Myristoylated Alanine-rich C Kinase Substrate-mediated Neurotensin Release via Protein Kinase C-δ Downstream of the Rho/ROK Pathway
2004; Elsevier BV; Volume: 280; Issue: 9 Linguagem: Inglês
10.1074/jbc.m409431200
ISSN1083-351X
AutoresJing Li, Kathleen L. O’Connor, George H. Greeley, Perry J. Blackshear, Courtney M. Townsend, B. Mark Evers,
Tópico(s)Neuropeptides and Animal Physiology
ResumoMyristoylated alanine-rich protein kinase C substrate (MARCKS) is a cellular substrate for protein kinase C (PKC). Recently, we have shown that PKC isoforms-α and -δ, as well as the Rho/Rho kinase (ROK) pathway, play a role in phorbol 12-myristate 13-acetate (PMA)-mediated secretion of the gut peptide neurotensin (NT) in the BON human endocrine cell line. Here, we demonstrate that activation of MARCKS protein is important for PMA- and bombesin (BBS)-mediated NT secretion in BON cells. Small interfering RNA (siRNA) to MARCKS significantly inhibited, whereas overexpression of wild-type MARCKS significantly increased PMA-mediated NT secretion. Endogenous MARCKS and green fluorescent protein-tagged wild-type MARCKS were translocated from membrane to cytosol upon PMA treatment, further confirming MARCKS activation. MARCKS phosphorylation was inhibited by PKC-δ siRNA, ROKα siRNA, and C3 toxin (a Rho protein inhibitor), suggesting that the PKC-δ and the Rho/ROK pathways are necessary for MARCKS activation. The phosphorylation of PKC-δ was inhibited by C3 toxin, demonstrating that the role of MARCKS in NT secretion was regulated by PKC-δ downstream of the Rho/ROK pathway. BON cell clones stably transfected with the receptor for gastrin releasing peptide, a physiologic stimulant of NT, and treated with BBS, the amphibian equivalent of gastrin releasing peptide, demonstrated a similar MARCKS phosphorylation as noted with PMA. BBS-mediated NT secretion was attenuated by MARCKS siRNA. Collectively, these findings provide evidence for novel signaling pathways, including the sequential regulation of MARCKS activity by Rho/ROK and PKC-δ proteins, in stimulated gut peptide secretion. Myristoylated alanine-rich protein kinase C substrate (MARCKS) is a cellular substrate for protein kinase C (PKC). Recently, we have shown that PKC isoforms-α and -δ, as well as the Rho/Rho kinase (ROK) pathway, play a role in phorbol 12-myristate 13-acetate (PMA)-mediated secretion of the gut peptide neurotensin (NT) in the BON human endocrine cell line. Here, we demonstrate that activation of MARCKS protein is important for PMA- and bombesin (BBS)-mediated NT secretion in BON cells. Small interfering RNA (siRNA) to MARCKS significantly inhibited, whereas overexpression of wild-type MARCKS significantly increased PMA-mediated NT secretion. Endogenous MARCKS and green fluorescent protein-tagged wild-type MARCKS were translocated from membrane to cytosol upon PMA treatment, further confirming MARCKS activation. MARCKS phosphorylation was inhibited by PKC-δ siRNA, ROKα siRNA, and C3 toxin (a Rho protein inhibitor), suggesting that the PKC-δ and the Rho/ROK pathways are necessary for MARCKS activation. The phosphorylation of PKC-δ was inhibited by C3 toxin, demonstrating that the role of MARCKS in NT secretion was regulated by PKC-δ downstream of the Rho/ROK pathway. BON cell clones stably transfected with the receptor for gastrin releasing peptide, a physiologic stimulant of NT, and treated with BBS, the amphibian equivalent of gastrin releasing peptide, demonstrated a similar MARCKS phosphorylation as noted with PMA. BBS-mediated NT secretion was attenuated by MARCKS siRNA. Collectively, these findings provide evidence for novel signaling pathways, including the sequential regulation of MARCKS activity by Rho/ROK and PKC-δ proteins, in stimulated gut peptide secretion. The digestion and subsequent absorption of ingested nutrients is coordinated by gut peptides localized to specific regions of the gastrointestinal tract mucosa and released in response to physiologic stimuli (1Guyton A.C. Guyton A.C. Textbook of Medical Physiology. W. B. Saunders, Philadelphia2000: 738-753Google Scholar, 2Townsend Jr., C.M. Bold R.J. Ishizuka J. Surg. Today. 1994; 24: 772-777Crossref PubMed Scopus (15) Google Scholar). Although the stimuli that effect gut peptide secretion have been well defined, the actual signal transduction pathways responsible for mediating these important cellular events are not well characterized. We are interested in the mechanisms governing the regulation and secretion of the gut peptide neurotensin (NT), 1The abbreviations used are: NT, neurotensin; MARCKS, myristoylated alanine-rich C-kinase substrate; PMA, phorbol 12-myristate 13-acetate; siRNA, small interfering RNA; PKD, protein kinase D; GRP, gastrin releasing peptide; GRPR, gastrin releasing peptide receptor; BBS, bombesin; ROK, Rho kinase; GTPγS, guanosine 5′-(γ-thio)triphosphate; GFP, green fluorescene protein; RIA, radioimmunoassay; BSA, bovine serum albumin; PBS, phosphate-buffered saline; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; GST, glutathione S-transferase. that are produced in enteroendocrine cells (N cells) localized predominantly in the distal small bowel (3Evers B.M. World J. Surg. 2002; 26: 799-805Crossref PubMed Scopus (26) Google Scholar). NT facilitates fatty acid translocation (4Armstrong M.J. Parker M.C. Ferris C.F. Leeman S.E. Am. J. Physiol. 1986; 251: G823-G829PubMed Google Scholar), affects gut motility (5Thor K. Rosell S. Gastroenterology. 1986; 90: 27-31Abstract Full Text PDF PubMed Scopus (79) Google Scholar), and stimulates growth of normal gut mucosa (6Chung D.H. Evers B.M. Shimoda I. Townsend Jr., C.M. Rajaraman S. Thompson J.C. Gastroenterology. 1992; 103: 1254-1259Abstract Full Text PDF PubMed Scopus (0) Google Scholar, 7Evers B.M. Izukura M. Chung D.H. Parekh D. Yoshinaga K. Greeley Jr., G.H. Uchida T. Townsend Jr., C.M. Thompson J.C. Gastroenterology. 1992; 103: 86-91Abstract Full Text PDF PubMed Scopus (70) Google Scholar) as well as certain pancreatic, colonic, and prostatic cancers bearing NT receptors (8Thomas R.P. Hellmich M.R. Townsend Jr., C.M. Evers B.M. Endocr. Rev. 2003; 24: 571-599Crossref PubMed Scopus (105) Google Scholar). We have established and characterized a novel human endocrine cell line, BON, which abundantly expresses the NT/neuromedin N gene, synthesizes and secretes NT peptide, and processes the NT/neuromedin N peptide in a manner analogous to that of N cells in the small bowel (9Evers B.M. Ishizuka J. Townsend Jr., C.M. Thompson J.C. Ann. N. Y. Acad. Sci. 1994; 733: 393-406Crossref PubMed Scopus (116) Google Scholar). Using the BON cell line, we have shown that protein kinase C (PKC), particularly PKC isoforms-α and -δ, play a role in the release of NT mediated by the phorbol ester, phorbol 12-myristate 13-acetate (PMA) (10Li J. Hellmich M.R. Greeley Jr., G.H. Townsend Jr., C.M. Evers B.M. Am. J. Physiol. 2002; 283: G1197-G1206Crossref PubMed Scopus (30) Google Scholar). Furthermore, we found that protein kinase D (PKD), a serine/threonine protein kinase that is structurally distinct from the PKC family members, and the Rho/Rho kinase (ROK) pathway is involved in PMA-mediated NT secretion as well as NT secretion mediated by bombesin (BBS), the amphibian equivalent of gastrin releasing peptide (GRP), which is a physiologic stimulant of NT release in vivo (11Li J. O'Connor K.L. Hellmich M.R. Greeley Jr., G.H. Townsend Jr., C.M. Evers B.M. J. Biol. Chem. 2004; 279: 28466-28474Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 12Guo Y.S. Townsend Jr., C.M. Greeley Jr., G.H. Gastrointestinal Endocrinology. Humana Press Inc., Totowa, NJ1999: 189-214Crossref Google Scholar). The MARCKS (myristoylated alanine-rich C kinase substrate) family of proteins was first identified as prominent substrates of PKC (13Blackshear P.J. J. Biol. Chem. 1993; 268: 1501-1504Abstract Full Text PDF PubMed Google Scholar, 14Aderem A. Biochem. Soc. Trans. 1995; 23: 587-591Crossref PubMed Scopus (72) Google Scholar). MARCKS has been implicated in cell motility, phagocytosis, membrane trafficking, and mitogenesis (15Ramsden J.J. Int. J. Biochem. Cell Biol. 2000; 32: 475-479Crossref PubMed Scopus (41) Google Scholar, 16Arbuzova A. Schmitz A.A. Vergeres G. Biochem. J. 2002; 362: 1-12Crossref PubMed Scopus (281) Google Scholar). Moreover, MARCKS has been implicated as a key molecule regulating mucin exocytosis (17Singer M. Martin L.D. Vargaftig B.B. Park J. Gruber A.D. Li Y. Adler K.B. Nat. Med. 2004; 10: 193-196Crossref PubMed Scopus (147) Google Scholar, 18Li Y. Martin L.D. Spizz G. Adler K.B. J. Biol. Chem. 2001; 276: 40982-40990Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 19Rogers D.F. Int. J. Biochem. Cell Biol. 2003; 35: 1-6Crossref PubMed Scopus (164) Google Scholar) and may play a role in phorbol ester-stimulated platelet secretion (20Elzagallaai A. Rose S.D. Trifaro J.M. Blood. 2000; 95: 894-902Crossref PubMed Google Scholar, 21Elzagallaai A. Rose S.D. Brandan N.C. Trifaro J.M. Br. J. Haematol. 2001; 112: 593-602Crossref PubMed Scopus (16) Google Scholar). Phosphorylation of MARCKS induces catecholamine release (22Rose S.D. Lejen T. Zhang L. Trifaro J.M. J. Biol. Chem. 2001; 276: 36757-36763Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 23Trifaro J. Rose S.D. Lejen T. Elzagallaai A. Biochimie (Paris). 2000; 82: 339-352Crossref PubMed Scopus (75) Google Scholar, 24Powis D.A. O'Brien K.J. Harrison S.M. Jarvie P.E. Dunkley P.R. Cell Calcium. 1996; 19: 419-429Crossref PubMed Scopus (17) Google Scholar) and noradrenaline release (25Goodall A.R. Turner N.A. Walker J.H. Ball S.G. Vaughan P.F. J. Neurochem. 1997; 68: 392-401Crossref PubMed Scopus (49) Google Scholar, 26Vaughan P.F. Walker J.H. Peers C. Mol. Neurobiol. 1998; 18: 125-155Crossref PubMed Scopus (99) Google Scholar, 27Walaas S.I. Sefland I. Neurochem. Int. 2000; 36: 581-593Crossref PubMed Scopus (9) Google Scholar). With regards to hormone secretion, phosphorylation of MARCKS protein is required for oxytocin exocytosis in bovine large luteal cells (28Salli U. Saito N. Stormshak F. Biol. Reprod. 2003; 69: 2053-2058Crossref PubMed Scopus (15) Google Scholar, 29Salli U. Supancic S. Stormshak F. Biol. Reprod. 2000; 63: 12-20Crossref PubMed Scopus (16) Google Scholar). Arginine vasopressin-induced induction in MARCKS phosphorylation may be involved in initiating the exocytosis of adrenocorticotropin (30Liu J.P. Engler D. Funder J.W. Robinson P.J. Mol. Cell. Endocrinol. 1994; 101: 247-256Crossref PubMed Scopus (19) Google Scholar, 31Liu J.P. Engler D. Funder J.W. Robinson P.J. Mol. Cell. Endocrinol. 1994; 105: 217-226Crossref PubMed Scopus (30) Google Scholar, 32Betancourt-Calle S. Bollag W.B. Jung E.M. Calle R.A. Rasmussen H. Mol. Cell. Endocrinol. 1999; 154: 1-9Crossref PubMed Scopus (22) Google Scholar). Furthermore, in isolated pancreatic islets, phosphorylation of MARCKS is increased by administration of either glucose or carbachol (33Calle R. Ganesan S. Smallwood J.I. Rasmussen H. J. Biol. Chem. 1992; 267: 18723-18727Abstract Full Text PDF PubMed Google Scholar). However, the role of MARCKS in gut peptide secretion is not known. Given the fact that the MARCKS proteins represent substrates for PKC, we speculated that PKC-mediated regulation of NT secretion might involve MARCKS activation. Therefore, the purpose of the present study was to determine: (i) the role of MARCKS in PMA- and BBS-mediated NT secretion in BON cells, and (ii) whether the PKC and PKD and/or the Rho/ROK pathway are involved in the regulation of MARCKS activity. Here, we show that MARCKS small interfering RNA (siRNA) significantly inhibited NT secretion, whereas overexpression of wild-type MARCKS resulted in a significant increase in PMA-mediated NT secretion in intact BON cells. The phosphorylation of MARCKS was inhibited by PKC-δ siRNA, ROKα siRNA, and C3 toxin (a Rho protein inhibitor). The phosphorylation of PKC-δ was inhibited by C3 toxin. MARCKS siRNA also inhibited BBS-stimulated NT secretion from the BON/GRPR cell lines. MARCKS phosphorylation, stimulated by BBS, was attenuated by PKC-δ and ROKα siRNA, demonstrating the dependence of MARCKS activity on PKC-δ and ROKα. Importantly, our results identify a role for MARCKS activation in the stimulated release of the intestinal peptide NT through sequential regulation by PKC-δ and Rho/ROK proteins. Reagents and Antibodies—Gö6976, GF109203X, Ro31-8220, Rott-lerin, Y27632, and HA1077 were from BIOMOL Research Laboratories Inc. (Plymouth Meeting, PA). Gö6983 was from Calbiochem (La Jolla, CA). BBS was obtained from Bachem (Torrance, CA). PMA and the mouse monoclonal anti-green fluorescent protein (GFP) antibody (clone GFP-20) and anti-β-actin antibodies were from Sigma. The anti-human MARCKS, and PKC-δ polyclonal antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-phospho-MARCKS (Ser-152/156) and PKC-δ (Thr-505) antibodies were from Cell Signaling Technology (Beverly, MA). The anti-ROKα antibody was from BD Pharmingen. Alexa Fluor 568 antibody for fluorescent staining was from Molecular Probes (Eugene, OR). The anti-secondary antibodies were from Pierce (Rockford, IL). The enhanced chemiluminescence (ECL) system for Western immunoblot analysis was from Amersham Biosciences. The concentrated protein assay dye reagent was from Bio-Rad. Tissue culture media and reagents were from Invitrogen. All other reagents were of molecular biology grade and purchased from Sigma. Expression Constructs and Small Interfering RNA (siRNA)—The GFP-tagged bovine MARCKS plasmids, including wild-type MARCKS, myristoylation mutant MARCKS (A2/G2), and phosphorylation mutant MARCKS (A/S) were described previously (34Spizz G. Blackshear P.J. J. Biol. Chem. 2001; 276: 32264-32273Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). MARCKS, ROKα, PKC-α, -δ, and PKD siRNA were synthesized by Custom SMARTPool siRNA Design Service of Dharmacon, Inc. (Lafayette, CO). The nonspecific control siRNA was purchased from Dharmacon. Recombinant GST-C3 and GST control proteins were purified from Escherichia coli using constructs provided by Dr. Keith Burridge (University of North Carolina, Chapel Hill, NC). Cell Culture, Transfection, and Stable Cell Lines—The BON cell line was derived from a human pancreatic carcinoid tumor and characterized in our laboratory (9Evers B.M. Ishizuka J. Townsend Jr., C.M. Thompson J.C. Ann. N. Y. Acad. Sci. 1994; 733: 393-406Crossref PubMed Scopus (116) Google Scholar). BON cells are maintained in a 1:1 mixture of Dulbecco's modified Eagle's medium and nutrient mixture, F12K, supplemented with 5% fetal bovine serum in 5% CO2 at 37 °C. Stable clones of BON cells transfected with the receptor for GRP (GRPR) tagged with GFP were established as described previously (11Li J. O'Connor K.L. Hellmich M.R. Greeley Jr., G.H. Townsend Jr., C.M. Evers B.M. J. Biol. Chem. 2004; 279: 28466-28474Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar) and cultured in the same medium as BON cells except supplemented with 10% fetal bovine serum and G418 (400 μg/ml). All plasmids, siRNA, and GST-C3 protein were transfected by electroporation (400 V, 500 microfarads for plasmids or siRNA; 450 V, 25 microfarads for GST-C3 protein) using GenePulser XCell (Bio-Rad). Cell Treatments and NT Radioimmunoassay (RIA)—All experiments were performed 24-48 h after seeding cells or transfection if not specifically indicated. Before each experiment, BON cells were washed with serum-free secretion medium (Dulbecco's modified Eagle's medium-F12K containing 1% dialyzed BSA) and starved in secretion medium. For NT release experiments, BON cells were treated with PMA in secretion medium for 30 min. For inhibitor treatments, cells were pretreated with inhibitors for 30 min, followed by combined treatments with PMA (10 nm) and inhibitors for another 30 min. BON/GRPR cells were treated with BBS (100 nm) in Krebs-Henseleit buffer, containing 0.294 g/liter of CaCl2, 5.9 g/liter of HEPES, 0.1% bovine serum albumin (pH 7.4) for 30 min. Medium was collected and stored at -80 °C until RIA for NT. RIA for NT was performed in duplicate samples as described previously (35Jones P.M. Howell S.L. Biochem. Soc. Trans. 1989; 17: 61-63Crossref PubMed Scopus (6) Google Scholar). Protein Preparation and Western Blotting—Protein preparation and Western blotting were performed as described previously (10Li J. Hellmich M.R. Greeley Jr., G.H. Townsend Jr., C.M. Evers B.M. Am. J. Physiol. 2002; 283: G1197-G1206Crossref PubMed Scopus (30) Google Scholar). In brief, equal amounts of protein were resolved on NuPAGE BisTris gels (Invitrogen) and electrophoretically transferred to polyvinylidene difluoride membranes; the membranes were incubated with primary antibodies overnight at 4 °C followed by secondary antibodies conjugated with horseradish peroxidase. Membranes were developed using the ECL detection system. Immunofluorescent Staining and Fluorescent Microscopy—BON cells were grown on glass coverslips in 24-well plates. Three days after seeding, cells were treated with vehicle (0.1% Me2SO) or PMA (10 nm) for 30 min. After treatment, cells were fixed with 4% paraformaldehyde for 20 min at 37 °C. After three washes with PBS, the cells were permeabilized with 0.3% Triton X-100 for 15 min at 37 °C and blocked with 1% BSA/PBS for 20 min. The cells were incubated with goat polyclonal anti-MARCKS antibody diluted 1:100 with 1% BSA/PBS for 1 h at room temperature or overnight at 4 °C. Cells were washed three times with PBS and incubated with Alexa 568-conjugated anti-goat secondary antibody diluted 1:500 in 1% BSA/PBS. The fluorescence of MARCKS immunoreactivity was observed under a fluorescent microscope. Real Time Confocal Microscopy—BON cells, transiently expressing GFP-tagged MARCKS, were cultured in 25-mm round coverslips in 6-well plates and imaged in real time before and after PMA treatment. Cells were placed inside a pre-warmed (37 °C) chamber on the stage of an LSM 510 META confocal system configured with an Axiovert 200M inverted microscope (Zeiss, Jena, Germany). GFP fluorescence images were acquired using a plan-apochromat ×63, 1.4 NA oil immersion objectives and the 488-nm line of an argon-ion laser for excitation. The image acquisition and processing was carried out using the Zeiss LSM510 work station (version 3.0) and the Zeiss Image Browser (version 3.1) software. Statistical Analysis—All experiments were repeated at least three times and data were reported as mean ± S.E. Data were analyzed using the Kruskal-Wallis test because of heterogeneous variability in each group. All tests were assessed at the 0.05 level of significance. All statistical computations were conducted using the SAS™ system, Release 8.2 (36SAS Institute Inc. SAS/STAT ® User's Guide. SAS Institute Inc., Cary, NC1999Google Scholar). MARCKS siRNA Inhibits PMA-mediated NT Secretion and MARCKS Expression in BON Cells—Based on findings that PKC-α and -δ regulated PMA-mediated NT secretion in BON cells (10Li J. Hellmich M.R. Greeley Jr., G.H. Townsend Jr., C.M. Evers B.M. Am. J. Physiol. 2002; 283: G1197-G1206Crossref PubMed Scopus (30) Google Scholar), we determined whether MARCKS, a major PKC substrate that has been implicated in the secretion and membrane trafficking of a number of cell types (17Singer M. Martin L.D. Vargaftig B.B. Park J. Gruber A.D. Li Y. Adler K.B. Nat. Med. 2004; 10: 193-196Crossref PubMed Scopus (147) Google Scholar, 28Salli U. Saito N. Stormshak F. Biol. Reprod. 2003; 69: 2053-2058Crossref PubMed Scopus (15) Google Scholar, 37Sasaki Y. J. Pharmacol. Sci. 2003; 93: 35-40Crossref PubMed Scopus (40) Google Scholar), was also involved in PMA-mediated NT secretion. We utilized the RNA interference technique to selectively reduce MARCKS expression (Fig. 1). BON cells were transfected with MARCKS siRNA and the control siRNA. Forty-eight h after transfection, cells were treated with PMA (10 nm) or the vehicle control (Me2SO) for 30 min. The medium was collected and NT secretion was assayed by RIA (Fig. 1A). Transfection with siRNA directed against MARCKS decreased PMA-stimulated NT secretion by more than 50% from BON cells compared with the control siRNA. Cells were lysed and Western blot analysis was performed to assess the expression of endogenous MARCKS (Fig. 1B, top). Compared with cells transfected with the control siRNA, MARCKS siRNA markedly suppressed endogenous MARCKS protein expression. The blot was reprobed with β-actin demonstrating equal loading (Fig. 1B, bottom). Overexpression of Wild-type MARCKS Increases PMA-mediated NT Secretion—To further confirm the role of MARCKS in NT secretion, BON cells were transiently transfected with GFP-tagged wild-type MARCKS, phosphorylation mutant (MARCKS-A/S) and myristoylation mutant (MARCKS-A2/G2), or the empty vector (pEGFP-N-1) as a control (Fig. 2). Cells were serum starved for 30 min and then treated with vehicle (Me2SO) or PMA (10 nm) for 30 min and the medium was collected for measurement of NT by RIA (Fig. 2A). In the absence of PMA treatment, overexpression of the three MARCKS constructs did not affect NT release compared with the empty vector. Importantly, PMA treatment of cells transfected with wild-type MARCKS significantly enhanced NT secretion compared with PMA treatment of BON cells transfected with the empty vector. In contrast, overexpression of the phosphorylation and myristoylation mutants and treatment with PMA did not enhance NT secretion compared with cells transfected with the control vector in the presence of PMA. Immunoblot analysis of cell lysate was performed to determine the phosphorylation of exogenous (exo) and endogenous (endo) MARCKS (Fig. 2B). The membrane was blotted with GFP to monitor the overexpression of MARCKS in BON cells; GFP-tagged MARCKS was detected after transfection with the three MARCKS constructs either in the presence or absence of PMA, but not with the control vector (Fig. 2B, lower panel). The membrane was stripped and reprobed with an antibody that recognizes phosphorylated serine 152/156 of MARCKS (Fig. 2B, upper panel). Phosphorylation of exogenous MARCKS was induced in BON cells transfected with either the wild-type MARCKS and myristoylation mutant MARCKS, but not in BON cells transfected with the phosphorylation mutant MARCKS. As expected, exogenous MARCKS was not detected in BON cells transfected with the control vector. Similar levels of endogenous (endo) phosphorylated MARCKS (∼65 kDa) were detected in BON cells transfected with all three MARCKS constructs, in the presence of PMA. Taken together, these data demonstrate activation of either overexpressed MARCKS or endogenous MARCKS and the enhancement of NT secretion by MARCKS overexpression. PMA Induces Translocation of Endogenous MARCKS from Membrane to the Cytosol in BON Cells—To further support the role of MARCKS in PMA-mediated NT secretion, we next analyzed the activation of endogenous MARCKS by PMA in BON cells by observing translocation using immunofluorescent imaging (Fig. 3). BON cells were cultured on coverslips and stained with anti-MARCKS antibody followed by Alexa 568-conjugated secondary antibody and translocation was observed by fluorescent microscopy. Endogenous MARCKS was expressed on the membrane of vehicle (Me2SO)-treated BON cells (Fig. 3, left) and translocated to the cytosol of PMA-treated BON cells (Fig. 3, right). These results demonstrate the activation of endogenous MARCKS protein in BON cells by PMA. PMA Induces Translocation of Wild-type MARCKS but Not Phosphorylation Mutant or Myristoylation Mutant MARCKS in BON Cells—To examine the intracellular translocation characteristics of exogenous MARCKS, GFP-tagged wild-type and mutant MARCKS were transfected separately into BON cells. GFP fluorescence was visualized in BON cells using time-lapse confocal microscopy to monitor translocation of MARCKS (Fig. 4). Serial images were taken before and after the addition of PMA (100 nm). As a control, the fluorescence in BON cells transfected with empty vector was first observed before and after the addition of PMA (Fig. 4, upper panel); no changes in GFP localization were noted after PMA treatment. Intense fluorescence was observed in BON cells transfected with wild-type MARCKS (Fig. 4, second panel). The addition of PMA induced a rapid translocation of wild-type MARCKS from the plasma membrane to the cytosol within 1 min after stimulation. These findings are consistent with our results showing translocation of endogenous MARCKS to the cytosol from the membrane after PMA stimulation in fixed cells (Fig. 3). In untreated BON cells transfected with phosphorylation mutant MARCKS (A/S), GFP fluorescence was present in the membrane, similar to wild-type MARCKS (Fig. 4, third panel). The addition of PMA did not result in translocation of the phosphorylation mutant MARCKS. In contrast, in untreated BON cells transfected with the myristoylation mutant MARCKS (A2/G2), GFP fluorescence was present in the cytosol (Fig. 4, lower panel). Treatment with PMA did not alter the location of the myristoylation mutant MARCKS. These results demonstrate that both the phosphorylation domain and the myristoylation domain are important for the function of MARCKS and the activation of wild-type MARCKS further supports the role of MARCKS in NT secretion. Activation of MARCKS Is Dependent on PKC-δ, but Not PKC-α or PKD—MARCKS is known to be a PKC substrate in some cell types (13Blackshear P.J. J. Biol. Chem. 1993; 268: 1501-1504Abstract Full Text PDF PubMed Google Scholar). Based on our previous studies in which we found that PKC-α and -δ play important roles in PMA-mediated NT secretion, we next determined whether PKC isoforms are involved in PMA-induced MARCKS activation in BON cells using the PKC inhibitors, Gö6976, Gö6983, GF109203X, and Ro31-8220, which inhibit activity of conventional and novel PKC isoforms (38Way K.J. Chou E. King G.L. Trends Pharmacol. Sci. 2000; 21: 181-187Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar, 39Gschwendt M. Dieterich S. Rennecke J. Kittstein W. Mueller H.J. Johannes F.J. FEBS Lett. 1996; 392: 77-80Crossref PubMed Scopus (566) Google Scholar), and Rottlerin, which is a selective PKC-δ inhibitor (38Way K.J. Chou E. King G.L. Trends Pharmacol. Sci. 2000; 21: 181-187Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar, 39Gschwendt M. Dieterich S. Rennecke J. Kittstein W. Mueller H.J. Johannes F.J. FEBS Lett. 1996; 392: 77-80Crossref PubMed Scopus (566) Google Scholar) (Fig. 5). BON cells were pretreated with these inhibitors (1 μm for each) for 30 min prior to a 10-min exposure to PMA (10 nm). As shown in Fig. 5A, all of the inhibitors blocked MARCKS phosphorylation at Ser-152/156 (upper panel), suggesting the requirement of upstream PKC isoforms in the activation of MARCKS. The membrane was reprobed with β-actin as a loading control (lower panel). We next used siRNA against PKC-α, PKC-δ, or PKD to further delineate the proteins that contribute to MARCKS activation (Fig. 5B). BON cells were transfected with PKC-α, PKC-δ, or PKD siRNA or the control siRNA. Forty-eight h after transfection, cells were treated with PMA (10 nm) or the vehicle control (Me2SO) for 30 min. Compared with cells transfected with the control siRNA, transfection of PKC-α, PKC-δ, and PKD siRNA significantly inhibited PKC-α (data not shown), PKC-δ (Fig. 5B, top panel), and PKD (data not shown) expression, respectively. Importantly, MARCKS phosphorylation was not decreased by either PKC-α or PKD siRNA (data not shown) but markedly decreased by PKC-δ siRNA (Fig. 5B, middle panel). Membranes were reprobed with β-actin demonstrating equal loading (Fig. 5B, bottom panel). Taken together, the results suggest that MARCKS protein is a downstream effector of PKC-δ in BON cells. Rho/ROK Pathway Is Involved in MARCKS Activation by PMA in BON Cells—ROK (a downstream effector of Rho proteins) regulates MARCKS activation in neuronal cells (37Sasaki Y. J. Pharmacol. Sci. 2003; 93: 35-40Crossref PubMed Scopus (40) Google Scholar). In a previous study, we demonstrated that the Rho/ROK pathway contributed to PMA-mediated NT secretion in BON cells (11Li J. O'Connor K.L. Hellmich M.R. Greeley Jr., G.H. Townsend Jr., C.M. Evers B.M. J. Biol. Chem. 2004; 279: 28466-28474Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Therefore, in this study, we determined whether the Rho/ROK pathway is involved in PMA-stimulated MARCKS phosphorylation (Fig. 6). ROK inhibitors, Y27632 and HA1077 (both at a concentration of 15 μm), were first used to assess the effect of ROK inhibition on MARCKS phosphorylation. Both inhibitors markedly attenuated MARCKS phosphorylation (Fig. 6A, top). These findings suggest the involvement of upstream ROK in the activation of MARCKS. Total MARCKS was probed to assess loading equality (Fig. 6A, bottom). To further confirm the involvement of the Rho/ROK pathway, the role of ROKα (an isoform of ROK proteins) on MARCKS phosphorylation was examined using ROKα siRNA (Fig. 6B). The inhibition of ROKα expression was shown by Western blot analysis (Fig. 6B, top panel). PMA-mediated MARCKS phosphorylation was decreased in BON cells transfected with ROKα siRNA compared with the control siRNA (Fig. 6B, middle panel). The blot was reprobed with β-actin as a loading control (Fig. 6B, bottom panel). These results further indicate the regulation of MARCKS activity by ROKα. Clostridium botulinum C3 toxin specifically ADP-ribosylates Rho and impairs its function (40Just I. Hofmann F. Genth H. Gerhard R. Int. J. Med. Microbiol. 2001; 291: 243-250Crossref PubMed Scopus (37) Google Scholar). We previously showed that C3 toxin inhibited PMA-mediated NT secretion in BON cells (11Li J. O'Connor K.L. Hellmich M.R. Greeley Jr., G.H. Townsend Jr., C.M. Evers B.M. J. Biol. Chem. 2004; 279: 28466-28474Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Therefore, to determine whether Rho proteins are involved in MARCKS activity, BON cells were transfected with C3 toxin overnight and treated with vehicle (Me2SO) or PMA (10 nm). Western blot analysis was performed using phospho-MARCKS antibody (Fig. 6C). C3 toxin attenuate
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