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Originally published In Press as doi:10.1074/jbc.M314307200 on May 28, 2004 Originally published In Press as doi:10.1074/jbc.M314307200 on April 27, 2004

J. Biol. Chem., Vol. 279, Issue 27, 28466-28474, July 2, 2004
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The Role of Protein Kinase D in Neurotensin Secretion Mediated by Protein Kinase C-{alpha}/-{delta} and Rho/Rho Kinase*

Jing Li{ddagger}, Kathleen L. O'Connor{ddagger}§, Mark R. Hellmich{ddagger}§, George H. Greeley, Jr.{ddagger}, Courtney M. Townsend, Jr.{ddagger}, and B. Mark Evers{ddagger}§

From the {ddagger}Department of Surgery and §Sealy Center for Cancer Cell Biology, The University of Texas Medical Branch, Galveston, Texas 77555-0536

Received for publication, December 30, 2003 , and in revised form, April 22, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Neurotensin (NT) is a gut peptide that plays an important role in gastrointestinal (GI) secretion, motility, and growth as well as the proliferation of NT receptor positive cancers. Secretion of NT is regulated by phorbol ester-sensitive protein kinase C (PKC) isoforms-{alpha} and -{delta} and may involve protein kinase D (PKD). The purpose of our present study was: (i) to define the role of PKD in NT release from BON endocrine cells and (ii) to delineate the upstream signaling mechanisms mediating this effect. Here, we demonstrate that small interfering RNA (siRNA) targeted against PKD dramatically inhibited both basal and PMA-stimulated NT secretion; NT release is significantly increased by overexpression of PKD. PKC-{alpha} and -{delta} siRNA attenuated PKD activity, whereas overexpression of PKC-{alpha} and -{delta} enhanced PKD activity. Rho kinase (ROK) siRNA significantly inhibited NT secretion, whereas overexpression of ROK{alpha} effectively increased NT release. Rho protein inhibitor C3 dramatically inhibited both NT secretion and PKD activity. In conclusion, our results demonstrate that PKD activation plays a central role in NT peptide secretion; upstream regulators of PKD include PKC-{alpha} and -{delta} and Rho/ROK. Importantly, our results identify novel signaling pathways, which culminate in gut peptide release.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Regulatory hormones, localized to specialized endocrine cells in the small bowel, control numerous physiological functions of the gastrointestinal (GI) tract including secretion, motility, and mucosal growth (1). The gut peptide neurotensin (NT),1 a tridecapeptide localized to enteroendocrine cells (N cells) of the distal small bowel (2, 3), facilitates fatty acid translocation (4), affects gut motility (5), and stimulates growth of normal gut mucosa (6, 7). In addition to its trophic effects on normal GI tissues, NT stimulates proliferation of certain pancreatic, colonic, and prostatic cancers bearing NT receptors (NTR) (8). Although the mechanisms for pancreatic hormone release have been well characterized (9), the signal transduction pathways regulating stimuli-induced gut hormone secretion are not entirely understood. One reason for this paucity in our understanding is the relative lack of useful in vitro models that recapitulate in vivo properties of intestinal endocrine cells.

The BON endocrine cell line was established from a human pancreatic carcinoid tumor and characterized in our laboratory (10). These cells have served as an invaluable in vitro model for hormone secretion studies. Similar to the terminally differentiated N cell of the small bowel, BON cells express high levels of NT/neuromedin N mRNA, synthesize and secrete NT peptide, and process the NT/neuromedin N precursor protein in a fashion identical to that of the normal intestine (11). BON cells exhibit morphological and biochemical characteristics consistent with the enteroendocrine cell phenotype, including the presence of numerous dense core granules and the expression and secretion of chromogranin A and other peptides (e.g. pancreastatin) (10, 12, 13). Thus, the BON cell line provides an excellent model to delineate the mechanisms underlying gut peptide secretion.

We have shown that protein kinase C (PKC), particularly isoforms PKC-{alpha} and -{delta}, plays a role in the stimulated release of NT (13–15). The PKCs comprise a family of intracellular serine/threonine-specific kinases that are, depending on the isoform, typically activated by Ca2+, lipid second messengers or protein activators and mediate the effects of a wide range of physiological stimuli, including growth factors, hormones, and neurotransmitters (16, 17). Protein kinase D (PKD), originally referred to as PKC-µ, is a serine/threonine protein kinase with unique structural, enzymological, and regulatory properties that are different from those of the PKC family members (18, 19). The most distinct characteristics of PKD are the presence of a catalytic domain distantly related to Ca2+-regulated kinases, a pleckstrin homology (PH) domain within the regulatory region, and a highly hydrophobic stretch of amino acids in its N-terminal region (18, 19). Expression of PKD has been demonstrated in some endocrine cells, including insulin- and gastrin-secreting cells (20–22). PKD activation occurs through several mediators via a PKC-dependent pathway (23–26).

Recent studies indicate a close association between PKC isoforms and members of the Rho family of small GTP-binding proteins including Rho (A, B, and C), Rac1, and Cdc42 (27, 28). The Rho family members, well-known regulators of the actin cytoskeleton and phosphoinositide metabolism (29), have been implicated in hormone secretion from endocrine cells (30–32). Rho kinases (ROK) are the first effectors of Rho to be discovered and, to date, two ROK isoforms have been identified: ROK{alpha} and ROK{beta} (33). There are now several examples where Rho GTPases participate in regulated secretory pathways, such as in mast cells (34), PC12 cells (35), and neurons (36). The potential role of Rho family members in gut peptide secretion is not known.

The purpose of our present study was 2-fold: (i) to determine whether PKD is involved in NT secretion, and (ii) to delineate upstream regulators of PKD in the BON endocrine cell line. We found that the novel PKD protein mediates phorbol ester- or bombesin (BBS)-stimulated NT secretion and is regulated by PKC-{alpha}, -{delta}, and the Rho/ROK pathway. Importantly, these findings describe a novel signaling mechanism for gut hormone release from specialized endocrine cells.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents and Antibodies—GF109203X (GFX), Ro31–8220, Y27632, and HA1077 were from BIOMOL Research Laboratories Inc. (Plymouth Meeting, PA). BBS was from Biochem (Torrance, CA). Syntide-2 and Gö6983 were from Calbiochem (La Jolla, CA). PMA and the mouse monoclonal anti-green fluorescent protein (GFP) antibody (clone GFP-20) were from Sigma. The anti-PKD, PKC-{alpha}, and PKC-{delta} polyclonal antibodies and anti-RhoA antibody were from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-phospho-PKD (Ser744/748 and Ser916) antibodies were from Cell Signaling Technology (Beverly, MA). The rat monoclonal anti-hemagglutinin (HA) antibody (clone 3F10) was from Roche Applied Science. Secondary antibodies were from Pierce. [{gamma}-32P]ATP was obtained from PerkinElmer Life Sciences. The enhanced chemiluminescence (ECL) system for Western immunoblot analysis was from Amersham Biosciences. Protein A-Sepharose 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 from Sigma.

Expression Constructs and Small Interfering RNA (siRNA)—The GFP-tagged PKD expression plasmids, wild-type (PKDWT) and PKD{Delta}PH (a constitutively active PKD with PH domain deleted) were provided by Dr. Franz-Josef Johannes (The Fraunhofer Institute for Interfacial Engineering) (37). The pSUPER PKD siRNA vector and the control vector pSUPER were provided by Dr. Alex Toker (Harvard Medical School, Boston, MA) (38). The PKC-{alpha} and -{delta} expression plasmids (pTB701-HA-PKC-{alpha} and pTB701-HA-PKC-{delta}) and the control plasmid (pTB701-HA) were provided by Dr. Yoshitaka Ono (Kobe University) (39). The HA-tagged ROK{alpha} expression plasmids, including the fulllength (ROK{alpha}F/L), the deletion mutant (ROK{alpha}1–543) and pXJ40 control plasmid, were provided by Dr. Thomas Leung (National University of Singapore, Singapore) (40). The vectors pCEFLAU5 encoding wild-type RhoA (RhoAWT), RhoAQ63L (constitutively active mutant), and RhoAN19 (dominant negative mutant) were provided by Dr. J. Silvio Gutkind (National Institutes of Health, Bethesda, MD) (41). The GFP-tagged human gastrin releasing peptide (GRP) receptor (GRPR-GFP) plasmid was from Dr. Dai H. Chung (The University of Texas Medical Branch, Galveston, TX). PKC-{alpha}, -{delta}, PKD and ROK{alpha} siRNA were synthesized by Custom SMARTPool siRNA Design Service of Dharmacon, Inc. (Lafayette, CO). The 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 Establishment of Stable Cell Line—The BON cell line was derived from a human pancreatic carcinoid tumor and characterized in our laboratory (10). 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. For transiently transfection, plasmids, siRNA and GST-C3 protein were transfected by electroporation (400 V, 500 µfarads for plasmids or siRNA; 450 V, 25 µfarads for GST-C3 protein) using GenePulser XCell (Bio-Rad). To establish BON cell clones that express GRPR, parental BON cells were transfected by electroporation with GRPR-GFP. Stably transfected clones were selected in medium containing 800 µg/ml of G418 (Cellgro). Individual G418-resistant clones were isolated using trypsin, transferred, and subcultured. Stable cell clones were screened by fluorescent microscopy. Once established, clones were maintained in media containing 400 µg/ml of G418.

NT Radioimmunoassay (RIA)—Parental BON cells were treated with PMA in secretion medium for 30 min. The BON/GRPR-GFP cells were treated with the GRPR ligand, BBS, 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 inhibitor treatments, cells were pretreated with inhibitors for 30 min, followed by combined treatments with PMA (10 nM) and inhibitors for another 10 min. Medium was collected and stored at -80 °C until RIA for NT. RIA for NT was performed in duplicate samples as described previously (42, 43).

Real Time Confocal Microscopy—BON cells, transiently expressing GFP-tagged PKD, 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 prewarmed (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-aprochromat 63x, 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 (v 3.0) and the Zeiss Image Browser (v3.1) software.

Protein Preparation and Western Blotting—Protein preparation and Western blotting were performed as described previously (14). In brief, equal amounts of protein were resolved on Novex Tris-glycine or Nu-PAGE Bis-Tris 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.

Immunoprecipitation and in Vitro Kinase Assays—Immunoprecipitation and in vitro kinase assays were performed as described previously (14). In brief, proteins (50 µg) were incubated with PKD or GFP antibodies (1:50) on a shaker for 2 h at 4 °C followed by another 2 h incubation with 30 µl of protein A-Sepharose beads at 4 °C. The immunocomplexes were suspended in 20 µl of kinase buffer and kinase reaction, with or without 2.5 µg of syntide-2 as a substrate, was started by adding 5 µCi of [{gamma}-32P]ATP and incubated for 10 min at 30 °C. Reactions were stopped by the addition of 2x Tris-glycine sample buffer. Samples were denatured by boiling for 5 min and separated by NuPAGE 4–12% Bis-Tris gels. Gels were incubated in Gel-Dry drying solution (Invitrogen) for 5 min and dried at 60 °C for 60 min followed by exposure to x-ray film.

RhoA Activity Assay—RhoA activity was assessed using the Rho binding domain of Rhotekin as described (44, 45). In brief, cells (3 x 106) were transfected with 50 µg of GST-C3 or GST protein and plated onto 60-mm dishes in growth medium, allowed to adhere and then serumstarved in secretion medium overnight. Cells were treated with PMA (10 nM) for 10 min and then extracted with radioimmune precipitation assay buffer. After centrifugation at 14,000 x g for 2 min, the extracts were incubated for 30 min at 4 °C with glutathione beads (Amersham Biosciences) coupled with bacterially expressed GST-RBD (Rho binding domain of Rhotekin) fusion protein (44), and then washed three times. The RhoA content was determined by immunoblotting samples using rabbit anti-RhoA antibody.

Statistical Analysis—All experiments were repeated at least two times and data are reported as means ± S.E. Data were analyzed using analysis of variance for a two-factor factorial experiment (Figs. 1B, 2A, 6, A–C, and 8C) or a one-factor experiment (Fig. 6D). Fisher's least significant difference procedure was used for multiple comparisons with Bonferroni adjustment for the number of comparisons. A p value < 0.05 was considered significant.



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  		FIG. 1.
PKD siRNA attenuates PKD expression, phosphorylation and NT secretion in BON cells. BON cells were transiently transfected with pSUPER PKD siRNA or the empty vector, pSUPER. A, top, cells were lysed, and Western blot analysis was performed for the expression of PKD in BON cells 48 or 96 h after transfection (upper panel); {beta}-actin protein expression was detected to monitor equal loading. Bottom, cells were allowed to recover for 36 h after transfection and then serum-starved in secretion medium overnight and treated with vehicle (0.1% Me2SO) or 10 nM PMA for 10 min. Western blot analysis was performed for the inhibition of PKD phosphorylation by PKD siRNA in BON cells. {beta}-Actin was determined to monitor equal loading. A representative result from three experiments is shown. B, cells were allowed to recover for 36 h after transfection and then serum-starved in secretion medium overnight and treated with vehicle (0.1% Me2SO) or 10 nM PMA for 30 min. The amount of NT released into the medium was determined by RIA. The relative amount of NT was compared with the basal levels in cells transfected with the empty vector (pSUPER). Experiments were performed in triplicate. Results are expressed as mean ± S.E. (n = 6); *, p < 0.05 versus empty vector (pSUPER); {dagger}, p < 0.05 versus empty vector plus PMA. {ddagger}, p < 0.05 versus PKD siRNA without PMA.

 



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  		FIG. 2.
Overexpression of wild-type PKD and constitutively active PKD significantly increases PMA-mediated NT secretion. A, BON cells were transiently transfected with GFP-tagged wild-type PKD, constitutively active PKD-GFP or the empty vector and allowed to recover for 24 h. Cells were then serum-starved in secretion medium overnight and treated with vehicle (0.1% Me2SO) or 10 nM PMA for 30 min. The amount of NT released into the medium was determined with an NT radioimmunoassay (RIA). The relative amount of NT was compared with the basal level in the empty vector-transfected cells. Experiments were performed in triplicate. Results are expressed as mean ± S.E. (n = 6); *, p < 0.05 versus empty vector (pEGFP-N-1); {dagger}, p < 0.05 versus empty vector plus PMA. B, top, BON cells were lysed and subjected to Western blotting using an anti-GFP antibody; middle, the membrane was blotted using an anti-phospho-PKD (Ser744/748) antibody. The upper signals demonstrate exogenous (Exo) phospho-PKD; the lower signals demonstrate endogenous (Endo) phospho-PKD. Bottom, PKD was blotted as a loading control. Representative results from five experiments are shown. C, in vitro kinase assays of exogenous PKD expressed in BON cells. PKD activity was measured by syntide-2 substrate phosphorylation and autophosphorylation using an anti-GFP antibody. The PKD substrate phosphorylation was shown by the lower signals (3 kDa). The top signals demonstrate autophosphorylation (140 kDa). Representative results from three experiments are shown.

 



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  		FIG. 6.
Rho/ROK pathway alters PMA-mediated NT secretion. A, BON cells were transfected with GST-C3 toxin (100 µg) or GST and cultured overnight. Cells were incubated in serum-free medium for 1 h and treated with 10 nM PMA for 30 min. Medium was collected, and the level of NT secreted into the medium was measured by RIA using duplicate samples. Results are expressed as means ± S.E. (n = 6). *, p < 0.05 versus GST; {dagger}, p < 0.05 versus PMA and GST. B, top, 48 h after transfection, BON cells were incubated in secretion medium for 30 min and treated with PMA (10 nM) for another 30 min. Medium was collected for NT RIA. Results are expressed as means ± S.E. (n = 6). *, p < 0.05 versus control siRNA; {dagger}, p < 0.05 versus PMA and control siRNA. Bottom, cells were lysed, and Western blotting analysis was performed to detect the inhibition of ROK{alpha} expression by ROK{alpha} siRNA using anti-ROK{alpha} antibody (upper panel). Actin was reprobed for the loading control (lower panel). C, BON cells were transiently transfected with the empty vector (pXJ40), the corresponding full-length ROK{alpha} or constitutively active ROK{alpha}-(1–543) vectors for 24 h. Cells were incubated with secretion medium overnight and incubated with vehicle or PMA (10 nM) for 30 min. Top, overexpression of ROK{alpha} was demonstrated by blotting with anti-HA antibody. Bottom, NT secretion was measured by RIA as described above. Results are expressed as means ± S.E. (n = 6). *, p < 0.05 versus empty vector (pXJ40); {dagger}, p < 0.05 versus PMA and empty vector. D, BON cells were incubated with secretion medium overnight and then preincubated with vehicle or the ROK inhibitors, Y27632 or HA1077, for 30 min before addition of PMA (10 nM). After another 30 min, the medium was collected and the level of NT secreted into the medium was measured by RIA using duplicate samples. Experiments were performed in triplicate; results are expressed as means ± S.E. (n = 6); *, p < 0.05 versus control (vehicle treatment); {dagger}, p < 0.05 versus 10 nM PMA. Experiments were performed in triplicate.

 



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  		FIG. 8.
PKD siRNA inhibited BBS-mediated NT secretion from BON/GRPR cells. A, BON cells stably expressing the human GRPR tagged with GFP (BON/GRPR-GFP) were generated and expression of the GRPR was determined by fluorescent microscopy. B, BON/GRPR-GFP cells were transfected with control siRNA or PKD siRNA (from Dharmacon). 48 h later, cells were treated with 0.1% Me2SO (0) or BBS at the indicated doses for 15 min. Western blots were performed using anti-PKD antibody to monitor the inhibition of PKD expression by PKD siRNA (upper panel); PKD siRNA inhibition of BBS-induced PKD activation was examined using phospho-PKD (Ser744/748) antibody (middle panel). {beta}-Actin was determined as a loading control (lower panel). C, NT release was measured by RIA. Results are expressed as means ± S.E. (n = 6). * and {dagger}, p < 0.05 versus vehicle (0.1% Me2SO) of control siRNA. {ddagger}, p < 0.05 versus 10 or 100 nM of BBS and control siRNA.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
siRNA Directed Against PKD Inhibits NT Secretion and PKD Phosphorylation in BON Cells—Based on findings that both PMA and bryostatin1 induce the activation of endogenous PKD in BON cells (14), we determined whether PKD is involved in PMA-mediated NT secretion. We took advantage of the RNA interference (RNAi) to selectively reduce PKD expression (Fig. 1). BON cells were transfected with the PKD siRNA vector (pSUPER.PKD siRNA) and the control vector (pSUPER). Cells were collected at 48 or 96 h after transfection, and lysates immunoblotted to assess expression of endogenous PKD (Fig. 1A, top). Compared with cells transfected with the control siRNA (pSUPER), transfection of PKD siRNA significantly reduced PKD levels. The blot was stripped and reprobed with {beta}-actin demonstrating equal loading. Moreover, the PMA-activated phosphorylation of PKD was reduced significantly by PKD siRNA compared with the PMA-treated control vector (Fig. 1A, bottom).

Next, BON cells were transfected with PKD siRNA or the control vector (pSUPER) and treated with PMA; NT secretion was assayed by RIA (Fig. 1B). Transfection with siRNA directed against PKD decreased both basal and PMA-stimulated NT secretion from BON cells compared with the control vector. Taken together, our results, using complementary techniques, strongly demonstrate an important role for PKD in NT secretion from BON cells.

Overexpression of Wild-type and Constitutively Active PKD Increases PMA-mediated NT Secretion—To further confirm the regulation of PKD on NT secretion, BON cells were transiently transfected with PKDWT, or the constitutively active PKD{Delta}PH, both linked to GFP, or the empty vector (pEGFP-N-1), as a control (Fig. 2). 24 h after, the cells were serum-starved, then treated with vehicle (Me2SO) or PMA (10 nM) for 30 min, and the medium collected for measurement of NT by RIA (Fig. 2A). In the absence of PMA treatment, overexpression of PKD{Delta}PH increased NT release compared with the empty vector (pEGFP-N-1) and PKDWT. The PH domain exerts an inhibitory effect on the catalytic domain of PKD (46); therefore, the increase in basal NT release suggests that the PH domain plays an inhibitory role in the regulation of its enzymatic activity. NT secretion was significantly increased by PMA treatment of BON cells transfected with the empty vector. Importantly, PMA treatment of cells transfected with either PKDWT or PKD{Delta}PH resulted in a significantly enhanced NT secretion compared with PMA treatment of BON cells transfected with the empty vector, but the increase in secretion from BON cells transfected with PKD{Delta}PH was lower than NT secretion noted from PKDWT transfected BON cells.

Overexpression of PKD plasmids was confirmed by immunoblot analysis for GFP tag (Fig. 2B). GFP-tagged PKD was evenly distributed in cells transfected with PKDWT or PKD{Delta}PH, either in the presence or absence of PMA, but not in the control vector (upper panel). The membrane was reprobed with an antibody that recognizes phosphorylated serine 744/748 (middle panel). Phosphorylation of exogenous PKD was not detected in BON cells transfected with the PKDWT, in the absence of PMA, but was induced in BON cells transfected with either the PKDWT and PKD{Delta}PH treated with PMA. These data indicate that the response of exogenous PKD to PMA is similar to endogenous PKD. A weak signal was detected in BON cells transfected with PKD{Delta}PH, in the absence of PMA treatment, demonstrating the constitutively active status of PKD with the PH domain deleted. This observation is in agreement with the increase of NT secretion found in BON cells transfected with PKD{Delta}PH without PMA stimulation (Fig. 2A). Similar levels of endogenous (endo) phosphorylated PKD (~115 kDa) were detected in all BON cells treated with PMA, showing that stimulation of endogenous PKD by PMA was not affected by exogenous PKD. Total endogenous PKD was reprobed as a loading control (Fig. 2B, bottom panel). Taken together, these results further support the findings that activation of overexpressed PKD enhances PMA-mediated NT secretion.

To confirm the above results of PKD phosphorylation, we directly assayed the activity of PKD using the same cell lysates as above by in vitro kinase assays of GFP immunoprecipitates (Fig. 2C). PKD activity in immunocomplexes was determined by phosphorylation of syntide-2, a synthetic peptide reported to be an excellent substrate for PKD (47) (lower bands), as well as by autophosphorylation (upper bands). Extracts from BON cells transfected with the empty vector showed a basal level of syntide-2 (with or without stimulation); PKDWT activity was increased in the absence of PMA compared with the basal level of syntide-2, suggesting the existence of weak kinase activity in the wild-type PKD even without PMA stimulation. The phosphorylation of syntide-2 was enhanced by PMA in BON cells transfected with PKDWT, further demonstrating the stimulation of PKD by PMA. BON cells expressing constitutively active PKD exhibited a similar level of syntide-2 phosphorylation as noted with PKDWT. With PMA stimulation, the activity of both PKDWT and PKD{Delta}PH was increased with PKDWT being more pronounced. These results are consistent with the findings of NT release following transfection with these constructs (Fig. 2A). Increased phosphorylation of syntide-2 was not noted in BON cells transfected with PKD{Delta}PH compared with BON cells transfected with PKDWT in the presence of PMA. This is consistent with the result in Fig. 2A in which NT secretion is lower in BON cells transfected with PKD{Delta}PH than BON cells transfected with PKDWT in the presence of PMA but inconsistent with the result in Fig. 2B in which PKD phosphorylation is much higher in BON cells transfected with PKD{Delta}PH than BON cells transfected with PKDWT. These results may be explained by different reaction conditions in the kinase assay in vitro compared with the in vivo conditions.

PMA Induces Translocation of PKDWT and PKD{Delta}PH in BON Cells—Translocation is considered to be a marker of PKC activation (48). To further confirm the role of PKD on NT secretion, translocation of PKD tagged with GFP was visualized in BON cells using time-lapse confocal microscopy (Fig. 3). Intense fluorescence was observed in BON cells transfected with PKDWT (top panel). The addition of PMA (100 nM) induced a rapid translocation of PKDWT from the cytosol to the plasma membrane within 1 min after stimulation. These results are consistent with our report that endogenous PKD translocated to the membrane from the cytosol after PMA stimulation in fixed cells (14). In quiescent BON cells transfected with PKD{Delta}PH, the fluorescence was present in the cytosol with intense fluorescence in the nuclei (middle panel). The addition of PMA induced a rapid translocation of PKD{Delta}PH from the cytosol to the plasma membrane; the intense fluorescence noted in the nuclei persisted after PMA treatment. In addition, vesicle-like structures were observed close to the membrane in BON cells transfected with either PKDWT or PKD{Delta}PH (Supplementary video of Fig. 3, middle panel) after addition of PMA. As a control, fluorescence in BON cells transfected with empty vector was observed before and after the addition of PMA (bottom panel); no changes in GFP localization were noted after PMA treatment. These results demonstrate the activation of PKD by PMA and further support the role of PKD in NT secretion.



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  		FIG. 3.
PMA induces translocation of wild-type PKD and constitutively active PKD in BON cells. BON cells were transiently transfected with GFP-tagged wild-type PKD, constitutively active PKD or the empty vector and allowed to recover for 24 h. Cells were then serum-starved in secretion medium overnight. BON cells were incubated in PBS, pH 7.4, and placed inside a prewarmed (37 °C) chamber and imaged in real time as described under "Experimental Procedures." Top, PKDWT in quiescent BON cells was distributed evenly throughout the cytosol of these cells with no apparent association with specific intracellular compartments. PKDWT rapidly translocated to the plasma membrane when PMA was added. Middle, the constitutively active PKD{Delta}PH localized to the cytosol as well as the nucleus. Moreover, PKD{Delta}PH was noted to translocate from the cytosol to the membrane by PMA stimulation. The PKD{Delta}PH in the nucleus failed to translocate to the membrane. Bottom, compared with wild-type PKD, the GFP control vector was noted throughout the cytosol and the nucleus. Translocation was not found in the cells transfected with the empty vector after PMA stimulation. A representative result from three experiments is shown.

 
Classic or Novel PKC and Rho Kinase Inhibitors Attenuate PMA-mediated PKD Activation—PKD is activated in a PKC-dependent fashion in some cell types (23–26). To determine whether PKC isoforms are involved in PMA-induced PKD activation in BON cells, we examined the effect of three PKC inhibitors, Gö6983, GFX, and Ro31–8220, which inhibit activity of classic and novel PKC isoforms but not PKD (49, 50) (Fig. 4). 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. 4A, Gö6983, GFX, and Ro31-8220 completely blocked PKD transphosphorylation at Ser744/748 (upper panel) and significantly attenuated PKD autophosphorylation at Ser916 (middle panel), suggesting the requirement of upstream PKC isoforms in the activation of PKD. We examined whether ROK inhibitors, Y27632 and HA1077 (both at a concentration of 15 µM), affect the activation of PKD. Both inhibitors blocked significantly attenuated PKD phosphorylation with the most pronounced inhibition noted with the Y27632 compound. These findings demonstrate the involvement of upstream ROK in the activation of PKD. Total PKD was probed to assess loading equality (bottom panel).



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  		FIG. 4.
PMA induces PKD activation through a PKC and ROK-dependent pathway. Cells were serum-starved in secretion medium overnight and pretreated with vehicle (0.1% Me2SO) or inhibitors for 30 min and then treated with 10 nM PMA in combination with inhibitors for another 10 min. A, PKD activation was determined by Western blot analysis using the phospho-PKD antibodies, including Ser744/748 (upper panel) and Ser916 (middle panel). PKD was examined as a loading control (bottom panel). B, PKD activation was determined by in vitro kinase assays using syntide-2 as substrate. A representative experiment from five experiments is shown.

 
To further confirm these results, in vitro kinase assays were performed (Fig. 4B). The same cell extracts above were immunoprecipitated with PKD antibody, and the catalytic activity was then assayed with syntide-2 as an exogenous substrate (47). PKD activity was increased by PMA stimulation compared with the activity without PMA treatment. Activation of PKD was attenuated by all three PKC inhibitors as well as the two ROK inhibitors, particularly the Y27632 compound, which is consistent with the results shown in Fig. 4A. Taken together, these results suggest that PKC and Rho kinase are required for PMA-induced PKD activation in BON cells.

PKD Activation Is PKC-{alpha}- and -{delta}-dependent—We have reported that PMA-mediated translocation of PKC-{alpha} and -{delta} from the cytosol to the membrane associated with PMA-mediated NT secretion (14), suggesting that PKC-{alpha} and -{delta} might act as upstream kinases for PKD. We first examined whether PKC-{alpha} contributed to PMA-induced PKD activation by overexpression of wild-type PKC-{alpha} (Fig. 5A). Phosphorylation of PKD was up-regulated in BON cells overexpressing wild-type PKC-{alpha} compared with the control vector in the presence of PMA (top). The membrane was stripped and reprobed with PKD antibody to monitor the loading equality (middle). The membrane was reprobed with anti-HA antibody to show the overexpression of PKC-{alpha} (bottom). Regulation of PKC-{alpha}-mediated PKD phosphorylation was further confirmed using PKC-{alpha} siRNA (Fig. 5B). BON cells were transfected with the PKC-{alpha} siRNA and the control siRNA; cells were lysed at 48 h after transfection. Western blotting using anti-PKC-{alpha} antibody was performed to confirm PKC-{alpha} inhibition (top). The blot was reprobed with anti-PKD (Ser744/748) antibody; PKD phosphorylation was markedly reduced by PKC-{alpha} siRNA (middle). The membrane was stripped and reprobed with PKD antibody to monitor the loading equality (bottom).



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  		FIG. 5.
PKD activity is PKC-{alpha}- and -{delta}-dependent. BON cells were transiently transfected with HA-tagged wild-type PKC-{alpha}, -{delta} or their empty vector (pTB701-HA) and allowed to recover for 24 h. Cells were then serum-starved in secretion medium overnight and treated with vehicle (0.1% Me2SO) or 10 nM PMA for 10 min. A, effects of wild-type PKC-{alpha} on PMA-induced PKD activation were examined using phospho-PKD (Ser744/748) antibody (upper panel). PKD was blotted as a loading control (middle panel). Overexpression of PKC-{alpha} was detected using anti-HA antibody (lower panel). B, top, Western blots were performed using anti-PKC-{alpha} antibody to monitor the inhibition of PKC-{alpha} expression by PKC-{alpha} siRNA; middle, inhibition of PKC-{alpha} siRNA on PMA-induced PKD activation was examined using phospho-PKD (Ser744/748) antibody. Bottom, PKD protein level was determined using anti-PKD antibody as a loading control. C, effects of wild-type PKC-{delta} on PMA-induced PKD activation were examined using phospho-PKD (Ser744/748) antibody (upper panel). PKD was blotted as a loading control (middle panel). Overexpression of PKC-{delta} was detected using anti-HA antibody (lower panel). D, top, Western blots were performed using anti-PKC-{delta} antibody to monitor the inhibition of PKC-{delta} expression by PKC-{delta} siRNA; middle, inhibition of PKC-{delta} siRNA on PMA-induced PKD activation was examined using phospho-PKD (Ser744/748) antibody. Bottom, PKD protein level was determined using anti-PKD antibody as a loading control.

 
Similar experiments were performed to determine the regulation of PKC-{delta} on PKD phosphorylation (Fig. 5C). Overexpression of wild-type PKC-{delta} dramatically up-regulated PKD phosphorylation compared with the control vector in the presence of PMA (top). The loading equality was controlled by probing for total PKD expression using the same membrane (middle). The membrane was reblotted with anti-HA antibody to confirm overexpression of PKC-{delta} (bottom). Treatment with PKC-{delta} siRNA, as shown by Fig. 5D, blocked PKC-{delta} expression (top) as well as PKD phosphorylation (middle). Total PKD expression was assessed as the loading control (bottom). This result further confirmed the upstream regulation of PKC-{delta} on PKD activity.

Rho/ROK Pathway Contributes to PMA-mediated NT Secretion—Based on the finding that PKD activation is decreased by ROK-specific inhibitors, we examined whether Rho/ROK pathway is involved in PMA-mediated NT secretion. BON cells were transfected with Clostridium botulinum C3 toxin, which specifically ADP-ribosylates Rho and impairs its function (51), and NT secretion was measured by RIA. C3 toxin significantly decreased PMA-stimulated NT secretion compared with control GST in the presence of PMA (Fig. 6A). To further confirm the involvement of Rho/ROK pathway, the role of ROK{alpha} (downstream effector of Rho proteins) on NT secretion was examined using ROK{alpha} siRNA (Fig. 6B). PMA-mediated NT secretion was decreased in BON cells transfected with ROK{alpha} siRNA compared with the control siRNA transfected BON cells. To extend these findings on the role of ROK{alpha} in PMA-mediated NT secretion, HA-tagged ROK{alpha}, including full-length (ROK{alpha}F/L), constitutively active (ROK{alpha}1–543), and control vector (pXJ40) were transfected into BON cells (Fig. 6C). The expression level was first examined by Western blotting using anti-HA antibody (top). Expression of ROK{alpha}F/L (~180 kDa) and ROK{alpha}1–543 (~60 kDa) was detected in BON cells. As expected, no signal was detected in BON cells transfected with the control vector. NT secretion was measured in BON cells transfected with ROK{alpha}F/L, ROK{alpha}1–543, and the control vector pXJ40 (bottom). Overexpression of ROK{alpha}F/L, but not ROK{alpha}1–543, significantly increased NT secretion compared with the control vector in the presence of 10 nM of PMA. The role of ROK mediating PMA-stimulated NT secretion was further supported using ROK inhibitors (Fig. 6D). BON cells were pretreated with varying dosages of Y27632 and HA1077 for 30 min and then treated with PMA (10 nM). Treatment with Y27632 decreased PMA-mediated NT secretion in a dose-dependent fashion. Compared with Y27632, HA1077 was less effective in blocking PMA-mediated NT secretion; significant inhibition was noted only at a concentration of 15 µM which correlates with the effects on PKD phosphorylation as shown in Fig. 4A. These results strongly support a role for Rho/ROK in PMA-mediated NT secretion in BON cells.

Rho Proteins Regulate PKD Phosphorylation—To determine whether PKD activation is induced by signaling pathway(s) initiated by Rho proteins, BON cells were transfected with C3 toxin and RhoA activity was examined as described under "Experimental Procedures" (Fig. 7A). C3 toxin blocked both basal and PMA-stimulated RhoA activity (upper panel) and significantly attenuated PKD activation induced by PMA (middle panels). Expression of the C3 toxin did not interfere with the expression of PKD (bottom panel). Furthermore, BON cells were transfected with expression vectors encoding wild-type RhoA (RhoAWT), dominant negative RhoA (RhoAN19) or a constitutively active form of RhoA (RhoAQ63L). As shown in Fig. 7B, cells transfected with RhoAQ63L, the constitutively active form, exhibited an increase in PMA-stimulated endogenous PKD activity compared with cells transfected with RhoAWT; PMA-induced PKD phosphorylation was blocked by the overexpression of RhoAN19, the dominant negative form, compared with cells transfected with RhoAWT treated with PMA. These results demonstrate the regulation of Rho/ROK pathway on PKD activation.



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  		FIG. 7.
PKD activity is Rho protein-dependent. A, cells were transfected with GST-C3 toxin or GST and serum-starved overnight and treated with 10 nM PMA for 10 min. Cells were then rapidly lysed, active Rho was pulled down with glutathione S-transferase (GST)-rhotekin Rho binding domain (or RBD), and RhoA was visualized by Western blotting (first panel); inhibition of PKD activation was examined using anti-phospho-PKD (Ser744/748) (second panel) and PKD (Ser916) (third panel) antibodies. PKD was blotted as a loading control (fourth panel). Representative results from three separate experiments are shown. B, BON cells were transiently transfected with pCEFLAU5 encoding RhoAWT, RhoAQ63L, or RhoAN19 for 24 h. Cells were then serum-starved in secretion medium overnight and treated with vehicle (0.1% Me2SO) or 10 nM PMA for 10 min. PKD activity was determined using anti-phospho-PKD (Ser744/748) antibody in Western blotting assays. A representative result from two experiments is shown.

 
PKD siRNA Decreases BBS-stimulated NT Secretion in BON/GRPR Cells—GRP or its amphibian equivalent BBS stimulate the release of all intestinal hormones, including NT (52). To determine whether the effects noted with PMA treatment also occurred with physiologic agents known to stimulate NT release, BON cells stably transfected with the human GRPR tagged with GFP were established. Increased expression of GRPR was noted on membranes in BON cells transfected with GRPR-GFP (Fig. 8A). To determine the role of PKD in BBS-stimulated NT secretion, PKD siRNA was transfected into BON/GRPR-GFP cells (Fig. 8B). 48 h after transfection, cells were treated with either vehicle control (Me2SO) or various concentrations of BBS. Inhibition of PKD expression was noted in BON/GRPR-GFP cells transfected with PKD siRNA compared with PKD expression in BON/GRPR-GFP cells transfected with control siRNA (Fig. 8B, upper panel). Treatment with BBS induced a dose-dependent increase in PKD Ser744/748 phosphorylation in BON/GRPR-GFP cells transfected with control siRNA; PKD Ser744/748 phosphorylation was significantly attenuated in BON/GRPR-GFP cells transfected with PKD siRNA compared with the control siRNA transfected BON/GRPR-GFP cells in the presence of BBS (Fig. 8B, middle panel). PKD Ser744/748 phosphorylation was not detected in the parental BON cells treated with BBS. Blots were reprobed for {beta}-actin as a loading control (Fig. 8B, lower panel). NT secretion was increased with BBS stimulation in a dose-dependent fashion in BON/GRPR-GFP cells transfected with control siRNA. BBS-stimulated NT secretion was significantly reduced in BON/GRPR-GFP cells transfected with PKD siRNA compared with that in BON/GRPR-GFP cells transfected with control siRNA either in the presence or absence of BBS (Fig. 8C). Similar results were obtained using another BON/GRPR-GFP cell clone (data not shown). Collectively, these findings demonstrate that PKD plays a role in NT secretion induced by BBS, a physiologic stimulant of NT release, suggesting that PKD may be a common regulator of stimulus-induced NT secretion.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The novel PKD protein is expressed in certain endocrine cells (14, 20–22); however, to date, there has been little evidence to suggest a role for PKD in hormone secretion. The findings in our present study are the first to identify a role for PKD in stimulated gut peptide release. Either PMA or bryostatin1, which are potent activators of PKC, resulted in the activation and cellular redistribution of PKD. Silencing PKD expression by RNA interference resulted in decreased basal and PMA-stimulated NT secretion. Overexpression of both wild-type and constitutively active PKD increased PMA-mediated NT secretion from BON cells. In addition, GRPR-expressing BON cells demonstrated increased PKD activation and NT secretion in response to the ligand BBS, thus further supporting a role for PKD in the physiological release of NT. Therefore, our current study, utilizing a combinatorial approach (i.e. specific inhibition and overexpression of PKD) demonstrates a critical role for PKD in the signal transduction pathway leading to NT peptide release.

PKD has been implicated in the organization of the Golgi apparatus, regulating the fission of vesicles from the trans-Golgi network (53). PKD is a resident protein of the Golgi compartment in certain cell types (22, 53, 54) where it is involved in constitutive transport processes. In our present study, we found that PKDWT-GFP was evenly distributed in the cytosol of untreated BON cells with no discernable Golgi localization. In the presence of PMA, translocation of PKDWT-GFP was noted to occur from the cytosol to the membrane. Consistent with our findings, Matthews et al. (55) noted that PKD was localized to membranes surrounding cytoplasmic granules and not in the Golgi of RBL 2H3 mast cells after treatment with phorbol ester. This report, in combination with our present study, suggests a role for PKD in stimulated peptide secretion that is distinct from its role in Golgi transport processes.

PKD activation can be mediated by a PKC-dependent signal transduction pathway that involves the phosphorylation of Ser744/748 of PKD within the activation loop of the catalytic domain of PKD and occurs by diverse stimuli including neuropeptides and growth factors (56) in non-endocrine cells. For example, activation of PKD can occur through activation of PKC-{epsilon} in HEK393 cells (22), PKC-{eta} in COS-7 cells (26), and PKC-{theta} in T cells (25). We demonstrate PKD phosphorylation was inhibited in the novel human endocrine cell line BON by pretreatment with PKC inhibitors and by PKC-{alpha} and -{delta} siRNA and enhanced by overexpression of wild-type PKC-{alpha} and -{delta}. Therefore, our findings indicate that downstream activation of PKD is PKC-dependent, particularly PKC-{alpha} and -{delta}, in the BON endocrine cell line. Similar to our results, Tan et al. (23) demonstrated PKC-{delta}-mediated PKD activation was stimulated by thrombin in aortic smooth muscle cells. Recently, Storz et al. (57) reported that PKC-{delta} participates in the activation of PKD in HeLa cells exposed to oxidative stress. To date, although it has been shown that PKD2 is activated via PKC-{alpha} through the CCKB/gastrin receptor in human gastric cancer cells (58), no direct interaction between PKC-{alpha} and PKD has been reported. PKD appears to be a scaffold protein and is an important regulator of diverse intracellular signaling pathways (19); therefore, it is possible that PKD forms a complex with PKC-{alpha} or PKC-{delta} or other proteins mediating NT secretion in BON cells. However, the exact relationship between PKC-{alpha} and PKC-{delta} with PKD will require further investigation.

The Rho family and their upstream or downstream factors have been implicated in various cellular functions such as regulation of actin filament reorganization and exocytosis (27, 28). In mast and chromaffin cells, one or more members of the Rho family play an active role in the exocytotic process (27, 28, 59, 60). In addition, Cdc42 and Rac control regulated secretion in pancreatic {beta}-cells (61). ROK, an effector of Rho proteins but not Rac or Cdc42, is also involved in exocytosis (35, 62). But the mechanisms how Rho family regulates the exocytotic process remains unknown. To our knowledge, our findings demonstrate, for the first time, that Rho/ROK pathway promoted PMA-mediated NT secretion as demonstrated by inhibition of PMA-stimulated NT secretion using the Rho inhibitor C. botulinum C3, ROK{alpha} siRNA, and ROK inhibitors Y27632 and HA1077 or, conversely, by the augmentation of NT secretion with the full-length ROK{alpha}. We also demonstrate for the first time a reduction of PKD activity in intact BON endocrine cells by C3 toxin, dominant negative RhoA and the ROK inhibitors. BON cells express endogenous RhoA and RhoC but not RhoB,2 therefore, our results do not exclude the possible involvement of RhoC since C3 toxin inhibits all three Rho proteins. However, taken together, our results provide strong evidence supporting the activation of Rho/ROK as a pathway leading to PKD activation, which is in agreement with studies demonstrating that activation of PKD occurs through upstream Rho stimulation in response to agonist activation of BBS receptors in COS-7 cells (63).

Increasingly, the interaction of Rho/ROK and PKC signaling pathways has been reported in a variety of cell types. For example, the interaction between PKC and Rho regulates enzyme secretion in pancreatic acini (64). In epithelial and endothelial cells, treatment with C3 toxin prevented PKC translocation and activation (65). Recently, Slater et al. (27, 28) demonstrated that Rho-GTP potently stimulates PKC-{alpha} activity in vitro. In other studies, PKC has been shown to activate Rho (66, 67). We found that treatment of BON cells with PMA stimulated PKC and Rho/ROK pathways, leading to PKD-regulated NT secretion. Rho has been predominantly described in the regulation of actin filament reorganization (27, 28), and PKCs are also known to be important regulators of the cytoskeleton (68). Therefore, it is possible that PKC-{alpha} and PKC-{delta} and Rho/ROK-mediated PKD activation leads to NT secretion through the reorganization of actin filaments. The precise sequence of events culminating in stimulated NT release remains to be fully elucidated.

In conclusion, the results of this study identify the novel PKD protein as a critical regulator of PMA- or BBS-mediated NT secretion from the BON or BON/GRPR cell lines, respectively. We demonstrate that PKD phosphorylation is regulated by upstream PKC-{alpha} and -{delta}, and the Rho/ROK pathway (Fig. 9 summarizes our proposed model for PKD-mediated NT secretion). Our findings identify novel signaling pathways contributing to stimulated peptide secretion from specialized endocrine cells of the GI tract. The coordinated release of intestinal peptides in response to extracellular mediators is essential for the regulation of intestinal digestion, secretion and motility; therefore, our results delineate critical signaling molecules that contribute to this important physiologic process. Interestingly, prominent regulators of PKD and NT secretion, namely PMA-sensitive PKCs (69) and RhoA (70) contribute to tumor progression. Since NT can stimulate proliferation of NTR positive tumors, it is interesting to speculate that pathways, such as RhoA and PKCs, may promote tumorigenesis through the dysregulated secretion of trophic factors such as NT.



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  		FIG. 9.
Model of PKD-regulated NT secretion in BON cells. The solid lines indicate pathways confirmed in this study. The dotted line indicates that PMA may directly activate Rho or, alternatively, may act through PKCs to activate Rho.

 

   FOOTNOTES
 
* This work was supported by Grants 2R37 AG10885, RO1 DK48489, PO1 DK35608, R21 CA10212, and RO1 DK58119 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

The on-line version of this article (available at http://www.jbc.org) contains a Supplementary Video. Back

To whom correspondence should be addressed: The University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-0536. Tel.: 409-772-5612; Fax: 409-747-4819; E-mail: mevers{at}utmb.edu.

1 The abbreviations used are: NT, neurotensin; PKC, protein kinase C; PKD, protein kinase D; HA, hemagglutinin; BBS, bombesin; PMA, phorbol 12-myristate 13-acetate; GST, glutathione S-transferase; RIA, radioimmunoassay; GFP, green fluorescent protein; siRNA, small interfering RNA; GRP, gastrin releasing peptide; WT, wild type. Back

2 J. Li and B. M. Evers, unpublished observations. Back


   ACKNOWLEDGMENTS
 
We thank Jingbo Qiao and Dai H. Chung for the GRPR construct, Robert Carraway (University of Massachusetts) for helpful discussions; Leoncio A. Vergara and Jason Reed for technical assistance; Tatsuo Uchida for statistical analysis; and Eileen Figueroa and Karen Martin for manuscript preparation.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Townsend, C. M., Jr., Bold, R. J., and Ishizuka, J. (1994) Surg. Today 24, 772-777[CrossRef][Medline] [Order article via Infotrieve]
  2. Evers, B. M., Wang, X., Zhou, Z., Townsend, C. M., Jr., McNeil, G. P., and Dobner, P. R. (1995) Mol. Cell. Biol. 15, 3870-3881[Abstract]
  3. Evers, B. M. (2002) World J. Surg. 26, 799-805[CrossRef][Medline] [Order article via Infotrieve]
  4. Armstrong, M. J., Parker, M. C., Ferris, C. F., and Leeman, S. E. (1986) Am. J. Physiol. 251, G823-G829[Medline] [Order article via Infotrieve]
  5. Thor, K., and Rosell, S. (1986) Gastroenterology 90, 27-31[Medline] [Order article via Infotrieve]
  6. Chung, D. H., Evers, B. M., Shimoda, I., Townsend, C. M., Jr., Rajaraman, S., and Thompson, J. C. (1992) Gastroenterology 103, 1254-1259[Medline] [Order article via Infotrieve]
  7. Evers, B. M., Izukura, M., Chung, D. H., Parekh, D., Yoshinaga, K., Greeley, G. H., Jr., Uchida, T., Townsend, C. M., Jr., and Thompson, J. C. (1992) Gastroenterology 103, 86-91[Medline] [Order article via Infotrieve]
  8. Thomas, R. P., Hellmich, M. R., Townsend, C. M., Jr., and Evers, B. M. (2003) Endocr. Rev. 24, 571-599[Abstract/Free Full Text]
  9. Goodge, K. A., and Hutton, J. C. (2000) Semin. Cell Dev. Biol. 11, 235-242[CrossRef][Medline] [Order article via Infotrieve]
  10. Evers, B. M., Ishizuka, J., Townsend, C. M., Jr., and Thompson, J. C. (1994) Ann. N. Y. Acad. Sci. 733, 393-406[Medline] [Order article via Infotrieve]
  11. Carraway, R. E., Mitra, S. P., Evers, B. M., and Townsend, C. M., Jr. (1994) Regul. Pept. 53, 17-29[CrossRef][Medline] [Order article via Infotrieve]
  12. Parekh, D., Ishizuka, J., Townsend, C. M., Jr., Haber, B., Beauchamp, R. D., Karp, G., Kim, S. W., Rajaraman, S., Greeley, G., Jr., and Thompson, J. C. (1994) Pancreas 9, 83-90[Medline] [Order article via Infotrieve]
  13. Zhang, T., Townsend, C. M., Jr., Udupi, V., Yanaihara, N., Rajaraman, S., Beauchamp, R. D., Ishizuka, J., Evers, B. M., Gomez, G., Thompson, J. C., et al. (1995) Endocrinology 136, 2252-2261[Abstract]
  14. Li, J., Hellmich, M. R., Greeley, G. H., Jr., Townsend, C. M., Jr., and Evers, B. M. (2002) Am. J. Physiol. Gastrointest Liver Physiol. 283, G1197-1206[Abstract/Free Full Text]
  15. Hellmich, M. R., Ives, K. L., Udupi, V., Soloff, M. S., Greeley, G. H., Jr., Christensen, B. N., and Townsend, C. M., Jr. (1999) J. Biol. Chem. 274, 23901-23909[Abstract/Free Full Text]
  16. Newton, A. C. (1995) J. Biol. Chem. 270, 28495-28498[Free Full Text]
  17. Toker, A. (1998) Front Biosci. 3, D1134-1147[Medline] [Order article via Infotrieve]
  18. Johannes, F. J., Prestle, J., Eis, S., Oberhagemann, P., and Pfizenmaier, K. (1994) J. Biol. Chem. 269, 6140-6148[Abstract/Free Full Text]
  19. Rykx, A., De Kimpe, L., Mikhalap, S., Vantus, T., Seufferlein, T., Vandenheede, J. R., and Van Lint, J. (2003) FEBS Lett. 546, 81-86[CrossRef][Medline] [Order article via Infotrieve]
  20. Tian, Y. M., Urquidi, V., and Ashcroft, S. J. (1996) Mol. Cell. Endocrinol. 119, 185-193[CrossRef][Medline] [Order article via Infotrieve]
  21. Yaney, G. C., Fairbanks, J. M., Deeney, J. T., Korchak, H. M., Tornheim, K., and Corkey, B. E. (2002) Am. J. Physiol. Endocrinol. Metab. 283, E880-888[Abstract/Free Full Text]
  22. Moore, E. D., Ring, M., Scriven, D. R., Smith, V. C., Meloche, R. M., and Buchan, A. M. (1999) J. Biol. Chem. 274, 22493-22501[Abstract/Free Full Text]
  23. Tan, M., Xu, X., Ohba, M., Ogawa, W., and Cui, M. Z. (2003) J. Biol. Chem. 278, 2824-2828[Abstract/Free Full Text]
  24. Brandlin, I., Eiseler, T., Salowsky, R., and Johannes, F. J. (2002) J. Biol. Chem. 277, 45451-45457[Abstract/Free Full Text]
  25. Yuan, J., Bae, D., Cantrell, D., Nel, A. E., and Rozengurt, E. (2002) Biochem. Biophys. Res. Commun. 291, 444-452[CrossRef][Medline] [Order article via Infotrieve]
  26. Brandlin, I., Hubner, S., Eiseler, T., Martinez-Moya, M., Horschinek, A., Hausser, A., Link, G., Rupp, S., Storz, P., Pfizenmaier, K., and Johannes, F. J. (2002) J. Biol. Chem. 277, 6490-6496[Abstract/Free Full Text]
  27. Slater, S. J., Seiz, J. L., Stagliano, B. A., and Stubbs, C. D. (2001) Biochemistry 40, 4437-4445[CrossRef][Medline] [Order article via Infotrieve]
  28. Slater, S. J., Cook, A. C., Seiz, J. L., Malinowski, S. A., Stagliano, B. A., and Stubbs, C. D. (2003) Biochemistry 42, 12105-12114[CrossRef][Medline] [Order article via Infotrieve]
  29. Ridley, A. J. (2001) Trends Cell Biol. 11, 471-477[CrossRef][Medline] [Order article via Infotrieve]
  30. Amin, R. H., Chen, H. Q., Veluthakal, R., Silver, R. B., Li, J., Li, G., and Kowluru, A. (2003) Endocrinology 144, 4508-4518