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Originally published In Press as doi:10.1074/jbc.M409431200 on December 28, 2004

J. Biol. Chem., Vol. 280, Issue 9, 8351-8357, March 4, 2005
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Myristoylated Alanine-rich C Kinase Substrate-mediated Neurotensin Release via Protein Kinase C-{delta} Downstream of the Rho/ROK Pathway*

Jing Li{ddagger}, Kathleen L. O'Connor{ddagger}§, George H. Greeley, Jr.{ddagger}, Perry J. Blackshear¶, Courtney M. Townsend, Jr.{ddagger}, and B. Mark Evers{ddagger}§||

From the Department of {ddagger}Surgery and §Sealy Center for Cancer Cell Biology, The University of Texas Medical Branch, Galveston, Texas 77555 and the Office of Clinical Research and Laboratory of Neurobiology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709

Received for publication, August 17, 2004 , and in revised form, December 16, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Myristoylated alanine-rich protein kinase C substrate (MARCKS) is a cellular substrate for protein kinase C (PKC). Recently, we have shown that PKC isoforms-{alpha} and -{delta}, 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-{delta} siRNA, ROK{alpha} siRNA, and C3 toxin (a Rho protein inhibitor), suggesting that the PKC-{delta} and the Rho/ROK pathways are necessary for MARCKS activation. The phosphorylation of PKC-{delta} was inhibited by C3 toxin, demonstrating that the role of MARCKS in NT secretion was regulated by PKC-{delta} 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-{delta} proteins, in stimulated gut peptide secretion.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 (1, 2). 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),1 that are produced in enteroendocrine cells (N cells) localized predominantly in the distal small bowel (3). NT facilitates fatty acid translocation (4), affects gut motility (5), and stimulates growth of normal gut mucosa (6, 7) as well as certain pancreatic, colonic, and prostatic cancers bearing NT receptors (8). 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 (9). Using the BON cell line, we have shown that protein kinase C (PKC), particularly PKC isoforms-{alpha} and -{delta}, play a role in the release of NT mediated by the phorbol ester, phorbol 12-myristate 13-acetate (PMA) (10). 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 (11, 12).

The MARCKS (myristoylated alanine-rich C kinase substrate) family of proteins was first identified as prominent substrates of PKC (13, 14). MARCKS has been implicated in cell motility, phagocytosis, membrane trafficking, and mitogenesis (15, 16). Moreover, MARCKS has been implicated as a key molecule regulating mucin exocytosis (17-19) and may play a role in phorbol ester-stimulated platelet secretion (20, 21). Phosphorylation of MARCKS induces catecholamine release (22-24) and noradrenaline release (25-27). With regards to hormone secretion, phosphorylation of MARCKS protein is required for oxytocin exocytosis in bovine large luteal cells (28, 29). Arginine vasopressin-induced induction in MARCKS phosphorylation may be involved in initiating the exocytosis of adrenocorticotropin (30-32). Furthermore, in isolated pancreatic islets, phosphorylation of MARCKS is increased by administration of either glucose or carbachol (33). 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-{delta} siRNA, ROK{alpha} siRNA, and C3 toxin (a Rho protein inhibitor). The phosphorylation of PKC-{delta} 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-{delta} and ROK{alpha} siRNA, demonstrating the dependence of MARCKS activity on PKC-{delta} and ROK{alpha}. Importantly, our results identify a role for MARCKS activation in the stimulated release of the intestinal peptide NT through sequential regulation by PKC-{delta} and Rho/ROK proteins.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
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-{beta}-actin antibodies were from Sigma. The anti-human MARCKS, and PKC-{delta} polyclonal antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-phospho-MARCKS (Ser-152/156) and PKC-{delta} (Thr-505) antibodies were from Cell Signaling Technology (Beverly, MA). The anti-ROK{alpha} 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 (34). MARCKS, ROK{alpha}, PKC-{alpha}, -{delta}, 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 (9). 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 (11) 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 (35).

Protein Preparation and Western Blotting—Protein preparation and Western blotting were performed as described previously (10). 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 x63, 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 SASTM system, Release 8.2 (36).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
MARCKS siRNA Inhibits PMA-mediated NT Secretion and MARCKS Expression in BON Cells—Based on findings that PKC-{alpha} and -{delta} regulated PMA-mediated NT secretion in BON cells (10), we determined whether MARCKS, a major PKC substrate that has been implicated in the secretion and membrane trafficking of a number of cell types (17, 28, 37), 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 {beta}-actin demonstrating equal loading (Fig. 1B, bottom).



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  		FIG. 1.
Inhibition of NT secretion by MARCKS siRNA in BON cells. A, BON cells were transfected with MARCKS siRNA or control siRNA. Forty-eight h after, BON cells were serum starved in secretion medium for 30 min and treated with vehicle (Me2SO) or PMA (10 nM) for another 30 min. Medium was collected and NT secretion was measured by RIA. Experiments were performed in triplicate. Results are expressed as mean ± S.E. (n = 6); *, p < 0.05 versus control siRNA; {dagger}, p < 0.05 versus control siRNA plus PMA. B, BON cells were lysed and Western blot analysis was performed for the expression of MARCKS (top); {beta}-actin protein expression was detected to monitor equal loading (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.



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  		FIG. 2.
Increase of NT secretion by overexpression of wild-type MARCKS. A, BON cells were transiently transfected with wild-type MARCKS (WT), phosphorylation mutant MARCKS (A/S), and myristoylation mutant MARCKS (A2/G2). Forty-eight h after transfection, BON cells were serum starved in secretion medium for 30 min and treated with vehicle (Me SO) or PMA (10 nM) for another 30 min. Medium was collected and NT secretion was measured by RIA. Results are expressed as mean ± S.E. (n2 = 6); *, p < 0.05 versus empty vector (pEGFP-N-1); {dagger}, p < 0.05 versus empty vector plus PMA. B, BON cells were lysed and Western blot analysis was performed to demonstrate the overexpression of MARCKS using anti-GFP antibody (bottom). The membrane was stripped and reblotted using an anti-phospho-MARCKS (Ser152/156) antibody. The upper signals demonstrate exogenous (Exo) phospho-MARCKS; the lower signals demonstrate endogenous (Endo) phospho-MARCKS. Representative results from three experiments are shown.

 
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.



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  		FIG. 3.
Translocation of endogenous MARCKS from membrane to the cytosol of BON cells stimulated by PMA. BON cells were grown on glass coverslips in 24-well plates for 3 days. Cells were incubated for 30 min with secretion medium, treated with vehicle (Me2SO) or PMA (10 nM) for 30 min. Cells were washed three times in PBS, fixed with 4% paraformaldehyde, and incubated with anti-MARCKS antibody (1:100) 1 h at room temperature or overnight at 4 °C followed by Alexa 568-conjugated secondary antibody (1:500) (red) for 30 min in the dark at room temperature. Cells were mounted with mounting medium supplemented with 4'-6-diamidino-2-phenylindole showing the nuclei (blue). Images were recorded by fluorescent microscopy and representative results from at least three separate experiments are shown. Endogenous MARCKS was expressed on the membrane (arrowheads) of vehicle (Me2SO)-treated BON cells and translocated to the cytosol (arrows) of PMA-treated BON cells.

 
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.



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  		FIG. 4.
Translocation of exogenous MARCKS in BON cells stimulated with PMA. BON cells were transiently transfected with GFP-tagged wild-type MARCKS (WT), phosphorylation mutant MARCKS (A/S), myristoylation mutant MARCKS (A2/G2), or the empty vector (pEGFP-N-1) for 3 days. Cells were then serum starved in secretion medium for 30 min. BON cells were incubated in PBS and placed inside a pre-warmed (37 °C) chamber and imaged in real time as described under "Experimental Procedures." The images were chosen from the serial pictures before or after the addition of PMA at 5, 10, and 15 min. Upper panel, BON cells transfected with the empty vector. Second panel, BON cells transfected with wild-type MARCKS. Third panel, BON cells transfected with phosphorylation mutant MARCKS (A/S). Lower panel, BON cells transfected with myristoylation mutant MARCKS (A2/G2). A representative result from three experiments is shown.

 
Activation of MARCKS Is Dependent on PKC-{delta}, but Not PKC-{alpha} or PKD—MARCKS is known to be a PKC substrate in some cell types (13). Based on our previous studies in which we found that PKC-{alpha} and -{delta} 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 (38, 39), and Rottlerin, which is a selective PKC-{delta} inhibitor (38, 39) (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 {beta}-actin as a loading control (lower panel).



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  		FIG. 5.
Regulation of MARCKS phosphorylation by PKC-{delta} in BON cells. A, cells were serum starved in secretion medium for 24 h and pretreated with vehicle (Me2SO) or inhibitors for 30 min and then treated with PMA (10 nM) in combination with inhibitors for another 10 min. MARCKS activation was determined by Western blot analysis using the phospho-MARCKS (Ser152/156) antibody (upper panel). MARCKS was examined as a loading control (bottom panel). B, BON cells were transfected with PKC-{delta} siRNA or the control siRNA. Upper panel, inhibition of PKC-{delta} expression by PKC-{delta} siRNA was shown by Western blotting using anti-PKC-{delta} antibody. Middle panel, MARCKS phosphorylation was examined using phospho-MARCKS (Ser152/156) antibody. Bottom panel, {beta}-actin was determined as a loading control. A representative result from three experiments is shown.

 
We next used siRNA against PKC-{alpha}, PKC-{delta}, or PKD to further delineate the proteins that contribute to MARCKS activation (Fig. 5B). BON cells were transfected with PKC-{alpha}, PKC-{delta}, 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-{alpha}, PKC-{delta}, and PKD siRNA significantly inhibited PKC-{alpha} (data not shown), PKC-{delta} (Fig. 5B, top panel), and PKD (data not shown) expression, respectively. Importantly, MARCKS phosphorylation was not decreased by either PKC-{alpha} or PKD siRNA (data not shown) but markedly decreased by PKC-{delta} siRNA (Fig. 5B, middle panel). Membranes were reprobed with {beta}-actin demonstrating equal loading (Fig. 5B, bottom panel). Taken together, the results suggest that MARCKS protein is a downstream effector of PKC-{delta} 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 (37). In a previous study, we demonstrated that the Rho/ROK pathway contributed to PMA-mediated NT secretion in BON cells (11). 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).



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  		FIG. 6.
Regulation of MARCKS activity by the Rho/ROK pathway mediated through PKC-{delta}. A, cells were serum starved in secretion medium for 24 h and pretreated with vehicle (Me2SO) or inhibitors for 30 min and then treated with PMA (10 nM) in combination with inhibitors for another 10 min. MARCKS phosphorylation was determined by Western blot analysis using the phospho-MARCKS (Ser-152/156) antibody (upper panel). MARCKS was examined as a loading control (bottom panel). B, BON cells were transfected with ROK{alpha} siRNA or the control siRNA. Upper panel, inhibition of ROK{alpha} expression by ROK{alpha} siRNA was shown by Western blotting using anti-ROK{alpha} antibody. Middle panel, MARCKS phosphorylation induced by PMA was examined using phospho-MARCKS (Ser-152/156) antibody. Bottom panel, {beta}-actin was determined as a loading control. C, BON cells were transfected with 100 µg of GST or GST-C3 toxin proteins overnight and serum starved for 30 min. MARCKS phosphorylation was determined by Western blot analysis using the phospho-MARCKS (Ser-152/156) antibody (upper panel). MARCKS was examined as a loading control (bottom panel). D, BON cells were transfected with 100 µg of GST or GST-C3 toxin proteins overnight and serum starved for 30 min. PKC-{delta} phosphorylation was shown using the phospho-PKC-{delta} (Thr-505) antibody (upper panel). The membrane was stripped and reprobed with PKC-{delta} antibody as a loading control (bottom panel). A representative result from three experiments is shown.

 
To further confirm the involvement of the Rho/ROK pathway, the role of ROK{alpha} (an isoform of ROK proteins) on MARCKS phosphorylation was examined using ROK{alpha} siRNA (Fig. 6B). The inhibition of ROK{alpha} expression was shown by Western blot analysis (Fig. 6B, top panel). PMA-mediated MARCKS phosphorylation was decreased in BON cells transfected with ROK{alpha} siRNA compared with the control siRNA (Fig. 6B, middle panel). The blot was reprobed with {beta}-actin as a loading control (Fig. 6B, bottom panel). These results further indicate the regulation of MARCKS activity by ROK{alpha}.

Clostridium botulinum C3 toxin specifically ADP-ribosylates Rho and impairs its function (40). We previously showed that C3 toxin inhibited PMA-mediated NT secretion in BON cells (11). 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 attenuated PMA-stimulated MARCKS phosphorylation (Fig. 6C, top panel). Expression of C3 toxin did not interfere with the expression of MARCKS (Fig. 6C, bottom panel). Taken together, these results demonstrate regulation of MARCKS activity through the Rho/ROK pathway.

To further delineate the regulated sequence of PKC-{delta} and Rho/ROK on MARCKS activity, we examined the effect of C3 toxin on PKC-{delta} phosphorylation (Fig. 6D). BON cells were transfected with C3 toxin overnight and treated with vehicle (Me2SO) or PMA (10 nM). Western blot analysis was performed using a phospho-PKC-{delta} (Thr-505) antibody. C3 toxin significantly attenuated PMA-stimulated PKC-{delta} phosphorylation (Fig. 6D, top panel). Expression of C3 toxin did not interfere with the expression of PKC-{delta} (Fig. 6D, bottom panel). These findings suggest the regulation of MARCKS phosphorylation by PKC-{delta} downstream of the Rho/ROK pathway.

MARCKS Protein Is Also Involved in BBS-stimulated NT Secretion from BON Cells Stably Transfected with GRPR—The hormone GRP (the human equivalent of BBS) stimulates the release of many gastrointestinal hormones, including NT (12). BBS-stimulated MARCKS phosphorylation was previously demonstrated in fibroblasts transfected with BBS/GRP receptor (41) or in intact Swiss 3T3 fibroblasts (42). To further determine whether MARCKS also contributes to NT secretion mediated by BBS, two clones of the stable cell line BON/GRPR were used. As shown in Fig. 7A, NT secretion from BON/GRPR cells was significantly increased by BBS (100 nM) treatment (Fig. 7A, upper panel). MARCKS phosphorylation was further demonstrated by Western blot analysis; total MARCKS was examined as a loading control (Fig. 7A, lower panel). Next, BON/GRPR cells were transfected with MARCKS siRNA and treated with BBS (100 nM) (Fig. 7B). NT secretion was increased by BBS treatment in BON/GRPR cells transfected with control siRNA. However, BBS-stimulated NT release was significantly attenuated in BON/GRPR cells transfected with MARCKS siRNA compared with BON/GRPR cells transfected with control siRNA in the presence of BBS (Fig. 7B, upper panel). Western blot analysis showed that the expression of MARCKS was decreased in BON/GRPR cells transfected with MARCKS siRNA compared with BON/GRPR cells transfected with the control siRNA (Fig. 7B, lower panel).



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  		FIG. 7.
MARCKS is involved in BBS-stimulated NT secretion from BON/GRPR cells. A, BON/GRPR cells were serum starved for 5 h and treated with BBS (100 nM) for 30 min. The medium was collected for NT assay (upper panel). Results are expressed as mean ± S.E. (n = 6). *, p < 0.05 versus control. The cells were lysed for MARCKS phosphorylation by Western blot using anti-MARCKS (Ser152/156) antibody; total MARCKS was determined as a loading control (lower panel, bottom). B, BON/GRPR cells were transfected with control siRNA or MARCKS siRNA. Seventy-two h later, cells were treated with or without BBS (100 nM) for 30 min. The medium was collected for NT assay (upper panel). Results are expressed as mean ± S.E. (n = 6). *, p < 0.05 versus control siRNA (-); {dagger}, p < 0.05 versus control siRNA and BBS (100 nM). Cells were lysed for MARCKS expression by Western blots using anti-MARCKS antibody. The membranes were reblotted with {beta}-actin as a loading control (lower panel). C, BON/GRPR cells were transfected with control siRNA, PKC-{delta}, or ROK{alpha} siRNA. Seventy-two h later, cells were treated with or without BBS (100 nM) for 30 min. Upper panel, Western blots were performed using anti-PKC-{delta} antibody to monitor the inhibition of expression by siRNA; MARCKS phosphorylation was detected using anti-MARCKS (Ser-152/156) antibody; {beta}-actin expression was determined as a loading control. Lower panel, Western blots were performed using anti-ROK{alpha} antibody to monitor the inhibition of expression of ROK{alpha} by siRNA; MARCKS phosphorylation was detected using anti-MARCKS (Ser152/156) antibody; {beta}-actin expression was determined as a loading control. (In all of the studies, representative data from one BON/GRPR clone are shown; similar results were noted with another clone (data not shown).)

 
To further determine whether PKC-{delta} and ROK are involved in regulation of MARCKS phosphorylation in BON/GRPR cells, PKC-{delta} or ROK{alpha} siRNA were transfected into the BON/GRPR cells and protein analyzed by Western blot (Fig. 7C). Expression of PKC-{delta} and MARCKS phosphorylation was decreased in BON/GRPR cells transfected with PKC-{delta} siRNA compared with control cells transfected with control siRNA treated with BBS; the blots were reprobed for {beta}-actin as a loading control (Fig. 7C, upper panel). Similar results were obtained in BON/GRPR cells transfected with ROK{alpha} siRNA. Expression of ROK{alpha} and MARCKS phosphorylation was decreased in BON/GRPR cells transfected with ROK{alpha} siRNA compared with control cells transfected with control siRNA treated with BBS (Fig. 7C, lower panel). All experiments were repeated using another BON/GRPR cell clone with similar results (data not shown). Taken together, these results, using the physiologic stimulant BBS, further demonstrate that MARCKS significantly contributes to stimulated NT secretion. MARCKS phosphorylation is PKC-{delta} and ROK{alpha}-dependent, which is similar to that noted with PMA-mediated NT secretion.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Proteins of the MARCKS family play critical roles in diverse cellular functions, including the regulation of exocytosis in certain cells (17-32, 43). Despite the obvious importance of MARCKS in the regulation of secretion, the intracellular signaling mechanisms linking MARCKS to secretion have not been entirely elucidated. In this study, we provide evidence that links the activation of MARCKS to stimulated gut peptide release. Treatment of BON endocrine cells with PMA, a potent activator of PKC, resulted in the activation and cellular redistribution of MARCKS. Silencing MARCKS expression by RNA interference significantly decreased PMA-stimulated NT secretion. Conversely, overexpression of wild-type MARCKS increased PMA-mediated NT secretion. We show that Rho inhibitor C3, ROK inhibitors, ROK siRNA, and PKC-{delta} siRNA inhibited the activity of MARCKS. Moreover, PKC-{delta} phosphorylation was decreased by C3 treatment, suggesting that the Rho/ROK pathway lies upstream of PKC-{delta} activation. In addition, we treated BON/GRPR cells with BBS, a physiologic stimulant of NT release, and demonstrate that, similar to PMA-mediated effects, MARCKS protein contributes to NT secretion and is regulated through PKC-{delta} and ROK{alpha}. Therefore, we conclude that the role of MARCKS in NT secretion was regulated by the Rho/ROK/PKC-{delta} pathway.

Several studies have reported a role of MARCKS in the regulation of hormone peptide release or neurosecretion. For example, wild-type MARCKS protein induces the peripheral movement of oxytocin vesicles in bovine large luteal cells in response to prostaglandin F2{alpha}, indicating the involvement of MARCKS in oxytocin secretion (28, 29, 43). The phosphorylation of MARCKS was spatiotemporally associated with the release of adrenocorticotropin in the ovine anterior pituitary by arginine vasopressin (30, 31), suggesting that MARCKS phosphorylation may be involved in this process. The involvement of MARCKS in aldosterone secretion from bovine adrenal glomerulosa cells was also analyzed (32). MARCKS phosphorylation was increased upon stimulation with PMA in chromaffin cells; MARCKS phosphorylation site domain sequence (MPSD), a peptide with amino acid sequence corresponding to the MARCKS phosphorylation site, inhibited Ca2+-evoked secretion, providing the first evidence for MARCKS involvement in chromaffin cell secretion (22). Many of these studies demonstrate the association of MARCKS phosphorylation with secretion, but do not provide direct evidence showing the role of MARCKS in hormone secretion. Moreover, some of these reports utilized permeabilized cells as secretion models. In our present study, NT secretion was analyzed in intact BON cells and siRNA directed to MARCKS was employed to suppress MARCKS expression and assess the functional consequences of this effect, thus providing direct and reliable evidence demonstrating the role of MARCKS in hormone peptide secretion.

MARCKS is a well known substrate of conventional as well as novel PKC isoforms (44). MARCKS mediates selected signal transduction events downstream of PKC pathways. For example, Salli and Stormshak (43) demonstrated that prostaglandin F2{alpha}-induced secretion of oxytocin involved the phosphorylation of MARCKS protein. In these cells, treatment with prostaglandin F2{alpha} increased membrane association of PKC-{alpha} but no direct evidence was shown to demonstrate phosphorylation of MARCKS by PKC-{alpha}. Uberall et al. (45) showed that expression of constitutively active mutants of either PKC-{alpha} or PKC-{epsilon} increased phosphorylation of endogenous MARCKS in intact mouse fibroblasts without stimulation, suggesting the regulation of MARCKS by PKC-{alpha} or PKC-{epsilon}. MARCKS phosphorylation, regulated by PKC-{epsilon}, was involved in prolactin secretion in rat pituitary cells (46, 47). In our present study, we found that PKC-{delta} regulates MARCKS phosphorylation in BON cells, as noted by the inhibition of MARCKS phosphorylation by PKC-{delta} siRNA, but not by PKC-{alpha} siRNA. Taken together, MARCKS phosphorylation can occur via different PKC subtypes, and this regulation appears to be cell type or function specific.

Our findings provide direct evidence to suggest the regulation of MARCKS activity by the interaction of PKC and Rho/ROK pathways, culminating in stimulated NT secretion. Both PKC (48-53) and Rho family (54-60) pathways have been separately implicated in the regulation of secretion from neuroendocrine cells; however, the interaction between the two molecules has not been previously reported in neuroendocrine cells. In human neuronal cells, MARCKS phosphorylation at Ser-159 (a site recognized by PKC) is induced by lysophosphatidic acid, interleukin-1{beta}, bradykinin, or GTP{gamma}S, which activates Rho protein with the resultant activation of ROK (37, 61, 62). In these studies MARCKS phosphorylation was sensitive to the ROK inhibitors HA1077 and H1152 or C3 toxin, suggesting MARCKS phosphorylation is Rho/ROK pathway-dependent. However, the phosphorylation induced by phorbol 12,13-dibutyrate was only minimally sensitive to the ROK inhibitors, suggesting that it is PKC-dependent and that the Rho/ROK pathway was not involved. Collectively, these studies demonstrate that MARCKS may be phosphorylated by either the PKC or the Rho/ROK pathways separately. In our present study, we demonstrated regulation of PMA-stimulated MARCKS phosphorylation at serine 152/156, which also represents a PKC phosphorylation site. This MARCKS phosphorylation was sensitive to the ROK inhibitors, Y27632 and HA1077, and Rho protein inhibitor C3 and ROK{alpha} siRNA as well as to PKC inhibitors and PKC-{delta} siRNA, suggesting the involvement of both Rho/ROK and PKC pathways. Furthermore, our data suggest that Rho/ROK-stimulated MARCKS phosphorylation is mediated by PKC-{delta} as demonstrated by the sensitivity of PKC-{delta} phosphorylation to C3 toxin. These data may help to explain how a PKC-consensus phosphorylation site in MARCKS (Ser-159) reported previously (37, 61, 62) could be regulated by Rho/ROK.

Phosphorylation of MARCKS in vitro by the novel serinethreonine kinase PKD has also been reported (63). In addition to PKC-{alpha} and -{delta}, we have also shown that PMA-induced activation of PKD stimulates NT secretion in BON cells (10, 11). In the present study, treatment with siRNA to PKD did not inhibit MARCKS phosphorylation, suggesting that PKD-mediated NT secretion is through separate downstream pathways and does not involve MARCKS protein. This supposition is further supported by our results demonstrating that PMA-stimulated NT secretion was decreased ~50% in BON cells transfected with MARCKS siRNA despite complete inhibition of MARCKS protein expression. Therefore, from the collective findings in our current study and our previous reports (10, 11), it appears that multiple signaling proteins are involved in the concerted release of NT peptide. These coordinated pathways involve both MARCKS-dependent as well as MARCKS-independent activation.

In conclusion, using a novel human endocrine cell line BON, we identify an important role for MARCKS protein in stimulated gut peptide release. Silencing MARCKS expression by RNA interference resulted in decreased NT secretion mediated by treatment with either PMA (in wild type BON cells) or BBS (in BON/GRPR cells). Overexpression of wild-type MARCKS increased NT secretion from BON cells. MARCKS phosphorylation was regulated by PKC-{delta} downstream of the Rho/ROK pathway. Our findings identify a novel mechanism of gut peptide release involving phosphorylation of MARCKS protein by the sequential regulation of Rho/ROK and PKC-{delta} signaling pathways.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants 2R37 AG10885, RO1 DK48489, PO1 DK35608, and R21 CA10212. This paper was presented, in part, at the annual meeting of the American Gastroenterological Association (May 18-24, 2004, New Orleans, LA) and was previously published in abstract form (64). 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

|| 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; 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; GTP{gamma}S, guanosine 5'-({gamma}-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. Back


   ACKNOWLEDGMENTS
 
We thank Jingbo Qiao and Dai H. Chung for the GRPR construct, Kim Chau, 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. Guyton, A. C. (2000) in Textbook of Medical Physiology (Guyton, A. C., ed) pp. 738-753, W. B. Saunders, Philadelphia
  2. Townsend, C. M., Jr., Bold, R. J., and Ishizuka, J. (1994) Surg. Today 24, 772-777[CrossRef][Medline] [Order article via Infotrieve]
  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
  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. 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]
  10. Li, J., Hellmich, M. R., Greeley, G. H., Jr., Townsend, C. M., Jr., and Evers, B. M. (2002) Am. J. Physiol. 283, G1197-G1206
  11. Li, J., O'Connor, K. L., Hellmich, M. R., Greeley, G. H., Jr., Townsend, C. M., Jr., and Evers, B. M. (2004) J. Biol. Chem. 279, 28466-28474[Abstract/Free Full Text]
  12. Guo, Y. S., and Townsend, C. M., Jr. (1999) in Gastrointestinal Endocrinology (Greeley, G. H., Jr., ed) pp. 189-214, Humana Press Inc., Totowa, NJ
  13. Blackshear, P. J. (1993) J. Biol. Chem. 268, 1501-1504[Free Full Text]
  14. Aderem, A. (1995) Biochem. Soc. Trans. 23, 587-591[Medline] [Order article via Infotrieve]
  15. Ramsden, J. J. (2000) Int. J. Biochem. Cell Biol. 32, 475-479[CrossRef][Medline] [Order article via Infotrieve]
  16. Arbuzova, A., Schmitz, A. A., and Vergeres, G. (2002) Biochem. J. 362, 1-12[CrossRef][Medline] [Order article via Infotrieve]
  17. Singer, M., Martin, L. D., Vargaftig, B. B., Park, J., Gruber, A. D., Li, Y., and Adler, K. B. (2004) Nat. Med. 10, 193-196[CrossRef][Medline] [Order article via Infotrieve]
  18. Li, Y., Martin, L. D., Spizz, G., and Adler, K. B. (2001) J. Biol. Chem. 276, 40982-40990[Abstract/Free Full Text]
  19. Rogers, D. F. (2003) Int. J. Biochem. Cell Biol. 35, 1-6[CrossRef][Medline] [Order article via Infotrieve]
  20. Elzagallaai, A., Rose, S. D., and Trifaro, J. M. (2000) Blood 95, 894-902[Abstract/Free Full Text]
  21. Elzagallaai, A., Rose, S. D., Brandan, N. C., and Trifaro, J. M. (2001) Br. J. Haematol. 112, 593-602[CrossRef][Medline] [Order article via Infotrieve]
  22. Rose, S. D., Lejen, T., Zhang, L., and Trifaro, J. M. (2001) J. Biol. Chem. 276, 36757-36763[Abstract/Free Full Text]
  23. Trifaro, J., Rose, S. D., Lejen, T., and Elzagallaai, A. (2000) Biochimie (Paris) 82, 339-352
  24. Powis, D. A., O'Brien, K. J., Harrison, S. M., Jarvie, P. E., and Dunkley, P. R. (1996) Cell Calcium 19, 419-429[CrossRef][Medline] [Order article via Infotrieve]
  25. Goodall, A. R., Turner, N. A., Walker, J. H., Ball, S. G., and Vaughan, P. F. (1997) J. Neurochem. 68, 392-401[Medline] [Order article via Infotrieve]
  26. Vaughan, P. F., Walker, J. H., and Peers, C. (1998) Mol. Neurobiol. 18, 125-155[CrossRef][Medline] [Order article via Infotrieve]
  27. Walaas, S. I., and Sefland, I. (2000) Neurochem. Int. 36, 581-593[CrossRef][Medline] [Order article via Infotrieve]
  28. Salli, U., Saito, N., and Stormshak, F. (2003) Biol. Reprod. 69, 2053-2058[Abstract/Free Full Text]
  29. Salli, U., Supancic, S., and Stormshak, F. (2000) Biol. Reprod. 63, 12-20[Abstract/Free Full Text]
  30. Liu, J. P., Engler, D., Funder, J. W., and Robinson, P. J. (1994) Mol. Cell. Endocrinol. 101, 247-256[CrossRef][Medline] [Order article via Infotrieve]
  31. Liu, J. P., Engler, D., Funder, J. W., and Robinson, P. J. (1994) Mol. Cell. Endocrinol. 105, 217-226[CrossRef][Medline] [Order article via Infotrieve]
  32. Betancourt-Calle, S., Bollag, W. B., Jung, E. M., Calle, R. A., and Rasmussen, H. (1999) Mol. Cell. Endocrinol. 154, 1-9[CrossRef][Medline] [Order article via Infotrieve]
  33. Calle, R., Ganesan, S., Smallwood, J. I., and Rasmussen, H. (1992) J. Biol. Chem. 267, 18723-18727[Abstract/Free Full Text]
  34. Spizz, G., and Blackshear, P. J. (2001) J. Biol. Chem. 276, 32264-32273[Abstract/Free Full Text]
  35. Jones, P. M., and Howell, S. L. (1989) Biochem. Soc. Trans. 17, 61-63[Medline] [Order article via Infotrieve]
  36. SAS Institute Inc. (1999) SAS/STAT® User's Guide, Version 8, SAS Institute Inc., Cary, NC
  37. Sasaki, Y. (2003) J. Pharmacol. Sci. 93, 35-40[CrossRef][Medline] [Order article via Infotrieve]
  38. Way, K. J., Chou, E., and King, G. L. (2000) Trends Pharmacol. Sci. 21, 181-187[CrossRef][Medline] [Order article via Infotrieve]
  39. Gschwendt, M., Dieterich, S., Rennecke, J., Kittstein, W., Mueller, H. J., and Johannes, F. J. (1996) FEBS Lett. 392, 77-80[CrossRef][Medline] [Order article via Infotrieve]
  40. Just, I., Hofmann, F., Genth, H., and Gerhard, R. (2001) Int. J. Med. Microbiol. 291, 243-250[CrossRef][Medline] [Order article via Infotrieve]
  41. Herget, T., and Rozengurt, E. (1994) Eur. J. Biochem. 225, 539-548[Medline] [Order article via Infotrieve]
  42. Clarke, P. R., Siddhanti, S. R., Cohen, P., and Blackshear, P. J. (1993) FEBS Lett. 336, 37-42[CrossRef][Medline] [Order article via Infotrieve]
  43. Salli, U., and Stormshak, F. (2001) Endocrine 16, 83-88[CrossRef][Medline] [Order article via Infotrieve]
  44. Herget, T., Oehrlein, S. A., Pappin, D. J., Rozengurt, E., and Parker, P. J. (1995) Eur. J. Biochem. 233, 448-457[Medline] [Order article via Infotrieve]
  45. Uberall, F., Giselbrecht, S., Hellbert, K., Fresser, F., Bauer, B., Gschwendt, M., Grunicke, H. H., and Baier, G. (1997) J. Biol. Chem. 272, 4072-4078[Abstract/Free Full Text]
  46. Akita, Y., Ohno, S., Yajima, Y., Konno, Y., Saido, T. C., Mizuno, K., Chida, K., Osada, S., Kuroki, T., and Kawashima, S. (1994) J. Biol. Chem. 269, 4653-4660[Abstract/Free Full Text]
  47. Akita, Y., Kawasaki, H., Ohno, S., Suzuki, K., and Kawashima, S. (2000) Electrophoresis 21, 452-459[CrossRef][Medline] [Order article via Infotrieve]
  48. Tfelt-Hansen, J., MacLeod, R. J., Chattopadhyay, N., Yano, S., Quinn, S., Ren, X., Terwilliger, E. F., Schwarz, P., and Brown, E. M. (2003) Am. J. Physiol. 285, E329-E337
  49. Mendez, C. F., Leibiger, I. B., Leibiger, B., Hoy, M., Gromada, J., Berggren, P. O., and Bertorello, A. M. (2003) J. Biol. Chem. 278, 44753-44757[Abstract/Free Full Text]
  50. Lee, I. S., Hur, E. M., Suh, B. C., Kim, M. H., Koh, D. S., Rhee, I. J., Ha, H., and Kim, K. T. (2003) Cell. Signal. 15, 529-537[CrossRef][Medline] [Order article via Infotrieve]
  51. Yaney, G. C., Fairbanks, J. M., Deeney, J. T., Korchak, H. M., Tornheim, K., and Corkey, B. E. (2002) Am. J. Physiol. 283, E880-E888
  52. Yaney, G. C., Korchak, H. M., and Corkey, B. E. (2000) Endocrinology 141, 1989-1998[Abstract/Free Full Text]
  53. Littman, E. D., Pitchumoni, S., Garfinkel, M. R., and Opara, E. C. (2000) Pancreas 20, 256-263[CrossRef][Medline] [Order article via Infotrieve]
  54. Gasman, S., Chasserot-Golaz, S., Malacombe, M., Way, M., and Bader, M. F. (2004) Mol. Biol. Cell 15, 520-531[Abstract/Free Full Text]
  55. Amin, R. H., Chen, H. Q., Veluthakal, R., Silver, R. B., Li, J., Li, G., and Kowluru, A. (2003) Endocrinology 144, 4508-4518[Abstract/Free Full Text]
  56. Nevins, A. K., and Thurmond, D. C. (2003) Am. J. Physiol. 285, C698-C710
  57. Li, Q., Ho, C. S., Marinescu, V., Bhatti, H., Bokoch, G. M., Ernst, S. A., Holz, R. W., and Stuenkel, E. L. (2003) J. Physiol. 550, 431-445[Abstract/Free Full Text]
  58. Daniel, S., Noda, M., Cerione, R. A., and Sharp, G. W. (2002) Biochemistry 41, 9663-9671[CrossRef][Medline] [Order article via Infotrieve]
  59. Frantz, C., Coppola, T., and Regazzi, R. (2002) Exp. Cell Res. 273, 119-126[CrossRef][Medline] [Order article via Infotrieve]
  60. Kowluru, A., Li, G., Rabaglia, M. E., Segu, V. B., Hofmann, F., Aktories, K., and Metz, S. A. (1997) Biochem. Pharmacol. 54, 1097-1108[CrossRef][Medline] [Order article via Infotrieve]
  61. Ikenoya, M., Hidaka, H., Hosoya, T., Suzuki, M., Yamamoto, N., and Sasaki, Y. (2002) J. Neurochem. 81, 9-16[CrossRef][Medline] [Order article via Infotrieve]
  62. Nagumo, H., Ikenoya, M., Sakurada, K., Furuya, K., Ikuhara, T., Hiraoka, H., and Sasaki, Y. (2001) Biochem. Biophys. Res. Commun. 280, 605-609[CrossRef][Medline] [Order article via Infotrieve]
  63. Tinsley, J. H., Teasdale, N. R., and Yuan, S. Y. (2004) Am. J. Physiol. 286, C105-C111
  64. Li, J., O'Connor, K. L., Hellmich, M. R., Greeley, G. H., Jr., and Evers, B. M. (2004) Gastroenterology 126, A147

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Copyright © 2005 by the American Society for Biochemistry and Molecular Biology.
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