Jingzhen Yuan

Activation of Protein Kinase D by Signaling through the α Subunit of the Heterotrimeric G Protein Gq

Protein kinase D (PKD/PKCμ) immunoprecipitated from COS-7 cells transiently transfected with either a constitutively active mutant of Rho (RhoQ63L) or the Rho-specific guanine nucleotide exchange factor pOnco-Lbc (Lbc) exhibited a marked increase in basal activity. Addition of aluminum fluoride to cells co-transfected with PKD and wild type Gα13 also induced PKD activation. Co-transfection of Clostridium botulinum C3 toxin blocked activation of PKD by RhoQ63L, Lbc, or aluminum fluoride-stimulated Gα13. Treatment with the protein kinase C inhibitors GF I or Ro 31-8220 prevented the increase in PKD activity induced by RhoQ63L, Lbc, or aluminum fluoride-stimulated Gα13. PKD activation in response to Gα13 signaling was also completely prevented by mutation of Ser-744 and Ser-748 to Ala in the kinase activation loop of PKD. Co-expression of C. botulinum C3 toxin and a COOH-terminal fragment of Gαq that acts in a dominant-negative fashion blocked PKD activation in response to agonist stimulation of bombesin receptor. Expression of the COOH-terminal region of Gα13 also attenuated PKD activation in response to bombesin receptor stimulation. Our results show that Gα13 contributes to PKD activation through a Rho- and protein kinase C-dependent signaling pathway and indicate that PKD activation is mediated by both Gαq and Gα13 in response to bombesin receptor stimulation.

Amino Acid-stimulated Ca2+ Oscillations Produced by the Ca2+-sensing Receptor Are Mediated by a Phospholipase C/Inositol 1,4,5-Trisphosphate-independent Pathway That Requires G12, Rho, Filamin-A, and the Actin Cytoskeleton

The G protein-coupled Ca2+-sensing receptor (CaR) is an allosteric protein that responds to two different agonists, Ca2+ and aromatic amino acids, with the production of sinusoidal or transient oscillations in intracellular Ca2+ concentration ([Ca2+]i). Here, we examined whether these differing patterns of [Ca2+]i oscillations produced by the CaR are mediated by separate signal transduction pathways. Using real time imaging of changes in phosphatidylinositol 4,5-biphosphate hydrolysis and generation of inositol 1,4,5-trisphosphate in single cells, we found that stimulation of CaR by an increase in the extracellular Ca2+ concentration ([Ca2+]o) leads to periodic synthesis of inositol 1,4,5-trisphosphate, whereas l-phenylalanine stimulation of the CaR does not induce any detectable change in the level this second messenger. Furthermore, we identified a novel pathway that mediates transient [Ca2+]i oscillations produced by the CaR in response to l-phenylalanine, which requires the organization of the actin cytoskeleton and involves the small GTPase Rho, heterotrimeric proteins of the G12 subfamily, the C-terminal region of the CaR, and the scaffolding protein filamin-A. Our model envisages that Ca2+ or amino acids stabilize unique CaR conformations that favor coupling to different G proteins and subsequent activation of distinct downstream signaling pathways.

Protein Kinase Cν/Protein Kinase D3 Nuclear Localization, Catalytic Activation, and Intracellular Redistribution in Response to G Protein-coupled Receptor Agonists

The protein kinase D (PKD) family consists of three serine/threonine kinases: PKCμ/PKD, PKD2, and PKCν/PKD3. Whereas PKD has been the focus of most studies, virtually nothing is known about the effect of G protein-coupled receptor agonists (GPCR) on the regulatory properties and intracellular distribution of PKD3. Consequently, we examined the mechanism that mediates its activation and intracellular distribution. GPCR agonists induced a rapid activation of PKD3 by a protein kinase C (PKC)-dependent pathway that leads to the phosphorylation of the activation loop of PKD3. Comparison of the steady-state distribution of endogenous or tagged PKD3 versus PKD and PKD2 in unstimulated cells indicated that whereas PKD and PKD2 are predominantly cytoplasmic, PKD3 is present both in the nucleus and cytoplasm. This distribution of PKD3 results from its continuous shuttling between both compartments by a mechanism that requires a nuclear import receptor and a competent CRM1-nuclear export pathway. Cell stimulation with the GPCR agonist neurotensin induced a rapid and reversible plasma membrane translocation of PKD3 that is PKC-dependent. Interestingly, the nuclear accumulation of PKD3 can be dramatically enhanced in response to its activation. Thus, this study demonstrates that the intracellular distribution of PKD isoenzymes are distinct, and suggests that their signaling properties are regulated by differential localization.

PKD, PKD2, and p38 MAPK mediate Hsp27 serine‐82 phosphorylation induced by neurotensin in pancreatic cancer PANC‐1 cells

It is widely recognized that Hsp27 is a downstream substrate of the p38 MAPK cascade whereas the role of PKD family members in mediating receptor‐stimulated Hsp27 Ser‐82 phosphorylation has not been evaluated. Here, we show that neurotensin induced a rapid and striking increase in Hsp27 Ser‐82 phosphorylation in PANC‐1 cells, which was closely correlated with stimulation of activation loop phosphorylation of PKDs and p38 MAPK Thr180/Tyr182 phosphorylation. Treatment of PANC‐1 cells with either the selective PKC inhibitor GF‐I or the p38 MAPK inhibitor SB202190 partially reduced neurotensin‐induced Hsp27 Ser‐82 phosphorylation. However, treatment of the cells with a combination of GF‐I and SB202190 virtually abolished neurotensin‐induced Hsp27 Ser‐82 phosphorylation. Overexpression of PKD in stably transfected PANC‐1 cells increased the magnitude and prolonged the duration of Hsp27 Ser‐82 phosphorylation in response to neurotensin. Either PKD or PKD2 gene silencing utilizing siRNAs targeting distinct PKD or PKD2 sequences reduced neurotensin‐stimulated Hsp27 Ser‐82 phosphorylation, but cotransfection of siRNAs targeting both, PKD and PKD2, markedly decreased neurotensin‐induced Hsp27 Ser‐82 phosphorylation. Knockdown of PKD and PKD2 abolished Hsp27 phosphorylation in cells treated with SB202190. Thus, neurotensin induces Hsp27 Ser‐82 phosphorylation through p38 MAPK‐ and PKC/PKD‐dependent pathways in PANC‐1 cells. Our results demonstrate, for the first time, that neurotensin induces a striking increase in Hsp27 phosphorylation on Ser‐82 in PANC‐1 cells through convergent p38 MAPK, PKD, and PKD2 signaling. J. Cell. Biochem. 103: 648–662, 2008.

Cooperation of Gq, Gi, and G12/13 in Protein Kinase D Activation and Phosphorylation Induced by Lysophosphatidic Acid

To examine the contribution of different G-protein pathways to lysophosphatidic acid (LPA)-induced protein kinase D (PKD) activation, we tested the effect of LPA on PKD activity in murine embryonic cell lines deficient in Gαq/11(Gαq/11 KO cells) or Gα12/13(Gα12/13 KO cells) and used cells lacking rhodopsin kinase (RK cells) as a control. In RK and Gα12/13 KO cells, LPA induced PKD activation through a phospholipase C/protein kinase C pathway in a concentration-dependent fashion with maximal stimulation (6-fold for RK cells and 4-fold for Gα12/13 KO cells in autophosphorylation activity) achieved at 3 μm. In contrast, LPA did not induce any significant increase in PKD activity in Gαq/11 KO cells. However, LPA induced a significantly increased PKD activity when Gαq/11 KO cells were transfected with Gαq. LPA-induced PKD activation was modestly attenuated by prior exposure of RK cells to pertussis toxin (PTx) but abolished by the combination treatments of PTx and Clostridium difficile toxin B. Surprisingly, PTx alone strikingly inhibited LPA-induced PKD activation in a concentration-dependent fashion in Gα12/13 KO cells. Similar results were obtained when activation loop phosphorylation at Ser-744 was determined using an antibody that detects the phosphorylated state of this residue. Our results indicate that Gq is necessary but not sufficient to mediate LPA-induced PKD activation. In addition to Gq, LPA requires additional G-protein pathways to elicit a maximal response with Gi playing a critical role in Gα12/13 KO cells. We conclude that LPA induces PKD activation through Gq, Gi, and G12 and propose that PKD activation is a point of convergence in the action of multiple G-protein pathways.

Intracellular redistribution of protein kinase D2 in response to G-protein-coupled receptor agonists ☆

The protein kinase D (PKD) family consists of three serine/threonine protein kinases: PKC mu/PKD, PKD2, and PKC nu/PKD3. While PKD has been the focus of most studies to date, no information is available on the intracellular distribution of PKD2. Consequently, we examined the mechanism that regulates its intracellular distribution in human pancreatic carcinoma Panc-1 cells. Analysis of the intracellular steady-state distribution of fluorescent-tagged PKD2 in unstimulated cells indicated that this kinase is predominantly cytoplasmic. Cell stimulation with the G protein-coupled receptor agonist neurotensin induced a rapid and reversible plasma membrane translocation of PKD2 by a mechanism that requires PKC activity. In contrast to the other PKD isoenzymes, PKD2 activation did not induce its redistribution from the cytoplasm to the nucleus. Thus, this study demonstrates that the regulation of the distribution of PKD2 is distinct from other PKD isoenzymes, and suggests that the differential spatio-temporal localization of these signaling molecules regulates their specific signaling properties.

Protein kinase D1 mediates NF-κB activation induced by cholecystokinin and cholinergic signaling in pancreatic acinar cells

The transcription factor NF-kappaB plays a critical role in inflammatory and cell death responses during acute pancreatitis. Previous studies in our laboratory demonstrated that protein kinase C (PKC) isoforms PKCdelta and epsilon are key regulators of NF-kappaB activation induced by cholecystokinin-8 (CCK-8), tumor necrosis factor-alpha, and ethanol. However, the downstream participants in regulating NF-kappaB activation in exocrine pancreas remain poorly understood. Here, we demonstrate that protein kinase D1 (PKD1) is a key downstream target of PKCdelta and PKCepsilon in pancreatic acinar cells stimulated by two major secretagogues, CCK-8 and the cholinergic agonist carbachol (CCh), and that PKD1 is necessary for NF-kappaB activation induced by CCK-8 and CCh. Both CCK-8 and CCh dose dependently induced a rapid and striking activation of PKD1 in rat pancreatic acinar cells, as measured by in vitro kinase assay and by phosphorylation at PKD1 activation loop (Ser744/748) or autophosphorylation site (Ser916). The phosphorylation and activation of PKD1 correlated with NF-kappaB activity stimulated by CCK-8 or CCh, as measured by NF-kappaB DNA binding. Either inhibition of PKCdelta or epsilon by isoform-specific inhibitory peptides, genetic deletion of PKCdelta and epsilon in pancreatic acinar cells, or knockdown of PKD1 by using small interfering RNAs in AR42J cells resulted in a marked decrease in PKD1 and NF-kappaB activation stimulated by CCK-8 or CCh. Conversely, overexpression of PKD1 resulted in augmentation of CCK-8- and CCh-stimulated NF-kappaB activation. Finally, the kinetics of PKD1 and NF-kappaB activation during cerulein-induced rat pancreatitis showed that both PKD1 and NF-kappaB activation were early events during acute pancreatitis and that their time courses of response were similar. Our results identify PKD1 as a novel early convergent point for PKCdelta and epsilon in the signaling pathways mediating NF-kappaB activation in pancreatitis.

Protein Kinase C δ-Mediated Processes in Cholecystokinin-8-Stimulated Pancreatic Acini

Objectives: To define the role of protein kinase C &dgr; (PKC &dgr;) in acinar cell responses to the hormone cholecystokinin-8 (CCK) using isoform-specific inhibitors and a previously unreported genetic deletion model. Methods: Pancreatic acinar cells were isolated from (1) rat, and pretreated with a PKC &dgr;-specific inhibitor or (2) PKC &dgr;-deficient and wild type mice. Isolated cells were stimulated with CCK (0.001-100 nmol/L) and cell responses were measured. Results: The PKC &dgr; inhibitor did not affect stimulated amylase secretion from rat pancreatic acinar cells. Cholecystokinin-8 stimulation induced a typical biphasic dose-response curve for amylase secretion in acinar cells isolated from both PKC &dgr;−/− and wild type mice, with maximal stimulation at 10-pmol/L CCK. Cholecystokinin-8 (100 nmol/L) induced zymogen and nuclear factor &kgr;B activation in both PKC &dgr;−/− and wild type mice, although it was up to 50% less in PKC &dgr;−/−. Conclusions: In contrast to previous studies, this study has used specific and complementary approaches to examine PKC &dgr;-mediated acinar cell responses. We could not confirm that it mediates amylase release but corroborated its role in the early stages of acute pancreatitis.

Inflammatory cells regulate p53 and caspases in acute pancreatitis

The inflammatory response during pancreatitis regulates necrotic and apoptotic rates of parenchymal cells. Neutrophil depletion by use of anti-polymorphonuclear serum (anti-PMN) increases apoptosis in experimental pancreatitis but the mechanism has not been determined. Our study was designed to investigate signaling mechanisms in pancreatic parenchymal cells regulating death responses with neutrophil depletion. Rats were neutrophil depleted with anti-PMN treatment. Then cerulein pancreatitis was induced, followed by measurements of apoptosis signaling pathways. There was greater activation of executioner caspases-3 in the pancreas with anti-PMN treatment compared with control. There were no differences between these groups of animals in mitochondrial cytochrome c release or in activities of initiator caspase-8 and -9. However, there was greater activation of caspase-2 with anti-PMN treatment during cerulein pancreatitis. The upstream regulation of caspases-2 includes p53, which was increased; the p53 negative regulator, Mdm2, was decreased by anti-PMN treatment during cerulein pancreatitis. In vitro experiments using isolated pancreatic acinar cells a pharmacological inhibitor of Mdm2 increased caspase-2/-3 activities, and an inhibitor of p53 decreased these activities during cholecystokinin-8 treatment. Furthermore, experiments using the AR42J cell line Mdm2 small interfering RNA (siRNA) increased caspase-2/-3 activities, and p53 siRNA decreased these activities during cholecystokinin-8 treatment. These results suggest that during acute pancreatitis the inflammatory response inhibits apoptosis. The mechanism of this inhibition involves caspase-2 and its upstream regulation by p53 and Mdm2. Because previous findings indicate that promotion of apoptosis decreases necrosis and severity of pancreatitis, these results suggest that strategies to inhibit Mdm2 or activate p53 will have beneficial effects for treatment of pancreatitis.

Su1892 Protein Kinase C Epsilon Promotes Necrosis During Experimental Pancreatitis by Upregulation of Expression of Inhibitors of Apoptosis Proteins and Stabilization of Receptor-Interacting Protein Kinase

infiltration, and interstitial edema were all reduced in cerulein-treated PKD3Δpanc mice compared to controls. Conclusion: PKD3Δpanc mice displayed significant attenuation in inflammation, acinar cell necrosis, and severity of pancreatitis. These novel findings indicate that this animal model can be used to demonstrate the role of PKD in the pathobiologic responses in the pancreatic acinar cell during pancreatitis.