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• W1963526979 abstract "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. 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. Cells possess a diverse array of surface proteins, lipids, and carbohydrates that provide active gateways for the selective intake and release of molecular information, which is important in regulating cell behavior. In fact, many disease processes relate to disorganized cell-surface communication systems. ADAMs 1The abbreviations used are: ADAM, a disintegrin and metalloprotease; PKC, protein kinase C; CHO, Chinese hamster ovary; FBS, fetal bovine serum; PMA, phorbol 12-myristate 13-acetate; mAb, monoclonal antibody; TRITC, tetramethylrhodamine B isothiocyanate; GFP, green fluorescent protein; FACS, fluorescence-activated cell sorting; EGFP, enhanced green fluorescent protein; PBS, phosphate-buffered saline; RIPA, radioimmunoprecipitation assay. belong to a large family of cell-surface proteins with over 30 members. The prototypical ADAM molecule is a transmembrane glycoprotein composed of several distinct domains, including a prodomain and a metalloprotease, disintegrin-like, cysteine-rich, epidermal growth factor-like, transmembrane, and cytoplasmic domain. ADAMs play important roles in cell adhesion, interacting with integrins and syndecans, and in the proteolysis of the ectodomains of cell-surface proteins, such as growth factors, growth factor receptors, and cytokines (1Blobel C.P. Curr. Opin. Cell Biol. 2000; 12: 606-612Crossref PubMed Scopus (225) Google Scholar, 2Blobel C.P. Inflamm. Res. 2002; 51: 83-84Crossref PubMed Scopus (64) Google Scholar, 3Seals D.F. Courtneidge S.A. Genes Dev. 2003; 17: 7-30Crossref PubMed Scopus (897) Google Scholar, 4Wolfsberg T.G. Primakoff P. Myles D.G. White J.M. J. Cell Biol. 1995; 131: 275-278Crossref PubMed Scopus (443) Google Scholar, 5Wolfsberg T.G. White J.M. Dev. Biol. 1996; 180: 389-401Crossref PubMed Scopus (217) Google Scholar). For example, ADAM17 (TACE) mediates release of tumor necrosis factor-α, transforming growth factor-α, β-amyloid, l-selectin, TRANCE, and amphiregulin precursor proteins (6Black R.A. Rauch C.T. Kozlosky C.J. Peschon J.J. Slack J.L. Wolfson M.F. Castner B.J. Stocking K.L. Reddy P. Srinivasan S. Nelson N. Boiani N. Schooley K.A. Gerhart M. Davis R. Fitzner J.N. Johnson R.S. Paxton R.J. March C.J. Cerretti D.P. Nature. 1997; 385: 729-733Crossref PubMed Scopus (2728) Google Scholar, 7Buxbaum J.D. Liu K.N. Luo Y. Slack J.L. Stocking K.L. Peschon J.J. Johnson R.S. Castner B.J. Cerretti D.P. Black R.A. J. Biol. Chem. 1998; 273: 27765-27767Abstract Full Text Full Text PDF PubMed Scopus (839) Google Scholar, 8Gschwind A. Hart S. Fischer O.M. Ullrich A. EMBO J. 2003; 22: 2411-2421Crossref PubMed Scopus (287) Google Scholar, 9Jackson L.F. Qiu T.H. Sunnarborg S.W. Chang A. Zhang C. Patterson C. Lee D.C. EMBO J. 2003; 22: 2704-2716Crossref PubMed Scopus (341) Google Scholar, 10Peschon J.J. Slack J.L. Reddy P. Stocking K.L. Sunnarborg S.W. Lee D.C. Russell W.E. Castner B.J. Johnson R.S. Fitzner J.N. Boyce R.W. Nelson N. Kozlosky C.J. Wolfson M.F. Rauch C.T. Cerretti D.P. Paxton R.J. March C.J. Black R.A. Science. 1998; 282: 1281-1284Crossref PubMed Scopus (1371) Google Scholar, 11Schlondorff J. Lum L. Blobel C.P. J. Biol. Chem. 2001; 276: 14665-14674Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). ADAMs 9, 10, and 12 have been shown to cleave membrane-anchored, heparin-binding epidermal growth factor (12Asakura M. Kitakaze M. Takashima S. Liao Y. Ishikura F. Yoshinaka T. Ohmoto H. Node K. Yoshino K. Ishiguro H. Asanuma H. Sanada S. Matsumura Y. Takeda H. Beppu S. Tada M. Hori M. Higashiyama S. Nat. Med. 2002; 8: 35-40Crossref PubMed Scopus (641) Google Scholar, 13Izumi Y. Hirata M. Hasuwa H. Iwamoto R. Umata T. Miyado K. Tamai Y. Kurisaki T. Sehara-Fujisawa A. Ohno S. Mekada E. EMBO J. 1998; 17: 7260-7272Crossref PubMed Scopus (475) Google Scholar, 14Weskamp G. Cai H. Brodie T.A. Higashyama S. Manova K. Ludwig T. Blobel C.P. Mol. Cell. Biol. 2002; 22: 1537-1544Crossref PubMed Scopus (175) Google Scholar, 15Yan Y. Shirakabe K. Werb Z. J. Cell Biol. 2002; 158: 221-226Crossref PubMed Scopus (279) Google Scholar). Important in vivo functions have been reported for several ADAMs (3Seals D.F. Courtneidge S.A. Genes Dev. 2003; 17: 7-30Crossref PubMed Scopus (897) Google Scholar). For example, the finding that ADAM9, -10, and -17 have, or mediate, α-secretase activity could be used to design new treatment strategies for Alzheimer's disease (7Buxbaum J.D. Liu K.N. Luo Y. Slack J.L. Stocking K.L. Peschon J.J. Johnson R.S. Castner B.J. Cerretti D.P. Black R.A. J. Biol. Chem. 1998; 273: 27765-27767Abstract Full Text Full Text PDF PubMed Scopus (839) Google Scholar, 14Weskamp G. Cai H. Brodie T.A. Higashyama S. Manova K. Ludwig T. Blobel C.P. Mol. Cell. Biol. 2002; 22: 1537-1544Crossref PubMed Scopus (175) Google Scholar, 16Lammich S. Kojro E. Postina R. Gilbert S. Pfeiffer R. Jasionowski M. Haass C. Fahrenholz F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3922-3927Crossref PubMed Scopus (986) Google Scholar, 17Allinson T.M. Parkin E.T. Turner A.J. Hooper N.M. J. Neurosci. Res. 2003; 74: 342-352Crossref PubMed Scopus (381) Google Scholar). Overexpression of ADAMs has been observed in many human cancers (18Iba K. Albrechtsen R. Gilpin B.J. Loechel F. Wewer U.M. Am. J. Pathol. 1999; 154: 1489-1501Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 19Le Pabic H. Bonnier D. Wewer U.M. Coutand A. Musso O. Baffet G. Clement B. Theret N. Hepatology. 2003; 37: 1056-1066Crossref PubMed Scopus (184) Google Scholar), 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 (20Van Eerdewegh P. Dowd M. Dupuis J. Falls K. Hayward B. Santangelo S.L. Genet. Epidemiol. 2001; 21: S67-S72Crossref PubMed Scopus (7) Google Scholar). 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 (21Shi Z. Xu W. Loechel F. Wewer U.M. Murphy L.J. J. Biol. Chem. 2000; 275: 18574-18580Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar), can be used as a first-trimester maternal serum marker for Down syndrome (22Laigaard J. Sorensen T. Frohlich C. Pedersen B.N. Christiansen M. Schiott K. Uldbjerg N. Albrechtsen R. Clausen H.V. Ottesen B. Wewer U.M. Prenat. Diagn. 2003; 23: 1086-1091Crossref PubMed Scopus (102) Google Scholar). 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 (10Peschon J.J. Slack J.L. Reddy P. Stocking K.L. Sunnarborg S.W. Lee D.C. Russell W.E. Castner B.J. Johnson R.S. Fitzner J.N. Boyce R.W. Nelson N. Kozlosky C.J. Wolfson M.F. Rauch C.T. Cerretti D.P. Paxton R.J. March C.J. Black R.A. Science. 1998; 282: 1281-1284Crossref PubMed Scopus (1371) Google Scholar). In contrast, ADAM9-deficient mice have an apparently normal phenotype (10Peschon J.J. Slack J.L. Reddy P. Stocking K.L. Sunnarborg S.W. Lee D.C. Russell W.E. Castner B.J. Johnson R.S. Fitzner J.N. Boyce R.W. Nelson N. Kozlosky C.J. Wolfson M.F. Rauch C.T. Cerretti D.P. Paxton R.J. March C.J. Black R.A. Science. 1998; 282: 1281-1284Crossref PubMed Scopus (1371) Google Scholar, 14Weskamp G. Cai H. Brodie T.A. Higashyama S. Manova K. Ludwig T. Blobel C.P. Mol. Cell. Biol. 2002; 22: 1537-1544Crossref PubMed Scopus (175) Google Scholar). ADAM12 deficiency confers increased perinatal mortality, although the reason for this is not yet well understood (23Kurisaki T. Masuda A. Sudo K. Sakagami J. Higashiyama S. Matsuda Y. Nagabukuro A. Tsuji A. Nabeshima Y. Asano M. Iwakura Y. Sehara-Fujisawa A. Mol. Cell. Biol. 2003; 23: 55-61Crossref PubMed Scopus (126) Google Scholar). Surviving ADAM12-null mice have defects in adipose tissue (23Kurisaki T. Masuda A. Sudo K. Sakagami J. Higashiyama S. Matsuda Y. Nagabukuro A. Tsuji A. Nabeshima Y. Asano M. Iwakura Y. Sehara-Fujisawa A. Mol. Cell. Biol. 2003; 23: 55-61Crossref PubMed Scopus (126) Google Scholar), and mice overexpressing ADAM12 under the muscle creatine kinase promoter exhibit increased adipogenesis (24Kawaguchi N. Xu X. Tajima R. Kronqvist P. Sundberg C. Loechel F. Albrechtsen R. Wewer U.M. Am. J. Pathol. 2002; 160: 1895-1903Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), supporting the idea that ADAM12 is involved in mesenchymal cell differentiation. ADAM12, originally named meltrin-α (25Yagami-Hiromasa T. Sato T. Kurisaki T. Kamijo K. Nabeshima Y. Fujisawa-Sehara A. Nature. 1995; 377: 652-656Crossref PubMed Scopus (439) Google Scholar), has been implicated in muscle cell function in vivo and in vitro (25Yagami-Hiromasa T. Sato T. Kurisaki T. Kamijo K. Nabeshima Y. Fujisawa-Sehara A. Nature. 1995; 377: 652-656Crossref PubMed Scopus (439) Google Scholar, 26Borneman A. Kuschel R. Fujisawa-Sehara A. J. Muscle Res. Cell Motil. 2000; 21: 475-480Crossref PubMed Scopus (36) Google Scholar, 27Galliano M.F. Huet C. Frygelius J. Polgren A. Wewer U.M. Engvall E. J. Biol. Chem. 2000; 275: 13933-13939Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 28Gilpin B.J. Loechel F. Mattei M.G. Engvall E. Albrechtsen R. Wewer U.M. J. Biol. Chem. 1998; 273: 157-166Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar, 29Kronqvist P. Kawaguchi N. Albrechtsen R. Xu X. Schroder H.D. Moghadaszadeh B. Nielsen F.C. Frohlich C. Engvall E. Wewer U.M. Am. J. Pathol. 2002; 161: 1535-1540Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 30Moghadaszadeh B. Albrechtsen R. Guo L.T. Zaik M. Kawaguchi N. Borup R.H. Kronqvist P. Schroder H.D. Davies K.E. Voit T. Nielsen F.C. Engvall E. Wewer U.M. Hum. Mol. Genet. 2003; 12: 2467-2479Crossref PubMed Scopus (61) Google Scholar). 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 (25Yagami-Hiromasa T. Sato T. Kurisaki T. Kamijo K. Nabeshima Y. Fujisawa-Sehara A. Nature. 1995; 377: 652-656Crossref PubMed Scopus (439) Google Scholar). 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 (31Cao Y. Zhao Z. Gruszczynska-Biegala J. Zolkiewska A. Mol. Cell. Biol. 2003; 23: 6725-6738Crossref PubMed Scopus (53) Google Scholar). 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 (25Yagami-Hiromasa T. Sato T. Kurisaki T. Kamijo K. Nabeshima Y. Fujisawa-Sehara A. Nature. 1995; 377: 652-656Crossref PubMed Scopus (439) Google Scholar, 27Galliano M.F. Huet C. Frygelius J. Polgren A. Wewer U.M. Engvall E. J. Biol. Chem. 2000; 275: 13933-13939Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 31Cao Y. Zhao Z. Gruszczynska-Biegala J. Zolkiewska A. Mol. Cell. Biol. 2003; 23: 6725-6738Crossref PubMed Scopus (53) Google Scholar). We reported, using a different cell-differentiation system (3T3-L1 preadipocytes) (32Kawaguchi N. Sundberg C. Kveiborg M. Moghadaszadeh B. Asmar M. Dietrich N. Thodeti C.K. Nielsen F.C. Moller P. Mercurio A.M. Albrechtsen R. Wewer U.M. J. Cell Sci. 2003; 116: 3893-3904Crossref PubMed Scopus (116) Google Scholar), 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 (12Asakura M. Kitakaze M. Takashima S. Liao Y. Ishikura F. Yoshinaka T. Ohmoto H. Node K. Yoshino K. Ishiguro H. Asanuma H. Sanada S. Matsumura Y. Takeda H. Beppu S. Tada M. Hori M. Higashiyama S. Nat. Med. 2002; 8: 35-40Crossref PubMed Scopus (641) Google Scholar, 18Iba K. Albrechtsen R. Gilpin B.J. Loechel F. Wewer U.M. Am. J. Pathol. 1999; 154: 1489-1501Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 19Le Pabic H. Bonnier D. Wewer U.M. Coutand A. Musso O. Baffet G. Clement B. Theret N. Hepatology. 2003; 37: 1056-1066Crossref PubMed Scopus (184) Google Scholar, 23Kurisaki T. Masuda A. Sudo K. Sakagami J. Higashiyama S. Matsuda Y. Nagabukuro A. Tsuji A. Nabeshima Y. Asano M. Iwakura Y. Sehara-Fujisawa A. Mol. Cell. Biol. 2003; 23: 55-61Crossref PubMed Scopus (126) Google Scholar, 24Kawaguchi N. Xu X. Tajima R. Kronqvist P. Sundberg C. Loechel F. Albrechtsen R. Wewer U.M. Am. J. Pathol. 2002; 160: 1895-1903Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 25Yagami-Hiromasa T. Sato T. Kurisaki T. Kamijo K. Nabeshima Y. Fujisawa-Sehara A. Nature. 1995; 377: 652-656Crossref PubMed Scopus (439) Google Scholar, 26Borneman A. Kuschel R. Fujisawa-Sehara A. J. Muscle Res. Cell Motil. 2000; 21: 475-480Crossref PubMed Scopus (36) Google Scholar, 27Galliano M.F. Huet C. Frygelius J. Polgren A. Wewer U.M. Engvall E. J. Biol. Chem. 2000; 275: 13933-13939Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 28Gilpin B.J. Loechel F. Mattei M.G. Engvall E. Albrechtsen R. Wewer U.M. J. Biol. Chem. 1998; 273: 157-166Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar, 29Kronqvist P. Kawaguchi N. Albrechtsen R. Xu X. Schroder H.D. Moghadaszadeh B. Nielsen F.C. Frohlich C. Engvall E. Wewer U.M. Am. J. Pathol. 2002; 161: 1535-1540Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 30Moghadaszadeh B. Albrechtsen R. Guo L.T. Zaik M. Kawaguchi N. Borup R.H. Kronqvist P. Schroder H.D. Davies K.E. Voit T. Nielsen F.C. Engvall E. Wewer U.M. Hum. Mol. Genet. 2003; 12: 2467-2479Crossref PubMed Scopus (61) Google Scholar, 33Kurisaki T. Masuda A. Osumi N. Nabeshima Y. Fujisawa-Sehara A. Mech. Dev. 1998; 73: 211-215Crossref PubMed Scopus (94) Google Scholar). However, little is known about the regulation of ADAM12 activation and translocation to the cell surface. Herein, we report that PKCϵ, a novel PKC isoform (34Ivaska J. Kermorgant S. Whelan R. Parsons M. Ng T. Parker P.J. Biochem. Soc. Trans. 2003; 31: 90-93Crossref PubMed Google Scholar), induces the translocation of ADAM12 to the cell surface in a manner that is dependent on its catalytic activity. 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 (CHOK1). 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 μg/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 μg/ml hygromycin and 1 μg/ml tetracycline, both obtained from Sigma-Aldrich (Vallensbaek, Denmark). The transfected MCF-7 cells were grown in medium (see above) containing 1 μg/ml tetracycline. In some experiments, cells were treated for 15 min with 0.1 μm phorbol 12-myristate 13-acetate (PMA; Calbiochem), 0.1 μm calphostin C (Calbiochem), or no additive. Antibodies against ADAM12 included mouse mAbs 6E6, 6C10, 8F8, and 4G2. To generate these mAbs, full-length recombinant human ADAM12 was produced in 293 cells, purified, and then used to immunize mice. Polyclonal antisera to human ADAM12 included rb122 raised against the recombinant cysteine-rich domain and rb134 raised against purified full-length recombinant ADAM12. Rabbit anti-human PKCα, -δ, and -ϵ were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The antibody recognizing MyoD (M3512) was obtained from DakoCytomation (Glostrup, Denmark), antibodies to myosin (fast skeletal muscle) were from Sigma-Aldrich, and the monoclonal antibodies to actin (mAb 1501R) and desmin (M724) were from Chemicon (Hampshire, UK) and DakoCytomation, respectively. Monoclonal antibodies to vinculin were generously provided by M. Glukhova (Institut Curie, Paris, France). Control mouse IgG, goat anti-mouse-horseradish peroxidase, goat anti-rabbit-horseradish peroxidase, swine anti-rabbit-fluorescein isothiocyanate, swine anti-rabbit-TRITC, and rabbit anti-mouse-TRITC were obtained from DakoCytomation. Goat anti-mouse conjugated to Alexa-546 was obtained from Molecular Probes (Leiden, The Netherlands). Streptavidin-phycoerythrin conjugate and mouse anti-GFP antibody (clone JL-8) were purchased from BD Biosciences (Brøndby, Denmark). The mouse monoclonal anti-c-myc antibody (clone 9E10) was purchased from Roche Molecular Biochemical. 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-Δcyt, a membrane-inserted ADAM12 protein lacking the cytoplasmic tail (35Hougaard S. Loechel F. Xu X. Tajima R. Albrechtsen R. Wewer U.M. Biochem. Biophys. Res. Commun. 2000; 275: 261-267Crossref PubMed Scopus (52) Google Scholar). RD cells were transiently transfected using cDNA constructs encoding EGFP-tagged full-length PKC isoforms α, δ, and ϵ (PKCαFL-E, PKCδFL-E, PKCϵFL-E, respectively); EGFP-tagged PKCϵ catalytic domain (PKCϵCD-E) or regulatory domain (PKCϵRD-E) (36Zeidman R. Lofgren B. Pahlman S. Larsson C. J. Cell Biol. 1999; 145: 713-726Crossref PubMed Scopus (110) Google Scholar); myc-tagged PSC1V3 subdomains (PKCϵPSC1V3-myc) (37Troller U. Raghunath A. Larsson C. Cell. Signalling. 2004; 16: 245-252Crossref PubMed Scopus (11) Google Scholar) and C2 subdomains (PKCϵC2-myc) generated by inserting the BglII/SalI fragment from PKCϵC2-EGFP (36Zeidman R. Lofgren B. Pahlman S. Larsson C. J. Cell Biol. 1999; 145: 713-726Crossref PubMed Scopus (110) Google Scholar) in BamHI/XhoI-digested pcDNA4-myc-His plasmid (Invitrogen); EGFP-tagged myristoylated full-length PKCα (Myr-PKCα-E) or PKCϵ (Myr PKCϵ-E) (38Zeidman R. Troller U. Raghunath A. Pahlman S. Larsson C. Mol. Biol. Cell. 2002; 13: 12-24Crossref PubMed Scopus (63) Google Scholar); GFP-tagged, full-length PKCϵ (GFP-PKCϵFL); or kinase-inactive PKCϵ mutants K552M (GFP-PKCϵFL-K/M) or K438R (PKCϵFLKD-E) (39Ling M. Troller U. Zeidman R. Lundberg C. Larsson C. Exp. Cell Res. 2004; 292: 135-150Crossref PubMed Scopus (32) Google Scholar). We found that both of these kinase-inactive PKCϵ mutants gave similar results, so for simplicity we used PKCϵ-KD to refer to these constructs. Myristoylated PKCϵKD-E is generated by inserting the BglII/SalI-excised fragment from PKCϵFLKD-E in the corresponding sites of our previously constructed MyrPKϵFL-E vector (38Zeidman R. Troller U. Raghunath A. Pahlman S. Larsson C. Mol. Biol. Cell. 2002; 13: 12-24Crossref PubMed Scopus (63) Google Scholar). 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 MgCl2, 1 mm Na3VO4, 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 μg 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 μl of protein-G beads alone to reduce nonspecific protein binding. The lysates were further incubated overnight with 50 μl 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 (α, δ, or ϵ), 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 MgCl2, 0.88 mm EDTA, 1 mm Na3VO4, 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 × 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 × g for 30 min. The resulting membrane-enriched pellets were dissolved in RIPA buffer and used for immunoprecipitation or immunoblotting. An equal amount of 2× sample buffer was added, and samples were analyzed by Western blotting for ADAM12 (rb 122), and PKCα, δ, and ϵ 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 μl 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 (18Iba K. Albrechtsen R. Gilpin B.J. Loechel F. Wewer U.M. Am. J. Pathol. 1999; 154: 1489-1501Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 40Iba K. Albrechtsen R. Gilpin B. Frohlich C. Loechel F. Zolkiewska A. Ishiguro K. Kojima T. Liu W. Langford J.K. Sanderson R.D. Brake-busch C. Fassler R. Wewer U.M. J. Cell Biol. 2000; 149: 1143-1156Crossref PubMed Scopus (223) Google Scholar, 41Thodeti C.K. Albrechtsen R. Grauslund M. Asmar M. Larsson C. Takada Y. Mercurio A.M. Couchman J.R. Wewer U.M. J. Biol. Chem. 2003; 278: 9576-9584Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). In brief, MaxiSorp 96-well plates (Nalge Nunc International) were coated (overnight at 4 °C) with 10 μg/ml of the IgG fraction of polyclonal anti-ADAM12 Ab (rb134) and the corresponding preimmune IgG in 0.1 m NaHCO3 buffer, pH 9.5. RD and MCF-7 cells were then treated with 0.1 μm PMA and/or 0.1 μm 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 (40Iba K. Albrechtsen R. Gilpin B. Frohlich C. Loechel F. Zolkiewska A. Ishiguro K. Kojima T. Liu W. Langford J.K. Sanderson R.D. Brake-busch C. Fassler R. Wewer U.M. J. Cell Biol. 2000; 149: 1143-1156Crossref PubMed Scopus (223) Google Scholar, 41Thodeti C.K. Albrechtsen R. Grauslund M. Asmar M. Larsson C. Takada Y. Mercurio A.M. Couchman J.R. Wewer U.M. J. Biol. Chem. 2003; 278: 9576-9584Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar) 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 IgG1 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 IgG1 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), res" @default.
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• W1963526979 date "2004-12-01" @default.
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• W1963526979 title "Regulation of ADAM12 Cell-surface Expression by Protein Kinase C ϵ" @default.
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