scholarly journals Association of bile-salt-dependent lipase with membranes of human pancreatic microsomes is under the control of ATP and phosphorylation

1997 ◽  
Vol 327 (2) ◽  
pp. 527-535 ◽  
Author(s):  
Eric PASQUALINI ◽  
Nathalie CAILLOL ◽  
Eric MAS ◽  
Nadine BRUNEAU ◽  
Doris LEXA ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Bile Salt ◽  
Specific Inhibitor ◽  
Dietary Lipid ◽  
Pancreatic Acinar Cells ◽  
Acetoxymethyl Ester ◽  
Secretion Pathway ◽  
Calphostin C ◽  
First Time

Bile-salt-dependent lipase (BSDL) is secreted by the pancreas into the duodenum, where it catalyses the hydrolysis of dietary lipid esters on activation by bile salts. The secretion pathway of BSDL is comparable with that of other digestive enzymes produced by pancreatic acinar cells. However, in contrast with these other enzymes, BSDL is partly associated with endoplasmic reticulum membranes as part of a folding complex, including a Grp94-related protein to which BSDL is transiently linked. The release of BSDL from membranes occurs once its glycosylation is completed [Bruneau and Lombardo (1995) J. Biol. Chem. 270, 13524-13533]. In the present study, investigations concerning the mechanism of association/dissociation of BSDL with membranes of microsomes were performed. For this purpose the role of ATP and that of the possible phosphorylation of BSDL were examined. For the first time, it is shown that human pancreatic BSDL is phosphorylated, probably at a serine residue, during its transport within the acinar cell. The phosphorylation of BSDL is provoked by calphostin C, an inhibitor of protein kinase C. In the presence of 1-(isoquinolinesulphonyl)2-methylpiperazine, a non-specific inhibitor of serine/threonine protein kinase A, C or G, or of calcium chelator 1,2-bis(O-aminophenoxy)ethane-N,N,Nʹ,Nʹ-tetra-acetic tetra(acetoxymethyl)ester, the phosphorylation of BSDL elicited by calphostin C is abolished. These data suggested that the phosphorylation of BSDL within human pancreatic microsomes is under the control of a cascade of protein kinases. We have also shown that the phosphorylation of BSDL appears to be involved in the release of the enzyme from microsome membranes. Nevertheless ATP, which modifies the conformation of BSDL, triggers this association, and an unhydrolysable ATP analogue was unable to promote it.

scholarly journals Phosphorylation of the rat pancreatic bile-salt-dependent lipase by casein kinase II is essential for secretion

1999 ◽  
Vol 345 (1) ◽  
pp. 121-128 ◽  
Author(s):  
Eric PASQUALINI ◽  
Nathalie CAILLOL ◽  
Anne VALETTE ◽  
Roland LLOUBES ◽  
Alain VERINE ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Bile Salt ◽  
Specific Inhibitor ◽  
Casein Kinase Ii ◽  
Casein Kinase ◽  
Pancreatic Acinar Cells ◽  
Amylase Secretion ◽  
Pancreatic Cell ◽  
Α Subunit ◽  
Intracellular Membranes

Bile-salt-dependent lipase (BSDL, EC 3.1.1.-) is an enzyme expressed by the pancreatic acinar cells and secreted as a component of the pancreatic juice of all examined species. During its secretion route BSDL is associated with intracellular membranes. This association allows the complete glycosylation of the enzyme or participates in the inhibition of the enzyme activity, which can deleterious for the acinar pancreatic cell. Thereafter, the human BSDL is phosphorylated by a serine/threonine protein kinase and released from intracellular membranes. In the present study, we show that the rat pancreatic BSDL, expressed by AR4-2J cells used as a model, is phosphorylated by a protein kinase that is insensitive to inhibitors of protein kinases A, C or G and that the phosphorylation process is favoured by okadaic acid (an inhibitor of protein phosphatases 1 and 2A). However, 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB), which is a specific inhibitor of casein kinase II, abolishes the phosphorylation in vitro of BSDL within micro- somes of AR4-2J pancreatic cells. We showed further that the α-subunit of casein kinase II co-locates with BSDL within the lumenal compartment of the Golgi. Genistein, which perturbs the trans-Golgi network, also inhibits the phosphorylation of BSDL, suggesting that this post-translational modification of BSDL probably occurred within this cell compartment. The inhibition of the phosphorylation of BSDL by DRB also decreases the rate at which the enzyme is secreted. Under the same conditions, the rate of α-amylase secretion was not modified. These data strongly suggest that phosphorylation is a post-translational event, which appears to be essential for the secretion of BSDL.

The role of protein kinase C-δ in PTH stimulation of IGF-binding protein-5 mRNA in UMR-106–01 cells

2002 ◽  
Vol 282 (3) ◽  
pp. E534-E541 ◽  
Author(s):  
Mary S. Erclik ◽  
Jane Mitchell
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Binding Protein ◽  
Specific Inhibitor ◽  
Dominant Negative Mutant ◽  
Nuclear Fraction ◽  
Mrna Levels ◽  
Kinase C ◽  
Umr 106

We have investigated the role of protein kinase C (PKC) signal transduction pathways in parathyroid hormone (PTH) regulation of insulin-like growth factor-binding protein-5 (IGFBP-5) gene expression in the rat osteoblast-like cell line UMR-106–01. Involvement of the PKC pathway was determined by the findings that bisindolylmaleimide I inhibited 40% of the PTH effect, and 1 μM bovine PTH-(3–34) stimulated a 10-fold induction of IGFBP-5 mRNA. PTH-(1–34) and PTH-(3–34) (100 nM) both stimulated PKC-δ translocation from the membrane to the nuclear fraction. Rottlerin, a PKC-δ-specific inhibitor, and a dominant negative mutant of PKC-δ were both able to significantly inhibit PTH-(1–34) and PTH-(3–34) induction of IGFBP-5 mRNA, suggesting a stimulatory role for PKC-δ in the effects of PTH. Phorbol 12-myristate 13-acetate (PMA) stimulated PKC-α translocation from the cytosol to the membrane and inhibited ∼50% of the PTH-(1–34), forskolin, and 8-bromoadenosine 3′,5′-cyclic monophosphate-stimulated IGFBP-5 mRNA levels, suggesting that PKC-α negatively regulates protein kinase A (PKA)-mediated induction of IGFBP-5 mRNA. These results suggest that the induction of IGFBP-5 by PTH is both PKA and PKC dependent and PKC-δ is the primary mediator of the effects of PTH via the PKC pathway.

The role of protein kinase C in GH secretion induced by GH-releasing factor and GH-releasing peptides in cultured ovine somatotrophs

1997 ◽  
Vol 154 (2) ◽  
pp. 219-230 ◽  
Author(s):  
D Wu ◽  
I J Clarke ◽  
C Chen
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Dependent Manner ◽  
Adenylyl Cyclase Activity ◽  
Down Regulation ◽  
Gh Secretion ◽  
Calphostin C ◽  
Kinase C ◽  
Pka Pathway

Abstract The involvement of protein kinase C (PKC) in the action of GH-releasing factor (GRF) and synthetic GH-releasing peptides (GHRP-2 and GHRP-6) was investigated in ovine somatotrophs in primary culture. In partially purified sheep somatotrophs, GRF and GHRP-2 caused translocation of PKC activity from the cytosol to the cell membranes and caused GH release in a dose- and time-dependent manner. GHRP-6 did not cause PKC translocation. The PKC inhibitors, calphostin C, staurosporine and chelerythrine, partially reduced GH release in response to GRF and GHRP-2 at doses which selectively inhibit PKC activity. These inhibitors totally abolished GH release caused by phorbol 12-myristate 13-acetate (PMA). Down-regulation of PKC by the treatment of cells with phorbol 12,13-dibutyrate for 16 h caused a significant (P<0·001) reduction in total PKC activity and totally abolished PKC translocation in response to a challenge with GRF, GHRP-2 or PMA. In addition, down-regulation abolished GH release in response to GRF, GHRP-2 or GHRP-6. Treatment of cells with H89, a selective PKA inhibitor, totally blocked GH release caused by either GRF or GHRP-2 and partially reduced PMA-induced GH release. H89 had no effect on PKC translocation caused by GRF, GHRP-2 or PMA and did not affect GH release caused by GHRP-6. These data suggest that GHRP-2 and GRF activate PKC in addition to stimulating adenylyl cyclase activity. Although the cAMP–protein kinase A (PKA) pathway is the major signalling pathway employed by GRF and GHRP-2, the activation of PKC may potentiate signalling via the cAMP–PKA pathway in ovine GH secretion. Importantly, the effect of PMA in increasing the secretion of GH from ovine somatotrophs is effected, in part, by up-regulation of the cAMP–PKA pathway. We conclude that there is cross-talk between the PKC pathway and the cAMP–PKA pathway in ovine somatotrophs during the action of GRF or GHRP. Journal of Endocrinology (1997) 154, 219–230

BCL2/BCL-XL inhibition induces apoptosis, disrupts cellular calcium homeostasis, and prevents platelet activation

Blood ◽  
2011 ◽  
Vol 117 (26) ◽  
pp. 7145-7154 ◽  
Author(s):  
Meike Vogler ◽  
Hassan A. Hamali ◽  
Xiao-Ming Sun ◽  
Edward T. W. Bampton ◽  
David Dinsdale ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Specific Inhibitor ◽  
Leukemia Cells ◽  
Calcium Stores ◽  
Selective Toxicity ◽  
Reticulated Platelets ◽  
Apoptotic Pathways ◽  
Induced Apoptosis ◽  
First Time

Abstract Apoptosis in megakaryocytes results in the formation of platelets. The role of apoptotic pathways in platelet turnover and in the apoptotic-like changes seen after platelet activation is poorly understood. ABT-263 (Navitoclax), a specific inhibitor of antiapoptotic BCL2 proteins, which is currently being evaluated in clinical trials for the treatment of leukemia and other malignancies, induces a dose-limiting thrombocytopenia. In this study, the relationship between BCL2/BCL-XL inhibition, apoptosis, and platelet activation was investigated. Exposure to ABT-263 induced apoptosis but repressed platelet activation by physiologic agonists. Notably, ABT-263 induced an immediate calcium response in platelets and the depletion of intracellular calcium stores, indicating that on BCL2/BCL-XL inhibition platelet activation is abrogated because of a diminished calcium signaling. By comparing the effects of ABT-263 and its analog ABT-737 on platelets and leukemia cells from the same donor, we show, for the first time, that these BCL2/BCL-XL inhibitors do not offer any selective toxicity but induce apoptosis at similar concentrations in leukemia cells and platelets. However, reticulated platelets are less sensitive to apoptosis, supporting the hypothesis that treatment with ABT-263 induces a selective loss of older platelets and providing an explanation for the transient thrombocytopenia observed on ABT-263 treatment.

Role of SLC12A10.2, a Na-Cl cotransporter-like protein, in a Cl uptake mechanism in zebrafish (Danio rerio)

2009 ◽  
Vol 296 (5) ◽  
pp. R1650-R1660 ◽  
Author(s):  
Yi-Fang Wang ◽  
Yung-Che Tseng ◽  
Jia-Jiun Yan ◽  
Junya Hiroi ◽  
Pung-Pung Hwang
Keyword(s):  
Specific Inhibitor ◽  
In Vivo Model ◽  
Zebrafish Embryos ◽  
Uptake Mechanism ◽  
Ion Regulation ◽  
Specific Expression ◽  
Net Uptake ◽  
First Time

The thiazide-sensitive Na+-Cl− cotransporter (NCC), a member of the SLC12 family, is mainly expressed in the apical membrane of the mammalian distal convoluted tubule (DCT) cells, is responsible for cotransporting Na+ and Cl− from the lumen into DCT cells and plays a major role in the mammalian renal NaCl reabsorption. The NCC has also been reported in fish, but the functional role in fish ion regulation is yet unclear. The present study used zebrafish as an in vivo model to test the hypothesis of whether the NCC plays a role in Na+ and/or Cl− uptake mechanisms. Four NCCs were cloned, and only one of them, zebrafish (z) slc12a10.2 was found to predominately and specifically be expressed in gills. Double in situ hybridization/immunocytochemistry in zebrafish skin/gills demonstrated that the specific expression of zslc12a10.2 mRNA in a novel group of ionocytes differed from those of the previously-reported H+-ATPase-rich (HR) cells and Na+-K+-ATPase-rich (NaR) cells. Gill mRNA expression of zslc12a10.2 was induced by a low-Cl environment that stimulated fish Cl− influx, while a low-Na environment suppressed this expression. Incubation with metolazone, a specific inhibitor of the NCC, impaired both Na+ and Cl− influx in 5-day postfertilization (dpf) zebrafish embryos. Translational knockdown of zslc12a10.2 with a specific morpholino caused significant decreases in both Cl− influx and Cl− content of 5-dpf zebrafish embryos, suggesting that the operation of zNCC-like 2 results in a net uptake of Cl− in zebrafish. On the contrary, zslc12a10.2 morphants showed increased Na+ influx and content that resulted from upregulation of mRNA expressions of Na+-H+ exchanger 3b and carbonic anhydrase 15a in HR cells. These results for the first time provide in vivo molecular physiological evidence for the possible role of the NCC in the Cl− uptake mechanism in zebrafish skin/gills.

scholarly journals Marek’s disease virus US3 protein kinase phosphorylates chicken HDAC 1 and 2 and regulates viral replication and pathogenesis

2021 ◽  
Vol 17 (2) ◽  
pp. e1009307
Author(s):  
Yifei Liao ◽  
Blanca Lupiani ◽  
Mohammad AI-Mahmood ◽  
Sanjay M. Reddy
Keyword(s):  
Protein Kinase ◽  
Disease Virus ◽  
Specific Inhibitor ◽  
Marek's Disease Virus ◽  
Marek's Disease ◽  
Marek’S Disease Virus ◽  
Marek’S Disease ◽  
Virus Growth ◽  
Histone Deacetylase 1

Marek’s disease virus (MDV) is a potent oncogenic alphaherpesvirus that elicits a rapid onset of malignant T-cell lymphomas in chickens. Three MDV types, including GaHV-2 (MDV-1), GaHV-3 (MDV-2) and MeHV-1 (HVT), have been identified and all encode a US3 protein kinase. MDV-1 US3 is important for efficient virus growth in vitro. To study the role of US3 in MDV replication and pathogenicity, we generated MDV-1 US3-null virus and chimeric viruses by replacing MDV-1 US3 with MDV-2 or HVT US3. Using MD as a natural virus-host model, we showed that both MDV-2 and HVT US3 partially rescued the growth deficiency of MDV-1 US3-null virus. In addition, deletion of MDV-1 US3 attenuated the virus resulting in higher survival rate and lower MDV specific tumor incidence, which could be partially compensated by MDV-2 and HVT US3. We also identified chicken histone deacetylase 1 (chHDAC1) as a common US3 substrate for all three MDV types while only US3 of MDV-1 and MDV-2 phosphorylate chHDAC2. We further determined that US3 of MDV-1 and HVT phosphorylate chHDAC1 at serine 406 (S406), while MDV-2 US3 phosphorylates S406, S410, and S415. In addition, MDV-1 US3 phosphorylates chHDAC2 at S407, while MDV-2 US3 targets S407 and S411. Furthermore, biochemical studies show that MDV US3 mediated phosphorylation of chHDAC1 and 2 affect their stability, transcriptional regulation activity, and interaction network. Using a class I HDACs specific inhibitor, we showed that MDV US3 mediated phosphorylation of chHDAC1 and 2 is involved in regulation of virus replication. Overall, we identified novel substrates for MDV US3 and characterized the role of MDV US3 in MDV pathogenesis.

scholarly journals Protein Kinase Cζ Attenuates Hypoxia-induced Proliferation of Fibroblasts by Regulating MAP Kinase Phosphatase-1 Expression

2006 ◽  
Vol 17 (4) ◽  
pp. 1995-2008 ◽  
Author(s):  
Megan D. Short ◽  
Stephanie M. Fox ◽  
Ching F. Lam ◽  
Kurt R. Stenmark ◽  
Mita Das
Keyword(s):  
Protein Kinase ◽  
Map Kinase ◽  
Fibroblast Proliferation ◽  
Atypical Protein Kinase ◽  
Downstream Target ◽  
Map Kinase Phosphatase ◽  
Pkc Isozymes ◽  
First Time ◽  
Repressor Activity

We have previously found that hypoxia stimulates proliferation of vascular fibroblasts through Gαi-mediated activation of ERK1/2. Here, we demonstrate that hypoxia also activates the atypical protein kinase Cζ (PKCζ) isozyme and stimulates the expression of ERK1/2-specific phosphatase, MAP kinase phosphatase-1 (MKP-1), which attenuates ERK1/2-mediated proliferative signals. Replication repressor activity is unique to PKCζ because the blockade of classical and novel PKC isozymes does not affect fibroblast proliferation. PKCζ is phosphorylated upon prolonged (24 h) exposure to hypoxia, whereas ERK1/2, the downstream kinases, are maximally activated in fibroblasts exposed to acute (10 min) hypoxia. However, PKCζ blockade results in persistent ERK1/2 phosphorylation and marked increase in hypoxia-induced replication. Similarly prolonged ERK1/2 phosphorylation and increase in hypoxia-stimulated proliferation are also observed upon blockade of MKP-1 activation. Because of the parallel suppressive actions of PKCζ and MKP-1 on ERK1/2 phosphorylation and proliferation, the role of PKCζ in the regulation of MKP-1 expression was evaluated. PKCζ attenuation reduces MKP-1 expression, whereas PKCζ overexpression increases MKP-1 levels. In conclusion, our results indicate for the first time that hypoxia activates PKCζ, which acts as a terminator of ERK1/2 activation through the regulation of downstream target, MKP-1 expression and thus serves to limit hypoxia-induced proliferation of fibroblasts.

Role of kinases and phosphatases in the regulation of fluid secretion and Cl-/HCO3- exchange in cholangiocytes

1997 ◽  
Vol 273 (2) ◽  
pp. G303-G313 ◽  
Author(s):  
D. Alvaro ◽  
A. Mennone ◽  
J. L. Boyer
Keyword(s):  
Bile Duct ◽  
Protein Kinase ◽  
Okadaic Acid ◽  
Protein Phosphatases ◽  
Specific Inhibitor ◽  
Fluid Secretion ◽  
Kinase C ◽  
Bile Duct Epithelium ◽  
Exchange Activity

The role of protein kinase A (PKA), protein kinase C (PKC), and protein phosphatases in the process of secretin stimulation of fluid and bicarbonate secretion from biliary epithelium was examined using a novel isolated bile duct unit (IBDU) model from rat liver. Sp-adenosine 3',5'-cyclic monophosphothiolate (Sp-cAMPS), 100 microM, a PKA-specific agonist, significantly increased secretion during a 30-min perfusion (+61%, P < 0.01). In contrast, preincubation and perfusion of Rp-cAMPS, 100 microM, a specific PKA inhibitor, reduced the ability of secretin to stimulate both fluid secretion (111 vs. 25%; P < 0.01) and Cl-/HCO3- exchanger activity (80 vs. 28%). Neither the PKC agonist phorbol 12-myristate 13-acetate, 10 microM, nor the PKC antagonist staurosporine showed any effect on either basal or secretin-stimulated fluid secretion or Cl-/HCO3- exchange activity in IBDU. Okadaic acid, a specific inhibitor of protein phosphatases 1 and 2A, also had no effect on basal fluid secretion or on the basal activity of the Cl-/HCO3- exchanger. However, okadaic acid resulted in persistence of secretion after removal of secretin, in contrast to the reduction in secretion observed in controls. These findings indicate that PKA but not PKC is involved in the signal transduction of secretin-stimulated fluid secretion and Cl-/HCO3- exchange activity in rat bile duct epithelium, a process inactivated by dephosphorylation by protein phosphatases 1 and/or 2A.

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