AHNAK-mediated Activation of Phospholipase C-γ1 through Protein Kinase C
2004; Elsevier BV; Volume: 279; Issue: 25 Linguagem: Inglês
10.1074/jbc.m311525200
ISSN1083-351X
AutoresIn Hye Lee, Je Ok You, Kwon‐Soo Ha, Duk Soo Bae, Pann‐Ghill Suh, Sue Goo Rhee, Yun Soo Bae,
Tópico(s)NF-κB Signaling Pathways
ResumoWe have recently shown that phospholipase C-γ (PLC-γ) is activated by the central repeated units (CRUs) of the AHNAK protein in the presence of arachidonic acid. Here we demonstrate that four central repeated units (4 CRUs) of AHNAK act as a scaffolding motif networking PLC-γ and PKC-α. Specifically, 4 CRUs of AHNAK bind and activate PKC-α, which in turn stimulates the release of arachidonic acid near where PLC-γ1 is localized. Moreover, 4 CRUs of AHNAK interacted with PLC-γ and the concerted action of 4 CRUs with arachidonic acid stimulated PLC-γ activity. Stimulation of NIH3T3 cells expressing 4 CRUs of AHNAK with phorbol 12-myristate 13-acetate resulted in the increased generation of total inositol phosphates (IPT) and mobilization of the intracellular calcium. Phorbol 12-myristate 13-acetate-dependent generation of IPT was completely blocked in NIH3T3 cells depleted of PLC-γ1 by RNA interference. Furthermore, bradykinin, which normally stimulated the PLC-β isozyme resulting in the generation of a monophasic IPT within 30 s in NIH3T3 cells, led to a biphasic pattern for generation of IPT in NIH3T3 cells expressing 4 CRUs of AHNAK. The secondary activation of PLC is likely because of the scaffolding activity of AHNAK, which is consistent with the role of 4 CRUs as a molecular linker between PLC-γ and PKC-α. We have recently shown that phospholipase C-γ (PLC-γ) is activated by the central repeated units (CRUs) of the AHNAK protein in the presence of arachidonic acid. Here we demonstrate that four central repeated units (4 CRUs) of AHNAK act as a scaffolding motif networking PLC-γ and PKC-α. Specifically, 4 CRUs of AHNAK bind and activate PKC-α, which in turn stimulates the release of arachidonic acid near where PLC-γ1 is localized. Moreover, 4 CRUs of AHNAK interacted with PLC-γ and the concerted action of 4 CRUs with arachidonic acid stimulated PLC-γ activity. Stimulation of NIH3T3 cells expressing 4 CRUs of AHNAK with phorbol 12-myristate 13-acetate resulted in the increased generation of total inositol phosphates (IPT) and mobilization of the intracellular calcium. Phorbol 12-myristate 13-acetate-dependent generation of IPT was completely blocked in NIH3T3 cells depleted of PLC-γ1 by RNA interference. Furthermore, bradykinin, which normally stimulated the PLC-β isozyme resulting in the generation of a monophasic IPT within 30 s in NIH3T3 cells, led to a biphasic pattern for generation of IPT in NIH3T3 cells expressing 4 CRUs of AHNAK. The secondary activation of PLC is likely because of the scaffolding activity of AHNAK, which is consistent with the role of 4 CRUs as a molecular linker between PLC-γ and PKC-α. AHNAK, a nuclear phosphoprotein with the estimated molecular mass of 700 Da, was originally identified in human neuroblastomas and skin epithelial cells (1Shtivenman E. Cohen F.E. Bishop J.M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5472-5476Crossref PubMed Scopus (107) Google Scholar, 2Shtivenman E. Bishop J.M. J. Cell Biol. 1993; 120: 625-630, 5472, 5476Crossref PubMed Scopus (70) Google Scholar, 3Hashimoto T. Gamou S. Shimizu N. Kitajima Y. Nishkawa T. Exp. Cell Res. 1995; 217: 258-266Crossref PubMed Scopus (58) Google Scholar). AHNAK contains three distinct structural regions: the NH2-terminal 251-amino acid region, a large central region of about 4300 amino acids with 36 repeated units, and the COOH-terminal 1002 amino acids region. The carboxyl-terminal region of AHNAK proteins was reported to play an important role in cellular localization and in interaction with L-type Ca2+ channels in cardiac cells (4Hohaus A. Person V. Behlke J. Schaper J. Morano I. Hasse H. FASEB J. 2002; 16: 1205-1216Crossref PubMed Scopus (109) Google Scholar) and with the calcium-binding S100B protein in rat embryo fibroblast cells (5Gentil B.J. Delphin C. Mbele G.O. Deloulme J.C. Ferro M. Garin J. Baudier J. J. Biol. Chem. 2001; 276: 23253-23261Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). In low calcium concentrations, AHNAK proteins are mainly localized in the nucleus, but the increase in intracellular calcium levels leads the protein to translocate to plasma membrane (3Hashimoto T. Gamou S. Shimizu N. Kitajima Y. Nishkawa T. Exp. Cell Res. 1995; 217: 258-266Crossref PubMed Scopus (58) Google Scholar). Phosphorylation of serine 5535 in the carboxyl-terminal AHNAK protein by nuclear PKB 1The abbreviations used are: PKB, protein kinase B; PLC, phospholipse C; PIP2, phosphatidylinositol 4,5-bisphosphate; IP3, inositol (1,4,5)-trisphosphate; DAG, 1,2-diacylglycerol; PTK, protein-tyrosine kinase; AA, arachidonic acid; MAPK, mitogen-activated protein kinase; PLA2, phospholipase A2; cPLA2, cytosolic PLA2; PMA, phorbol 12-myristate 13-acetate; GFP, green fluorescent protein; PDGF, platelet-derived growth factor; HA, hemagglutinin; GST, glutathione S-transferase; TRITC, tetramethylrhodamine isothiocyanate; siRNA, small interfering RNA; CRU, central repeated unit; BK, bradykinin; DMEM, Dulbecco's modified Eagle's medium; IPT, total inositol phosphate; PBS, phosphate-buffered saline; [Ca2+]i, intracellular Ca2+; SH3, Src homology domain 3. was shown to be essential for its export from the nucleus (6Sussman J. Stokoe D. Ossina N. Shitivenman E. J. Cell Biol. 2001; 154: 1019-1030Crossref PubMed Scopus (70) Google Scholar). The central repeated unit in AHNAK is 128 amino acids in length and displays a heptasequence motif, (D/E)φΩφK(A/G)P, where φ and Ω represent hydrophobic and hydrophilic amino acid residues, respectively. Shtivenman et al. (1Shtivenman E. Cohen F.E. Bishop J.M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5472-5476Crossref PubMed Scopus (107) Google Scholar, 2Shtivenman E. Bishop J.M. J. Cell Biol. 1993; 120: 625-630, 5472, 5476Crossref PubMed Scopus (70) Google Scholar, 6Sussman J. Stokoe D. Ossina N. Shitivenman E. J. Cell Biol. 2001; 154: 1019-1030Crossref PubMed Scopus (70) Google Scholar) suggested that this sequence exists as a β-strand and polyionic rod with hydrophobic amino acids facing inward and hydrophilic amino acids facing outward. It is suggested that the central repeats likely support the structural integrity of AHNAK (1Shtivenman E. Cohen F.E. Bishop J.M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5472-5476Crossref PubMed Scopus (107) Google Scholar, 2Shtivenman E. Bishop J.M. J. Cell Biol. 1993; 120: 625-630, 5472, 5476Crossref PubMed Scopus (70) Google Scholar, 6Sussman J. Stokoe D. Ossina N. Shitivenman E. J. Cell Biol. 2001; 154: 1019-1030Crossref PubMed Scopus (70) Google Scholar). Activation of phosphoinositide-specific phospholipase C (PLC) is a key event in cellular signal transduction involved in cell growth, proliferation, metabolism, and secretion (7Rhee S.G. Annu. Rev. Biochem. 2001; 70: 281-312Crossref PubMed Scopus (1227) Google Scholar). PLC catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to produce inositol (1,4,5)-trisphosphate (IP3) and 1,2-diacylglycerol (DAG). To date, a total of 11-different isozymes of PLC have been identified in mammalian cells, and these can be classified into four subfamilies, β (β1-β4), γ (γ1 and γ2), δ (δ1-δ4), and ϵ isozymes. Based on their primary structures, this classification has been correlated with their different activation mechanisms (7Rhee S.G. Annu. Rev. Biochem. 2001; 70: 281-312Crossref PubMed Scopus (1227) Google Scholar). Although protein-tyrosine kinase (PTK)-mediated PLC-γ isozyme activation is well established, lipid-derived second messengers such as phosphatidic acid, phosphatidylinositol (3,4,5)-triphosphate, and arachidonic acid (AA) were also proposed to activate the isozyme (7Rhee S.G. Annu. Rev. Biochem. 2001; 70: 281-312Crossref PubMed Scopus (1227) Google Scholar). Furthermore, concerted action of arachidonic acid with tau or with repeated units of AHNAK was also shown to stimulate the activation of PLC-γ isozymes (8Hwang S.C. Jhon D.Y. Bae Y.S. Kim J.H. Rhee S.G. J. Biol. Chem. 1996; 271: 18342-18349Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 9Sekiya F. Bae Y.S. Jhon D.Y. Hwang S.C. Rhee S.G. J. Biol. Chem. 1999; 274: 13900-13907Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). The 11 PKC isozymes thus far identified display diverse tissue expression, subcellular localization, cofactor requirements, and functional diversity (10Kikkawa U. Kishimoto A. Nishizuka Y. Annu. Rev. Biochem. 1989; 58: 31-44Crossref PubMed Scopus (588) Google Scholar, 11Mellor H. Parker P.J. Biochem. J. 1998; 332: 281-312Crossref PubMed Scopus (1361) Google Scholar, 12Mochly-Rosen D. Gordon A.S. FASEB J. 1998; 12: 35-42Crossref PubMed Scopus (510) Google Scholar, 13Jaken S. Parker P.J. BioEssays. 2000; 22: 245-254Crossref PubMed Scopus (234) Google Scholar). The PKC isozymes can be classified into three groups according to their regulatory properties, which are in turn governed by the presence of specific domains in the proteins. The conventional PKCs include PKCα, βI, βII, and γ, and these isoforms can be activated by Ca2+ and/or by DAG and phorbol esters. The novel PKCs, δ, ϵ, θ, and η, can also be activated by DAG and phorbol esters but are Ca2+-independent. The atypical PKCs, which include PKCξ and PKCι, are unresponsive to Ca2+ and DAG/phorbol esters. It has been established that PKC isozymes activate the Raf-MAPK cascade and NF-κB as downstream molecules in cell signaling (14Moscat J. Diaz-Meco M.T. Rennert P. EMBO Rep. 2003; 4: 31-36Crossref PubMed Scopus (103) Google Scholar). Phospholipase A2 (PLA2) enzymes hydrolyze fatty acid from the sn-2 position of phospholipid with the concomitant production of lysophospholipid. Mammalian cells contain structurally diverse forms of PLA2 including secretory PLA2, calcium-independent PLA2, and the 85-kDa cytosolic PLA2 (cPLA2). It has been reported that AA is produced in response to diverse stimuli including interleukin-1, tumor necrosis factor, epidermal growth factor, okadaic acid, the phagocytic particle zymosan, and phorbol 12-myristate 13-acetate (PMA) (15Kramer R.M. Sharp J.D. FEBS Lett. 1997; 410: 49-53Crossref PubMed Scopus (235) Google Scholar, 16Gijón M.A. Leslie C.C. J. Leukocyte Biol. 1999; 65: 330-336Crossref PubMed Scopus (253) Google Scholar, 17Gijón M.A. Spencer D.M. Siddiqu A.R. Bonventre J.V. Leslie C.C. J. Biol. Chem. 2000; 275: 20146-20156Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). These reports indicate that AA, a product of PLA2, plays the role not only of an important initiator of inflammatory processes but also of a regulator of signaling process (16Gijón M.A. Leslie C.C. J. Leukocyte Biol. 1999; 65: 330-336Crossref PubMed Scopus (253) Google Scholar). Although 4 central repeated units (CRUs) of the AHNAK protein bind and activate PLC-γ in vitro, the cellular function of the CRUs in AHNAK is not clear. Our results suggest that the 4 CRUs of AHNAK concomitantly interact with PKC-α and PLC-γ in response to PMA. It is likely that PKC-α in a ternary complex translocates to the membrane and then induces the release of AA through cPLA2 activation. Once released, AA likely activates PLC-γ through a concerted action with AHNAK. Taken together, these results indicate that 4 CRUs of AHANK act as a scaffolding protein networking for PLC-γ and PKC-α. Materials—[5,6,8,9,11,12,14,15-3H]Arachidonic acid (189 Ci/mmol) and myo-[2-3H]inositol were purchased from PerkinElmer Life Sciences. GF109203X, PMA, bradykinin (BK), and AG1478 were purchased from Calbiochem. Fluo-4/AM was obtained from Molecular Probes. MACSelect KK MicroBeads were obtained from Miltenyi Biotec, and SuperFect was purchased from Qiagen. The anti-HA, anti-PKC-β3, and anti-PKC-α monoclonal antibodies were purchased from Roche Diagnostics, Santa Cruz Biotechnology, and Upstate Biotechnology, respectively. Cell Cultures—NIH3T3 cells were cultured at 37 °C in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% calf serum. COS-7 and TV1 null cells were cultured in DMEM supplemented with 10% fetal bovine serum. Plasmids—Human AHNAK cDNA was obtained by screening the human male BAC library. The division of AHNAK into N (amino acid residues 1-257), C1 (amino acid residues 4640-5386), and C2 (amino acid residues 5258-5643) domains was based on a previously published report (4Hohaus A. Person V. Behlke J. Schaper J. Morano I. Hasse H. FASEB J. 2002; 16: 1205-1216Crossref PubMed Scopus (109) Google Scholar). The cDNA fragments were amplified by PCR and inserted into the pcDNA3-HA vector by digestion with EcoRI and XhoI. The gene for 4 CRUs of AHNAK (amino acid residues 3860-4412) was subcloned into pcDNA3-HA by digestion with EcoRI and XhoI. The gene for 4 CRUs of AHNAK was also obtained by PCR and inserted into pEGFP-N1 and pDsRed-C1 (Clontech) by digestion with XhoI and HindIII and HindIII and BamHI, respectively. All constructs were checked by restriction site mapping and sequencing. Transfection—NIH3T3 cells were plated at a density of 1.5 × 105 cells/well in six-well plates. The cells were transfected with 4 μg of pcDNA3, pcDNA3-HA-AHNAK-N, pcDNA3-HA-4CRUs, pcDNA3-HA-C1, pcDNA3-HA-C2, pDsRed-C1, or pDsRed-C1-4CRUs using SuperFect reagent according to the manufacturer's protocol and maintained in the completed medium for 24 h. The cells were serum-starved for 12 h and then stimulated with various agonists for the indicated times. Release of Arachidonic Acid—NIH3T3 cells (2.5 × 105) were plated at 6-well plates. The cells were cultured for 24 h and labeled by incubation for 16 h in 1 ml of serum-free medium containing [3H]arachidonic acid (0.5 μCi/ml) and 0.1% fatty acid-free bovine serum albumin. The cells were then washed twice with DMEM containing 0.1% bovine serum albumin and incubated with 100 nm PMA in the absence or presence of 10 μm A23187 for 10 min. Radioactivity in the supernatant fractions and the cell lysates containing 1% Triton X-100 were measured by liquid scintillation counting. The percentage of arachidonic acid release was calculated as the (medium cpm/(cells + medium) cpm) × 100 and then normalized to the value of unstimulated controls (18Lin L.L. Wartmann M. Lin A.Y. Kinopf J.L. Seth A. Davis R.J. Cell. 1993; 72: 269-278Abstract Full Text PDF PubMed Scopus (1659) Google Scholar, 19Panini S.R. Yang L. Rusinol A.E. Sinensky M.S. Bonventre J.V. Leslie C.C. J. Lipid Res. 2001; 42: 1678-1686Abstract Full Text Full Text PDF PubMed Google Scholar). Measurement of Total Inositol Phosphate (IPT) in Cells—NIH3T3 cells were labeled with inositol-free DMEM supplemented with myo-[2-3H]inositol (1 μCi/ml, 25 mCi/mmol) (DuPont Biotechnology Systems) for 14 h and then washed with inositol-free DMEM (20Bae Y.S. Cantley L.G. Chen C.S. Kim S.R. Kwon K.S. Rhee S.G. J. Biol. Chem. 1998; 273: 4465-4469Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). The cells were subsequently incubated in DMEM containing 20 mm LiCl for 30 min and stimulated with 100 nm PMA for 10 min or stimulated with 1 μm bradykinin for the indicated time. To inhibit PKC activity, the cells were treated with 5 μm GF109203X for 10 min followed by 100 nm PMA for 10 min. To inhibit Src and epidermal growth factor receptor activity, the cells were pretreated with 10 μm PP-1, 1 μm AG1478 for 30 min and then stimulated with 1 μm bradykinin for the indicated time periods. The incubation was terminated by adding perchloric acid to a final concentration of 5% (w/v). The cells were scraped into Eppendorf tubes and centrifuged at 15,000 × g for 20 min at 4 °C. The supernatant was equilibrated with 2 m KOH, 1 mm EDTA and applied to a SAX column connected to a high performance liquid chromatograph (Hewlett Packard series 1100). Bound inositol phosphates were eluted by applying a linear gradient (0-1.0 m ammonium phosphate) at a flow rate of 2 ml/min. Radioactivity in the resulting fraction, corresponding to liberated [3H]IPT, was measured using a liquid scintillation counter. Magnetic Enrichment of NIH3T3 Cells Expressing AHNAK—NIH3T3 cells were co-transfected pMACS KK with pcDNA3-HA or pcDNA3-HA-4CRUs using FuGENE 6 (Roche Diagnostics) as described in the manufacturer's protocol. After 24 h incubation, the cells were washed with phosphate-buffered saline (PBS) without EDTA. The cells were again incubated with 500 μl of trypsin solution per 100-mm dish until dissociated from culture dish and from each other. Trypsinization was stopped by adding 100 μl of 100% fetal bovine serum. The cells were then incubated with 80 μl of MACSelect KK MicroBeads per 100-mm dish for 15 min at room temperature and PBS containing 2 mm EDTA and 0.5% fetal bovine serum was added to a final volume of 2 ml. The cells expressing MACS KK protein were separated from magnetic columns, plated on 6-well plates, and allowed to recover for 24 h. The cells were subsequently incubated for 18 h in inositol-free DMEM supplemented with myo-[3H]inositol (1.5 μCi/ml, 25 mCi/mmol) (DuPont), and processed as described above. Measurement of Intracellular Calcium ([Ca2+]i)—[Ca2+]i was measured using a laser scanning confocal microscope (21Lee Z.W. Kweon S.M. Kim B.C. Leem S.H. Shin I.C. Kim J.H. Ha K.S. J. Biol. Chem. 1998; 273: 12710-12715Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). NIH3T3 cells were grown on coverslips and transfected with pDsRed-C1-4CRUs. Transfected or nontransfected NIH3T3 cells were serum-starved for 12 h, incubated with 2 μm Fluo-4/AM in serum-free medium for 40 min, and washed three times with Ca2+-free Locke's solution (158.4 mm NaCl, 5.6 mm KCl, 1.2 mm MgCl2, 5 mm HEPES buffer adjusted to pH 7.3, 10 mm glucose, and 0.2 mm EGTA). The coverslips containing the stained cells were mounted on a perfusion chamber (21Lee Z.W. Kweon S.M. Kim B.C. Leem S.H. Shin I.C. Kim J.H. Ha K.S. J. Biol. Chem. 1998; 273: 12710-12715Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar) and subjected to confocal laser scanning microscopic analysis (Olympus LV300). Prior to observing the release of real calcium in response to PMA, NIH3T3 cells expressing red fluorescence protein-tagged AHNAK were pre-selected through scanning with 543- and 560-nm emission filters and scanned every second with a 488-nm excitation argon laser and a 515-nm long pass emission filter. A solution containing 100 nm PMA was then added to the cells with an automatic pumping system (21Lee Z.W. Kweon S.M. Kim B.C. Leem S.H. Shin I.C. Kim J.H. Ha K.S. J. Biol. Chem. 1998; 273: 12710-12715Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). About 150 images resulting from the scanning were analyzed for changes of [Ca2+]i at a single cell level. The results were expressed as relative fluorescence intensity. Construction of Small Interfering RNA (SiRNA) for PLC-γ1—Specific sequences of 19-nucleotide sequence (ctactactctgaggagacc, residues 1695 to 1713) of the human PLC-γ1 cDNA were selected for synthesis of a siRNA. pSUPER vector for siRNA was purchased from Oligoengine (22Brummelkamp T.R. Bernards R. Agami R. Science. 2002; 296: 550-553Crossref PubMed Scopus (3971) Google Scholar). The phosphorylated oligonucleotides were annealed and cloned into the pSUPER vector with BglII (5′ end) and HindIII (3′ end). Cells were transfected with the resulting construct and cultured for 48 h in complete medium. The transfected cells were deprived of serum for 12 h, incubated for 10 min in the absence or presence of PMA or for the indicated times in the absence or presence of bradykinin, and then analyzed by measurement of total inositol phosphates. The depletion of endogenous PLC-γ1 by the siRNA was confirmed by immunoblot analysis. Immunofluorescence—COS-7 cells were grown on coverslips and transfected with pEGFP-N1-4CRUs. The cells were serum-starved for 12 h, stimulated with 100 nm PMA for 10 min, washed with cold PBS, fixed with 3.5% paraformaldehyde in PBS for 10 min at room temperature, and permeabilized in 0.5% Triton X-100. Nonspecific sites were blocked by treating the cells with PBS containing 0.05% gelatin and 0.5% bovine serum albumin for 1 h. The cells were incubated with primary antibodies against PKC-α or PLC-γ in PBS for 1 h at room temperature, washed with PBS, cells were incubated with the secondary antibody (TRITC-conjugated goat anti-mouse-IgG), and then mounted on glass slides using a drop of Aqua-Poly/mount. Images were recorded using a confocal laser scanning microscope (Carl Zeiss 510). GST Fusion Protein Binding Assays—Cultures of Escherichia coli BL21 containing pGEX4T1 and pGEX4T1-4CRUs were induced with 0.4 mm isopropyl-β-d-thiogalactopyranoside for 3 h at 30 °C. The harvested bacteria were suspended in PBS containing 1% Triton X-100 and protease inhibitors (0.1 μm 4-(2-aminoethyl)benzenesulfonyl fluoride, 1 μg/ml aprotinin, and 1 μg/ml leupeptin) and lysed by sonication. After centrifugation at 15,000 × g for 20 min, the supernatant was incubated with glutathione-agarose beads for 3 h at 4 °C. The samples were washed three times with PBS containing 1% Triton X-100 and subjected to immunoblot analyses. Immunoprecipitation and Immunoblotting—Lysates (1-2 × 106 cells) were mixed with antibodies (0.5-1 μg) for 4 h, followed by addition of 40 μl of protein G-Sepharose for 2 h at 4 °C. Immune complexes were washed five times with lysis buffer (50 mm Tris, pH 7.4, 1% Triton X-100, 0.5% Nonidet P-40, 150 mm NaCl, 0.1 μm 4-(2-aminoethyl)benzenesulfonyl fluoride, 1 mm Na3VO4, 1 mm NaF, 1 μg/ml aprotinin, 1 μg/ml leupeptin, and 10% glycerol). After boiling 2 times in sample buffer, samples were subjected to SDS-PAGE and electrotransferred to nitrocellulose membranes. Membranes were immunoblotted with the indicated primary antibodies, followed by horseradish peroxidase-conjugated goat secondary antibodies. Bands were visualized by chemiluminescence. PMA Stimulates PLC-γ Activation in NIH3T3 Cells Expressing Four Central Repeated Units of AHNAK—In a previous report (9Sekiya F. Bae Y.S. Jhon D.Y. Hwang S.C. Rhee S.G. J. Biol. Chem. 1999; 274: 13900-13907Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar), we have shown that AHNAK binds and activates PLC-γ in the presence of AA. To verify the cellular function of the AHNAK protein as an activator of PLC-γ, we first identified an agonist that can generate AA in cells. It is known that PMA, a known PKC activator, can activate cPLA2 and then release AA in various cell types (23Qiu Z.-H. Gijón M.A. de Carvalho M.S. Spencer D.M. Leslie C.C. J. Biol. Chem. 1998; 273: 8203-8211Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). NIH3T3 cells were labeled with [3H]AA, and the cPLA2 activity was monitored by measuring the release of [3H]AA to the supernatant in the absence or presence of PMA stimulation. Stimulation of the cell with PMA resulted in the maximal release of [3H]AA within 30 min followed by a gradual decrease to basal level (Fig. 1). To determine whether AHNAK can activate PLC-γ in response to PMA, we transiently expressed the hemagglutinin (HA)-tagged AHNAK (HA-AHNAK) derivative in NIH3T3 cells and measured the production of IPT as an indicator of PLC activity. The large size of AHNAK makes it virtually impossible to express the full-length of the protein, AHNAK was divided into three domains based on sequence analysis and functional studies previously reported (4Hohaus A. Person V. Behlke J. Schaper J. Morano I. Hasse H. FASEB J. 2002; 16: 1205-1216Crossref PubMed Scopus (109) Google Scholar, 9Sekiya F. Bae Y.S. Jhon D.Y. Hwang S.C. Rhee S.G. J. Biol. Chem. 1999; 274: 13900-13907Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar); HA-N (amino acids 1-257), HA-4CRUs (amino acids 3859-4412), HA-C1 (amino acids 4640-5386), and HA-C2 (amino acids 5258-5643) (Fig. 2A). We found that PMA stimulated IPT production in cells expressing HA-4CRUs by 150% but not in those expressing other regions of AHNAK (Fig. 2B). Moreover, inhibition of PKC activity by pretreatment of the cells with GF109203X abrogated production of IPT in cells expressing HA-4CRUs. These results suggest that PKC might be involved in the AHNAK-dependent activation of PLC. The rather modest level increase in IPT generation by PMA in NIH3T3 cells is mostly likely because HA-4CRUs transfection efficiency is low. To confirm the effect of AHNAK on PLC activation, we transfected NIH3T3 cells with HA-4CRUs and pMACS KK, which encodes truncated mouse H-2Kk protein, thus allowing enrichment of cells expressing HA-4CRUs using the MACSelect KK magnetic bead. Incubation of the selected cells expressing the HA-4CRU protein with PMA resulted in an increase of IPT generation by 7-fold (Fig. 2D). We next examined whether PMA can trigger intracellular Ca2+ mobilization ([Ca2+]i) in NIH3T3 cells expressing 4 CRUs of AHNAK. NIH3T3 cells were transfected with RFP-4CRU and the intracellular Ca2+ concentration was measured by laser-based confocal microscopy with Fluo-4 dye. PMA failed to mobilize intracellular calcium in untransfected cells, whereas stimulation of NIH3T3 cells expressing RFP-4CRU with PMA resulted in a rapid and transient increase of [Ca2+]i (Fig. 2E). These results suggest that AHNAK is involved in production of IPT and mobilizes intracellular Ca2+ in response to PMA. To prove the selectivity for PLC-γ1 in AHNAK-mediated IPT generation, we subjected NIH3T3 cells to transient transfection with pSUPER-PLC-γ1 encoding a siRNA specific for the PLC-γ1 gene (22Brummelkamp T.R. Bernards R. Agami R. Science. 2002; 296: 550-553Crossref PubMed Scopus (3971) Google Scholar). The cells transfected with the siRNA vector exhibited a marked reduction in the abundance of the endogenous PLC-γ1 protein compared with cells transfected with the empty pSUPER vector (Fig. 3A). NIH3T3 cells transfected with the pSUPER-PLC-γ1 vector failed to generate IPT in response to PMA, whereas the cells transfected with pSUPER alone exhibited a marked increase of IPT generation in response to PMA stimulation, suggesting that generation of IPT results from AHNAK-mediated activation of PLC-γ1 (Fig. 3A). The central role of PLC-γ1 in AHNAK-mediated IPT generation was further demonstrated in PLC-γ1-null fibroblasts (TV1-null) (24Ji Q.-S. Winnier G.E. Niswender K.D. Horstman D. Wisdom R. Magnuson M.A. Carpenter G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2999-3003Crossref PubMed Scopus (220) Google Scholar). Expression of 4 CRUs of AHNAK in PLC-γ1-null fibroblasts failed in IPT generation in response to PMA, whereas add-back expression of PLC-γ1 in the fibroblast cells expressing 4 CRUs of AHNAK restored PMA-dependent IPT generation (Fig. 3B). These results demonstrate that the PLC-γ1 isozyme plays the central role in PMA-induced IPT generation in the presence of AHNAK. Central Repeated Units of the AHNAK Protein Interact with PLC-γ in Response to PMA—We previously demonstrated a direct interaction between AHNAK and PLC-γ1 in an AA-dependent manner in vitro but did not establish whether PMA can induce 4 CRUs of the AHNAK-PLC-γ1 complex formation in cells. To address this question, we performed co-immunoprecipitation experiments with monoclonal antibodies against HA. Cells expressing the HA-4CRUs protein were either unstimulated or stimulated with PMA for 10 min and then lysed in a buffer containing nonionic detergent. The lysates were immunoprecipitated with an anti-HA monoclonal antibody and examined by immunoblotting with monoclonal antibodies against PLC-γ1, PLC-β1, PLC-β3, and PLC-δ1, respectively. PLC-β3 is the predominantly expressed PLC-β isozyme in NIH3T3 cells. The 4 CRUs of the AHNAK protein interacted with PLC-γ in a PMA-dependent manner, whereas other isozymes failed to interact with AHNAK (Fig. 4A). To investigate whether platelet-derived growth factor (PDGF) stimulates the interaction of 4 CRUs of AHNAK with PLC-γ1, NIH3T3 cells expressing the HA-4CRU protein were either unstimulated or stimulated with PDGF for 10 min and then lysed in a buffer containing nonionic detergent. The lysates were immunoprecipitated with antibodies against HA and examined by immunoblotting with monoclonal antibodies against PLC-γ1. The result indicates that 4 CRUs of the AHNAK protein failed to interact with PLC-γ1 in response to PDGF suggesting that AHNAK mediates PTK-independent PLC-γ activation (Fig. 4B). Moreover, we have observed colocalization of PLC-γ1 with AHNAK in response to PMA. Endogenous PLC-γ and GFP-4CRU were located in the cytoplasm and perinuclear regions in resting cells, whereas GFP-4CRU was co-localized with PLC-γ in plasma membrane (arrow) and the perinuclear region in response to PMA stimulation (Fig. 4C). Asterisks indicate NIH3T3 cells untransfected with 4 CRUs of AHNAK. Plasma membrane localization of PLC-γ in response to PMA was not observed in untransfected NIH3T3 cells (indicated by asterisks). Results from immunoprecipitation experiments and confocal microscopy indicate that PMA induces formation of the AHNAK-PLCγ complex in the plasma membrane. Central Repeated Units of AHNAK Protein Interact with PKC Isozyme—Because NIH3T3 cells appear to mainly express the PKC-α isozyme (25Goodnight J. Mischak H. Kolch W. Mushinaski J.F. J. Biol. Chem. 1995; 270: 9991-10001Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar), we employed co-immunoprecipitation experiments and confocal microscopy experiments to investigate molecular interaction between 4 CRUs of AHNAK and PKC-α. We have observed that PKC-α interacts with 4 CRUs of AHNAK in response to PMA stimulation in a GST pull-down assay and co-immunoprecipitation experiments (Fig. 5, A and B). To examine co-localization of 4 CRUs of AHNAK with PKC-α in cells, we performed confocal microscopy with cells expressing GFP-4CRUs. PKC-α was stained with antibody against PKC-α. GFP-4CRUs and PKC-α were dispersed in the cytoplasm in the absence of PMA, whereas GFP-4CRU (arrow) was co-localized with endogenous PKC-α (arrow) in plasma membrane upon PMA stimulation (Fig. 5C). S
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