neurotransmitters and growth factors can activate P21 activated kinase 2 in pancreatic acinar cells by novel mechanisms

BBAMCR-17571; No. of pages: 12; 4C: Biochimica et Biophysica Acta xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbamcr

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Bernardo Nuche-Berenguer, R.T. Jensen ⁎

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Digestive Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-1804, USA

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Article history: Received 18 January 2015 Received in revised form 28 April 2015 Accepted 7 May 2015 Available online xxxx

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Keywords: PAK2 activation Pancreatic acini CCK Signaling Pancreatic growth factor PKC

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P-21-activated kinases (PAKs) are serine/threonine kinases comprising six isoforms divided in two groups, group-I (PAK1–3)/group-II (PAK4–6) which play important roles in cell cytoskeletal dynamics, survival, secretion and proliferation and are activated by diverse stimuli. However, little is known about PAKs ability to be activated by gastrointestinal (GI) hormones/neurotransmitters/growth-factors. We used rat pancreatic acini to explore the ability of GI-hormones/neurotransmitters/growth-factors to activate Group-I-PAKs and the signaling cascades involved. Only PAK2 was present in acini. PAK2 was activated by some pancreatic growth-factors [EGF, PDGF, bFGF], by secretagogues activating phospholipase-C (PLC) [CCK, carbachol, bombesin] and by post-receptor stimulants activating PKC [TPA], but not agents only mobilizing cellular calcium or increasing cyclic AMP. CCKactivation of PAK2 required both high- and low-affinity-CCK1-receptor-state activation. It was partially reduced by PKC- or Src-inhibition, but not with PI3K-inhibitors (wortmannin, LY294002) or thapsigargin. IPA-3, which prevents PAK2 binding to small-GTPases partially inhibited PAK2-activation, as well as reduced CCK-induced ERK1/2 activation and amylase release induced by CCK or bombesin. This study demonstrates pancreatic acini, possess only one Group-I-PAK, PAK2. CCK and other GI-hormones/neurotransmitters/growth-factors activate PAK2 via small GTPases (CDC42/Rac1), PKC and SFK but not cytosolic calcium or PI3K. CCK-activation of PAK2 showed several novel features being dependent on both receptor-activation states, having PLC- and PKCdependent/independent components and small-GTPase-dependent/independent components. These results show that PAK2 is important in signaling cascades activated by numerous pancreatic stimuli which mediate their various physiological/pathophysiological responses and thus could be a promising target for the development of therapies in some pancreatic disorders such as pancreatitis. © 2015 Published by Elsevier B.V.

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Gastrointestinal hormones/neurotransmitters and growth factors can activate P21 activated kinase 2 in pancreatic acinar cells by novel mechanisms☆

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Abbreviations: CCK, COOH-terminal octapeptide of cholecystokinin; TPA, 12-Otetradecanoylphorbol-13-acetate; PAKs, p21-activated kinases; SFK, Src family of kinases; PKC, protein kinase C; PYK2, proline-rich tyrosine kinase 2; FAK, focal adhesion kinase; PKD, protein kinase D; PI3K, phosphatidylinositol-3-kinase; HRP, horseradish peroxidase; HGF, hepatocyte growth factor; IGF-1, insulin-like growth factor 1; bFGF, basic fibroblast growth factor; VIP, vasoactive intestinal peptide; CCK-JMV, CCK-JMV-180; EGF, epidermal growth factor; PDGF, platelet-derived growth factor; 8-Br-cAMP, 8-bromo-cyclic adenosine monophosphate; PLC, phospholipase C; ET, endothelin; CDC42, cell control division protein; RAC, Ras-related C3 botulinum toxin substrate; GFX, GFX109203X, PKC inhibitor; MAPK/ ERK, mitogen-activated protein kinase; PKA, protein kinase A; IPA-3, 1,1′-dithiodi-2naphthtol; Pir 3,5, 6,6′-dithiodi-2-naphthtol; PP2, 4-amino-5-(4-chlorophenyl)-7-(tbutyl)pyrazolo[3,4-d]pyrimidine; PP3, 4-amino-7-phenylpyrazol[3,4-d]pyrimidine; LY294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one. ☆ This work is partially supported by the Intramural Research Program of the NIDDK, NIH. ⁎ Corresponding author at: NIH/NIDDK/DDB, Bldg. 10, Rm. 9C-103, 10 Center Dr., MSC 1804, Bethesda, MD 20892-1804, USA. E-mail address: [email protected] (R.T. Jensen).

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1. Introduction

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The p21-activated kinases (PAKs) are a family of serine threonine kinases that are one of the main effectors for the small Rho GTPases, Cdc42 and Rac [1–3]. The PAK family consists of six members that are segregated into two subgroups (Group I and Group II) based on sequence homology [1,3–6]. Group-I-PAKs (PAK 1–3) are the most extensively studied and play an important role in signaling for many cellular processes, such as regulation of cell survival, apoptosis, cell motility, tumorigenesis, protein synthesis, glucose homeostasis, secretion and cellular proliferation [1–5,7–14]. The Group-I-PAKs normal tissue expression varies greatly between the three isoforms [15,16]. PAK2 is expressed in a wide variety of different tissues and can be thought of as ubiquitously expressed. In contrast, PAK1 has a slightly more reserved distribution, with high levels of PAK1 in tissues such as muscle, spleen, heart and liver. PAK3 has a much more restricted expression, being predominantly expressed in the brain, although recent reports have identify it in enteroendocrine cells [17]. Studies demonstrate that PAKs play an important signaling role in various tissues for a wide

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http://dx.doi.org/10.1016/j.bbamcr.2015.05.011 0167-4889/© 2015 Published by Elsevier B.V.

Please cite this article as: B. Nuche-Berenguer, R.T. Jensen, Gastrointestinal hormones/neurotransmitters and growth factors can activate P21 activated kinase 2 in pancreatic acinar cells by n..., Biochim. Biophys. Acta (2015), http://dx.doi.org/10.1016/j.bbamcr.2015.05.011

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2.1. Materials

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Male Sprague–Dawley rats (150–250 g) were obtained from the Small Animals Section, Veterinary Resources Branch, National Institutes of Health (NIH), Bethesda, MD. PAK1 recombinant protein was from Abnova (Walnut, CA). Recombinant PAK2 protein active was from Active Motif (Carlsbad, CA). Pak 3 recombinant human protein was from Life Tech. (Grand Island, NY). Pak 4 recombinant human protein was from MyBIosource (San Diego, CA). Rabbit anti-PAK1, rabbit antiphospho-PAK1/2 (Thr402/423), and nonfat dry milk were purchased from Cell Signaling Technology, Inc. (Beverly, MA). Stabilized goat anti-rabbit IgG peroxidase conjugated was from Pierce Biotechnology, Inc. (Rockford, IL). Goat PAK2 antibody, and anti-goat-HRP-conjugate antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Pak 3 antibody was from Abcam (Cambridge, MA). Tris/HCl pH 8.0 and 7.5 were from Mediatech, Inc. (Herndon, VA). 2-Mercaptoethanol, protein assay solution, sodium lauryl sulfate (SDS) and Tris/Glycine/ SDS (10 ×) were from Bio-Rad Laboratories (Hercules, CA). MgCl2, CaCl2, Tris/HCl 1 M pH 7.5 and Tris/Glycine buffer (10 ×) were from Quality Biological, Inc. (Gaithersburg, MD). Pak6 recombinant protein, minimal essential media (MEM) vitamin solution, amino acids 100 ×, Dulbecco's phosphate buffered saline (DPBS), glutamine (200 mM), Tris–Glycine gels, L-glutamine, and fetal bovine serum (FBS) were from Invitrogen (Carlsbad, CA). COOH-terminal octapeptide of cholecystokinin (CCK), hepatocyte growth factor (HGF), bombesin, insulin-like growth factor 1 (IGF-1), basic fibroblast growth factor (bFGF), vasoactive intestinal peptide (VIP), endothelin and secretin were from Bachem Bioscience Inc. (King of Prussia, PA). CCK-JMV-180 (CCK-JMV) was obtained from Research Plus Inc. (Bayonne, NJ). Epidermal growth factor (EGF), thapsigargin, platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), PAK5 active protein, deoxycholic acid and P21-activated kinase inhibitor 3 (IPA-3) were from Calbiochem (La Jolla, CA). Carbachol, insulin, dimethyl sulfoxide (DMSO), 12-O-tetradecanoylphobol-13-acetate (TPA), L-glutamic acid, glucose, fumaric acid, pyruvic acid, trypsin inhibitor, HEPES, TWEEN® 20, Triton X-100, GFX (GFX109203X), phenylmethanesulfonylfluoride

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2.2.1. Pancreatic acini preparation Pancreatic acini were obtained by collagenase digestion as previously described [34]. Standard incubation solution contained 25.5 mM HEPES (pH 7.45), 98 mM NaCl, 6 mM KCl, 2.5 mM NaH2PO4, 5 mM sodium pyruvate, 5 mM sodium glutamate, 5 mM sodium fumarate, 11.5 mM glucose, 0.5 mM CaCl2, 1 mM MgCl2, 1 mM glutamine, 1% (w/v) albumin, 0.01% (w/v) trypsin inhibitor, 1% (v/v) vitamin mixture and 1% (v/v) amino acid mixture.

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2.2. Methods

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2.2.2. Acini stimulation After collagenase digestion, dispersed acini were pre-incubated in standard incubation solution for 2 h at 37 °C as described previously [34,39]. After pre-incubation 1 ml aliquots of dispersed acini were incubated at 37 °C with or without stimulants. Cells were lysed in lysis buffer (50 mM Tris/HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 1% deoxycholate, 0.1% sodium azide, 1 mM EGTA, 0.4 mM EDTA, 0.2 mM sodium orthovanadate, 1 mM PMSF, and one protease inhibitor tablet per 10 ml). After sonication, lysates were centrifuged at 10,000 × g for 15 min at 4 °C and protein concentration was measured using the Bio-Rad protein assay reagent.

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2.2.3. Inhibition experiments We used small inhibitor molecules to identify upstream activators of the CCK/TPA-mediated activation of PAK2. Isolated acini were preincubated for 15 min with IPA-3 that prevents binding of CDC42 to group I PAKs and its inactive analog PIR 3,5 as a control; with the PI3K inhibitors wortmannin or LY294002, also for 15 min and with the Src inhibitor, PP2, and its inactive analog PP3 for 1 h. Cells were then treated for 3 min with physiological (0.3 nM) or supraphysiological (100 nM) concentrations of CCK and also with TPA (5 min, 1 μM), with untreated cells for each pretreatment used as controls. After incubation, cells were processed as stated above.

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2.3. Western blotting

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It was performed as described previously [40]. Whole cell lysates were subjected to SDS-PAGE using 4–20% Tris–Glycine gels. After electrophoresis, proteins were transferred to nitrocellulose membranes. Membranes were blocked in blocking buffer (50 mM Tris/HCl pH 8.0, 2 mM CaCl2, 80 mM NaCl, 0.05% Tween® 20, 5% non fat dry milk) at room temperature for 1 h. Membranes were then incubated with primary antibody overnight at 4 °C under constant agitation at antibody dilutions suggested by the supplier. After primary antibody incubation membranes were washed twice in blocking buffer for 4 min and then incubated with HRP-conjugated secondary antibody (anti-mouse,

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2. Materials and methods

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(PMSF), ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), sucrose, sodium-orthovanadate, and sodium azide were from Sigma-Aldrich, Inc. (St. Louis, MO). Albumin standard and Super Signal West (Pico, Dura) chemiluminescent substrate were from Pierce (Rockford, IL). Phadebas Amylase test was from Magle Life Science (Lund, Sweden). Protease inhibitor tablets were from Roche (Basel, Switzerland). Purified collagenase (type CLSPA) was from Worthington Biochemicals (Freehold, NJ). Nitrocellulose membranes were from Schleicher and Schuell Bioscience, Inc. (Keene, NH). L364,718 (3S(−)-N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4benzodiazepine-3-yl-1H-indole-2-carboxamide)) was from Merck, Sharp and Dohme (West Point, PA). YM022 ((R)-1-[2,3-dihydro-1(2′-methyl-phenacyl)-2-oxo-5-phenyl-1H-1,4-benzodiazepin-3-yl]-3(3-methylphenyl)urea), SR27897 (1-[[2-(4-(2-chlorophenyl)-thiazol2-yl)aminocarbonyl] indolyl]acetic acid) and PIR 3,5 (IPA-3 inactive control) were from Tocris Bioscience (Ellisville, MO). Albumin bovine fraction V was from MP Biomedical (Solon, OH). NaCl, KCl and NaH2PO4 were from Mallinckrodt (Paris, KY).

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range of cellular stimuli including bioactive lipids [18,19], oncogenes [20], chemokines [21], growth factors [22–29], cellular stress [9,10], radiation [30], and with activation of some G-protein-coupled receptors [26,31]. However, there is little information on the activation of Group-I-PAKs by gastrointestinal (GI) hormones/neurotransmitters and GI growth factors in normal GI tissues. Pancreatic acinar cells possess receptors for a large number of GI hormones/neurotransmitters and growth factors and are an excellent model to study their cellular basis of action, because the agents also alter cellular function activating numerous cellular signaling cascades [32–34]. At present it is unclear if any Group-I-PAKs occur in pancreatic acinar cells, although it is known that at least 2 types (PAK1, 3) occur in pancreatic islets [16,35,36]. It is also unknown whether activation of any of the GI hormones/neurotransmitter receptors or GI growth factor receptors in these cells activates acinar cell PAKs, or if present, any information on the cellular signaling mechanisms involved. To address this question in the case of the PAKs, in the present study we sought to determine whether the three members of the Group-IPAKs were present in rat pancreatic acinar cells and if they are involved in mediating the cellular signaling of gastrointestinal hormones/neurotransmitters and of various gastrointestinal growth factors, which are known to alter pancreatic acinar cell function. Particular attention was paid to the GI hormone/neurotransmitter, cholecystokinin (CCK), because this is a well-studied physiological regulator of pancreatic acinar cell function with effects on secretion, growth, and enzyme synthesis as well as playing a prominent role in a number of pancreatic acinar cell pathophysiological processes [37,38].

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2.3.1. Amylase release In order to study the effect of PAK2 activation in the stimulation of amylase release in rat pancreatic acinar cells, isolated acini were preincubated with either no additions, or with 40 μM IPA3 or its inactive analog Pir 3,5, and then subsequently incubated with CCK at either a low (0.01 nM) or maximal (0.1 nM) concentrations and with a maximal bombesin concentration (10 nM). Amylase release was measured using the procedure published previously [41,42]. Amylase activity was determined after a 30 min incubation using the Phadebas reagent and was expressed as percentage of the total cellular amylase released into the extracellular medium during the incubation [41,42].

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3. Results

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3.1. Specificity of the antibodies used in the study and presence of PAK2 in pancreatic acini

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To assess the possible presence of Group I PAKs (i.e., PAK 1, 2, 3) in pancreatic acinar cells and also to determine the specificity of the antibodies to be used in this study, whole pancreatic acinar lysates, in parallel with human recombinant samples of the 6 PAK family members (PAK1–PAK6) and with control cell lysates (C6 and COS cells) were blotted with antibodies reported to be specific for PAK1, PAK2 and PAK3 (Fig. 1, A–C). Each of the three antibodies was specific for their respective PAK. We observed only PAK2 was present in pancreatic acinar cells (Fig. 1, B, Lane 1). Moreover, using a phospho (T423/402) PAK1/2 antibody (Fig. 1D) in stimulated pancreatic acinar cells and in rat glioma cell line C6 cells, reported to have both PAK1 and PAK2 [15] we detected a double band corresponding to PAK1 and PAK2 in the C6 cells (Fig. 1D, Lane 3), but only detected a single band corresponding to phospho (T402) PAK2 (Fig. 1D, Lane 2) in the pancreatic acinar cells. These results demonstrate that, under the experimental conditions used in our study the only Group-I-PAKs activated by the various stimuli in the pancreatic acini was phospho-PAK2.

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3.2. Ability of various pancreatic secretagogues and pancreatic growth factors to stimulate PAK2 phosphorylation (pT402) in rat pancreatic acini

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In order to establish whether PAK2 is activated by known pancreatic secretagogues or growth factors [33], rat pancreatic acini were incubated in the absence and presence of several gastrointestinal hormones (CCK, carbachol, bombesin, secretin, VIP, endothelin) known to interact with specific G protein-coupled receptors in pancreatic acini [33]. As a measurement of PAK2 activity, we analyzed the phosphorylation of T402PAK2, which has been shown to be essential for PAK2 protein kinase activity, as well as reflecting its degree of activation and has been

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2.3.2. Statistical analysis All experiments were performed at least 5 times. Data are presented as mean ± SEM and were analyzed with the non-parametric Kruskal– Wallis analysis using the GraphPad 5.0 software. p values b0.05 were considered significant. Curve fitting, EC50 and tmax were also calculated using the GraphPad 5.0 software.

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anti-rabbit, anti-goat) according to the species of the first antibody for 1 h at room temperature under constant agitation. Membranes were then washed again twice in blocking buffer for 4 min, twice in washing buffer (50 mM Tris/HCl pH 8.0, 2 mM CaCl2, 80 mM NaCl, 0.05% Tween® 20) for 4 min, incubated for 4 min with chemiluminescence detection reagents and finally exposed to Kodak Biomax film (XAR, MS or MR). The intensity of the protein bands was measured using Kodak ID Image Analysis, which were assessed in the linear detection range. When re-probing was necessary membranes were incubated in a stripping buffer (Pierce, Rockford, IL) for 30 min at room temperature, washed twice for 10 min in washing buffer, blocked for 1 h in blocking buffer at room temperature and re-probed as described above.

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Fig. 1. Specificity of the antibodies used in the study. Lysates from pancreatic acini and equal amounts (5 μg) of human recombinant PAK family members (PAK 1–6) or control cell lysates (C6, COS) were detected by Western blotting with specific anti-PAK1, antiPAK2 and anti-PAK3 antibodies (Cell signaling = CS), Panels A, B and C; or with a specific anti-phospho Pak1 (T423)/PAK2 (T402) antibody (Cell signaling), Panel D. These results are representative of 4 other experiments.

widely used to assess its activation in other studies [43–46]. The pancreatic secretagogues that activate phospholipase C (bombesin, carbachol and CCK) stimulated an increase in phospho-(T402)PAK2 (222 ± 53, 220 ± 56, 408 ± 104 of control, respectively, all p b 0.05 vs control) (Fig. 2A, Rows 4–6; Table 1). VIP and secretin, which activate adenylate cyclase in rat pancreatic acini at the concentrations used [33,47], did not increase phosphorylation of (T402) PAK2 (Fig. 2A, Rows 2 and 3; Table 1). Endothelin-1 (ET-1), which interacts with ET-1 and ET-3

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B. Nuche-Berenguer, R.T. Jensen / Biochimica et Biophysica Acta xxx (2015) xxx–xxx Table 1 t1:1 T402 PAK2 kinase phosphorylation characteristics and interactions of PAK2 in rat pancre- t1:2 atic acinia. t1:3 Q1 Variable

pY416 phosphorylation

Stimulation by pancreatic secretagoguesb Yes No

CCK, carbachol, bombesin Secretin, Endothelin-1, VIP

Stimulation by pancreatic growth factorsb Yes EGF, bFGF, PDGF No Insulin, IGF-1, HGF

A2387, TPA Thapsigargin

Potency/time-course EC50 of CCK (nM)c EC50 of CCK-JMV (nM)c CCK-stimulated PAK2 activation inhibited (0.3 nM)e Yes: Inhibitor: SFK (PP2), GTPase to PAK2 (IPA-3), GFX (PKC) No: Inhibitor PI3K (wortmannin, LY294002), Ca++ (thapsigargin)

0.44 ± 0.05 0.18 ± 0.14 CCK-stimulated PAK2 activation inhibited (100 nM) Yes: Inhibitor: GTPase to PAK2 (IPA-3), GFX (PKC) No: Inhibitor SFK (PP2), PI3K (wortmannin, LY294002), Ca++ (thapsigargin)

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Results are calculated from the data shown in Figs. 2–5. Concentration and incubation times are reported in Fig. 2. Concentrations and incubation time are reported in Fig. 3. Concentration and incubation times are reported in Fig. 5. Concentration and incubation times are reported in Fig. 6.

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receptors in the pancreatic acinar cell, but does not activate PLC cascades nor activate adenylate cyclase [48] did not produce any effect upon T402 PAK2 phosphorylation (Fig. 2A, Row 7; Table 1).

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3.3. Dose–response effect of CCK and CCK-JMV on T402 PAK2 phosphorylation in rat pancreatic acini

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As CCK has an important role in both the normal physiology and the pathophysiology of the pancreas [33,38,52,53], we focused our study in the activation of PAK2 exerted by this hormone in rat pancreatic acini. Increasing concentrations of CCK produced an increase in T402 phosphorylation of PAK2, detectable at 0.01 nM concentration (Fig. 3), maximal stimulation occurred with 100 nM CCK (452 ± 70% of control) and CCK's half-maximal effect (EC50) occurred with 0.44 nM ± 0.05 nM (Fig. 3, Table 1). The CCK1 receptor in pancreatic acini can exist in two different activation states, a low- and a high-affinity state, and the

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Of six growth factors known to alter pancreatic acinar cell function, which were tested [33], EGF (195 ± 28 of control p b 0.05), bFGF (228 ± 37 of control p b 0.05) and PDGF (240 ± 44 of control p b 0.05) were able to stimulate PAK2 threonine 402 phosphorylation, whereas IGF and HGF had no effect (Fig. 2B, Table 1). Insulin caused a 210 ± 44 increase in T402 PAK2 phosphorylation, however this effect did not quite reach statistical significance (p = 0.08) (Fig. 2B, Row 2; Table 1). In order to determine if the CCK stimulating effect on pT402 PAK2 phosphorylation was due to the presence/activation of CCK1 or CCK2 receptors in the acinar cell suspension, isolated acinar cells were first incubated with either CCK, or the selective CCKB agonist gastrin, or a known selective CCK1 receptor agonist (A71378) [49] (Fig. 2C, Lanes 1–4). Gastrin did not produce any increase in pT402 PAK2 phosphorylation, and the CCK activation of PAK2 was mimicked by the incubation of the cells with the specific CCK 1 receptor agonist, A71378 (Fig. 1C, Lanes 2–4). Moreover, when the acinar cells were incubated with CCK and two different CCK1 receptor antagonists [L364, 718 or SR27897] [50,51] the increment in pT402 PAK2 phosphorylation observed in the sole presence of CCK was largely inhibited, but not in the presence of the CCK2 receptor antagonists YM022 [50] (Fig. 2, Panel C, Lanes 5–10). These results demonstrate that the observed effect of CCK in pT402 PAK2 phosphorylation is only due to the activation of CCK1 receptors.

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Fig. 2. Ability of various pancreatic secretagogues and pancreatic growth factors to stimulate PAK2 phosphorylation (pT402) in rat pancreatic acini. Panel A: Ability of CCK, carbachol, bombesin, secretin, VIP or endothelin I to activate pT402 PAK2 in isolated pancreatic acini. Isolated pancreatic acini were incubated in the absence or presence of CCK (100 nM), carbachol (10 μM), bombesin (1 nM), secretin (10 nM), VIP (10 nM) or endothelin 1 (10 nM) for 1 min, and then lysed. The cell lysates were subjected to Western blotting and analyzed using anti-pT402 PAK2 and, as loading control, anti-total PAK2. Bands were visualized using chemiluminescence and quantified by densitometry. Top: Results of a representative blot of 5 independent experiments are shown. Bottom: Means ± S.E. of 5 independent experiments. Results are expressed as % of basal stimulation of the control group taking total PAK2 as a loading control. *p b 0.05 compared to the control. Panel B: Ability of insulin, IGF-1, HGF, EGF, bFGF and PDGF to activate PAK2 in the pancreatic acini. Isolated pancreatic acini were incubated in the absence or presence of insulin (1 μM, 10 min), EGF (10 nM, 5 min), PDGF (100 ng/ml, 10 min), bFGF (100 ng/ml, 5 min), IGF (100 nM, 10 min) and HGF (1 nM) for 10 min, and then lysed. The lysates were subjected to Western blotting and analyzed using anti-pT402 PAK2 antibody and, as loading control, anti-Total PAK2. Bands were visualized using chemiluminescence and quantified by densitometry. Results of a representative blot of 5 independent experiments are shown. * p b 0.05 compared to the control. Panel C: Ability of selective CCKA or CCKB receptor agonist/antagonists to alter PAK2 activation in pancreatic acini. Isolated pancreatic acini were incubated in the absence or presence of CCK (100 nM), the CCKB agonist, gastrin (10 nM) or the CCKA receptor agonist, A71378 (30 nM) for 1 min (left blot), or preincubated for 5 min in the presence of the CCKB antagonist YM022 (1 μM), the CCKA antagonists SR27897 (1 μM) or L364,718 (1 μM) and then after the additional presence of CCK (100 nM) for 3 min (right blot), the cells were lysed. PAK2 kinase activity was determined as outlined in Materials and methods and Western blots were analyzed using anti-pT402 PAK2 and, as loading control, anti-Total PAK2.

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Fig. 4. Time course of CCK and EGF stimulation of PAK2 T402 phosphorylation in rat pancreatic acini. Isolated pancreatic acini were incubated in the absence or presence of CCK (100 nM) or EGF (10 nM) for the indicated times, and then lysed. Western blots were analyzed using anti-pT402 PAK2 and, as loading control, anti-Total PAK2. Bands were visualized using chemiluminescence and quantified by densitometry. Top: Results of a representative blot of 5 independent experiments are shown. Bottom: Means ± S.E. of 5 independent experiments. Results are expressed as % of basal stimulation of the control group. * p b 0.001 compared to the control group (i.e., 0 time).

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3.4. Time course of CCK and EGF stimulation of T402 PAK2 phosphorylation in rat pancreatic acini Stimulation of T402 PAK2 by CCK was time-dependent, with a significant increment after 1-minute incubation time (275 ± 17% of control, p b 0.05), and maximum after 3 min (298 ± 26% of control, p b 0.05) (Fig. 4). The increase in phosphorylation of phospho-T402 PAK2 by CCK was maintained in time and lasted over 15 min (all p b 0.05) (Fig. 4). Because some of the growth factors studied stimulated T402 PAK2 phosphorylation, the ability of EGF, to stimulate PAK2 T402 phosphorylation at different times, was studied (Fig. 4). EGF produced, as in the case of CCK, a rapid and maximum increase (1 min: 300 ± 30% of control) in PAK2 pT402 phosphorylation (Fig. 4), and the increase was not maintained for longer than 3 min. EGF stimulation of pT402 PAK2 phosphorylation had a rapid decrease compared with CCK (Fig. 4).

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3.5. Effect of post-receptor stimulants, PKC inhibition or Ca2+ depletion on 322 T402 PAK2 kinase phosphorylation by CCK and TPA 323

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activation of the different states activates different cell signaling cascades [38,54–56]. In order to determine the contribution of each activation receptor state to the activation of PAK2 by CCK, pancreatic acini were incubated in the presence of increasing concentrations of CCKJMV, known to be an agonist of the CCK1 high affinity state and an antagonist of the low affinity CCK1 receptor state in the rat pancreatic acini [54,56,57]. CCK-JMV stimulated threonine 402 phosphorylation of PAK2 in a monophasic manner with concentrations from 100 nM to 1000 nM (Fig. 3) with an EC50 of 0.18 ± 0.14 nM (Fig. 3, Table 1), and therefore was 2.5-times less potent than CCK. CCK-JMV caused 26% of the maximal stimulation of pT402 PAK2 phosphorylation caused by CCK (Fig. 3). These results support the conclusion that CCK stimulation of PAK2 kinase activation is mediated 26% by the high affinity state CCK1 receptor and 74% by activation of the low affinity CCK1 receptor state. At a CCK concentration of 100 nM, a concentration that activates both CCK receptor states, CCK produced the maximal stimulation of T402 PAK2 phosphorylation, thus it was selected for use in the rest of the study.

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Fig. 3. Dose–response effect of CCK and CCK-JMV on PAK2 kinase phosphorylation in rat pancreas acini. Isolated pancreatic acini were incubated in the absence or presence of CCK and CCK-JMV (at the indicated concentrations) for 3 min, and then lysed. Western blots were analyzed using anti-pT402 PAK2 antibody and, as loading control, anti-Total PAK2. Bands were visualized using chemiluminescence and quantified by densitometry. Top: Results of a representative blot of 5 independent experiments are shown. Bottom: Means ± S.E. of 5 independent experiments. Results are expressed as % of maximal CCK stimulation (CCK 100 nM: 904 ± 140% of control). * p b 0.05 compared to the control group.

When the ability of post-receptor stimulants to stimulate threonine 402 phosphorylation of PAK2 was studied, the PKC activating agent, TPA, produced a significant increment in threonine 402 phosphorylation of PAK2 (TPA = 635 ± 97% of control p b 0.05). However, in calcium containing medium neither the cellular calcium-mobilizing agent, thapsigargin (TG) (180 ± 40% of control) nor the calcium ionophore, A23187 (209 ± 35%), significantly increased PAK2 phosphorylation. The combination of the calcium mobilization agent, thapsigargin, or the calcium ionophore, A23187, with the PKC activator, TPA, did not cause greater activation than with any of these agents alone (Fig. 5, Panel A, Rows 1–7; Table 1). We next examined whether CCK-induced activation of PKC or increase in intracellular calcium were needed for its ability to cause pT402 threonine phosphorylation of PAK2 in pancreatic acini (Fig. 5, Panel B, Rows 1–12). To determine the role of intracellular calcium changes, [Cai], pancreatic acinar cells were pretreated for 1 h in a calcium-free medium with thapsigargin (10 μM), an agent that specifically inhibits the endoplasmic reticulum Ca2+-ATPase and depletes calcium from intracellular compartments in a calcium free medium [34]. These conditions have been previously shown to completely inhibit the [Ca2 +i] increase stimulated by CCK in rat pancreatic acinar cells [34]. In order to examine the implication of PKC in the CCK stimulated threonine 402 PAK2 phosphorylation, isolated acinar cells were treated for 1 h with a general PKC inhibitor GFX109203X [34] prior to incubation with CCK or TPA. When PKCs activation was inhibited, the increment in the threonine phosphorylation of PAK2 was significantly reduced in the case of CCK or completely inhibited in the case of TPA (CCK alone: 515 ± 73% of control, p b 0.05 vs control; CCK + GFX: 245 ± 50% of control, p b 0.05 vs CCK alone; TPA alone: 319 ± 45% of control, p b 0.05 vs control; TPA + GFX: 100 ± 26% of control, p b 0.05 vs TPA alone) (Fig. 5, Panel B, Rows 1–6). Inhibition of CCK-stimulated increases in intracellular Ca2+ did not significantly decrease the pT402 threonine phosphorylation of PAK2 by CCK (CCK alone: 515 ± 73% of control, p b 0.05 vs control; TG + CCK: 403 ± 86% of control) (Fig. 5,

Please cite this article as: B. Nuche-Berenguer, R.T. Jensen, Gastrointestinal hormones/neurotransmitters and growth factors can activate P21 activated kinase 2 in pancreatic acinar cells by n..., Biochim. Biophys. Acta (2015), http://dx.doi.org/10.1016/j.bbamcr.2015.05.011

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B. Nuche-Berenguer, R.T. Jensen / Biochimica et Biophysica Acta xxx (2015) xxx–xxx

Panel B, Rows 7–12). The combination of both GFX and thapsigargin had an additive effect in the inhibition of basal T402 PAK2 phosphorylation (26 ± 11% of control, p b 0.05 vs control) and also in the phosphorylation induced by CCK (CCK alone: 515 ± 73% of control, p b 0.05 vs control; TG + GFX + CCK: 138 ± 31% of control, p b 0.05 vs CCK-alone) and with TPA, where no signal of phospho-T402 PAK2 was detected in any of the experiments (Fig. 5, Panel B, compare Rows 9–12, 4–6, 7–9).

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3.6. SFKs and CDC42/Rac1, but not PI3K are upstream activators of PAK2 in 365 pancreatic acinar cells 366 367 368

3.7. Effects of PAK2 inhibition in ERK1/2 activity

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Once we determined the involvement of PKC in activation of PAK2, we wanted to know whether CCK- and TPA-induced PAK2 activation was dependent on stimulation of the small GTPases (CDC42 and Rac1), SFK or PI3K in pancreatic acini (Fig. 6, Panels A–C, Rows 1–12). To determine their possible involvement, isolated acinar cells were treated, prior to incubation with CCK or TPA, either for 1 h with the SFK inhibitor PP2 (10 μM) [58] or for 15 min with the PI3K inhibitors wortmannin [57] (1 μM) or the PAK2 inhibitor, IPA-3 (40 μM) that binds covalently to the Group-I-PAKs regulatory domain, preventing binding to Cdc42 and/or Rac1 [15,59] and that has proven specificity for Group I PAKS [59]. We used IPA-3 at this incubation time (t = 15 min) and concentration (40 μM) because in a previous study [8] it was shown to efficaciously inhibit PAK activity under these incubation conditions. The PI3K inhibitor, LY294002 (100 μM), and the inactive analogs for PP2 and IPA-3, PP3 (10 μM) and Pir-3,5 (40 μM) were also used to insure specificity. When PI3K activation was inhibited with wortmannin or LY294002, the basal pT402 threonine phosphorylation of PAK2 was not affected (Fig. 6, Panel C, Lanes 5 and 9). Furthermore, the increment in the threonine phosphorylation of PAK2 was not significantly reduced in the case of both CCK concentrations or TPA (CCK 0.3 nM + Wort: 82 ± 15% of CCK 0.3 nM alone, p b 0.05; CCK 100 nM + Wort: 99 ± 21% of CCK 100 nM alone, p b 0.05; TPA + Wort: 79 ± 20% of TPA alone, p b 0.05) (Fig. 6, Panel C, Rows 6–8). Preincubation with 100 μM LY294002 confirmed the results obtained with wortmannin (CCK 0.3 nM + Wort: 86 ± 39% of CCK 0.3 nM alone, p b 0.05; CCK 100 nM + Wort: 89 ± 26% of CCK 100 nM alone, p b 0.05; TPA + Wort: 90 ± 18% of TPA alone, p b 0.05) (Fig. 6, Panel C, Rows 10–12). Inhibition of SFK did not significantly decrease either the basal pT402 threonine phosphorylation of PAK2 (95 ± 8% of control) or its 100 nM CCK-stimulated phosphorylation (CCK 100 nM + PP2: 90 ± 11% of CCK 100 nM alone). However, preincubation with PP2 decreased 0.3 nM CCK- and TPA-stimulated phosphorylation (CCK 0.3 nM + PP2: 51 ± 14% of CCK 0.3 nM alone, p b 0.05; TPA + PP2: 38 ± 3% of TPA alone, p b 0.05) (Fig. 6, Panel B, Rows 1–12). The inactive control PP3 did not have any effect upon PAK2 phosphorylation or upon the positive control (Fig. 6, Panel B, Rows 9–12). Prevention of the binding of the small GTPases, CDC42 and/or Rac1 by preincubation with the specific PAK inhibitor IPA-3 [15], resulted in a reduction of basal activation levels of PAK2 (32 ± 6% of control, p b 0.05 vs control), and also decreased its CCK-induced activation (CCK 0.3 nM + IPA3: 30 ± 9% of CCK 0.3 nM alone, p b 0.05; CCK 100 nM + IPA3: 45 ± 4% of CCK 100 nM alone, p b 0.05) and TPAinduced activation (TPA + IPA3: 39 ± 9% of TPA alone, p b 0.05) (Fig. 6, Panel A, Rows 1–12). That this IPA-3 inhibitory effect was not due to a nonspecific effect on PAK2 activation is supported by the results with the inactive control Pir-3, 5 which did not have any effect upon PAK2 activation (Fig. 6, Panel A, Rows 9–12). This concentration of IPA-3 produced a 70% of PAK2 inhibition.

Fig. 5. Effect of post-receptor stimulants, PKC inhibition or Ca2+ depletion on pT402 PAK2 phosphorylation by CCK and TPA. Panel A: Effect of the calcium ionophore A23187 (1 μM), TPA (1 μM), Thapsigargin (TG) (1 μM), or CCK (100 nM), on PAK2 phosphorylation [all for 5 min, except CCK for 3 min]. Top: Results of a representative blot of 5 independent experiments are shown. Bottom: Means ± S.E. of 5 independent experiments. Results are expressed as % of basal stimulation of the control group. * p b 0.05 compared to the control group. Panel B: Effect of PKC inhibition or Ca2+ depletion on pT402 PAK2 CCK- and TPA-induced phosphorylation. Isolated acini were preincubated for 2 h, alone or in the additional presence of GF109203X (5 μM, 1 h), Thapsigargin (TG) (1 μM, 1 h) or both and then incubated with or without 100 nM CCK or 1 μM TPA for 3 or 5 min, respectively. Top: Results of a representative blot of 5 independent experiments are shown. Bottom: Means ± S.E. of 5 independent experiments. Results are expressed as % of basal stimulation of the control group. * p b 0.05 compared to the control group, ^ p b 0.05 vs its own control, $ p b 0.05 vs CCK-alone, # p b 0.05 vs TPA alone.

369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415

Preincubation with 40 μM IPA-3, a concentration that reduced PAK 417 activity by a 70% did not affect the basal phosphorylation of p42/44 418 (89 ± 18% of basal, p b 0.05) (Fig. 7). However, IPA-3 preincubation 419

Please cite this article as: B. Nuche-Berenguer, R.T. Jensen, Gastrointestinal hormones/neurotransmitters and growth factors can activate P21 activated kinase 2 in pancreatic acinar cells by n..., Biochim. Biophys. Acta (2015), http://dx.doi.org/10.1016/j.bbamcr.2015.05.011

B. Nuche-Berenguer, R.T. Jensen / Biochimica et Biophysica Acta xxx (2015) xxx–xxx

reduced significantly the CCK induced phosphorylation of p42/44 (64 ± 4% of CCK 0.3 nM alone, p b 0.05) (Fig. 7).

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3.8. Effects of PAK2 inhibition in amylase release

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To investigate the possible effect of PAK2 inhibition on enzyme secretion, acini were incubated with our with two pancreatic secretagogues (i.e., CCK at two concentrations and bombesin) with or without the active PAK2 inhibitor or its inactive control analog (Table 2). The two CCK concentrations or bombesin alone resulted in a stimulation of enzyme secretion, as previously reported in numerous studies [60–62] with 0.01 nM CCK causing a 313 ± 57% increase in amylase release, 0.1 nM CCK a 413 ± 11% and bombesin

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Fig. 7. Effect of the PAK2 inhibitor, IPA-3 and its inactive analog PIR 3,5 on the ability of a physiological 0.3 nM concentration of CCK to activate ERK1/2 . Rat pancreatic acinar cells were processed as stated in Fig. 6. Membranes were analyzed using anti p-Y202/204 p42/ 44. An antibody detecting total amount of ERK1/2 was used to verify loading of equal amounts of protein. The bands were visualized using chemoluminescence and quantification of phosphorylation was assessed using scanning densitometry. Both a representative experiment of 4 others (top panel) and the means of all the experiments (bottom panel) are shown. * p b 0.05 vs control, # p b 0.05 vs IPA-3 alone, ∞ p b 0.05 vs Pir 3,5 alone and $ p b 0.05 comparing CCK stimulation vs stimulants pre-incubated with IPA-3 or Pir 3,5, respectively.

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causing an increase of 401 ± 21%(Table 2). Preincubation with 40 μM IPA3, which inhibited PAK2 activity by approximately 70% (Fig. 6A) reduced the increase in amylase release induced by both concentrations of CCK by a 63% of maximal CCK stimulation and that induced by bombesin by 55% of the maximal bombesininduced stimulation. Preincubation with the IPA3 inactive analog Pir3,5 (40 μM) had no effect on stimulated enzyme secretion (Table 2) supporting the specificity of IPA-3 in inhibiting CCK- and bombesin-induced amylase release. Fig. 6. Effect of the inhibition of Src, PI3K and the binding of GTPases to PAK2 on the ability of physiological (0.3 nM) and supraphysiological concentration of CCK (100 nM) or TPA to stimulate PAK2.Both a representative experiment of 4 others and the means of all the experiments are shown in each panel.Panel A: Rat pancreatic acinar cells were pretreated with no additions or with IPA-3 (40 μM) or Pir 3,5 (40 μM) for 15 min and then incubated with no additions (control), with 0.3 nM or 100 nM CCK for 3 min or with 1 μM TPA for 5 min and then lysed and analyzed anti-pT402 PAK2 and, as a loading control, anti-total PAK2. * p b 0.05 vs control, # p b 0.05 vs IPA-3 alone, ∞ p b 0.05 vs Pir 3,5 alone and $ p b 0.05 comparing stimulants (CCK or TPA) vs stimulants pre-incubated with IPA-3.Panel B: Rat pancreatic acinar cells were pretreated with no additions or with PP2 (10 μM) or PP3 (10 μM) for 1 h and then incubated with no additions (control), with 0.3 nM, 100 nM CCK for 3 min or with 1 μM TPA for 5 min and then lysed and analyzed antipT402 PAK2 and, as a loading control, anti-total PAK2. * p b 0.05 vs control, # p b 0.05 vs PP2 alone, ∞ p b 0.05 vs PP3 alone and $ p b 0.05 comparing stimulants (CCK or TPA) vs stimulants pre-incubated with PP2.Panel C: Rat pancreatic acinar cells were pretreated with no additions or with wortmannin (1 μM) or Ly294002 (100 μM) for 30 min and then incubated with no additions (control), with 0.3 nM or 100 nM CCK for 3 min or with 1 μM TPA for 5 min and then lysed and analyzed anti-pT402 PAK2 and, as a loading control, antitotal PAK2. * p b 0.05 vs control, # p b 0.05 vs wortmannin alone and ∞ p b 0.05 vs LY294002 alone.

Please cite this article as: B. Nuche-Berenguer, R.T. Jensen, Gastrointestinal hormones/neurotransmitters and growth factors can activate P21 activated kinase 2 in pancreatic acinar cells by n..., Biochim. Biophys. Acta (2015), http://dx.doi.org/10.1016/j.bbamcr.2015.05.011

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8

Secretagogue

Amylase secretion (% total)

t2:4 Q2

(0.01 nM) (0.1 nM) (10 nM)

40 μM Pir 3,5

3.8 ± 0.3 11.9 ± 2.2* 15.7 ± 0.4* 15.2 ± 0.8*

5.2 ± 0.5 7.5 ± 1.3^ 9.6 ± 0.6^ 10.3 ± 0.9^

4.3 ± 0.9 12.6 ± 0.8* 14.7 ± 1.0* 14.4 ± 1.2*

Pancreatic acini were incubated with no additions, the indicated secretagogue alone or with the active PAK2 inhibitor, IPA-3 (40 μM) or its inactive analog Pir 3,5 (40 μM). Amylase release, expressed as percent of cellular total amylase was determined after 30 min incubation. Results are the result of the average of 5–10 experiments. *p b 0.05 compared to control, and $ p b 0.05 comparing stimulants (CCK or bombesin) vs stimulants at the same concentration pre-incubated with IPA-3.

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4. Discussion

441 Q9 442

The purpose of this study was to determine if Group-I-PAK kinases are present in pancreatic acinar cells, and if so, are they activated by GI hormones/neurotransmitters and growth factors, and to determine the transduction cascades involved. In various tissues, PAKs are activated by a wide range of stimuli [2,2,9,10,19–21,30,36,63–67] and a number of G-protein-coupled-receptors [26,68]. However, little is known of their ability to be activated by GI hormones/neurotransmitters or if so, the cellular signaling cascades involved. To address this question we studied pancreatic acinar cells because these cells respond to a large number of GI hormones/neurotransmitters and GI growth factors [33,69]. Particular attention was paid to the GI hormone/neurotransmitter cholecystokinin, CCK, because it is both a physiological regulator of pancreatic function [37] and plays an important role in a number of pathophysiological processes, such as pancreatitis and response to injury [37,38,70,71]. In regards to the pancreas, except in the case of islet function [16,24, 35], there are no studies examining the presence of PAKs in exocrine tissues and their activation or signaling has not been investigated. However, previous studies report CCK activates the small GTP binding proteins, Rho and Rac1, which are one of the main stimulants of PAKs in other tissues [13]. Furthermore, activation of Ras and Rho in acinar cells is important in enzyme secretion as well as in mediating various pathologic effects on the morphology of the acinar cell occurring in CCK-induced experimental models of acute pancreatitis [72–74]. In the case of pancreatic cancer, a number of studies have reported PAK1 and PAK4 activation promote pancreatic acinar cell growth and effects on survival [75,76] and that PAK1 expression is an important prognostic marker of pancreatic cancer [76]. In this study we established that PAK2 is the only Group-I-PAK present in rat pancreatic acini. This result differs from findings in pancreatic islets, which are reported to contain PAK1 in murine [16,24] and human islets [35] and from results in embryonic islet β cells, which contain PAK3 [17]. Previous results have demonstrated that numerous GI growth factors (HGF, EGF, bFGF, insulin, IGF and PDGF) are able to interact with specific receptors on pancreatic acinar cells and stimulate growth and/ or protein synthesis as well as alter other cellular functions [33,33,54, 57,77–79]. We observed activation of PAK2 by EGF, bFGF and PDGF, but not by insulin, IGF-1 and HGF. This result has both similarities and differences from studies of the effects of the growth factors in other tissues. In contrast to our results, in NIH-3T3 [22], prostate cancer [80] or epithelial cells [13,81], neither EGF nor FGF activated PAK1/PAK2, whereas HGF did [22]. Moreover, in contrast to our results, Group-IPAKs mediate IGF-1 signaling in mesothelial cells [23] and insulin signaling in mouse endocrine L cells [24]. Similar to our results, PDGF stimulated of PAK1 and PAK2 in NIH-3T3 cells, whereas insulin did not [22]. Also, in epidermal cells EGF activated PAK2 [25] as well as stimulating PAK1 in Cos7 cells [26]. Similarly, in PC-12 cells [27–29] bFGF stimulated cell growth by activating PAK1/PAK2. These results demonstrate that

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40 μM IPA3

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IPA3 inhibitor

growth factors' ability to activate Group-I-PAKs can vary markedly in different cells. Our studies show that only the secretagogues stimulating PLCactivated cascades resulting in changes of cytosolic calcium and PKC activation (CCK, carbachol, bombesin), but not those activating adenylate cyclase (VIP, secretin) [33,69,82,83], or other signaling cascades (endothelin) [47,48], activate PAK2 in pancreatic acini. The failure of the latter agents to activate PAK2 was not due to the experimental conditions used because each was used at concentrations and under experimental conditions which are known to alter acinar cell function [33,47, 48,84]. Our results in pancreatic acini are similar to findings in vascular smooth cells in which Group-I-PAKs are activated in a PLC-dependent manner by angiotensin II [68,85], as well as Group-I-PAKs activation by muscarinic cholinergic agents in fibroblasts or neuroblastoma cells [26] and in smooth muscle cells [86]. Similar to pancreatic acini, endothelin did not activate Group-I-PAKs in myocytes [87]. Also, in various tissues, gastrin, which interacts with the high affinity CCK2 receptor, which is also coupled to PLC, stimulated Group-I-PAKs [31,88,89]. Our results are consistent with observations that CCK, in pancreatic acini, stimulates, in a PLC-dependent manner [72], Rac1, which is a PAK activator in other tissues [1,13]. In contrast to our results with secretagogues activating cAMP failing to activate PAK2, in human mesangial cells, endothelin activates CDC42, in a PKA-dependent manner [22], and PKA activation is required for PAKs activation by other GPCRs [90]. These results demonstrate that signaling cascades activating Group-I-PAKs show wide differences in different cells, even with stimuli, which activate similar cellular-signaling cascades. CCK stimulates several kinases in pancreatic acini and their kinetics of activation can vary considerably [34,58,61,91–93]. We found that the kinetics of CCK-stimulated PAK2 activation was similar to those observed in pancreatic acini with the SFK, Yes, PKC theta, PYK2 and PKD [41,54,77,94] in that it showed a rapid increase, which subsequently partially decreased (by 40%), but was sustained over time. However, it differed from CCK's activation of p125FAK or Lyn [39,78], which after initial stimulation decreases markedly to almost basal levels. The time course of CCK-activation of PAK2 in our study differs from that reported in most other tissues for PAK activation by various stimuli, where it is generally much slower [18,26,95]. However, the CCK time-course for PAK2 activation in pancreatic acini is similar to that of EGF-induced PAK2-activation in COS7 cells [26]. These results demonstrate the kinetics of activation of PAKs in different cells shows wide variation, even by different stimuli in the same cell. Numerous studies demonstrate that CCK can activate both high- and low-affinity receptor-states, which can mediate different cellular responses [34,38,96]. Our results show that activation of both receptorstates is required for maximal PAK2 activation, because the CCK dose– response curve for PAK2 activation included the concentration ranges that activate, both high and low affinity receptor-states [33,56,96]. Moreover, incubation with CCK-JMV, a full agonist at the high-affinity receptor state and an antagonist of the low-affinity state [56,96], demonstrated that 26% of PAK2 maximal CCK-stimulation is due to activation of the high-affinity CCK1 receptor-state and 74% to activation of the low-affinity CCK1 receptor-state. Our results with PAK2 activation are similar to the CCK-induced activation of the Src kinases (Lyn, Yes) [41,78], the focal adhesion kinases (p125FAK, PYK2) [34,39], paxillin [34,40] and PKD [54] in pancreatic acini [78], that are all mediated by activation of both high- and low-affinity receptor-states, although with differences in their relative importance. In contrast to these results, CCK-mediated activation in pancreatic acini of phospholipase D or PI3K requires activation of only the high-affinity receptor-state [97], whereas in the case of PKC-δ [32] or CRK-II [98], only the low-affinity CCK1 receptor-state is required for activation. Also, a previous study [99] reported CCK-stimulated SFK activity is mediated almost entirely by activation of the low-CCK1 receptor state in pancreatic acini. Our results provide additional support to the proposal [94] that not only are the different CCK1 receptor-states on pancreatic acini differentially

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Table 2 Effect of PAK2 inhibition on amylase secretion.

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t2:1 t2:2

B. Nuche-Berenguer, R.T. Jensen / Biochimica et Biophysica Acta xxx (2015) xxx–xxx

Please cite this article as: B. Nuche-Berenguer, R.T. Jensen, Gastrointestinal hormones/neurotransmitters and growth factors can activate P21 activated kinase 2 in pancreatic acinar cells by n..., Biochim. Biophys. Acta (2015), http://dx.doi.org/10.1016/j.bbamcr.2015.05.011

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significantly inhibited the activation of PAK2 at a physiological CCK concentration (0.3 nM), but not at supramaximal concentrations (100 nM), suggests that the activation of the high-affinity CCK receptor state requires SFKs for full PAK2 activation. The finding that under experimental conditions that inhibited completely SFKs [86], only partially inhibition of PAK2 activation was seen, demonstrates that CCK, at physiological concentrations, activates PAK2 by both SFK-dependent and independent mechanisms. Our findings with the involvement of SFK's in PAK2 activation by CCK are novel in that they demonstrate both an agonist concentration-dependent effect and receptor-state activation effect, as well as support for both SFK-dependent and independent PAK2 activation. The lack of effect of PI3K inhibition in PAK2 activation in pancreatic acini was not due to an inadequate inhibitory dose, because the inhibitor concentrations used were similar to that previously reported in other tissues that are known to inhibit PI3K activation of Akt. Similarly to reported in other tissues [14,104], the inhibition of the binding of small GTPases to the activation domain of PAK2 by IPA3 incubation markedly inhibited the PAK2 activation, although, under our experimental conditions, the inhibition was not complete. The specificity of the IPA-3 inhibitory effect was supported by the lack of effect of the inactive control Pir 3,5 [59,68,104]. These results demonstrate that, although the small GTPases are the principal upstream activator of PAK2 in these cells, there are additional mechanisms of activation that involve PKC and/or SFKs, both which are important CCK signaling pathways in pancreatic acinar cells [58,77,105]. These results differ from that observed in COS7 [102], HeLa [9], A2058 human melanoma [19] and colorectal cells [31] where the activation of PAK by different stimuli requires activation of PI3K. Also similar to our results, SFK activation is needed for increased PAK1 activity in hippocampal neurons [103]. However, in smooth muscle cells SFK activation was not needed for the angiotensin II-mediated activation of PAK [85]. These findings that activation of SFKs is frequently upstream of PAKs activation are consistent with studies demonstrating SFKs are important in the activation of the PAK activators, Rac and CDC42, by a number of stimulants [103,106,107]. These results reveal that the GI hormone/neurotransmitter, CCK, demonstrates a number of unique features related to its receptor activationstate in activating signaling cascades to stimulate PAK2 activation. CCK stimulates both enzyme secretion by pancreatic acinar cells, as well as their growth [33,38]. The activation of Rac by CCK has been shown to be important for the pancreatic enzyme secretion [72] and

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coupled to activation of phospholipase A2 and C, SFKs, PKCs and focal adhesion kinases [33], they are also differentially coupled to activation of small GTPases, Rac1/CDC42 leading to the activation of PAK2. Activation of the pancreatic CCK1 receptor stimulates phospholipase C (PLC), resulting in the generation of inositol phosphates and diacylglycerol which in turn results in mobilization of cellular calcium and activation of PKCs, respectively [33,34,38]. The experiments performed in this study, including incubation with calcium ionophore, A23187 and thapsigargin (TG) [34,100]; with the PKC activator, TPA; the inhibition of increases of cytosolic Ca++ or PKC activity, and the combination of Ca++ and PKC-modulating experiments, showed that activation of PKC, but not changes in cytosolic calcium, by CCK1 receptor stimulation, is an important mediator for the activation of PAK2 in pancreatic acini and that 40% of the PKC-independent activation of PAK2 was also PLCindependent. In some experiments A23187 had an apparent stimulatory effect, however due to high variability, it was not statistically significant. However, as the combination of A23187, with the PKC activator, TPA, did not cause greater activation than with any of these agents alone, we conclude that A23187 have no effect on PAK2. These results differ from the stimulatory effect of angiotensin II on Group-1-PAKs which in vascular smooth cells requires both limbs of the PLC cascade (PKC and calcium) [68], whereas in other cell systems only increases in Ca2+ are required [85]. Similarly, activation of COS cells by muscarinic cholinergic agents or LPA, stimulated Group-I-PAKs activation in a completely PKC-independent manner [19]. In contrast, our results are similar to activation of Group-I-PAKs in neutrophils receptors by FMLP, which requires activation of PKC, but not of cytosolic calcium [101]. These results demonstrate that even with stimuli, which activate PLC in a number of different tissues, the relative importance of activation of the two limbs of the PLC cascade (increases in Ca2+, PKC) in stimulating PAK activation varies markedly. In addition to small GTPases (Rac1, CDC42) PAK's activity can be affected by activation of a number of signaling cascades [9,19,31,85,102, 103] and the participation of the various cascades can vary markedly in different cells. However, there is almost no information on the signaling cascades by which gastrointestinal hormones/neurotransmitters activate PAKs. Our results demonstrate that in addition to PKC being involved as an upstream activator of CCK- and TPA-induced activation of PAK2, activation of SFKs at physiological CCK doses is important, but not activation of PI3K. Our finding that the SFK inhibitor PP2,

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Fig. 8. Schematic diagram of signaling role of PAK2 in pancreatic acinar cells. In rat pancreatic acinar cells maximal activation of PAK2 by CCK requires activation of CDC42/Rac1 and phospholipase C resulting in PKC activation. PAK2 activation involves both PLC-dependent and independent cascades with the PLC-dependent cascade mediated by PKC activation, with changes in Ca++-not involved. PAK2 also requires SFK-dependent and SFK-independent signaling. However, changes in PI3K are not involved, but a major component of PAK2 activation mediated by CCK-activation is CDC42/Rac1. On the other hand PAK2 is a mediator of CCK-mediated ERK1/2 activation.

Please cite this article as: B. Nuche-Berenguer, R.T. Jensen, Gastrointestinal hormones/neurotransmitters and growth factors can activate P21 activated kinase 2 in pancreatic acinar cells by n..., Biochim. Biophys. Acta (2015), http://dx.doi.org/10.1016/j.bbamcr.2015.05.011

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1. This paper has not been previously published nor is under submission elsewhere for consideration for publication. 2. The authors have no conflicts of interest.

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the action of the MAP kinase, ERK1/2 for the pancreatic growth effect in acinar cells [38,108]. With some cells such as the stimulation by LPS of RATs fibroblasts [109], prolactin in breast cancer cells [110] or 12HETE in CHO cells [111], the activation of Group-I-PAKs is needed for the activation of ERK. These observations raise the possibility that activation of PAK2 by Rac could also be essential for CCK-mediated enzyme secretion and activation of ERK in pancreatic acini, subsequently resulting in growth. To determine whether this in fact occurs, we examined the effect of the specific Group-I-PAKs inhibitor on the ability of CCK to activate both ERK1/2 and stimulate enzyme secretion. We found that activation of PAK2 by CCK was needed for both enzyme secretion stimulated by CCK or bombesin as well as the ability of CCK to activate the MAP kinase, ERK1/2. Our ERK results are consistent with studies in other cells reporting that activation of PAKs by growth factors [22], gastrin [31], prolactin [112] or biological active lipids [18] result in ERK activation. In contrast to our results, the activation of ERK1/2 in skeletal muscle cells is independent of PAK1 [113]. In addition, to these results demonstrating activation of PAK2 is important in mediation of two of CCK's physiological effects on pancreatic acini, they also raise the possibility that PAK2 activation may be important in the pathophysiological effects of CCK. The latter hypothesis is by studies which demonstrate that in the CCK model of acute pancreatitis, the activation of Rac as well as Rho is essential for the characteristic morphologic effects which occur and the actin-distribution which accompanies these changes [73,114]. In conclusion, our study demonstrates pancreatic acinar cells possess only one member of the Group-I-PAKs, PAK2, which differs from the finding in pancreatic islets in a number of previous studies [16,17, 24]. Furthermore, a number of GI growth factors as well as GI hormones/neurotransmitters, which activate PLC, can activate the PAK2. The results of our cell signaling studies are summarized in Fig. 8. CCK stimulated PAK2 activation is novel in that it involves both PLCdependent and -independent cascades and the PLC-dependent cascade are mediated by PKC activation with no involvement of changes in cytosolic calcium. Furthermore, activation of SFK is partially required for the PKC-mediated PAK2 activation, but in contrast to a number of other cells, PI3K activation is not needed. CCK also activates PAK2 through a CDC42/Rac1, dependent cascade. PAK2 is also important in mediating ERK1/2 activation and amylase release. Our results support the conclusion that activation of PAKs in pancreatic acinar cells by GI hormones/ neurotransmitters and GI growth factors may be an important signaling cascade in both physiological and pathophysiological responses in the case of pancreatic acinar cells. The importance of PAKs in the pancreatic acinar cell pathologic conditions is supported by recent studies demonstrating that inhibition of Rac activation, one of the PAK's main upstream activators, decreases the severity of pancreatitis [114] or by studies demonstrating activation of PAK4 in the pancreatic acinar cell line, AR42J, is associated with activation of intracellular trypsinogen, one of the main mediators of acute pancreatitis [36]. These support the idea that PAK's could be a potential target for the treatment of acute pancreatitis.

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