| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Biochemistry and Biophysics |
Department of Medicine, Division of Digestive Diseases, School of Medicine and Molecular Biology Institute, University of California, Los Angeles, California 90095
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Neuropeptides including NT,3
gastrin, cholecystokinin, and mammalian bombesin-like peptides act as potent cellular mitogens for a variety of cell types (3, 4, 5)
. These peptides have been implicated as autocrine/paracrine growth factors for some human cancer cells, notably SCLC (5
, 6)
. We have reported recently that multiple neuropeptides, including NT, induce rapid Ca2+ mobilization from intracellular stores in human ductal pancreatic cancer cell lines (7)
. In particular, NT stimulated activation of ERK1 and ERK2, and DNA synthesis in PANC-1 cells, an extensively studied model of human ductal pancreatic cancers (7, 8, 9, 10, 11, 12, 13)
. Interestingly, NT-binding sites have been detected by autoradiography of frozen sections in 
75% of human pancreatic cancer specimen (14)
. More recently, the expression of mRNA for the NTR-1 has been shown to be markedly increased in pancreatic cancer samples as compared with normal controls (15
, 16)
. It is therefore important to characterize the NT-mediated signal transduction pathways in human pancreatic cancers, which may identify potential targets for therapeutic interventions.
Neuropeptides, including NT, bind to heptahelical GPCRs and trigger the activation of multiple signal transduction pathways including the phospholipase C-mediated generation of the second messengers, inositol (1
, 4
, 5)
trisphosphate, which induces the rapid release of Ca2+ from internal stores and DAG, which activates PKC. PKC plays a central role in signal transduction pathways that mediate action of growth factors, neuropeptides, tumor promoters, and cellular oncogenes (17
, 18)
. Molecular cloning has demonstrated the existence of multiple related PKC isoforms, which are subdivided in three groups based on their structural homology and mode of activation (reviewed in Ref. (19)
. The conventional isoenzymes (
, ß1, ß2, and 
) require DAG and calcium as coactivators in addition to phosphatidyl-L-serine, whereas activation of the novel isoforms (
, 
, 
, and 
) is calcium-independent. Both conventional and novel PKCs function as major intracellular receptors for phorbol esters. In contrast, the atypical PKCs (
, and 
/
) are neither DAG- nor calcium-dependent. Phorbol ester-mediated PKC activation has been reported to either promote or inhibit growth of human pancreatic cancer cells (20, 21, 22)
. In contrast, the effect of GPCR-mediated PKC activation on pancreatic cancer cell growth has not been examined. Furthermore, the immediate downstream signaling events stimulated by PKCs in human pancreatic cancer cells as well as in other cancer cells remain poorly characterized.
PKD, also known as PKCµ, is a serine/threonine protein kinase with distinct structural features and unique enzymological properties (23
, 24)
. In particular, the catalytic domain of the PKD shows very low homology to the conserved kinase subdomain of the PKCs and displays distinct substrate specificity (23
, 25 , 26)
. In contrast to all of the known PKCs, the NH2-terminal region of PKD contains a pleckstrin homology domain that regulates enzyme activity (27)
and lacks a sequence with homology to the typical PKC autoinhibitory pseudosubstrate motif (23)
. However, the proximal NH2-terminal region of PKD contains a tandem repeat of cysteine-rich, zinc finger-like motifs that binds phorbol esters with high affinity (23
, 28)
and mediates PKD translocation to the cell membrane (29)
. PKD/PKCµ can be activated in vitro by DAG/phorbol esters in the presence of phosphatidyl-L-serine (26
, 30
, 31)
, indicating that PKD/PKCµ is a phorbol ester/DAG-stimulated kinase (32)
. Two other members have been included recently in this family of serine/threonine kinases, PKC
(33)
and PKD2 (34)
, lending additional support to the notion that the PKDs comprise a novel family of protein kinases.
Recently, a second mechanism of PKD activation has been identified that involves PKD phosphorylation (31
, 35, 36, 37)
. Specifically, treatment of intact fibroblasts with biologically active phorbol esters (31)
, bryostatin (38)
, growth factors, and GPCR agonists (39, 40, 41)
induces PKD activation that persists during cell disruption and immunoprecipitation. Treatment with PKC-selective inhibitors prevents PKD activation by all of these factors (31
, 32 , 38
, 41)
. Furthermore, cotransfection of PKD with constitutively active mutants of PKC and PKC dramatically increases the catalytic activity of PKD (31
, 42)
and leads to complex formation between PKD and PKC (37)
. It has been proposed previously that phosphorylation of Ser744 and Ser748 within the activation loop of PKD plays a critical role in mediating PKC-dependent PKD activation (35
, 37) . It has been demonstrated that PKD Ser744/Ser748 phosphorylation is rapidly induced by stimulation with PDB or via receptor-mediated pathways in a variety of model systems (43)
. Also, Ser744/Ser748 phosphorylation requires PKC but not PKD activity, indicative of transphosphorylation (43)
. In addition, PKCs play an important role in regulating the rapid release of activated PKD from the cell membrane (44)
. These findings reveal a link between PKCs and PKD, and implied that PKD lies downstream of PKCs in a novel signal transduction pathway (32
, 37)
. The existence of a functional receptor-mediated PKC/PKD pathway in human cancers was first noted in SCLC cells (45)
. However, nothing is known about the PKC/PKD signaling pathway in human pancreatic cancer cells.
In the present study, we demonstrate that multiple PKC isoforms are expressed in PANC-1 and other human ductal pancreatic cancer cell lines along with PKD/PKCµ. We then identify that PKD is rapidly activated in these cells in response to biologically active PDB. Using PANC-1 cell as a model system, we additionally demonstrate that NT induced rapid and striking activation, multisite phosphorylation, and membrane translocation of PKD in these cells. Our results also show that NT induced DNA synthesis in PANC-1 cells through a PKC-dependent pathway. Thus, PKCs play a critical role in mediating PKD activation and mitogenic signaling from the NT receptor in PANC-1 cells.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Immunoprecipitation.
PANC-1 cells cultured for 4–6 days were washed twice in ice-cold PBS (Sigma Chemical Co.) and lysed in 50 mM of Tris-HCl (pH 7.6), 2 mM of EGTA, 2 mM of EDTA, 1 mM of DTT, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM of 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, and 1% Triton X-100 (lysis buffer). The immunoprecipitation was carried out with the PA-1 antipeptide antiserum (1:100) in the absence or the presence of the immunizing peptide (10 µg/ml) as described before (26
, 31)
.
Kinase Assay of PKD.
PKD autophosphorylation was determined in an IVK assay by mixing 20 µl of PKD immunocomplexes with 10 µl of a phosphorylation mixture containing (final concentration) 100 µM [-32P]ATP (specific activity 400–600 cpm/pmol), 30 mM Tris-HCl (pH 7.4), 10 mM MgCl2, and 1 mM DTT, and followed by SDS-PAGE analysis (26
, 31)
. The gels were dried, and the 110-kDa radioactive band corresponding to autophosphorylated PKD was visualized by autoradiography. Autoradiographs were scanned in a GS-710 Calibrated Imaging Densitometer (Bio-Rad), and the labeled band was quantified with the Quantity One software program.
Western Blot Analysis.
Serum-starved cultures of PANC-1 cells grown on 100-mm dishes were washed twice with PBS then replaced with DMEM and treated as described in individual experiments. To detect different PKC isoenzymes and PKD, the cells were directly lysed in 2 x SDS-PAGE sample buffer, followed by SDS-PAGE on 8% gels and transfer to Immobilon-P membranes (Millipore). For Western blot analysis of PKD IP, the samples were washed twice in lysis buffer followed by twice in kinase buffer. Western blots were then performed on membranes incubated overnight with antibodies that recognize the different PKC isoforms and PKD (C-20) at a dilution of 1 µg/ml, or an antiserum that specifically recognizes the dual-phosphorylated state of S744/S748 in PKD (pS744/S748) at a concentration of 1 µg/ml, or for 3 h with an antiserum that specifically recognizes the phosphorylated state of serine 916 position in PKD (pS916) at a dilution of 1:500 in PBS containing 5% nonfat dried milk and 0.1% Tween 20. The immunoreactive bands were detected with enhanced chemiluminescence reagents (Amersham).
Immunocytochemistry.
PANC-1 cells plated in Lab-Tek (Nalge Nunc International, Naperville, IL) double-chambers per each slide were fixed for 15 min at 25°C in 10% buffered formalin phosphate and permeabilized with 0.2% Triton X-100 in PBS for 5 min at 25°C. The fixed cells were incubated for 18 h at 25°C in BB (PBS-1% gelatin-0.05% Tween 20) and then stained at 37°C for 60 min with rabbit polyclonal PKD antibody (C-20) diluted in BB. Subsequently, the cells were washed with PBS containing 0.05% Tween 20 at 25°C and stained at 37°C for 60 min with fluorescein-conjugated goat-anti rabbit antibody diluted in BB. After washing with PBS containing 0.05% Tween 20 and PBS, the samples were mounted with a gelvatol-glycerol solution containing 2.5% 1,4-diazobicyclo-[2.2.2]octane.
Confocal Imaging.
The samples were examined with a Leica TCS-SP upright laser-scanning confocal microscope (Leica, Heidelberg, Germany) using a 63 x 1.2 w HCX PL APO oil immersion objective. Images were collected using an argon gas laser with excitation filter set at 488 nm and emission filter set between 500 and 550 nm. The images were captured as uncompressed 8-bit TIFF files using Leica TCS-NT software (version 1.6.587). One hundred cells were analyzed per experiment, and each experiment was performed at least in duplicate.
[3H]Thymidine Incorporation.
PANC-1 cells (5 x 104) were plated and grown in 3.5-cm tissue culture plates for 5 days in DMEM (Life Technologies, Inc.) with 4 mM of glutamine, 1 mM of Na-pyruvate, and 10% FBS. The cells were washed twice and incubated in serum-free medium for 6 h. To start the experiment, fresh medium containing the specified concentration of agonist/antagonist or solvent was added after washing twice with PBS (four cultures for each condition), and the cells were incubated for 16 h then pulsed for 6 h with [3H]thymidine (0.25 µCi/ml). The cells were fixed with 5% trichloroacetic acid and washed twice with ethanol. Finally, cell residues were dissolved in 0.1 N NaOH with 1% SDS and counted in a liquid scintillation counter.
Materials.
[-32P]ATP (370 MBq/ml) was obtained from Amersham International. Bisindolylmaleimide I (GF-1), Bisindolylmaleimide V (GF-V), Ro 31–8220, wortmannin, PD 98059, and genistein were purchased from Calbiochem. NT, PDB, and rapamycin were obtained from Sigma Chemical Co. Protein-A agarose was from Boehringer Mannheim-Roche. PA-1 antiserum was raised against the synthetic peptide EEREMKALSERVSIL that corresponds to the COOH-terminal region of the predicted amino acid sequence of PKD, as described previously (26
, 31)
. Antibodies (PKD C-20, PKC C-20, PKC C-15, PKCC-15, PKC C-15, and PKC C-15) used in Western blot analysis were obtained from Santa Cruz Biotechnology (Palo Alto, CA). The phospho-S744/S748 PKD/PKCµ antibody was obtained from Cell Signaling Technology (Beverly, MA). An antiserum that specifically recognizes pS916 was generously provided by Dr. Doreen Cantrell (Imperial Cancer Research Institute, London, United Kingdom). Fluorescence-conjugated goat-anti rabbit antibody was obtained from Sigma Chemical Co. Other items were from standard suppliers or as indicated in text.
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
, a major immunoreactive band corresponding to the predicted molecular masses of PKC, PKCß1, PKC, PKC, and PKC were detected in PANC-1 cells. Isoform-specific matched controls were used to detect the presence of a band corresponding to different PKCs (results not shown). PKC, which inhibits growth and induces apoptosis in certain cancer cells (46
, 47)
, is notably not detected by Western blot analysis in these cells as compared with a control lysate from IEC-6 cells, known to express PKC (48)
. A similar pattern of expression of PKC isoforms was noted in other human pancreatic ductal adenocarcinoma cell lines, HPAF-II and MIAPaCa-2 (results not shown). Our results differ from an earlier report that indicated expression of only PKC and PKC in human pancreatic cancer cell lines, including PANC-1 (22
, 49)
. We have demonstrated here that the expression of PKCs in human pancreatic cancer cell line PANC-1 is not restrictive, similar to that observed in HPAC cells (20)
.
|
(left panel), a pair of immunoreactive band of 110 kDa, which corresponds to the molecular mass of PKD (23)
, was detected by SDS-PAGE in PANC-1 cells.
A pair of prominent band migrating with an identical molecular mass (110 kDa) was also obtained when lysates from PANC-1 cells were immunoprecipitated with the PA-1 antiserum (see "Materials and Methods"), and the IPs were analyzed by Western blot using the polyclonal PKD antiserum, C-20. Detection of the 110 kDa doublet was completely blocked by the inclusion of the synthetic peptide EEREMKALSERVSIL (immunizing peptide) that corresponds to the COOH-terminal region of the predicted amino acid sequence of PKD during the immunoprecipitation (Fig. 1B
, middle panel). The band migrating with a lower molecular mass, the detection of which was also completely extinguished by the immunizing peptide, could represent either a proteolytic fragment of PKD or PKD2, a novel homologue with predicted molecular mass of 105 kDa (34)
. These results clearly demonstrate the expression of PKD in PANC-1 cells.
Next, we examined whether phorbol esters can induce PKD activation in intact PANC-1 cells. We treated serum-starved cultures of cells with PDB for 10 min, lysed the cells, and immunoprecipitated PKD with the PA-1 antiserum. The resulting immunocomplexes were incubated with [-32P]ATP, and the incorporation of 32P into PKD was analyzed by SDS-PAGE and autoradiography. As shown in Fig. 1B
, right panel, stimulation of intact cells with 200 nM of PDB induced a striking increase in PKD activity (measured by PKD autophosphorylation), which was maintained during cell lysis and immunoprecipitation. We verified that the immunoprecipitation of the kinase activity in PKD immunocomplexes was also abolished by the inclusion of the immunizing peptide. These results demonstrate that PDB stimulation of intact human pancreatic cancer cells induces the conversion of PKD from a low basal catalytic activity to a state of high activity that persists during cell disruption and protein isolation by the immunoprecipitation procedure.
NT Induces PKD Activation in PANC-1 Cells.
We reported recently that NT stimulated rapid Ca2+ fluxes, ERK activation, and DNA synthesis in PANC-1 cells through NTR-1 (7)
. To additionally elucidate the signaling events downstream of NTR-1 in PANC-1 cells, we examined whether NT can stimulate PKD activation in intact PANC-1 cells. We treated serum-starved cultures of PANC-1 cells with NT for increasing times, lysed the cells, immunoprecipitated PKD with the PA-1 antiserum, and performed IVK assay. As shown in Fig. 2A
, top panel, PKD isolated from unstimulated PANC-1 cells had low basal catalytic activity. Treatment of PANC-1 cells with NT induced a rapid and striking increase in PKD kinase activity that was maintained during cell lysis and immunoprecipitation. PKD activation was detectable within 1 min and reached a maximum (9-fold) after 15 min of NT stimulation. These results demonstrate that PKD activation is one of the early events induced by NT in pancreatic cancer cells.
|
, middle panel). An increase in PKD autophosphorylation was detectable within 2.5 min and reached a maximum (10-fold) after 10–15 min of NT stimulation. NT-induced PKD activation within cells declined gradually toward baseline levels. In contrast, PKD activation induced by PDB persisted for at least 180 min of treatment (results not shown). Thus, NT-induced PKD activation is transient when compared with that induced by PDB. Western blotting with PKD specific antiserum (C-20) confirmed that similar amounts of PKD protein were loaded into the gel after treatment with NT for different times (Fig. 2A
, bottom panel).
Stimulation of intact PANC-1 cells with increasing concentrations of NT for 10 min induced a striking dose-dependent increase in PKD activation, as judged by assays of IVK activity after immunoprecipitation or by Western blotting with pS916 antibody to detect autophosphorylation in intact cells (Fig. 2B)
. Again, Western blot analysis with a PKD-specific antibody (C-20) confirmed that equal amounts of PKD protein were loaded into the gel after treatment with increasing concentrations of NT for 10 min. Half-maximal PKD activation by NT was achieved at 10 nM (Fig. 2B)
.
NT Increases Transient and Rapid Translocation of PKD from the Cytosol to the Plasma Membrane in PANC-1 Cells.
In unstimulated cells, PKD has been localized to the cytosol and to several intracellular compartments including Golgi and mitochondria (29
, 50, 51, 52, 53, 54, 55)
. It has been demonstrated recently that treatment of Swiss 3T3 fibroblasts with the GPCR agonist bombesin induces a striking and transient translocation of PKD from the cytosol to the plasma membrane (29
, 44
, 55)
. Here, we analyzed the effect of NT stimulation on the intracellular distribution of endogenous PKD in PANC-1 cells. As illustrated by the confocal image presented in Fig. 3A
, endogenous PKD expressed in unstimulated PANC-1 cells was distributed throughout the cytosol and mostly excluded from the nucleus with basal fluorescent signal localized to the plasma membrane. The basal level of fluorescence seen in unstimulated cells (Fig. 3A)
represents plasma membrane localized serum-activated PKD. Stimulation with NT for 5 min induced rapid translocation of PKD to the plasma membrane, causing a striking increase in localized fluorescence in the plasma membrane at the cell periphery (Fig. 3B)
. The association of endogenous PKD with the plasma membrane was transient. The reverse translocation from the plasma membrane to the cytosol was nearly complete within 15 min of NT stimulation (Fig. 3C)
. The results presented in Fig. 3
demonstrate that NT induces a prominent and transient translocation of endogenous PKD to the plasma membrane in PANC-1 cells.
|
, treatment with GF-1 potently blocked PKD activation induced by subsequent addition of NT in a concentration-dependent manner. In contrast, GF-1 added directly to the IVK assay, even at concentrations (0.1–1 µM) that abrogated NT-induced PKD activation in intact PANC-1 cells did not inhibit PKD activity. Fig. 4B
depicts similar results obtained with a structurally distinct PKC inhibitor Ro 31–8220 instead of GF-1. Ro 31–8220 blocked NT-induced PKD activation at a lower concentration than GF-1 (half-maximal inhibition obtained with Ro was at 0.25 µM as compared with 0.5 µM of GF-1). These results imply that GF-1 and Ro 31–8220 do not inhibit PKD activity directly but interfere with NT-mediated PKD activation in intact PANC-1 cells by blocking PKC.
|
, the increase in PKD activity induced by acute exposure to PDB or NT was completely abrogated by previous chronic treatment with PDB for 36 h. This result provides additional evidence indicating the requirement of PKC(s) for PKD activation.
|
. In contrast, GF-1, a selective PKC inhibitor, abrogated NT-induced PKD activation as measured both by the IVK assay and pS916 immunoreactivity in Western blot (Fig. 5B)
. These results demonstrate the specificity of the PKC inhibitors and indicate that these kinases are not upstream regulators of PKD. So, activation of PKD by NT in PANC-1 cells is dependent on phorbol ester-regulated PKC(s).
It has been reported recently that Ser744 and Ser748 located in the activation loop of PKD are transphosphorylated in vivo resulting in PKD activation (43)
. To determine whether a similar mechanism operates in human pancreatic cancer cells, we examined whether NT stimulated Ser744/Ser748 phosphorylation in PANC-1 cells. As shown in Fig. 5C
(upper panel), NT induced a rapid and transient increase in phospho-Ser744/Ser748 immunoreactivity (indicative of Ser744/Ser748 phosphorylation). Western blot also demonstrated that treatment with this agonist induced an electrophoretic mobility shift of PKD Ser744/Ser748, which is indicative of their phosphorylation and subsequent activation. Next, we assessed the role of PKC in the phosphorylation of Ser744/Ser748 of PKD after stimulation with NT and PDB in PANC-1 cells. NT-induced phosphorylation of Ser744/Ser748 was potently blocked by the PKC inhibitors GF-1 and Ro 31–8220 in PANC-1 cells (Fig. 5C
, bottom panel). A similar result was obtained when the cells were stimulated with PDB instead of NT (Fig. 5C
, bottom panel). These results demonstrate that activation of PKD by NT is a rapid and transient event, and support a model of PKC-PKD phosphorylation cascade in PANC-1 cells.
PKD Reverse Translocation from the Plasma Membrane Is PKC-dependent in PANC-1 Cells.
We have shown in Fig. 3
that NT induced a rapid translocation of PKD from the cytosol to the plasma membrane. As shown in Fig. 3C
, the rapid translocation of PKD to the plasma membrane is transient. PKD returns to the cytosol after 15 min of exposure to NT. Next, we examined whether PKCs are required for these processes. PANC-1 cells grown in serum were treated with PKC inhibitors GF-1 (Fig. 6, A–C)
and Ro 31–8220 (Fig. 6, D–F)
and subsequently challenged with NT for 5 min (Fig. 6, B and E)
and 15 min (Fig. 6, C and F)
. Cells treated with GF-1 (Fig. 6A)
or Ro 31–8220 (Fig. 6D)
alone demonstrated basal level of immunofluorescence indicative of serum-stimulated PKD localized to the plasma membrane. On stimulation with NT for 5 min, there was a significant increase in the level of localized fluorescence in the plasma membrane at the cell periphery (Fig. 6, B and E)
. These results indicate that PKC is not required for NT-induced translocation of PKD to the plasma membrane.
|
in contrast to Fig. 3C
. This demonstrates that PKC activity is required for the rapid reverse translocation of PKD from the plasma membrane to the cytosol, which is consistent with recent data shown in other nontransformed cells (44)
.
Effect of the PKC Inhibitor GF-1 on NT-induced DNA Synthesis in PANC-1 Cells.
The effect of receptor-mediated PKC activation on the regulation of DNA synthesis has not been examined previously in human pancreatic cancer cells. We have reported earlier that NT promotes DNA synthesis in PANC-1 cells in a dose-dependent manner signaling via NTR-1 receptor subtype (7)
. Now, we examined whether NT receptor-mediated DNA synthesis is dependent on PKC activity in these cells.
Cultures of PANC-1 cells grown in medium containing 10% FBS were washed and transferred to serum-free medium for 6 h. To start the experiment, fresh serum-free medium containing NT, 10% FBS, or solvent were added to parallel cultures (n = 4 for each condition). After 16 h of incubation, the cultures were pulse labeled for 6 h with [3H]thymidine. As shown in Fig. 7A
, NT induced a significant increase in the incorporation of [3H]thymidine into acid-insoluble pools. These results demonstrate that NT, at concentrations that induced PKC/PKD activation, also induced DNA synthesis in PANC-1 cells.
|
. NT alone stimulated DNA synthesis to the same level as depicted in Fig. 8A (result not shown). In striking contrast, GF-1 blocked NT-induced DNA synthesis in PANC-1 cells (Fig. 7B)
. The addition of GF-1 prevented the NT-induced increase in the incorporation of [3H]thymidine in a dose-dependent fashion (Fig. 7C)
. Increasing concentrations of GF-1 attenuated NT-induced DNA synthesis as compared with cells treated with highest concentration of GF-V (Fig. 7C)
. The results demonstrate that NT receptor-mediated activation of DNA synthesis is PKC-dependent in PANC-1 cells. |
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
PKD/PKCµ, a novel serine/threonine kinase, has been identified as a unique downstream effector of PKC-mediated signal transduction in variety of cell types. PKD is also expressed in PANC-1 cells. As a first step to elucidate the mechanism of activation of PKD in human pancreatic cancer cells, we examined the expression of various PKC isoforms in these cells. Our results demonstrate that multiple PKCs are expressed in PANC-1 ductal adenocarcinoma cells. This result differs from the restrictive pattern of PKC isoforms expression in human pancreatic cancer cells proposed in earlier studies (49 , 70) .
Next, we examined the regulation of PKD in PANC-1 cells. Our results demonstrate that treatment of intact cells with the tumor-promoting phorbol ester PDB induces rapid PKD activation. To additionally investigate the physiological significance of PKD activation in pancreatic cancer cells, we then examined the regulation of PKD in PANC-1 cells via receptor-mediated pathways. PANC-1 cells have been used extensively as a model of human pancreatic ductal adenocarcinoma in recent reports (7, 8, 9, 10, 11, 12, 13) . We demonstrated that PKD activation occurs within minutes of NT stimulation in intact PANC-1 cells and, thus, is one of the early events induced by NT in these cells. This suggests that NT signaling through NTR-1 induced a rapid and physiological PKD activation. We found that treatment of these cells with the PKC inhibitors GF-1 and Ro 31–8220 before stimulation with NT strikingly prevents PKD activation. Importantly, these PKC inhibitors do not reduce PKD activity when added directly to the IVK assays, even at concentrations higher than those used in intact PANC-1 cells to block NT-induced PKD activation. PKD is not constitutively active in these cells but can be strikingly activated within these cells via PKC. These results suggest that NT promotes a rapid and concentration-dependent PKD activation in intact PANC-1 cells through a PKC-dependent pathway.
It is well established that long-term exposure to potent biologically active phorbol esters induces down-regulation of conventional and novel PKC isoforms in mammalian cells (48) . So, we examined whether chronic PDB stimulation abrogates the NT-induced PKD activation in PANC-1 cells. The result indicates that chronic PDB exposure leads to complete inhibition of NT-induced PKD activation in these cells. Inhibition of other signaling pathways, including the FRAP-p70 ribosomal S6 kinase, which is constitutively active in PANC-1 cells (71) , or the p42mapk/p44mapk pathway, which is markedly stimulated by phorbol esters in HPAC cells via PKC (72) , did not affect PKD activation by NT. Recently, new experimental evidence indicates that activation loop transphosphorylation at Ser744 and Ser748 occurs during PKD activation in vivo and requires PKC activity (43) . We examined Ser744/Ser748 phosphorylation of PKD in NT-stimulated PANC-1 cells. Our results demonstrate that NT-induced Ser744/Ser748 phosphorylation of PKD is rapid and transient, indicating one of the early events induced by NT in PANC-1 cells. The PKC inhibitors GF-1 and Ro 31–8220 potently blocked NT-induced phosphorylation of Ser744/Ser748 in PKD. These findings strongly support the notion of a PKC-PKD phosphorylation cascade in signal transduction by NT in pancreatic cancer cells.
Activated PKD is transiently localized to the cell membranes but then returns to the cytosol where its activity is retained for extended periods (29 , 44 , 55) . We investigated the plasma membrane translocation of endogenous PKD in PANC-1 cells. Our results demonstrate that NT-induced PKD translocation to the plasma membrane is a rapid and transient phenomena. The striking increase in plasma membrane fluorescence after NT stimulation declines quite rapidly in these cells. Treatment with the PKC inhibitors GF-1 and Ro 31–8220 did not prevent NT-induced rapid translocation of PKD to the plasma membrane but blocked the reverse translocation of PKD from the plasma membrane to the cytosol. PKC activity is required for reverse translocation of activated PKD from plasma membrane to the cytosol where activated PKD now can target multiple proteins to mediate biological functions. Thus, our result is consistent with recent evidence suggesting PKCs play an important role in regulating the rapid release of activated PKD from the cell membrane thereby mediating multiple cellular functions (44) .
The effect of phorbol ester-induced PKC activation on the growth of human pancreatic cancer cells has been a subject of controversy. PKC was proposed as a potent inhibitor of the G1-S transition in phorbol ester-treated proliferating human pancreatic cancer cells (DanG; Ref. 22
). In contrast, overexpression of PKC in HPAC cells was associated with increased tumorigenicity in an orthotopic model that was attenuated with antisense therapy toward PKC (21)
. We examined PKC-dependence on NT-induced DNA synthesis in serum-starved PANC-1 cells. We found that NT-induced DNA synthesis in PANC-1 cells is dependent on PKC. We conclude that receptor-mediated, unlike phorbol ester-induced stimulation of PKCs in human pancreatic cancer cells is growth-stimulatory. Recently, it has been demonstrated that overexpression of PKD in Swiss 3T3 fibroblasts selectively potentiates mitogenesis induced by GPCR agonists like bombesin and vasopressin (73)
. In view of our results that NT induced PKC/PKD activation and DNA synthesis in human pancreatic cancer cells, it is conceivable that PKD mediates some aspect(s) of PKC-dependent mitogenesis in these cells.
In conclusion, our results provide experimental evidence of NT-induced rapid and transient activation of PKD in PANC-1 cells, a model system for human pancreatic ductal adenocarcinoma. A salient feature of the results presented here is that PKC plays a critical role in mediating this response. Our results, showing that PKD can function downstream of PKC in these cells, raise the possibility that PKD mediates some of the biological responses elicited by PKC in the human pancreatic cancer cell line PANC-1. The results also demonstrate that NT promotes DNA synthesis in these cells, which is PKC dependent. Our findings indicate that receptor-mediated PKC stimulation promotes growth of human pancreatic cancer cell line PANC-1 and suggest that elucidation of the signal transduction pathways that mediate NT-induced mitogenesis may identify novel targets for therapeutic intervention.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 Supported in part by the Ronald S. Hirshberg Memorial Foundation for Pancreatic Cancer Research and NIH Grant DK 55003 (to E. R.). S. G. would like to acknowledge the Specialty Training and Advanced Research (STAR) fellowship from the Department of Medicine, University of California, Los Angeles. 
2 To whom requests for reprints should be addressed, at 900 Veteran Avenue, Warren Hall Room 11-124, Department of Medicine, School of Medicine, University of California, Los Angeles, CA 90095-1786. Phone: (310) 794-6610; Fax: (310) 267-2399; E-mail: erozengurt{at}mednet.ucla.edu
3 The abbreviations used are: NT, neurotensin; SCLC, small cell lung cancer; GPCR, G protein-coupled receptor; NTR-1, NT receptor subtype 1; ERK, extracellular signal-regulated kinase; PKC, protein kinase C; PKD, protein kinase D; PDB, phorbol-12,13-dibutyrate; DAG, diacylglycerol; GF-1, GF 109203X; FBS, fetal bovine serum; IP, immunoprecipitate; BB, blocking buffer; IVK, in vitro kinase; FRAP, FK506-binding protein rapamycin-associated protein; MAPK, MAP kinase or mitogen-activated protein kinase.
Received 10/ 1/01. Accepted 1/ 9/02.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
in the tumorigenicity of pancreatic cancer. Surgery (St. Louis), 124: 218–223; discussion 223–224, 1998.
inhibits growth of pancreatic cancer cells via p21(cip)-mediated G(1) arrest. J. Cell Sci., 113: 3025-3035, 2000.[Abstract]
, a new member of the protein kinase C family, composes a fourth subfamily with PKCµ. Biochim. Biophys. Acta., 1450: 99-106, 1999.[Medline]
and protein kinase C. J. Biol. Chem., 273: 7038-7043, 1998. inhibits anchorage-dependent and -independent growth, enhances differentiation, and increases apoptosis in CaCo-2 cells. Gastroenterology, 120: 1700-1712, 2001.[Medline]
. Mol. Cell Biol., 17: 3418-3428, 1997. on human pancreatic carcinoma cell lines are associated with differential regulation of protein kinase C isoenzymes. Gut, 39: 255-261, 1996.-mediated regulation of Golgi organization is through the direct activation of protein kinase D. Cell, 98: 59-68, 1999.[Medline]
in human pancreatic carcinoma cells. Endocrinology, 137: 3340-3347, 1996.[Abstract]
This article has been cited by other articles:
|
|
 |
|
  I. Ohno, G. Eibl, I. Odinokova, M. Edderkaoui, R. D. Damoiseaux, M. Yazbec, R. Abrol, W. A. Goddard III, O. Yokosuka, S. J. Pandol, et al. Rottlerin stimulates apoptosis in pancreatic cancer cells through interactions with proteins of the Bcl-2 family Am J Physiol Gastrointest Liver Physiol, January 1, 2010; 298(1): G63 - G73. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Yoo, C. Chung, L. Slice, J. Sinnett-Smith, and E. Rozengurt Protein kinase D mediates synergistic expression of COX-2 induced by TNF-{alpha} and bradykinin in human colonic myofibroblasts Am J Physiol Cell Physiol, December 1, 2009; 297(6): C1576 - C1587. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Kisfalvi, G. Eibl, J. Sinnett-Smith, and E. Rozengurt Metformin Disrupts Crosstalk between G Protein-Coupled Receptor and Insulin Receptor Signaling Systems and Inhibits Pancreatic Cancer Growth Cancer Res., August 15, 2009; 69(16): 6539 - 6545. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Sinnett-Smith, R. Jacamo, R. Kui, Y. M. Wang, S. H. Young, O. Rey, R. T. Waldron, and E. Rozengurt Protein Kinase D Mediates Mitogenic Signaling by Gq-coupled Receptors through Protein Kinase C-independent Regulation of Activation Loop Ser744 and Ser748 Phosphorylation J. Biol. Chem., May 15, 2009; 284(20): 13434 - 13445. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. A. Chen, J. Li, S. R. Silva, L. N. Jackson, Y. Zhou, H. Watanabe, K. L. Ives, M. R. Hellmich, and B. M. Evers PKD3 Is the Predominant Protein Kinase D Isoform in Mouse Exocrine Pancreas and Promotes Hormone-induced Amylase Secretion J. Biol. Chem., January 23, 2009; 284(4): 2459 - 2471. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. R. Sharlow, K. V. Giridhar, C. R. LaValle, J. Chen, S. Leimgruber, R. Barrett, K. Bravo-Altamirano, P. Wipf, J. S. Lazo, and Q. J. Wang Potent and Selective Disruption of Protein Kinase D Functionality by a Benzoxoloazepinolone J. Biol. Chem., November 28, 2008; 283(48): 33516 - 33526. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Jacamo, J. Sinnett-Smith, O. Rey, R. T. Waldron, and E. Rozengurt Sequential Protein Kinase C (PKC)-dependent and PKC-independent Protein Kinase D Catalytic Activation via Gq-coupled Receptors: DIFFERENTIAL REGULATION OF ACTIVATION LOOP SER744 AND SER748 PHOSPHORYLATION J. Biol. Chem., May 9, 2008; 283(19): 12877 - 12887. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Kisfalvi, O. Rey, S. H. Young, J. Sinnett-Smith, and E. Rozengurt Insulin Potentiates Ca2+ Signaling and Phosphatidylinositol 4,5-Bisphosphate Hydrolysis Induced by Gq Protein-Coupled Receptor Agonists through an mTOR-Dependent Pathway Endocrinology, July 1, 2007; 148(7): 3246 - 3257. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Johannessen, M. P. Delghandi, A. Rykx, M. Dragset, J. R. Vandenheede, J. Van Lint, and U. Moens Protein Kinase D Induces Transcription through Direct Phosphorylation of the cAMP-response Element-binding Protein J. Biol. Chem., May 18, 2007; 282(20): 14777 - 14787. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Schulz, C. Rocken, M. P A Ebert, and S. Schulz Immunocytochemical identification of low-affinity NTS2 neurotensin receptors in parietal cells of human gastric mucosa. J. Endocrinol., October 1, 2006; 191(1): 121 - 128. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Souaze, V. Viardot-Foucault, N. Roullet, M. Toy-Miou-Leong, A. Gompel, E. Bruyneel, E. Comperat, M. C Faux, M. Mareel, W. Rostene, et al. Neurotensin receptor 1 gene activation by the Tcf/{beta}-catenin pathway is an early event in human colonic adenomas Carcinogenesis, April 1, 2006; 27(4): 708 - 716. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. Rey, R. Papazyan, R. T. Waldron, S. H. Young, J. Lippincott-Schwartz, R. Jacamo, and E. Rozengurt The Nuclear Import of Protein Kinase D3 Requires Its Catalytic Activity J. Biol. Chem., February 24, 2006; 281(8): 5149 - 5157. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Rozengurt, O. Rey, and R. T. Waldron Protein Kinase D Signaling J. Biol. Chem., April 8, 2005; 280(14): 13205 - 13208. [Full Text] [PDF]
|
|
|
|
|
|
S. Guha, G. Eibl, K. Kisfalvi, R. S. Fan, M. Burdick, H. Reber, O. J. Hines, R. Strieter, and E. Rozengurt Broad-Spectrum G Protein-Coupled Receptor Antagonist, [D-Arg1,D-Trp5,7,9,Leu11]SP: A Dual Inhibitor of Growth and Angiogenesis in Pancreatic Cancer Cancer Res., April 1, 2005; 65(7): 2738 - 2745. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. Rey, J. R. Reeve Jr., E. Zhukova, J. Sinnett-Smith, and E. Rozengurt G Protein-coupled Receptor-mediated Phosphorylation of the Activation Loop of Protein Kinase D: DEPENDENCE ON PLASMA MEMBRANE TRANSLOCATION AND PROTEIN KINASE C{epsilon} J. Biol. Chem., August 13, 2004; 279(33): 34361 - 34372. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Cabrera-Poch, L. Sanchez-Ruiloba, M. Rodriguez-Martinez, and T. Iglesias Lipid Raft Disruption Triggers Protein Kinase C and Src-dependent Protein Kinase D Activation and Kidins220 Phosphorylation in Neuronal Cells J. Biol. Chem., July 2, 2004; 279(27): 28592 - 28602. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. B. Kennett, J. D. Roberts, and K. Olden Requirement of Protein Kinase C{micro} Activation and Calpain-mediated Proteolysis for Arachidonic Acid-stimulated Adhesion of MDA-MB-435 Human Mammary Carcinoma Cells to Collagen Type IV J. Biol. Chem., January 30, 2004; 279(5): 3300 - 3307. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. P. Thomas, M. R. Hellmich, C. M. Townsend Jr., and B. M. Evers Role of Gastrointestinal Hormones in the Proliferation of Normal and Neoplastic Tissues Endocr. Rev., October 1, 2003; 24(5): 571 - 599. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. Rey, J. Yuan, S. H. Young, and E. Rozengurt Protein Kinase C{nu}/Protein Kinase D3 Nuclear Localization, Catalytic Activation, and Intracellular Redistribution in Response to G Protein-coupled Receptor Agonists J. Biol. Chem., June 20, 2003; 278(26): 23773 - 23785. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Guha, J. A. Lunn, C. Santiskulvong, and E. Rozengurt Neurotensin Stimulates Protein Kinase C-dependent Mitogenic Signaling in Human Pancreatic Carcinoma Cell Line PANC-1 Cancer Res., May 15, 2003; 63(10): 2379 - 2387. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Li, M. R. Hellmich, G. H. Greeley Jr., C. M. Townsend Jr., and B. M. Evers Phorbol ester-mediated neurotensin secretion is dependent on the PKC-alpha and -delta isoforms Am J Physiol Gastrointest Liver Physiol, November 1, 2002; 283(5): G1197 - G1206. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
). Control cells (-) received equivalent amounts of solvent. The cultures were subsequently stimulated with 50 nM NT for 10 min at 37°C. Cells were lysed with lysis buffer and immunoprecipitated with PA-1 antiserum. The PKD activity in the immunocomplexes was determined by IVK assay as described in "Materials and Methods," followed by SDS-PAGE and autoradiography (in vivo). Parallel cultures were treated with 50 nM NT for 10 min at 37°C (bottom blot and 
), lysed with lysis buffer and immunoprecipitated with PA-1 antiserum. The PKD activity was then determined by IVK assay in the absence or presence of indicated concentrations of GF-1 (in vitro). Graphed data represent average values (n = 3) of the level of PKD activation obtained from scanning densitometry and are expressed as percentages of the maximum increase in phosphorylation obtained with 50 nM NT for 10 min at 37°C; bars, ±SE. B, Ro 31–8220 inhibits PKD activity. Serum-starved PANC-1 cells were incubated for 1 h with different concentrations of a structurally unrelated PKC inhibitor Ro 31–8220 (top blot and 
