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• W2012810520 abstract "We examined whether protein kinase D (PKD) overexpression in Swiss 3T3 cells potentiates the proliferative response to either the G protein-coupled receptor agonists bombesin and vasopressin or the biologically active phorbol ester phorbol 12,13-dibutyrate (PDBu). In order to generate Swiss 3T3 cells stably overexpressing PKD, cultures of these cells were infected with retrovirus encoding murine PKD and green fluorescent protein (GFP) expressed as two separate proteins translated from the same mRNA. GFP was used as a marker for selection of PKD-positive cells. PKD overexpressed in Swiss 3T3 cells was dramatically activated by cell treatment with bombesin or PDBu as judged by in vitrokinase autophosphorylation assays and exogenous substrate phosphorylation. Concomitantly, these stimuli induced PKD phosphorylation at Ser744, Ser748, and Ser916. PKD activation and phosphorylation were prevented by exposure of the cells to protein kinase C-specific inhibitors. Addition of bombesin, vasopressin, or PDBu to cultures of Swiss 3T3 cells overexpressing PKD induced a striking increase in DNA synthesis and cell number compared with cultures of Swiss 3T3-GFP cells. In contrast, stimulation of DNA synthesis in response to epidermal growth factor, which acts via protein kinase C/PKD-independent pathways, was not enhanced. Our results demonstrate that overexpression of PKD selectively potentiates mitogenesis induced by bombesin, vasopressin, or PDBu in Swiss 3T3 cells. We examined whether protein kinase D (PKD) overexpression in Swiss 3T3 cells potentiates the proliferative response to either the G protein-coupled receptor agonists bombesin and vasopressin or the biologically active phorbol ester phorbol 12,13-dibutyrate (PDBu). In order to generate Swiss 3T3 cells stably overexpressing PKD, cultures of these cells were infected with retrovirus encoding murine PKD and green fluorescent protein (GFP) expressed as two separate proteins translated from the same mRNA. GFP was used as a marker for selection of PKD-positive cells. PKD overexpressed in Swiss 3T3 cells was dramatically activated by cell treatment with bombesin or PDBu as judged by in vitrokinase autophosphorylation assays and exogenous substrate phosphorylation. Concomitantly, these stimuli induced PKD phosphorylation at Ser744, Ser748, and Ser916. PKD activation and phosphorylation were prevented by exposure of the cells to protein kinase C-specific inhibitors. Addition of bombesin, vasopressin, or PDBu to cultures of Swiss 3T3 cells overexpressing PKD induced a striking increase in DNA synthesis and cell number compared with cultures of Swiss 3T3-GFP cells. In contrast, stimulation of DNA synthesis in response to epidermal growth factor, which acts via protein kinase C/PKD-independent pathways, was not enhanced. Our results demonstrate that overexpression of PKD selectively potentiates mitogenesis induced by bombesin, vasopressin, or PDBu in Swiss 3T3 cells. G protein-coupled receptor diacylglycerol Dulbecco's modified Eagle's medium epidermal growth factor green fluorescent protein internal ribosome entry site polyacrylamide gel electrophoresis phosphate-buffered saline phorbol 12,13-dibutyrate protein kinase C protein kinase D fluorescence-activated cell sorter fetal bovine serum murine stem cell virus Neuropeptides stimulate DNA synthesis and cell proliferation in cultured cells and are implicated as growth factors in a variety of fundamental processes including development, inflammation, tissue regeneration, and tumorigenesis (1Rozengurt E. Science. 1986; 234: 161-166Crossref PubMed Scopus (852) Google Scholar, 2Rozengurt E. J. Cell. Physiol. 1998; 177: 507-517Crossref PubMed Scopus (154) Google Scholar, 3Rozengurt E. Curr. Opin. Oncol. 1999; 11: 116-122Crossref PubMed Scopus (66) Google Scholar). In particular, the potent mitogens of the bombesin family (4Rozengurt E. Sinnett-Smith J. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 2936-2940Crossref PubMed Scopus (371) Google Scholar, 5Zachary I. Rozengurt E. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 7616-7620Crossref PubMed Scopus (159) Google Scholar) bind to a G protein-coupled receptor (GPCR)1 (6Battey J.F. Way J.M. Corjay M.H. Shapira H. Kusano K. Harkins R. Wu W.J.M. Slattery T. Mann E. Feldman R.I. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 395-399Crossref PubMed Scopus (286) Google Scholar, 7Zachary I. Rozengurt E. J. Biol. Chem. 1987; 262: 3947-3950Abstract Full Text PDF PubMed Google Scholar) that promotes Gαq-mediated activation of β-isoforms of phospholipase C (8Offermanns S. Heiler E. Spicher K. Schultz G. FEBS Lett. 1994; 349: 201-204Crossref PubMed Scopus (64) Google Scholar, 9Hellmich M.R. Battey J.F. Northup J.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 751-756Crossref PubMed Scopus (62) Google Scholar) to produce 2 second messengers as follows: inositol 1,4,5-trisphosphate that mobilizes Ca2+ from internal stores and diacylglycerol that activates PKC (1Rozengurt E. Science. 1986; 234: 161-166Crossref PubMed Scopus (852) Google Scholar, 10Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6188) Google Scholar, 11Nishizuka Y. FASEB J. 1995; 9: 484-496Crossref PubMed Scopus (2368) Google Scholar, 12Exton J.H. Annu. Rev. Pharmacol. Toxicol. 1996; 36: 481-509Crossref PubMed Scopus (300) Google Scholar). There are multiple related PKC isoforms, which can be classified into three distinct subgroups on the basis of structural and regulatory differences as follows: the conventional PKCs (α, βI, βII, and γ), which are stimulated by calcium, diacylglycerol (DAG), and phospholipids; the novel PKCs (δ, ε, η, and θ), which are activated by DAG and phospholipids; and the atypical PKCs (ζ and λ), whose regulation is less characterized but that have been proposed to be regulated by D-3 phosphoinositides (13Dekker L.V. Parker P.J. Trends Biochem. Sci. 1994; 19: 73-77Abstract Full Text PDF PubMed Scopus (920) Google Scholar, 14Newton A.C. J. Biol. Chem. 1995; 270: 28495-28498Abstract Full Text Full Text PDF PubMed Scopus (1472) Google Scholar, 15Mellor H. Parker P.J. Biochem. J. 1998; 332: 281-292Crossref PubMed Scopus (1361) Google Scholar). The DAG-regulated PKC isoforms all bind phorbol esters and are major cellular targets for this class of tumor promoter (16Hurley J.H. Newton A.C. Parker P.J. Blumberg P.M. Nishizuka Y. Protein Sci. 1997; 6: 477-480Crossref PubMed Scopus (321) Google Scholar). PKCs, which have been implicated in the regulation of a wide range of biological responses including cell proliferation and carcinogenesis (17Livneh E. Fishman D.D. Eur. J. Biochem. 1997; 248: 1-9Crossref PubMed Scopus (208) Google Scholar, 18Toker A. Front. Biosci. 1998; 3: D1134-D1147Crossref PubMed Google Scholar), play a pivotal role in neuropeptide-mediated mitogenesis (2Rozengurt E. J. Cell. Physiol. 1998; 177: 507-517Crossref PubMed Scopus (154) Google Scholar,19Rozengurt E. Eur. J. Clin. Invest. 1991; 21: 123-134Crossref PubMed Scopus (114) Google Scholar). Despite the recognized importance of PKCs in mitogenic signal transduction, the downstream targets that mediate PKC-induced cell proliferation remain largely undefined. Protein kinase D (PKD)/protein kinase Cµ is a serine/threonine protein kinase with structural, enzymological, and regulatory properties different from the PKC family members (20Valverde A.M. Sinnett-Smith J. Van Lint J. Rozengurt E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8572-8576Crossref PubMed Scopus (361) Google Scholar, 21Johannes F.J. Prestle J. Eis S. Oberhagemann P. Pfizenmaier K. J. Biol. Chem. 1994; 269: 6140-6148Abstract Full Text PDF PubMed Google Scholar). The most distinct characteristics of PKD are the presence of a catalytic domain distantly related to Ca2+-regulated kinases, a pleckstrin homology region that regulates enzyme activity, and a highly hydrophobic stretch of amino acids in its N-terminal region (22Rozengurt E. Sinnett-Smith J. Zugaza J.L. Biochem. Soc. Trans. 1997; 25: 565-571Crossref PubMed Scopus (60) Google Scholar, 23Iglesias T. Rozengurt E. J. Biol. Chem. 1998; 273: 410-416Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 24Waldron R.T. Iglesias T. Rozengurt E. J. Biol. Chem. 1999; 274: 9224-9230Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). This N-terminal region also contains a tandem repeat of cysteine-rich, zinc finger-like motifs (CRD), which confer high affinity binding of phorbol esters, and plays a negative role in the regulation of catalytic kinase activity of PKD (25Matthews S. Iglesias T. Cantrell D. Rozengurt E. FEBS Lett. 1999; 457: 515-521Crossref PubMed Scopus (70) Google Scholar, 26Iglesias T. Matthews S. Rozengurt E. FEBS Lett. 1998; 437: 19-23Crossref PubMed Scopus (60) Google Scholar, 27Iglesias T. Rozengurt E. FEBS Lett. 1999; 454: 53-56Crossref PubMed Scopus (45) Google Scholar, 28Matthews S.A. Iglesias T. Rozengurt E. Cantrell D. EMBO J. 2000; 19: 2935-2945Crossref PubMed Scopus (114) Google Scholar). The recent identification of additional cDNA clones, similar in overall structure, primary amino acid sequence, and enzymological properties to PKD/PKCµ (29Hayashi A. Seki N. Hattori A. Kozuma S. Saito T. Biochim. Biophys. Acta. 1999; 1450: 99-106Crossref PubMed Scopus (174) Google Scholar, 30Sturany S. Van Lint J. Mueller F. Wilda M. Hameister H. Hoecker M. Brey A. Gern U. Vandenheede J. Gress T. Adler G. Seufferlein T. J. Biol. Chem. 2001; 276: 3310-3318Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar), supports the notion that PKD isoenzymes constitute a separate family of serine protein kinases. PKD can be activated within intact cells by pharmacological agents like biologically active phorbol esters and cell-permeant DAGs as well as by physiological stimuli including GPCR agonists, growth factors, and antigen-receptor engagement (31Zugaza J.L. Sinnett-Smith J. Van Lint J. Rozengurt E. EMBO J. 1996; 15: 6220-6230Crossref PubMed Scopus (221) Google Scholar, 32Zugaza J.L. Waldron R.T. Sinnett-Smith J. Rozengurt E. J. Biol. Chem. 1997; 272: 23952-23960Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 33Matthews S.A. Pettit G.R. Rozengurt E. J. Biol. Chem. 1997; 272: 20245-20250Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 34Van Lint J. Ni Y. Valius M. Merlevede W. Vandenheede J.R. J. Biol. Chem. 1998; 273: 7038-7043Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 35Paolucci L. Rozengurt E. Cancer Res. 1999; 59: 572-577PubMed Google Scholar, 36Paolucci L. Sinnett-Smith J. Rozengurt E. Am. J. Physiol. 2000; 278: C33-C39Crossref PubMed Google Scholar, 37Matthews S.A. Rozengurt E. Cantrell D. J. Exp. Med. 2000; 191: 2075-2082Crossref PubMed Scopus (99) Google Scholar, 38Chiu T. Rozengurt E. FEBS Lett. 2001; 489: 101-106Crossref PubMed Scopus (21) Google Scholar, 39Chiu T. Rozengurt E. Am. J. Physiol. 2001; 280: C929-C942Crossref PubMed Google Scholar). Treatment with PKC-selective inhibitors prevented PKD activation by all these factors (22Rozengurt E. Sinnett-Smith J. Zugaza J.L. Biochem. Soc. Trans. 1997; 25: 565-571Crossref PubMed Scopus (60) Google Scholar, 31Zugaza J.L. Sinnett-Smith J. Van Lint J. Rozengurt E. EMBO J. 1996; 15: 6220-6230Crossref PubMed Scopus (221) Google Scholar, 32Zugaza J.L. Waldron R.T. Sinnett-Smith J. Rozengurt E. J. Biol. Chem. 1997; 272: 23952-23960Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 33Matthews S.A. Pettit G.R. Rozengurt E. J. Biol. Chem. 1997; 272: 20245-20250Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Furthermore, cotransfection of PKD with constitutively active mutants of PKC ε and η dramatically increased the catalytic activity of PKD (24Waldron R.T. Iglesias T. Rozengurt E. J. Biol. Chem. 1999; 274: 9224-9230Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 31Zugaza J.L. Sinnett-Smith J. Van Lint J. Rozengurt E. EMBO J. 1996; 15: 6220-6230Crossref PubMed Scopus (221) Google Scholar) and led to complex formation between PKD and PKC η (24Waldron R.T. Iglesias T. Rozengurt E. J. Biol. Chem. 1999; 274: 9224-9230Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). In all cases, PKD activation appears to involve the phosphorylation of Ser744 and Ser748 within the activation loop of the catalytic domain of PKD (40Iglesias T. Waldron R.T. Rozengurt E. J. Biol. Chem. 1998; 273: 27662-27667Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 41Waldron R.T. Iglesias T. Rozengurt E. Electrophoresis. 1999; 20: 382-390Crossref PubMed Scopus (58) Google Scholar, 42Waldron R.T. Rozengurt E. Free Radic. Biol. Med. 1999; 27: 67Google Scholar, 43Yuan J.Z. Slice L. Walsh J.H. Rozengurt E. J. Biol. Chem. 2000; 275: 2157-2164Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). These findings revealed a link between PKCs and PKD and implied that PKD lies downstream of PKCs in a novel signal transduction pathway activated by multiple growth-promoting factors (22Rozengurt E. Sinnett-Smith J. Zugaza J.L. Biochem. Soc. Trans. 1997; 25: 565-571Crossref PubMed Scopus (60) Google Scholar, 41Waldron R.T. Iglesias T. Rozengurt E. Electrophoresis. 1999; 20: 382-390Crossref PubMed Scopus (58) Google Scholar). PKD has been implicated in the regulation of a variety of cellular functions including EGF receptor and c-Jun signaling (44Bagowski C.P. Stein-Gerlach M. Choidas A. Ullrich A. EMBO J. 1999; 18: 5567-5576Crossref PubMed Scopus (78) Google Scholar, 45Hurd C. Rozengurt E. Biochem. Biophys. Res. Commun. 2001; 282: 404-408Crossref PubMed Scopus (32) Google Scholar), Na+/H+ antiport activity (46Haworth R.S. Sinnett-Smith J. Rozengurt E. Avkiran M. Am. J. Physiol. 1999; 277: C1202-C1209Crossref PubMed Google Scholar), Golgi organization and function (47Prestle J. Pfizenmaier K. Brenner J. Johannes F.J. J. Cell Biol. 1996; 134: 1401-1410Crossref PubMed Scopus (100) Google Scholar, 48Jamora C. Yamanouye N. Van Lint J. Laudenslager J. Vandenheede J.R. Faulkner D.J. Malhotra V. Cell. 1999; 98: 59-68Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar), NFκB-mediated gene expression (49Johannes F.J. Horn J. Link G. Haas E. Siemienski K. Wajant H. Pfizenmaier K. Eur. J. Biochem. 1998; 257: 47-54Crossref PubMed Scopus (64) Google Scholar), and cell migration (50Bowden E.T. Barth M. Thomas D. Glazer R.I. Mueller S.C. Oncogene. 1999; 18: 4440-4449Crossref PubMed Scopus (308) Google Scholar). However, the precise role of PKD in neuropeptide-induced DNA synthesis and cell proliferation has not been elucidated. In the present study, we examined whether increased PKD expression potentiates the proliferative response to the GPCR agonists bombesin and vasopressin and the biologically active phorbol ester PDBu. We used high efficiency retroviral mediated transfer of PKD into Swiss 3T3 cells, a cell line that undergoes reversible arrest in the G0 phase of the cell cycle and has been used extensively as a model system to elucidate signal transduction pathways in the action of mitogenic GPCR agonists (1Rozengurt E. Science. 1986; 234: 161-166Crossref PubMed Scopus (852) Google Scholar, 2Rozengurt E. J. Cell. Physiol. 1998; 177: 507-517Crossref PubMed Scopus (154) Google Scholar, 19Rozengurt E. Eur. J. Clin. Invest. 1991; 21: 123-134Crossref PubMed Scopus (114) Google Scholar). Our results show that PKD overexpression strikingly and selectively potentiates the stimulation of DNA synthesis and cell division induced by bombesin, vasopressin, or PDBu in Swiss 3T3 cells. These findings support the hypothesis that PKD mediates neuropeptide-induced mitogenesis in these cells. Stock cultures of untransfected and transfected Swiss 3T3 cells were maintained at 37 °C in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum in a humidified atmosphere containing 10% CO2 and 90% air. For experimental purposes, cells were plated in 100-mm dishes at 6 × 105 cells/dish or 35-mm dishes at 1 × 105 cells/dish and grown in DMEM containing 10% fetal bovine serum for 7–9 days until they became confluent and quiescent (51Seufferlein T. Withers D.J. Mann D. Rozengurt E. Mol. Biol. Cell. 1996; 7: 1865-1875Crossref PubMed Scopus (62) Google Scholar). Phoenix packaging cells (kindly provided by Dr. G. Nolan, Stanford University, Stanford, CA) were cultured in the same media in a humidified atmosphere containing 5% CO2. pMSCVneo retroviral vector was engineered to include a single cassette that expresses the murine PKD and green fluorescent protein (GFP) from the same promoter. The MSCV-IRES-GFP plasmid was constructed by substituting theneo gene in the vector with the IRES-GFP fragment. PKD cDNA (spanning from position +50 to +2910, from the published GenBankTM sequence) was inserted into EcoRI site of the MSCV-IRES-GFP plasmid, upstream of IRES. This fragment of PKD cDNA, which contains all coding sequences but lacks the polyadenylation signal, was amplified by polymerase chain reaction. Polymerase chain reaction was initiated from bipartite primers 5′-CTGGAATTCCTCCCGGAAAGTTTGGTGGTT (sense) and 5′-ATCGAATTCGTGTTTTGACAGATTAGAGG (antisense) that introducedEcoRI restriction sites at 5′- and 3′-ends of the fragment. The nucleotide sequence of the amplified PKD coding region was confirmed by sequencing which revealed the presence of A and not T at position 2172, correcting previously published data. This correction leads to the appearance of Arg rather than Trp at position 606 in the amino acid sequence of the wild-type PKD. For retrovirus production, logarithmically growing Phoenix ecotropic cells were transfected with either MSCV-PKD-IRES-GFP or MSCV-IRES-GFP using Fugene 6 Transfection Reagent as per protocol of the manufacturer. Virus-containing supernatants were collected 48 h after transfection and used immediately. Logarithmically growing Swiss 3T3 cells were incubated with the virus-containing supernatants in the presence of 5 µg/ml Polybrene for 5 h. Cells were collected 48–72 h later, and GFP-positive fractions were FACS-sorted using a Becton Dickinson FACStar PLUS machine. GFP-positive cells were propagated, and multiple aliquots were frozen. A fresh batch of cells was restarted every 2 months. Following sorting, GFP-positive Swiss 3T3 cells were maintained as described above. Confluent, quiescent Swiss 3T3-GFP cells and Swiss 3T3-PKD.GFP cells were lysed in 2× SDS-polyacrylamide gel electrophoresis sample buffer (20 mmTris/HCl, pH 6.8, 6% SDS, 2 mm EDTA, 4% 2-mercaptoethanol, 10% glycerol) and boiled for 10 min. After SDS-PAGE, proteins were transferred to Immobilon-P membranes as described previously (31Zugaza J.L. Sinnett-Smith J. Van Lint J. Rozengurt E. EMBO J. 1996; 15: 6220-6230Crossref PubMed Scopus (221) Google Scholar) and blocked by overnight incubation with 5% non-fat dried milk in PBS, pH 7.2. Membranes were incubated at room temperature for 2 h with antisera specifically recognizing either the C terminus of PKD at a dilution of 1 µg/ml or the phospho-specific pS916 antibody recognizing PKD phosphorylated at Ser916 (1:500), or the phospho-specific pS748 antibody recognizing PKD phosphorylated at Ser748 (1:500), in PBS containing 3% non-fat dried milk, or the phospho-specific pSpS744/748 antibody recognizing PKD phosphorylated at Ser744 and Ser748 (1:1000) in PBS, 0.1% Tween 20 containing 5% bovine serum albumin. Our recent results indicate that the phospho-specific pSpS744/748 antibody recognizes primarily PKD phosphorylated at Ser744 (52Waldron R.T. Rey O. Iglesias T. Tugal T. Cantrell D. Rozengurt E. J. Biol. Chem. 2001; 276: 32606-32615Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). Immunoreactive bands were visualized using horseradish peroxidase-conjugated anti-rabbit IgG and enhanced chemiluminescence methods. Autoradiograms were scanned using a GS-710 scanner (Bio-Rad), and the labeled bands were quantified using the Quantity One software program (Bio-Rad). Cultures of Swiss 3T3-PKD.GFP cells, treated as described in the individual experiments, were washed and lysed in 50 mmTris/HCl, pH 7.6, 2 mm EGTA, 2 mm EDTA, 1 mm dithiothreitol, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mm 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, and 1% Triton X-100 (lysis buffer A). Cell lysates were clarified by centrifugation at 15,000 × g for 10 min at 4 °C. PKD was immunoprecipitated at 4 °C for 2–4 h with the PA-1 antiserum (1:100), as described previously (31Zugaza J.L. Sinnett-Smith J. Van Lint J. Rozengurt E. EMBO J. 1996; 15: 6220-6230Crossref PubMed Scopus (221) Google Scholar). The immune complexes were recovered using protein-A coupled to agarose. The immune complexes were then washed twice with lysis buffer and then twice with kinase buffer consisting of 30 mm Tris/HCl, pH 7.4, 10 mm MgCl2, 1 mm dithiothreitol. Autophosphorylation reactions were initiated by combining 20 µl of immune complexes with 5 µl of a phosphorylation mixture containing 100 µm [γ-32P]ATP (specific activity, 400–600 cpm/pmol) in kinase buffer. Following incubation at 30 °C for 10 min, the reactions were terminated by addition of 1 ml of ice-cold kinase buffer and placed on ice. Immune complexes were recovered by centrifugation, and the proteins were extracted for SDS-PAGE analysis by addition of 2× SDS-PAGE sample buffer (200 mm Tris/HCl, pH 6.8, 0.1 mm sodium orthovanadate, 1 mm EDTA, 6% SDS, 2 mm EDTA, 4% 2-mercaptoethanol, 10% glycerol). Dried SDS-PAGE gels were subjected to autoradiography to visualize radiolabeled protein bands. For assays of exogenous substrate phosphorylation, immune complexes were processed as for autophosphorylation reactions, and then substrate (syntide-2, final concentration 2.5 mg/ml) was added in the presence of 100 µm [γ-32P]ATP (400–600 cpm/pmol) in kinase buffer (final reaction volume, 30 µl). After incubation at 30 °C for 10 min, the reactions were terminated by adding 100 µl of 75 mm H3PO4, and 75 µl of the mixed supernatant was spotted to Whatman P-81 phosphocellulose paper. Papers were washed thoroughly in 75 mmH3PO4, dried, and radioactivity incorporated into syntide-2 was determined by detection of Cerenkov radiation in a scintillation counter. Confluent and quiescent cultures of Swiss 3T3-PKD.GFP cells or Swiss 3T3-GFP cells were washed twice with DMEM and incubated with DMEM/Waymouth's medium (1:1, v/v) containing [3H]thymidine (0.2 µCi/ml, 1 µm) and various agonists as described in the figure legends. After 40 h of incubation at 37 °C, cultures were washed twice with PBS and incubated in 5% trichloroacetic acid at 4 °C for 20 min to remove acid-soluble radioactivity, washed with ethanol, and solubilized in 1 ml of 0.1 m NaOH, 0.1% SDS. The acid-insoluble radioactivity was determined by scintillation counting in 6 ml of Beckman Readysafe. Swiss 3T3-PKD.GFP cells, Swiss 3T3-GFP cells, and untransfected Swiss 3T3 cells were seeded in 35-mm Nunc Petri dishes at a density of 2 × 104 with 2 ml of DMEM containing 10% FBS. At day 0 (24 h after plating), cultures were washed twice with DMEM and replaced with DMEM/Waymouth's medium (1:1, v/v) containing 1% FBS and 1 µg/ml insulin (to preserve cell viability in low serum) and supplemented with bombesin, vasopressin, PDBu, or EGF, as described in the legend to Fig. 6. Cell number was determined by removing the cells from the dish with a trypsin/EDTA solution (0.5% trypsin in a Ca2+- and Mg2+-free PBS with EDTA) and counting a portion of the resulting cell suspension in a Coulter counter. Cell counts were obtained at day 0 (24 h after plating) and at days 3, 5, and 7 after plating. pMSCVneo retroviral vector was fromCLONTECH (Palo Alto, CA). Restriction enzymes were purchased from New England Biolabs (Beverly, MA). Fugene 6 Transfection Reagent was obtained from Roche Molecular Biochemicals. Polybrene was from Aldrich. PD 98059, SB202190, rapamycin, wortmannin, PP-2, HA 1077, and Ro 81-3220 were from Calbiochem. Insulin, EGF, vasopressin, bombesin, PDBu, GF I (also known as GF109203X), and cytochalasin D were obtained from Sigma. [3H]Thymidine and [γ-32P]ATP (370 MBq/ml) were from Amersham Pharmacia Biotech. Protein A-agarose was from Roche Molecular Biochemicals. PA-1 was produced as described previously (53Van Lint J.V. Sinnett-Smith J. Rozengurt E. J. Biol. Chem. 1995; 270: 1455-1461Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). pS916 and pS748 antisera were generously provided by Dr. Doreen Cantrell, Imperial Cancer Research Fund, London, UK. Phospho-PKD/PKCµ Ser744/748antibody (catalog number 2054) was obtained from Cell Signaling Technology, Beverly, MA. All other materials were of the highest grade available. In order to generate Swiss 3T3 cells stably overexpressing PKD, cultures of these cells were infected with MSCV retrovirus encoding murine PKD linked via an internal ribosome entry site (IRES) to GFP. This bicistronic retroviral vector expresses PKD and GFP as two separate proteins. Since transcription of both genes is driven by the same promoter, cells expressing higher levels of GFP also express higher levels of PKD. Consequently, GFP was used as a marker for selection of PKD-positive cells (termed Swiss 3T3-PKD.GFP cells). After infection, cells expressing higher levels of GFP were sorted by FACS, collected, and propagated for further studies (Fig.1A, left). A major advantage of this expression system is that it eliminates phenotypic variation(s) resulting from clonal selection. As a control, parallel cultures of Swiss 3T3 cells were infected with MSCV retrovirus encoding only GFP and then FACS-sorted, collected, and propagated to generate Swiss 3T3-GFP cells (Fig. 1 B, right). As expected, both Swiss 3T3-PKD.GFP cells and Swiss 3T3-GFP cells express GFP, as revealed by fluorescence microscopy (Fig. 1 B). In order to examine the expression of PKD in the FACS-sorted cells, lysates of these cell populations were analyzed by SDS-PAGE and Western blotting using an antibody directed against the C-terminal region of this enzyme. As shown in Fig. 1 C, lysates of Swiss 3T3-PKD.GFP cells exhibited a marked increase (8.7 ± 1.8-fold increase; n = 12) in the expression of an immunoreactive band of 115 kDa, which corresponds to the molecular mass of PKD (20Valverde A.M. Sinnett-Smith J. Van Lint J. Rozengurt E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8572-8576Crossref PubMed Scopus (361) Google Scholar), as compared with Swiss 3T3-GFP cells. The detection of this band was completely blocked when the immunoblots were incubated with the antibody in the presence of the immunizing peptide EEREMKALSERVSIL that corresponds to the C-terminal region of the predicted amino acid sequence of PKD. A prominent PKD band was also obtained when lysates from Swiss 3T3-PKD.GFP cells were immunoprecipitated with the PA-1 antiserum (see “Experimental Procedures”), and the immunoprecipitates were analyzed by Western blotting using a different antibody directed against PKD (results not shown). Detection of this 115-kDa band was blocked by the inclusion of the immunizing peptide during the immunoprecipitation. Thus, using high efficiency retrovirally mediated transfection we generated Swiss 3T3 cells overexpressing PKD. We reported that cell stimulation with neuropeptide agonists that signal through heptahelical receptors coupled to Gαq, including bombesin and vasopressin, induces rapid PKC-dependent conversion of PKD from an inactive to an active state (32Zugaza J.L. Waldron R.T. Sinnett-Smith J. Rozengurt E. J. Biol. Chem. 1997; 272: 23952-23960Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 35Paolucci L. Rozengurt E. Cancer Res. 1999; 59: 572-577PubMed Google Scholar, 39Chiu T. Rozengurt E. Am. J. Physiol. 2001; 280: C929-C942Crossref PubMed Google Scholar, 43Yuan J.Z. Slice L. Walsh J.H. Rozengurt E. J. Biol. Chem. 2000; 275: 2157-2164Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 54Abedi H. Rozengurt E. Zachary I. FEBS Lett. 1998; 247: 209-212Crossref Scopus (52) Google Scholar). Here, we examined whether the activity of PKD overexpressed in Swiss 3T3 cells is regulated in a similar manner. To determine whether bombesin induces PKD activation in intact Swiss 3T3-PKD.GFP cells, cultures of these cells were treated with increasing concentrations of this agonist for 10 min and lysed, and PKD was immunoprecipitated with PA-1 antiserum. The resulting immunocomplexes were incubated with [γ-32P]ATP, and the incorporation of 32P into PKD was analyzed by SDS-PAGE and autoradiography. As shown in Fig.2A, PKD isolated from unstimulated Swiss 3T3-PKD.GFP cells had very low catalytic activity, indicating that overexpression did not lead to constitutive activation of this enzyme. Stimulation of these cells with bombesin induced a striking dose-dependent increase in PKD kinase activity that was maintained during cell lysis and immunoprecipitation. Half-maximal and maximal increases in catalytic PKD activity were achieved at 0.3 and 3 nm. PKD activation was also induced when Swiss 3T3-PKD.GFP cells were stimulated with PDBu instead of bombesin. Next, we examined whether PKD activation induced by bombesin or PDBu in Swiss 3T3-PKD.GFP cells occurs through a PKC-dependent pathway. Cultures of these retrovirally transfected cells were treated with either Ro 31-8220 or bisindolylmaleimide GF I, selective inhibitors of phorbol-ester sensitive isoforms of PKC (55Yeo E.J. Exton J.H. J. Biol. Chem. 1995; 270: 3980-3988Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 56Toullec D. Pianetti P. Coste H. Bellevergue P. Grandperret T. Ajakane M. Baudet V. Boissin P. Boursier E. Loriolle F. Duhamel L. Charon D. Kirilovsky J. J. Biol. Chem. 1991; 266: 15771-15781Abstract Full Text PDF PubMed Google Scholar, 57Martiny-Baron G. Kazanietz M.G. Mischak H. Blumberg P.M. Kochs G. Hug H. Marmé D. Schächtele C. J. Biol. Chem. 1993; 268: 9194-9197Abstract Full Text PDF Pu" @default.
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• W2012810520 title "Protein Kinase D Potentiates DNA Synthesis and Cell Proliferation Induced by Bombesin, Vasopressin, or Phorbol Esters in Swiss 3T3 Cells" @default.
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