1 and C2 subdomains of the regulatory domain in cells transfected with myc-tagged PKC⑀PSC1V3 or C2 using the PKC⑀FL-myc construct as a positive control (Fig. 8C). ADAM12 interacted with both the C1 and C2 domains of PKC (Fig. 8C). Western blotting of cell lysates with the GFP antibody confirmed that cells were effectively transfected with all the PKC⑀ constructs (Fig. 7C, lanes  4 -6). These results demonstrate that both the C1 and C2 domains of PKC⑀ contain binding sites for ADAM12. DISCUSSION The proteins that form the ADAM family are known to play important roles in diverse developmental and disease processes (1)(2)(3)(4)(5). In this study, we set out to determine how cell-surface expression of ADAM12 is regulated. We took advantage of RD cells, a human rhabdomyosarcoma cell line that expresses a variety of muscle-specific transcription factors and structural proteins, as well as ADAM12. Using a panel of monoclonal and polyclonal antibodies, we found that prominent intracellular ADAM12 immunostaining could be detected in certain RD cells at different stages of differentiation. The finding that ADAM12 is present in a subpopulation of cells that was not positive for MyoD (an early marker of myogenic differentiation) supports previous studies showing that ADAM12 is produced by "activated" rather than "well differentiated" cells (18, 19, 25-27, 29, 33). Consistent with this is our finding that a RD subline, RD12 (42), that does not readily differentiate expresses much less ADAM12 (as assessed by immunostaining and Western blot analysis) than another subline, RD18, that readily differentiates. 2 Another intriguing finding was that the presence of detectable amounts of ADAM12 at the cell surface of RD cells seemed to be temporally restricted. It is noteworthy that staining for cell-surface-located ADAM12 was most intense in cells with "an early differentiated" morphology (i.e. cells with an elongated, mostly mononuclear, myotube-like shape) (Fig. 1H). These data, together with those described in the literature, strongly suggest that even if ADAM12 were constitutively synthesized and continuously present inside cells, the function of ADAM12 might be tightly controlled by the regulation of the protein's translocation to the cell surface. This conclusion led us to hypothesize that ADAM12 is only present on the cell surface at very specific stages of cell differentiation, which in turn motivated us to dissect the molecular mechanisms of ADAM12's translocation to the cell surface.
In this article, we report that PKC⑀ is involved in translocating ADAM12 to the cell surface in human RD cells in a manner dependent on the catalytic activity of PKC⑀. This conclusion is based on the following findings: 1) brief treatment of RD cells with a general PKC activator (PMA) increased the expression of ADAM12 at the cell surface, whereas a general PKC inhibitor (calphostin C) inhibited the effect of PMA; 2) ADAM12 could be co-immunoprecipitated with PKC⑀, but not PKC␣ or PKC␦, from membrane-enriched fractions of PMAtreated cells; 3) transfecting RD cells with constitutively activated MyrPKC⑀ resulted in translocation of ADAM12 to the cell surface, as assessed by immunostaining, and no ADAM12 translocation was observed upon PMA treatment in cells transfected with a kinase-inactive PKC⑀ mutant (PKC⑀KD). Furthermore, we have demonstrated that the regulatory domain of PKC⑀ contains binding sites for the interaction between ADAM12 and PKC⑀. These data imply that translocation of ADAM12 to the cell surface, and hence its critical biological role in ectodomain shedding and/or interactions with cell ad-hesion receptors, is likely to be regulated by PKC⑀.
PKCs are serine/threonine protein kinases that are involved in several aspects of cellular function; one intriguing example is that PKC␣ and PKC⑀ regulate ␤1 integrin trafficking during cell migration (43,44). In addition, PKC⑀ is known to have oncogenic potential and has been related to tumor aggressiveness by regulating the growth and survival of tumor cells (45,46). Izumi et al. (13) demonstrated that ADAM9 and PKC␦ are involved in PMA-induced ectodomain shedding of membraneanchored, heparin-binding epidermal growth factor. The interaction between ADAM9 as a specific binding protein and substrate for PKC␦ was further found to require the catalytic domain of PKC␦ and a stretch of 25 amino acids downstream of the transmembrane domain of ADAM9. Using yeast two hybrid screening, PKC␦ was also found to interact with ADAM12 (12). In the present study, we also found that ADAM12 could interact with PKC␦. They co-immunoprecipitated when crude cell lysate from RD cells was used, 2 but PKC␦ did not interact with ADAM12 in membrane-enriched fractions from untreated or PMA-treated cells (Fig. 3, D and E). It should be mentioned that in our system, membrane translocation of PKC␦ seemed to be insensitive to PMA treatment, thus excluding detailed studies of a putative role of PKC␦ in ADAM12 translocation. We therefore conclude that PKC⑀ might be particularly important for ADAM12 function. Our data further suggest that PKC⑀ interacts via its regulatory domain, rather than via its catalytic domain. There is precedent for this in previous studies showing that the regulatory domain contains binding sites responsible for protein-protein interactions; one prominent example of an interacting protein is the RACKs (receptors for activated protein kinase C) (47). Thus, although the regulatory domain of PKC⑀ contains the binding site for the interaction with ADAM12, catalytic activity seems to be required for the translocation of ADAM12 to the cell membrane. This conclusion is based on the finding that transfecting cells with PKC⑀, but not with a kinase-dead PKC⑀, enhanced ADAM12 translocation to the cell surface upon PMA treatment. This observation leads to questions concerning the role of phosphorylation in, or of, the cytoplasmic tails of ADAMs in general, and of ADAM12 in particular, by PKCs and other kinases. ADAM17 (TACE) is phosphorylated by Erk at threonine 735 (48), ADAM9 has been shown to become phosphorylated by purified PKC␦ in an vitro assay in response to PMA (13), and phosphorylation of 2 M. Kveiborg, R. Albrechtsen, and U. M. Wewer, unpublished data.

FIG. 7.
Co-translocalization of ADAM12 and PKC⑀ to the cell surface upon PMA treatment. RD cells were transfected with constructs encoding either GFP-tagged full-length PKC⑀ (PKC⑀) or kinase-inactive PKC⑀ (PKC⑀KD). Transfected cells were either untreated (ϪPMA) or treated (ϩPMA) with PMA for 30 min. The Triton X-100-permeabilized cells were immunostained with a rabbit polyclonal antiserum against ADAM12 (rb 122) followed by rhodamine-conjugated secondary antibody (B, D, F, H). PKC⑀ localization was visualized by GFP fluorescence (A, C, E, G). Arrows in C, D, and G indicate cell surface localization of PKC⑀ or ADAM12, respectively.
ADAM15v2 protein markedly enhanced binding with Lck in T lymphocytes (49). ADAM12 has been demonstrated to become phosphorylated at the C-terminal Tyr901 in vitro and in cultured cells by v-Src (50). In the present study, we did not determine whether ADAM12 is phosphorylated by PKC⑀. However, we found that in the absence of PMA, ADAM12 interacted with cytosolic (inactive) PKC␣, -␦, and -⑀ in co-immunoprecipitation experiments using crude cell lysates. In contrast, when membrane-enriched fractions were analyzed by co-immunoprecipitation experiments, ADAM12 interacted only with PKC⑀, and this interaction was enhanced after PMA treatment. This result suggests that phosphorylation of the cytoplasmic tail of ADAM12 by PKC⑀ may not be required for their interaction but does not exclude the possibility that ADAM12 becomes phosphorylated during the translocation process.
The major biological functions of the extracellular adhesion and metalloprotease domains of ADAMs have been studied, but less is known about the function of the cytoplasmic tails. We have previously reported that transfection with the full-length form of human ADAM12 results in little ADAM12 at the cell surface, whereas very efficient cell-surface translocation is observed with transfection of ADAM12 with the cytoplasmic domain deleted (35). These studies, and those reported by Cao et al. (51), suggest that the cytoplasmic tail actively regulates the amount of ADAM12 at the cell surface. In line with this idea is the finding that overexpression of the ADAM12 cytoplasmic tail in C2C12 cells inhibited cell fusion in a dominant-negative fashion (27). It follows that the dynamics and the components of the signaling complexes in which ADAM12 resides in the biosynthetic pathway might critically determine its cell-surface translocation, and hence determine when, where, and how much ADAM12 activity is available. In this regard, it is interesting that ADAM17 must be membrane-anchored (and therefore at the cell surface) to shed tumor necrosis factor-␣, p75TNFR, and interleukin 1R-II, but that the cytoplasmic tail, per se, is not required for this proteolytic activity (52). This further supports our hypothesis that one major function of the cytoplasmic tails of ADAMs is to regulate the balance between an intra-and extracellular location. ADAM12 links to the cytoskeleton by interacting, via its cytoplasmic tail, with ␣-acti- . C, RD cells were transiently transfected with constructs encoding myc-tagged, full-length PKC⑀ (PKC⑀FL-myc), the PSC1V3 subdomains (PKC⑀PSC1V3myc), or the C2 subdomain (PKC⑀C2-myc) of the PKC⑀ regulatory subdomains. Cells were left untreated or were treated with PMA for 30 min. Extracts of membrane fractions (B) or cell lysates (C) were prepared and used for direct Western blot analysis using antibodies against GFP or myc or subjected to immunoprecipitation with monoclonal antibodies to ADAM12 followed by Western blotting with antibodies against GFP. The expected molecular sizes are: PKC⑀FL-E, 113 kDa; PKC⑀RD-E, 70 kDa; PKC⑀CD-E, 68kDa; PKC⑀PSC1V3-myc, 28kDa; and PKC⑀C2myc, 18 kDa. nin (27,53). It also interacts with Src homology 3 (SH3) domains in several molecules involved in intracellular signal transduction, including Src, Grb2, phosphatidylinositol 3-kinase, PACSIN, and Fish (50, 54 -56). The interaction between ADAM12 and PACSIN was recently shown to regulate ADAM12 protease activity (56). It may be that some of the other interaction partners have similar effects on the protease activity. It is likely that, in addition to PKC⑀, such cytosolic proteins may be directly or indirectly involved in translocating ADAM12 to the cell surface. Thus, we suggest the following model for the effect of PKC⑀ on ADAM12: ADAM12 binds PKC⑀; the interaction is independent of the PKC⑀ kinase activity and is mediated via the regulatory domain. Upon activation, PKC⑀ phosphorylates ADAM12 or other protein(s), thereby modulating protein conformation(s), which in turn leads to translocation of ADAM12 to the cell surface. Future studies are needed to determine which cytosolic protein partners enhance or inhibit translocation of ADAM12 to the cell surface. Finally, it is important to find out whether similar regulatory mechanisms are applicable to other ADAMs and cell systems. An understanding of ADAM membrane-trafficking is likely to be central to understanding how the biological functions of these molecules are regulated.