PRAK, a novel protein kinase regulated by the p38 MAP kinase.

PubMed Central

New, L; Jiang, Y; Zhao, M; Liu, K; Zhu, W; Flood, L J; Kato, Y; Parry, G C; Han, J

1998-01-01

We have identified and cloned a novel serine/ threonine kinase, p38-regulated/activated protein kinase (PRAK). PRAK is a 471 amino acid protein with 20-30% sequence identity to the known MAP kinase-regulated protein kinases RSK1/2/3, MNK1/2 and MAPKAP-K2/3. PRAK was found to be expressed in all human tissues and cell lines examined. In HeLa cells, PRAK was activated in response to cellular stress and proinflammatory cytokines. PRAK activity was regulated by p38alpha and p38beta both in vitro and in vivo and Thr182 was shown to be the regulatory phosphorylation site. Activated PRAK in turn phosphorylated small heat shock protein 27 (HSP27) at the physiologically relevant sites. An in-gel kinase assay demonstrated that PRAK is a major stress-activated kinase that can phosphorylate small heat shock protein, suggesting a potential role for PRAK in mediating stress-induced HSP27 phosphorylation in vivo. PMID:9628874

Decreased glucagon responsiveness by bile acids: a role for protein kinase Calpha and glucagon receptor phosphorylation.

PubMed

Ikegami, Tadashi; Krilov, Lada; Meng, Jianping; Patel, Bhumika; Chapin-Kennedy, Kelli; Bouscarel, Bernard

2006-11-01

Dihydroxy bile acids like chenodeoxycholic acid (CDCA) induce heterologous glucagon receptor desensitization. We previously demonstrated that protein kinase C (PKC) was activated by certain bile acids and mediated the CDCA-induced decrease in glucagon responsiveness. The aim of the present study was to explore the role of PKC in the phosphorylation and desensitization of the glucagon receptor by CDCA. Desensitization was evaluated by measuring adenylyl cyclase activity. Receptor phosphorylation was assayed by metabolic labeling with [gamma-(32)P] ATP. Protein kinase C (PKC) translocation and activation was visualized by fluorescence microscopy. CDCA decreased cAMP production induced by glucagon in a dose-dependent manner without affecting cAMP synthesis through stimulation of either stimulatory GTP-binding protein (Gs) by NaF or adenylyl cyclase by forskolin. The CDCA-induced inhibition of adenylyl cyclase activity was potentiated by the phosphatase inhibitor, okadaic acid. The desensitizing effect of CDCA was bile acid-specific and was significantly reduced in the presence of PKC inhibitors and after PKC down-regulation by phorbol 12-myristate 13-acetate. CDCA increased glucagon receptor phosphorylation more than 3-fold at concentrations as low as 25 mum. Furthermore, CDCA significantly stimulated human recombinant PKCalpha autophosphorylation in vitro, as well as PKCalpha translocation to the plasma membrane and phosphorylation in vivo at concentrations as low as 25 mum. CDCA also stimulated PKCdelta translocation to the perinuclear region. Activated PKCalpha, PKCzeta, and to a lesser extent, PKCdelta, phosphorylated the glucagon receptor in vitro. This study demonstrates that certain bile acids, such as CDCA, stimulate phosphorylation and heterologous desensitization of the glucagon receptor, involving at least PKCalpha activation.

Protein Kinase Mitogen-activated Protein Kinase Kinase Kinase Kinase 4 (MAP4K4) Promotes Obesity-induced Hyperinsulinemia.

PubMed

Roth Flach, Rachel J; Danai, Laura V; DiStefano, Marina T; Kelly, Mark; Menendez, Lorena Garcia; Jurczyk, Agata; Sharma, Rohit B; Jung, Dae Young; Kim, Jong Hun; Kim, Jason K; Bortell, Rita; Alonso, Laura C; Czech, Michael P

2016-07-29

Previous studies revealed a paradox whereby mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4) acted as a negative regulator of insulin sensitivity in chronically obese mice, yet systemic deletion of Map4k4 did not improve glucose tolerance. Here, we report markedly reduced glucose-responsive plasma insulin and C-peptide levels in whole body Map4k4-depleted mice (M4K4 iKO) as well as an impaired first phase of insulin secretion from islets derived from M4K4 iKO mice ex vivo After long-term high fat diet (HFD), M4K4 iKO mice pancreata also displayed reduced β cell mass, fewer proliferating β cells and reduced islet-specific gene mRNA expression compared with controls, although insulin content was normal. Interestingly, the reduced plasma insulin in M4K4 iKO mice exposed to chronic (16 weeks) HFD was not observed in response to acute HFD challenge or short term treatment with the insulin receptor antagonist S961. Furthermore, the improved insulin sensitivity in obese M4K4 iKO mice was abrogated by high exogenous insulin over the course of a euglycemic clamp study, indicating that hypoinsulinemia promotes insulin sensitivity in chronically obese M4K4 iKO mice. These results demonstrate that protein kinase Map4k4 drives obesity-induced hyperinsulinemia and insulin resistance in part by promoting insulin secretion from β cells in mice. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Regulation of Ca(2+)/calmodulin-dependent protein kinase kinase alpha by cAMP-dependent protein kinase: I. Biochemical analysis.

PubMed

Okuno, S; Kitani, T; Fujisawa, H

2001-10-01

Ca(2+)/calmodulin-dependent protein kinases (CaM-kinases) I and IV are activated upon phosphorylation of their Thr(177) and Thr(196), respectively, by the upstream Ca(2+)/calmodulin-dependent protein kinases CaM-kinase kinase alpha and beta, and deactivated upon dephosphorylation by protein phosphatases such as CaM-kinase phosphatase. Recent studies demonstrated that the activity of CaM-kinase kinase alpha is decreased upon phosphorylation by cAMP-dependent protein kinase (PKA), and the relationship between the inhibition and phosphorylation of CaM-kinase kinase alpha by PKA has been studied. In the present study, we demonstrate that the activity of CaM-kinase kinase alpha toward PKIV peptide, which contains the sequence surrounding Thr(196) of CaM-kinase IV, is increased by incubation with PKA in the presence of Ca(2+)/calmodulin but decreased in its absence, while the activity toward CaM-kinase IV is decreased by incubation with PKA in both the presence and absence of Ca(2+)/calmodulin. Six phosphorylation sites on CaM-kinase kinase alpha, Ser(24) for autophosphorylation, and Ser(52), Ser(74), Thr(108), Ser(458), and Ser(475) for phosphorylation by PKA, were identified by amino acid sequence analysis of the phosphopeptides purified from the tryptic digest of the phosphorylated enzymes. The presence of Ca(2+)/calmodulin suppresses phosphorylation on Ser(52), Ser(74), Thr(108), and Ser(458) by PKA, but accelerates phosphorylation on Ser(475). The changes in the activity of the enzyme upon phosphorylation appear to occur as a result of conformational changes induced by phosphorylation on several sites.

Activation of AMP-activated Protein Kinase by Metformin Induces Protein Acetylation in Prostate and Ovarian Cancer Cells*

PubMed Central

Galdieri, Luciano; Gatla, Himavanth; Vancurova, Ivana; Vancura, Ales

2016-01-01

AMP-activated protein kinase (AMPK) is an energy sensor and master regulator of metabolism. AMPK functions as a fuel gauge monitoring systemic and cellular energy status. Activation of AMPK occurs when the intracellular AMP/ATP ratio increases and leads to a metabolic switch from anabolism to catabolism. AMPK phosphorylates and inhibits acetyl-CoA carboxylase (ACC), which catalyzes carboxylation of acetyl-CoA to malonyl-CoA, the first and rate-limiting reaction in de novo synthesis of fatty acids. AMPK thus regulates homeostasis of acetyl-CoA, a key metabolite at the crossroads of metabolism, signaling, chromatin structure, and transcription. Nucleocytosolic concentration of acetyl-CoA affects histone acetylation and links metabolism and chromatin structure. Here we show that activation of AMPK with the widely used antidiabetic drug metformin or with the AMP mimetic 5-aminoimidazole-4-carboxamide ribonucleotide increases the inhibitory phosphorylation of ACC and decreases the conversion of acetyl-CoA to malonyl-CoA, leading to increased protein acetylation and altered gene expression in prostate and ovarian cancer cells. Direct inhibition of ACC with allosteric inhibitor 5-(tetradecyloxy)-2-furoic acid also increases acetylation of histones and non-histone proteins. Because AMPK activation requires liver kinase B1, metformin does not induce protein acetylation in liver kinase B1-deficient cells. Together, our data indicate that AMPK regulates the availability of nucleocytosolic acetyl-CoA for protein acetylation and that AMPK activators, such as metformin, have the capacity to increase protein acetylation and alter patterns of gene expression, further expanding the plethora of metformin's physiological effects. PMID:27733682

Interleukin-1 beta induced synthesis of protein kinase C-delta and protein kinase C-epsilon in EL4 thymoma cells: possible involvement of phosphatidylinositol 3-kinase.

PubMed

Varley, C L; Royds, J A; Brown, B L; Dobson, P R

2001-01-01

We present evidence here that the proinflammatory cytokine, interleukin-1 beta (IL-1 beta) stimulates a significant increase in protein kinase C (PKC)-epsilon and PKC-delta protein levels and increases PKC-epsilon, but not PKC-delta, transcripts in EL4 thymoma cells. Incubation of EL4 cells with IL-1 beta induced protein synthesis of PKC-epsilon (6-fold increase) by 7 h and had a biphasic effect on PKC-delta levels with peaks at 4 h (2-fold increase) and 24 h (4-fold increase). At the level of mRNA, PKC-epsilon, but not PKC-delta levels, were induced after incubation of EL4 cells with IL-1 beta. The signalling mechanisms utilized by IL-1 beta to induce the synthesis of these PKC isoforms were investigated. Two phosphatidylinositol (PI) 3-kinase-specific inhibitors, wortmannin and LY294002, inhibited IL-1 beta-induced synthesis of PKC-epsilon. However, the PI 3-kinase inhibitors had little effect on the IL-1 beta-induced synthesis of PKC-delta in these cells. Our results indicate that IL-1 beta induced both PKC-delta and PKC-epsilon expression over different time periods. Furthermore, our evidence suggests that IL-1 beta induction of PKC-epsilon, but not PKC-delta, may occur via the PI 3-kinase pathway. Copyright 2001 S. Karger AG, Basel

Nordihydroguaiaretic acid protects against high-fat diet-induced fatty liver by activating AMP-activated protein kinase in obese mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Myoung-Su; Kim, Daeyoung; Jo, Keunae

Research highlights: {yields} NDGA decreases high-fat diet-induced body weight gain and adiposity. {yields} NDGA reduces high-fat diet-induced triglyceride accumulation in liver. {yields} NDGA improves lipid storage in vitro through altering lipid regulatory proteins. {yields} Inhibition of lipid storage in vivo and in vitro is mediated by AMPK activation. -- Abstract: Nonalcoholic fatty liver disease, one of the most common causes of chronic liver disease, is strongly associated with metabolic syndrome. Nordihydroguaiaretic acid (NDGA) has been reported to inhibit lipoprotein lipase; however, the effect of NDGA on hepatic lipid metabolism remains unclear. We evaluated body weight, adiposity, liver histology, and hepaticmore » triglyceride content in high-fat diet (HFD)-fed C57BL/6J mice treated with NDGA. In addition, we characterized the underlying mechanism of NDGA's effects in HepG2 hepatocytes by Western blot and RT-PCR analysis. NDGA (100 or 200 mg/kg/day) reduced weight gain, fat pad mass, and hepatic triglyceride accumulation, and improved serum lipid parameters in mice fed a HFD for 8 weeks. NDGA significantly increased AMP-activated protein kinase (AMPK) phosphorylation in the liver and in HepG2 hepatocytes. NDGA downregulated the level of mature SREBP-1 and its target genes (acetyl-CoA carboxylase and fatty acid synthase), but, it upregulated expression of genes involved in fatty acid oxidation, such as peroxisome proliferator-activated receptor (PPAR){alpha}, PPAR{gamma} coactivator-1, carnitine palmitoyl transferase-1, and uncoupling protein-2. The specific AMPK inhibitor compound C attenuated the effects of NDGA on expression of lipid metabolism-related proteins in HepG2 hepatocytes. The beneficial effects of NDGA on HFD-induced hepatic triglyceride accumulation are mediated through AMPK signaling pathways, suggesting a potential target for preventing NAFLD.« less

Interleukin-2-induced survival of natural killer (NK) cells involving phosphatidylinositol-3 kinase-dependent reduction of ceramide through acid sphingomyelinase, sphingomyelin synthase, and glucosylceramide synthase.

PubMed

Taguchi, Yoshimitsu; Kondo, Tadakazu; Watanabe, Mitsumasa; Miyaji, Michihiko; Umehara, Hisanori; Kozutsumi, Yasunori; Okazaki, Toshiro

2004-11-15

Interleukin 2 (IL-2) rescued human natural killer (NK) KHYG-1 cells from apoptosis along with a reduction of ceramide. Conversely, an increase of ceramide inhibited IL-2-rescued survival. IL-2 deprivation-induced activation of acid sphingomyelinase (SMase) and inhibition of glucosylceramide synthase (GCS) and sphingomyelin synthase (SMS) were normalized by IL-2 supplementation. A phosphatidyl inositol-3 (PI-3) kinase inhibitor, LY294002, inhibited IL-2-rescued survival, but a mitogen-activated protein kinase inhibitor, PD98059, and an inhibitor of Janus tyrosine kinase/signal transducer and activator of transcription pathway, AG490, did not. LY294002 inhibited IL-2-induced reduction of ceramide through activation of acid SMase and inhibition of GCS and SMS, suggesting the positive involvement of PI-3 kinase in ceramide reduction through enzymatic regulation. Indeed, a constitutively active PI-3 kinase enhanced growth rate and ceramide reduction through inhibition of acid SMase and activation of GCS and SMS. Further, LY294002 inhibited IL-2-induced changes of transcriptional level as well as mRNA and protein levels in acid SMase and GCS but did not affect the stability of the mRNAs. These results suggest that PI-3 kinase-dependent reduction of ceramide through regulation of acid SMase, GCS, and SMS plays a role in IL-2-rescued survival of NK cells.

Nonsteroidal anti-inflammatory drug flufenamic acid is a potent activator of AMP-activated protein kinase.

PubMed

Chi, Yuan; Li, Kai; Yan, Qiaojing; Koizumi, Schuichi; Shi, Liye; Takahashi, Shuhei; Zhu, Ying; Matsue, Hiroyuki; Takeda, Masayuki; Kitamura, Masanori; Yao, Jian

2011-10-01

Flufenamic acid (FFA) is a nonsteroidal anti-inflammatory drug (NSAID). It has anti-inflammatory and antipyretic properties. In addition, it modulates multiple channel activities. The mechanisms underlying the pharmacological actions of FFA are presently unclear. Given that AMP-activated protein kinase (AMPK) has both anti-inflammatory and channel-regulating functions, we examined whether FFA induces AMPK activation. 1) Exposure of several different types of cells to FFA resulted in an elevation of AMPKα phosphorylation at Thr172. This effect of FFA was reproduced by functionally and structurally similar mefenamic acid, tolfenamic acid, niflumic acid, and meclofenamic acid. 2) FFA-induced activation of AMPK was largely abolished by the treatment of cells with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (an intracellular Ca(2+) chelator) or depletion of extracellular Ca(2+), whereas it was mimicked by stimulation of cells with the Ca(2+) ionophore 5-(methylamino)-2-({(2R,3R,6S,8S,9R,11R)-3,9,11-trimethyl-8-[(1S)-1-methyl-2-oxo-2-(1H-pyrrol-2-yl)ethyl]-1,7-dioxaspiro[5.5]undec-2-yl}methyl)-1,3-benzoxazole-4-carboxylic acid (A23187) or ionomycin. 3) FFA triggered a rise in intracellular Ca(2+), which was abolished by cyclosporine, a blocker of mitochondrial permeability transition pore. Cyclosporine also abolished FFA-induced activation of AMPK. 4) Inhibition of Ca(2+)/calmodulin-dependent kinase kinase β (CaMKKβ) with 7-oxo-7H-benzimidazo[2,1-a]benz[de]isoquinoline-3-carboxylic acid acetate (STO-609) or down-regulation of CaMKKβ with short interfering RNA largely abrogated FFA-induced activation of AMPK. 5) FFA significantly suppressed nuclear factor-κB activity and inducible nitric-oxide synthase expression triggered by interleukin-1β and tumor necrosis factor α. This suppression was also largely abrogated by STO-609. Taken together, we conclude that FFA induces AMPK activation through the Ca(2+)-CaMKKβ pathway

Activation of the AMP-activated protein kinase-p38 MAP kinase pathway mediates apoptosis induced by conjugated linoleic acid in p53-mutant mouse mammary tumor cells.

PubMed

Hsu, Yung-Chung; Meng, Xiaojing; Ou, Lihui; Ip, Margot M

2010-04-01

Conjugated linoleic acid (CLA) inhibits tumorigenesis and tumor growth in most model systems, an effect mediated in part by its pro-apoptotic activity. We previously showed that trans-10,cis-12 CLA induced apoptosis of p53-mutant TM4t mouse mammary tumor cells through both mitochondrial and endoplasmic reticulum stress pathways. In the current study, we investigated the role of AMP-activated protein kinase (AMPK), a key player in fatty acid metabolism, in CLA-induced apoptosis in TM4t cells. We found that t10,c12-CLA increased phosphorylation of AMPK, and that CLA-induced apoptosis was enhanced by the AMPK agonist 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) and inhibited by the AMPK inhibitor compound C. The increased AMPK activity was not due to nutrient/energy depletion since ATP levels did not change in CLA-treated cells, and knockdown of the upstream kinase LKB1 did not affect its activity. Furthermore, our data do not demonstrate a role for the AMPK-modulated mTOR pathway in CLA-induced apoptosis. Although CLA decreased mTOR levels, activity was only modestly decreased. Moreover, rapamycin, which completely blocked the activity of mTORC1 and mTORC2, did not induce apoptosis, and attenuated rather than enhanced CLA-induced apoptosis. Instead, the data suggest that CLA-induced apoptosis is mediated by the AMPK-p38 MAPK-Bim pathway: CLA-induced phosphorylation of AMPK and p38 MAPK, and increased expression of Bim, occurred with a similar time course as apoptosis; phosphorylation of p38 MAPK was blocked by compound C; the increased Bim expression was blocked by p38 MAPK siRNA; CLA-induced apoptosis was attenuated by the p38 inhibitor SB-203580 and by siRNAs directed against p38 MAPK or Bim. Copyright 2009 Elsevier Inc. All rights reserved.

Gallic acid attenuates calcium calmodulin-dependent kinase II-induced apoptosis in spontaneously hypertensive rats.

PubMed

Jin, Li; Piao, Zhe Hao; Liu, Chun Ping; Sun, Simei; Liu, Bin; Kim, Gwi Ran; Choi, Sin Young; Ryu, Yuhee; Kee, Hae Jin; Jeong, Myung Ho

2018-03-01

Hypertension causes cardiac hypertrophy and leads to heart failure. Apoptotic cells are common in hypertensive hearts. Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) is associated with apoptosis. We recently demonstrated that gallic acid reduces nitric oxide synthase inhibition-induced hypertension. Gallic acid is a trihydroxybenzoic acid and has been shown to have beneficial effects, such as anti-cancer, anti-calcification and anti-oxidant activity. The purpose of this study was to determine whether gallic acid regulates cardiac hypertrophy and apoptosis in essential hypertension. Gallic acid significantly lowered systolic and diastolic blood pressure in spontaneously hypertensive rats (SHRs). Wheat germ agglutinin (WGA) and H&E staining revealed that gallic acid reduced cardiac enlargement in SHRs. Gallic acid treatment decreased cardiac hypertrophy marker genes, including atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), in SHRs. The four isoforms, α, β, δ and γ, of CaMKII were increased in SHRs and were significantly reduced by gallic acid administration. Gallic acid reduced cleaved caspase-3 protein as well as bax, p53 and p300 mRNA levels in SHRs. CaMKII δ overexpression induced bax and p53 expression, which was attenuated by gallic acid treatment in H9c2 cells. Gallic acid treatment reduced DNA fragmentation and the TUNEL positive cells induced by angiotensin II. Taken together, gallic acid could be a novel therapeutic for the treatment of hypertension through suppression of CaMKII δ-induced apoptosis. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Emerging roles of protein kinase CK2 in abscisic acid signaling.

PubMed

Vilela, Belmiro; Pagès, Montserrat; Riera, Marta

2015-01-01

The phytohormone abscisic acid (ABA) regulates many aspects of plant growth and development as well as responses to multiple stresses. Post-translational modifications such as phosphorylation or ubiquitination have pivotal roles in the regulation of ABA signaling. In addition to the positive regulator sucrose non-fermenting-1 related protein kinase 2 (SnRK2), the relevance of the role of other protein kinases, such as CK2, has been recently highlighted. We have recently established that CK2 phosphorylates the maize ortholog of open stomata 1 OST1, ZmOST1, suggesting a role of CK2 phosphorylation in the control of ZmOST1 protein degradation (Vilela et al., 2015). CK2 is a pleiotropic enzyme involved in multiple developmental and stress-responsive pathways. This review summarizes recent advances that taken together suggest a prominent role of protein kinase CK2 in ABA signaling and related processes.

Lithium and Tamoxifen Modulate Behavior and Protein Kinase C Activity in the Animal Model of Mania Induced by Ouabain

PubMed Central

Dal-Pont, Gustavo C; Resende, Wilson R; Varela, Roger B; Peterle, Bruna R; Gava, Fernanda F; Mina, Francielle G; Cararo, José H; Carvalho, André F; Quevedo, João

2017-01-01

Abstract Background The intracerebroventricular injection of ouabain, a specific inhibitor of the Na+/K+-adenosine-triphosphatase (Na+/K+-ATPase) enzyme, induces hyperactivity in rats in a putative animal model of mania. Several evidences have suggested that the protein kinase C signaling pathway is involved in bipolar disorder. In addition, it is known that protein kinase C inhibitors, such as lithium and tamoxifen, are effective in treating acute mania. Methods In the present study, we investigated the effects of lithium and tamoxifen on the protein kinase C signaling pathway in the frontal cortex and hippocampus of rats submitted to the animal model of mania induced by ouabain. We showed that ouabain induced hyperlocomotion in the rats. Results Ouabain increased the protein kinase C activity and the protein kinase C and MARCKS phosphorylation in frontal cortex and hippocampus of rats. Lithium and tamoxifen reversed the behavioral and protein kinase C pathway changes induced by ouabain. These findings indicate that the Na+/K+-ATPase inhibition can lead to protein kinase C alteration. Conclusions The present study showed that lithium and tamoxifen modulate changes in the behavior and protein kinase C signalling pathway alterations induced by ouabain, underlining the need for more studies of protein kinase C as a possible target for treatment of bipolar disorder. PMID:29020306

Adipocyte Fatty Acid Binding Protein Potentiates Toxic Lipids-Induced Endoplasmic Reticulum Stress in Macrophages via Inhibition of Janus Kinase 2-dependent Autophagy

PubMed Central

Hoo, Ruby L. C.; Shu, Lingling; Cheng, Kenneth K. Y.; Wu, Xiaoping; Liao, Boya; Wu, Donghai; Zhou, Zhiguang; Xu, Aimin

2017-01-01

Lipotoxicity is implicated in the pathogenesis of obesity-related inflammatory complications by promoting macrophage infiltration and activation. Endoplasmic reticulum (ER) stress and adipocyte fatty acid binding protein (A-FABP) play key roles in obesity and mediate inflammatory activity through similar signaling pathways. However, little is known about their interplay in lipid-induced inflammatory responses. Here, we showed that prolonged treatment of palmitic acid (PA) increased ER stress and expression of A-FABP, which was accompanied by reduced autophagic flux in macrophages. Over-expression of A-FABP impaired PA-induced autophagy associating with enhanced ER stress and pro-inflammatory cytokine production, while genetic ablation or pharmacological inhibition of A-FABP reversed the conditions. PA-induced expression of autophagy-related protein (Atg)7 was attenuated in A-FABP over-expressed macrophages, but was elevated in A-FABP-deficient macrophages. Mechanistically, A-FABP potentiated the effects of PA by inhibition of Janus Kinase (JAK)2 activity, thus diminished PA-induced Atg7 expression contributing to impaired autophagy and further augmentation of ER stress. These findings suggest that A-FABP acts as autophagy inhibitor to instigate toxic lipids-induced ER stress through inhibition of JAK2-dependent autophagy, which in turn triggers inflammatory responses in macrophages. A-FABP-JAK2 axis may represent an important pathological pathway contributing to obesity-related inflammatory diseases. PMID:28094778

Protein kinase inhibitor peptide (PKI): a family of endogenous neuropeptides that modulate neuronal cAMP-dependent protein kinase function.

PubMed

Dalton, George D; Dewey, William L

2006-02-01

Signal transduction cascades involving cAMP-dependent protein kinase are highly conserved among a wide variety of organisms. Given the universal nature of this enzyme it is not surprising that cAMP-dependent protein kinase plays a critical role in numerous cellular processes. This is particularly evident in the nervous system where cAMP-dependent protein kinase is involved in neurotransmitter release, gene transcription, and synaptic plasticity. Protein kinase inhibitor peptide (PKI) is an endogenous thermostable peptide that modulates cAMP-dependent protein kinase function. PKI contains two distinct functional domains within its amino acid sequence that allow it to: (1) potently and specifically inhibit the activity of the free catalytic subunit of cAMP-dependent protein kinase and (2) export the free catalytic subunit of cAMP-dependent protein kinase from the nucleus. Three distinct PKI isoforms (PKIalpha, PKIbeta, PKIgamma) have been identified and each isoform is expressed in the brain. PKI modulates neuronal synaptic activity, while PKI also is involved in morphogenesis and symmetrical left-right axis formation. In addition, PKI also plays a role in regulating gene expression induced by cAMP-dependent protein kinase. Future studies should identify novel physiological functions for endogenous PKI both in the nervous system and throughout the body. Most interesting will be the determination whether functional differences exist between individual PKI isoforms which is an intriguing possibility since these isoforms exhibit: (1) cell-type specific tissue expression patterns, (2) different potencies for the inhibition of cAMP-dependent protein kinase activity, and (3) expression patterns that are hormonally, developmentally and cell-cycle regulated. Finally, synthetic peptide analogs of endogenous PKI will continue to be invaluable tools that are used to elucidate the role of cAMP-dependent protein kinase in a variety of cellular processes throughout the nervous

Pyroglutamic acid stimulates DNA synthesis in rat primary hepatocytes through the mitogen-activated protein kinase pathway.

PubMed

Inoue, Shinjiro; Okita, Yoichi; de Toledo, Andreia; Miyazaki, Hiroyuki; Hirano, Eiichi; Morinaga, Tetsuo

2015-01-01

We purified pyroglutamic acid from human placental extract and identified it as a potent stimulator of rat primary hepatocyte DNA synthesis. Pyroglutamic acid dose-dependently stimulated DNA synthesis, and this effect was inhibited by PD98059, a dual specificity mitogen-activated protein kinase kinase 1 (MAP2K1) inhibitor. Therefore, pyroglutamic acid stimulated DNA synthesis in rat primary hepatocytes via MAPK signaling.

Identifying protein kinase target preferences using mass spectrometry

PubMed Central

Douglass, Jacqueline; Gunaratne, Ruwan; Bradford, Davis; Saeed, Fahad; Hoffert, Jason D.; Steinbach, Peter J.; Pisitkun, Trairak

2012-01-01

A general question in molecular physiology is how to identify candidate protein kinases corresponding to a known or hypothetical phosphorylation site in a protein of interest. It is generally recognized that the amino acid sequence surrounding the phosphorylation site provides information that is relevant to identification of the cognate protein kinase. Here, we present a mass spectrometry-based method for profiling the target specificity of a given protein kinase as well as a computational tool for the calculation and visualization of the target preferences. The mass spectrometry-based method identifies sites phosphorylated in response to in vitro incubation of protein mixtures with active recombinant protein kinases followed by standard phosphoproteomic methodologies. The computational tool, called “PhosphoLogo,” uses an information-theoretic algorithm to calculate position-specific amino acid preferences and anti-preferences from the mass-spectrometry data (http://helixweb.nih.gov/PhosphoLogo/). The method was tested using protein kinase A (catalytic subunit α), revealing the well-known preference for basic amino acids in positions −2 and −3 relative to the phosphorylated amino acid. It also provides evidence for a preference for amino acids with a branched aliphatic side chain in position +1, a finding compatible with known crystal structures of protein kinase A. The method was also employed to profile target preferences and anti-preferences for 15 additional protein kinases with potential roles in regulation of epithelial transport: CK2, p38, AKT1, SGK1, PKCδ, CaMK2δ, DAPK1, MAPKAPK2, PKD3, PIM1, OSR1, STK39/SPAK, GSK3β, Wnk1, and Wnk4. PMID:22723110

Comparative Molecular Dynamics Simulations of Mitogen-Activated Protein Kinase-Activated Protein Kinase 5

PubMed Central

Lindin, Inger; Wuxiuer, Yimingjiang; Ravna, Aina Westrheim; Moens, Ugo; Sylte, Ingebrigt

2014-01-01

The mitogen-activated protein kinase-activated protein kinase MK5 is a substrate of the mitogen-activated protein kinases p38, ERK3 and ERK4. Cell culture and animal studies have demonstrated that MK5 is involved in tumour suppression and promotion, embryogenesis, anxiety, cell motility and cell cycle regulation. In the present study, homology models of MK5 were used for molecular dynamics (MD) simulations of: (1) MK5 alone; (2) MK5 in complex with an inhibitor; and (3) MK5 in complex with the interaction partner p38α. The calculations showed that the inhibitor occupied the active site and disrupted the intramolecular network of amino acids. However, intramolecular interactions consistent with an inactive protein kinase fold were not formed. MD with p38α showed that not only the p38 docking region, but also amino acids in the activation segment, αH helix, P-loop, regulatory phosphorylation region and the C-terminal of MK5 may be involved in forming a very stable MK5-p38α complex, and that p38α binding decreases the residual fluctuation of the MK5 model. Electrostatic Potential Surface (EPS) calculations of MK5 and p38α showed that electrostatic interactions are important for recognition and binding. PMID:24651460

Activation of G protein-coupled bile acid receptor, TGR5, induces smooth muscle relaxation via both Epac- and PKA-mediated inhibition of RhoA/Rho kinase pathway.

PubMed

Rajagopal, Senthilkumar; Kumar, Divya P; Mahavadi, Sunila; Bhattacharya, Sayak; Zhou, Ruizhe; Corvera, Carlos U; Bunnett, Nigel W; Grider, John R; Murthy, Karnam S

2013-03-01

The present study characterized the TGR5 expression and the signaling pathways coupled to this receptor that mediates the relaxation of gastric smooth muscle. TGR5 was detected in gastric muscle cells by RT-PCR and Western blotting. Treatment of cells with the TGR5-selective ligand oleanolic acid (OA) activated Gαs, but not Gαq, Gαi1, Gαi2, or Gαi3, and increased cAMP levels. OA did not elicit contraction, but caused relaxation of carbachol-induced contraction of gastric muscle cells from wild-type mice, but not tgr5(-/-) mice. OA, but not a selective exchange protein activated by cAMP (Epac) ligand (8-pCPT-2'-O-Me-cAMP), caused phosphorylation of RhoA and the phosphorylation was blocked by the PKA inhibitor, myristoylated PKI, and by the expression of phosphorylation-deficient mutant RhoA (S188A). Both OA and Epac ligand stimulated Ras-related protein 1 (Rap1) and inhibited carbachol (CCh)-induced Rho kinase activity. Expression of RhoA (S188A) or PKI partly reversed the inhibition of Rho kinase activity by OA but had no effect on inhibition by Epac ligand. However, suppression of Rap1 with siRNA blocked the inhibition of Rho kinase by Epac ligand, and partly reversed the inhibition by OA; the residual inhibition was blocked by PKI. Muscle relaxation in response to OA, but not Epac ligand, was partly reversed by PKI. We conclude that activation of TGR5 causes relaxation of gastric smooth muscle and the relaxation is mediated through inhibition of RhoA/Rho kinase pathway via both cAMP/Epac-dependent stimulation of Rap1 and cAMP/PKA-dependent phosphorylation of RhoA at Ser(188). TGR5 receptor activation on smooth muscle reveals a novel mechanism for the regulation of gut motility by bile acids.

Inhibitory effects of omega-3 fatty acids on early brain injury after subarachnoid hemorrhage in rats: Possible involvement of G protein-coupled receptor 120/β-arrestin2/TGF-β activated kinase-1 binding protein-1 signaling pathway.

PubMed

Yin, Jia; Li, Haiying; Meng, Chengjie; Chen, Dongdong; Chen, Zhouqing; Wang, Yibin; Wang, Zhong; Chen, Gang

2016-06-01

Omega-3 fatty acids have been reported to improve neuron functions during aging and in patients affected by mild cognitive impairment, and mediate potent anti-inflammatory via G protein-coupled receptor 120 (GPR120) signal pathway. Neuron dysfunction and inflammatory response also contributed to the progression of subarachnoid hemorrhage (SAH)-induced early brain injury (EBI). This study was to examine the effects of omega-3 fatty acids on SAH-induced EBI. Two weeks before SAH, 30% Omega-3 fatty acids was administered by oral gavage at 1g/kg body weight once every 24h. Specific siRNA for GPR120 was exploited. Terminal deoxynucleotidyl transferase dUTP nick end labeling, fluoro-Jade B staining, and neurobehavioral scores and brain water content test showed that omega-3 fatty acids effectively suppressed SAH-induced brain cell apoptosis and neuronal degradation, behavioral impairment, and brain edema. Western blot, immunoprecipitation, and electrophoretic mobility shift assays results showed that omega-3 fatty acids effectively suppressed SAH-induced elevation of inflammatory factors, including cyclooxygenase-2, monocyte chemoattractant protein-1, and inducible nitric oxide synthase. In addition, omega-3 fatty acids could inhibit phosphorylation of transforming growth factor β activated kinase-1 (TAK1), MEK4, c-Jun N-terminal kinase, and IkappaB kinase as well as activation of nuclear factor kappa B through regulating GPR120/β-arrestin2/TAK1 binding protein-1 pathway. Furthermore, siRNA-induced GPR120 silencing blocked the protective effects of omega-3 fatty acids. Here, we show that stimulation of GPR120 with omega-3 fatty acids pretreatment causes anti-apoptosis and anti-inflammatory effects via β-arrestin2/TAK1 binding protein-1/TAK1 pathway in the brains of SAH rats. Fish omega-3 fatty acids as part of a daily diet may reduce EBI in an experimental rat model of SAH. Copyright © 2016 Elsevier Ltd. All rights reserved.

The human mu opioid receptor: modulation of functional desensitization by calcium/calmodulin-dependent protein kinase and protein kinase C.

PubMed

Mestek, A; Hurley, J H; Bye, L S; Campbell, A D; Chen, Y; Tian, M; Liu, J; Schulman, H; Yu, L

1995-03-01

Opioids are some of the most efficacious analgesics used in humans. Prolonged administration of opioids, however, often causes the development of drug tolerance, thus limiting their effectiveness. To explore the molecular basis of those mechanisms that may contribute to opioid tolerance, we have isolated a cDNA for the human mu opioid receptor, the target of such opioid narcotics as morphine, codeine, methadone, and fentanyl. The receptor encoded by this cDNA is 400 amino acids long with 94% sequence similarity to the rat mu opioid receptor. Transient expression of this cDNA in COS-7 cells produced high-affinity binding sites to mu-selective agonists and antagonists. This receptor displays functional coupling to a recently cloned G-protein-activated K+ channel. When both proteins were expressed in Xenopus oocytes, functional desensitization developed upon repeated stimulation of the mu opioid receptor, as observed by a reduction in K+ current induced by the second mu receptor activation relative to that induced by the first. The extent of desensitization was potentiated by both the multifunctional calcium/calmodulin-dependent protein kinase and protein kinase C. These results demonstrate that kinase modulation is a molecular mechanism by which the desensitization of mu receptor signaling may be regulated at the cellular level, suggesting that this cellular mechanism may contribute to opioid tolerance in humans.

Lysophosphatidic acid induces reactive oxygen species generation by activating protein kinase C in PC-3 human prostate cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lin, Chu-Cheng; Lin, Chuan-En; Lin, Yueh-Chien

2013-11-01

Highlights: •LPA induces ROS generation through LPA{sub 1} and LPA{sub 3}. •LPA induces ROS generation by activating PLC. •PKCζ mediates LPA-induced ROS generation. -- Abstract: Prostate cancer is one of the most frequently diagnosed cancers in males, and PC-3 is a cell model popularly used for investigating the behavior of late stage prostate cancer. Lysophosphatidic acid (LPA) is a lysophospholipid that mediates multiple behaviors in cancer cells, such as proliferation, migration and adhesion. We have previously demonstrated that LPA enhances vascular endothelial growth factor (VEGF)-C expression in PC-3 cells by activating the generation of reactive oxygen species (ROS), which ismore » known to be an important mediator in cancer progression. Using flow cytometry, we showed that LPA triggers ROS generation within 10 min and that the generated ROS can be suppressed by pretreatment with the NADPH oxidase (Nox) inhibitor diphenylene iodonium. In addition, transfection with LPA{sub 1} and LPA{sub 3} siRNA efficiently blocked LPA-induced ROS production, suggesting that both receptors are involved in this pathway. Using specific inhibitors and siRNA, phospholipase C (PLC) and protein kinase C (PKC) were also suggested to participate in LPA-induced ROS generation. Overall, we demonstrated that LPA induces ROS generation in PC-3 prostate cancer cells and this is mediated through the PLC/PKC/Nox pathway.« less

A role for Pyk2 and Src in linking G-protein-coupled receptors with MAP kinase activation.

PubMed

Dikic, I; Tokiwa, G; Lev, S; Courtneidge, S A; Schlessinger, J

1996-10-10

The mechanisms by which mitogenic G-protein-coupled receptors activate the MAP kinase signalling pathway are poorly understood. Candidate protein tyrosine kinases that link G-protein-coupled receptors with MAP kinase include Src family kinases, the epidermal growth factor receptor, Lyn and Syk. Here we show that lysophosphatidic acid (LPA) and bradykinin induce tyrosine phosphorylation of Pyk2 and complex formation between Pyk2 and activated Src. Moreover, tyrosine phosphorylation of Pyk2 leads to binding of the SH2 domain of Src to tyrosine 402 of Pyk2 and activation of Src. Transient overexpression of a dominant interfering mutant of Pyk2 or the protein tyrosine kinase Csk reduces LPA- or bradykinin-induced activation of MAP kinase. LPA- or bradykinin-induced MAP kinase activation was also inhibited by overexpression of dominant interfering mutants of Grb2 and Sos. We propose that Pyk2 acts with Src to link Gi- and Gq-coupled receptors with Grb2 and Sos to activate the MAP kinase signalling pathway in PC12 cells.

Nitric oxide-activated calcium/calmodulin-dependent protein kinase regulates the abscisic acid-induced antioxidant defence in maize

PubMed Central

Zhang, Aying; Jiang, Mingyi

2012-01-01

Nitric oxide (NO), hydrogen peroxide (H2O2), and calcium (Ca2+)/calmodulin (CaM) are all required for abscisic acid (ABA)-induced antioxidant defence. Ca2+/CaM-dependent protein kinase (CCaMK) is a strong candidate for the decoder of Ca2+ signals. However, whether CCaMK is involved in ABA-induced antioxidant defence is unknown. The results of the present study show that exogenous and endogenous ABA induced increases in the activity of ZmCCaMK and the expression of ZmCCaMK in leaves of maize. Subcellular localization analysis showed that ZmCCaMK is located in the nucleus, the cytoplasm, and the plasma membrane. The transient expression of ZmCCaMK and the RNA interference (RNAi) silencing of ZmCCaMK analysis in maize protoplasts revealed that ZmCCaMK is required for ABA-induced antioxidant defence. Moreover, treatment with the NO donor sodium nitroprusside (SNP) induced the activation of ZmCCaMK and the expression of ZmCCaMK. Pre-treatments with an NO scavenger and inhibitor blocked the ABA-induced increases in the activity and the transcript level of ZmCCaMK. Conversely, RNAi silencing of ZmCCaMK in maize protoplasts did not affect the ABA-induced NO production, which was further confirmed using a mutant of OsCCaMK, the homologous gene of ZmCCaMK in rice. Moreover, H2O2 was also required for the ABA activation of ZmCCaMK, and pre-treatments with an NO scavenger and inhibitor inhibited the H2O2-induced increase in the activity of ZmCCaMK. Taken together, the data clearly suggest that ZmCCaMK is required for ABA-induced antioxidant defence, and H2O2-dependent NO production plays an important role in the ABA-induced activation of ZmCCaMK. PMID:22865912

Ethanol inhibits thrombin-induced secretion by human platelets at a site distinct from phospholipase C or protein kinase C.

PubMed Central

Benistant, C; Rubin, R

1990-01-01

Ethanol is known to inhibit the activation of platelets in response to several physiological agonists, but the mechanism of this action is unclear. The addition of physiologically relevant concentrations of ethanol (25-150 mM) to suspensions of washed human platelets resulted in the inhibition of thrombin-induced secretion of 5-hydroxy[14C]tryptamine. Indomethacin was included in the incubation buffer to prevent feedback amplification by arachidonic acid metabolites. Ethanol had no effect on the activation of phospholipase C by thrombin, as determined by the formation of inositol phosphates and the mobilization of intracellular Ca2+. Moreover, ethanol did not interfere with the thrombin-induced formation of diacylglycerol or phosphatidic acid. Stimulation of platelets with phorbol ester (5-50 nM) resulted in 5-hydroxy[14C]tryptamine release comparable with those with threshold doses of thrombin. However, ethanol did not inhibit phorbol-ester-induced secretion. Ethanol also did not interfere with thrombin- or phorbol-ester-induced phosphorylation of myosin light chain (20 kDa) or a 47 kDa protein, a known substrate for protein kinase C. By electron microscopy, ethanol had no effect on thrombin-induced shape change and pseudopod formation, but prevented granule centralization and fusion. The results indicate that ethanol does not inhibit platelet secretion by interfering with the activation of phosphoinositide-specific phospholipase C or protein kinase C by thrombin. Rather, the data demonstrate an inhibition of a Ca2(+)-mediated event such as granule centralization. Images p495-a PMID:2117442

Inhibition of protein kinase C α/βII and activation of c-Jun NH2-terminal kinase mediate glycyrrhetinic acid induced apoptosis in non-small cell lung cancer NCI-H460 cells.

PubMed

Song, Junho; Ko, Hyun-suk; Sohn, Eun Jung; Kim, Bonglee; Kim, Jung Hyo; Kim, Hee Jeong; Kim, Chulwoo; Kim, Jai-eun; Kim, Sung-Hoon

2014-02-15

Though glycyrrhetinic acid (GA) from Glycyrrhiza glabra was known to exert antioxidant, antifilarial, hepatoprotective, anti-inflammatory and anti-tumor effects, the antitumor mechanism of GA was not clearly elucidated in non-small cell lung cancer cells (NSCLCCs). Thus, in the present study, the underlying apoptotic mechanism of GA was examined in NCI-H460 NSCLCCs. GA significantly suppressed the viability of NCI-H460 and A549 non-small lung cancer cells. Also, GA significantly increased the sub G1 population by cell cycle analysis and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) positive cells in a concentration dependent manner in NCI-H460 non-small lung cancer cells. Consistently, GA cleaved poly (ADP-ribosyl) polymerase (PARP), caspase 9/3, attenuated the expression of Bcl-XL, Bcl-2, Cyclin D1 and Cyclin E in NCI-H460 cells. Interestingly, GA attenuated the phosphorylation of protein kinase C (PKC) α/βII and extracellular activated protein kinase (ERK) as well as activated the phosphorylation of PKC δ and c-Jun NH2-terminal kinase in NCI-H460 cells. Conversely, PKC promoter phorbol 12-myristate 13-acetate (PMA) and JNK inhibitor SP600125 reversed the cleavages of caspase 3 and PARP induced by GA in NCI-H460 cells. Overall, our findings suggest that GA induces apoptosis via inhibition of PKC α/βII and activation of JNK in NCI-H460 non-small lung cancer cells as a potent anticancer candidate for lung cancer treatment. Copyright © 2013 Elsevier Ltd. All rights reserved.

Increased dietary protein attenuates C-reactive protein and creatine kinase responses to exercise-induced energy deficit

USDA-ARS?s Scientific Manuscript database

We determined if dietary protein (P) modulates responses of C-reactive protein (CRP) and creatine kinase (CK), biomarkers of inflammation and muscle damage, during exercise-induced energy deficit (DEF). Thirteen healthy men (22 +/- 1 y, VO2peak 60 +/- 2 ml.kg-1.min-1) balanced energy expenditure (EE...

Role of protein kinase C alpha and mitogen-activated protein kinases in endothelin-1-stimulation of cytosolic phospholipase A2 in iris sphincter smooth muscle.

PubMed

Abdel-Latif, A A; Husain, S; Yousufzai, S Y

2000-11-01

We have investigated the roles of protein kinase C (PKC) and mitogen-activated protein kinases (MAPK) in the phosphorylation and activation of cytosolic phospholipase A2 (cPLA2) in endothelin-1- (ET-1) stimulated cat iris sphincter smooth muscle (CISM) cells. We found that in these cells both PKC and p38 MAP kinases play a critical role in ET-1-induced cPLA, phosphorylation and arachidonic acid (AA) release. Our findings indicate that stimulation of the endothelin-A- (ET(A)) receptor leads to: (1) activation of Gq protein which stimulates phospholipase C to hydrolyze the polyphosphoinositide PIP, into diacylglycerol (DAG) and inositol trisphosphate (IP3), the DAG may then activate PKC to phosphorylate and activate cPLA2; and (2) activation of Gi protein, which, through a series of kinases, leads to the stimulation of p38 MAPK and subsequently to phosphorylation and activation of cPLA2. The ability of the activated ET(A)-receptor, which is coupled to both Gq and Gi proteins, to recruit and activate this complex signal transduction mechanism remains to be clarified.

[Protein kinase A inhibitor H-89 blocks polyploidization of SP600125-induced CMK cells by regulating phosphorylation of ribosomal protein S6 kinase 1].

PubMed

Zhao, Song; Yang, Jingang; Li, Changling; Xing, Sining; Yu, Ying; Liu, Shuo; Pu, Feifei; Ma, Dongchu

2016-10-01

Objective To investigate the regulatory effect of post-translation modification of ribosomal protein S6 kinase 1 (S6K1) on the polyploidization of megakaryocytes. Methods SP600125, a c-Jun N-terminal kinase (JNK) inhibitor, and H-89, a cAMP-dependent protein kinase (PKA) inhibitor, were used to treat CMK cells separately or in combination. With propidium iodide (PI) to dye DNA in the treated cells, the relative DNA content was detected by flow cytometry, and then the DNA polyploidy was analyzed. The change of expression and phosphorylation of ribosomal protein S6 kinase 1 (S6K1), an important mammalian target of rapamycin (mTOR) downstream target molecule, was analyzed by Western blotting. Molecular docking study and kinase activity assay were performed to analyze the combination of H-89 with S6K1 and the effect of H-89 on the activity of S6K1 kinase. Results SP600125 induced CMK cell polyploidization in a time-dependent and dose-dependent manner. At the same time, it increased the phosphorylation of S6K1 at Thr421/Ser424 and decreased the phosphorylation of S6K1 at Thr389. H-89 not only blocked polyploidization, but also decreased the phosphorylation of S6K1 at Thr421/Ser424 and increased the phosphorylation of S6K1 at Thr389. Molecular docking and kinase activity assay showed that H-89 occupied the ATP binding sites of S6K1 and inhibited its activity. Noticeably, both H-89 and SP600125 inhibited the activity of PKA. Moreover, the two drugs further inhibited the activity of PKA when used together. Therefore, these data indicated that H-89 blocked the SP600125-induced polyploidization of CMK cells mainly by changing S6K1 phosphorylation state, rather than its inhibitory effect on PKA. Conclusion H-89 can block the polyploidization of SP600125-induced CMK cells by regulating S6K1 phosphorylation state.

The pearl millet mitogen-activated protein kinase PgMPK4 is involved in responses to downy mildew infection and in jasmonic- and salicylic acid-mediated defense.

PubMed

Melvin, Prasad; Prabhu, S Ashok; Veena, Mariswamy; Shailasree, Sekhar; Petersen, Morten; Mundy, John; Shetty, Shekar H; Kini, K Ramachandra

2015-02-01

Plant mitogen-activated protein kinases (MPKs) transduce signals required for the induction of immunity triggered by host recognition of pathogen-associated molecular patterns. We isolated a full-length cDNA of a group B MPK (PgMPK4) from pearl millet. Autophosphorylation assay of recombinant PgMPK4 produced in Escherichia coli confirmed it as a kinase. Differential accumulation of PgMPK4 mRNA and kinase activity was observed between pearl millet cultivars 852B and IP18292 in response to inoculation with the downy mildew oomycete pathogen Sclerospora graminicola. This increased accumulation of PgMPK4 mRNA, kinase activity as well as nuclear-localization of PgMPK protein(s) was only detected in the S. graminicola resistant cultivar IP18292 with a ~tenfold peak at 9 h post inoculation. In the susceptible cultivar 852B, PgMPK4 mRNA and immuno-detectable nuclear PgMPK could be induced by application of the chemical elicitor β-amino butyric acid, the non-pathogenic bacteria Pseudomonas fluorescens, or by the phytohormones jasmonic acid (JA) or salicylic acid (SA). Furthermore, kinase inhibitor treatments indicated that PgMPK4 is involved in the JA- and SA-mediated expression of three defense genes, lipoxygenase, catalase 3 and polygalacturonase-inhibitor protein. These findings indicate that PgMPK/s contribute to pearl millet defense against the downy mildew pathogen by activating the expression of defense proteins.

Combining affinity enrichment, cross-linking with photo-amino acids, and mass spectrometry for probing protein kinase D2 interactions.

PubMed

Häupl, Björn; Ihling, Christian H; Sinz, Andrea

2017-04-07

We present a novel approach that relies on the affinity capture of protein interaction partners from a complex mixture, followed by covalent fixation via UV-induced activation of incorporated diazirine photo-reactive amino acids (photo-methionine and photo-leucine). The captured protein complexes are enzymatically digested and interacting proteins are identified and quantified by label-free LC/MS analysis. Using HeLa cell lysates with photo-methionine and photo-leucine-labeled proteins, we were able to capture and preserve protein interactions that are otherwise elusive in conventional pull-down experiments. Our approach is exemplified for mapping the protein interaction network of protein kinase D2, but has the potential be applied to any protein system. Data are available via ProteomeXchange with identifiers PXD005346 (photo-amino acid incorporation) and PXD005349 (enrichment experiments). This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Role of arachidonic acid and protein kinase C during maturation-inducing hormone-dependent meiotic resumption and ovulation in ovarian follicles of Atlantic croaker

USGS Publications Warehouse

Patino, R.; Yoshizaki, G.; Bolamba, D.; Thomas, P.

2003-01-01

The roles of arachidonic acid (AA) and protein kinase C (PKC) during in vitro maturation-inducing hormone (MIH)-dependent meiotic resumption (maturation) and ovulation were studied in ovarian follicles of Atlantic croaker (Micropogonias undulatus). The requirement for cyclooxygenase (COX) metabolites of AA was examined using a nonspecific COX inhibitor, indomethacin (IM), as well as two COX products, prostaglandin (PG) F2?? and PGE2, whereas the role of lipoxygenase (LOX) was investigated using a specific LOX inhibitor, nordihydroguaiaretic acid (NDGA). The involvement of PKC was examined using phorbol 12-myristate 13-acetate (PMA), a PKC activator, as well as GF109203X (GF), a specific inhibitor of PKC and 1-(5-isoquin- olinesulfonyl)-2-methylpiperazine (H7), nonspecific inhibitor of protein kinases. Genomic mechanisms were examined with the transcription-inhibitor actinomycin D (ActD) and the functionality of heterologous (oocyte-granulosa) gap junctions (GJ) with a dye transfer assay. The AA (100 ??M) and PGF2?? (5 ??M) did not induce maturation, and NDGA (10 ??M) did not affect MIH-dependent maturation. However, IM (100 ??M) partially inhibited MIH-dependent maturation. Conversely, AA and both PGs induced, and IM and NDGA inhibited, MIH-dependent ovulation in matured follicles. The PMA (1 ??g/ml) did not induce maturation but caused ovulation in matured follicles, whereas PKC inhibitors (GF, 5 ??M; H7, 50??M) did not affect MIH-dependent maturation but inhibited MIH- and PMA-dependent ovulation. The PMA-dependent ovulation was inhibited by IM but not by NDGA. In addition, ActD (5 ??M) blocked MIH-dependent, but not PMA-dependent, ovulation, and PGF2?? restored MIH-dependent ovulation in ActD-blocked follicles. The AA and PGs did not induce, and GF did not inhibit, MIH-dependent heterologous GJ uncoupling. In conclusion, AA and PKC mediate MIH-dependent ovulation but not meiotic resumption or heterologous GJ uncoupling in croaker follicles, but a permissive role

Stimulation of hepatocytic AMP-activated protein kinase by okadaic acid and other autophagy-suppressive toxins

PubMed Central

2004-01-01

Autophagic activity in isolated rat hepatocytes is strongly suppressed by OA (okadaic acid) and other PP (protein phosphatase)-inhibitory toxins as well as by AICAR (5-aminoimidazole-4-carboxamide riboside), a direct activator of AMPK (AMP-activated protein kinase). To investigate whether AMPK is a mediator of the effects of the toxin, a phosphospecific antibody directed against the activation of phosphorylation of the AMPK α (catalytic)-subunit at Thr172 was used to assess the activation status of this enzyme. AICAR as well as all the toxins tested (OA, microcystin-LR, calyculin A, cantharidin and tautomycin) induced strong, dose-dependent AMPKα phosphorylation, correlating with AMPK activity in situ (in intact hepatocytes) as measured by the AMPK-dependent phosphorylation of acetyl-CoA carboxylase at Ser79. All treatments induced the appearance of multiple, phosphatase-sensitive, low-mobility forms of the AMPK α-subunit, consistent with phosphorylation at several sites other than Thr172. The flavonoid naringin, an effective antagonist of OA-induced autophagy suppression, inhibited the AMPK phosphorylation and mobility shifting induced by AICAR, OA or microcystin, but not the changes induced by calyculin A or cantharidin. AMPK may thus be activated both by a naringin-sensitive and a naringin-resistant mechanism, probably involving the PPs PP2A and PP1 respectively. Neither the Thr172-phosphorylating protein kinase LKB1 nor the Thr172-dephosphorylating PP, PP2C, were mobility-shifted after treatment with toxins or AICAR, whereas a slight mobility shifting of the regulatory AMPK β-subunit was indicated. Immunoblotting with a phosphospecific antibody against pSer108 at the β-subunit revealed a naringin-sensitive phosphorylation induced by OA, microcystin and AICAR and a naringin-resistant phosphorylation induced by calyculin A and cantharidin, suggesting that β-subunit phosphorylation could play a role in AMPK activation. Naringin antagonized the autophagy

COMPARATIVE PATHOGENESIS OF HALOACETIC ACID AND PROTEIN KINASE INHIBITOR EMBRYOTOXICITY IN MOUSE WHOLE EMBRYO CULTURE

EPA Science Inventory

Comparative pathogenesis of haloacetic acid and protein kinase inhibitor embryotoxicity in mouse whole embryo culture.

Ward KW, Rogers EH, Hunter ES 3rd.

Curriculum in Toxicology, University of North Carolina at Chapel Hill, 27599-7270, USA.

Haloacetic acids ...

Oleanolic acid supplement attenuates liquid fructose-induced adipose tissue insulin resistance through the insulin receptor substrate-1/phosphatidylinositol 3-kinase/Akt signaling pathway in rats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Ying; Wang, Jianwei, E-mail: wangjianwei1968@gmail.com; Gu, Tieguang

Oleanolic acid, a triterpenoid contained in more than 1620 plants including various fruits and foodstuffs, has numerous metabolic effects, such as hepatoprotection. However, its underlying mechanisms remain poorly understood. Adipose tissue insulin resistance (Adipo-IR) may contribute to the development and progress of metabolic abnormalities through release of excessive free fatty acids from adipose tissue. This study investigated the effect of oleanolic acid on Adipo-IR. The results showed that supplement with oleanolic acid (25 mg/kg, once daily, by oral gavage) over 10 weeks attenuated liquid fructose-induced increase in plasma insulin concentration and the homeostasis model assessment of insulin resistance (HOMA-IR) indexmore » in rats. Simultaneously, oleanolic acid reversed the increase in the Adipo-IR index and plasma non-esterified fatty acid concentrations during the oral glucose tolerance test assessment. In white adipose tissue, oleanolic acid enhanced mRNA expression of the genes encoding insulin receptor, insulin receptor substrate (IRS)-1 and phosphatidylinositol 3-kinase. At the protein level, oleanolic acid upregulated total IRS-1 expression, suppressed the increased phosphorylated IRS-1 at serine-307, and restored the increased phosphorylated IRS-1 to total IRS-1 ratio. In contrast, phosphorylated Akt to total Akt ratio was increased. Furthermore, oleanolic acid reversed fructose-induced decrease in phosphorylated-Akt/Akt protein to plasma insulin concentration ratio. However, oleanolic acid did not affect IRS-2 mRNA expression. Therefore, these results suggest that oleanolic acid supplement ameliorates fructose-induced Adipo-IR in rats via the IRS-1/phosphatidylinositol 3-kinase/Akt pathway. Our findings may provide new insights into the mechanisms of metabolic actions of oleanolic acid. - Highlights: • Adipose insulin resistance (Adipo-IR) contributes to metabolic abnormalities. • We investigated the effect of oleanolic acid (OA) on adipo

A Ser/Thr protein kinase phosphorylates MA-ACS1 (Musa acuminata 1-aminocyclopropane-1-carboxylic acid synthase 1) during banana fruit ripening.

PubMed

Choudhury, Swarup Roy; Roy, Sujit; Sengupta, Dibyendu N

2012-08-01

1-Aminocyclopropane-1-carboxylic acid synthase (ACS) catalyzes the rate-limiting step in ethylene biosynthesis during ripening. ACS isozymes are regulated both transcriptionally and post-translationally. However, in banana, an important climacteric fruit, little is known about post-translational regulation of ACS. Here, we report the post-translational modification of MA-ACS1 (Musa acuminata ACS1), a ripening inducible isozyme in the ACS family, which plays a key role in ethylene biosynthesis during banana fruit ripening. Immunoprecipitation analyses of phospholabeled protein extracts from banana fruit using affinity-purified anti-MA-ACS1 antibody have revealed phosphorylation of MA-ACS1, particularly in ripe fruit tissue. We have identified the induction of a 41-kDa protein kinase activity in pulp at the onset of ripening. The 41-kDa protein kinase has been identified as a putative protein kinase by MALDI-TOF/MS analysis. Biochemical analyses using partially purified protein kinase fraction from banana fruit have identified the protein kinase as a Ser/Thr family of protein kinase and its possible involvement in MA-ACS1 phosphorylation during ripening. In vitro phosphorylation analyses using synthetic peptides and site-directed mutagenized recombinant MA-ACS1 have revealed that serine 476 and 479 residues at the C-terminal region of MA-ACS1 are phosphorylated. Overall, this study provides important novel evidence for in vivo phosphorylation of MA-ACS1 at the molecular level as a possible mechanism of post-translational regulation of this key regulatory protein in ethylene signaling pathway in banana fruit during ripening.

Activation of G protein-coupled bile acid receptor, TGR5, induces smooth muscle relaxation via both Epac- and PKA-mediated inhibition of RhoA/Rho kinase pathway

PubMed Central

Rajagopal, Senthilkumar; Kumar, Divya P.; Mahavadi, Sunila; Bhattacharya, Sayak; Zhou, Ruizhe; Corvera, Carlos U.; Bunnett, Nigel W.; Grider, John R.

2013-01-01

The present study characterized the TGR5 expression and the signaling pathways coupled to this receptor that mediates the relaxation of gastric smooth muscle. TGR5 was detected in gastric muscle cells by RT-PCR and Western blotting. Treatment of cells with the TGR5-selective ligand oleanolic acid (OA) activated Gαs, but not Gαq, Gαi1, Gαi2, or Gαi3, and increased cAMP levels. OA did not elicit contraction, but caused relaxation of carbachol-induced contraction of gastric muscle cells from wild-type mice, but not tgr5−/− mice. OA, but not a selective exchange protein activated by cAMP (Epac) ligand (8-pCPT-2′-O-Me-cAMP), caused phosphorylation of RhoA and the phosphorylation was blocked by the PKA inhibitor, myristoylated PKI, and by the expression of phosphorylation-deficient mutant RhoA (S188A). Both OA and Epac ligand stimulated Ras-related protein 1 (Rap1) and inhibited carbachol (CCh)-induced Rho kinase activity. Expression of RhoA (S188A) or PKI partly reversed the inhibition of Rho kinase activity by OA but had no effect on inhibition by Epac ligand. However, suppression of Rap1 with siRNA blocked the inhibition of Rho kinase by Epac ligand, and partly reversed the inhibition by OA; the residual inhibition was blocked by PKI. Muscle relaxation in response to OA, but not Epac ligand, was partly reversed by PKI. We conclude that activation of TGR5 causes relaxation of gastric smooth muscle and the relaxation is mediated through inhibition of RhoA/Rho kinase pathway via both cAMP/Epac-dependent stimulation of Rap1 and cAMP/PKA-dependent phosphorylation of RhoA at Ser188. TGR5 receptor activation on smooth muscle reveals a novel mechanism for the regulation of gut motility by bile acids. PMID:23275618

Fed levels of amino acids are required for the somatotropin-induced increase in muscle protein synthesis.

PubMed

Wilson, Fiona A; Suryawan, Agus; Orellana, Renán A; Nguyen, Hanh V; Jeyapalan, Asumthia S; Gazzaneo, Maria C; Davis, Teresa A

2008-10-01

Chronic somatotropin (pST) treatment in pigs increases muscle protein synthesis and circulating insulin, a known promoter of protein synthesis. Previously, we showed that the pST-mediated rise in insulin could not account for the pST-induced increase in muscle protein synthesis when amino acids were maintained at fasting levels. This study aimed to determine whether the pST-induced increase in insulin promotes skeletal muscle protein synthesis when amino acids are provided at fed levels and whether the response is associated with enhanced translation initiation factor activation. Growing pigs were treated with pST (0 or 180 microg x kg(-1) x day(-1)) for 7 days, and then pancreatic-glucose-amino acid clamps were performed. Amino acids were raised to fed levels in the presence of either fasted or fed insulin concentrations; glucose was maintained at fasting throughout. Muscle protein synthesis was increased by pST treatment and by amino acids (with or without insulin) (P<0.001). In pST-treated pigs, fed, but not fasting, amino acid concentrations further increased muscle protein synthesis rates irrespective of insulin level (P<0.02). Fed amino acids, with or without raised insulin concentrations, increased the phosphorylation of S6 kinase (S6K1) and eukaryotic initiation factor (eIF) 4E-binding protein 1 (4EBP1), decreased inactive 4EBP1.eIF4E complex association, and increased active eIF4E.eIF4G complex formation (P<0.02). pST treatment did not alter translation initiation factor activation. We conclude that the pST-induced stimulation of muscle protein synthesis requires fed amino acid levels, but not fed insulin levels. However, under the current conditions, the response to amino acids is not mediated by the activation of translation initiation factors that regulate mRNA binding to the ribosomal complex.

Mechanism of alpha-lipoic acid in attenuating kanamycin-induced ototoxicity☆

PubMed Central

Wang, Aimei; Hou, Ning; Bao, Dongyan; Liu, Shuangyue; Xu, Tao

2012-01-01

In view of the theory that alpha-lipoic acid effectively prevents cochlear cells from injury caused by various factors such as cisplatin and noise, this study examined whether alpha-lipoic acid can prevent kanamycin-induced ototoxicity. To this end, healthy BALB/c mice were injected subcutaneously with alpha-lipoic acid and kanamycin for 14 days. Auditory brainstem response test showed that increased auditory brainstem response threshold shifts caused by kanamycin were significantly inhibited. Immunohistochemical staining and western blot analysis showed that the expression of phosphorylated p38 mitogen-activated protein kinase and phosphorylated c-Jun N-terminal kinase in mouse cochlea was significantly decreased. The experimental findings suggest that phosphorylated p38 and phosphorylated c-Jun N-terminal kinase mediated kanamycin-induced ototoxic injury in BALB/c mice. Alpha-lipoic acid effectively attenuated kanamycin ototoxicity by inhibiting the kanamycin-induced high expression of phosphorylated p38 and phosphorylated c-Jun N-terminal kinase. PMID:25317129

Ezrin is a cyclic AMP-dependent protein kinase anchoring protein.

PubMed Central

Dransfield, D T; Bradford, A J; Smith, J; Martin, M; Roy, C; Mangeat, P H; Goldenring, J R

1997-01-01

cAMP-dependent protein kinase (A-kinase) anchoring proteins (AKAPs) are responsible for the subcellular sequestration of the type II A-kinase. Previously, we identified a 78 kDa AKAP which was enriched in gastric parietal cells. We have now purified the 78 kDa AKAP to homogeneity from gastric fundic mucosal supernates using type II A-kinase regulatory subunit (RII) affinity chromatography. The purified 78 kDa AKAP was recognized by monoclonal antibodies against ezrin, the canalicular actin-associated protein. Recombinant ezrin produced in either Sf9 cells or bacteria also bound RII. Recombinant radixin and moesin, ezrin-related proteins, also bound RII in blot overlay. Analysis of recombinant truncations of ezrin mapped the RII binding site to a region between amino acids 373 and 439. This region contained a 14-amino-acid amphipathic alpha-helical putative RII binding region. A synthetic peptide containing the amphipathic helical region (ezrin409-438) blocked RII binding to ezrin, but a peptide with a leucine to proline substitution at amino acid 421 failed to inhibit RII binding. In mouse fundic mucosa, RII immunoreactivity redistributed from a predominantly cytosolic location in resting parietal cells, to a canalicular pattern in mucosa from animals stimulated with gastrin. These results demonstrate that ezrin is a major AKAP in gastric parietal cells and may function to tether type II A-kinase to a region near the secretory canaliculus. PMID:9009265

Megakaryocytic differentiation induced by constitutive activation of mitogen-activated protein kinase kinase.

PubMed Central

Whalen, A M; Galasinski, S C; Shapiro, P S; Nahreini, T S; Ahn, N G

1997-01-01

The K562 erythroleukemia cell line was used to study the molecular mechanisms regulating lineage commitment of hematopoietic stem cells. Phorbol esters, which initiate megakaryocyte differentiation in this cell line, caused a rapid increase in extracellular-signal-regulated kinase (ERK), which remained elevated for 2 h and returned to near-basal levels by 24 h. In the absence of extracellular stimuli, ERK could be activated by expression of constitutively active mutants of mitogen-activated protein (MAP) kinase kinase (MKK), resulting in cell adhesion and spreading, increased cell size, inhibition of cell growth, and induction of the platelet-specific integrin alphaIIb beta3, all hallmarks of megakaryocytic differentiation. In contrast, expression of wild-type MKK had little effect. In addition, constitutively active MKK suppressed the expression of an erythroid marker, alpha-globin, indicating the ability to suppress cellular responses necessary for alternative cell lineages. The MKK inhibitor PD98059 blocked MKK/ERK activation and cellular responses to phorbol ester, demonstrating that activation of MKK is necessary and sufficient to induce a differentiation program along the megakaryocyte lineage. Thus, the MAP kinase cascade, which promotes cell growth and proliferation in many cell types, instead inhibits cell proliferation and initiates lineage-specific differentiation in K562 cells, establishing a model system to investigate the mechanisms by which this signal transduction pathway specifies cell fate and developmental processes. PMID:9121442

Megakaryocytic differentiation induced by constitutive activation of mitogen-activated protein kinase kinase.

PubMed

Whalen, A M; Galasinski, S C; Shapiro, P S; Nahreini, T S; Ahn, N G

1997-04-01

The K562 erythroleukemia cell line was used to study the molecular mechanisms regulating lineage commitment of hematopoietic stem cells. Phorbol esters, which initiate megakaryocyte differentiation in this cell line, caused a rapid increase in extracellular-signal-regulated kinase (ERK), which remained elevated for 2 h and returned to near-basal levels by 24 h. In the absence of extracellular stimuli, ERK could be activated by expression of constitutively active mutants of mitogen-activated protein (MAP) kinase kinase (MKK), resulting in cell adhesion and spreading, increased cell size, inhibition of cell growth, and induction of the platelet-specific integrin alphaIIb beta3, all hallmarks of megakaryocytic differentiation. In contrast, expression of wild-type MKK had little effect. In addition, constitutively active MKK suppressed the expression of an erythroid marker, alpha-globin, indicating the ability to suppress cellular responses necessary for alternative cell lineages. The MKK inhibitor PD98059 blocked MKK/ERK activation and cellular responses to phorbol ester, demonstrating that activation of MKK is necessary and sufficient to induce a differentiation program along the megakaryocyte lineage. Thus, the MAP kinase cascade, which promotes cell growth and proliferation in many cell types, instead inhibits cell proliferation and initiates lineage-specific differentiation in K562 cells, establishing a model system to investigate the mechanisms by which this signal transduction pathway specifies cell fate and developmental processes.

Chrysophanic acid reduces testosterone-induced benign prostatic hyperplasia in rats by suppressing 5α-reductase and extracellular signal-regulated kinase

PubMed Central

Kim, Hye-Lin; Jung, Yunu; Kang, JongWook; Jeong, Mi-Young; Sethi, Gautam; Ahn, Kwang Seok; Um, Jae-Young

2017-01-01

Benign prostatic hyperplasia (BPH) is one of the most common chronic diseases in male population, of which incidence increases gradually with age. In this study, we investigated the effect of chrysophanic acid (CA) on BPH. BPH was induced by a 4-week injection of testosterone propionate (TP). Four weeks of further injection with vehicle, TP, TP + CA, TP + finasteride was carried on. In the CA treatment group, the prostate weight was reduced and the TP-induced histological changes were restored as the normal control group. CA treatment suppressed the TP-elevated prostate specific antigen (PSA) expression. In addition, 5α-reductase, a crucial factor in BPH development, was suppressed to the normal level close to the control group by CA treatment. The elevated expressions of androgen receptor (AR), estrogen receptor α and steroid receptor coactivator 1 by TP administration were also inhibited in the CA group when compared to the TP-induced BPH group. Then we evaluated the changes in three major factors of the mitogen-activated protein kinase chain during prostatic hyperplasia; extracellular signal-regulated kinase (ERK), c-Jun-N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38). While ERK was elevated in the process of BPH, JNK and p38 was not changed. This up-regulated ERK was also reduced as normal by CA treatment. Further in vitro studies with RWPE-1 cells confirmed TP-induced proliferation and elevated AR, PSA and p-ERK were all reduced by CA treatment. Overall, these results suggest a potential pharmaceutical feature of CA in the treatment of BPH. PMID:27880726

A chimeric cyclic interferon-α2b peptide induces apoptosis by sequential activation of phosphatidylinositol 3-kinase, protein kinase Cδ and p38 MAP kinase.

PubMed

Blank, V C; Bertucci, L; Furmento, V A; Peña, C; Marino, V J; Roguin, L P

2013-06-10

We have previously demonstrated that tyrosine phosphorylation of STAT1/3 and p38 mitogen-activated protein kinase (p38 MAPK) activation are involved in the apoptotic response triggered by a chimeric cyclic peptide of the interferon-α2b (IFN-α2b) in WISH cells. Since the peptide also induced serine phosphorylation of STAT proteins, in the present study we examined the kinase involved in serine STAT1 phosphorylation and the signaling effectors acting upstream such activation. We first found that p38 MAPK is involved in serine STAT1 phosphorylation, since a reduction of phophoserine-STAT1 levels was evident after incubating WISH cells with cyclic peptide in the presence of a p38 pharmacological inhibitor or a dominant-negative p38 mutant. Next, we demonstrated that the peptide induced activation of protein kinase Cδ (PKCδ). Based on this finding, the role of this kinase was then evaluated. After incubating WISH cells with a PKCδ inhibitor or after decreasing PKCδ expression levels by RNA interference, both peptide-induced serine STAT1 and p38 phosphorylation levels were significantly decreased, indicating that PKCδ functions as an upstream regulator of p38. We also showed that PKCδ and p38 activation stimulated by the peptide was inhibited by a specific pharmacological inhibitor of phosphatidylinositol 3-kinase (PI3K) or by a dominant-negative p85 PI3K-regulatory subunit, suggesting that PI3K is upstream in the signaling cascade. In addition, the role of PI3K and PKCδ in cyclic peptide-induced apoptosis was examined. Both signaling effectors were found to regulate the antiproliferative activity and the apoptotic response triggered by the cyclic peptide in WISH cells. In conclusion, we herein demonstrated that STAT1 serine phosphorylation is mediated by the sequential activation of PI3K, PKCδ and p38 MAPK. This signaling cascade contributes to the antitumor effect induced by the chimeric IFN-α2b cyclic peptide in WISH cells. Copyright © 2013 Elsevier Inc

Role of Phosphatidylinositol-3 Kinase Pathway in NMDA Preconditioning: Different Mechanisms for Seizures and Hippocampal Neuronal Degeneration Induced by Quinolinic Acid.

PubMed

Constantino, Leandra C; Binder, Luisa B; Vandresen-Filho, Samuel; Viola, Giordano G; Ludka, Fabiana K; Lopes, Mark W; Leal, Rodrigo B; Tasca, Carla I

2018-04-20

N-methyl D-aspartate (NMDA) preconditioning is evoked by the administration of a subtoxic dose of NMDA and is protective against neuronal excitotoxicity. This effect may involve a diversity of targets and cell signaling cascades associated to neuroprotection. Phosphatidylinositol-3 kinase/protein kinase B (PI3K/Akt) and mitogen-activated protein kinases (MAPKs) such as extracellular regulated protein kinase 1/2 (ERK1/2) and p38 MAPK pathways play a major role in neuroprotective mechanisms. However, their involvement in NMDA preconditioning was not yet fully investigated. The present study aimed to evaluate the effect of NMDA preconditioning on PI3K/Akt, ERK1/2, and p38 MAPK pathways in the hippocampus of mice and characterize the involvement of PI3K on NMDA preconditioning-evoked prevention of seizures and hippocampal cell damage induced by quinolinic acid (QA). Thus, mice received wortmannin (a PI3K inhibitor) and 15 min later a subconvulsant dose of NMDA (preconditioning) or saline. After 24 h of this treatment, an intracerebroventricular QA infusion was administered. Phosphorylation levels and total content of Akt, glycogen synthase protein kinase-3β (GSK-3β), ERK1/2, and p38 MAPK were not altered after 24 h of NMDA preconditioning with or without wortmmanin pretreatment. Moreover, after QA administration, behavioral seizures, hippocampal neuronal degeneration, and Akt activation were evaluated. Inhibition of PI3K pathway was effective in abolishing the protective effect of NMDA preconditioning against QA-induced seizures, but did not modify neuronal protection promoted by preconditioning as evaluated by Fluoro-Jade B staining. The study confirms that PI3K participates in the mechanism of protection induced by NMDA preconditioning against QA-induced seizures. Conversely, NMDA preconditioning-evoked protection against neuronal degeneration is not altered by PI3K signaling pathway inhibition. These results point to differential mechanisms regarding protection

Proteomic Analysis of Responsive Proteins Induced in Japanese Birch Plantlet Treated with Salicylic Acid

PubMed Central

Suzuki, Hiromu; Takashima, Yuya; Ishiguri, Futoshi; Yoshizawa, Nobuo; Yokota, Shinso

2014-01-01

The present study was performed to unravel the mechanisms of systemic acquired resistance (SAR) establishment and resistance signaling pathways against the canker-rot fungus (Inonotus obliquus strain IO-U1) infection in Japanese birch plantlet No.8. Modulation of protein-profile induced by salicylic acid (SA)-administration was analyzed, and SA-responsive proteins were identified. In total, 5 specifically expressed, 3 significantly increased, and 3 significantly decreased protein spots were identified using liquid chromatography/tandem mass spectrometry (LC/MS/MS) and the sequence tag method. These proteins were malate dehydrogenase, succinate dehydrogenase, phosphoglycerate kinase, diaminopimalate decarboxylase, arginase, chorismate mutase, cyclophilin, aminopeptidase, and unknown function proteins. These proteins are considered to be involved in SAR-establishment mechanisms in the Japanese birch plantlet No 8. PMID:28250384

Protein kinase C negatively regulates Akt activity and modifies UVC-induced apoptosis in mouse keratinocytes.

PubMed

Li, Luowei; Sampat, Keeran; Hu, Nancy; Zakari, Julia; Yuspa, Stuart H

2006-02-10

Skin keratinocytes are subject to frequent chemical and physical injury and have developed elaborate cell survival mechanisms to compensate. Among these, the Akt/protein kinase B (PKB) pathway protects keratinocytes from the toxic effects of ultraviolet light (UV). In contrast, the protein kinase C (PKC) family is involved in several keratinocyte death pathways. During an examination of potential interactions among these two pathways, we found that the insulin-like growth factor (IGF-1) activates both the PKC and the Akt signaling pathways in cultured primary mouse keratinocytes as indicated by increased phospho-PKC and phospho-Ser-473-Akt. IGF-1 also selectively induced translocation of PKCdelta and PKCepsilon from soluble to particulate fractions in mouse keratinocytes. Furthermore, the PKC-specific inhibitor, GF109203X, increased IGF-1-induced phospho-Ser-473-Akt and Akt kinase activity and enhanced IGF-1 protection from UVC-induced apoptosis. Selective activation of PKC by 12-O-tetradecanoylphorbol-13-acetate (TPA) reduced phospho-Ser-473-Akt, suggesting that activation of PKC inhibits Akt activity. TPA also attenuated IGF-1 and epidermal growth factor-induced phospho-Ser-473-Akt, reduced Akt kinase activity, and blocked IGF-1 protection from UVC-induced apoptosis. The inhibition of Akt activity by TPA was reduced by inhibitors of protein phosphatase 2A, and TPA stimulated the association of phosphatase 2A with Akt. Individual PKC isoforms were overexpressed in cultured keratinocytes by transduction with adenoviral vectors or inhibited with PKC-selective inhibitors. These studies indicated that PKCdelta and PKCepsilon were selectively potent at causing dephosphorylation of Akt and modifying cell survival, whereas PKCalpha enhanced phosphorylation of Akt on Ser-473. Our results suggested that activation of PKCdelta and PKCepsilon provide a negative regulation for Akt phosphorylation and kinase activity in mouse keratinocytes and serve as modulators of cell

PEBP1, a RAF kinase inhibitory protein, negatively regulates starvation-induced autophagy by direct interaction with LC3.

PubMed

Noh, Hae Sook; Hah, Young-Sool; Zada, Sahib; Ha, Ji Hye; Sim, Gyujin; Hwang, Jin Seok; Lai, Trang Huyen; Nguyen, Huynh Quoc; Park, Jae-Yong; Kim, Hyun Joon; Byun, June-Ho; Hahm, Jong Ryeal; Kang, Kee Ryeon; Kim, Deok Ryong

2016-11-01

Autophagy plays a critical role in maintaining cell homeostasis in response to various stressors through protein conjugation and activation of lysosome-dependent degradation. MAP1LC3B/LC3B (microtubule- associated protein 1 light chain 3 β) is conjugated with phosphatidylethanolamine (PE) in the membranes and regulates initiation of autophagy through interaction with many autophagy-related proteins possessing an LC3-interacting region (LIR) motif, which is composed of 2 hydrophobic amino acids (tryptophan and leucine) separated by 2 non-conserved amino acids (WXXL). In this study, we identified a new putative LIR motif in PEBP1/RKIP (phosphatidylethanolamine binding protein 1) that was originally isolated as a PE-binding protein and also a cellular inhibitor of MAPK/ERK signaling. PEBP1 was specifically bound to PE-unconjugated LC3 in cells, and mutation (WXXL mutated to AXXA) of this LIR motif disrupted its interaction with LC3 proteins. Interestingly, overexpression of PEBP1 significantly inhibited starvation-induced autophagy by activating the AKT and MTORC1 (mechanistic target of rapamycin [serine/threonine kinase] complex 1) signaling pathway and consequently suppressing the ULK1 (unc-51 like autophagy activating kinase 1) activity. In contrast, ablation of PEBP1 expression dramatically promoted the autophagic process under starvation conditions. Furthermore, PEBP1 lacking the LIR motif highly stimulated starvation-induced autophagy through the AKT-MTORC1-dependent pathway. PEBP1 phosphorylation at Ser153 caused dissociation of LC3 from the PEBP1-LC3 complex for autophagy induction. PEBP1-dependent suppression of autophagy was not associated with the MAPK pathway. These findings suggest that PEBP1 can act as a negative mediator in autophagy through stimulation of the AKT-MTORC1 pathway and direct interaction with LC3.

Arctigenin protects against steatosis in WRL68 hepatocytes through activation of phosphoinositide 3-kinase/protein kinase B and AMP-activated protein kinase pathways.

PubMed

Chen, Kung-Yen; Lin, Jui-An; Yao, Han-Yun; Hsu, An-Chih; Tai, Yu-Ting; Chen, Jui-Tai; Hsieh, Mao-Chih; Shen, Tang-Long; Hsu, Ren-Yi; Wu, Hong-Tan; Wang, Guey Horng; Ho, Bing-Ying; Chen, Yu-Pei

2018-04-01

Arctigenin (ATG), a lignin extracted from Arctium lappa (L.), exerts antioxidant and anti-inflammatory effects. We hypothesized that ATG exerts a protective effect on hepatocytes by preventing nonalcoholic fatty liver disease (NAFLD) progression associated with lipid oxidation-associated lipotoxicity and inflammation. We established an in vitro NAFLD cell model by using normal WRL68 hepatocytes to investigate oleic acid (OA) accumulation and the potential bioactive role of ATG. The results revealed that ATG inhibited OA-induced lipid accumulation, lipid peroxidation, and inflammation in WRL68 hepatocytes, as determined using Oil Red O staining, thiobarbituric acid reactive substance assay, and inflammation antibody array assays. Quantitative RT-PCR analysis demonstrated that ATG significantly mitigated the expression of acetylcoenzyme A carboxylase 1 and sterol regulatory element-binding protein-1 and significantly increased the expression of carnitine palmitoyltransferase 1 and peroxisome proliferator-activated receptor alpha. The 40 targets of the Human Inflammation Antibody Array indicated that ATG significantly inhibited the elevation of the U937 lymphocyte chemoattractant, ICAM-1, IL-1β, IL-6, IL-6sR, IL-7, and IL-8. ATG could activate the phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) and AMP-activated protein kinase (AMPK) pathways and could increase the phosphorylation levels of Akt and AMPK to mediate cell survival, lipid metabolism, oxidation stress, and inflammation. Thus, we demonstrated that ATG could inhibit NAFLD progression associated with lipid oxidation-associated lipotoxicity and inflammation, and we provided insights into the underlying mechanisms and revealed potential targets to enable a thorough understanding of NAFLD progression. Copyright © 2018 Elsevier Inc. All rights reserved.

Protein Kinases in Mammary Gland Development and Carcinogenesis

DTIC Science & Technology

1999-09-01

studies identical at the amino acid level to calcium/calmodulin-dependent may provide insight into mechanisms of growth control and DNA protein kinase I...human homologues of these kinases(19, 20 ). Amino acid conservation in the coding region between mouse and human Hunk is greater than 90% identical. While...genes (13, 14). Over the past 4 years , several of the mRNA and protein levels (39-46). These findings clearly dem- these breast cancer susceptibility

Mitogen-Activated Protein Kinase 14 Promotes AKI

PubMed Central

Husi, Holger; Gonzalez-Lafuente, Laura; Valiño-Rivas, Lara; Fresno, Manuel; Sanz, Ana Belen; Mullen, William; Albalat, Amaya; Mezzano, Sergio; Vlahou, Tonia; Mischak, Harald

2017-01-01

An improved understanding of pathogenic pathways in AKI may identify novel therapeutic approaches. Previously, we conducted unbiased liquid chromatography-tandem mass spectrometry–based protein expression profiling of the renal proteome in mice with acute folate nephropathy. Here, analysis of the dataset identified enrichment of pathways involving NFκB in the kidney cortex, and a targeted data mining approach identified components of the noncanonical NFκB pathway, including the upstream kinase mitogen-activated protein kinase kinase kinase 14 (MAP3K14), the NFκB DNA binding heterodimer RelB/NFκB2, and proteins involved in NFκB2 p100 ubiquitination and proteasomal processing to p52, as upregulated. Immunohistochemistry localized MAP3K14 expression to tubular cells in acute folate nephropathy and human AKI. In vivo, kidney expression levels of NFκB2 p100 and p52 increased rapidly after folic acid injection, as did DNA binding of RelB and NFκB2, detected in nuclei isolated from the kidneys. Compared with wild-type mice, MAP3K14 activity–deficient aly/aly (MAP3K14aly/aly) mice had less kidney dysfunction, inflammation, and apoptosis in acute folate nephropathy and less kidney dysfunction and a lower mortality rate in cisplatin-induced AKI. The exchange of bone marrow between wild-type and MAP3K14aly/aly mice did not affect the survival rate of either group after folic acid injection. In cultured tubular cells, MAP3K14 small interfering RNA targeting decreased inflammation and cell death. Additionally, cell culture and in vivo studies identified the chemokines MCP-1, RANTES, and CXCL10 as MAP3K14 targets in tubular cells. In conclusion, MAP3K14 promotes kidney injury through promotion of inflammation and cell death and is a promising novel therapeutic target. PMID:27620989

Novel receptor-like protein kinases induced by Erwinia carotovora and short oligogalacturonides in potato.

PubMed

Montesano, M; Kõiv, V; Mäe, A; Palva, E T

2001-11-01

summary Identification of potato genes responsive to cell wall-degrading enzymes of Erwinia carotovora resulted in the isolation of cDNA clones for four related receptor-like protein kinases. One of the putative serine-threonine protein kinases might have arisen through alternative splicing. These potato receptor-like kinases (PRK1-4) were highly equivalent (91-99%), most likely constituting a family of related receptors. All PRKs and four other plant RLKs share in their extracellular domain a conserved bi-modular pattern of cysteine repeats distinct from that in previously characterized plant RLKs, suggesting that they represent a new class of receptors. The corresponding genes were rapidly induced by E. carotovora culture filtrate (CF), both in the leaves and tubers of potato. Furthermore, the genes were transiently induced by short oligogalacturonides. The structural identity of PRKs and their induction pattern suggested that they constitute part of the early response of potato to E. carotovora infection.

Hypochlorous and peracetic acid induced oxidation of dairy proteins.

PubMed

Kerkaert, Barbara; Mestdagh, Frédéric; Cucu, Tatiana; Aedo, Philip Roger; Ling, Shen Yan; De Meulenaer, Bruno

2011-02-09

Hypochlorous and peracetic acids, both known disinfectants in the food industry, were compared for their oxidative capacity toward dairy proteins. Whey proteins and caseins were oxidized under well controlled conditions at pH 8 as a function of the sanitizing concentration. Different markers for protein oxidation were monitored. The results established that the protein carbonyl content was a rather unspecific marker for protein oxidation, which did not allow one to differentiate the oxidant used especially at the lower concentrations. Cysteine, tryptophan, and methionine were proven to be the most vulnerable amino acids for degradation upon hypochlorous and peracetic acid treatment, while tyrosine was only prone to degradation in the presence of hypochlorous acid. Hypochlorous acid induced oxidation gave rise to protein aggregation, while during peracetic acid induced oxidation, no high molecular weight aggregates were observed. Protein aggregation upon hypochlorous acid oxidation could primarily be linked to tryptophan and tyrosine degradation.

Phosphoproteomic Analysis of Protein Kinase C Signaling in Saccharomyces cerevisiae Reveals Slt2 Mitogen-activated Protein Kinase (MAPK)-dependent Phosphorylation of Eisosome Core Components*

PubMed Central

Mascaraque, Victoria; Hernáez, María Luisa; Jiménez-Sánchez, María; Hansen, Rasmus; Gil, Concha; Martín, Humberto; Cid, Víctor J.; Molina, María

2013-01-01

The cell wall integrity (CWI) pathway of the model organism Saccharomyces cerevisiae has been thoroughly studied as a paradigm of the mitogen-activated protein kinase (MAPK) pathway. It consists of a classic MAPK module comprising the Bck1 MAPK kinase kinase, two redundant MAPK kinases (Mkk1 and Mkk2), and the Slt2 MAPK. This module is activated under a variety of stimuli related to cell wall homeostasis by Pkc1, the only member of the protein kinase C family in budding yeast. Quantitative phosphoproteomics based on stable isotope labeling of amino acids in cell culture is a powerful tool for globally studying protein phosphorylation. Here we report an analysis of the yeast phosphoproteome upon overexpression of a PKC1 hyperactive allele that specifically activates CWI MAPK signaling in the absence of external stimuli. We found 82 phosphopeptides originating from 43 proteins that showed enhanced phosphorylation in these conditions. The MAPK S/T-P target motif was significantly overrepresented in these phosphopeptides. Hyperphosphorylated proteins provide putative novel targets of the Pkc1–cell wall integrity pathway involved in diverse functions such as the control of gene expression, protein synthesis, cytoskeleton maintenance, DNA repair, and metabolism. Remarkably, five components of the plasma-membrane-associated protein complex known as eisosomes were found among the up-regulated proteins. We show here that Pkc1-induced phosphorylation of the eisosome core components Pil1 and Lsp1 was not exerted directly by Pkc1, but involved signaling through the Slt2 MAPK module. PMID:23221999

Effect of mitogen-activated protein kinases on chemokine synthesis induced by substance P in mouse pancreatic acinar cells

PubMed Central

Ramnath, Raina Devi; Sun, Jia; Adhikari, Sharmila; Bhatia, Madhav

2007-01-01

Abstract Substance P, acting via its neurokinin 1 receptor (NK1 R), plays an important role in mediating a variety of inflammatory processes. Its interaction with chemokines is known to play a crucial role in the pathogenesis of acute pancreatitis. In pancreatic acinar cells, substance P stimulates the release of NFκB-driven chemokines. However, the signal transduction pathways by which substance P-NK1 R interaction induces chemokine production are still unclear. To that end, we went on to examine the participation of mitogen-activated protein kinases (MAPKs) in substance P-induced synthesis of pro-inflammatory chemokines, monocyte chemoanractant protein-1 (MCP-I), macrophage inflammatory protein-lα (MIP-lα) and macrophage inflammatory protein-2 (MIP-2), in pancreatic acini. In this study, we observed a time-dependent activation of ERK1/2, c-Jun N-terminal kinase (JNK), NFκB and activator protein-1 (AP-1) when pancreatic acini were stimulated with substance P. Moreover, substance P-induced ERK 1/2, JNK, NFκB and AP-1 activation as well as chemokine synthesis were blocked by pre-treatment with either extracellular signal-regulated protein kinase kinase 1 (MEK1) inhibitor or JNK inhibitor. In addition, substance P-induced activation of ERK 112, JNK, NFκB and AP-1-driven chemokine production were attenuated by CP96345, a selective NK1 R antagonist, in pancreatic acinar cells. Taken together, these results suggest that substance P-NK1 R induced chemokine production depends on the activation of MAPKs-mediated NFκB and AP-1 signalling pathways in mouse pancreatic acini. PMID:18205703

Activation of the kinase activity of ATM by retinoic acid is required for CREB-dependent differentiation of neuroblastoma cells.

PubMed

Fernandes, Norvin D; Sun, Yingli; Price, Brendan D

2007-06-01

The ATM protein kinase is mutated in ataxia telangiectasia, a genetic disease characterized by defective DNA repair, neurodegeneration, and growth factor signaling defects. The activity of ATM kinase is activated by DNA damage, and this activation is required for cells to survive genotoxic events. In addition to this well characterized role in DNA repair, we now demonstrate a novel role for ATM in the retinoic acid (RA)-induced differentiation of SH-SY5Y neuroblastoma cells into post-mitotic, neuronal-like cells. RA rapidly activates the activity of ATM kinase, leading to the ATM-dependent phosphorylation of the CREB protein, extrusion of neuritic processes, and differentiation of SH-SY5Y cells into neuronal-like cells. When ATM protein expression was suppressed by short hairpin RNA, the ATM-dependent phosphorylation of CREB was blocked. Furthermore, ATM-negative cells failed to differentiate into neuronal-like cells when exposed to retinoic acid; instead, they underwent cell death. Expression of a constitutively active CREBVP16 construct, or exposure to forskolin to induce CREB phosphorylation, rescued ATM negative cells and restored differentiation. Furthermore, when dominant negative CREB proteins with mutations in either the CREB phosphorylation site (CREBS133A) or the DNA binding domain (KCREB) were introduced into SH-SY5Y cells, retinoic acid-induced differentiation was blocked and the cells underwent cell death. The results demonstrate that ATM is required for the retinoic acid-induced differentiation of SH-SY5Y cells through the ATM dependent-phosphorylation of serine 133 of CREB. These results therefore define a novel mechanism for activation of the activity of ATM kinase by RA, and implicate ATM in the regulation of CREB function during RA-induced differentiation.

Receptor-interacting protein kinases modulate noise-induced sensory hair cell death

PubMed Central

Zheng, H-W; Chen, J; Sha, S-H

2014-01-01

Receptor-interacting protein (RIP) kinases promote the induction of necrotic cell death pathways. Here we investigated signaling pathways in outer hair cells (OHCs) of adult male CBA/J mice exposed to noise that causes permanent threshold shifts, with a particular focus on RIP kinase-regulated necroptosis. One hour after noise exposure, nuclei of OHCs in the basal region of the cochlea displayed both apoptotic and necrotic features. RIP1 and RIP3 protein levels increased and caspase-8 was activated. Treatment with pan-caspase inhibitor ZVAD blocked the activation of caspase-8 and reduced the number of apoptotic nuclei, while increasing levels of RIP1, RIP3, and necrotic OHCs. Conversely, treatment with necrosis inhibitor necrostatin-1 (Nec-1) or RIP3 siRNA (siRIP3) diminished noise-induced increases in RIP1 and RIP3, and decreased necrotic OHC nuclei. This treatment also increased the number of apoptotic nuclei without increasing activation of caspase-8. Consistent with the elevation of levels of RIP1 and RIP3, noise-induced active AMPKα levels increased with ZVAD treatment, but decreased with Nec-1 and siRIP3 treatment. Furthermore, treatment with siRIP3 did not alter the activation of caspase-8, but instead increased activation of caspase-9 and promoted endonuclease G translocation into OHC nuclei. Finally, auditory brainstem response functional measurements and morphological assessment of OHCs showed that ZVAD treatment reduces noise-induced deficits. This protective function is potentiated when combined with siRIP3 treatment. In conclusion, noise-induced OHC apoptosis and necrosis are modulated by caspases and RIP kinases, respectively. Inhibition of either pathway shifts the prevalence of OHC death to the alternative pathway. PMID:24874734

Protein Kinase Cδ Suppresses Autophagy to Induce Kidney Cell Apoptosis in Cisplatin Nephrotoxicity

PubMed Central

Pan, Jian; Xiang, Xudong; Liu, Yu; Dong, Guie; Livingston, Man J.; Chen, Jian-Kang; Yin, Xiao-Ming

2017-01-01

Nephrotoxicity is a major adverse effect in cisplatin chemotherapy, and renoprotective approaches are unavailable. Recent work unveiled a critical role of protein kinase Cδ (PKCδ) in cisplatin nephrotoxicity and further demonstrated that inhibition of PKCδ not only protects kidneys but enhances the chemotherapeutic effect of cisplatin in tumors; however, the underlying mechanisms remain elusive. Here, we show that cisplatin induced rapid activation of autophagy in cultured kidney tubular cells and in the kidneys of injected mice. Cisplatin also induced the phosphorylation of mammalian target of rapamycin (mTOR), p70S6 kinase downstream of mTOR, and serine/threonine-protein kinase ULK1, a component of the autophagy initiating complex. In vitro, pharmacologic inhibition of mTOR, directly or through inhibition of AKT, enhanced autophagy after cisplatin treatment. Notably, in both cells and kidneys, blockade of PKCδ suppressed the cisplatin-induced phosphorylation of AKT, mTOR, p70S6 kinase, and ULK1 resulting in upregulation of autophagy. Furthermore, constitutively active and inactive forms of PKCδ respectively enhanced and suppressed cisplatin-induced apoptosis in cultured cells. In mechanistic studies, we showed coimmunoprecipitation of PKCδ and AKT from lysates of cisplatin-treated cells and direct phosphorylation of AKT at serine-473 by PKCδ in vitro. Finally, administration of the PKCδ inhibitor rottlerin with cisplatin protected against cisplatin nephrotoxicity in wild-type mice, but not in renal autophagy–deficient mice. Together, these results reveal a pathway consisting of PKCδ, AKT, mTOR, and ULK1 that inhibits autophagy in cisplatin nephrotoxicity. PKCδ mediates cisplatin nephrotoxicity at least in part by suppressing autophagy, and accordingly, PKCδ inhibition protects kidneys by upregulating autophagy. PMID:27799485

Coordinated activation of AMP-activated protein kinase, extracellular signal-regulated kinase, and autophagy regulates phorbol myristate acetate-induced differentiation of SH-SY5Y neuroblastoma cells.

PubMed

Zogovic, Nevena; Tovilovic-Kovacevic, Gordana; Misirkic-Marjanovic, Maja; Vucicevic, Ljubica; Janjetovic, Kristina; Harhaji-Trajkovic, Ljubica; Trajkovic, Vladimir

2015-04-01

We explored the interplay between the intracellular energy sensor AMP-activated protein kinase (AMPK), extracellular signal-regulated kinase (ERK), and autophagy in phorbol myristate acetate (PMA)-induced neuronal differentiation of SH-SY5Y human neuroblastoma cells. PMA-triggered expression of neuronal markers (dopamine transporter, microtubule-associated protein 2, β-tubulin) was associated with an autophagic response, measured by the conversion of microtubule-associated protein light chain 3 (LC3)-I to autophagosome-bound LC3-II, increase in autophagic flux, and expression of autophagy-related (Atg) proteins Atg7 and beclin-1. This coincided with the transient activation of AMPK and sustained activation of ERK. Pharmacological inhibition or RNA interference-mediated silencing of AMPK suppressed PMA-induced expression of neuronal markers, as well as ERK activation and autophagy. A selective pharmacological blockade of ERK prevented PMA-induced neuronal differentiation and autophagy induction without affecting AMPK phosphorylation. Conversely, the inhibition of autophagy downstream of AMPK/ERK, either by pharmacological agents or LC3 knockdown, promoted the expression of neuronal markers, thus indicating a role of autophagy in the suppression of PMA-induced differentiation of SH-SY5Y cells. Therefore, PMA-induced neuronal differentiation of SH-SY5Y cells depends on a complex interplay between AMPK, ERK, and autophagy, in which the stimulatory effects of AMPK/ERK signaling are counteracted by the coinciding autophagic response. Phorbol myristate acetate (PMA) induces the expression of dopamine transporter, microtubule-associated protein 2, and β-tubulin, and subsequent neuronal differentiation of SH-SY5Y neuroblastoma cells through AMP-activated protein kinase (AMPK)-dependent activation of extracellular signal-regulated kinase (ERK). The activation of AMPK/ERK axis also induces the expression of beclin-1 and Atg7, and increases LC3 conversion, thereby triggering

Fed levels of amino acids are required for the somatotropin-induced increase in muscle protein synthesis

PubMed Central

Wilson, Fiona A.; Suryawan, Agus; Orellana, Renán A.; Nguyen, Hanh V.; Jeyapalan, Asumthia S.; Gazzaneo, Maria C.; Davis, Teresa A.

2008-01-01

Chronic somatotropin (pST) treatment in pigs increases muscle protein synthesis and circulating insulin, a known promoter of protein synthesis. Previously, we showed that the pST-mediated rise in insulin could not account for the pST-induced increase in muscle protein synthesis when amino acids were maintained at fasting levels. This study aimed to determine whether the pST-induced increase in insulin promotes skeletal muscle protein synthesis when amino acids are provided at fed levels and whether the response is associated with enhanced translation initiation factor activation. Growing pigs were treated with pST (0 or 180 μg·kg−1·day−1) for 7 days, and then pancreatic-glucose-amino acid clamps were performed. Amino acids were raised to fed levels in the presence of either fasted or fed insulin concentrations; glucose was maintained at fasting throughout. Muscle protein synthesis was increased by pST treatment and by amino acids (with or without insulin) (P < 0.001). In pST-treated pigs, fed, but not fasting, amino acid concentrations further increased muscle protein synthesis rates irrespective of insulin level (P < 0.02). Fed amino acids, with or without raised insulin concentrations, increased the phosphorylation of S6 kinase (S6K1) and eukaryotic initiation factor (eIF) 4E-binding protein 1 (4EBP1), decreased inactive 4EBP1·eIF4E complex association, and increased active eIF4E·eIF4G complex formation (P < 0.02). pST treatment did not alter translation initiation factor activation. We conclude that the pST-induced stimulation of muscle protein synthesis requires fed amino acid levels, but not fed insulin levels. However, under the current conditions, the response to amino acids is not mediated by the activation of translation initiation factors that regulate mRNA binding to the ribosomal complex. PMID:18682537

Protein kinases: mechanisms and downstream targets in inflammation-mediated obesity and insulin resistance.

PubMed

Nandipati, Kalyana C; Subramanian, Saravanan; Agrawal, Devendra K

2017-02-01

Obesity-induced low-grade inflammation (metaflammation) impairs insulin receptor signaling. This has been implicated in the development of insulin resistance. Insulin signaling in the target tissues is mediated by stress kinases such as p38 mitogen-activated protein kinase, c-Jun NH2-terminal kinase, inhibitor of NF-kB kinase complex β (IKKβ), AMP-activated protein kinase, protein kinase C, Rho-associated coiled-coil containing protein kinase, and RNA-activated protein kinase. Most of these kinases phosphorylate several key regulators in glucose homeostasis. The phosphorylation of serine residues in the insulin receptor and IRS-1 molecule results in diminished enzymatic activity in the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. This has been one of the key mechanisms observed in the tissues that are implicated in insulin resistance especially in type 2 diabetes mellitus (T2-DM). Identifying the specific protein kinases involved in obesity-induced chronic inflammation may help in developing the targeted drug therapies to minimize the insulin resistance. This review is focused on the protein kinases involved in the inflammatory cascade and molecular mechanisms and their downstream targets with special reference to obesity-induced T2-DM.

SH2/SH3 adaptor proteins can link tyrosine kinases to a Ste20-related protein kinase, HPK1.

PubMed

Anafi, M; Kiefer, F; Gish, G D; Mbamalu, G; Iscove, N N; Pawson, T

1997-10-31

Ste20-related protein kinases have been implicated as regulating a range of cellular responses, including stress-activated protein kinase pathways and the control of cytoskeletal architecture. An important issue involves the identities of the upstream signals and regulators that might control the biological functions of mammalian Ste20-related protein kinases. HPK1 is a protein-serine/threonine kinase that possesses a Ste20-like kinase domain, and in transfected cells activates a protein kinase pathway leading to the stress-activated protein kinase SAPK/JNK. Here we have investigated candidate upstream regulators that might interact with HPK1. HPK1 possesses an N-terminal catalytic domain and an extended C-terminal tail with four proline-rich motifs. The SH3 domains of Grb2 bound in vitro to specific proline-rich motifs in the HPK1 tail and functioned synergistically to direct the stable binding of Grb2 to HPK1 in transfected Cos1 cells. Epidermal growth factor (EGF) stimulation did not affect the binding of Grb2 to HPK1 but induced recruitment of the Grb2.HPK1 complex to the autophosphorylated EGF receptor and to the Shc docking protein. Several activated receptor and cytoplasmic tyrosine kinases, including the EGF receptor, stimulated the tyrosine phosphorylation of the HPK1 serine/threonine kinase. These results suggest that HPK1, a mammalian Ste20-related protein-serine/threonine kinase, can potentially associate with protein-tyrosine kinases through interactions mediated by SH2/SH3 adaptors such as Grb2. Such interaction may provide a possible mechanism for cross-talk between distinct biochemical pathways following the activation of tyrosine kinases.

Dermatophytes activate skin keratinocytes via mitogen-activated protein kinase signaling and induce immune responses.

PubMed

Achterman, Rebecca R; Moyes, David L; Thavaraj, Selvam; Smith, Adam R; Blair, Kris M; White, Theodore C; Naglik, Julian R

2015-04-01

Dermatophytes cause superficial and cutaneous fungal infections in immunocompetent hosts and invasive disease in immunocompromised hosts. However, the host mechanisms that regulate innate immune responses against these fungi are largely unknown. Here, we utilized commercially available epidermal tissues and primary keratinocytes to assess (i) damage induction by anthropophilic, geophilic, and zoophilic dermatophyte strains and (ii) the keratinocyte signaling pathways, transcription factors, and proinflammatory responses induced by a representative dermatophyte, Trichophyton equinum. Initially, five dermatophyte species were tested for their ability to invade, cause tissue damage, and induce cytokines, with Microsporum gypseum inducing the greatest level of damage and cytokine release. Using T. equinum as a representative dermatophyte, we found that the mitogen-activated protein kinase (MAPK) pathways were predominantly affected, with increased levels of phospho-p38 and phospho-Jun N-terminal protein kinase (JNK) but decreased levels of phospho-extracellular signal-regulated kinases 1 and 2 (ERK1/2). Notably, the NF-κB and PI3K pathways were largely unaffected. T. equinum also significantly increased expression of the AP-1-associated transcription factor, c-Fos, and the MAPK regulatory phosphatase, MKP1. Importantly, the ability of T. equinum to invade, cause tissue damage, activate signaling and transcription factors, and induce proinflammatory responses correlated with germination, indicating that germination may be important for dermatophyte virulence and host immune activation. Copyright © 2015, Achterman et al.

Activity of adenylyl cyclase and protein kinase A contributes to morphine-induced spinal apoptosis.

PubMed

Lim, Grewo; Wang, Shuxing; Lim, Jeong-Ae; Mao, Jianren

2005-12-02

Our previous study has shown that chronic morphine exposure induces neuronal apoptosis within the spinal cord dorsal horn; however, the mechanisms of morphine-induced apoptosis remain unclear. Here we examined whether adenylyl cyclase (AC) and protein kinase A (PKA) would play a role in this process. Intrathecal morphine regimen (10 microg, twice daily x 7 days) that resulted in antinociceptive tolerance induced spinal apoptosis as revealed by in situ terminal deoxynucleotidyl transferase (TdT)-UTP-biotin nick end labeling (TUNEL). The TUNEL-positive cells were detected primarily in the superficial laminae of the spinal cord dorsal horn, which was associated with an increase in the expression of activated caspase-3 and mitogen-activated protein kinase (MAPK) within the same spinal region. Co-administration of morphine with a broad AC inhibitor (ddA), a PKA inhibitor (H89), or a MAPK inhibitor (PD98059) substantially reduced the number of TUNEL-positive cells, as compared with the morphine alone group. The results indicate that the spinal AC and PKA pathway through intracellular MAPK may be contributory to the cellular mechanisms of morphine-induced apoptosis.

CIKS, a connection to IκB kinase and stress-activated protein kinase

PubMed Central

Leonardi, Antonio; Chariot, Alain; Claudio, Estefania; Cunningham, Kirk; Siebenlist, Ulrich

2000-01-01

Pathogens, inflammatory signals, and stress cause acute transcriptional responses in cells. The induced expression of genes in response to these signals invariably involves transcription factors of the NF-κB and AP-1/ATF families. Activation of NF-κB factors is thought to be mediated primarily via IκB kinases (IKK), whereas that of AP-1/ATF can be mediated by stress-activated protein kinases (SAPKs; also named Jun kinases or JNKs). IKKα and IKKβ are two catalytic subunits of a core IKK complex that also contains the regulatory subunit NEMO (NF-κB essential modulator)/IKKγ. The latter protein is essential for activation of the IKKs, but its mechanism of action is not known. Here we describe the molecular cloning of CIKS (connection to IKK and SAPK/JNK), a previously unknown protein that directly interacts with NEMO/IKKγ in cells. When ectopically expressed, CIKS stimulates IKK and SAPK/JNK kinases and it transactivates an NF-κB-dependent reporter. Activation of NF-κB is prevented in the presence of kinase-deficient, interfering mutants of the IKKs. CIKS may help to connect upstream signaling events to IKK and SAPK/JNK modules. CIKS could coordinate the activation of two stress-induced signaling pathways, functions reminiscent of those noted for tumor necrosis factor receptor-associated factor adaptor proteins. PMID:10962033

CIKS, a connection to Ikappa B kinase and stress-activated protein kinase.

PubMed

Leonardi, A; Chariot, A; Claudio, E; Cunningham, K; Siebenlist, U

2000-09-12

Pathogens, inflammatory signals, and stress cause acute transcriptional responses in cells. The induced expression of genes in response to these signals invariably involves transcription factors of the NF-kappaB and AP-1/ATF families. Activation of NF-kappaB factors is thought to be mediated primarily via IkappaB kinases (IKK), whereas that of AP-1/ATF can be mediated by stress-activated protein kinases (SAPKs; also named Jun kinases or JNKs). IKKalpha and IKKbeta are two catalytic subunits of a core IKK complex that also contains the regulatory subunit NEMO (NF-kappaB essential modulator)/IKKgamma. The latter protein is essential for activation of the IKKs, but its mechanism of action is not known. Here we describe the molecular cloning of CIKS (connection to IKK and SAPK/JNK), a previously unknown protein that directly interacts with NEMO/IKKgamma in cells. When ectopically expressed, CIKS stimulates IKK and SAPK/JNK kinases and it transactivates an NF-kappaB-dependent reporter. Activation of NF-kappaB is prevented in the presence of kinase-deficient, interfering mutants of the IKKs. CIKS may help to connect upstream signaling events to IKK and SAPK/JNK modules. CIKS could coordinate the activation of two stress-induced signaling pathways, functions reminiscent of those noted for tumor necrosis factor receptor-associated factor adaptor proteins.

The Antiviral Alkaloid Berberine Reduces Chikungunya Virus-Induced Mitogen-Activated Protein Kinase Signaling

PubMed Central

Thaa, Bastian; Amrun, Siti Naqiah; Simarmata, Diane; Rausalu, Kai; Nyman, Tuula A.; Merits, Andres; McInerney, Gerald M.; Ng, Lisa F. P.

2016-01-01

ABSTRACT Chikungunya virus (CHIKV) has infected millions of people in the tropical and subtropical regions since its reemergence in the last decade. We recently identified the nontoxic plant alkaloid berberine as an antiviral substance against CHIKV in a high-throughput screen. Here, we show that berberine is effective in multiple cell types against a variety of CHIKV strains, also at a high multiplicity of infection, consolidating the potential of berberine as an antiviral drug. We excluded any effect of this compound on virus entry or on the activity of the viral replicase. A human phosphokinase array revealed that CHIKV infection specifically activated the major mitogen-activated protein kinase (MAPK) signaling pathways extracellular signal-related kinase (ERK), p38 and c-Jun NH2-terminal kinase (JNK). Upon treatment with berberine, this virus-induced MAPK activation was markedly reduced. Subsequent analyses with specific inhibitors of these kinases indicated that the ERK and JNK signaling cascades are important for the generation of progeny virions. In contrast to specific MAPK inhibitors, berberine lowered virus-induced activation of all major MAPK pathways and resulted in a stronger reduction in viral titers. Further, we assessed the in vivo efficacy of berberine in a mouse model and measured a significant reduction of CHIKV-induced inflammatory disease. In summary, we demonstrate the efficacy of berberine as a drug against CHIKV and highlight the importance of the MAPK signaling pathways in the alphavirus infectious cycle. IMPORTANCE Chikungunya virus (CHIKV) is a mosquito-borne virus that causes severe and persistent muscle and joint pain and has recently spread to the Americas. No licensed drug exists to counter this virus. In this study, we report that the alkaloid berberine is antiviral against different CHIKV strains and in multiple human cell lines. We demonstrate that berberine collectively reduced the virus-induced activation of cellular mitogen

Protein kinases: mechanisms and downstream targets in inflammation mediated obesity and insulin resistance

PubMed Central

Nandipati, Kalyana C; Subramanian, Saravanan; Agrawal, Devendra K

2016-01-01

Obesity induced low-grade inflammation (metaflammation) impairs insulin receptor signaling (IRS). This has been implicated in the development of insulin resistance. Insulin signaling in the target tissues is mediated by stress kinases such as p38 mitogen-activated protein kinase (MAPK), c-Jun NH2-terminal kinase (JNK), inhibitor of NF-kB kinase complex beta (IKKβ), AMP activated protein kinase (AMPK), protein kinase C (PKC), Rho associated coiled-coil containing protein kinase (ROCK) and RNA-activated protein kinase (PKR), etc. Most of these kinases phosphorylate several key regulators in glucose homeostasis. The phosphorylation of serine residues in the insulin receptor (IR) and IRS-1 molecule results in diminished enzymatic activity in the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. This has been one of the key mechanisms observed in the tissues that are implicated in insulin resistance especially in Type II Diabetes Mellitus (T2-DM). Identifying the specific protein kinases involved in obesity induced chronic inflammation may help in developing the targeted drug therapies to minimize the insulin resistance. This review is focused on the protein kinases involved in the inflammatory cascade and molecular mechanisms and their downstream targets with special reference to obesity induced T2-DM. PMID:27868170

Protein Kinase G Induces an Immune Response in Cows Exposed to Mycobacterium avium Subsp. paratuberculosis

PubMed Central

Bach, Eviatar; Chaffer, Marcelo; Lai, Wanika; Keefe, Greg; Begg, Douglas J.

2018-01-01

To establish infection, pathogens secrete virulence factors, such as protein kinases and phosphatases, to modulate the signal transduction pathways used by host cells to initiate immune response. The protein MAP3893c is annotated in the genome sequence of Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of Johne's disease, as the serine/threonine protein kinase G (PknG). In this work, we report that PknG is a functional kinase that is secreted within macrophages at early stages of infection. The antigen is able to induce an immune response from cattle exposed to MAP in the form of interferon gamma production after stimulation of whole blood with PknG. These findings suggest that PknG may contribute to the pathogenesis of MAP by phosphorylating macrophage signalling and/or adaptor molecules as observed with other pathogenic mycobacterial species. PMID:29581962

Protein Kinase G Induces an Immune Response in Cows Exposed to Mycobacterium avium Subsp. paratuberculosis.

PubMed

Bach, Horacio; Richard-Greenblatt, Melissa; Bach, Eviatar; Chaffer, Marcelo; Lai, Wanika; Keefe, Greg; Begg, Douglas J

2018-01-01

To establish infection, pathogens secrete virulence factors, such as protein kinases and phosphatases, to modulate the signal transduction pathways used by host cells to initiate immune response. The protein MAP3893c is annotated in the genome sequence of Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of Johne's disease, as the serine/threonine protein kinase G (PknG). In this work, we report that PknG is a functional kinase that is secreted within macrophages at early stages of infection. The antigen is able to induce an immune response from cattle exposed to MAP in the form of interferon gamma production after stimulation of whole blood with PknG. These findings suggest that PknG may contribute to the pathogenesis of MAP by phosphorylating macrophage signalling and/or adaptor molecules as observed with other pathogenic mycobacterial species.

Escitalopram Ameliorates Tau Hyperphosphorylation and Spatial Memory Deficits Induced by Protein Kinase A Activation in Sprague Dawley Rats.

PubMed

Ren, Qing-Guo; Wang, Yan-Juan; Gong, Wei-Gang; Xu, Lin; Zhang, Zhi-Jun

2015-01-01

Here, we investigated the effect of escitalopram pretreatment on protein kinase A (PKA)-induced tau hyperphosphorylation and spatial memory deficits in rats using western blot and behavioral tests, respectively. We demonstrated that escitalopram effectively ameliorated tau hyperphosphorylation and the spatial memory deficits induced by PKA activation. We measured the total and activity-dependent Ser9-phosphorylated levels of glycogen synthase kinase (GSK)-3β in hippocampal extracts. No significant change in the total level of GSK-3β was observed between the different groups. However, compared with forskolin injection alone, pretreatment with escitalopram increased the level of Ser9-phosphorylated GSK-3β. We also demonstrated that escitalopram increased Akt phosphorylation at Ser473 (the active form of Akt). Furthermore, we identified other important kinases and phosphatases, such as protein phosphatase 2A, extracellular signal-regulated kinases 1 and 2, and MAP kinase kinase-1/2, that have previously been reported to play a crucial role in tau phosphorylation; however, we did not detect any significant change in the activation of these kinases or phosphatases in our study. We unexpectedly demonstrated that forskolin caused anxiety-like behavior in rats, and pretreatment with escitalopram did not significantly ameliorate the anxiety-like behavior induced by forskolin. These data provide the first evidence that escitalopram ameliorates forskolin-induced tau hyperphosphorylation and spatial memory impairment in rats; these effects do not occur via the anti-anxiety activity of escitalopram but may involve the Akt/GSK-3β signaling pathway.

Neutral endopeptidase promotes phorbol ester-induced apoptosis in prostate cancer cells by inhibiting neuropeptide-induced protein kinase C delta degradation.

PubMed

Sumitomo, M; Shen, R; Goldberg, J S; Dai, J; Navarro, D; Nanus, D M

2000-12-01

Phorbol esters induce apoptosis in androgen-sensitive LNCaP cells, which express neutral endopeptidase (NEP), but not in androgen-independent prostate cancer (PC) cells, which lack NEP expression. We investigated the role of NEP in PC cell susceptibility to 12-O-tetradecanoylphorbol-13-acetate (TPA). Western analysis showed that expression of NEP and protein kinase Cdelta (PKCdelta) correlated with PC cell sensitivity to TPA-induced growth arrest and apoptosis in LNCaP cells and in TSU-Prl cells expressing an inducible wild-type NEP protein. Inhibition of NEP enzyme activity using the specific NEP inhibitor CGS24592, or inhibition of PKCdelta using Rottlerin at concentrations that inhibit PKCdelta but not PKCalpha, significantly inhibited TPA-induced growth inhibition and cell death. Furthermore, pulse-chase experiments showed PKCdelta is stabilized in LNCaP cells and in TSU-Pr1 cells overexpressing wild-type NEP compared with PC cells lacking NEP expression. This results from NEP inactivation of its neuropeptide substrates (bombesin and endothelin-1), which in the absence of NEP stimulate cSrc kinase activity and induce rapid degradation of PKCdelta protein. These results indicate that expression of enzymatically active NEP by PC cells is necessary for TPA-induced apoptosis, and that NEP inhibits neuropeptide-induced, cSrc-mediated PKCdelta degradation.

Tonoplast-Bound Protein Kinase Phosphorylates Tonoplast Intrinsic Protein 1

PubMed Central

Johnson, Kenneth D.; Chrispeels, Maarten J.

1992-01-01

Tonoplast intrinsic protein (TIP) is a member of a family of putative membrane channels found in bacteria, animals, and plants. Plants have seed-specific, vegetative/reproductive organ-specific, and water-stress-induced forms of TIP. Here, we report that the seed-specific TIP is a phosphoprotein whose phosphorylation can be monitored in vivo by allowing bean cotyledons to take up [32P]orthophosphate and in vitro by incubating purified tonoplasts with γ-labeled [32P]ATP. Characterization of the in vitro phosphorylation of TIP indicates that a membrane-bound protein kinase phosphorylates TIP in a Ca2+-dependent manner. The capacity of the isolated tonoplast membranes to phosphorylate TIP declined markedly during seed germination, and this decline occurred well before the development-mediated decrease in TIP occurs. Phosphoamino acid analysis of purified, radiolabeled TIP showed that serine is the major, if not only, phosphorylated residue, and cyanogen bromide cleavage yielded a single radioactive peptide peak on a reverse-phase high-performance liquid chromatogram. Estimation of the molecular mass of the cyanogen bromide phosphopeptide by laser desorption mass spectroscopy led to its identification as the hydrophilic N-terminal domain of TIP. The putative phosphate-accepting serine residue occurs in a consensus phosphorylation site for serine/threonine protein kinases. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 PMID:16653198

The crystal structure of choline kinase reveals a eukaryotic protein kinase fold

DOE Office of Scientific and Technical Information (OSTI.GOV)

Peisach, D.; Gee, P.; Kent, K.

2010-03-08

Choline kinase catalyzes the ATP-dependent phosphorylation of choline, the first committed step in the CDP-choline pathway for the biosynthesis of phosphatidylcholine. The 2.0 {angstrom} crystal structure of a choline kinase from C. elegans (CKA-2) reveals that the enzyme is a homodimeric protein with each monomer organized into a two-domain fold. The structure is remarkably similar to those of protein kinases and aminoglycoside phosphotransferases, despite no significant similarity in amino acid sequence. Comparisons to the structures of other kinases suggest that ATP binds to CKA-2 in a pocket formed by highly conserved and catalytically important residues. In addition, a choline bindingmore » site is proposed to be near the ATP binding pocket and formed by several structurally flexible loops.« less

Regulation of basal gastric acid secretion by the glycogen synthase kinase GSK3.

PubMed

Rotte, Anand; Pasham, Venkanna; Eichenmüller, Melanie; Yang, Wenting; Qadri, Syed M; Bhandaru, Madhuri; Lang, Florian

2010-10-01

According to previous observations, basal gastric acid secretion is downregulated by phosphoinositol-3-(PI3)-kinase, phosphoinositide-dependent kinase (PDK1), and protein kinase B (PKBβ/Akt2) signaling. PKB/Akt phosphorylates glycogen synthase kinase GSK3. The present study explored whether PKB/Akt-dependent GSK3-phosphorylation modifies gastric acid secretion. Utilizing 2',7'-bis-(carboxyethyl)-5(6')-carboxyfluorescein (BCECF)-fluorescence, basal gastric acid secretion was determined from Na(+)-independent pH recovery (∆pH/min) following an ammonium pulse, which reflects H(+)/K(+)-ATPase activity. Experiments were performed in gastric glands from gene-targeted mice (gsk3 ( KI )) with PKB/serum and glucocorticoid-inducible kinase (SGK)-insensitive GSKα,β, in which the serines within the PKB/SGK phosphorylation site were replaced by alanine (GSK3α(21A/21A), GSK3β(9A/9A)). The cytosolic pH in isolated gastric glands was similar in gsk3 ( KI ) and their wild-type littermates (gsk3 ( WT )). However, ∆pH/min was significantly larger in gsk3 ( KI ) than in gsk3 ( WT ) mice and ∆pH/min was virtually abolished by the H(+)/K(+)-ATPase inhibitor omeprazole (100 μM) in gastric glands from both gsk3 ( KI ) and gsk3 ( WT ). Plasma gastrin levels were lower in gsk3 ( KI ) than in gsk3 ( WT ). Both, an increase of extracellular K(+) concentration to 35 mM [replacing Na(+)/N-methyl-D: -glucamine (NMDG)] and treatment with forskolin (5 μM), significantly increased ∆pH/min to virtually the same value in both genotypes. The protein kinase A (PKA) inhibitor H89 (150 nM) and the H(2)-receptor antagonist ranitidine (100 μM) decreased ∆pH/min in gsk3 ( KI ) but not gsk3 ( WT ) and again abrogated the differences between the genotypes. The protein abundance of phosphorylated but not of total PKA was significantly larger in gsk3 ( KI ) than in gsk3 ( WT ). Basal gastric acid secretion is enhanced by the disruption of PKB/SGK-dependent phosphorylation and the

Cyclic phosphatidic acid and lysophosphatidic acid induce hyaluronic acid synthesis via CREB transcription factor regulation in human skin fibroblasts.

PubMed

Maeda-Sano, Katsura; Gotoh, Mari; Morohoshi, Toshiro; Someya, Takao; Murofushi, Hiromu; Murakami-Murofushi, Kimiko

2014-09-01

Cyclic phosphatidic acid (cPA) is a naturally occurring phospholipid mediator and an analog of the growth factor-like phospholipid lysophosphatidic acid (LPA). cPA has a unique cyclic phosphate ring at the sn-2 and sn-3 positions of its glycerol backbone. We showed before that a metabolically stabilized cPA derivative, 2-carba-cPA, relieved osteoarthritis pathogenesis in vivo and induced hyaluronic acid synthesis in human osteoarthritis synoviocytes in vitro. This study focused on hyaluronic acid synthesis in human fibroblasts, which retain moisture and maintain health in the dermis. We investigated the effects of cPA and LPA on hyaluronic acid synthesis in human fibroblasts (NB1RGB cells). Using particle exclusion and enzyme-linked immunosorbent assays, we found that both cPA and LPA dose-dependently induced hyaluronic acid synthesis. We revealed that the expression of hyaluronan synthase 2 messenger RNA and protein is up-regulated by cPA and LPA treatment time dependently. We then characterized the signaling pathways up-regulating hyaluronic acid synthesis mediated by cPA and LPA in NB1RGB cells. Pharmacological inhibition and reporter gene assays revealed that the activation of the LPA receptor LPAR1, Gi/o protein, phosphatidylinositol-3 kinase (PI3K), extracellular-signal-regulated kinase (ERK), and cyclic adenosine monophosphate response element-binding protein (CREB) but not nuclear factor κB induced hyaluronic acid synthesis by the treatment with cPA and LPA in NB1RGB cells. These results demonstrate for the first time that cPA and LPA induce hyaluronic acid synthesis in human skin fibroblasts mainly through the activation of LPAR1-Gi/o followed by the PI3K, ERK, and CREB signaling pathway. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

Phorbol 12,13-dibutyrate-induced protein kinase C activation triggers sustained contracture in human myometrium in vitro.

PubMed

Massenavette, Laurence; Paul, Wilène; Corriveau, Stéphanie; Pasquier, Jean-Charles; Rousseau, Éric

2017-09-01

Although physiologic transition from rhythmic contractions to uterine retraction postpartum remains a poorly understood process, it has been shown that the latter is essential in the prevention of hemorrhage and its negative consequences. To investigate the transition from oscillatory contractions to tonic contracture in human myometrium after delivery, a mechanism purported to facilitate postpartum hemostasis. Protein kinase C (PKC) plays a key regulatory role in human uterine contractions because it can prevent dephosphorylation of regulatory proteins and sensitize the contractile machinery to low Ca 2+ . Thus, activation of PKC by phorbol 12,13-dibutyrate (PDBu) may act as a strong uterotonic agent. Uterine biopsies were obtained from consenting women undergoing elective caesarian delivery at term without labor (N = 19). Isometric tension measurements were performed on uterine strips (n = 114). The amplitudes and area under the curve of phasic contractions and tonic responses were measured and compared. A total of 1 μM PDBu was added to the isolated organ baths, and maximal tension of the uterine contracture was determined in the absence and presence of either 1 μM of staurosporine, 100 nM nifedipine, or 10 μM cyclopiazonic acid to assess the role of PKC and calcium sensitivity on uterine contractility. On the addition of PDBu on either basal or oxytocin-induced activity, consistent contractures were obtained concomitant with complete inhibition of phasic contractions. After a 30-minute incubation period, the mean amplitude of the PDBu-induced tone represented 65.3% of the amplitude of spontaneous contraction. Staurosporine, a protein kinase inhibitor, induced a 91.9% inhibition of PDBu contractures, a process not affected by nifedipine or cyclopiazonic acid, thus indicating that this mechanism is largely Ca 2+ independent. Pharmacologic activation of PKC leads to a significant contracture of the myometrium. Together, these data suggest that the up

Thrombin-induced p38 mitogen-activated protein kinase activation is mediated by epidermal growth factor receptor transactivation pathway

PubMed Central

Kanda, Yasunari; Mizuno, Katsushige; Kuroki, Yasutomi; Watanabe, Yasuhiro

2001-01-01

Thrombin is a potent mitogen for vascular smooth muscle cells (VSMC) and has been implicated its pathogenic role in vascular remodelling. However, the signalling pathways by which thrombin mediates its mitogenic response are not fully understood.We have previously reported that thrombin activates p38 mitogen-activated protein kinase (p38 MAPK) by a tyrosine kinase-dependent mechanism, and that p38 MAPK has a role in thrombin-induced mitogenic response in rat VSMC.In the present study, we examine the involvement of epidermal growth factor (EGF) receptor in thrombin-induced p38 MAPK activation. We found that thrombin induced EGF receptor tyrosine phosphorylation (transactivation) in A10 cells, a clonal VSMC cell line. A selective inhibitor of EGF receptor kinase (AG1478) inhibited the p38 MAPK activation in a dose-dependent manner, whereas it had no effect on the response to platelet-derived growth factor (PDGF). EGF receptor phosphorylation induced by thrombin was inhibited by BAPTA-AM and GF109203X, which suggest a requirement for intracellular Ca2+ increase and protein kinase C.We next examined the effect of AG1478 on thrombin-induced DNA synthesis. AG1478 inhibited thrombin-induced DNA synthesis in a dose-dependent manner. In contrast, PDGF-induced DNA synthesis was not affected by AG1478.In conclusion, these data suggest that the EGF receptor transactivation and subsequent p38 MAPK activation is required for thrombin-induced proliferation of VSMC. PMID:11309236

Cholecystokinin (CCK) stimulates S6 phosphorylation and induced activation of S6 protein kinase in rat pancreatic acini

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sung, C.; Okabayashi, Y.; Williams, J.

CCK and insulin stimulate pancreatic protein synthesis at a post transcriptional step. To better understand this regulation the authors evaluated the phosphorylation state of ribosomal protein S6 and the presence of a specific S6 protein kinase in pancreatic acini from diabetic rats. Both CCK and insulin increased S6 phosphorylation by up to 400% in intact TSP-labelled acini. The phorbol ester 12-0-tetradecanoylphorbol 13-acetate also stimulated both protein synthesis and S6 phosphorlyation suggesting a role for protein kinase C in mediating the effect of CCK. By contrast, the CaS ionophore ionomycin had no effect on either parameter. Recently, insulin has been shownmore » to activate a unique S6 kinase in various cells. To test for its presence, cytosolic extracts were prepared from acini stimulated with CCK and insulin by homogenization in US -glycerophosphate buffer and assayed for the kinase using el-TSP ATP and rat pancreatic ribosomes followed by SDS-polyacrylamide gel electrophoresis. CCK and insulin both increased S6 kinase activity which required neither CaS or phospholipid. The dose response for CCk was similar to S6 phosphorlyation in the intact acini. TPA did not stimulate the S6 kinase. Thus, CCK may induce S6 phosphorylation both via C kinase and by activation of a unique S6 kinase.« less

Cefradine blocks solar-ultraviolet induced skin inflammation through direct inhibition of T-LAK cell-originated protein kinase

PubMed Central

Ke, Changshu; Zhang, Guiping; Xiao, Juanjuan; Wu, Dan; Zeng, Xiaoyu; Chen, Jingwen; Guo, Jinguang; Zhou, Jie; Shi, Fei; Zhu, Feng

2016-01-01

Skin inflammation, and skin cancer induced by excessive solar ultraviolet (SUV) is a great threat to human health. SUV induced skin inflammation through activating p38 mitogen-activated protein kinase (p38) and c-Jun N-termeinal kinases (JNKs). T-LAK cell-originated protein kinase (TOPK) plays an important role in this process. Herein, the clinical data showed TOPK, phospho-p38, phospho-JNKs were highly expressed in human solar dermatitis. Ex vivo studies showed that SUV induced the phosphorylation of p38 and JNKs in HaCat and JB6 cells in a dose and time dependent manner. Molecule docking model indicated cefradine, an FDA-approved cephalosporin antibiotic, directly binds with TOPK. The result of in vitro binding assay verified cefradine can directly bind with TOPK. In vitro kinase results showed cefradine can inhibit TOPK activity. Ex vivo studies further showed cefradine inhibited SUV-induced the phosphorylation level of p38, JNKs and H2AX through inhibiting TOPK activity in a dose and time dependent manner, and cefradine inhibited the secretion of IL6 and TNF-α in HaCat and JB6 cells. In vivo studies showed that cefradine down-regulated SUV-induced the phosphorylation of p38, JNKs and H2AX and inhibited the secretion of IL6 and TNF-α in Babl/c mice. These results indicated that cefradine can inhibit SUV-induced skin inflammation by blocking TOPK signaling pathway, and TOPK is an effective target for suppressing inflammation induced by SUV irradiation. PMID:27016423

The involvement of protein kinase C-ε in isoflurane induced preconditioning of human embryonic stem cell--derived Nkx2.5(+) cardiac progenitor cells.

PubMed

Song, In-Ae; Oh, Ah-Young; Kim, Jin-Hee; Choi, Young-Min; Jeon, Young-Tae; Ryu, Jung-Hee; Hwang, Jung-Won

2016-02-20

Anesthetic preconditioning can improve survival of cardiac progenitor cells exposed to oxidative stress. We investigated the role of protein kinase C and isoform protein kinase C-ε in isoflurane-induced preconditioning of cardiac progenitor cells exposed to oxidative stress. Cardiac progenitor cells were obtained from undifferentiated human embryonic stem cells. Immunostaining with anti-Nkx2.5 was used to confirm the differentiated cardiac progenitor cells. Oxidative stress was induced by H2O2 and FeSO4. For anesthetic preconditioning, cardiac progenitor cells were exposed to 0.25, 0.5, and 1.0 mM of isoflurane. PMA and chelerythrine were used for protein kinase C activation and inhibition, while εψRACK and εV1-2 were used for protein kinase C -ε activation and inhibition, respectively. Isoflurane-preconditioning decreased the death rate of Cardiac progenitor cells exposed to oxidative stress (death rates isoflurane 0.5 mM 12.7 ± 9.3%, 1.0 mM 12.0 ± 7.7% vs. control 31.4 ± 10.2%). Inhibitors of both protein kinase C and protein kinase C -ε abolished the preconditioning effect of isoflurane 0.5 mM (death rates 27.6 ± 13.5% and 25.9 ± 8.7% respectively), and activators of both protein kinase C and protein kinase C - ε had protective effects from oxidative stress (death rates 16.0 ± 3.2% and 10.6 ± 3.8% respectively). Both PKC and PKC-ε are involved in isoflurane-induced preconditioning of human embryonic stem cells -derived Nkx2.5(+) Cardiac progenitor cells under oxidative stress.

Endothelin-1 activates p38 mitogen-activated protein kinase and cytosolic phospholipase A2 in cat iris sphincter smooth muscle cells.

PubMed

Husain, S; Abdel-Latif, A A

1999-08-15

We have shown previously that cytosolic phospholipase A(2) (cPLA(2)) is responsible for endothelin-1-induced release of arachidonic acid for prostaglandin synthesis in cat iris sphincter smooth muscle (CISM) cells [Husain and Abdel-Latif (1998) Biochim. Biophys. Acta 1392, 127-144]. Here we show that p38 mitogen-activated protein (MAP) kinase, but not p42/p44 MAP kinases, plays an important role in the phosphorylation and activation of cPLA(2) in endothelin-1-stimulated CISM cells. This conclusion is supported by the following findings. Both p38 MAP kinase and p42/p44 MAP kinases were present in the CISM cells and both were activated by endothelin-1. SB203580, a potent specific inhibitor of p38 MAP kinase, but not the p42/p44 MAP kinases specific inhibitor, PD98059, markedly suppressed endothelin-1-enhanced cPLA(2) phosphorylation, cPLA(2) activity and arachidonic acid release. The addition of endothelin-1 resulted in the phosphorylation and activation of cPLA(2). Endothelin-1 stimulated p38 MAP kinase activity in a time- and concentration-dependent manner, and these effects were mediated through the endothelin-A receptor subtype. The protein kinase C (PKC) inhibitor, RO 31-8220, had no inhibitory effect on endothelin-1-induced p38 MAP kinase activation, suggesting that endothelin-1 activation of p38 MAP kinase is independent of PKC. Pertussis toxin inhibited both endothelin-1 and mastoparan stimulation of p38 MAP kinase activity and arachidonic acid release. The inhibitory effects of pertussis toxin are not mediated through cAMP formation. Mastoparan-stimulated [(3)H]arachidonic acid release and cPLA(2) activation was inhibited by SB203580, but not by RO 31-8220. These data suggest that endothelin-1 binds to the endothelin-A receptor to activate the Gi-protein which, through a series of kinases, leads to the activation of p38 MAP kinase and subsequently to phosphorylation and activation of cPLA(2). Activation of cPLA(2) leads to the liberation of arachidonic acid

Endothelin-1 activates p38 mitogen-activated protein kinase and cytosolic phospholipase A2 in cat iris sphincter smooth muscle cells.

PubMed Central

Husain, S; Abdel-Latif, A A

1999-01-01

We have shown previously that cytosolic phospholipase A(2) (cPLA(2)) is responsible for endothelin-1-induced release of arachidonic acid for prostaglandin synthesis in cat iris sphincter smooth muscle (CISM) cells [Husain and Abdel-Latif (1998) Biochim. Biophys. Acta 1392, 127-144]. Here we show that p38 mitogen-activated protein (MAP) kinase, but not p42/p44 MAP kinases, plays an important role in the phosphorylation and activation of cPLA(2) in endothelin-1-stimulated CISM cells. This conclusion is supported by the following findings. Both p38 MAP kinase and p42/p44 MAP kinases were present in the CISM cells and both were activated by endothelin-1. SB203580, a potent specific inhibitor of p38 MAP kinase, but not the p42/p44 MAP kinases specific inhibitor, PD98059, markedly suppressed endothelin-1-enhanced cPLA(2) phosphorylation, cPLA(2) activity and arachidonic acid release. The addition of endothelin-1 resulted in the phosphorylation and activation of cPLA(2). Endothelin-1 stimulated p38 MAP kinase activity in a time- and concentration-dependent manner, and these effects were mediated through the endothelin-A receptor subtype. The protein kinase C (PKC) inhibitor, RO 31-8220, had no inhibitory effect on endothelin-1-induced p38 MAP kinase activation, suggesting that endothelin-1 activation of p38 MAP kinase is independent of PKC. Pertussis toxin inhibited both endothelin-1 and mastoparan stimulation of p38 MAP kinase activity and arachidonic acid release. The inhibitory effects of pertussis toxin are not mediated through cAMP formation. Mastoparan-stimulated [(3)H]arachidonic acid release and cPLA(2) activation was inhibited by SB203580, but not by RO 31-8220. These data suggest that endothelin-1 binds to the endothelin-A receptor to activate the Gi-protein which, through a series of kinases, leads to the activation of p38 MAP kinase and subsequently to phosphorylation and activation of cPLA(2). Activation of cPLA(2) leads to the liberation of arachidonic acid

Maintained activity of glycogen synthase kinase-3{beta} despite of its phosphorylation at serine-9 in okadaic acid-induced neurodegenerative model

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lim, Yong-Whan; Yoon, Seung-Yong, E-mail: ysy@amc.seoul.kr; Institute for Biomacromolecules, University of Ulsan College of Medicine, Seoul

2010-04-30

Glycogen synthase kinase-3{beta} (GSK3{beta}) is recognized as one of major kinases to phosphorylate tau in Alzheimer's disease (AD), thus lots of AD drug discoveries target GSK3{beta}. However, the inactive form of GSK3{beta} which is phosphorylated at serine-9 is increased in AD brains. This is also inconsistent with phosphorylation status of other GSK3{beta} substrates, such as {beta}-catenin and collapsin response mediator protein-2 (CRMP2) since their phosphorylation is all increased in AD brains. Thus, we addressed this paradoxical condition of AD in rat neurons treated with okadaic acid (OA) which inhibits protein phosphatase-2A (PP2A) and induces tau hyperphosphorylation and cell death. Interestingly,more » OA also induces phosphorylation of GSK3{beta} at serine-9 and other substrates including tau, {beta}-catenin and CRMP2 like in AD brains. In this context, we observed that GSK3{beta} inhibitors such as lithium chloride and 6-bromoindirubin-3'-monoxime (6-BIO) reversed those phosphorylation events and protected neurons. These data suggest that GSK3{beta} may still have its kinase activity despite increase of its phosphorylation at serine-9 in AD brains at least in PP2A-compromised conditions and that GSK3{beta} inhibitors could be a valuable drug candidate in AD.« less

Identification of proteins regulated by ferulic acid in a middle cerebral artery occlusion animal model-a proteomics approach.

PubMed

Sung, Jin-Hee; Cho, Eun-Hae; Cho, Jae-Hyeon; Won, Chung-Kil; Kim, Myeong-Ok; Koh, Phil-Ok

2012-11-01

Ferulic acid plays a neuroprotective role in cerebral ischemia. The aim of this study was to identify the proteins that are differentially expressed following ferulic acid treatment during ischemic brain injury using a proteomics technique. Middle cerebral artery occlusion (MCAO) was performed to induce a focal cerebral ischemic injury in adult male rats, and ferulic acid (100 mg/kg) or vehicle was administered immediately after MCAO. Brain tissues were collected 24 hr after MCAO. The proteins in the cerebral cortex were separated using two-dimensional gel electrophoresis and were identified by mass spectrometry. We detected differentially expressed proteins between vehicle- and ferulic acid-treated animals. Adenosylhomocysteinase, isocitrate dehydrogenase [NAD(+)], mitogen-activated protein kinase kinase 1 and glyceraldehyde-3-phosphate dehydrogenase were decreased in the vehicle-treated group, and ferulic acid prevented the injury-induced decreases in these proteins. However, pyridoxal phosphate phosphatase and heat shock protein 60 were increased in the vehicle-treated group, while ferulic acid prevented the injury-induced increase in these proteins. It is accepted that these enzymes are involved in cellular metabolism and differentiation. Thus, these findings suggest evidence that ferulic acid plays a neuroprotective role against focal cerebral ischemia through the up- and down-modulation of specific enzymes.

Regulation of Ca(2+)/calmodulin-dependent protein kinase kinase alpha by cAMP-dependent protein kinase: II. Mutational analysis.

PubMed

Kitani, T; Okuno, S; Fujisawa, H

2001-10-01

We previously reported that rat brain Ca(2+)/calmodulin-dependent protein kinase (CaM-kinase) IV is inactivated by cAMP-dependent protein kinase (PKA) [Kameshita, I. and Fujisawa, H. (1991) Biochem. Biophys. Res. Commun. 180, 191-196]. In the preceding paper, we demonstrated that changes in the activity of CaM-kinase IV by PKA results from the phosphorylation of CaM-kinase kinase alpha by PKA and identified six phosphorylation sites, Ser(24) for autophosphorylation, and Ser(52), Ser(74), Thr(108), Ser(458), and Ser(475) for phosphorylation by PKA. In the present study, a causal relationship between the phosphorylation and change in the activity toward PKIV peptide has been studied using mutant enzymes with amino acid substitutions at the six phosphorylation sites. The following conclusions can be drawn from the experimental results: (i) Phosphorylation of Ser74 and/or unidentified sites causes an increase in activity; (ii) phosphorylation of Thr(108) or Ser(458) causes a decrease in the activity; (iii) the inhibitory effect of the phosphorylation of Thr(108) is canceled by the stimulatory effect of the phosphorylation, but that of Ser(458) is not; and (iv) the inhibitory effects of Thr(108) and Ser(458) are synergistic. In contrast to the activity toward PKIV peptide, the activity toward CaM-kinase IV appears to be decreased by the phosphorylation of Thr(108), but not significantly affected by the phosphorylation of Ser(458).

Phosphorylation of the Yeast Choline Kinase by Protein Kinase C

PubMed Central

Choi, Mal-Gi; Kurnov, Vladlen; Kersting, Michael C.; Sreenivas, Avula; Carman, George M.

2005-01-01

The Saccharomyces cerevisiae CKI1-encoded choline kinase catalyzes the committed step in phosphatidylcholine synthesis via the Kennedy pathway. The enzyme is phosphorylated on multiple serine residues, and some of this phosphorylation is mediated by protein kinase A. In this work, we examined the hypothesis that choline kinase is also phosphorylated by protein kinase C. Using choline kinase as a substrate, protein kinase C activity was dose- and time-dependent, and dependent on the concentrations of choline kinase (Km = 27 μg/ml) and ATP (Km = 15 μM). This phosphorylation, which occurred on a serine residue, was accompanied by a 1.6-fold stimulation of choline kinase activity. The synthetic peptide SRSSS25QRRHS (Vmax/Km = 17.5 mM-1 μmol min-1 mg-1) that contains the protein kinase C motif for Ser25 was a substrate for protein kinase C. A Ser25 to Ala (S25A) mutation in choline kinase resulted in a 60% decrease in protein kinase C phosphorylation of the enzyme. Phosphopeptide mapping analysis of the S25A mutant enzyme confirmed that Ser25 was a protein kinase C target site. In vivo, the S25A mutation correlated with a decrease (55%) in phosphatidylcholine synthesis via the Kennedy pathway whereas an S25D phosphorylation site mimic correlated with an increase (44%) in phosphatidylcholine synthesis. Whereas the S25A (protein kinase C site) mutation did not affect the phosphorylation of choline kinase by protein kinase A, the S30A (protein kinase A site) mutation caused a 46% reduction in enzyme phosphorylation by protein kinase C. A choline kinase synthetic peptide (SQRRHS30LTRQ) containing Ser30 was a substrate (Vmax/Km = 3.0 mM−1 μmol min−1 mg−1) for protein kinase C. Comparison of phosphopeptide maps of the wild type and S30A mutant choline kinase enzymes phosphorylated by protein kinase C confirmed that Ser30 was also a target site for protein kinase C. PMID:15919656

Calcium Channels, Rho-Kinase, Protein Kinase-C, and Phospholipase-C Pathways Mediate Mercury Chloride-Induced Myometrial Contractions in Rats.

PubMed

Koli, Swati; Prakash, Atul; Choudhury, Soumen; Mandil, Rajesh; Garg, Satish K

2018-05-21

Adverse effects of mercury on female reproduction are reported; however, its effect on myogenic activity of uterus and mechanism thereof is obscure. Present study was undertaken to unravel the mechanistic pathways of mercuric chloride (HgCl 2 )-induced myometrial contraction in rats. Isometric tension in myometrial strips of rats following in vitro exposure to HgCl 2 was recorded using data acquisition system-based physiograph. HgCl 2 produced concentration-dependent (10 nM-100 μM) uterotonic effect which was significantly (p < 0.05) reduced in Ca 2+ -free solution and inhibited in the presence of nifedipine (1 μM), a L-type Ca 2+ channel blocker, thus suggesting the importance of extracellular Ca 2+ and its entry through L-type calcium channels in HgCl 2 -induced myometrial contractions in rats. Cumulative concentration-response curve of HgCl 2 was significantly (p < 0.05) shifted towards right in the presence of Y-27632 (10 μM), a Rho-kinase inhibitor, suggesting the involvement of Ca 2+ -sensitization pathway in mediating HgCl 2 -induced myometrial contraction. HgCl 2 -induced myometrial contraction was also significantly (p < 0.05) inhibited in the presence of methoctramine or para-fluoro-hexahydro-siladifenidol, a selective M 2 and M 3 receptor antagonists, respectively, which evidently suggest that mercury also interacts with M 2 and M 3 muscarinic receptors to produce myometrial contractions. U-73122 and GF-109203X, the respective inhibitors of PLC and PKC-dependent pathways, downstream to the receptor activation, also significantly (p < 0.05) attenuated the uterotonic effect of HgCl 2 on rat uterus. Taken together, present study evidently reveals that HgCl 2 interacts with muscarinic receptors and activates calcium signaling cascades involving calcium channels, Rho-kinase, protein kinase-C, and phospholipase-C pathways to exert uterotonic effect in rats. Graphical Abstract Graphical abstract depicting the mechanism of mercury-induced

β-Guanidinopropionic acid extends the lifespan of Drosophila melanogaster via an AMP-activated protein kinase-dependent increase in autophagy.

PubMed

Yang, Si; Long, Li-Hong; Li, Di; Zhang, Jian-Kang; Jin, Shan; Wang, Fang; Chen, Jian-Guo

2015-12-01

Previous studies have demonstrated that AMP-activated protein kinase (AMPK) controls autophagy through the mammalian target of rapamycin (mTOR) and Unc-51 like kinase 1 (ULK1/Atg1) signaling, which augments the quality of cellular housekeeping, and that β-guanidinopropionic acid (β-GPA), a creatine analog, leads to a chronic activation of AMPK. However, the relationship between β-GPA and aging remains elusive. In this study, we hypothesized that feeding β-GPA to adult Drosophila produces the lifespan extension via activation of AMPK-dependent autophagy. It was found that dietary administration of β-GPA at a concentration higher than 900 mm induced a significant extension of the lifespan of Drosophila melanogaster in repeated experiments. Furthermore, we found that Atg8 protein, the homolog of microtubule-associated protein 1A/1B-light chain 3 (LC3) and a biomarker of autophagy in Drosophila, was significantly upregulated by β-GPA treatment, indicating that autophagic activity plays a role in the effect of β-GPA. On the other hand, when the expression of Atg5 protein, an essential protein for autophagy, was reduced by RNA interference (RNAi), the effect of β-GPA on lifespan extension was abolished. Moreover, we found that AMPK was also involved in this process. β-GPA treatment significantly elevated the expression of phospho-T172-AMPK levels, while inhibition of AMPK by either AMPK-RNAi or compound C significantly attenuated the expression of autophagy-related proteins and lifespan extension in Drosophila. Taken together, our results suggest that β-GPA can induce an extension of the lifespan of Drosophila via AMPK-Atg1-autophagy signaling pathway. © 2015 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

Identification and functional characterization of the Arabidopsis Snf1-related protein kinase SnRK2.4 phosphatidic acid-binding domain.

PubMed

Julkowska, Magdalena M; McLoughlin, Fionn; Galvan-Ampudia, Carlos S; Rankenberg, Johanna M; Kawa, Dorota; Klimecka, Maria; Haring, Michel A; Munnik, Teun; Kooijman, Edgar E; Testerink, Christa

2015-03-01

Phosphatidic acid (PA) is an important signalling lipid involved in various stress-induced signalling cascades. Two SnRK2 protein kinases (SnRK2.4 and SnRK2.10), previously identified as PA-binding proteins, are shown here to prefer binding to PA over other anionic phospholipids and to associate with cellular membranes in response to salt stress in Arabidopsis roots. A 42 amino acid sequence was identified as the primary PA-binding domain (PABD) of SnRK2.4. Unlike the full-length SnRK2.4, neither the PABD-YFP fusion protein nor the SnRK2.10 re-localized into punctate structures upon salt stress treatment, showing that additional domains of the SnRK2.4 protein are required for its re-localization during salt stress. Within the PABD, five basic amino acids, conserved in class 1 SnRK2s, were found to be necessary for PA binding. Remarkably, plants overexpressing the PABD, but not a non-PA-binding mutant version, showed a severe reduction in root growth. Together, this study biochemically characterizes the PA-SnRK2.4 interaction and shows that functionality of the SnRK2.4 PABD affects root development. © 2014 The Authors. Plant, Cell & Environment published by John Wiley & Sons Ltd.

14-3-3 proteins mediate inhibitory effects of cAMP on salt-inducible kinases (SIKs).

PubMed

Sonntag, Tim; Vaughan, Joan M; Montminy, Marc

2018-02-01

The salt-inducible kinase (SIK) family regulates cellular gene expression via the phosphorylation of cAMP-regulated transcriptional coactivators (CRTCs) and class IIA histone deacetylases, which are sequestered in the cytoplasm by phosphorylation-dependent 14-3-3 interactions. SIK activity toward these substrates is inhibited by increases in cAMP signaling, although the underlying mechanism is unclear. Here, we show that the protein kinase A (PKA)-dependent phosphorylation of SIKs inhibits their catalytic activity by inducing 14-3-3 protein binding. SIK1 and SIK3 contain two functional PKA/14-3-3 sites, while SIK2 has four. In keeping with the dimeric nature of 14-3-3s, the presence of multiple binding sites within target proteins dramatically increases binding affinity. As a result, loss of a single 14-3-3-binding site in SIK1 and SIK3 abolished 14-3-3 association and rendered them insensitive to cAMP. In contrast, mutation of three sites in SIK2 was necessary to fully block cAMP regulation. Superimposed on the effects of PKA phosphorylation and 14-3-3 association, an evolutionary conserved domain in SIK1 and SIK2 (the so called RK-rich region; 595-624 in hSIK2) is also required for the inhibition of SIK2 activity. Collectively, these results point to a dual role for 14-3-3 proteins in repressing a family of Ser/Thr kinases as well as their substrates. © 2017 Federation of European Biochemical Societies.

Identification of a two-component fatty acid kinase responsible for host fatty acid incorporation by Staphylococcus aureus

PubMed Central

Parsons, Joshua B.; Broussard, Tyler C.; Bose, Jeffrey L.; Rosch, Jason W.; Jackson, Pamela; Subramanian, Chitra; Rock, Charles O.

2014-01-01

Extracellular fatty acid incorporation into the phospholipids of Staphylococcus aureus occurs via fatty acid phosphorylation. We show that fatty acid kinase (Fak) is composed of two dissociable protein subunits encoded by separate genes. FakA provides the ATP binding domain and interacts with two distinct FakB proteins to produce acyl-phosphate. The FakBs are fatty acid binding proteins that exchange bound fatty acid/acyl-phosphate with fatty acid/acyl-phosphate presented in detergent micelles or liposomes. The ΔfakA and ΔfakB1 ΔfakB2 strains were unable to incorporate extracellular fatty acids into phospholipid. FakB1 selectively bound saturated fatty acids whereas FakB2 preferred unsaturated fatty acids. Affymetrix array showed a global perturbation in the expression of virulence genes in the ΔfakA strain. The severe deficiency in α-hemolysin protein secretion in ΔfakA and ΔfakB1 ΔfakB2 mutants coupled with quantitative mRNA measurements showed that fatty acid kinase activity was required to support virulence factor transcription. These data reveal the function of two conserved gene families, their essential role in the incorporation of host fatty acids by Gram-positive pathogens, and connects fatty acid kinase to the regulation of virulence factor transcription in S. aureus. PMID:25002480

Phosphorylation of 1-Aminocyclopropane-1-Carboxylic Acid Synthase by MPK6, a Stress-Responsive Mitogen-Activated Protein Kinase, Induces Ethylene Biosynthesis in ArabidopsisW⃞

PubMed Central

Liu, Yidong; Zhang, Shuqun

2004-01-01

Mitogen-activated protein kinases (MAPKs) are implicated in regulating plant growth, development, and response to the environment. However, the underlying mechanisms are unknown because of the lack of information about their substrates. Using a conditional gain-of-function transgenic system, we demonstrated that the activation of SIPK, a tobacco (Nicotiana tabacum) stress-responsive MAPK, induces the biosynthesis of ethylene. Here, we report that MPK6, the Arabidopsis thaliana ortholog of tobacco SIPK, is required for ethylene induction in this transgenic system. Furthermore, we found that selected isoforms of 1-aminocyclopropane-1-carboxylic acid synthase (ACS), the rate-limiting enzyme of ethylene biosynthesis, are substrates of MPK6. Phosphorylation of ACS2 and ACS6 by MPK6 leads to the accumulation of ACS protein and, thus, elevated levels of cellular ACS activity and ethylene production. Expression of ACS6DDD, a gain-of-function ACS6 mutant that mimics the phosphorylated form of ACS6, confers constitutive ethylene production and ethylene-induced phenotypes. Increasing numbers of stress stimuli have been shown to activate Arabidopsis MPK6 or its orthologs in other plant species. The identification of the first plant MAPK substrate in this report reveals one mechanism by which MPK6/SIPK regulates plant stress responses. Equally important, this study uncovers a signaling pathway that modulates the biosynthesis of ethylene, an important plant hormone, in plants under stress. PMID:15539472

Saw palmetto extract suppresses insulin-like growth factor-I signaling and induces stress-activated protein kinase/c-Jun N-terminal kinase phosphorylation in human prostate epithelial cells.

PubMed

Wadsworth, Teri L; Carroll, Julie M; Mallinson, Rebecca A; Roberts, Charles T; Roselli, Charles E

2004-07-01

A common alternative therapy for benign prostatic hyperplasia (BPH) is the extract from the fruit of saw palmetto (SPE). BPH is caused by nonmalignant growth of epithelial and stromal elements of the prostate. IGF action is important for prostate growth and development, and changes in the IGF system have been documented in BPH tissues. The main signaling pathways activated by the binding of IGF-I to the IGF-I receptor (IGF-IR) are the ERK arm of the MAPK cascade and the phosphoinositol-3-kinase (PI3K)/protein kinase B (PKB/Akt) cascade. We tested the hypothesis that SPE suppresses growth and induces apoptosis in the P69 prostate epithelial cell line by inhibiting IGF-I signaling. Treatment with 150 microg/ml SPE for 24 h decreased IGF-I-induced proliferation of P69 cells and induced cleavage of the enzyme poly(ADP-ribose)polymerase (PARP), an index of apoptosis. Treatment of serum-starved P69 cells with 150 microg/ml SPE for 6 h reduced IGF-I-induced phosphorylation of Akt (assessed by Western blot) and Akt activity (assessed by an Akt kinase assay). Western blot analysis showed that SPE reduced IGF-I-induced phosphorylation of the adapter protein insulin receptor substrate-1 and decreased downstream effects of Akt activation, including increased cyclin D1 levels and phosphorylation of glycogen synthase kinase-3 and p70(s6k). There was no effect on IGF-I-induced phosphorylation of MAPK, IGF-IR, or Shc. Treatment of starved cells with SPE alone induced phosphorylation the proapoptotic protein JNK. SPE treatment may relieve symptoms of BPH, in part, by inhibiting specific components of the IGF-I signaling pathway and inducing JNK activation, thus mediating antiproliferative and proapoptotic effects on prostate epithelia.

Cyclophilin B induces integrin-mediated cell adhesion by a mechanism involving CD98-dependent activation of protein kinase C-delta and p44/42 mitogen-activated protein kinases.

PubMed

Melchior, Aurélie; Denys, Agnès; Deligny, Audrey; Mazurier, Joël; Allain, Fabrice

2008-02-01

Initially identified as a cyclosporin-A binding protein, cyclophilin B (CyPB) is an inflammatory mediator that induces adhesion of T lymphocytes to fibronectin, by a mechanism dependent on CD147 and alpha 4 beta 1 integrins. Recent findings have suggested that another cell membrane protein, CD98, may cooperate with CD147 to regulate beta1 integrin functions. Based on these functional relationships, we examined the contribution of CD98 in the pro-adhesive activity of CyPB, by utilizing the responsive promonocyte cell line THP-1. We demonstrated that cross-linking CD98 with CD98-AHN-18 antibody mimicked the responses induced by CyPB, i.e. homotypic aggregation, integrin-mediated adhesion to fibronectin and activation of p44/42 MAPK. Consistent with previous data, immunoprecipitation confirmed the existence of a heterocomplex wherein CD147, CD98 and beta1 integrins were associated. We then demonstrated that CyPB-induced cell adhesion and p44/42 MAPK activation were dependent on the participation of phosphoinositide 3-kinase and subsequent activation of protein kinase C-delta. Finally, silencing the expression of CD98 by RNA interference potently reduced CyPB-induced cell responses, thus confirming the role of CD98 in the pro-adhesive activity of CyPB. Altogether, our results support a model whereby CyPB induces integrin-mediated adhesion via interaction with a multimolecular unit formed by the association between CD147, CD98 and beta1 integrins.

Protein Kinase B Activation and Lamellipodium Formation Are Independent Phosphoinositide 3-Kinase-Mediated Events Differentially Regulated by Endogenous Ras

PubMed Central

van Weering, David H. J.; de Rooij, Johan; Marte, Barbara; Downward, Julian; Bos, Johannes L.; Burgering, Boudewijn M. T.

1998-01-01

Regulation of phosphoinositide 3-kinase (PI 3-kinase) can occur by binding of the regulatory p85 subunit to tyrosine-phosphorylated proteins and by binding of the p110 catalytic subunit to activated Ras. However, the way in which these regulatory mechanisms act to regulate PI 3-kinase in vivo is unclear. Here we show that several growth factors (basic fibroblast growth factor [bFGF], platelet-derived growth factor [PDGF], and epidermal growth factor [EGF; to activate an EGF receptor-Ret chimeric receptor]) all activate PI 3-kinase in vivo in the neuroectoderm-derived cell line SKF5. However, these growth factors differ in their ability to activate PI 3-kinase-dependent signaling. PDGF and EGF(Ret) treatment induced PI 3-kinase-dependent lamellipodium formation and protein kinase B (PKB) activation. In contrast, bFGF did not induce lamellipodium formation but activated PKB, albeit to a small extent. PDGF and EGF(Ret) stimulation resulted in binding of p85 to tyrosine-phosphorylated proteins and strong Ras activation. bFGF, however, induced only strong activation of Ras. In addition, while RasAsn17 abolished bFGF activation of PKB, PDGF- and EGF(Ret)-induced PKB activation was only partially inhibited and lamellipodium formation was unaffected. Interestingly, in contrast to activation of only endogenous Ras (bFGF), ectopic expression of activated Ras did result in lamellipodium formation. From this we conclude that, in vivo, p85 and Ras synergize to activate PI 3-kinase and that strong activation of only endogenous Ras exerts a small effect on PI 3-kinase activity, sufficient for PKB activation but not lamellipodium formation. This differential sensitivity to PI 3-kinase activation could be explained by our finding that PKB activation and lamellipodium formation are independent PI 3-kinase-induced events. PMID:9528752

Methylation of eukaryotic elongation factor 2 induced by basic fibroblast growth factor via mitogen-activated protein kinase.

PubMed

Jung, Gyung Ah; Shin, Bong Shik; Jang, Yeon Sue; Sohn, Jae Bum; Woo, Seon Rang; Kim, Jung Eun; Choi, Go; Lee, Kyung Mi; Min, Bon Hong; Lee, Kee Ho; Park, Gil Hong

2011-10-31

Protein arginine methylation is important for a variety of cellular processes including transcriptional regulation, mRNA splicing, DNA repair, nuclear/cytoplasmic shuttling and various signal transduction pathways. However, the role of arginine methylation in protein biosynthesis and the extracellular signals that control arginine methylation are not fully understood. Basic fibroblast growth factor (bFGF) has been identified as a potent stimulator of myofibroblast dedifferentiation into fibroblasts. We demonstrated that symmetric arginine dimethylation of eukaryotic elongation factor 2 (eEF2) is induced by bFGF without the change in the expression level of eEF2 in mouse embryo fibroblast NIH3T3 cells. The eEF2 methylation is preceded by ras-raf-mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK1/2)- p21Cip/WAF1 activation, and suppressed by the mitogenactivated protein kinase (MAPK) inhibitor PD98059 and p21Cip/WAF1 short interfering RNA (siRNA). We determined that protein arginine methyltransferase 7 (PRMT7) is responsible for the methylation, and that PRMT5 acts as a coordinator. Collectively, we demonstrated that eEF2, a key factor involved in protein translational elongation is symmetrically arginine-methylated in a reversible manner, being regulated by bFGF through MAPK signaling pathway.

Methylation of eukaryotic elongation factor 2 induced by basic fibroblast growth factor via mitogen-activated protein kinase

PubMed Central

Jung, Gyung Ah; Shin, Bong Shik; Jang, Yeon Sue; Sohn, Jae Bum; Woo, Seon Rang; Kim, Jung Eun; Choi, Go; Lee, Kyung-Mi; Min, Bon Hong

2011-01-01

Protein arginine methylation is important for a variety of cellular processes including transcriptional regulation, mRNA splicing, DNA repair, nuclear/cytoplasmic shuttling and various signal transduction pathways. However, the role of arginine methylation in protein biosynthesis and the extracellular signals that control arginine methylation are not fully understood. Basic fibroblast growth factor (bFGF) has been identified as a potent stimulator of myofibroblast dedifferentiation into fibroblasts. We demonstrated that symmetric arginine dimethylation of eukaryotic elongation factor 2 (eEF2) is induced by bFGF without the change in the expression level of eEF2 in mouse embryo fibroblast NIH3T3 cells. The eEF2 methylation is preceded by ras-raf-mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK1/2)-p21Cip/WAF1 activation, and suppressed by the mitogen-activated protein kinase (MAPK) inhibitor PD98059 and p21Cip/WAF1 short interfering RNA (siRNA). We determined that protein arginine methyltransferase 7 (PRMT7) is responsible for the methylation, and that PRMT5 acts as a coordinator. Collectively, we demonstrated that eEF2, a key factor involved in protein translational elongation is symmetrically arginine-methylated in a reversible manner, being regulated by bFGF through MAPK signaling pathway. PMID:21778808

Valproate induced hepatic steatosis by enhanced fatty acid uptake and triglyceride synthesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bai, Xupeng; Hong, Weipeng; Cai, Peiheng

Steatosis is the characteristic type of VPA-induced hepatotoxicity and may result in life-threatening hepatic lesion. Approximately 61% of patients treated with VPA have been diagnosed with hepatic steatosis through ultrasound examination. However, the mechanisms underlying VPA-induced intracellular fat accumulation are not yet fully understood. Here we demonstrated the involvement of fatty acid uptake and lipogenesis in VPA-induced hepatic steatosis in vitro and in vivo by using quantitative real-time PCR (qRT-PCR) analysis, western blotting analysis, fatty acid uptake assays, Nile Red staining assays, and Oil Red O staining assays. Specifically, we found that the expression of cluster of differentiation 36 (CD36),more » an important fatty acid transport, and diacylglycerol acyltransferase 2 (DGAT2) were significantly up-regulated in HepG2 cells and livers of C57B/6J mice after treatment with VPA. Furthermore, VPA treatment remarkably enhanced the efficiency of fatty acid uptake mediated by CD36, while this effect was abolished by the interference with CD36-specific siRNA. Also, VPA treatment significantly increased DGAT2 expression as a result of the inhibition of mitogen-activated protein kinase kinase (MEK) – extracellular regulated kinase (ERK) pathway; however, DGAT2 knockdown significantly alleviated VPA-induced intracellular lipid accumulation. Additionally, we also found that sterol regulatory element binding protein-1c (SREBP-1c)-mediated fatty acid synthesis may be not involved in VPA-induced hepatic steatosis. Overall, VPA-triggered over-regulation of CD36 and DGAT2 could be helpful for a better understanding of the mechanisms underlying VPA-induced hepatic steatosis and may offer novel therapeutic strategies to combat VPA-induced hepatotoxicity. - Highlights: • VPA induced hepatic steatosis and modulated genes associated with lipid metabolism. • CD36-mediated fatty acid uptake contributed to VPA-induced lipid accumulation. • PA increased the

Spermidine-Induced Improvement of Reconsolidation of Memory Involves Calcium-Dependent Protein Kinase in Rats

ERIC Educational Resources Information Center

Girardi, Bruna Amanda; Ribeiro, Daniela Aymone; Signor, Cristiane; Muller, Michele; Gais, Mayara Ana; Mello, Carlos Fernando; Rubin, Maribel Antonello

2016-01-01

In this study, we determined whether the calcium-dependent protein kinase (PKC) signaling pathway is involved in the improvement of fear memory reconsolidation induced by the intrahippocampal administration of spermidine in rats. Male Wistar rats were trained in a fear conditioning apparatus using a 0.4-mA footshock as an unconditioned stimulus.…

Formononetin-induced apoptosis of human prostate cancer cells through ERK1/2 mitogen-activated protein kinase inactivation.

PubMed

Ye, Y; Hou, R; Chen, J; Mo, L; Zhang, J; Huang, Y; Mo, Z

2012-04-01

Formononetin is a main active component of red clover plants (Trifolium pratense L.), and is considered as a phytoestrogen. Our previous studies demonstrated that formononetin caused cell cycle arrest at the G0/G1 phase by inactivating insulin-like growth factor 1(IGF1)/IGF1R-phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway in MCF-7 cells. In the present study, we investigated the molecular mechanisms involved in the effect of formononetin on prostate cancer cells. Our results suggested that higher concentrations of formononetin inhibited the proliferation of prostate cancer cells (LNCaP and PC-3), while the most striking effect was observed in LNCaP cells. We further found that formononetin inactivated extracellular signal-regulated kinase1/2 (ERK1/2) mitogen-activated protein kinase (MAPK) signaling pathway in a dose-dependent manner, which resulted in increased the expression levels of BCL2-associated X (Bax) mRNA and protein, and induced apoptosis in LNCaP cells. Thus, we concluded that the induced apoptosis effect of formononetin on human prostate cancer cells was related to ERK1/2 MAPK-Bax pathway. Considering that red clover plants were widely used clinically, our results provided the foundation for future development of different concentrations formononetin for treatment of prostate cancer. © Georg Thieme Verlag KG Stuttgart · New York.

Activated STAT5 proteins induce activation of the PI 3-kinase/Akt and Ras/MAPK pathways via the Gab2 scaffolding adapter.

PubMed

Nyga, Rémy; Pecquet, Christian; Harir, Noria; Gu, Haihua; Dhennin-Duthille, Isabelle; Régnier, Aline; Gouilleux-Gruart, Valérie; Lassoued, Kaïss; Gouilleux, Fabrice

2005-08-15

The active forms of STAT5A (signal transducer and activator of transcription 5A) and STAT5B are able to relieve the cytokine dependence of haematopoietic cells and to induce leukaemia in mice. We have demonstrated previously that activation of the PI3K (phosphoinositide 3-kinase) signalling cascade plays a major role in cell growth and survival induced by these proteins. Interaction between STAT5 and p85, the regulatory subunit of the PI3K, has been suggested to be required for this activation. We show in the present study that the scaffolding protein Gab2 [Grb2 (growth-factor-receptor-bound protein 2)-associated binder-2] is an essential component of this interaction. Gab2 is persistently tyrosine-phosphorylated in Ba/F3 cells expressing caSTAT5 (constitutively activated STAT5), independent of JAK2 (Janus kinase 2) activation where it interacts with STAT5, p85 and Grb2, but not with Shp2 [SH2 (Src homology 2)-domain-containing tyrosine phosphatase] proteins. Interaction of STAT5 with Gab2 was also observed in Ba/F3 cells stimulated with interleukin-3 or expressing the oncogenic fusion protein Tel-JAK2. The MAPKs (mitogen-activated protein kinases) ERK1 (extracellular-signal-regulated kinase 1) and ERK2 were constitutively activated in the caSTAT5-expressing cells and were found to be required for caSTAT5-induced cell proliferation. Overexpression of Gab2-3YF, a mutant of Gab2 incapable of binding PI3K, inhibited the proliferation and survival of caSTAT5-expressing cells as well as ERK1/2 and Akt/protein kinase B phosphorylation. Taken together, our results indicate that Gab2 is required for caSTAT5-induced cell proliferation by regulating both the PI3K/Akt and the Ras/MAPK pathways.

Activated STAT5 proteins induce activation of the PI 3-kinase/Akt and Ras/MAPK pathways via the Gab2 scaffolding adapter

PubMed Central

2005-01-01

The active forms of STAT5A (signal transducer and activator of transcription 5A) and STAT5B are able to relieve the cytokine dependence of haematopoietic cells and to induce leukaemia in mice. We have demonstrated previously that activation of the PI3K (phosphoinositide 3-kinase) signalling cascade plays a major role in cell growth and survival induced by these proteins. Interaction between STAT5 and p85, the regulatory subunit of the PI3K, has been suggested to be required for this activation. We show in the present study that the scaffolding protein Gab2 [Grb2 (growth-factor-receptor-bound protein 2)-associated binder-2] is an essential component of this interaction. Gab2 is persistently tyrosine-phosphorylated in Ba/F3 cells expressing caSTAT5 (constitutively activated STAT5), independent of JAK2 (Janus kinase 2) activation where it interacts with STAT5, p85 and Grb2, but not with Shp2 [SH2 (Src homology 2)-domain-containing tyrosine phosphatase] proteins. Interaction of STAT5 with Gab2 was also observed in Ba/F3 cells stimulated with interleukin-3 or expressing the oncogenic fusion protein Tel–JAK2. The MAPKs (mitogen-activated protein kinases) ERK1 (extracellular-signal-regulated kinase 1) and ERK2 were constitutively activated in the caSTAT5-expressing cells and were found to be required for caSTAT5-induced cell proliferation. Overexpression of Gab2-3YF, a mutant of Gab2 incapable of binding PI3K, inhibited the proliferation and survival of caSTAT5-expressing cells as well as ERK1/2 and Akt/protein kinase B phosphorylation. Taken together, our results indicate that Gab2 is required for caSTAT5-induced cell proliferation by regulating both the PI3K/Akt and the Ras/MAPK pathways. PMID:15833084

Rapid activation by 3,5,3'-L-triiodothyronine of adenosine 5'-monophosphate-activated protein kinase/acetyl-coenzyme a carboxylase and akt/protein kinase B signaling pathways: relation to changes in fuel metabolism and myosin heavy-chain protein content in rat gastrocnemius muscle in vivo.

PubMed

de Lange, Pieter; Senese, Rosalba; Cioffi, Federica; Moreno, Maria; Lombardi, Assunta; Silvestri, Elena; Goglia, Fernando; Lanni, Antonia

2008-12-01

T3 stimulates metabolic rate in many tissues and induces changes in fuel use. The pathways by which T3 induces metabolic/structural changes related to altered fuel use in skeletal muscle have not been fully clarified. Gastrocnemius muscle (isolated at different time points after a single injection of T3 into hypothyroid rats), displayed rapid inductions of AMP-activated protein kinase (AMPK) phosphorylation (threonine 172; within 6 h) and acetyl-coenzyme A carboxylase phosphorylation (serine 79; within 12 h). As a consequence, increases occurred in mitochondrial fatty acid oxidation and carnitine palmitoyl transferase activity. Concomitantly, T3 stimulated signaling toward increased glycolysis through a rapid increase in Akt/protein kinase B (serine 473) phosphorylation (within 6 h) and a directly related increase in the activity of phosphofructokinase. The kinase specificity of the above effects was verified by treatment with inhibitors of AMPK and Akt activity (compound C and wortmannin, respectively). In contrast, glucose transporter 4 translocation to the membrane (activated by T3 within 6 h) was maintained when either AMPK or Akt activity was inhibited. The metabolic changes were accompanied by a decline in myosin heavy-chain Ib protein [causing a shift toward the fast-twitch (glycolytic) phenotype]. The increases in AMPK and acetyl-coenzyme A carboxylase phosphorylation were transient events, both levels declining from 12 h after the T3 injection, but Akt phosphorylation remained elevated until at least 48h after the injection. These data show that in skeletal muscle, T3 stimulates both fatty acid and glucose metabolism through rapid activations of the associated signaling pathways involving AMPK and Akt/protein kinase B.

Valproate induced hepatic steatosis by enhanced fatty acid uptake and triglyceride synthesis.

PubMed

Bai, Xupeng; Hong, Weipeng; Cai, Peiheng; Chen, Yibei; Xu, Chuncao; Cao, Di; Yu, Weibang; Zhao, Zhongxiang; Huang, Min; Jin, Jing

2017-06-01

Steatosis is the characteristic type of VPA-induced hepatotoxicity and may result in life-threatening hepatic lesion. Approximately 61% of patients treated with VPA have been diagnosed with hepatic steatosis through ultrasound examination. However, the mechanisms underlying VPA-induced intracellular fat accumulation are not yet fully understood. Here we demonstrated the involvement of fatty acid uptake and lipogenesis in VPA-induced hepatic steatosis in vitro and in vivo by using quantitative real-time PCR (qRT-PCR) analysis, western blotting analysis, fatty acid uptake assays, Nile Red staining assays, and Oil Red O staining assays. Specifically, we found that the expression of cluster of differentiation 36 (CD36), an important fatty acid transport, and diacylglycerol acyltransferase 2 (DGAT2) were significantly up-regulated in HepG2 cells and livers of C57B/6J mice after treatment with VPA. Furthermore, VPA treatment remarkably enhanced the efficiency of fatty acid uptake mediated by CD36, while this effect was abolished by the interference with CD36-specific siRNA. Also, VPA treatment significantly increased DGAT2 expression as a result of the inhibition of mitogen-activated protein kinase kinase (MEK) - extracellular regulated kinase (ERK) pathway; however, DGAT2 knockdown significantly alleviated VPA-induced intracellular lipid accumulation. Additionally, we also found that sterol regulatory element binding protein-1c (SREBP-1c)-mediated fatty acid synthesis may be not involved in VPA-induced hepatic steatosis. Overall, VPA-triggered over-regulation of CD36 and DGAT2 could be helpful for a better understanding of the mechanisms underlying VPA-induced hepatic steatosis and may offer novel therapeutic strategies to combat VPA-induced hepatotoxicity. Copyright © 2017 Elsevier Inc. All rights reserved.

Protein phosphatase and kinase activities possibly involved in exocytosis regulation in Paramecium tetraurelia.

PubMed Central

Kissmehl, R; Treptau, T; Hofer, H W; Plattner, H

1996-01-01

In Paramecium tetraurelia cells synchronous exocytosis induced by aminoethyldextran (AED) is accompanied by an equally rapid dephosphorylation of a 63 kDa phosphoprotein (PP63) within 80 ms. In vivo, rephosphorylation occurs within a few seconds after AED triggering. In homogenates (P)P63 can be solubilized in all three phosphorylation states (phosphorylated, dephosphorylated and rephosphorylated) and thus tested in vitro. By using chelators of different divalent cations, de- and rephosphorylation of PP63 and P63 respectively can be achieved by an endogenous protein phosphatase/kinase system. Dephosphorylation occurs in the presence of EDTA, whereas in the presence of EGTA this was concealed by phosphorylation by endogenous kinase(s), thus indicating that phosphorylation of P63 is calcium-independent. Results obtained with protein phosphatase inhibitors (okadaic acid, calyculin A) allowed us to exclude a protein serine/threonine phosphatase of type I (with selective sensitivity in Paramecium). Protein phosphatase 2C is also less likely to be a candidate because of its requirement for high Mg2+ concentrations. According to previous evidence a protein serine/threonine phosphatase of type 2B (calcineurin; CaN) is possibly involved. We have now found that bovine brain CaN dephosphorylates PP63 in vitro. Taking into account the specific requirements of this phosphatase in vitro, with p-nitrophenyl phosphate as a substrate, we have isolated a cytosolic phosphatase of similar characteristics by combined preparative gel electrophoresis and affinity-column chromatography. In Paramecium this phosphatase also dephosphorylates PP63 in vitro (after 32P labelling in vivo). Using various combinations of ion exchange, affinity and hydrophobic interaction chromatography we have also isolated three different protein kinases from the soluble fraction, i.e. a cAMP-dependent protein kinase (PKA), a cGMP-dependent protein kinase (PKG) and a casein kinase. Among the kinases tested, PKA

Genistein inhibits voltage-gated sodium currents in SCG neurons through protein tyrosine kinase-dependent and kinase-independent mechanisms.

PubMed

Jia, Zhanfeng; Jia, Yueqin; Liu, Boyi; Zhao, Zhiying; Jia, Qingzhong; Liang, Huiling; Zhang, Hailin

2008-08-01

Voltage-gated sodium channels play a crucial role in the initiation and propagation of neuronal action potentials. Genistein, an isoflavone phytoestrogen, has long been used as a broad-spectrum inhibitor of protein tyrosine kinases (PTK). In addition, genistein-induced modulation of ion channels has been described previously in the literature. In this study, we investigated the effect of genistein on voltage-gated sodium channels in rat superior cervical ganglia (SCG) neurons. The results show that genistein inhibits Na(+) currents in a concentration-dependent manner, with a concentration of half-maximal effect (IC(50)) at 9.1 +/- 0.9 microM. Genistein positively shifted the voltage dependence of activation but did not affect inactivation of the Na(+) current. The inactive genistein analog daidzein also inhibited Na(+) currents, but was less effective than genistein. The IC(50) for daidzein-induced inhibition was 20.7 +/- 0.1 microM. Vanadate, an inhibitor of protein tyrosine phosphatases, partially but significantly reversed genistein-induced inhibition of Na(+) currents. Other protein tyrosine kinase antagonists such as tyrphostin 23, an erbstatin analog, and PP2 all had small but significant inhibitory effects on Na(+) currents. Among all active and inactive tyrosine kinase inhibitors tested, genistein was the most potent inhibitor of Na(+) currents. These results suggest that genistein inhibits Na(+) currents in rat SCG neurons through two distinct mechanisms: protein tyrosine kinase-independent, and protein tyrosine kinase-dependent mechanisms. Furthermore, the Src kinase family may be involved in the basal phosphorylation of the Na(+) channel.

Overexpression of an Arabidopsis cysteine-rich receptor-like protein kinase, CRK5, enhances abscisic acid sensitivity and confers drought tolerance.

PubMed

Lu, Kai; Liang, Shan; Wu, Zhen; Bi, Chao; Yu, Yong-Tao; Wang, Xiao-Fang; Zhang, Da-Peng

2016-09-01

Receptor-like kinases (RLKs) have been reported to regulate many developmental and defense process, but only a few members have been functionally characterized. In the present study, our observations suggest that one of the RLKs, a membrane-localized cysteine-rich receptor-like protein kinase, CRK5, is involved in abscisic acid (ABA) signaling in Arabidopsis thaliana Overexpression of CRK5 increases ABA sensitivity in ABA-induced early seedling growth arrest and promotion of stomatal closure and inhibition of stomatal opening. Interestingly, and importantly, overexpression of CRK5 enhances plant drought tolerance without affecting plant growth at the mature stages and plant productivity. Transgenic lines overexpressing a mutated form of CRK5, CRK5 (K372E) with the change of the 372nd conserved amino acid residue from lysine to glutamic acid in its kinase domain, result in wild-type ABA and drought responses, supporting the role of CRK5 in ABA signaling. The loss-of-function mutation of the CRK5 gene does not affect the ABA response, while overexpression of two homologs of CRK5, CRK4 and CRK19, confers ABA responses, suggesting that these CRK members function redundantly. We further showed that WRKY18, WRKY40 and WRKY60 transcription factors repress the expression of CRK5, and that CRK5 likely functions upstream of ABI2 in ABA signaling. These findings help in understanding the complex ABA signaling network. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Prostaglandin E2 and the protein kinase A pathway mediate arachidonic acid induction of c-fos in human prostate cancer cells

NASA Technical Reports Server (NTRS)

Chen, Y.; Hughes-Fulford, M.

2000-01-01

Arachidonic acid (AA) is the precursor for prostaglandin E2 (PGE2) synthesis and increases growth of prostate cancer cells. To further elucidate the mechanisms involved in AA-induced prostate cell growth, induction of c-fos expression by AA was investigated in a human prostate cancer cell line, PC-3. c-fos mRNA was induced shortly after addition of AA, along with a remarkable increase in PGE2 production. c-fos expression and PGE2 production induced by AA was blocked by a cyclo-oxygenase inhibitor, flurbiprofen, suggesting that PGE2 mediated c-fos induction. Protein kinase A (PKA) inhibitor H-89 abolished induction of c-fos expression by AA, and partially inhibited PGE2 production. Protein kinase C (PKC) inhibitor GF109203X had no significant effect on c-fos expression or PGE2 production. Expression of prostaglandin (EP) receptors, which mediate signal transduction from PGE2 to the cells, was examined by reverse transcription polymerase chain reaction in several human prostate cell lines. EP4 and EP2, which are coupled to the PKA signalling pathway, were expressed in all cells tested. Expression of EP1, which activates the PKC pathway, was not detected. The current study showed that induction of the immediate early gene c-fos by AA is mediated by PGE2, which activates the PKA pathway via the EP2/4 receptor in the PC-3 cells.

Eicosapentaenoic acid-enriched phosphatidylcholine isolated from Cucumaria frondosa exhibits anti-hyperglycemic effects via activating phosphoinositide 3-kinase/protein kinase B signal pathway.

PubMed

Hu, Shiwei; Xu, Leilei; Shi, Di; Wang, Jingfeng; Wang, Yuming; Lou, Qiaoming; Xue, Changhu

2014-04-01

Eicosapentaenoic acid-enriched phosphatidylcholine was isolated from the sea cucumber Cucumaria frondosa (Cucumaria-PC) and its effects on streptozotocin (STZ)-induced hyperglycemic rats were investigated. Male Sprague-Dawley rats were randomly divided into normal control, model control (STZ), low- and high-dose Cucumaria-PC groups (STZ + Cucumaria-PC at 25 and 75 mg/Kg·b·wt, intragastrically, respectively). Blood glucose, insulin, glycogen in liver and gastrocnemius were determined over 60 days. Insulin signaling in the rats' gastrocnemius was determined by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. The results showed that Cucumaria-PC significantly decreased blood glucose level, increased insulin secretion and glycogen synthesis in diabetic rats. RT-PCR analysis revealed that Cucumaria-PC significantly promoted the expressions of glycometabolism-related genes of insulin receptor (IR), insulin receptor substrate-1 (IRS-1), phosphoinositide 3-kinase (PI3K), protein kinase B (PKB), and glucose transporter 4 (GLUT4) in gastrocnemius. Western blotting assay demonstrated that Cucumaria-PC remarkably enhanced the proteins abundance of IR-β, PI3K, PKB, GLUT4, as well as phosphorylation of Tyr-IR-β, p85-PI3K, Ser473-PKB (P < 0.05 and P < 0.01). These findings suggested that Cucumaria-PC exhibited significant anti-hyperglycemic activities through up-regulating PI3K/PKB signal pathway mediated by insulin. Nutritional supplementation with Cucumaria-PC, if validated for human studies, may offer an adjunctive therapy for diabetes mellitus. Copyright © 2013 The Society for Biotechnology, Japan. All rights reserved.

Lysophosphatidic acid and adenylyl cyclase inhibitor increase proliferation of senescent human diploid fibroblasts by inhibiting adenosine monophosphate-activated protein kinase.

PubMed

Rhim, Ji-Heon; Jang, Ik-Soon; Song, Kye-Yong; Ha, Moon-Kyung; Cho, Sung-Chun; Yeo, Eui-Ju; Park, Sang Chul

2008-08-01

This study was designed to elucidate the molecular mechanism underlying lysophosphatidic acid (LPA) and adenylyl cyclase inhibitor SQ22536 (ACI)-induced senescent human diploid fibroblast (HDF) proliferation. Because adenosine monophosphate (AMP)-activated protein kinase (AMPK) is known to inhibit cell proliferation, we examined the phosphorylation status of AMPK and p53 and the expression level of p21(waf1/cip1) after treating HDFs with LPA and ACI. Phosphorylation of AMPKalpha on threonine-172 (p-Thr172-AMPKalpha) increases its catalytic activity but phosphorylation on serine-485/491 (p-Ser485/491-AMPKalpha) reduces the accessibility of the Thr172 phosphorylation site thereby inhibiting its catalytic activity. LPA increased p-Ser485/491-AMPKalpha, presumably by activating cAMP-dependent protein kinase (PKA). However, ACI reduced p-Thr172-AMPKalpha by inhibiting the LKB signaling. Our data demonstrated that both LPA and ACI inhibit the catalytic activity of AMPKalpha and p53 by differentially regulating phosphorylation of AMPKalpha, causing increased senescent cell proliferation. These findings suggest that the proliferation potential of senescent HDFs can be modulated through the regulation of the AMPK signaling pathway.

Tau-Induced Ca2+/Calmodulin-Dependent Protein Kinase-IV Activation Aggravates Nuclear Tau Hyperphosphorylation.

PubMed

Wei, Yu-Ping; Ye, Jin-Wang; Wang, Xiong; Zhu, Li-Ping; Hu, Qing-Hua; Wang, Qun; Ke, Dan; Tian, Qing; Wang, Jian-Zhi

2018-04-01

Hyperphosphorylated tau is the major protein component of neurofibrillary tangles in the brains of patients with Alzheimer's disease (AD). However, the mechanism underlying tau hyperphosphorylation is not fully understood. Here, we demonstrated that exogenously expressed wild-type human tau40 was detectable in the phosphorylated form at multiple AD-associated sites in cytoplasmic and nuclear fractions from HEK293 cells. Among these sites, tau phosphorylated at Thr205 and Ser214 was almost exclusively found in the nuclear fraction at the conditions used in the present study. With the intracellular tau accumulation, the Ca 2+ concentration was significantly increased in both cytoplasmic and nuclear fractions. Further studies using site-specific mutagenesis and pharmacological treatment demonstrated that phosphorylation of tau at Thr205 increased nuclear Ca 2+ concentration with a simultaneous increase in the phosphorylation of Ca 2+ /calmodulin-dependent protein kinase IV (CaMKIV) at Ser196. On the other hand, phosphorylation of tau at Ser214 did not significantly change the nuclear Ca 2+ /CaMKIV signaling. Finally, expressing calmodulin-binding protein-4 that disrupts formation of the Ca 2+ /calmodulin complex abolished the okadaic acid-induced tau hyperphosphorylation in the nuclear fraction. We conclude that the intracellular accumulation of phosphorylated tau, as detected in the brains of AD patients, can trigger nuclear Ca 2+ /CaMKIV signaling, which in turn aggravates tau hyperphosphorylation. Our findings provide new insights for tauopathies: hyperphosphorylation of intracellular tau and an increased Ca 2+ concentration may induce a self-perpetuating harmful loop to promote neurodegeneration.

Differential activation of the Ras/extracellular-signal-regulated protein kinase pathway is responsible for the biological consequences induced by the Axl receptor tyrosine kinase.

PubMed

Fridell, Y W; Jin, Y; Quilliam, L A; Burchert, A; McCloskey, P; Spizz, G; Varnum, B; Der, C; Liu, E T

1996-01-01

To understand the mechanism of Axl signaling, we have initiated studies to delineate downstream components in interleukin-3-dependent 32D cells by using a chimeric receptor containing the recombinant epidermal growth factor (EGF) receptor extracellular and transmembrane domains and the Axl kinase domain (EAK [for EGF receptor-Axl kinase]). We have previously shown that upon exogenous EGF stimulation, 32D-EAK cells are capable of proliferation in the absence of interleukin-3. With this system, we determined that EAK-induced cell survival and mitogenesis are dependent upon the Ras/extracellular-signal-regulated protein kinase (ERK) cascade. Although the phosphatidylinositol-3 kinase pathway is activated upon EAK signaling, it appears to be dispensable for the biological actions of the Axl kinase. Furthermore, we demonstrated that different threshold levels of Ras/ERK activation are needed to induce a block to apoptosis or proliferation in 32D cells. Recently, we have identified an Axl ligand, GAS6. Surprisingly, GAS6-stimulated 32D-Axl cells exhibited no blockage to apoptosis or mitogenic response which is correlated with the absence of Ras/ERK activation. Taken together, these data suggest that different extracellular domains dramatically alter the intracellular response of the Axl kinase. Furthermore, our data suggest that the GAS6-Axl interaction does not induce mitogenesis and that its exact role remains to be determined.

Phosphorylation of a NAC Transcription Factor by a Calcium/Calmodulin-Dependent Protein Kinase Regulates Abscisic Acid-Induced Antioxidant Defense in Maize [Phosphorylation of a NAC Transcription Factor by ZmCCaMK Regulates Abscisic Acid-Induced Antioxidant Defense in Maize

DOE PAGES

Zhu, Yuan; Yan, Jingwei; Liu, Weijuan; ...

2016-05-10

Calcium/calmodulin-dependent protein kinase (CCaMK) has been shown to play an important role in abscisic acid (ABA)-induced antioxidant defense and enhance the tolerance of plants to drought stress. However, its downstream molecular events are poorly understood. Here, we identify a NAC transcription factor, ZmNAC84, in maize, which physically interacts with ZmCCaMK in vitro and in vivo. ZmNAC84 display a partially overlapping expression pattern with ZmCCaMK after ABA treatment and H 2O 2 is required for ABA-induced ZmNAC84 expression. Functional analysis reveals that ZmNAC84 is essential for ABA-induced antioxidant defense in a ZmCCaMK-dependent manner. Furthermore, ZmCCaMK directly phosphorylates S113 of ZmNAC84 inmore » vitro, and S113 is essential for the ABA-induced stimulation of antioxidant defense by ZmCCaMK. Moreover, overexpression of ZmNAC84 in tobacco can improve drought tolerance, and alleviate drought-induced oxidative damage of transgenic plants. These results define a mechanism for ZmCCaMK function in ABA-induced antioxidant defense, where ABA-produced H 2O 2 first induces expression of ZmCCaMK and ZmNAC84 and activates ZmCCaMK, and subsequently the activated ZmCCaMK phosphorylates ZmNAC84 at S113, thereby inducing antioxidant defense by activating downstream genes.« less

Phosphorylation of a NAC Transcription Factor by a Calcium/Calmodulin-Dependent Protein Kinase Regulates Abscisic Acid-Induced Antioxidant Defense in Maize [Phosphorylation of a NAC Transcription Factor by ZmCCaMK Regulates Abscisic Acid-Induced Antioxidant Defense in Maize

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhu, Yuan; Yan, Jingwei; Liu, Weijuan

Calcium/calmodulin-dependent protein kinase (CCaMK) has been shown to play an important role in abscisic acid (ABA)-induced antioxidant defense and enhance the tolerance of plants to drought stress. However, its downstream molecular events are poorly understood. Here, we identify a NAC transcription factor, ZmNAC84, in maize, which physically interacts with ZmCCaMK in vitro and in vivo. ZmNAC84 display a partially overlapping expression pattern with ZmCCaMK after ABA treatment and H 2O 2 is required for ABA-induced ZmNAC84 expression. Functional analysis reveals that ZmNAC84 is essential for ABA-induced antioxidant defense in a ZmCCaMK-dependent manner. Furthermore, ZmCCaMK directly phosphorylates S113 of ZmNAC84 inmore » vitro, and S113 is essential for the ABA-induced stimulation of antioxidant defense by ZmCCaMK. Moreover, overexpression of ZmNAC84 in tobacco can improve drought tolerance, and alleviate drought-induced oxidative damage of transgenic plants. These results define a mechanism for ZmCCaMK function in ABA-induced antioxidant defense, where ABA-produced H 2O 2 first induces expression of ZmCCaMK and ZmNAC84 and activates ZmCCaMK, and subsequently the activated ZmCCaMK phosphorylates ZmNAC84 at S113, thereby inducing antioxidant defense by activating downstream genes.« less

Neurotoxicity induced by dexamethasone in the human neuroblastoma SH-SY5Y cell line can be prevented by folic acid.

PubMed

Budni, J; Romero, A; Molz, S; Martín-de-Saavedra, M D; Egea, J; Del Barrio, L; Tasca, C I; Rodrigues, A L S; López, M G

2011-09-08

Folic acid (folate) is a vitamin of the B-complex group that is essential for cell replication. Folate is a major determinant of one-carbon metabolism, in which S-adenosylmethionine donates methyl groups that are crucial for neurological function. Many roles for folic acid have been reported, including neuroprotective and antidepressant properties. On the other hand, increased concentrations of corticoids have proven neurotoxic effects and hypersecretion of glucocorticoids has been linked to different mood disorders. The purpose of this study was to investigate the potential protective effect of folic acid on dexamethasone-induced cellular death in SH-SY5Y neuroblastoma cell line and the possible intracellular signaling pathway involved in such effect. Exposure to 1 mM dexamethasone for 48 h caused a significant reduction of cell viability measured as 3-[4,5 dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) reduction. Exposure of SH-SY5Y cells for 72 h to increasing concentrations of folate (1-300 μM) was not cytotoxic. However, pretreatment with folate (10-300 μM) reduced dexamethasone-induced toxicity in a significant manner. To explore the putative intracellular signaling pathways implicated in the protective effect of folate we used different protein kinase inhibitors. The protective effect of folic acid on dexamethasone-induced neurotoxicity was reversed by the phosphatidylinositol-3 kinase/Akt (PI3K/Akt, LY294002), Ca²⁺/Calmodulin-dependent protein kinase II (CaMKII, KN-93), and protein kinase A (PKA, H-89) inhibitors, but not the mitogen-activated protein/extracellular signal-regulated kinase (MEK1/2, PD98059) and protein kinase C (PKC, chelerythrine) inhibitors. In conclusion, the results of this study show that folic acid can protect against dexamethasone-induced neurotoxicity and its protective mechanism is related to a signaling pathway that involves PI3K/Akt, CaMKII, and PKA. Copyright © 2011. Published by Elsevier Ltd.

Abscisic acid-activated SNRK2 protein kinases function in the gene-regulation pathway of ABA signal transduction by phosphorylating ABA response element-binding factors.

PubMed

Kobayashi, Yuhko; Murata, Michiharu; Minami, Hideyuki; Yamamoto, Shuhei; Kagaya, Yasuaki; Hobo, Tokunori; Yamamoto, Akiko; Hattori, Tsukaho

2005-12-01

The plant hormone abscisic acid (ABA) induces gene expression via the ABA-response element (ABRE) present in the promoters of ABA-regulated genes. A group of bZIP proteins have been identified as ABRE-binding factors (ABFs) that activate transcription through this cis element. A rice ABF, TRAB1, has been shown to be activated via ABA-dependent phosphorylation. While a large number of signalling factors have been identified that are involved in stomatal regulation by ABA, relatively less is known about the ABA-signalling pathway that leads to gene expression. We have shown recently that three members of the rice SnRK2 protein kinase family, SAPK8, SAPK9 and SAPK10, are activated by ABA signal as well as by hyperosmotic stress. Here we show that transient overexpression in cultured cell protoplasts of these ABA-activated SnRK2 protein kinases leads to the activation of an ABRE-regulated promoter, suggesting that these kinases are involved in the gene-regulation pathway of ABA signalling. We further show several lines of evidence that these ABA-activated SnRK2 protein kinases directly phosphorylate TRAB1 in response to ABA. Kinetic analysis of SAPK10 activation and TRAB1 phosphorylation indicated that the latter immediately followed the former. TRAB1 was found to be phosphorylated not only in response to ABA, but also in response to hyperosmotic stress, which was interpreted as the consequence of phosphorylation of TRAB1 by hyperosmotically activated SAPKs. Physical interaction between TRAB1 and SAPK10 in vivo was demonstrated by a co-immunoprecipitation experiment. Finally, TRAB1 was phosphorylated in vitro by the ABA-activated SnRK2 protein kinases at Ser102, which is phosphorylated in vivo in response to ABA and is critical for the activation function.

H- ras deletion protects against angiotensin II-induced arterial hypertension and cardiac remodeling through protein kinase G-Iβ pathway activation.

PubMed

Martín-Sánchez, Paloma; Luengo, Alicia; Griera, Mercedes; Orea, María Jesús; López-Olañeta, Marina; Chiloeches, Antonio; Lara-Pezzi, Enrique; de Frutos, Sergio; Rodríguez-Puyol, Manuel; Calleros, Laura; Rodríguez-Puyol, Diego

2018-02-01

Ras proteins regulate cell survival, growth, differentiation, blood pressure, and fibrosis in some organs. We have demonstrated that H- ras gene deletion produces mice hypotension via a soluble guanylate cyclase-protein kinase G (PKG)-dependent mechanism. In this study, we analyzed the consequences of H- ras deletion on cardiac remodeling induced by continuous angiotensin II (AngII) infusion and the molecular mechanisms implied. Left ventricular posterior wall thickness and mass and cardiomyocyte cross-sectional area were similar between AngII-treated H-Ras knockout (H -ras -/- ) and control wild-type (H -ras +/+ ) mice, as were extracellular matrix protein expression. Increased cardiac PKG-Iβ protein expression in H -ras -/- mice suggests the involvement of this protein in heart protection. Ex vivo experiments on cardiac explants could support this mechanism, as PKG blockade blunted protection against AngII-induced cardiac hypertrophy and fibrosis markers in H -ras -/- mice. Genetic modulation studies in cardiomyocytes and cardiac and embryonic fibroblasts revealed that the lack of H-Ras down-regulates the B-RAF/MEK/ERK pathway, which induces the glycogen synthase kinase-3β-dependent activation of the transcription factor, cAMP response element-binding protein, which is responsible for PKG-Iβ overexpression in H -ras -/- mouse embryonic fibroblasts. This study demonstrates that H- ras deletion protects against AngII-induced cardiac remodeling, possibly via a mechanism in which PKG-Iβ overexpression could play a partial role, and points to H-Ras and/or downstream proteins as potential therapeutic targets in cardiovascular disease.-Martín-Sánchez, P., Luengo, A., Griera, M., Orea, M. J., López-Olañeta, M., Chiloeches, A., Lara-Pezzi, E., de Frutos, S., Rodríguez-Puyol, M., Calleros, L., Rodríguez-Puyol, D. H- ras deletion protects against angiotensin II-induced arterial hypertension and cardiac remodeling through protein kinase G-Iβ pathway activation.

Zinc protoporphyrin inhibition of lipopolysaccharide-, lipoteichoic acid-, and peptidoglycan-induced nitric oxide production through stimulating iNOS protein ubiquitination.

PubMed

Chow, Jyh-Ming; Lin, Hui-Yi; Shen, Shing-Chuan; Wu, Ming-Shun; Lin, Cheng-Wei; Chiu, Wen-Ta; Lin, Chien-Huang; Chen, Yen-Chou

2009-06-15

In the present study, zinc protoporphyrin (ZnPP), but not ferric protoporphyrin (FePP), tin protoporphyrin (SnPP), or zinc chloride (ZnCl(2)), at the doses of 0.5, 1, and 2 microM, dose-dependently inhibited lipopolysaccharide- (LPS), lipoteichoic acid (LTA), and peptidoglycan (PGN)-induced inducible nitric oxide (iNOS) and nitric oxide (NO) production with an increase in heme oxygenase 1 (HO-1) protein in RAW264.7 macrophages in a serum-free condition. NO inhibition and HO-1 induction by ZnPP were blocked by the separate addition of fetal bovine serum (FBS) and bovine serum albumin (BSA). A decrease in the iNOS/NO ratio and an increase in HO-1 protein by ZnPP were identified in three different conditions including ZnPP pretreatment, ZnPP co-treatment, and ZnPP post-treatment with LPS and LTA. Activation of c-Jun N-terminal kinases (JNKs) and extracellular regulated kinases (ERKs) were detected in LPS-, LTA-, and PGN-treated RAW264.7 cells, and iNOS/NO production was blocked by adding the JNK inhibitor, SP600125, but not the ERK inhibitor, PD98059. However, ZnPP addition potentiated ERK and JNK protein phosphorylation stimulated by LPS, LTA, and PGN. Increases in total protein ubiquitination and ubiquitinated iNOS proteins were detected in ZnPP-treated macrophages elicited by LPS according to Western and immunoprecipitation/Western blotting assays, respectively. The decrease in LPS-induced iNOS protein by ZnPP was reversed by adding the proteasome inhibitors MG132 and lactacystin. The reduction in HO-1 protein induced by ZnPP via transfection of HO-1 small interfering RNA did not affect the inhibitory effect of ZnPP against LPS-induced iNOS/NO production and protein ubiquitination induced by ZnPP in macrophages. Data of the present study provide the first evidence to support ZnPP effectively inhibiting inflammatory iNOS/NO production through activation of protein ubiquitination in a HO-1-independent manner in macrophages.

Zinc protoporphyrin inhibition of lipopolysaccharide-, lipoteichoic acid-, and peptidoglycan-induced nitric oxide production through stimulating iNOS protein ubiquitination

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chow, J.-M.; Lin, H.-Y.; Shen, S.-C.

2009-06-15

In the present study, zinc protoporphyrin (ZnPP), but not ferric protoporphyrin (FePP), tin protoporphyrin (SnPP), or zinc chloride (ZnCl{sub 2}), at the doses of 0.5, 1, and 2 {mu}M, dose-dependently inhibited lipopolysaccharide- (LPS), lipoteichoic acid (LTA), and peptidoglycan (PGN)-induced inducible nitric oxide (iNOS) and nitric oxide (NO) production with an increase in heme oxygenase 1 (HO-1) protein in RAW264.7 macrophages in a serum-free condition. NO inhibition and HO-1 induction by ZnPP were blocked by the separate addition of fetal bovine serum (FBS) and bovine serum albumin (BSA). A decrease in the iNOS/NO ratio and an increase in HO-1 protein bymore » ZnPP were identified in three different conditions including ZnPP pretreatment, ZnPP co-treatment, and ZnPP post-treatment with LPS and LTA. Activation of c-Jun N-terminal kinases (JNKs) and extracellular regulated kinases (ERKs) were detected in LPS-, LTA-, and PGN-treated RAW264.7 cells, and iNOS/NO production was blocked by adding the JNK inhibitor, SP600125, but not the ERK inhibitor, PD98059. However, ZnPP addition potentiated ERK and JNK protein phosphorylation stimulated by LPS, LTA, and PGN. Increases in total protein ubiquitination and ubiquitinated iNOS proteins were detected in ZnPP-treated macrophages elicited by LPS according to Western and immunoprecipitation/Western blotting assays, respectively. The decrease in LPS-induced iNOS protein by ZnPP was reversed by adding the proteasome inhibitors MG132 and lactacystin. The reduction in HO-1 protein induced by ZnPP via transfection of HO-1 small interfering RNA did not affect the inhibitory effect of ZnPP against LPS-induced iNOS/NO production and protein ubiquitination induced by ZnPP in macrophages. Data of the present study provide the first evidence to support ZnPP effectively inhibiting inflammatory iNOS/NO production through activation of protein ubiquitination in a HO-1-independent manner in macrophages.« less

[Effect of inhibitors serine/threonine protein kinases and protein phosphatases on mitosis progression of synchronized tobacco by-2 cells].

PubMed

Sheremet, Ia A; Emets, A I; Azmi, A; Vissenberg, K; Verbelen, J-P; Blium, Ia B

2012-01-01

In order to investigate the role of various serine/ threonine protein kinases and protein phosphatases in the regulation of mitosis progression in plant cells the influence of cyclin-dependent (olomoucine) and Ca2+ -calmodulin-dependent (W7) protein kinases inhibitors, as well as protein kinase C inhibitors (H7 and staurosporine) and protein phosphatases inhibitor (okadaic acid) on mitosis progression in synchronized tobacco BY-2 cells has been studied. It was found that BY-2 culture treatment with inhibitors of cyclin dependent protein kinases and protein kinase C causes prophase delay, reduces the mitotic index and displaces of mitotic peak as compare with control cells. Inhibition of Ca2+ -calmodulin dependent protein kinases enhances the cell entry into prophase and delays their exit from mitosis. Meanwhile inhibition of serine/threonine protein phosphatases insignificantly enhances of synchronized BY-2 cells entering into all phases of mitosis.

Inhibition of protein kinase A and GIRK channel reverses fentanyl-induced respiratory depression.

PubMed

Liang, Xiaonan; Yong, Zheng; Su, Ruibin

2018-06-11

Opioid-induced respiratory depression is a major obstacle to improving the clinical management of moderate to severe chronic pain. Opioids inhibit neuronal activity via various pathways, including calcium channels, adenylyl cyclase, and potassium channels. Currently, the underlying molecular pathway of opioid-induced respiratory depression is only partially understood. This study aimed to investigate the mechanisms of opioid-induced respiratory depression in vivo by examining the effects of different pharmacological agents on fentanyl-induced respiratory depression. Respiratory parameters were detected using whole body plethysmography in conscious rats. We show that pre-treatment with the protein kinase A (PKA) inhibitor H89 reversed the fentanyl-related effects on respiratory rate, inspiratory time, and expiratory time. Pre-treatment with the G protein-gated inwardly rectifying potassium (GIRK) channel blocker Tertiapin-Q dose-dependently reversed the fentanyl-related effects on respiratory rate and inspiratory time. A phosphodiesterase 4 (PDE4) inhibitor and cyclic adenosine monophosphate (cAMP) analogs did not affect fentanyl-induced respiratory depression. These findings suggest that PKA and GIRK may be involved in fentanyl-induced respiratory depression and could represent useful therapeutic targets for the treatment of fentanyl-induced ventilatory depression. Copyright © 2018 Elsevier B.V. All rights reserved.

Cyanidin-3-O-β-glucoside regulates fatty acid metabolism via an AMP-activated protein kinase-dependent signaling pathway in human HepG2 cells

PubMed Central

2012-01-01

Background Hepatic metabolic derangements are key components in the development of fatty liver disease. AMP-activated protein kinase (AMPK) plays a central role in controlling hepatic lipid metabolism through modulating the downstream acetyl CoA carboxylase (ACC) and carnitine palmitoyl transferase 1 (CPT-1) pathway. In this study, cyanidin-3-O-β-glucoside (Cy-3-g), a typical anthocyanin pigment was used to examine its effects on AMPK activation and fatty acid metabolism in human HepG2 hepatocytes. Results Anthocyanin Cy-3-g increased cellular AMPK activity in a calmodulin kinase kinase dependent manner. Furthermore, Cy-3-g substantially induced AMPK downstream target ACC phosphorylation and inactivation, and then decreased malonyl CoA contents, leading to stimulation of CPT-1 expression and significant increase of fatty acid oxidation in HepG2 cells. These effects of Cy-3-g are largely abolished by pharmacological and genetic inhibition of AMPK. Conclusion This study demonstrates that Cy-3-g regulates hepatic lipid homeostasis via an AMPK-dependent signaling pathway. Targeting AMPK activation by anthocyanin may represent a promising approach for the prevention and treatment of obesity-related nonalcoholic fatty liver disease. PMID:22243683

Increased choline kinase activity in 1,2-dimethylhydrazine-induced rat colon cancer.

PubMed

Nakagami, K; Uchida, T; Ohwada, S; Koibuchi, Y; Morishita, Y

1999-11-01

Cancer cells acquire particular characteristics that benefit their proliferation. We previously reported that human colon cancers examined had increased choline kinase activity and phosphocholine levels. The elevated phosphocholine levels were in part due to both activation of choline kinase and increased choline kinase alpha protein levels. In this report, we analyzed choline kinase, which catalyzes the phosphorylation of choline to produce phosphocholine, in rat 1,2-dimethylhydrazine (DMH)-induced colon cancer. This study is the first to demonstrate increased choline kinase alpha enzymatic activity, protein levels, and mRNA levels in DMH-induced colon cancer as well as human colon cancer, although phosphocholine was not increased in DMH-induced rat cancer. The increase in the mRNA level was partly due to an increase in the transcription of the choline kinase alpha gene. The increased choline kinase activity may be a specific characteristic acquired by cancer cells that benefits their proliferation.

PH domain leucine-rich repeat protein phosphatase 2 (PHLPP2) regulates G-protein-coupled receptor kinase 5 (GRK5)-induced cardiac hypertrophy in vitro.

PubMed

Yeh, Szu-Tsen; Zambrano, Cristina M; Koch, Walter J; Purcell, Nicole H

2018-05-25

PH domain leucine-rich repeat protein phosphatase (PHLPP) is a serine/threonine phosphatase that has been shown to regulate cell growth and survival through dephosphorylation of several members of the AGC family of kinases. G-protein-coupled receptor kinase 5 (GRK5) is an AGC kinase that regulates phenylephrine (PE)-induced cardiac hypertrophy through its noncanonical function of directly targeting proteins to the nucleus to regulate transcription. Here we investigated the possibility that the PHLPP2 isoform can regulate GRK5-induced cardiomyocyte hypertrophy in neonatal rat ventricular myocytes (NRVMs). We show that removal of PHLPP2 by siRNA induces hypertrophic growth of NRVMs as measured by cell size changes at baseline, potentiated PE-induced cell size changes, and re-expression of fetal genes atrial natriuretic factor and brain natriuretic peptide. Endogenous GRK5 and PHLPP2 were found to interact in NRVMs, and PE-induced nuclear accumulation of GRK5 was enhanced upon down-regulation of PHLPP2. Conversely, overexpression of PHLPP2 blocked PE-induced hypertrophic growth, re-expression of fetal genes, and nuclear accumulation of GRK5, which depended on its phosphatase activity. Finally, using siRNA against GRK5, we found that GRK5 was necessary for the hypertrophic response induced by PHLPP2 knockdown. Our findings demonstrate for the first time a novel regulation of GRK5 by the phosphatase PHLPP2, which modulates hypertrophic growth. Understanding the signaling pathways affected by PHLPP2 has potential for new therapeutic targets in the treatment of cardiac hypertrophy and failure. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Terreic acid, a quinone epoxide inhibitor of Bruton’s tyrosine kinase

PubMed Central

Kawakami, Yuko; Hartman, Stephen E.; Kinoshita, Eiji; Suzuki, Hidefumi; Kitaura, Jiro; Yao, Libo; Inagaki, Naoki; Franco, Alessandra; Hata, Daisuke; Maeda-Yamamoto, Mari; Fukamachi, Hiromi; Nagai, Hiroichi; Kawakami, Toshiaki

1999-01-01

Bruton’s tyrosine kinase (Btk) plays pivotal roles in mast cell activation as well as in B cell development. Btk mutations lead to severe impairments in proinflammatory cytokine production induced by cross-linking of high-affinity IgE receptor on mast cells. By using an in vitro assay to measure the activity that blocks the interaction between protein kinase C and the pleckstrin homology domain of Btk, terreic acid (TA) was identified and characterized in this study. This quinone epoxide specifically inhibited the enzymatic activity of Btk in mast cells and cell-free assays. TA faithfully recapitulated the phenotypic defects of btk mutant mast cells in high-affinity IgE receptor-stimulated wild-type mast cells without affecting the enzymatic activities and expressions of many other signaling molecules, including those of protein kinase C. Therefore, this study confirmed the important roles of Btk in mast cell functions and showed the usefulness of TA in probing into the functions of Btk in mast cells and other immune cell systems. Another insight obtained from this study is that the screening method used to identify TA is a useful approach to finding more efficacious Btk inhibitors. PMID:10051623

Protein Kinase Cϵ (PKCϵ) Promotes Synaptogenesis through Membrane Accumulation of the Postsynaptic Density Protein PSD-95*

PubMed Central

Sen, Abhik; Hongpaisan, Jarin; Wang, Desheng; Nelson, Thomas J.; Alkon, Daniel L.

2016-01-01

Protein kinase Cϵ (PKCϵ) promotes synaptic maturation and synaptogenesis via activation of synaptic growth factors such as BDNF, NGF, and IGF. However, many of the detailed mechanisms by which PKCϵ induces synaptogenesis are not fully understood. Accumulation of PSD-95 to the postsynaptic density (PSD) is known to lead to synaptic maturation and strengthening of excitatory synapses. Here we investigated the relationship between PKCϵ and PSD-95. We show that the PKCϵ activators dicyclopropanated linoleic acid methyl ester and bryostatin 1 induce phosphorylation of PSD-95 at the serine 295 residue, increase the levels of PSD-95, and enhance its membrane localization. Elimination of the serine 295 residue in PSD-95 abolished PKCϵ-induced membrane accumulation. Knockdown of either PKCϵ or JNK1 prevented PKCϵ activator-mediated membrane accumulation of PSD-95. PKCϵ directly phosphorylated PSD-95 and JNK1 in vitro. Inhibiting PKCϵ, JNK, or calcium/calmodulin-dependent kinase II activity prevented the effects of PKCϵ activators on PSD-95 phosphorylation. Increase in membrane accumulation of PKCϵ and phosphorylated PSD-95 (p-PSD-95S295) coincided with an increased number of synapses and increased amplitudes of excitatory post-synaptic potentials (EPSPs) in adult rat hippocampal slices. Knockdown of PKCϵ also reduced the synthesis of PSD-95 and the presynaptic protein synaptophysin by 30 and 44%, respectively. Prolonged activation of PKCϵ increased synapse number by 2-fold, increased presynaptic vesicle density, and greatly increased PSD-95 clustering. These results indicate that PKCϵ promotes synaptogenesis by activating PSD-95 phosphorylation directly through JNK1 and calcium/calmodulin-dependent kinase II and also by inducing expression of PSD-95 and synaptophysin. PMID:27330081

Involvement of protein kinase B and mitogen-activated protein kinases in experimental normothermic liver ischaemia-reperfusion injury.

PubMed

Cursio, R; Filippa, N; Miele, C; Van Obberghen, E; Gugenheim, J

2006-06-01

This study evaluated the role of protein kinase B (PKB), phosphatidylinositol 3-kinase (PI3-K), Bcl-2-associated death protein (BAD) and mitogen-activated protein kinases (MAPKs) in normothermic ischaemia-reperfusion (IR)-induced apoptosis in rat liver. Rats were divided into two groups that received either phosphate-buffered saline (control) or the caspase inhibitor Z-Asp-2,6-dichorobenzoyloxymethylketone (Z-Asp-cmk), injected intravenously 2 min before the induction of 120 min of normothermic liver ischaemia. Liver apoptosis was assessed by the terminal deoxyribonucleotidyltransferase-mediated dUTP nick end labelling (TUNEL) method. PI3-K, PKB, BAD and MAPK activities were measured in ischaemic and non-ischaemic lobes at various times after reperfusion. The number of TUNEL-positive cells was significantly decreased after pretreatment with Z-Asp-cmk. In controls, PI3-K and PKB activities and BAD phosphorylation were inhibited in ischaemic liver lobes. The MAPKs (extracellular signal-regulated kinases, c-Jun N-terminal kinase and p38) showed different patterns of activation during IR. PKB activity was not modified by pretreatment with Z-Asp-cmk. Induction of apoptosis during IR liver injury might be triggered by inactivation of the antiapoptotic PI3-K-PKB pathway and activation of the proapoptotic MAPKs. Copyright (c) 2006 British Journal of Surgery Society Ltd. Published by John Wiley & Sons, Ltd.

Phospholipase D1 modulates protein kinase C-epsilon in retinal pigment epithelium cells during inflammatory response.

PubMed

Tenconi, Paula E; Giusto, Norma M; Salvador, Gabriela A; Mateos, Melina V

2016-12-01

Inflammation is a key factor in the pathogenesis of several retinal diseases. In view of the essential role of the retinal pigment epithelium in visual function, elucidating the molecular mechanisms elicited by inflammation in this tissue could provide new insights for the treatment of retinal diseases. The aim of the present work was to study protein kinase C signaling and its modulation by phospholipases D in ARPE-19 cells exposed to lipopolysaccharide. This bacterial endotoxin induced protein kinase C-α/βII phosphorylation and protein kinase-ε translocation to the plasma membrane in ARPE-19 cells. Pre-incubation with selective phospholipase D inhibitors demonstrated that protein kinase C-α phosphorylation depends on phospholipase D1 and 2 while protein kinase C-ε activation depends only on phospholipase D1. The inhibition of α and β protein kinase C isoforms with Go 6976 did not modify the reduced mitochondrial function induced by lipopolysaccharide. On the contrary, the inhibition of protein kinase C-α, β and ε with Ro 31-8220 potentiated the decrease in mitochondrial function. Moreover, inhibition of protein kinase C-ε reduced Bcl-2 expression and Akt activation and increased Caspase-3 cleavage in cells treated or not with lipopolysaccharide. Our results demonstrate that through protein kinase C-ε regulation, phospholipase D1 protects retinal pigment epithelium cells from lipopolysaccharide-induced damage. Copyright © 2016 Elsevier Ltd. All rights reserved.

Salutary effect of Aurintricarboxylic acid (ATA) on endotoxin- and sepsis-induced changes in muscle protein synthesis and inflammation

PubMed Central

Laufenberg, Lacee; Kazi, Abid A.; Lang, Charles H.

2014-01-01

Small molecule nonpeptidyl molecules are potentially attractive drug candidates as adjunct therapies in the treatment of sepsis-induced metabolic complications. As such, the current study investigates the use of aurintricarboxylic acid (ATA), which stimulates insulin-like growth factor (IGF)-I receptor and AKT signaling, for its ability to ameliorate the protein metabolic effects of endotoxin (LPS) + interferon (IFN)γ in C2C12 myotubes and sepsis in skeletal muscle. ATA dose- and time-dependently increases mTOR-dependent protein synthesis. Pretreatment with ATA prevents the LPS/IFNγ-induced decrease in protein synthesis at least in part by maintaining mTOR kinase activity, while post-treatment with ATA is able to increase protein synthesis when added up to 6 h after LPS/IFNγ. ATA also reverses the amino acid resistance which is detected in response to nutrient deprivation. Conversely, ATA decreases the basal rate of protein degradation and prevents the LPS/IFNγ-increase in proteolysis, and the latter change is associated reduced atrogin-1 and MuRF1 mRNA. The ability of ATA to antagonize LPS/IFNγ-induced changes in protein metabolism were associated with its ability to prevent the increases in IL-6 and NOS2 and decreases in IGF-I. In vivo studies indicate ATA acutely increases skeletal muscle, but not cardiac, protein synthesis, and attenuates the loss of lean body mass over 5 days. These data suggest ATA and other small molecule agonists of endogenous anabolic hormones may prove beneficial in treating sepsis by decreasing the inflammatory response and improving muscle protein balance. PMID:24430547

Conformationally Induced Off-On Cell Membrane Chemosensor Targeting Receptor Protein-Tyrosine Kinases for in Vivo and in Vitro Fluorescence Imaging of Cancers.

PubMed

Jiao, Yang; Yin, Jiqiu; He, Haiyang; Peng, Xiaojun; Gao, Qianmiao; Duan, Chunying

2018-05-09

Molecules capable of monitoring receptor protein-tyrosine kinase expression could potentially serve as useful tools for cancer diagnosis due to the overexpression of tyrosine kinases during tumor growth and metastasis. In this work, a conformationally induced "off-on" tyrosine kinase cell membrane fluorescent sensor (SP1) was designed and evaluated for the detection and imaging of receptor protein-tyrosine kinases in vivo and in vitro. SP1 consists of sunitinib and pyrene linked via hexamethylenediamine and displays quenched fluorescence as a dimer. The fluorescence of SP1 is restored in the presence of receptor protein-tyrosine kinases upon strong interaction with SP1 at the target terminal. The unique signal response mechanism enables SP1 use for fluorescence microscopy imaging of receptor protein-tyrosine kinases in the cell membranes of living cells, allowing for the rapid differentiation of cancer cells from normal cells. SP1 can be used to visualize the chick embryo chorioallantoic membrane and mouse model tumors, suggesting its possible application for early cancer diagnosis.

Calcium homeostasis and protein kinase/phosphatase balance participate in nicotine-induced memory improvement in passive avoidance task in mice.

PubMed

Michalak, Agnieszka; Biala, Grazyna

2017-01-15

Long-term potentiation (LTP) and long-term depression (LTD) depend on specific postsynaptic Ca 2+ /calmodulin concentration. LTP results from Ca 2+ influx through the activated NMDA receptors or voltage-gated calcium channels (VGCCs) and is linked with activation of protein kinases including mitogen-activated protein kinase (MAPK). Weaker synaptic stimulation, as a result of low Ca 2+ influx, leads to activation of Ca 2+ /calmodulin-dependent phosphatase (calcineurin - CaN) and triggers LTD. Interestingly, both memory formation and drug addiction share similar neuroplastic changes. Nicotine, which is one of the most common addictive drugs, manifests its memory effects through nicotinic acetylcholine receptors (nAChRs). Because nAChRs may also gate Ca 2+ , it is suggested that calcium signaling pathways are involved in nicotine-induced memory effects. Within the scope of the study was to evaluate the importance of calcium homeostasis and protein kinase/phosphatase balance in nicotine-induced short- and long-term memory effects. To assess memory function in mice passive avoidance test was used. The presented results confirm that acute nicotine (0.1mg/kg) improves short- and long-term memory. Pretreatment with L-type VGCC blockers (amlodipine, nicardipine verapamil) increased nicotine-induced memory improvement in the context of short- and long-term memory. Pretreatment with FK-506 (a potent CaN inhibitor) enhanced short- but not long-term memory effects of nicotine, while SL-327 (a selective MAPK/ERK kinase inhibitor) attenuated both nicotine-induced short- and long-term memory improvement. Acute nicotine enhances both types of memory via L-type VGCC blockade and via ERK1/2 activation. Only short- but not long-term memory enhancement induced by nicotine is dependent on CaN inhibition. Copyright © 2016 Elsevier B.V. All rights reserved.

Kinase Pathway Database: An Integrated Protein-Kinase and NLP-Based Protein-Interaction Resource

PubMed Central

Koike, Asako; Kobayashi, Yoshiyuki; Takagi, Toshihisa

2003-01-01

Protein kinases play a crucial role in the regulation of cellular functions. Various kinds of information about these molecules are important for understanding signaling pathways and organism characteristics. We have developed the Kinase Pathway Database, an integrated database involving major completely sequenced eukaryotes. It contains the classification of protein kinases and their functional conservation, ortholog tables among species, protein–protein, protein–gene, and protein–compound interaction data, domain information, and structural information. It also provides an automatic pathway graphic image interface. The protein, gene, and compound interactions are automatically extracted from abstracts for all genes and proteins by natural-language processing (NLP).The method of automatic extraction uses phrase patterns and the GENA protein, gene, and compound name dictionary, which was developed by our group. With this database, pathways are easily compared among species using data with more than 47,000 protein interactions and protein kinase ortholog tables. The database is available for querying and browsing at http://kinasedb.ontology.ims.u-tokyo.ac.jp/. PMID:12799355

Inhibition of the AMP-activated protein kinase-α2 accentuates agonist-induced vascular smooth muscle contraction and high blood pressure in mice.

PubMed

Wang, Shuangxi; Liang, Bin; Viollet, Benoit; Zou, Ming-Hui

2011-05-01

The aim of the present study was to determine the effects and molecular mechanisms by which AMP-activated protein kinase (AMPK) regulates smooth muscle contraction and blood pressure in mice. In cultured human vascular smooth muscle cells, we observed that activation of AMPK by 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside inhibited agonist-induced phosphorylation of myosin light chain (MLC) and myosin phosphatase targeting subunit 1 (MYPT1). Conversely, AMPK inhibition with pharmacological or genetic means potentiated agonist-induced the phosphorylation of MLC and MYPT1, whereas it inhibited both Ras homolog gene family member A and Rho-associated kinase activity. In addition, AMPK activation or Rho-associated kinase inhibition with Y27632 abolished agonist-induced phosphorylation of MLC and MYPT1. Gene silencing of p190-guanosine triphosphatase-activating protein abolished the effects of AMPK activation on MLC, MYPT1, and Ras homolog gene family member A in human smooth muscle cells. Ex vivo analyses revealed that agonist-induced contractions of the mesenteric artery and aortas were stronger in both AMPKα1(-/-) and AMPKα2(-/-) knockout mice than in wild-type mice. Inhibition of Rho-associated kinase with Y27632 normalized agonist-induced contractions of AMPKα1(-/-) and AMPKα2(-/-) vessels. AMPKα2(-/-) mice had higher blood pressure along with decreased serine phosphorylation of p190-guanosine triphosphatase-activating protein. Finally, inhibition of the Ras homolog gene family member A/Rho-associated kinase pathway with Y27632, which suppressed MYPT1 and MLC phosphorylation, lowered blood pressure in AMPKα2(-/-) mice. In conclusion, AMPK decreases vascular smooth muscle cell contractility by inhibiting p190-GTP-activating protein-dependent Ras homolog gene family member A activation, indicating that AMPK may be a new therapeutic target in lowering high blood pressure.

A dual-specificity isoform of the protein kinase inhibitor PKI produced by alternate gene splicing.

PubMed

Kumar, Priyadarsini; Walsh, Donal A

2002-03-15

We have previously shown that the protein kinase inhibitor beta (PKIbeta) form of the cAMP-dependent protein kinase inhibitor exists in multiple isoforms, some of which are specific inhibitors of the cAMP-dependent protein kinase, whereas others also inhibit the cGMP-dependent enzyme [Kumar, Van Patten and Walsh (1997), J. Biol. Chem. 272, 20011-20020]. We have now demonstrated that the switch from a cAMP-dependent protein kinase (PKA)-specific inhibitor to one with dual specificity arises as a consequence of alternate gene splicing. We have confirmed using bacterially produced pure protein that a single inhibitor species has dual specificity for both PKA and cGMP-dependent protein kinase (PKG), inhibiting each with very high and closely similar inhibitory potencies. The gene splicing converted a protein with 70 amino acids into one of 109 amino acids, and did not change the inhibitory potency to PKA, but changed it from a protein that had no detectable PKG inhibitory activity to one that now inhibited PKG in the nanomolar range.

Isolation and characterization of a novel wheat cysteine-rich receptor-like kinase gene induced by Rhizoctonia cerealis

NASA Astrophysics Data System (ADS)

Yang, Kun; Rong, Wei; Qi, Lin; Li, Jiarui; Wei, Xuening; Zhang, Zengyan

2013-10-01

Cysteine-rich receptor kinases (CRKs) belong to the receptor-like kinase family. Little is known about CRK genes in wheat. We isolated a wheat CRK gene TaCRK1 from Rhizoctonia cerealis-resistant wheat CI12633 based on a differentially expressed sequence identified by RNA-Sequencing (RNA-Seq) analysis. TaCRK1 was more highly expressed in CI12633 than in susceptible Wenmai 6. Transcription of TaCRK1 in wheat was induced in CI12633 after R. cerealis infection and exogenous abscisic acid (ABA) treatment. The deduced TaCRK1 protein contained a signal peptide, two DUF26 domains, a transmembrane domain, and a serine/threonine protein kinase domain. Transient expression of a green fluorescence protein fused with TaCRK1 in wheat and onion indicated that TaCRK1 may localize to plasma membranes. Characterization of TaCRK1 silencing induced by virus-mediated method in CI12633 showed that the downregulation of TaCRK1 transcript did not obviously impair resistance to R. cerealis. This study paves the way to further CRK research in wheat.

Taurolithocholate-induced MRP2 retrieval involves MARCKS phosphorylation by protein kinase Cϵ in HUH-NTCP Cells.

PubMed

Schonhoff, Christopher M; Webster, Cynthia R L; Anwer, M Sawkat

2013-07-01

Taurolithocholate (TLC) acutely inhibits the biliary excretion of multidrug-resistant associated protein 2 (Mrp2) substrates by inducing Mrp2 retrieval from the canalicular membrane, whereas cyclic adenosine monophosphate (cAMP) increases plasma membrane (PM)-MRP2. The effect of TLC may be mediated via protein kinase Cϵ (PKCϵ). Myristoylated alanine-rich C kinase substrate (MARCKS) is a membrane-bound F-actin crosslinking protein and is phosphorylated by PKCs. MARCKS phosphorylation has been implicated in endocytosis, and the underlying mechanism appears to be the detachment of phosphorylated myristoylated alanine-rich C kinase substrate (pMARCKS) from the membrane. The aim of the present study was to test the hypothesis that TLC-induced MRP2 retrieval involves PKCϵ-mediated MARCKS phosphorylation. Studies were conducted in HuH7 cells stably transfected with sodium taurocholate cotransporting polypeptide (HuH-NTCP cells) and in rat hepatocytes. TLC increased PM-PKCϵ and decreased PM-MRP2 in both HuH-NTCP cells and hepatocytes. cAMP did not affect PM-PKCϵ and increased PM-MRP2 in these cells. In HuH-NTCP cells, dominant-negative (DN) PKCϵ reversed TLC-induced decreases in PM-MRP2 without affecting cAMP-induced increases in PM-MRP2. TLC, but not cAMP, increased MARCKS phosphorylation in HuH-NTCP cells and hepatocytes. TLC and phorbol myristate acetate increased cytosolic pMARCKS and decreased PM-MARCKS in HuH-NTCP cells. TLC failed to increase MARCKS phosphorylation in HuH-NTCP cells transfected with DN-PKCϵ, and this suggested PKCϵ-mediated phosphorylation of MARCKS by TLC. In HuH-NTCP cells transfected with phosphorylation-deficient MARCKS, TLC failed to increase MARCKS phosphorylation or decrease PM-MRP2. Taken together, these results support the hypothesis that TLC-induced MRP2 retrieval involves TLC-mediated activation of PKCϵ followed by MARCKS phosphorylation and consequent detachment of MARCKS from the membrane. Copyright © 2013 American Association

Suppressor of cytokine signaling 1 interacts with oncogenic lymphocyte-specific protein tyrosine kinase.

PubMed

Venkitachalam, Srividya; Chueh, Fu-Yu; Leong, King-Fu; Pabich, Samantha; Yu, Chao-Lan

2011-03-01

Lymphocyte-specific protein tyrosine kinase (Lck) plays a key role in T cell signal transduction and is tightly regulated by phosphorylation and dephosphorylation. Lck can function as an oncoprotein when overexpressed or constantly activated by mutations. Our previous studies showed that Lck-induced cellular transformation could be suppressed by enforced expression of suppressor of cytokine signaling 1 (SOCS1), a SOCS family member involved in the negative feedback control of cytokine signaling. We observed attenuated Lck kinase activity in SOCS1-expressing cells, suggesting an important role of SOCS in regulating Lck functions. It remains largely unknown whether and how SOCS proteins interact with the oncogenic Lck kinase. Here, we report that among four SOCS family proteins, SOCS1, SOCS2, SOCS3 and CIS (cytokine-inducible SH2 domain containing protein), SOCS1 has the highest affinity in binding to the oncogenic Lck kinase. We identified the positive regulatory phosphotyrosine 394 residue in the kinase domain as the key interacting determinant in Lck. Additionally, the Lck kinase domain alone is sufficient to bind SOCS1. While the SH2 domain in SOCS1 is important in its association with the oncogenic Lck kinase, other functional domains may also contribute to overall binding affinity. These findings provide important mechanistic insights into the role of SOCS proteins as tumor suppressors in cells transformed by oncogenic protein tyrosine kinases.

Suppressor of cytokine signaling 1 interacts with oncogenic lymphocyte-specific protein tyrosine kinase

PubMed Central

VENKITACHALAM, SRIVIDYA; CHUEH, FU-YU; LEONG, KING-FU; PABICH, SAMANTHA; YU, CHAO-LAN

2011-01-01

Lymphocyte-specific protein tyrosine kinase (Lck) plays a key role in T cell signal transduction and is tightly regulated by phosphorylation and dephosphorylation. Lck can function as an oncoprotein when overexpressed or constantly activated by mutations. Our previous studies showed that Lck-induced cellular transformation could be suppressed by enforced expression of suppressor of cytokine signaling 1 (SOCS1), a SOCS family member involved in the negative feedback control of cytokine signaling. We observed attenuated Lck kinase activity in SOCS1-expressing cells, suggesting an important role of SOCS in regulating Lck functions. It remains largely unknown whether and how SOCS proteins interact with the oncogenic Lck kinase. Here we report that, among four SOCS family proteins, SOCS1, SOCS2, SOCS3 and CIS (cytokine–inducible SH2 domain containing protein), SOCS1 has the highest affinity in binding to the oncogenic Lck kinase. We identify the positive regulatory phospho-tyrosine 394 residue in the kinase domain as the key interacting determinant in Lck. Additionally, the Lck kinase domain alone is sufficient to bind SOCS1. While the SH2 domain in SOCS1 is important in its association with the oncogenic Lck kinase, other functional domains may also contribute to overall binding affinity. These findings provide important mechanistic insights into the role of SOCS proteins as tumor suppressors in cells transformed by oncogenic protein tyrosine kinases. PMID:21234523

Rebamipide induces the gastric mucosal protective factor, cyclooxygenase-2, via activation of 5'-AMP-activated protein kinase.

PubMed

Lee, Sunyoung; Jeong, Seongkeun; Kim, Wooseong; Kim, Dohoon; Yang, Yejin; Yoon, Jeong-Hyun; Kim, Byung Joo; Min, Do Sik; Jung, Yunjin

2017-01-29

Rebamipide, an amino acid derivative of 2(1H)-quinolinone, has been used for mucosal protection, healing of gastroduodenal ulcers, and treatment of gastritis. Induction of cyclooxygenase (COX)-2, a gastric mucosal protective factor, by rebamipide has been suggested as the major mechanism of the drug action. However, how rebamipide induces COX-2 at the molecular level needs further investigation. In this study, the molecular mechanism underlying the induction of COX-2 by rebamipide was investigated. In gastric carcinoma cells and macrophage cells, rebamipide induced phosphorylation of AMP-activated protein kinase (AMPK), leading to phosphorylation of acetyl-CoA carboxylase (ACC), a substrate of AMPK. The induction of COX-2 by rebamipide was dependent on AMPK activation because compound C, an AMPK inhibitor, abolished COX-2 induction by rebamipide. In a mouse ulcer model, rebamipide protected against hydrochloric acid/ethanol-induced gastric ulcer, and these protective effects were deterred by co-administration of compound C. In parallel, in the gastric tissues, rebamipide increased the phosphorylation AMPK, whereas compound C reduced the levels of COX-2 and phosphorylated ACC, which were increased by rebamipide. Taken together, the activation of AMPK by rebamipide may be a molecular mechanism that contributes to induction of COX-2, probably resulting in protection against gastric ulcers. Copyright © 2016 Elsevier Inc. All rights reserved.

Anti-inflammatory properties of Gö 6850: a selective inhibitor of protein kinase C.

PubMed

Jacobson, P B; Kuchera, S L; Metz, A; Schächtele, C; Imre, K; Schrier, D J

1995-11-01

Protein kinase C (PKC) regulates a variety of signal transduction events implicated in the pathogenesis of inflammation, including the biosynthesis of inflammatory cytokines and superoxide and the activation of phospholipase A2. Because of the significant role of PKC in these inflammatory processes, we evaluated a specific and potent inhibitor of C kinase for efficacy in several in vitro and in vivo murine models of inflammation. Unlike the relatively nonspecific kinase inhibitor staurosporine, the bisindolylmaleimide 3-[1-[-3-(dimethylaminopropyl]-1H-indol-3-yl]- 4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione monohydrochloride (Gö 6850) demonstrated increased selectivity for C kinase in purified enzyme assays (respective IC50 values (microM) for Gö 6850 and staurosporine: protein kinase C (0.032, 0.009); myosin light-chain kinase (0.6, 0.01); protein kinase G (4.6, 0.018); protein kinase A (33, 0.04); tyrosine kinase1 (94, 0.4); tyrosine kinase2 (> 100, > 1)). Topically applied Gö 6850 inhibited phorbol myristate acetate-induced edema, neutrophil influx and vascular permeability in murine epidermis in a dose- and time-dependent manner at levels comparable to indomethacin. In a murine model of delayed type hypersensitivity, Gö 6850 inhibited dinitrofluorobenzene-induced contact dermatitis with and ID50 value of 150 micrograms/ear. Cellular studies in mouse peritoneal macrophages demonstrated that Gö 6850 was a potent inhibitor of phorbol myristate acetate-induced prostaglandin E2 production. Superoxide production in phorbol myristate acetate-stimulated murine neutrophils was also inhibited by Gö 6850 (IC50 = 88 nM).(ABSTRACT TRUNCATED AT 250 WORDS)

Mulberry leaf polyphenol extract induced apoptosis involving regulation of adenosine monophosphate-activated protein kinase/fatty acid synthase in a p53-negative hepatocellular carcinoma cell.

PubMed

Yang, Tzi-Peng; Lee, Huei-Jane; Ou, Ting-Tsz; Chang, Ya-Ju; Wang, Chau-Jong

2012-07-11

The polyphenols in mulberry leaf possess the ability to inhibit cell proliferation, invasion, and metastasis of tumors. It was reported that the p53 status plays an important role in switching apoptosis and the cell cycle following adenosine monophosphate-activated protein kinase (AMPK) activation. In this study, we aimed to detect the effect of the mulberry leaf polyphenol extract (MLPE) on inducing cell death in p53-negative (Hep3B) and p53-positive (Hep3B with transfected p53) hepatocellular carcinoma cells and also to clarify the role of p53 in MLPE-treated cells. After treatment of the Hep3B cells with MLPE, apoptosis was induced via the AMPK/PI3K/Akt and Bcl-2 family pathways. Transient transfection of p53 into Hep3B cells led to switching autophagy instead of apoptosis by MLPE treatment. We demonstrated that acridine orange staining and protein expressions of LC-3 and beclin-1 were increased in p53-transfected cells. These results implied induction of apoptosis or autophagy in MLPE-treated hepatocellular carcinoma cells can be due to the p53 status. We also found MLPE can not only activate AMPK but also diminish fatty acid synthase, a molecular target for cancer inhibition. At present, our results indicate MLPE can play an active role in mediating the cell death of hepatocellular carcinoma cells and the p53 might play an important role in regulating the death mechanisms.

Corticosteroids inhibit sphingosine 1-phosphate-induced interleukin-6 secretion from human airway smooth muscle via mitogen-activated protein kinase phosphatase 1-mediated repression of mitogen and stress-activated protein kinase 1.

PubMed

Che, Wenchi; Parmentier, Johannes; Seidel, Petra; Manetsch, Melanie; Ramsay, Emma E; Alkhouri, Hatem; Ge, Qi; Armour, Carol L; Ammit, Alaina J

2014-02-01

Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid that plays an important proinflammatory role in asthmatic airways. Corticosteroids are first-line antiinflammatories in asthma; however, their repressive effects on S1P-induced cytokine secretion have not been investigated. To address this, our in vitro study reveals the molecular mechanisms by which corticosteroids inhibit S1P-induced IL-6 expression in the pivotal immunomodulatory cell type, airway smooth muscle (ASM). We first uncover the cellular signaling pathways responsible: S1P activates a cyclic adenosine monophosphate/cAMP response-element-binding protein (CREB)/CRE-dependent pathway to induce IL-6 transcription, concomitant with stimulation of the mitogen-activated protein kinase (MAPK) superfamily and downstream mitogen and stress-activated protein kinase 1 (MSK1) and histone H3 phosphorylation. In this way, S1P stimulates parallel signaling pathways to induce IL-6 secretion via CRE-driven transcription of the IL-6 gene promoter in a relaxed chromatin environment achieved through histone H3 phosphorylation. Second, we investigated how corticosteroids mediate their repressive effects. The corticosteroid dexamethasone inhibits S1P-induced IL-6 protein secretion and mRNA expression, but CREB/CRE transrepression, inhibition of IL-6 mRNA stability, or subcellular relocation of MSK1 were not responsible for the repressive effects of dexamethasone. Rather, we show that dexamethasone rapidly induces up-regulation of the MAPK deactivator MAPK phosphatase 1 (MKP-1) and that MKP-1 blocks the MAPK-driven activation of MSK1 and phosphorylation of histone H3. This was confirmed by treatment with triptolide, an inhibitor of MKP-1 up-regulation, where repressive effects of corticosteroids were reversed. Our study reveals the molecular mechanism underlying the antiinflammatory capacity of corticosteroids to repress proinflammatory functions induced by the potent bioactive sphingolipid S1P in the lung.

Signaling by Kit protein-tyrosine kinase--the stem cell factor receptor.

PubMed

Roskoski, Robert

2005-11-11

Signaling by stem cell factor and Kit, its receptor, plays important roles in gametogenesis, hematopoiesis, mast cell development and function, and melanogenesis. Moreover, human and mouse embryonic stem cells express Kit transcripts. Stem cell factor exists as both a soluble and a membrane-bound glycoprotein while Kit is a receptor protein-tyrosine kinase. The complete absence of stem cell factor or Kit is lethal. Deficiencies of either produce defects in red and white blood cell production, hypopigmentation, and sterility. Gain-of-function mutations of Kit are associated with several human neoplasms including acute myelogenous leukemia, gastrointestinal stromal tumors, and mastocytomas. Kit consists of an extracellular domain, a transmembrane segment, a juxtamembrane segment, and a protein kinase domain that contains an insert of about 80 amino acid residues. Binding of stem cell factor to Kit results in receptor dimerization and activation of protein kinase activity. The activated receptor becomes autophosphorylated at tyrosine residues that serve as docking sites for signal transduction molecules containing SH2 domains. The adaptor protein APS, Src family kinases, and Shp2 tyrosyl phosphatase bind to phosphotyrosine 568. Shp1 tyrosyl phosphatase and the adaptor protein Shc bind to phosphotyrosine 570. C-terminal Src kinase homologous kinase and the adaptor Shc bind to both phosphotyrosines 568 and 570. These residues occur in the juxtamembrane segment of Kit. Three residues in the kinase insert domain are phosphorylated and attract the adaptor protein Grb2 (Tyr703), phosphatidylinositol 3-kinase (Tyr721), and phospholipase Cgamma (Tyr730). Phosphotyrosine 900 in the distal kinase domain binds phosphatidylinositol 3-kinase which in turn binds the adaptor protein Crk. Phosphotyrosine 936, also in the distal kinase domain, binds the adaptor proteins APS, Grb2, and Grb7. Kit has the potential to participate in multiple signal transduction pathways as a result of

The DNA-dependent protein kinase: a multifunctional protein kinase with roles in DNA double strand break repair and mitosis

PubMed Central

Jette, Nicholas; Lees-Miller, Susan P.

2015-01-01

The DNA-dependent protein kinase (DNA-PK) is a serine/threonine protein kinase composed of a large catalytic subunit (DNA-PKcs) and the Ku70/80 heterodimer. Over the past two decades, significant progress has been made in elucidating the role of DNA-PK in non-homologous end joining (NHEJ), the major pathway for repair of ionizing radiation-induced DNA double strand breaks in human cells and recently, additional roles for DNA-PK have been reported. In this review, we will describe the biochemistry, structure and function of DNA-PK, its roles in DNA double strand break repair and its newly described roles in mitosis and other cellular processes. PMID:25550082

Protein Kinase Cϵ (PKCϵ) Promotes Synaptogenesis through Membrane Accumulation of the Postsynaptic Density Protein PSD-95.

PubMed

Sen, Abhik; Hongpaisan, Jarin; Wang, Desheng; Nelson, Thomas J; Alkon, Daniel L

2016-08-05

Protein kinase Cϵ (PKCϵ) promotes synaptic maturation and synaptogenesis via activation of synaptic growth factors such as BDNF, NGF, and IGF. However, many of the detailed mechanisms by which PKCϵ induces synaptogenesis are not fully understood. Accumulation of PSD-95 to the postsynaptic density (PSD) is known to lead to synaptic maturation and strengthening of excitatory synapses. Here we investigated the relationship between PKCϵ and PSD-95. We show that the PKCϵ activators dicyclopropanated linoleic acid methyl ester and bryostatin 1 induce phosphorylation of PSD-95 at the serine 295 residue, increase the levels of PSD-95, and enhance its membrane localization. Elimination of the serine 295 residue in PSD-95 abolished PKCϵ-induced membrane accumulation. Knockdown of either PKCϵ or JNK1 prevented PKCϵ activator-mediated membrane accumulation of PSD-95. PKCϵ directly phosphorylated PSD-95 and JNK1 in vitro Inhibiting PKCϵ, JNK, or calcium/calmodulin-dependent kinase II activity prevented the effects of PKCϵ activators on PSD-95 phosphorylation. Increase in membrane accumulation of PKCϵ and phosphorylated PSD-95 (p-PSD-95(S295)) coincided with an increased number of synapses and increased amplitudes of excitatory post-synaptic potentials (EPSPs) in adult rat hippocampal slices. Knockdown of PKCϵ also reduced the synthesis of PSD-95 and the presynaptic protein synaptophysin by 30 and 44%, respectively. Prolonged activation of PKCϵ increased synapse number by 2-fold, increased presynaptic vesicle density, and greatly increased PSD-95 clustering. These results indicate that PKCϵ promotes synaptogenesis by activating PSD-95 phosphorylation directly through JNK1 and calcium/calmodulin-dependent kinase II and also by inducing expression of PSD-95 and synaptophysin. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Ghrelin augments murine T-cell proliferation by activation of the phosphatidylinositol-3-kinase, extracellular signal-regulated kinase and protein kinase C signaling pathways

PubMed Central

Lee, Jun Ho; Patel, Kalpesh; Tae, Hyun Jin; Lustig, Ana; Kim, Jie Wan; Mattson, Mark P.; Taub, Dennis D.

2014-01-01

Thymic atrophy occurs during normal aging, and is accelerated by exposure to chronic stressors that elevate glucocorticoid levelsand impair the naïve T cell output. The orexigenic hormone ghrelin was recently shown to attenuate age-associated thymic atrophy. Here, we report that ghrelin enhances the proliferation of murine CD4+ primary T cells and a CD4+ T-cell line. Ghrelin induced activation of the ERK1/2 and Akt signaling pathways, via upstream activation of phosphatidylinositol-3-kinase and protein kinase C, to enhance T-cell proliferation. Moreover, ghrelin induced expression of the cell cycle proteins cyclin D1, cyclin E, cyclin-dependent kinase 2 (CDK2) and retinoblastoma phosphorylation. Finally, ghrelin activated the above-mentioned signaling pathways and stimulated thymocyte proliferation in young and older mice in vivo. PMID:25447526

Spatial Distribution of Protein Kinase A Activity during Cell Migration Is Mediated by A-kinase Anchoring Protein AKAP Lbc*

PubMed Central

Paulucci-Holthauzen, Adriana A.; Vergara, Leoncio A.; Bellot, Larry J.; Canton, David; Scott, John D.; O'Connor, Kathleen L.

2009-01-01

Protein kinase A (PKA) has been suggested to be spatially regulated in migrating cells due to its ability to control signaling events that are critical for polarized actin cytoskeletal dynamics. Here, using the fluorescence resonance energy transfer-based A-kinase activity reporter (AKAR1), we find that PKA activity gradients form with the strongest activity at the leading edge and are restricted to the basal surface in migrating cells. The existence of these gradients was confirmed using immunocytochemistry using phospho-PKA substrate antibodies. This observation holds true for carcinoma cells migrating randomly on laminin-1 or stimulated to migrate on collagen I with lysophosphatidic acid. Phosphodiesterase inhibition allows the formation of PKA activity gradients; however, these gradients are no longer polarized. PKA activity gradients are not detected when a non-phosphorylatable mutant of AKAR1 is used, if PKA activity is inhibited with H-89 or protein kinase inhibitor, or when PKA anchoring is perturbed. We further find that a specific A-kinase anchoring protein, AKAP-Lbc, is a major contributor to the formation of these gradients. In summary, our data show that PKA activity gradients are generated at the leading edge of migrating cells and provide additional insight into the mechanisms of PKA regulation of cell motility. PMID:19106088

Autoregulation of kinase dephosphorylation by ATP binding in AGC protein kinases.

PubMed

Chan, Tung O; Pascal, John M; Armen, Roger S; Rodeck, Ulrich

2012-02-01

AGC kinases, including the three Akt (protein kinase B) isoforms, protein kinase A (PKA) and all protein kinase C (PKC) isoforms, require activation loop phosphorylation (threonine 308 in Akt1) as well as phosphorylation of a C-terminal residue (serine 473 in Akt1) for catalytic activity and phosphorylation of downstream targets. Conversely, phosphatases reverse these phosphorylations. Virtually all cellular processes are affected by AGC kinases, a circumstance that has led to intense scrutiny of the molecular mechanisms that regulate phosphorylation of these kinases. Here, we review a new layer of control of phosphorylation in Akt, PKA and PKC pointing to ATP binding pocket occupancy as a means to decelerate dephosphorylation of these and, potentially, other kinases. This additional level of kinase regulation opens the door to search for new functional motifs for the rational design of non- ATP-competitive kinase inhibitors that discriminate within and between protein kinase families.

Glycyrrhetinic acid attenuates lipopolysaccharide-induced fulminant hepatic failure in D-galactosamine-sensitized mice by up-regulating expression of interleukin-1 receptor-associated kinase-M

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yin, Xinru

Glycyrrhetinic acid (GA), the main active ingredient of licorice, reportedly has anti-inflammatory and hepatoprotective properties, but its molecular mechanisms remain be elusive. In the present study, Balb/c mice were pretreated with GA (10, 30, or 100 mg/kg) 1 h before lipopolysaccharide (LPS)/D-galactosamine (D-GalN) administration. In other in vitro experiment, RAW264.7 macrophages were pretreated with GA before LPS exposure. The mortality, hepatic tissue histology, serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were analyzed. Toll like receptor 4 (TLR4), interleukin-1 receptor-associated kinases (IRAKs), activation of mitogen-activated protein kinases (MAPKs) and NF-κB, and production of TNF-α were assessed by flow cytometry, westernmore » blotting, and enzyme-linked immunosorbent assay (ELISA), respectively. Our results showed that pretreatment with GA protected mice against LPS/D-GalN-induced fulminant hepatic failure (FHF), including a dose-dependent alleviation of mortality and ALT/AST elevation, ameliorating hepatic pathological damage, and decreasing TNF-α release. Moreover, GA inhibited LPS-induced activation of MAPKs and NF-κB in response to LPS, but the expression of TLR4 was not affected in vivo and in vitro. Notably, GA pretreatment in vivo suppressed IRAK-1 activity while inducing IRAK-M expression. Silencing of IRAK-M expression with siRNA blocked these beneficial effects of GA on the activation of MAPKs and NF-κB as well as TNF-α production in LPS-primed macrophages. Taken together, we conclude that GA could prevent LPS/D-GalN-induced FHF. The underlying mechanisms may be related to up-regulation of IRAK-M, which in turn caused deactivation of IRAK-1 and subsequent MAPKs and NF-κB, resulting in inhibiting TNF-α production. - Highlights: • Glycyrrhetinic acid protected from LPS/D-GalN-induced liver injury in mice. • Glycyrrhetinic acid inhibited LPS-induced TNF-α production in vivo and in vitro. �

Role of activator protein-1 on the effect of arginine-glycine-aspartic acid containing peptides on transforming growth factor-beta1 promoter activity.

PubMed

Ruiz-Torres, M P; Perez-Rivero, G; Diez-Marques, M L; Griera, M; Ortega, R; Rodriguez-Puyol, M; Rodríguez-Puyol, D

2007-01-01

While arginine-glycine-aspartic acid-based peptidomimetics have been employed for the treatment of cardiovascular disorders and cancer, their use in other contexts remains to be explored. Arginine-glycine-aspartic acid-serine induces Transforming growth factor-beta1 transcription in human mesangial cells, but the molecular mechanisms involved have not been studied extensively. We explored whether this effect could be due to Activator protein-1 activation and studied the potential pathways involved. Addition of arginine-glycine-aspartic acid-serine promoted Activator protein-1 binding to its cognate sequence within the Transforming growth factor-beta1 promoter as well as c-jun and c-fos protein abundance. Moreover, this effect was suppressed by curcumin, a c-Jun N terminal kinase inhibitor, and was absent when the Activator protein-1 cis-regulatory element was deleted. Activator protein-1 binding was dependent on the activity of integrin linked kinase, as transfection with a dominant negative mutant suppressed both Activator protein-1 binding and c-jun and c-fos protein increment. Integrin linked kinase was, in turn, dependent on Phosphoinositol-3 kinase activity. Arginine-glycine-aspartic acid-serine stimulated Phosphoinositol-3 kinase activity, and Transforming growth factor-beta1 promoter activation was abrogated by the use of Phosphoinositol-3 kinase specific inhibitors. In summary, we propose that arginine-glycine-aspartic acid-serine activates Integrin linked kinase via the Phosphoinositol-3 kinase pathway and this leads to activation of c-jun and c-fos and increased Activator protein-1 binding and Transforming growth factor-beta1 promoter activity. These data may contribute to understand the molecular mechanisms involved in the cellular actions of arginine-glycine-aspartic acid-related peptides and enhance their relevance as these products evolve into clinical therapeutic use.

The new indirubin derivative inhibitors of glycogen synthase kinase-3, 6-BIDECO and 6-BIMYEO, prevent tau phosphorylation and apoptosis induced by the inhibition of protein phosphatase-2A by okadaic acid in cultured neurons.

PubMed

Martin, Ludovic; Magnaudeix, Amandine; Wilson, Cornelia M; Yardin, Catherine; Terro, Faraj

2011-11-01

Alterations in glycogen synthase kinase-3β (GSK3β) and protein phosphatase-2A (PP2A) have been proposed to be involved in the abnormal tau phosphorylation and aggregation linked to Alzheimer's disease (AD). Interconnections between GSK3β and PP2A signaling pathways are well established. Targeting tau kinases was proposed to represent a therapeutic strategy for AD. However, which tau kinases should be blocked and to what extent, keeping in mind that kinases have physiological roles? Because most kinase inhibitors are relatively specific and many of them interfere with the cell cycle, it is necessary to develop more specific tau kinase inhibitors devoid of cell toxicity. Here, we used the PP2A inhibition by okadaic acid (OKA) in primary cultured cortical neurons as an in vitro model of increased tau phosphorylation and apoptosis. We tested the effects of two newly characterized indirubin derivative inhibitors of GSK3, 6-BIDECO (6-bromoindirubin-3'-[O-(N,N-diethylcarbamyl)-oxime] and 6-BIMYEO (6-bromoindirubin-3'-[O-(2-morpholin-1-ylethyl)-oxime] hydrochloride) on OKA-induced tau phosphorylation and neuronal apoptosis. Both compounds exhibit higher selectivity toward GSK3 compared with other tau kinases (for 6-BIDECO, IC50 is 0.03 μM for GSK3, >10 μM for CDK1, and 10 μM for CDK5; for 6-BIMYEO, IC50 is 0.11 μM for GSK3, 1.8 μM for CDK1, and 0.9 μM for CDK5). We show that 6-BIDECO and 6-BIMYEO used at micromolar concentrations are not neurotoxic and potently reversed tau phosphorylation and apoptosis induced by OKA. The neuroprotection by these compounds should be further validated in animal models of AD. Copyright © 2011 Wiley-Liss, Inc.

Regulation of cotton (Gossypium hirsutum) drought responses by mitogen-activated protein (MAP) kinase cascade-mediated phosphorylation of GhWRKY59.

PubMed

Li, Fangjun; Li, Maoying; Wang, Ping; Cox, Kevin L; Duan, Liusheng; Dever, Jane K; Shan, Libo; Li, Zhaohu; He, Ping

2017-09-01

Drought is a key limiting factor for cotton (Gossypium spp.) production, as more than half of the global cotton supply is grown in regions with high water shortage. However, the underlying mechanism of the response of cotton to drought stress remains elusive. By combining genome-wide transcriptome profiling and a loss-of-function screen using virus-induced gene silencing, we identified Gossypium hirsutum GhWRKY59 as an important transcription factor that regulates the drought stress response in cotton. Biochemical and genetic analyses revealed a drought stress-activated mitogen-activated protein (MAP) kinase cascade consisting of GhMAP3K15-Mitogen-activated Protein Kinase Kinase 4 (GhMKK4)-Mitogen-activated Protein Kinase 6 (GhMPK6) that directly phosphorylates GhWRKY59 at residue serine 221. Interestingly, GhWRKY59 is required for dehydration-induced expression of GhMAPK3K15, constituting a positive feedback loop of GhWRKY59-regulated MAP kinase activation in response to drought stress. Moreover, GhWRKY59 directly binds to the W-boxes of DEHYDRATION-RESPONSIVE ELEMENT-BINDING PROTEIN 2 (GhDREB2), which encodes a dehydration-inducible transcription factor regulating the plant hormone abscisic acid (ABA)-independent drought response. Our study identified a complete MAP kinase cascade that phosphorylates and activates a key WRKY transcription factor, and elucidated a regulatory module, consisting of GhMAP3K15-GhMKK4-GhMPK6-GhWRKY59-GhDREB2, that is involved in controlling the cotton drought response. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

Kinase Associated-1 Domains Drive MARK/PAR1 Kinases to Membrane Targets by Binding Acidic Phospholipids

DOE Office of Scientific and Technical Information (OSTI.GOV)

Moravcevic, Katarina; Mendrola, Jeannine M.; Schmitz, Karl R.

Phospholipid-binding modules such as PH, C1, and C2 domains play crucial roles in location-dependent regulation of many protein kinases. Here, we identify the KA1 domain (kinase associated-1 domain), found at the C terminus of yeast septin-associated kinases (Kcc4p, Gin4p, and Hsl1p) and human MARK/PAR1 kinases, as a membrane association domain that binds acidic phospholipids. Membrane localization of isolated KA1 domains depends on phosphatidylserine. Using X-ray crystallography, we identified a structurally conserved binding site for anionic phospholipids in KA1 domains from Kcc4p and MARK1. Mutating this site impairs membrane association of both KA1 domains and intact proteins and reveals the importancemore » of phosphatidylserine for bud neck localization of yeast Kcc4p. Our data suggest that KA1 domains contribute to coincidence detection, allowing kinases to bind other regulators (such as septins) only at the membrane surface. These findings have important implications for understanding MARK/PAR1 kinases, which are implicated in Alzheimer's disease, cancer, and autism.« less

The role of protein kinase C in the opening of blood-brain barrier induced by electromagnetic pulse.

PubMed

Qiu, Lian-Bo; Ding, Gui-Rong; Li, Kang-Chu; Wang, Xiao-Wu; Zhou, Yan; Zhou, Yong-Chun; Li, Yu-Rong; Guo, Guo-Zhen

2010-06-29

The aim of this study was to determine the role of protein kinase C signaling in electromagnetic pulse (EMP)-induced blood-brain barrier (BBB) permeability change in rats. The protein level of total PKC and two PKC isoforms (PKC-alpha, and PKC-beta II) were determined in brain cerebral cortex microvessels by Western blot after exposing rats to EMP at 200kV/m for 200 pulses with 1Hz repetition rate. It was found that the protein level of PKC and PKC-betaII (but not PKC-alpha) in cerebral cortex microvessels increased significantly at 0.5h and 1h after EMP exposure compared with sham-exposed animals and then recovered at 3h. A specific PKC antagonist (H7) almost blocked EMP-induced BBB permeability change. EMP-induced BBB tight junction protein ZO-1 translocation was also inhibited. Our data indicated that PKC signaling was involved in EMP-induced BBB permeability change and ZO-1 translocation in rat.

Autoregulation of kinase dephosphorylation by ATP binding to AGC protein kinases

PubMed Central

Pascal, John M; Armen, Roger S

2012-01-01

AGC kinases, including the three Akt (protein kinase B) isoforms, protein kinase A (PKA) and all protein kinase C (PKC) isoforms, require activation loop phosphorylation (threonine 308 in Akt1) as well as phosphorylation of a C-terminal residue (serine 473 in Akt1) for catalytic activity and phosphorylation of downstream targets. Conversely, phosphatases reverse these phosphorylations. Virtually all cellular processes are affected by AGC kinases, a circumstance that has led to intense scrutiny of the molecular mechanisms that regulate phosphorylation of these kinases. Here, we review a new layer of control of phosphorylation in Akt, PKA and PKC pointing to ATP binding pocket occupancy as a means to decelerate dephosphorylation of these and, potentially, other kinases. This additional level of kinase regulation opens the door to search for new functional motifs for the rational design of non-ATP-competitive kinase inhibitors that discriminate within and between protein kinase families. PMID:22262182

Activation of G-proteins by receptor-stimulated nucleoside diphosphate kinase in Dictyostelium.

PubMed Central

Bominaar, A A; Molijn, A C; Pestel, M; Veron, M; Van Haastert, P J

1993-01-01

Recently, interest in the enzyme nucleoside diphosphate kinase (EC2.7.4.6) has increased as a result of its possible involvement in cell proliferation and development. Since NDP kinase is one of the major sources of GTP in cells, it has been suggested that the effects of an altered NDP kinase activity on cellular processes might be the result of altered transmembrane signal transduction via guanine nucleotide-binding proteins (G-proteins). In the cellular slime mould Dictyostelium discoideum, extracellular cAMP induces an increase of phospholipase C activity via a surface cAMP receptor and G-proteins. In this paper it is demonstrated that part of the cellular NDP kinase is associated with the membrane and stimulated by cell surface cAMP receptors. The GTP produced by the action of NDP kinase is capable of activating G-proteins as monitored by altered G-protein-receptor interaction and the activation of the effector enzyme phospholipase C. Furthermore, specific monoclonal antibodies inhibit the effect of NDP kinase on G-protein activation. These results suggest that receptor-stimulated NDP kinase contributes to the mediation of hormone action by producing GTP for the activation of GTP-binding proteins. Images PMID:8389692

Spatio-temporal imaging of EGF-induced activation of protein kinase A by FRET in living cells

NASA Astrophysics Data System (ADS)

Wang, Jin Jun; Chen, Xiao-Chuan; Xing, Da

2004-07-01

Intracellular molecular interaction is important for the study of cell physiology, yet current relevant methods require fixation or microinjection and lack temporal or spatial resolution. We introduced a new method -- fluorescence resonance energy transfer (FRET) to detect molecular interaction in living cells. On the basis of FRET principle, A-kinase activity reporter (AKAR) protein was designed to consist of the fusions of cyan fluorescent protein (CFP), a phosphoamino acid binding domain, a consensus substrate for protein kinase-A (PKA), and yellow fluorescent protein (YFP). In this study, the designed pAKAR plasmid was used to transfect a human lung cancer cell line (ASTC-a-1). When the AKAR-transfected cells were treated by forskolin (Fsk), we were able to observe the efficient transfer of energy from excited CFP to YFP within the AKAR molecule by fluorescence microcopy, whereas no FRET was detected in the transfected cells without the treatment of Fsk. When the cells were treated by Epidermal growth factor (EGF), the change of FRET was observed at different subcellular locations, reflecting PKA activation inside the cells upon EGF stimulation. The successful design of a fluorescence reporter of PKA activation and its application demonstrated the superiority of this technology in the research of intracellular protein-protein interaction.

Matriptase is required for the active form of hepatocyte growth factor induced Met, focal adhesion kinase and protein kinase B activation on neural stem/progenitor cell motility.

PubMed

Fang, Jung-Da; Lee, Sheau-Ling

2014-07-01

Hepatocyte growth factor (HGF) is a chemoattractant and inducer for neural stem/progenitor (NS/P) cell migration. Although the type II transmembrane serine protease, matriptase (MTP) is an activator of the latent HGF, MTP is indispensable on NS/P cell motility induced by the active form of HGF. This suggests that MTP's action on NS/P cell motility involves mechanisms other than proteolytic activation of HGF. In the present study, we investigate the role of MTP in HGF-stimulated signaling events. Using specific inhibitors of phosphatidylinositol-3-kinase (PI3K), protein kinase B (Akt) or focal adhesion kinase (FAK), we demonstrated that in NS/P cells HGF-activated c-Met induces PI3k-Akt signaling which then leads to FAK activation. This signaling pathway ultimately induces MMP2 expression and NS/P cell motility. Knocking down of MTP in NS/P cells with specific siRNA impaired HGF-stimulation of c-Met, Akt and FAK activation, blocked HGF-induced production of MMP2 and inhibited HGF-stimulated NS/P cell motility. MTP-knockdown NS/P cells cultured in the presence of recombinant protein of MTP protease domain or transfected with the full-length wild-type but not the protease-defected MTP restored HGF-responsive events in NS/P cells. In addition to functioning as HGF activator, our data revealed novel function of MTP on HGF-stimulated c-Met signaling activation. Copyright © 2014. Published by Elsevier B.V.

A Eukaryotic-Type Serine/Threonine Protein Kinase Is Required for Biofilm Formation, Genetic Competence, and Acid Resistance in Streptococcus mutans

PubMed Central

Hussain, Haitham; Branny, Pavel; Allan, Elaine

2006-01-01

We report an operon encoding a eukaryotic-type serine/threonine protein kinase (STPK) and its cognate phosphatase (STPP) in Streptococcus mutans. Mutation of the gene encoding the STPK produced defects in biofilm formation, genetic competence, and acid resistance, determinants important in caries pathogenesis. PMID:16452447

Identification of Open Stomata1-Interacting Proteins Reveals Interactions with Sucrose Non-fermenting1-Related Protein Kinases2 and with Type 2A Protein Phosphatases That Function in Abscisic Acid Responses

DOE PAGES

Waadt, Rainer; Manalansan, Bianca; Rauniyar, Navin; ...

2015-09-04

The plant hormone abscisic acid (ABA) controls growth and development and regulates plant water status through an established signaling pathway. In the presence of ABA, pyrabactin resistance/regulatory component of ABA receptor proteins inhibit type 2C protein phosphatases (PP2Cs). This, in turn, enables the activation of Sucrose Nonfermenting1-Related Protein Kinases2 (SnRK2). Open Stomata1 (OST1)/SnRK2.6/SRK2E is a major SnRK2-type protein kinase responsible for mediating ABA responses. Arabidopsis (Arabidopsis thaliana) expressing an epitope-tagged OST1 in the recessive ost1-3 mutant background was used for the copurification and identification of OST1-interacting proteins after osmotic stress and ABA treatments. Furthemore, these analyses, which were confirmed usingmore » bimolecular fluorescence complementation and coimmunoprecipitation, unexpectedly revealed homo- and heteromerization of OST1 with SnRK2.2, SnRK2.3, OST1, and SnRK2.8. Furthermore, several OST1-complexed proteins were identified as type 2A protein phosphatase (PP2A) subunits and as proteins involved in lipid and galactolipid metabolism. More detailed analyses suggested an interaction network between ABA-activated SnRK2-type protein kinases and several PP2A-type protein phosphatase regulatory subunits. pp2a double mutants exhibited a reduced sensitivity to ABA during seed germination and stomatal closure and an enhanced ABA sensitivity in root growth regulation. Our analyses add PP2A-type protein phosphatases as another class of protein phosphatases to the interaction network of SnRK2-type protein kinases.« less

[Design of new anti-tumor agents interrupting deregulated signaling pathways induced by tyrosine kinase proteins. Inhibition of protein-protein interaction involving Grb2].

PubMed

Vidal, Michel; Liu, Wang Qing; Gril, Brunile; Assayag, Franck; Poupon, Marie-France; Garbay, Christiane

2004-01-01

Cellular signaling pathways induced by growth-factor receptors are frequently deregulated in cancer. Anti-tumor agents that inhibit their enzymatic tyrosine kinase activity have been designed and are now used in human chemotherapy. We propose here an alternative way to interrupt over-expressed signaling by inhibiting protein-protein interactions that involve either the over-expressed proteins or proteins located downstream. The adaptor protein Grb2 over-expressed in connection with HER2/ErbB2/neu in Ras signaling pathway was chosen as a target. Peptides with very high affinity for Grb2 were rationally designed from structural data. Their capacity to interrupt the signaling pathway, their anti-proliferative activity as well as their potential anti-tumor properties are described.

Characterization of a protein kinase activity associated with purified capsids of the granulosis virus infecting Plodia interpunctella.

PubMed

Wilson, M E; Consigli, R A

1985-06-01

A cyclic-nucleotide independent protein kinase activity has been demonstrated in highly purified preparations of the granulosis virus infecting the Indian meal moth, Plodia interpunctella. A divalent cation was required for activity. Manganese was the preferred cation and a pH of 8.0 resulted in optimal incorporation of 32P radiolabel into acid-precipitable protein. Although both ATP and GTP could serve as phosphate donors, ATP was utilized more efficiently by the enzyme. The kinase activity was localized to purified capsids; and the basic, internal core protein, VP12, was found to be the predominant viral acceptor. Histones and protamine sulfate could also serve as acceptors for the capsid-associated kinase activity. Using acid hydrolysis and phosphoamino acid analysis of phosphorylated nucleocapsid protein and nuclear magnetic resonance of phosphorylated VP12, it was determined that the enzyme catalyzes the transfer of phosphate to both serine and arginine residues of acceptor proteins. We believe this kinase activity may play a significant role in the viral replication cycle.

Flow-dependent regulation of endothelial nitric oxide synthase: role of protein kinases

NASA Technical Reports Server (NTRS)

Boo, Yong Chool; Jo, Hanjoong

2003-01-01

Vascular endothelial cells are directly and continuously exposed to fluid shear stress generated by blood flow. Shear stress regulates endothelial structure and function by controlling expression of mechanosensitive genes and production of vasoactive factors such as nitric oxide (NO). Though it is well known that shear stress stimulates NO production from endothelial nitric oxide synthase (eNOS), the underlying molecular mechanisms remain unclear and controversial. Shear-induced production of NO involves Ca2+/calmodulin-independent mechanisms, including phosphorylation of eNOS at several sites and its interaction with other proteins, including caveolin and heat shock protein-90. There have been conflicting results as to which protein kinases-protein kinase A, protein kinase B (Akt), other Ser/Thr protein kinases, or tyrosine kinases-are responsible for shear-dependent eNOS regulation. The functional significance of each phosphorylation site is still unclear. We have attempted to summarize the current status of understanding in shear-dependent eNOS regulation.

Activation of the AMP-Activated Protein Kinase by Eicosapentaenoic Acid (EPA, 20:5 n-3) Improves Endothelial Function In Vivo

PubMed Central

Wu, Yong; Zhang, Cheng; Dong, Yunzhou; Wang, Shuangxi; Song, Ping; Viollet, Benoit; Zou, Ming-Hui

2012-01-01

The aim of the present study was to test the hypothesis that the cardiovascular-protective effects of eicosapentaenoic acid (EPA) may be due, in part, to its ability to stimulate the AMP-activated protein kinase (AMPK)-induced endothelial nitric oxide synthase (eNOS) activation. The role of AMPK in EPA-induced eNOS phosphorylation was investigated in bovine aortic endothelial cells (BAEC), in mice deficient of either AMPKα1 or AMPKα2, in eNOS knockout (KO) mice, or in Apo-E/AMPKα1 dual KO mice. EPA-treatment of BAEC increased both AMPK-Thr172 phosphorylation and AMPK activity, which was accompanied by increased eNOS phosphorylation, NO release, and upregulation of mitochondrial uncoupling protein-2 (UCP-2). Pharmacologic or genetic inhibition of AMPK abolished EPA-enhanced NO release and eNOS phosphorylation in HUVEC. This effect of EPA was absent in the aortas isolated from either eNOS KO mice or AMPKα1 KO mice fed a high-fat, high-cholesterol (HFHC) diet. EPA via upregulation of UCP-2 activates AMPKα1 resulting in increased eNOS phosphorylation and consequent improvement of endothelial function in vivo. PMID:22532857

Quantitative phosphoproteomics identifies SnRK2 protein kinase substrates and reveals the effectors of abscisic acid action.

PubMed

Wang, Pengcheng; Xue, Liang; Batelli, Giorgia; Lee, Shinyoung; Hou, Yueh-Ju; Van Oosten, Michael J; Zhang, Huiming; Tao, W Andy; Zhu, Jian-Kang

2013-07-02

Sucrose nonfermenting 1 (SNF1)-related protein kinase 2s (SnRK2s) are central components of abscisic acid (ABA) signaling pathways. The snrk2.2/2.3/2.6 triple-mutant plants are nearly completely insensitive to ABA, suggesting that most of the molecular actions of ABA are triggered by the SnRK2s-mediated phosphorylation of substrate proteins. Only a few substrate proteins of the SnRK2s are known. To identify additional substrate proteins of the SnRK2s and provide insight into the molecular actions of ABA, we used quantitative phosphoproteomics to compare the global changes in phosphopeptides in WT and snrk2.2/2.3/2.6 triple mutant seedlings in response to ABA treatment. Among the 5,386 unique phosphorylated peptides identified in this study, we found that ABA can increase the phosphorylation of 166 peptides and decrease the phosphorylation of 117 peptides in WT seedlings. In the snrk2.2/2.3/2.6 triple mutant, 84 of the 166 peptides, representing 58 proteins, could not be phosphorylated, or phosphorylation was not increased under ABA treatment. In vitro kinase assays suggest that most of the 58 proteins can serve as substrates of the SnRK2s. The SnRK2 substrates include proteins involved in flowering time regulation, RNA and DNA binding, miRNA and epigenetic regulation, signal transduction, chloroplast function, and many other cellular processes. Consistent with the SnRK2 phosphorylation of flowering time regulators, the snrk2.2/2.3/2.6 triple mutant flowered significantly earlier than WT. These results shed new light on the role of the SnRK2 protein kinases and on the downstream effectors of ABA action, and improve our understanding of plant responses to adverse environments.

The isothiocyanate class of bioactive nutrients covalently inhibit the MEKK1 protein kinase

PubMed Central

Cross, Janet V; Foss, Frank W; Rady, Joshua M; Macdonald, Timothy L; Templeton, Dennis J

2007-01-01

Background Dietary isothiocyanates (ITCs) are electrophilic compounds that have diverse biological activities including induction of apoptosis and effects on cell cycle. They protect against experimental carcinogenesis in animals, an activity believed to result from the transcriptional induction of "Phase 2" enzymes. The molecular mechanism of action of ITCs is unknown. Since ITCs are electrophiles capable of reacting with sulfhydryl groups on amino acids, we hypothesized that ITCs induce their biological effects through covalent modification of proteins, leading to changes in cell regulatory events. We previously demonstrated that stress-signaling kinase pathways are inhibited by other electrophilic compounds such as menadione. We therefore tested the effects of nutritional ITCs on MEKK1, an upstream regulator of the SAPK/JNK signal transduction pathway. Methods The activity of MEKK1 expressed in cells was monitored using in vitro kinase assays to measure changes in catalytic activity. The activity of endogenous MEKK1, immunopurified from ITC treated and untreated LnCAP cells was also measured by in vitro kinase assay. A novel labeling and affinity reagent for detection of protein modification by ITCs was synthesized and used in competition assays to monitor direct modification of MEKK1 by ITC. Finally, immunoblots with phospho-specific antibodies were used to measure the activity of MAPK protein kinases. Results ITCs inhibited the MEKK1 protein kinase in a manner dependent on a specific cysteine residue in the ATP binding pocket. Inhibition of MEKK1 catalytic activity was due to direct, covalent and irreversible modification of the MEKK1 protein itself. In addition, ITCs inhibited the catalytic activity of endogenous MEKK1. This correlated with inhibition of the downstream target of MEKK1 activity, i.e. the SAPK/JNK kinase. This inhibition was specific to SAPK, as parallel MAPK pathways were unaffected. Conclusion These results demonstrate that MEKK1 is directly

Apelin Increases Cardiac Contractility via Protein Kinase Cε- and Extracellular Signal-Regulated Kinase-Dependent Mechanisms

PubMed Central

Perjés, Ábel; Skoumal, Réka; Tenhunen, Olli; Kónyi, Attila; Simon, Mihály; Horváth, Iván G.; Kerkelä, Risto; Ruskoaho, Heikki; Szokodi, István

2014-01-01

Background Apelin, the endogenous ligand for the G protein-coupled apelin receptor, is an important regulator of the cardiovascular homoeostasis. We previously demonstrated that apelin is one of the most potent endogenous stimulators of cardiac contractility; however, its underlying signaling mechanisms remain largely elusive. In this study we characterized the contribution of protein kinase C (PKC), extracellular signal-regulated kinase 1/2 (ERK1/2) and myosin light chain kinase (MLCK) to the positive inotropic effect of apelin. Methods and Results In isolated perfused rat hearts, apelin increased contractility in association with activation of prosurvival kinases PKC and ERK1/2. Apelin induced a transient increase in the translocation of PKCε, but not PKCα, from the cytosol to the particulate fraction, and a sustained increase in the phosphorylation of ERK1/2 in the left ventricle. Suppression of ERK1/2 activation diminished the apelin-induced increase in contractility. Although pharmacological inhibition of PKC attenuated the inotropic response to apelin, it had no effect on ERK1/2 phosphorylation. Moreover, the apelin-induced positive inotropic effect was significantly decreased by inhibition of MLCK, a kinase that increases myofilament Ca2+ sensitivity. Conclusions Apelin increases cardiac contractility through parallel and independent activation of PKCε and ERK1/2 signaling in the adult rat heart. Additionally MLCK activation represents a downstream mechanism in apelin signaling. Our data suggest that, in addition to their role in cytoprotection, modest activation of PKCε and ERK1/2 signaling improve contractile function, therefore these pathways represent attractive possible targets in the treatment of heart failure. PMID:24695532

Discovery of cellular substrates for protein kinase A using a peptide array screening protocol.

PubMed

Smith, F Donelson; Samelson, Bret K; Scott, John D

2011-08-15

Post-translational modification of proteins is a universal form of cellular regulation. Phosphorylation on serine, threonine, tyrosine or histidine residues by protein kinases is the most widespread and versatile form of covalent modification. Resultant changes in activity, localization or stability of phosphoproteins drives cellular events. MS and bioinformatic analyses estimate that ~30% of intracellular proteins are phosphorylated at any given time. Multiple approaches have been developed to systematically define targets of protein kinases; however, it is likely that we have yet to catalogue the full complement of the phosphoproteome. The amino acids that surround a phosphoacceptor site are substrate determinants for protein kinases. For example, basophilic enzymes such as PKA (protein kinase A), protein kinase C and calmodulin-dependent kinases recognize basic side chains preceding the target serine or threonine residues. In the present paper we describe a strategy using peptide arrays and motif-specific antibodies to identify and characterize previously unrecognized substrate sequences for protein kinase A. We found that the protein kinases PKD (protein kinase D) and MARK3 [MAP (microtubule-associated protein)-regulating kinase 3] can both be phosphorylated by PKA. Furthermore, we show that the adapter protein RIL [a product of PDLIM4 (PDZ and LIM domain protein 4)] is a PKA substrate that is phosphorylated on Ser(119) inside cells and that this mode of regulation may control its ability to affect cell growth. © The Authors Journal compilation © 2011 Biochemical Society

Protein kinase Cε regulates nuclear translocation of extracellular signal-regulated kinase, which contributes to bradykinin-induced cyclooxygenase-2 expression.

PubMed

Nakano, Rei; Kitanaka, Taku; Namba, Shinichi; Kitanaka, Nanako; Sugiya, Hiroshi

2018-06-04

The proinflammatory mediator bradykinin stimulated cyclooxygenase-2 (COX-2) expression and subsequently prostaglandin E 2 synthesis in dermal fibroblasts. The involvement of B2 receptors and Gαq in the role of bradykinin was suggested by using pharmacological inhibitors. The PKC activator PMA stimulated COX-2 mRNA expression. Bradykinin failed to induce COX-2 mRNA expression in the presence of PKC inhibitors, whereas the effect of bradykinin was observed in the absence of extracellular Ca 2+ . Bradykinin-induced COX-2 mRNA expression was inhibited in cells transfected with PKCε siRNA. These observations suggest that the novel PKCε is concerned with bradykinin-induced COX-2 expression. Bradykinin-induced PKCε phosphorylation and COX-2 mRNA expression were inhibited by an inhibitor of 3-phosphoinositide-dependent protein kinase-1 (PDK-1), and bradykinin-induced PDK-1 phosphorylation was inhibited by phospholipase D (PLD) inhibitors, suggesting that PLD/PDK-1 pathway contributes to bradykinin-induced PKCε activation. Pharmacological and knockdown studies suggest that the extracellular signal-regulated kinase 1 (ERK1) MAPK signaling is involved in bradykinin-induced COX-2 expression. Bradykinin-induced ERK phosphorylation was attenuated in the cells pretreated with PKC inhibitors or transfected with PKCε siRNA. We observed the interaction between PKCε and ERK by co-immunoprecipitation experiments. These observations suggest that PKCε activation contributes to the regulation of ERK1 activation. Bradykinin stimulated the accumulation of phosphorylated ERK in the nuclear fraction, that was inhibited in the cells treated with PKC inhibitors or transfected with PKCε siRNA. Consequently, we concluded that bradykinin activates PKCε via the PLD/PDK-1 pathway, which subsequently induces activation and translocation of ERK1 into the nucleus, and contributes to COX-2 expression for prostaglandin E 2 synthesis in dermal fibroblasts.

Membrane skeletal proteins and their integral membrane protein anchors are targets for tyrosine and threonine kinases in Euglena.

PubMed

Fazio, M J; Da Silva, A C; Rosiere, T K; Bouck, G B

1995-01-01

Proteins of the membrane skeleton of Euglena gracilis were extensively phosphorylated in vivo and in vitro after incubation with [32P]-orthophosphate or gamma-[32P] ATP. Endogenous protein threonine/serine activity phosphorylated the major membrane skeletal proteins (articulins) and the putative integral membrane protein (IP39) anchor for articulins. The latter was also the major target for endogenous protein tyrosine kinase activity. A cytoplasmic domain of IP39 was specifically phosphorylated, and removal of this domain with papain eliminated the radiolabeled phosphoamino acids and eliminated or radically shifted the PI of the multiple isoforms of IP39. In gel kinase assays IP39 autophosphorylated and a 25 kDa protein which does not autophosphorylate was identified as a threonine/serine (casein) kinase. Plasma membranes from the membrane skeletal protein complex contained threonine/serine (casein) kinase activity, and cross-linking experiments suggested that IP39 was the likely source for this membrane activity. pH optima, cation requirements and heparin sensitivity of the detergent solubilized membrane activity were determined. Together these results suggest that protein kinases may be important modulators of protein assembly and function of the membrane skeleton of these protistan cells.

Fatty acid synthase inhibition activates AMP-activated protein kinase in SKOV3 human ovarian cancer cells.

PubMed

Zhou, Weibo; Han, Wan Fang; Landree, Leslie E; Thupari, Jagan N; Pinn, Michael L; Bililign, Tsion; Kim, Eun Kyoung; Vadlamudi, Aravinda; Medghalchi, Susan M; El Meskini, Rajaa; Ronnett, Gabriele V; Townsend, Craig A; Kuhajda, Francis P

2007-04-01

Fatty acid synthase (FAS), the enzyme responsible for the de novo synthesis of fatty acids, is highly expressed in ovarian cancers and most common human carcinomas. Inhibition of FAS and activation of AMP-activated protein kinase (AMPK) have been shown to be cytotoxic to human cancer cells in vitro and in vivo. In this report, we explore the cytotoxic mechanism of action of FAS inhibition and show that C93, a synthetic FAS inhibitor, increases the AMP/ATP ratio, activating AMPK in SKOV3 human ovarian cancer cells, which leads to cytotoxicity. As a physiologic consequence of AMPK activation, acetyl-CoA carboxylase (ACC), the rate-limiting enzyme of fatty acid synthesis, was phosphorylated and inhibited whereas glucose oxidation was increased. Despite these attempts to conserve energy, the AMP/ATP ratio increased with worsening cellular redox status. Pretreatment of SKOV3 cells with compound C, an AMPK inhibitor, substantially rescued the cells from C93 cytotoxicity, indicating its dependence on AMPK activation. 5-(Tetradecyloxy)-2-furoic acid, an ACC inhibitor, did not activate AMPK despite inhibiting fatty acid synthesis pathway activity and was not significantly cytotoxic to SKOV3 cells. This indicates that substrate accumulation from FAS inhibition triggering AMPK activation, not end-product depletion of fatty acids, is likely responsible for AMPK activation. C93 also exhibited significant antitumor activity and apoptosis against SKOV3 xenografts in athymic mice without significant weight loss or cytotoxicity to proliferating cellular compartments such as bone marrow, gastrointestinal tract, or skin. Thus, pharmacologic FAS inhibition selectively activates AMPK in ovarian cancer cells, inducing cytotoxicity while sparing most normal human tissues from the pleiotropic effects of AMPK activation.

Mechanistic insights into the urea-induced denaturation of kinase domain of human integrin linked kinase.

PubMed

Syed, Sunayana Begum; Khan, Faez Iqbal; Khan, Sabab Hasan; Srivastava, Saurabha; Hasan, Gulam Mustafa; Lobb, Kevin A; Islam, Asimul; Ahmad, Faizan; Hassan, Md Imtaiyaz

2018-05-01

Integrin-linked kinase (ILK), a ubiquitously expressed intracellular Ser/Thr protein kinase, plays a major role in the oncogenesis and tumour progression. The conformational stability and unfolding of kinase domain of ILK (ILK 193-446 ) was examined in the presence of increasing concentrations of urea. The stability parameters of the urea-induced denaturation were measured by monitoring changes in [θ] 222 (mean residue ellipticity at 222nm), difference absorption coefficient at 292nm (Δε 292 ) and intrinsic fluorescence emission intensity at pH7.5 and 25±0.1°C. The urea-induced denaturation was found to be reversible. The protein unfolding transition occurred in the urea concentration range 3.0-7.0M. A coincidence of normalized denaturation curves of optical properties ([θ] 222 , Δε 292 and λ max , the wavelength of maximum emission intensity) suggested that urea-induced denaturation of kinase domain of ILK is a two-state process. We further performed molecular dynamics simulation for 100ns to see the effect of urea on structural stability of kinase domain of ILK at atomic level. Structural changes with increasing concentrations of urea were analysed, and we observed a significant increase in the root mean square deviation, root mean square fluctuations, solvent accessible surface area and radius of gyration. A correlation was observed between in vitro and in silico studies. Copyright © 2018 Elsevier B.V. All rights reserved.

Fish oil modulates glycogen synthase kinase-3 signaling pathway in diabetes-induced hippocampal neurons apoptosis.

PubMed

Sun, Li-Juan; Hou, Xiang-Hong; Xue, Sen-Hai; Yan, Feng; Dai, Yu-Jie; Zhao, Chang-Hai; Wang, Feng; Yang, Rui-Hua

2014-07-29

Previous research has demonstrated that diabetes induces learning and memory deficits. However, the mechanism of memory impairment induced by diabetes is poorly understood. Dietary fatty acids, especially polyunsaturated fatty acids, have been shown to enhance learning and memory and prevent memory deficits in various experimental conditions. The present study investigated the effects of fish oil supplementation on the neuron apoptosis in the hippocampus of streptozotocin (STZ)-induced diabetes rats, further explored the effect of fish oil on the phosphorylation of protein kinase B and glycogen synthase kinase-3 beta. The effects of diabetes and fish oil treatment on the spatial learning and memory were also evaluated using the Morris Water Maze. STZ-induced diabetes impaired spatial learning and memory of rats, which was associated with the apoptosis of hippocampal neurons and oxidative stress. Fish oil administration ameliorated cognitive deficit, reduced oxidative stress, increased AKT phosphorylation, decreased GSK-3β phosphorylation, and decreased pro-apoptotic molecules expression, which protected the hippocampal neurons from apoptosis in diabetic rats. These results suggested a potential role for fish oil as an adjuvant therapy for the prevention and treatment of diabetic complications. Copyright © 2014 Elsevier B.V. All rights reserved.

Induced overexpression of protein kinase D1 stimulates mitogenic signaling in human pancreatic carcinoma PANC-1 cells.

PubMed

Kisfalvi, Krisztina; Hurd, Cliff; Guha, Sushovan; Rozengurt, Enrique

2010-05-01

Neurotensin (NT) stimulates protein kinase D1 (PKD1), extracellular signal regulated kinase (ERK), c-Jun N-terminal Kinase (JNK), and DNA synthesis in the human pancreatic adenocarcinoma cell line PANC-1. To determine the effect of PKD1 overexpression on these biological responses, we generated inducible stable PANC-1 clones that express wild-type (WT) or kinase-dead (K618N) forms of PKD1 in response to the ecdysone analog ponasterone-A (PonA). NT potently stimulated c-Jun Ser(63) phosphorylation in both wild type and clonal derivatives of PANC-1 cells. PonA-induced expression of WT, but not K618N PKD1, rapidly blocked NT-mediated c-Jun Ser(63) phosphorylation either at the level of or upstream of MKK4, a dual-specificity kinase that leads to JNK activation. This is the first demonstration that PKD1 suppresses NT-induced JNK/cJun activation in PANC-1 cells. In contrast, PKD1 overexpression markedly increased the duration of NT-induced ERK activation in these cells. The reciprocal influence of PKD1 signaling on pro-mitogenicERK and pro-apopotic JNK/c-Jun pathways prompted us to examine whether PKD1 overexpression promotes DNA synthesis and proliferation of PANC-1 cells. Our results show that PKD1 overexpression increased DNA synthesis and cell numbers of PANC-1 cells cultured in regular dishes or in polyhydroxyethylmethacrylate [Poly-(HEMA)]-coated dishes to eliminate cell adhesion (anchorage-independent growth). Furthermore, PKD1 overexpression markedly enhanced DNA synthesis induced by NT (1-10 nM). These results indicate that PKD1 mediates mitogenic signaling in PANC-1 and suggests that this enzyme could be a novel target for the development of therapeutic drugs that restrict the proliferation of these cells.

GSK-3 mediates the okadaic acid-induced modification of collapsin response mediator protein-2 in human SK-N-SH neuroblastoma cells.

PubMed

Ni, Mei-Hui; Wu, Chih-Ching; Chan, Wen-Hsiung; Chien, Kun-Yi; Yu, Jau-Song

2008-04-15

Collapsin response mediator protein-2 (CRMP-2), a phosphoprotein involved in axonal outgrowth and microtubule dynamics, is aberrantly phosphorylated in Alzheimer's disease (AD) brain. Alteration of glycogen synthase kinase-3 (GSK-3) activity is associated with the pathogenesis of AD. Here, we show that CRMP-2 is one of the major substrates for GSK-3 in pig brain extracts. Both GSK-3alpha and 3beta phosphorylate purified pig brain CRMP-2 and significantly alter its mobility in SDS-gels, resembling the CRMP-2 modification observed in AD brain. Interestingly, this modification can be detected in SK-N-SH neuroblastoma cells treated with a phosphatase inhibitor, okadaic acid (OA), and GSK-3 inhibitors completely block this OA-induced event. Knockdown of both GSK-3alpha and 3beta, but not either kinase alone, impairs OA-induced modification of CRMP-2. Mutation of Ser-518 or Ser-522 of CRMP-2, which are highly phosphorylated in AD brain, to Ala blocks the OA-induced modification of CRMP-2 in SK-N-SH cells. Ser-522 prephosphorylated by Cdk5 is required for subsequent GSK-3alpha-mediated phosphorylation of CRMP-2 in vitro. Collectively, our results demonstrate for the first time that OA can induce phosphorylation of CRMP-2 in SK-N-SH cells at sites aberrantly phosphorylated in AD brain, and both GSK-3alpha and 3beta and Ser-522 kinase(s) are involved in this process.

Mitogen-activated protein kinase inhibitors suppress prostaglandin F(2alpha)-induced myosin-light chain phosphorylation and contraction in iris sphincter smooth muscle.

PubMed

Yousufzai, S Y; Gao, G; Abdel-Latif, A A

2000-10-27

The purpose of this study was to investigate the potential role of mitogen-activated protein (MAP) kinase in contraction by monitoring MAP kinase phosphorylation (activation) and contraction during agonist stimulation of cat iris sphincter smooth muscle. Changes in tension in response to prostaglandin F(2alpha), latanoprost, a prostaglandin F(2alpha) analog used as an anti-glaucoma drug, and carbachol were recorded isometrically, and MAP kinase activation was monitored by Western blot using a phosphospecific p42/p44 MAP kinase antibody. We found that treatment of the muscle with 2'-Amino-3'-methoxyflavone (PD98059) (10 microM), a specific inhibitor of MAP kinase kinase (MEK), inhibited significantly prostaglandin F(2alpha)- and latanoprost-induced phosphorylation and contraction, but had little effect on those evoked by carbachol. Prostaglandin F(2alpha) increased MAP kinase phosphorylation in a concentration-dependent manner with EC(50) value of 1.1 x 10(-8) M and increased contraction with EC(50) of 0.92 x 10(-9) M. The MAP kinase inhibitors PD98059, Apigenin and 1,4-Diamino-2,3-dicyano-1, 4bis(2-aminophenylthio)butadiene (UO126) inhibited prostaglandin F(2alpha)-induced contraction in a concentration-dependent manner with IC(50) values of 2.4, 3.0 and 4.8 microM, respectively. PD98059 had no effect on prostaglandin F(2alpha)- or on carbachol-stimulated inositol-1,4,5-trisphosphate (IP(3)) production. In contrast, the MAP kinase inhibitor inhibited prostaglandin F(2alpha)-induced myosin-light chain (MLC) phosphorylation, but had no effect on that of carbachol. N-[2-(N-(4-Chloro-cinnamyl)-N-methylaminomethyl)phenyl]-N-[2- hydroxyethyl]-4-methoxybenzenesulfonamide (KN-93) (10 microM), a Ca(2+)-calmodulin-dependent protein kinase inhibitor, and Wortmannin (10 microM), an MLC kinase inhibitor, inhibited significantly (by 80%) prostaglandin F(2alpha)- and carbachol-induced contraction. It can be concluded that in this smooth muscle p42/p44 MAP kinases are involved in

Protein kinase A-dependent increase in WAVE2 expression induced by the focal adhesion protein vinexin.

PubMed

Mitsushima, Masaru; Sezaki, Takuhito; Akahane, Rie; Ueda, Kazumitsu; Suetsugu, Shiro; Takenawa, Tadaomi; Kioka, Noriyuki

2006-03-01

The focal adhesion protein vinexin is a member of a family of adaptor proteins that are thought to participate in the regulation of cell adhesion, cytoskeletal reorganization, and growth factor signaling. Here, we show that vinexin beta increases the amount of and reduces the mobility on SDS-PAGE of Wiskott-Aldrich syndrome protein family verprolin-homologous protein (WAVE) 2 protein, which is a key factor modulating actin polymerization in migrating cells. This mobility retardation disappeared after in vitro phosphatase treatment. Co-immunoprecipitation assays revealed the interaction of vinexin beta with WAVE2 as well as WAVE1 and N-WASP. Vinexin beta interacts with the proline-rich region of WAVE2 through the first and second SH3 domains of vinexin beta. Mutations disrupting the interaction impaired the ability of vinexin beta to increase the amount of WAVE2 protein. Treatments with proteasome inhibitors increased the amount of WAVE2, but did not have an additive effect with vinexin beta. Inhibition of protein kinase A (PKA) activity suppressed the vinexin-induced increase in WAVE2 protein, while activation of PKA increased WAVE2 expression without vinexin beta. These results suggest that vinexin beta regulates the proteasome-dependent degradation of WAVE2 in a PKA-dependent manner.

In vitro phosphorylation of the movement protein of tomato mosaic tobamovirus by a cellular kinase.

PubMed

Matsushita, Y; Hanazawa, K; Yoshioka, K; Oguchi, T; Kawakami, S; Watanabe, Y; Nishiguchi, M; Nyunoya, H

2000-08-01

The movement protein (MP) of tomato mosaic virus (ToMV) was produced in E. coli as a soluble fusion protein with glutathione S-transferase. When immobilized on glutathione affinity beads, the recombinant protein was phosphorylated in vitro by incubating with cell extracts of Nicotiana tabacum and tobacco suspension culture cells (BY-2) in the presence of [gamma-(32)P]ATP. Phosphorylation occurred even after washing the beads with a detergent-containing buffer, indicating that the recombinant MP formed a stable complex with some protein kinase(s) during incubation with the cell extract. Phosphoamino acid analysis revealed that the MP was phosphorylated on serine and threonine residues. Phosphorylation of the MP was decreased by addition of kinase inhibitors such as heparin, suramin and quercetin, which are known to be effective for casein kinase II (CK II). The phosphorylation level was not changed by other types of inhibitor. In addition, as shown for animal and plant CK II, [gamma-(32)P]GTP was efficiently used as a phosphoryl donor. Phosphorylation was not affected by amino acid replacements at serine-37 and serine-238, but was completely inhibited by deletion of the carboxy-terminal 9 amino acids, including threonine-256, serine-257, serine-261 and serine-263. These results suggest that the MP of ToMV could be phosphorylated in plant cells by a host protein kinase that is closely related to CK II.

Identification of Open Stomata1-Interacting Proteins Reveals Interactions with Sucrose Non-fermenting1-Related Protein Kinases2 and with Type 2A Protein Phosphatases That Function in Abscisic Acid Responses1[OPEN

PubMed Central

Waadt, Rainer; Manalansan, Bianca; Rauniyar, Navin; Munemasa, Shintaro; Booker, Matthew A.; Brandt, Benjamin; Waadt, Christian; Nusinow, Dmitri A.; Kay, Steve A.; Kunz, Hans-Henning; Schumacher, Karin; DeLong, Alison; Yates, John R.; Schroeder, Julian I.

2015-01-01

The plant hormone abscisic acid (ABA) controls growth and development and regulates plant water status through an established signaling pathway. In the presence of ABA, pyrabactin resistance/regulatory component of ABA receptor proteins inhibit type 2C protein phosphatases (PP2Cs). This, in turn, enables the activation of Sucrose Nonfermenting1-Related Protein Kinases2 (SnRK2). Open Stomata1 (OST1)/SnRK2.6/SRK2E is a major SnRK2-type protein kinase responsible for mediating ABA responses. Arabidopsis (Arabidopsis thaliana) expressing an epitope-tagged OST1 in the recessive ost1-3 mutant background was used for the copurification and identification of OST1-interacting proteins after osmotic stress and ABA treatments. These analyses, which were confirmed using bimolecular fluorescence complementation and coimmunoprecipitation, unexpectedly revealed homo- and heteromerization of OST1 with SnRK2.2, SnRK2.3, OST1, and SnRK2.8. Furthermore, several OST1-complexed proteins were identified as type 2A protein phosphatase (PP2A) subunits and as proteins involved in lipid and galactolipid metabolism. More detailed analyses suggested an interaction network between ABA-activated SnRK2-type protein kinases and several PP2A-type protein phosphatase regulatory subunits. pp2a double mutants exhibited a reduced sensitivity to ABA during seed germination and stomatal closure and an enhanced ABA sensitivity in root growth regulation. These analyses add PP2A-type protein phosphatases as another class of protein phosphatases to the interaction network of SnRK2-type protein kinases. PMID:26175513

Chimeric calcium/calmodulin-dependent protein kinase in tobacco: differential regulation by calmodulin isoforms

NASA Technical Reports Server (NTRS)

Liu, Z.; Xia, M.; Poovaiah, B. W.

1998-01-01

cDNA clones of chimeric Ca2+/calmodulin-dependent protein kinase (CCaMK) from tobacco (TCCaMK-1 and TCCaMK-2) were isolated and characterized. The polypeptides encoded by TCCaMK-1 and TCCaMK-2 have 15 different amino acid substitutions, yet they both contain a total of 517 amino acids. Northern analysis revealed that CCaMK is expressed in a stage-specific manner during anther development. Messenger RNA was detected when tobacco bud sizes were between 0.5 cm and 1.0 cm. The appearance of mRNA coincided with meiosis and became undetectable at later stages of anther development. The reverse polymerase chain reaction (RT-PCR) amplification assay using isoform-specific primers showed that both of the CCaMK mRNAs were expressed in anther with similar expression patterns. The CCaMK protein expressed in Escherichia coli showed Ca2+-dependent autophosphorylation and Ca2+/calmodulin-dependent substrate phosphorylation. Calmodulin isoforms (PCM1 and PCM6) had differential effects on the regulation of autophosphorylation and substrate phosphorylation of tobacco CCaMK, but not lily CCaMK. The evolutionary tree of plant serine/threonine protein kinases revealed that calmodulin-dependent kinases form one subgroup that is distinctly different from Ca2+-dependent protein kinases (CDPKs) and other serine/threonine kinases in plants.

Orexin A Inhibits Propofol-Induced Neurite Retraction by a Phospholipase D/Protein Kinase Cε-Dependent Mechanism in Neurons

PubMed Central

Björnström, Karin; Turina, Dean; Strid, Tobias; Sundqvist, Tommy; Eintrei, Christina

2014-01-01

Background The intravenous anaesthetic propofol retracts neurites and reverses the transport of vesicles in rat cortical neurons. Orexin A (OA) is an endogenous neuropeptide regulating wakefulness and may counterbalance anaesthesia. We aim to investigate if OA interacts with anaesthetics by inhibition of the propofol-induced neurite retraction. Methods In primary cortical cell cultures from newborn rats’ brains, live cell light microscopy was used to measure neurite retraction after propofol (2 µM) treatment with or without OA (10 nM) application. The intracellular signalling involved was tested using a protein kinase C (PKC) activator [phorbol 12-myristate 13-acetate (PMA)] and inhibitors of Rho-kinase (HA-1077), phospholipase D (PLD) [5-fluoro-2-indolyl des-chlorohalopemide (FIPI)], PKC (staurosporine), and a PKCε translocation inhibitor peptide. Changes in PKCε Ser729 phosphorylation were detected with Western blot. Results The neurite retraction induced by propofol is blocked by Rho-kinase and PMA. OA blocks neurite retraction induced by propofol, and this inhibitory effect could be prevented by FIPI, staurosporine and PKCε translocation inhibitor peptide. OA increases via PLD and propofol decreases PKCε Ser729 phosphorylation, a crucial step in the activation of PKCε. Conclusions Rho-kinase is essential for propofol-induced neurite retraction in cortical neuronal cells. Activation of PKC inhibits neurite retraction caused by propofol. OA blocks propofol-induced neurite retraction by a PLD/PKCε-mediated pathway, and PKCε maybe the key enzyme where the wakefulness and anaesthesia signal pathways converge. PMID:24828410

Triptolide-induced mitochondrial damage dysregulates fatty acid metabolism in mouse sertoli cells.

PubMed

Cheng, Yisen; Chen, Gaojian; Wang, Li; Kong, Jiamin; Pan, Ji; Xi, Yue; Shen, Feihai; Huang, Zhiying

2018-08-01

Triptolide is a major active ingredient of tripterygium glycosides, used for the therapy of immune and inflammatory diseases. However, its clinical applications are limited by severe male fertility toxicity associated with decreased sperm count, mobility and testicular injures. In this study, we determined that triptoide-induced mitochondrial dysfunction triggered reduction of lactate and dysregulation of fatty acid metabolism in mouse Sertoli cells. First, triptolide induced mitochondrial damage through the suppressing of proliferator-activated receptor coactivator-1 alpha (PGC-1α) activity and protein. Second, mitochondrial damage decreased lactate production and dysregulated fatty acid metabolism. Finally, mitochondrial dysfunction was initiated by the inhibition of sirtuin 1 (SIRT1) with the regulation of AMP-activated protein kinase (AMPK) in Sertoli cells after triptolide treatment. Meanwhile, triptolide induced mitochondrial fatty acid oxidation dysregulation by increasing AMPK phosphorylation. Taken together, we provide evidence that the mechanism of triptolide-induced testicular toxicity under mitochondrial injury may involve a metabolic change. Copyright © 2018 Elsevier B.V. All rights reserved.

Overexpression of heart-type fatty acid binding protein enhances fatty acid-induced podocyte injury.

PubMed

Gao, Qing; Sarkar, Alhossain; Chen, Yizhi; Xu, Bo; Zhu, Xiaojuan; Yuan, Yang; Guan, Tianjun

2018-02-01

Deregulated lipid metabolism is a characteristic of metabolic diseases including type 2 diabetes and obesity, and likely contributes to podocyte injury and end-stage kidney disease. Heart-type fatty acid binding protein (H-FABP) was reported to be associated with lipid metabolism. The present study investigated whether H-FABP contributes to podocyte homeostasis. Podocytes were transfected by lentiviral vector to construct a cell line which stably overexpressed H-FABP. Small interfering RNA capable of effectively silencing H-FABP was introduced into podocytes to construct a cell line with H-FABP knockdown. Certain groups were treated with palmitic acid (PA) and the fat metabolism, as well as inflammatory and oxidative stress markers were measured. PA accelerated lipid metabolism derangement, inflammatory reaction and oxidative stress in podocytes. Overexpression of H-FABP enhanced the PA-induced disequilibrium in podocytes. The mRNA and protein expression levels of acyl-coenzyme A oxidase 3 and monocyte chemotactic protein 1, and the protein expression levels of 8-hydroxy-2'-deoxyguanosine and 4-hydroxynonenal were upregulated in the H-FABP overexpression group, while the mRNA and protein expression of peroxisome proliferator activated receptor α was downregulated. Knockdown of H-FABP inhibited the PA-induced injury and lipid metabolism derangement, as well as the inflammatory reaction and oxidative stress in podocytes. These results indicated that overexpression of H-FABP enhances fatty acid-induced podocyte injury, while H-FABP inhibition may represent a potential therapeutic strategy for the prevention of lipid metabolism-associated podocyte injury.

Signaling of the ITK (interleukin 2-inducible T cell kinase)-SYK (spleen tyrosine kinase) fusion kinase is dependent on adapter SLP-76 and on the adapter function of the kinases SYK and ZAP70.

PubMed

Hussain, Alamdar; Mohammad, Dara K; Gustafsson, Manuela O; Uslu, Merve; Hamasy, Abdulrahman; Nore, Beston F; Mohamed, Abdalla J; Smith, C I Edvard

2013-03-08

The inducible T cell kinase-spleen tyrosine kinase (ITK-SYK) oncogene consists of the Tec homology-pleckstrin homology domain of ITK and the kinase domain of SYK, and it is believed to be the cause of peripheral T cell lymphoma. We and others have recently demonstrated that this fusion protein is constitutively tyrosine-phosphorylated and is transforming both in vitro and in vivo. To gain a deeper insight into the molecular mechanism(s) underlying its activation and signaling, we mutated a total of eight tyrosines located in the SYK portion of the chimera into either phenylalanine or to the negatively charged glutamic acid. Although mutations in the interdomain-B region affected ITK-SYK kinase activity, they only modestly altered downstream signaling events. In contrast, mutations that were introduced in the kinase domain triggered severe impairment of downstream signaling. Moreover, we show here that SLP-76 is critical for ITK-SYK activation and is particularly required for the ITK-SYK-dependent phosphorylation of SYK activation loop tyrosines. In Jurkat cell lines, we demonstrate that expression of ITK-SYK fusion requires an intact SLP-76 function and significantly induces IL-2 secretion and CD69 expression. Furthermore, the SLP-76-mediated induction of IL-2 and CD69 could be further enhanced by SYK or ZAP-70, but it was independent of their kinase activity. Notably, ITK-SYK expression in SYF cells phosphorylates SLP-76 in the absence of SRC family kinases. Altogether, our data suggest that ITK-SYK exists in the active conformation state and is therefore capable of signaling without SRC family kinases or stimulation of the T cell receptor.

Signaling of the ITK (Interleukin 2-inducible T Cell Kinase)-SYK (Spleen Tyrosine Kinase) Fusion Kinase Is Dependent on Adapter SLP-76 and on the Adapter Function of the Kinases SYK and ZAP70*

PubMed Central

Hussain, Alamdar; Mohammad, Dara K.; Gustafsson, Manuela O.; Uslu, Merve; Hamasy, Abdulrahman; Nore, Beston F.; Mohamed, Abdalla J.; Smith, C. I. Edvard

2013-01-01

The inducible T cell kinase-spleen tyrosine kinase (ITK-SYK) oncogene consists of the Tec homology-pleckstrin homology domain of ITK and the kinase domain of SYK, and it is believed to be the cause of peripheral T cell lymphoma. We and others have recently demonstrated that this fusion protein is constitutively tyrosine-phosphorylated and is transforming both in vitro and in vivo. To gain a deeper insight into the molecular mechanism(s) underlying its activation and signaling, we mutated a total of eight tyrosines located in the SYK portion of the chimera into either phenylalanine or to the negatively charged glutamic acid. Although mutations in the interdomain-B region affected ITK-SYK kinase activity, they only modestly altered downstream signaling events. In contrast, mutations that were introduced in the kinase domain triggered severe impairment of downstream signaling. Moreover, we show here that SLP-76 is critical for ITK-SYK activation and is particularly required for the ITK-SYK-dependent phosphorylation of SYK activation loop tyrosines. In Jurkat cell lines, we demonstrate that expression of ITK-SYK fusion requires an intact SLP-76 function and significantly induces IL-2 secretion and CD69 expression. Furthermore, the SLP-76-mediated induction of IL-2 and CD69 could be further enhanced by SYK or ZAP-70, but it was independent of their kinase activity. Notably, ITK-SYK expression in SYF cells phosphorylates SLP-76 in the absence of SRC family kinases. Altogether, our data suggest that ITK-SYK exists in the active conformation state and is therefore capable of signaling without SRC family kinases or stimulation of the T cell receptor. PMID:23293025

Actions of Tamoxifen and Estrogen on Osteoblast Protein Kinase C Expression.

DTIC Science & Technology

1996-07-01

extended period of time over which estrogen deficiency -induced bone loss occurs. Postmenopausal bone loss occurs gradually over several years, and changes...Identification of luteal estrogen-modulated lipid- stimulated kinase as protein kinase C5. J Biol Chem 267:17061-17068. 24. Cutler RE Jr, Maizels ET

Antagonistic Regulation of Cystic Fibrosis Transmembrane Conductance Regulator Cell Surface Expression by Protein Kinases WNK4 and Spleen Tyrosine Kinase ▿

PubMed Central

Mendes, Ana Isabel; Matos, Paulo; Moniz, Sónia; Luz, Simão; Amaral, Margarida D.; Farinha, Carlos M.; Jordan, Peter

2011-01-01

Members of the WNK (with-no-lysine [K]) subfamily of protein kinases regulate various ion channels involved in sodium, potassium, and chloride homeostasis by either inducing their phosphorylation or regulating the number of channel proteins expressed at the cell surface. Here, we describe findings demonstrating that the cell surface expression of the cystic fibrosis transmembrane conductance regulator (CFTR) is also regulated by WNK4 in mammalian cells. This effect of WNK4 is independent of the presence of kinase and involves interaction with and inhibition of spleen tyrosine kinase (Syk), which phosphorylates Tyr512 in the first nucleotide-binding domain 1 (NBD1) of CFTR. Transfection of catalytically active Syk into CFTR-expressing baby hamster kidney cells reduces the cell surface expression of CFTR, whereas that of WNK4 promotes it. This is shown by biotinylation of cell surface proteins, immunofluorescence microscopy, and functional efflux assays. Mutation of Tyr512 to either glutamic acid or phenylalanine is sufficient to alter CFTR surface levels. In human airway epithelial cells, downregulation of endogenous Syk and WNK4 confirms their roles as physiologic regulators of CFTR surface expression. Together, our results show that Tyr512 phosphorylation is a novel signal regulating the prevalence of CFTR at the cell surface and that WNK4 and Syk perform an antagonistic role in this process. PMID:21807898

Pea DNA topoisomerase I is phosphorylated and stimulated by casein kinase 2 and protein kinase C.

PubMed

Tuteja, Narendra; Reddy, Malireddy Kodandarami; Mudgil, Yashwanti; Yadav, Badam Singh; Chandok, Meena Rani; Sopory, Sudhir Kumar

2003-08-01

DNA topoisomerase I catalyzes the relaxation of superhelical DNA tension and is vital for DNA metabolism; therefore, it is essential for growth and development of plants. Here, we have studied the phosphorylation-dependent regulation of topoisomerase I from pea (Pisum sativum). The purified enzyme did not show autophosphorylation but was phosphorylated in an Mg(2+)-dependent manner by endogenous protein kinases present in pea nuclear extracts. This phosphorylation was abolished with calf intestinal alkaline phosphatase and lambda phosphatase. It was also phosphorylated by exogenous casein kinase 2 (CK2), protein kinase C (PKC; from animal sources), and an endogenous pea protein, which was purified using a novel phorbol myristate acetate affinity chromatography method. All of these phosphorylations were inhibited by heparin (inhibitor of CK2) and calphostin (inhibitor of PKC), suggesting that pea topoisomerase I is a bona fide substrate for these kinases. Spermine and spermidine had no effect on the CK2-mediated phosphorylation, suggesting that it is polyamine independent. Phospho-amino acid analysis showed that only serine residues were phosphorylated, which was further confirmed using antiphosphoserine antibody. The topoisomerase I activity increased after phosphorylation with exogenous CK2 and PKC. This study shows that these kinases may contribute to the physiological regulation of DNA topoisomerase I activity and overall DNA metabolism in plants.

Glycogen Synthase Kinase 3 Inactivation Induces Cell Senescence through Sterol Regulatory Element Binding Protein 1-Mediated Lipogenesis in Chang Cells.

PubMed

Kim, You-Mie; Song, Insun; Seo, Yong-Hak; Yoon, Gyesoon

2013-12-01

Enhanced lipogenesis plays a critical role in cell senescence via induction of expression of the mature form of sterol regulatory element binding protein 1 (SREBP1), which contributes to an increase in organellar mass, one of the indicators of senescence. We investigated the molecular mechanisms by which signaling molecules control SREBP1-mediated lipogenesis and senescence. We developed cellular models for stress-induced senescence, by exposing Chang cells, which are immortalized human liver cells, to subcytotoxic concentrations (200 µM) of deferoxamine (DFO) and H2O2. In this model of stress-induced cell senescence using DFO and H2O2, the phosphorylation profile of glycogen synthase kinase 3α (GSK3α) and β corresponded closely to the expression profile of the mature form of SREBP-1 protein. Inhibition of GSK3 with a subcytotoxic concentration of the selective GSK3 inhibitor SB415286 significantly increased mature SREBP1 expression, as well as lipogenesis and organellar mass. In addition, GSK3 inhibition was sufficient to induce senescence in Chang cells. Suppression of GSK3 expression with siRNAs specific to GSK3α and β also increased mature SREBP1 expression and induced senescence. Finally, blocking lipogenesis with fatty acid synthase inhibitors (cerulenin and C75) and siRNA-mediated silencing of SREBP1 and ATP citrate lyase (ACL) significantly attenuated GSK3 inhibition-induced senescence. GSK3 inactivation is an important upstream event that induces SREBP1-mediated lipogenesis and consequent cell senescence.

A rice kinase-protein interaction map.

PubMed

Ding, Xiaodong; Richter, Todd; Chen, Mei; Fujii, Hiroaki; Seo, Young Su; Xie, Mingtang; Zheng, Xianwu; Kanrar, Siddhartha; Stevenson, Rebecca A; Dardick, Christopher; Li, Ying; Jiang, Hao; Zhang, Yan; Yu, Fahong; Bartley, Laura E; Chern, Mawsheng; Bart, Rebecca; Chen, Xiuhua; Zhu, Lihuang; Farmerie, William G; Gribskov, Michael; Zhu, Jian-Kang; Fromm, Michael E; Ronald, Pamela C; Song, Wen-Yuan

2009-03-01

Plants uniquely contain large numbers of protein kinases, and for the vast majority of the 1,429 kinases predicted in the rice (Oryza sativa) genome, little is known of their functions. Genetic approaches often fail to produce observable phenotypes; thus, new strategies are needed to delineate kinase function. We previously developed a cost-effective high-throughput yeast two-hybrid system. Using this system, we have generated a protein interaction map of 116 representative rice kinases and 254 of their interacting proteins. Overall, the resulting interaction map supports a large number of known or predicted kinase-protein interactions from both plants and animals and reveals many new functional insights. Notably, we found a potential widespread role for E3 ubiquitin ligases in pathogen defense signaling mediated by receptor-like kinases, particularly by the kinases that may have evolved from recently expanded kinase subfamilies in rice. We anticipate that the data provided here will serve as a foundation for targeted functional studies in rice and other plants. The application of yeast two-hybrid and TAPtag analyses for large-scale plant protein interaction studies is also discussed.

Induction of viral, 7-methyl-guanosine cap-independent translation and oncolysis by mitogen-activated protein kinase-interacting kinase-mediated effects on the serine/arginine-rich protein kinase.

PubMed

Brown, Michael C; Bryant, Jeffrey D; Dobrikova, Elena Y; Shveygert, Mayya; Bradrick, Shelton S; Chandramohan, Vidyalakshmi; Bigner, Darell D; Gromeier, Matthias

2014-11-01

Protein synthesis, the most energy-consuming process in cells, responds to changing physiologic priorities, e.g., upon mitogen- or stress-induced adaptations signaled through the mitogen-activated protein kinases (MAPKs). The prevailing status of protein synthesis machinery is a viral pathogenesis factor, particularly for plus-strand RNA viruses, where immediate translation of incoming viral RNAs shapes host-virus interactions. In this study, we unraveled signaling pathways centered on the ERK1/2 and p38α MAPK-interacting kinases MNK1/2 and their role in controlling 7-methyl-guanosine (m(7)G) "cap"-independent translation at enterovirus type 1 internal ribosomal entry sites (IRESs). Activation of Raf-MEK-ERK1/2 signals induced viral IRES-mediated translation in a manner dependent on MNK1/2. This effect was not due to MNK's known functions as eukaryotic initiation factor (eIF) 4G binding partner or eIF4E(S209) kinase. Rather, MNK catalytic activity enabled viral IRES-mediated translation/host cell cytotoxicity through negative regulation of the Ser/Arg (SR)-rich protein kinase (SRPK). Our investigations suggest that SRPK activity is a major determinant of type 1 IRES competency, host cell cytotoxicity, and viral proliferation in infected cells. We are targeting unfettered enterovirus IRES activity in cancer with PVSRIPO, the type 1 live-attenuated poliovirus (PV) (Sabin) vaccine containing a human rhinovirus type 2 (HRV2) IRES. A phase I clinical trial of PVSRIPO with intratumoral inoculation in patients with recurrent glioblastoma (GBM) is showing early promise. Viral translation proficiency in infected GBM cells is a core requirement for the antineoplastic efficacy of PVSRIPO. Therefore, it is critically important to understand the mechanisms controlling viral cap-independent translation in infected host cells. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Protein kinase C mediates platelet secretion and thrombus formation through protein kinase D2.

PubMed

Konopatskaya, Olga; Matthews, Sharon A; Harper, Matthew T; Gilio, Karen; Cosemans, Judith M E M; Williams, Christopher M; Navarro, Maria N; Carter, Deborah A; Heemskerk, Johan W M; Leitges, Michael; Cantrell, Doreen; Poole, Alastair W

2011-07-14

Platelets are highly specialized blood cells critically involved in hemostasis and thrombosis. Members of the protein kinase C (PKC) family have established roles in regulating platelet function and thrombosis, but the molecular mechanisms are not clearly understood. In particular, the conventional PKC isoform, PKCα, is a major regulator of platelet granule secretion, but the molecular pathway from PKCα to secretion is not defined. Protein kinase D (PKD) is a family of 3 kinases activated by PKC, which may represent a step in the PKC signaling pathway to secretion. In the present study, we show that PKD2 is the sole PKD member regulated downstream of PKC in platelets, and that the conventional, but not novel, PKC isoforms provide the upstream signal. Platelets from a gene knock-in mouse in which 2 key phosphorylation sites in PKD2 have been mutated (Ser707Ala/Ser711Ala) show a significant reduction in agonist-induced dense granule secretion, but not in α-granule secretion. This deficiency in dense granule release was responsible for a reduced platelet aggregation and a marked reduction in thrombus formation. Our results show that in the molecular pathway to secretion, PKD2 is a key component of the PKC-mediated pathway to platelet activation and thrombus formation through its selective regulation of dense granule secretion.

Roles of PTEN-induced putative kinase 1 and dynamin-related protein 1 in transient global ischemia-induced hippocampal neuronal injury

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Shang-Der, E-mail: chensd@adm.cgmh.org.tw; Center for Translational Research in Biomedical Sciences, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Taiwan; Lin, Tsu-Kung

Recent studies showed that increased mitochondrial fission is an early event of cell death during cerebral ischemia and dynamin-related protein 1 (Drp1) plays an important role in mitochondrial fission, which may be regulated by PTEN-induced putative kinase 1 (PINK1), a mitochondrial serine/threonine-protein kinase thought to protect cells from stress-induced mitochondrial dysfunction and regulate mitochondrial fission. However, the roles of PINK1 and Drp1 in hippocampal injury caused by transient global ischemia (TGI) remain unknown. We therefore tested the hypothesis that TGI may induce PINK1 causing downregulation of Drp1 phosphorylation to enhance hippocampal neuronal survival, thus functioning as an endogenous neuroprotective mechanism.more » We found progressively increased PINK1 expression in the hippocampal CA1 subfield1-48 h following TGI, reaching the maximal level at 4 h. Despite lack of changes in the expression level of total Drp1 and phosphor-Drp1 at Ser637, TGI induced a time-dependent increase of Drp1 phosphorlation at Ser616 that peaked after 24 h. Notably, PINK1-siRNA increased p-Drp1(Ser616) protein level in hippocampal CA1 subfield 24 h after TGI. The PINK1 siRNA also aggravated the TGI-induced oxidative DNA damage with an increased 8-hydroxy-deoxyguanosine (8-OHdG) content in hippocampal CA1 subfield. Furthermore, PINK1 siRNA also augmented TGI-induced apoptosis as evidenced by the increased numbers of TUNEL-positive staining and enhanced DNA fragmentation. These findings indicated that PINK1 is an endogenous protective mediator vital for neuronal survival under ischemic insult through regulating Drp1 phosphorylation at Ser616. - Highlights: • Transient global ischemia increases expression of PINK1 and p-Drp1 at Ser616 in hippocampal CA1 subfield. • PINK1-siRNA decreases PINK1 expression but increases p-Drp1 at Ser616 in hippocampal CA1 subfield. • PINK1-siRNA augments oxidative stress and neuronal damage in hippocampal CA1 subfield.« less

Spatiotemporal characterization of mTOR kinase activity following kainic acid induced status epilepticus and analysis of rat brain response to chronic rapamycin treatment.

PubMed

Macias, Matylda; Blazejczyk, Magdalena; Kazmierska, Paulina; Caban, Bartosz; Skalecka, Agnieszka; Tarkowski, Bartosz; Rodo, Anna; Konopacki, Jan; Jaworski, Jacek

2013-01-01

Mammalian target of rapamycin (mTOR) is a protein kinase that senses nutrient availability, trophic factors support, cellular energy level, cellular stress, and neurotransmitters and adjusts cellular metabolism accordingly. Adequate mTOR activity is needed for development as well as proper physiology of mature neurons. Consequently, changes in mTOR activity are often observed in neuropathology. Recently, several groups reported that seizures increase mammalian target of rapamycin (mTOR) kinase activity, and such increased activity in genetic models can contribute to spontaneous seizures. However, the current knowledge about the spatiotemporal pattern of mTOR activation induced by proconvulsive agents is rather rudimentary. Also consequences of insufficient mTOR activity on a status epilepticus are poorly understood. Here, we systematically investigated these two issues. We showed that mTOR signaling was activated by kainic acid (KA)-induced status epilepticus through several brain areas, including the hippocampus and cortex as well as revealed two waves of mTOR activation: an early wave (2 h) that occurs in neurons and a late wave that predominantly occurs in astrocytes. Unexpectedly, we found that pretreatment with rapamycin, a potent mTOR inhibitor, gradually (i) sensitized animals to KA treatment and (ii) induced gross anatomical changes in the brain.

Spatiotemporal Characterization of mTOR Kinase Activity Following Kainic Acid Induced Status Epilepticus and Analysis of Rat Brain Response to Chronic Rapamycin Treatment

PubMed Central

Macias, Matylda; Blazejczyk, Magdalena; Kazmierska, Paulina; Caban, Bartosz; Skalecka, Agnieszka; Tarkowski, Bartosz; Rodo, Anna; Konopacki, Jan; Jaworski, Jacek

2013-01-01

Mammalian target of rapamycin (mTOR) is a protein kinase that senses nutrient availability, trophic factors support, cellular energy level, cellular stress, and neurotransmitters and adjusts cellular metabolism accordingly. Adequate mTOR activity is needed for development as well as proper physiology of mature neurons. Consequently, changes in mTOR activity are often observed in neuropathology. Recently, several groups reported that seizures increase mammalian target of rapamycin (mTOR) kinase activity, and such increased activity in genetic models can contribute to spontaneous seizures. However, the current knowledge about the spatiotemporal pattern of mTOR activation induced by proconvulsive agents is rather rudimentary. Also consequences of insufficient mTOR activity on a status epilepticus are poorly understood. Here, we systematically investigated these two issues. We showed that mTOR signaling was activated by kainic acid (KA)-induced status epilepticus through several brain areas, including the hippocampus and cortex as well as revealed two waves of mTOR activation: an early wave (2 h) that occurs in neurons and a late wave that predominantly occurs in astrocytes. Unexpectedly, we found that pretreatment with rapamycin, a potent mTOR inhibitor, gradually (i) sensitized animals to KA treatment and (ii) induced gross anatomical changes in the brain. PMID:23724051

Cellular reprogramming through mitogen-activated protein kinases.

PubMed

Lee, Justin; Eschen-Lippold, Lennart; Lassowskat, Ines; Böttcher, Christoph; Scheel, Dierk

2015-01-01

Mitogen-activated protein kinase (MAPK) cascades are conserved eukaryote signaling modules where MAPKs, as the final kinases in the cascade, phosphorylate protein substrates to regulate cellular processes. While some progress in the identification of MAPK substrates has been made in plants, the knowledge on the spectrum of substrates and their mechanistic action is still fragmentary. In this focused review, we discuss the biological implications of the data in our original paper (Sustained mitogen-activated protein kinase activation reprograms defense metabolism and phosphoprotein profile in Arabidopsis thaliana; Frontiers in Plant Science 5: 554) in the context of related research. In our work, we mimicked in vivo activation of two stress-activated MAPKs, MPK3 and MPK6, through transgenic manipulation of Arabidopsis thaliana and used phosphoproteomics analysis to identify potential novel MAPK substrates. Here, we plotted the identified putative MAPK substrates (and downstream phosphoproteins) as a global protein clustering network. Based on a highly stringent selection confidence level, the core networks highlighted a MAPK-induced cellular reprogramming at multiple levels of gene and protein expression-including transcriptional, post-transcriptional, translational, post-translational (such as protein modification, folding, and degradation) steps, and also protein re-compartmentalization. Additionally, the increase in putative substrates/phosphoproteins of energy metabolism and various secondary metabolite biosynthesis pathways coincides with the observed accumulation of defense antimicrobial substances as detected by metabolome analysis. Furthermore, detection of protein networks in phospholipid or redox elements suggests activation of downstream signaling events. Taken in context with other studies, MAPKs are key regulators that reprogram cellular events to orchestrate defense signaling in eukaryotes.

A Direct Redox Regulation of Protein Kinase C Isoenzymes Mediates Oxidant-induced Neuritogenesis in PC12 Cells*

PubMed Central

Gopalakrishna, Rayudu; Gundimeda, Usha; Schiffman, Jason Eric; McNeill, Thomas H.

2008-01-01

In this study, we have used the PC12 cell model to elucidate the mechanisms by which sublethal doses of oxidants induce neuritogenesis. The xanthine/xanthine oxidase (X/XO) system was used for the steady state generation of superoxide, and CoCl2 was used as a representative transition metal redox catalyst. Upon treatment of purified protein kinase C (PKC) with these oxidants, there was an increase in its cofactor-independent activation. Redox-active cobalt competed with the redoxinert zinc present in the zinc-thiolates of the PKC regulatory domain and induced the oxidation of these cysteine-rich regions. Both CoCl2 and X/XO induced neurite outgrowth in PC12 cells, as determined by an overexpression of neuronal marker genes. Furthermore, these oxidants induced a translocation of PKC from cytosol to membrane and subsequent conversion of PKC to a cofactor-independent form. Isoenzyme-specific PKC inhibitors demonstrated that PKCε plays a crucial role in neuritogenesis. Moreover, oxidant-induced neurite outgrowth was increased with a conditional overexpression of PKCε and decreased with its knock-out by small interfering RNA. Parallel with PKC activation, an increase in phosphorylation of the growth-associated neuronal protein GAP-43 at Ser41 was observed. Additionally, there was a sustained activation of extracellular signal-regulated kinases 1 and 2, which was correlated with activating phosphorylation (Ser133) of cAMP-responsive element-binding protein. All of these signaling events that are causally linked to neuritogenesis were blocked by antioxidant N-acetylcysteine (both l and d-forms) and by a variety of PKC-specific inhibitors. Taken together, these results strongly suggest that sublethal doses of oxidants induce neuritogenesis via a direct redox activation of PKCε. PMID:18375950

Epoxyeicosatrienoic Acids Prevent Cisplatin-Induced Renal Apoptosis through a p38 Mitogen-Activated Protein Kinase–Regulated Mitochondrial Pathway

PubMed Central

Liu, Yingmei; Lu, Xiaodan; Nguyen, Sinh; Olson, Jean L.; Webb, Heather K.

2013-01-01

Soluble epoxide hydrolase (sEH) catalyzes the conversion of epoxyeicosatrienoic acids into less active eicosanoids, and inhibitors of sEH have anti-inflammatory and antiapoptotic properties. Based on previous observations that sEH inhibition attenuates cisplatin-induced nephrotoxicity by modulating nuclear factor-κB signaling, we hypothesized that this strategy would also attenuate cisplatin-induced renal apoptosis. Inhibition of sEH with AR9273 [1-adamantan-1-yl-3-(1-methylsulfonyl-piperidin-4-yl-urea)] reduced cisplatin-induced apoptosis through mechanisms involving mitochondrial apoptotic pathways and by reducing reactive oxygen species. Renal mitochondrial Bax induction following cisplatin treatment was significantly decreased by treatment of mice with AR9273 and these antiapoptotic effects involved p38 mitogen-activated protein kinase signaling. Similar mechanisms contributed to reduced apoptosis in Ephx2−/− mice treated with cisplatin. Moreover, in pig kidney proximal tubule cells, cisplatin-induced mitochondrial trafficking of Bax and cytochrome c, caspase-3 activation, and oxidative stress are significantly attenuated in the presence of epoxyeicosatrienoic acids (EETs). Collectively, these in vivo and in vitro studies demonstrate a role for EETs in limiting cisplatin-induced renal apoptosis. Inhibition of sEH represents a novel therapeutic strategy for protection against cisplatin-induced renal damage. PMID:24092818

Matrix-specific protein kinase A signaling regulates p21 activated kinase activation by flow in endothelial cells

PubMed Central

Funk, Steven Daniel; Yurdagul, Arif; Green, Jonette M.; Jhaveri, Krishna A.; Schwartz, Martin Alexander; Orr, A. Wayne

2010-01-01

Rationale Atherosclerosis is initiated by blood flow patterns that activate inflammatory pathways in endothelial cells. Activation of inflammatory signaling by fluid shear stress is highly dependent on the composition of the subendothelial extracellular matrix. The basement membrane proteins laminin and collagen found in normal vessels suppress flow-induced p21 activated kinase (PAK) and NF-κB activation. By contrast, the provisional matrix proteins fibronectin and fibrinogen found in wounded or inflamed vessels support flow-induced PAK and NF-κB activation. PAK mediates both flow-induced permeability and matrix-specific activation of NF-κB. Objective To elucidate the mechanisms regulating matrix-specific PAK activation. Methods and Results We now show that matrix composition does not affect the upstream pathway by which flow activates PAK (integrin activation, Rac). Instead basement membrane proteins enhance flow-induced protein kinase A (PKA) activation, which suppresses PAK. Inhibiting PKA restored flow-induced PAK and NF-κB activation in cells on basement membrane proteins, whereas stimulating PKA inhibited flow-induced activation of inflammatory signaling in cells on fibronectin. PKA suppressed inflammatory signaling through PAK inhibition. Activating PKA by injection of the PGI2 analog iloprost reduced PAK activation and inflammatory gene expression at sites of disturbed flow in vivo, whereas inhibiting PKA by PKI injection enhanced PAK activation and inflammatory gene expression. Inhibiting PAK prevented the enhancement of inflammatory gene expression by PKI. Conclusions Basement membrane proteins inhibit inflammatory signaling in endothelial cells via PKA-dependent inhibition of PAK. PMID:20224042

Ionizing radiation induces cellular senescence of articular chondrocytes via negative regulation of SIRT1 by p38 kinase.

PubMed

Hong, Eun-Hee; Lee, Su-Jae; Kim, Jae-Sung; Lee, Kee-Ho; Um, Hong-Duck; Kim, Jae-Hong; Kim, Song-Ja; Kim, Jong-Il; Hwang, Sang-Gu

2010-01-08

Radiotherapy is increasingly used in the treatment of joint diseases, but limited information is available on the effects of radiation on cartilage. Here, we characterize the molecular mechanisms leading to cellular senescence in irradiated primary cultured articular chondrocytes. Ionizing radiation (IR) causes activation of ERK, in turn generating intracellular reactive oxygen species (ROS) with induction of senescence-associated beta-galactosidase (SA-beta-gal) activity. ROS activate p38 kinase, which further promotes ROS generation, forming a positive feedback loop to sustain ROS-p38 kinase signaling. The ROS inhibitors, nordihydroguaiaretic acid and GSH, suppress phosphorylation of p38 and cell numbers positive for SA-beta-gal following irradiation. Moreover, inhibition of the ERK and p38 kinase pathways leads to blockage of IR-induced SA-beta-gal activity via reduction of ROS generation. Although JNK is activated by ROS, this pathway is not associated with cellular senescence of chondrocytes. Interestingly, IR triggers down-regulation of SIRT1 protein expression but not the transcript level, indicative of post-transcriptional cleavage of the protein. SIRT1 degradation is markedly blocked by SB203589 or MG132 after IR treatment, suggesting that cleavage occurs as a result of binding with p38 kinase, followed by processing via the 26 S proteasomal degradation pathway. Overexpression or activation of SIRT1 significantly reduces the IR-induced senescence phenotype, whereas inhibition of SIRT1 activity induces senescence. Based on these findings, we propose that IR induces cellular senescence of articular chondrocytes by negative post-translational regulation of SIRT1 via ROS-dependent p38 kinase activation.

Purine inhibitors of protein kinases, G proteins and polymerases

DOEpatents

Gray, Nathanael S.; Schultz, Peter; Kim, Sung-Hou; Meijer, Laurent

2001-07-03

The present invention relates to purine analogs that inhibit, inter alia, protein kinases, G-proteins and polymerases. In addition, the present invention relates to methods of using such purine analogs to inhibit protein kinases, G-proteins, polymerases and other cellular processes and to treat cellular proliferative diseases.

Ca(2+)-sensitive tyrosine kinase Pyk2/CAK beta-dependent signaling is essential for G-protein-coupled receptor agonist-induced hypertrophy.

PubMed

Hirotani, Shinichi; Higuchi, Yoshiharu; Nishida, Kazuhiko; Nakayama, Hiroyuki; Yamaguchi, Osamu; Hikoso, Shungo; Takeda, Toshihiro; Kashiwase, Kazunori; Watanabe, Tetsuya; Asahi, Michio; Taniike, Masayuki; Tsujimoto, Ikuko; Matsumura, Yasushi; Sasaki, Terukatsu; Hori, Masatsugu; Otsu, Kinya

2004-06-01

G-protein-coupled receptor agonists including endothelin-1 (ET-1) and phenylephrine (PE) induce hypertrophy in neonatal ventricular cardiomyocytes. Others and we previously reported that Rac1 signaling pathway plays an important role in this agonist-induced cardiomyocyte hypertrophy. In this study reported here, we found that a Ca(2+)-sensitive non-receptor tyrosine kinase, proline-rich tyrosine kinase 2 (Pyk2)/cell adhesion kinase beta (CAKbeta), is involved in ET-1- and PE-induced cardiomyocyte hypertrophy medicated through Rac1 activation. ET-1, PE or the Ca(2+) inophore, ionomycin, stimulated a rapid increase in tyrosine phosphorylation of Pyk2. The tyrosine phosphorylation of Pyk2 was suppressed by the Ca(2+) chelator, BAPTA. ET-1- or PE-induced increases in [(3)H]-leucine incorporation and expression of atrial natriuretic factor and the enhancement of sarcomere organization. Infection of cardiomyocytes with an adenovirus expressing a mutant Pyk2 which lacked its kinase domain or its ability to bind to c-Src, eliminated ET-1- and PE-induced hypertrophic responses. Inhibition of Pyk2 activation also suppressed Rac1 activation and reactive oxygen species (ROS) production. These findings suggest that the signal transduction pathway leading to hypertrophy involves Ca(2+)-induced Pyk2 activation followed by Rac1-dependent ROS production.

Tangeretin reduces ultraviolet B (UVB)-induced cyclooxygenase-2 expression in mouse epidermal cells by blocking mitogen-activated protein kinase (MAPK) activation and reactive oxygen species (ROS) generation.

PubMed

Yoon, Ji Hye; Lim, Tae-Gyu; Lee, Kyung Mi; Jeon, Ae Ji; Kim, Su Yeon; Lee, Ki Won

2011-01-12

The present study examined the effects of tangeretin, a polymethoxylated flavonone present in citrus fruits, on ultraviolet B (UVB)-induced cyclooxygenase-2 (COX-2) expression in JB6 P+ mouse skin epidermal cells. Tangeretin suppressed UVB-induced COX-2 expression and transactivation of nuclear factor-κB and activator protein-1 in JB6 P+ cells. Moreover, tangeretin blocked UVB-induced phosphorylation of Akt and mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated protein kinase, c-Jun N-terminal kinase, and p38, and attenuated the phosphorylation of MAPK kinases 1/2, 3/6, and 4. Tangeretin also limited the endogenous generation of reactive oxygen species (ROS), thereby protecting the cells against oxidative stress. However, tangeretin did not scavenge the stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and influence the nicotinamide adenine dinucleotide phosphate oxidase activity. These results suggest that the anti-inflammatory effects of tangeretin stem from its modulation of cell signaling and suppression of intracellular ROS generation. Tangeretin may have a potent chemopreventive effect in skin cancer.

Overexpression of heart-type fatty acid binding protein enhances fatty acid-induced podocyte injury

PubMed Central

Gao, Qing; Sarkar, Alhossain; Chen, Yizhi; Xu, Bo; Zhu, Xiaojuan; Yuan, Yang; Guan, Tianjun

2018-01-01

Deregulated lipid metabolism is a characteristic of metabolic diseases including type 2 diabetes and obesity, and likely contributes to podocyte injury and end-stage kidney disease. Heart-type fatty acid binding protein (H-FABP) was reported to be associated with lipid metabolism. The present study investigated whether H-FABP contributes to podocyte homeostasis. Podocytes were transfected by lentiviral vector to construct a cell line which stably overexpressed H-FABP. Small interfering RNA capable of effectively silencing H-FABP was introduced into podocytes to construct a cell line with H-FABP knockdown. Certain groups were treated with palmitic acid (PA) and the fat metabolism, as well as inflammatory and oxidative stress markers were measured. PA accelerated lipid metabolism derangement, inflammatory reaction and oxidative stress in podocytes. Overexpression of H-FABP enhanced the PA-induced disequilibrium in podocytes. The mRNA and protein expression levels of acyl-coenzyme A oxidase 3 and monocyte chemotactic protein 1, and the protein expression levels of 8-hydroxy-2′-deoxyguanosine and 4-hydroxynonenal were upregulated in the H-FABP overexpression group, while the mRNA and protein expression of peroxisome proliferator activated receptor α was downregulated. Knockdown of H-FABP inhibited the PA-induced injury and lipid metabolism derangement, as well as the inflammatory reaction and oxidative stress in podocytes. These results indicated that overexpression of H-FABP enhances fatty acid-induced podocyte injury, while H-FABP inhibition may represent a potential therapeutic strategy for the prevention of lipid metabolism-associated podocyte injury. PMID:29434805

Conserved herpesvirus protein kinases

PubMed Central

Gershburg, Edward; Pagano, Joseph S.

2008-01-01

Conserved herpesviral protein kinases (CHPKs) are a group of enzymes conserved throughout all subfamilies of Herpesviridae. Members of this group are serine/threonine protein kinases that are likely to play a conserved role in viral infection by interacting with common host cellular and viral factors; however along with a conserved role, individual kinases may have unique functions in the context of viral infection in such a way that they are only partially replaceable even by close homologues. Recent studies demonstrated that CHPKs are crucial for viral infection and suggested their involvement in regulation of numerous processes at various infection steps (primary infection, nuclear egress, tegumentation), although the mechanisms of this regulation remain unknown. Notwithstanding, recent advances in discovery of new CHPK targets, and studies of CHPK knockout phenotypes have raised their attractiveness as targets for antiviral therapy. A number of compounds have been shown to inhibit the activity of human cytomegalovirus (HCMV)-encoded UL97 protein kinase and exhibit a pronounced antiviral effect, although the same compounds are inactive against Epstein-Barr Virus (EBV)-encoded protein kinase BGLF4, illustrating the fact that low homology between the members of this group complicates development of compounds targeting the whole group, and suggesting that individualized, structure-based inhibitor design will be more effective. Determination of CHPK structures will greatly facilitate this task. PMID:17881303

Effects of protein kinase C activators on phorbol ester-sensitive and -resistant EL4 thymoma cells.

PubMed

Sansbury, H M; Wisehart-Johnson, A E; Qi, C; Fulwood, S; Meier, K E

1997-09-01

Phorbol ester-sensitive EL4 murine thymoma cells respond to phorbol 12-myristate 13-acetate with activation of ERK mitogen-activated protein kinases, synthesis of interleukin-2, and death, whereas phorbol ester-resistant variants of this cell line do not exhibit these responses. Additional aspects of the resistant phenotype were examined, using a newly-established resistant cell line. Phorbol ester induced morphological changes, ERK activation, calcium-dependent activation of the c-Jun N-terminal kinase (JNK), interleukin-2 synthesis, and growth inhibition in sensitive but not resistant cells. A series of protein kinase C activators caused membrane translocation of protein kinase C's (PKCs) alpha, eta, and theta in both cell lines. While PKC eta was expressed at higher levels in sensitive than in resistant cells, overexpression of PKC eta did not restore phorbol ester-induced ERK activation to resistant cells. In sensitive cells, PKC activators had similar effects on cell viability and ERK activation, but differed in their abilities to induce JNK activation and interleukin-2 synthesis. PD 098059, an inhibitor of the mitogen activated protein (MAP)/ERK kinase kinase MEK, partially inhibited ERK activation and completely blocked phorbol ester-induced cell death in sensitive cells. Thus MEK and/or ERK activation, but not JNK activation or interleukin-2 synthesis, appears to be required for phorbol ester-induced toxicity. Alterations in phorbol ester response pathways, rather than altered expression of PKC isoforms, appear to confer phorbol ester resistance to EL4 cells.

Intracellular Signaling by Hydrolysis of Phospholipids and Activation of Protein Kinase C

NASA Astrophysics Data System (ADS)

Nishizuka, Yasutomi

1992-10-01

Hydrolysis of inositol phospholipids by phospholipase C is initiated by either receptor stimulation or opening of Ca2+ channels. This was once thought to be the sole mechanism to produce the diacylglycerol that links extracellular signals to intracellular events through activation of protein kinase C. It is becoming clear that agonist-induced hydrolysis of other membrane phospholipids, particularly choline phospholipids, by phospholipase D and phospholipase A_2 may also take part in cell signaling. The products of hydrolysis of these phospholipids may enhance and prolong the activation of protein kinase C. Such prolonged activation of protein kinase C is essential for long-term cellular responses such as cell proliferation and differentiation.

Gadd45a Protein Promotes Skeletal Muscle Atrophy by Forming a Complex with the Protein Kinase MEKK4.

PubMed

Bullard, Steven A; Seo, Seongjin; Schilling, Birgit; Dyle, Michael C; Dierdorff, Jason M; Ebert, Scott M; DeLau, Austin D; Gibson, Bradford W; Adams, Christopher M

2016-08-19

Skeletal muscle atrophy is a serious and highly prevalent condition that remains poorly understood at the molecular level. Previous work found that skeletal muscle atrophy involves an increase in skeletal muscle Gadd45a expression, which is necessary and sufficient for skeletal muscle fiber atrophy. However, the direct mechanism by which Gadd45a promotes skeletal muscle atrophy was unknown. To address this question, we biochemically isolated skeletal muscle proteins that associate with Gadd45a as it induces atrophy in mouse skeletal muscle fibers in vivo We found that Gadd45a interacts with multiple proteins in skeletal muscle fibers, including, most prominently, MEKK4, a mitogen-activated protein kinase kinase kinase that was not previously known to play a role in skeletal muscle atrophy. Furthermore, we found that, by forming a complex with MEKK4 in skeletal muscle fibers, Gadd45a increases MEKK4 protein kinase activity, which is both sufficient to induce skeletal muscle fiber atrophy and required for Gadd45a-mediated skeletal muscle fiber atrophy. Together, these results identify a direct biochemical mechanism by which Gadd45a induces skeletal muscle atrophy and provide new insight into the way that skeletal muscle atrophy occurs at the molecular level. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

The Energy Landscape Analysis of Cancer Mutations in Protein Kinases

PubMed Central

Dixit, Anshuman; Verkhivker, Gennady M.

2011-01-01

The growing interest in quantifying the molecular basis of protein kinase activation and allosteric regulation by cancer mutations has fueled computational studies of allosteric signaling in protein kinases. In the present study, we combined computer simulations and the energy landscape analysis of protein kinases to characterize the interplay between oncogenic mutations and locally frustrated sites as important catalysts of allostetric kinase activation. While structurally rigid kinase core constitutes a minimally frustrated hub of the catalytic domain, locally frustrated residue clusters, whose interaction networks are not energetically optimized, are prone to dynamic modulation and could enable allosteric conformational transitions. The results of this study have shown that the energy landscape effect of oncogenic mutations may be allosteric eliciting global changes in the spatial distribution of highly frustrated residues. We have found that mutation-induced allosteric signaling may involve a dynamic coupling between structurally rigid (minimally frustrated) and plastic (locally frustrated) clusters of residues. The presented study has demonstrated that activation cancer mutations may affect the thermodynamic equilibrium between kinase states by allosterically altering the distribution of locally frustrated sites and increasing the local frustration in the inactive form, while eliminating locally frustrated sites and restoring structural rigidity of the active form. The energy landsape analysis of protein kinases and the proposed role of locally frustrated sites in activation mechanisms may have useful implications for bioinformatics-based screening and detection of functional sites critical for allosteric regulation in complex biomolecular systems. PMID:21998754

[Dracorhodin perchlorate inhibit high glucose induce serum and glucocorticoid induced protein kinase 1 and fibronectin expression in human mesangial cells].

PubMed

Xie, Yifeng; Wang, Quansheng; Liu, Jianguo; Xie, Jiwen; Xue, Kaming; Tang, Qing

2010-08-01

To investigate the effect of dracorhodin perchlorate (DP) on inhibiting high glucose-induced serum and glucocorticoid induced protein kinase 1 (SGK1) and fibronectin (FN) expression in human mesangial cells (HMC), and its mechanism of prevention and treatment on renal fibrosis in diabetic nephropathy (DN) . The HMC were divided into normal glucose group (NG group, 5.5 mmol x L(-1) D-glucose), normal glucose +low DP group (NG + LDP group, 5.5 mmol x L(-1) D-glucose +7.5 micromol x L(-1) DP), normal glucose +high DP group (NG + HDP group, 5.5 mmol x L(-1) D-glucose + 15 micromol x L(-1) DP), high glucose group (HG group,25 mmol x L(-1) D-glucose), high glucose +low DP group (HG + LDP group, 25 mmol x L(-1) D-glucose + 7.5 micromol x L(-1) DP)and high glucose +high DP group (HG +HDP group, 25 mmol x L(-1) D-glucose + 15 micromol x L(-1) DP). Each group was examined at 24 hours. The levels of SGK1 and FN mRNA was detected by real-time fluorescence quantitative PCR,and the expression of SGK1 and FN protein was detected by Western blot or indirect immunofluorescence. A basal level of SGK1 and FN in HMC were detected in NG group, and the level of SGK1 and FN mRNA and protein were not evidently different compared to that of NG group adding 7.5 micromol x L(-1) DP for 24 hours. On the other hand, the levels of SGK1 and FN mRNA and protein were obviously decreased by adding 15 micromol x L(-1) DP for 24 hours. Compared to NG group, the levels of SGK1 and FN mRNA and protein were increased in HG group after stimulating for 24 hours (P < 0.01). Compared to HG group, the level of SGK1 and FN mRNA and protein were evidently reduced in HG + LDP and HG + HDP groups by adding 7.5 micromol x L(-1) DP and 15 micromol x L(-1) DP for 24 hours (P < 0.01). Dracorhodin perchlorate can inhibit high glucose-induced serum and glucocorticoid induced protein kinase 1 (SGK1) and fibronectin(FN) expression in human mesangial cells, and this may be part of the mechanism of preventing and treating

Calcium-mediated signaling and calmodulin-dependent kinase regulate hepatocyte-inducible nitric oxide synthase expression.

PubMed

Zhang, Baochun; Crankshaw, Will; Nesemeier, Ryan; Patel, Jay; Nweze, Ikenna; Lakshmanan, Jaganathan; Harbrecht, Brian G

2015-02-01

Induced nitric oxide synthase (iNOS) is induced in hepatocytes by shock and inflammatory stimuli. Excessive NO from iNOS mediates shock-induced hepatic injury and death, so understanding the regulation of iNOS will help elucidate the pathophysiology of septic shock. In vitro, cytokines induce iNOS expression through activation of signaling pathways including mitogen-activated protein kinases and nuclear factor κB. Cytokines also induce calcium (Ca(2+)) mobilization and activate calcium-mediated intracellular signaling pathways, typically through activation of calmodulin-dependent kinases (CaMK). Calcium regulates NO production in macrophages but the role of calcium and calcium-mediated signaling in hepatocyte iNOS expression has not been defined. Primary rat hepatocytes were isolated, cultured, and induced to produce NO with proinflammatory cytokines. Calcium mobilization and Ca(2+)-mediated signaling were altered with ionophore, Ca(2+) channel blockers, and inhibitors of CaMK. The Ca(2+) ionophore A23187 suppressed cytokine-stimulated NO production, whereas Ethylene glycol tetraacetic acid and nifedipine increased NO production, iNOS messenger RNA, and iNOS protein expression. Inhibition of CaMK with KN93 and CBD increased NO production but the calcineurin inhibitor FK 506 decreased iNOS expression. These data demonstrate that calcium-mediated signaling regulates hepatocyte iNOS expression and does so through a mechanism independent of calcineurin. Changes in intracellular calcium levels may regulate iNOS expression during hepatic inflammation induced by proinflammatory cytokines. Copyright © 2015 Elsevier Inc. All rights reserved.

Redox Regulation of Protein Kinases

PubMed Central

Truong, Thu H.; Carroll, Kate S.

2015-01-01

Protein kinases represent one of the largest families of genes found in eukaryotes. Kinases mediate distinct cellular processes ranging from proliferation, differentiation, survival, and apoptosis. Ligand-mediated activation of receptor kinases can lead to the production of endogenous H2O2 by membrane-bound NADPH oxidases. In turn, H2O2 can be utilized as a secondary messenger in signal transduction pathways. This review presents an overview of the molecular mechanisms involved in redox regulation of protein kinases and its effects on signaling cascades. In the first half, we will focus primarily on receptor tyrosine kinases (RTKs), whereas the latter will concentrate on downstream non-receptor kinases involved in relaying stimulant response. Select examples from the literature are used to highlight the functional role of H2O2 regarding kinase activity, as well as the components involved in H2O2 production and regulation during cellular signaling. In addition, studies demonstrating direct modulation of protein kinases by H2O2 through cysteine oxidation will be emphasized. Identification of these redox-sensitive residues may help uncover signaling mechanisms conserved within kinase subfamilies. In some cases, these residues can even be exploited as targets for the development of new therapeutics. Continued efforts in this field will further basic understanding of kinase redox regulation, and delineate the mechanisms involved in physiologic and pathological H2O2 responses. PMID:23639002

Pea DNA Topoisomerase I Is Phosphorylated and Stimulated by Casein Kinase 2 and Protein Kinase C

PubMed Central

Tuteja, Narendra; Reddy, Malireddy Kodandarami; Mudgil, Yashwanti; Yadav, Badam Singh; Chandok, Meena Rani; Sopory, Sudhir Kumar

2003-01-01

DNA topoisomerase I catalyzes the relaxation of superhelical DNA tension and is vital for DNA metabolism; therefore, it is essential for growth and development of plants. Here, we have studied the phosphorylation-dependent regulation of topoisomerase I from pea (Pisum sativum). The purified enzyme did not show autophosphorylation but was phosphorylated in an Mg2+-dependent manner by endogenous protein kinases present in pea nuclear extracts. This phosphorylation was abolished with calf intestinal alkaline phosphatase and lambda phosphatase. It was also phosphorylated by exogenous casein kinase 2 (CK2), protein kinase C (PKC; from animal sources), and an endogenous pea protein, which was purified using a novel phorbol myristate acetate affinity chromatography method. All of these phosphorylations were inhibited by heparin (inhibitor of CK2) and calphostin (inhibitor of PKC), suggesting that pea topoisomerase I is a bona fide substrate for these kinases. Spermine and spermidine had no effect on the CK2-mediated phosphorylation, suggesting that it is polyamine independent. Phospho-amino acid analysis showed that only serine residues were phosphorylated, which was further confirmed using antiphosphoserine antibody. The topoisomerase I activity increased after phosphorylation with exogenous CK2 and PKC. This study shows that these kinases may contribute to the physiological regulation of DNA topoisomerase I activity and overall DNA metabolism in plants. PMID:12913165

Double-Stranded RNA-Dependent Protein Kinase Regulates the Motility of Breast Cancer Cells

PubMed Central

Xu, Mei; Chen, Gang; Wang, Siying; Liao, Mingjun; Frank, Jacqueline A.; Bower, Kimberly A.; Zhang, Zhuo; Shi, Xianglin; Luo, Jia

2012-01-01

Double-stranded RNA (dsRNA)-dependent protein kinase (PKR) is an interferon-induced protein kinase that plays a central role in the anti-viral process. Due to its pro-apoptotic and anti-proliferative action, there is an increased interest in PKR modulation as an anti-tumor strategy. PKR is overexpressed in breast cancer cells; however, the role of PKR in breast cancer cells is unclear. The expression/activity of PKR appears inversely related to the aggressiveness of breast cancer cells. The current study investigated the role of PKR in the motility/migration of breast cancer cells. The activation of PKR by a synthesized dsRNA (PIC) significantly decreased the motility of several breast cancer cell lines (BT474, MDA-MB231 and SKBR3). PIC inhibited cell migration and blocked cell membrane ruffling without affecting cell viability. PIC also induced the reorganization of the actin cytoskeleton and impaired the formation of lamellipodia. These effects of PIC were reversed by the pretreatment of a selective PKR inhibitor. PIC also activated p38 mitogen-activated protein kinase (MAPK) and its downstream MAPK-activated protein kinase 2 (MK2). PIC-induced activation of p38 MAPK and MK2 was attenuated by the PKR inhibitor and the PKR siRNA, but a selective p38 MAPK inhibitor (SB203580) or other MAPK inhibitors did not affect PKR activity, indicating that PKR is upstream of p38 MAPK/MK2. Cofilin is an actin severing protein and regulates membrane ruffling, lamellipodia formation and cell migration. PIC inhibited cofilin activity by enhancing its phosphorylation at Ser3. PIC activated LIM kinase 1 (LIMK1), an upstream kinase of cofilin in a p38 MAPK-dependent manner. We concluded that the activation of PKR suppressed cell motility by regulating the p38 MAPK/MK2/LIMK/cofilin pathway. PMID:23112838

Protein kinase C-α and arginase I mediate pneumolysin-induced pulmonary endothelial hyperpermeability.

PubMed

Lucas, Rudolf; Yang, Guang; Gorshkov, Boris A; Zemskov, Evgeny A; Sridhar, Supriya; Umapathy, Nagavedi S; Jezierska-Drutel, Agnieszka; Alieva, Irina B; Leustik, Martin; Hossain, Hamid; Fischer, Bernhard; Catravas, John D; Verin, Alexander D; Pittet, Jean-François; Caldwell, Ruth B; Mitchell, Timothy J; Cederbaum, Stephen D; Fulton, David J; Matthay, Michael A; Caldwell, Robert W; Romero, Maritza J; Chakraborty, Trinad

2012-10-01

Antibiotics-induced release of the pore-forming virulence factor pneumolysin (PLY) in patients with pneumococcal pneumonia results in its presence days after lungs are sterile and is a major factor responsible for the induction of permeability edema. Here we sought to identify major mechanisms mediating PLY-induced endothelial dysfunction. We evaluated PLY-induced endothelial hyperpermeability in human lung microvascular endothelial cells (HL-MVECs) and human lung pulmonary artery endothelial cells in vitro and in mice instilled intratracheally with PLY. PLY increases permeability in endothelial monolayers by reducing stable and dynamic microtubule content and modulating VE-cadherin expression. These events, dependent upon an increased calcium influx, are preceded by protein kinase C (PKC)-α activation, perturbation of the RhoA/Rac1 balance, and an increase in myosin light chain phosphorylation. At later time points, PLY treatment increases the expression and activity of arginase in HL-MVECs. Arginase inhibition abrogates and suppresses PLY-induced endothelial barrier dysfunction by restoring NO generation. Consequently, a specific PKC-α inhibitor and the TNF-derived tonoplast intrinsic protein peptide, which blunts PLY-induced PKC-α activation, are able to prevent activation of arginase in HL-MVECs and to reduce PLY-induced endothelial hyperpermeability in mice. Arginase I (AI)(+/-)/arginase II (AII)(-/-) C57BL/6 mice, displaying a significantly reduced arginase I expression in the lungs, are significantly less sensitive to PLY-induced capillary leak than their wild-type or AI(+/+)/AII(-/-) counterparts, indicating an important role for arginase I in PLY-induced endothelial hyperpermeability. These results identify PKC-α and arginase I as potential upstream and downstream therapeutic targets in PLY-induced pulmonary endothelial dysfunction.

Protein Kinase C-α and Arginase I Mediate Pneumolysin-Induced Pulmonary Endothelial Hyperpermeability

PubMed Central

Yang, Guang; Gorshkov, Boris A.; Zemskov, Evgeny A.; Sridhar, Supriya; Umapathy, Nagavedi S.; Jezierska-Drutel, Agnieszka; Alieva, Irina B.; Leustik, Martin; Hossain, Hamid; Fischer, Bernhard; Catravas, John D.; Verin, Alexander D.; Pittet, Jean-François; Caldwell, Ruth B.; Mitchell, Timothy J.; Cederbaum, Stephen D.; Fulton, David J.; Matthay, Michael A.; Caldwell, Robert W.; Romero, Maritza J.; Chakraborty, Trinad

2012-01-01

Antibiotics-induced release of the pore-forming virulence factor pneumolysin (PLY) in patients with pneumococcal pneumonia results in its presence days after lungs are sterile and is a major factor responsible for the induction of permeability edema. Here we sought to identify major mechanisms mediating PLY-induced endothelial dysfunction. We evaluated PLY-induced endothelial hyperpermeability in human lung microvascular endothelial cells (HL-MVECs) and human lung pulmonary artery endothelial cells in vitro and in mice instilled intratracheally with PLY. PLY increases permeability in endothelial monolayers by reducing stable and dynamic microtubule content and modulating VE-cadherin expression. These events, dependent upon an increased calcium influx, are preceded by protein kinase C (PKC)-α activation, perturbation of the RhoA/Rac1 balance, and an increase in myosin light chain phosphorylation. At later time points, PLY treatment increases the expression and activity of arginase in HL-MVECs. Arginase inhibition abrogates and suppresses PLY-induced endothelial barrier dysfunction by restoring NO generation. Consequently, a specific PKC-α inhibitor and the TNF-derived tonoplast intrinsic protein peptide, which blunts PLY-induced PKC-α activation, are able to prevent activation of arginase in HL-MVECs and to reduce PLY-induced endothelial hyperpermeability in mice. Arginase I (AI)+/−/arginase II (AII)−/− C57BL/6 mice, displaying a significantly reduced arginase I expression in the lungs, are significantly less sensitive to PLY-induced capillary leak than their wild-type or AI+/+/AII−/− counterparts, indicating an important role for arginase I in PLY-induced endothelial hyperpermeability. These results identify PKC-α and arginase I as potential upstream and downstream therapeutic targets in PLY-induced pulmonary endothelial dysfunction. PMID:22582175

Staurosporine induces rapid homotypic intercellular adhesion of U937 cells via multiple kinase activation

PubMed Central

Cho, Jae Youl; Katz, David R; Chain, Benjamin M

2003-01-01

Staurosporine is a broad-specificity kinase inhibitor, which has acted as lead compound for the development of some novel cytotoxic compounds for treatment of cancer. This study investigates the unexpected observation that staurosporine can also induce homotypic cellular aggregation. In this study, staurosporine is shown to activate rapid homotypic aggregation of U937 cells, at concentrations below those required to induce cell death. This activity is a particular feature of staurosporine, and is not shared by a number of other kinase inhibitors. The proaggregating activity of staurosporine is inhibited by deoxyglucose, cytochalasin B and colchicine. Staurosporine-induced aggregation can be distinguished from that induced by the phorbol 12-myristate 13-acetate by faster kinetics and insensitivity to cycloheximide. Staurosporine induces translocation of conventional and novel, but not atypical isoforms of protein kinase C (PKC). Aggregation induced by staurosporine is inhibited by a number of inhibitors of PKC isoforms, and by inhibitors of protein tyrosine kinases. Staurosporine also induces rapid phosphorylation of ERK and p38, and inhibitors of both these enzymes block aggregation. Staurosporine induces dysregulated activation of multiple kinase signaling pathways in U937 cells, and the combined activity of several of these pathways is essential for the induction of aggregation. PMID:12970105

Evidence that N-acetylcysteine inhibits TNF-alpha-induced cerebrovascular endothelin-1 upregulation via inhibition of mitogen- and stress-activated protein kinase.

PubMed

Sury, Matthias D; Frese-Schaper, Manuela; Mühlemann, Miranda K; Schulthess, Fabienne T; Blasig, Ingolf E; Täuber, Martin G; Shaw, Sidney G; Christen, Stephan

2006-11-01

N-acetylcysteine (NAC) is neuroprotective in animal models of acute brain injury such as caused by bacterial meningitis. However, the mechanism(s) by which NAC exerts neuroprotection is unclear. Gene expression of endothelin-1 (ET-1), which contributes to cerebral blood flow decline in acute brain injury, is partially regulated by reactive oxygen species, and thus a potential target of NAC. We therefore examined the effect of NAC on tumor necrosis factor (TNF)-alpha-induced ET-1 production in cerebrovascular endothelial cells. NAC dose dependently inhibited TNF-alpha-induced preproET-1 mRNA upregulation and ET-1 protein secretion, while upregulation of inducible nitric oxide synthase (iNOS) was unaffected. Intriguingly, NAC had no effect on the initial activation (i.e., IkappaB degradation, nuclear p65 translocation, and Ser536 phosphorylation) of NF-kappaB by TNF-alpha. However, transient inhibition of NF-kappaB DNA binding suggested that NAC may inhibit ET-1 upregulation by inhibiting (a) parallel pathway(s) necessary for full transcriptional activation of NF-kappaB-mediated ET-1 gene expression. Similar to NAC, the MEK1/2 inhibitor U0126, the p38 inhibitor SB203580, and the protein kinase inhibitor H-89 selectively inhibited ET-1 upregulation without affecting nuclear p65 translocation, suggesting that NAC inhibits ET-1 upregulation via inhibition of mitogen- and stress-activated protein kinase (MSK). Supporting this notion, cotreatment with NAC inhibited the TNF-alpha-induced rise in MSK1 and MSK2 kinase activity, while siRNA knock-down experiments showed that MSK2 is the predominant isoform involved in TNF-alpha-induced ET-1 upregulation.

Endoplasmic reticulum stress sensor protein kinase R-like endoplasmic reticulum kinase (PERK) protects against pressure overload-induced heart failure and lung remodeling.

PubMed

Liu, Xiaoyu; Kwak, Dongmin; Lu, Zhongbing; Xu, Xin; Fassett, John; Wang, Huan; Wei, Yidong; Cavener, Douglas R; Hu, Xinli; Hall, Jennifer; Bache, Robert J; Chen, Yingjie

2014-10-01

Studies have reported that development of congestive heart failure is associated with increased endoplasmic reticulum stress. Double stranded RNA-activated protein kinase R-like endoplasmic reticulum kinase (PERK) is a major transducer of the endoplasmic reticulum stress response and directly phosphorylates eukaryotic initiation factor 2α, resulting in translational attenuation. However, the physiological effect of PERK on congestive heart failure development is unknown. To study the effect of PERK on ventricular structure and function, we generated inducible cardiac-specific PERK knockout mice. Under unstressed conditions, cardiac PERK knockout had no effect on left ventricular mass, or its ratio to body weight, cardiomyocyte size, fibrosis, or left ventricular function. However, in response to chronic transverse aortic constriction, PERK knockout mice exhibited decreased ejection fraction, increased left ventricular fibrosis, enhanced cardiomyocyte apoptosis, and exacerbated lung remodeling in comparison with wild-type mice. PERK knockout also dramatically attenuated cardiac sarcoplasmic reticulum Ca(2+)-ATPase expression in response to aortic constriction. Our findings suggest that PERK is required to protect the heart from pressure overload-induced congestive heart failure. © 2014 American Heart Association, Inc.

Role of oxidative stress in methamphetamine-induced dopaminergic toxicity mediated by protein kinase Cδ

PubMed Central

Nguyen, Xuan-Khanh Thi; Li, Zhengyi; Bing, Guoying; Bach, Jae-Hyung; Park, Dae Hun; Nakayama, Keiichi; Ali, Syed F.; Kanthasamy, Anumantha G.; Cadet, Jean Lud; Nabeshima, Toshitaka; Kim, Hyoung-Chun

2014-01-01

This study examined the role of protein kinase C (PKC) isozymes in methamphetamine (MA)-induced dopaminergic toxicity. Multiple-dose administration of MA did not significantly alter PKCα, PKCβI, PKCβII, or PKCζ expression in the striatum, but did significantly increase PKCδ expression. Gö6976 (a co-inhibitor of PKCα and -β), hispidin (PKCβ inhibitor), and PKCζ pseudosubstrate inhibitor (PKCζ inhibitor) did not significantly alter MA-induced behavioral impairments. However, rottlerin (PKCδ inhibitor) significantly attenuated behavioral impairments in a dose-dependent manner. In addition, MA-induced behavioral impairments were not apparent in PKCδ knockout (–/–) mice. MA-induced oxidative stress (i.e., lipid peroxidation and protein oxidation) was significantly attenuated in rottlerin-treated mice and was not apparent in PKCδ (–/–) mice. Consistent with this, MA-induced apoptosis (i.e., terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive apoptotic cells) was significantly attenuated in rottlerin-treated mice. Furthermore, MA-induced increases in the dopamine (DA) turnover rate and decreases in tyrosine hydroxylase (TH) activity and the expression of TH, dopamine transporter (DAT), and vesicular monoamine transporter 2 (VMAT2) were not significantly observed in rottlerin-treated or PKCδ (–/–) mice. Our results suggest that PKCδ gene expression is a key mediator of oxidative stress and dopaminergic damage induced by MA. Thus, inhibition of PKCδ may be a useful target for protection against MA-induced neurotoxicity. PMID:22512859

Role of oxidative stress in methamphetamine-induced dopaminergic toxicity mediated by protein kinase Cδ.

PubMed

Shin, Eun-Joo; Duong, Chu Xuan; Nguyen, Xuan-Khanh Thi; Li, Zhengyi; Bing, Guoying; Bach, Jae-Hyung; Park, Dae Hun; Nakayama, Keiichi; Ali, Syed F; Kanthasamy, Anumantha G; Cadet, Jean Lud; Nabeshima, Toshitaka; Kim, Hyoung-Chun

2012-06-15

This study examined the role of protein kinase C (PKC) isozymes in methamphetamine (MA)-induced dopaminergic toxicity. Multiple-dose administration of MA did not significantly alter PKCα, PKCβI, PKCβII, or PKCζ expression in the striatum, but did significantly increase PKCδ expression. Gö6976 (a co-inhibitor of PKCα and -β), hispidin (PKCβ inhibitor), and PKCζ pseudosubstrate inhibitor (PKCζ inhibitor) did not significantly alter MA-induced behavioral impairments. However, rottlerin (PKCδ inhibitor) significantly attenuated behavioral impairments in a dose-dependent manner. In addition, MA-induced behavioral impairments were not apparent in PKCδ knockout (-/-) mice. MA-induced oxidative stress (i.e., lipid peroxidation and protein oxidation) was significantly attenuated in rottlerin-treated mice and was not apparent in PKCδ (-/-) mice. Consistent with this, MA-induced apoptosis (i.e., terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive apoptotic cells) was significantly attenuated in rottlerin-treated mice. Furthermore, MA-induced increases in the dopamine (DA) turnover rate and decreases in tyrosine hydroxylase (TH) activity and the expression of TH, dopamine transporter (DAT), and vesicular monoamine transporter 2 (VMAT2) were not significantly observed in rottlerin-treated or PKCδ (-/-) mice. Our results suggest that PKCδ gene expression is a key mediator of oxidative stress and dopaminergic damage induced by MA. Thus, inhibition of PKCδ may be a useful target for protection against MA-induced neurotoxicity. Copyright © 2012 Elsevier B.V. All rights reserved.

Mitochondria-Translocated PGK1 Functions as a Protein Kinase to Coordinate Glycolysis and the TCA Cycle in Tumorigenesis.

PubMed

Li, Xinjian; Jiang, Yuhui; Meisenhelder, Jill; Yang, Weiwei; Hawke, David H; Zheng, Yanhua; Xia, Yan; Aldape, Kenneth; He, Jie; Hunter, Tony; Wang, Liwei; Lu, Zhimin

2016-03-03

It is unclear how the Warburg effect that exemplifies enhanced glycolysis in the cytosol is coordinated with suppressed mitochondrial pyruvate metabolism. We demonstrate here that hypoxia, EGFR activation, and expression of K-Ras G12V and B-Raf V600E induce mitochondrial translocation of phosphoglycerate kinase 1 (PGK1); this is mediated by ERK-dependent PGK1 S203 phosphorylation and subsequent PIN1-mediated cis-trans isomerization. Mitochondrial PGK1 acts as a protein kinase to phosphorylate pyruvate dehydrogenase kinase 1 (PDHK1) at T338, which activates PDHK1 to phosphorylate and inhibit the pyruvate dehydrogenase (PDH) complex. This reduces mitochondrial pyruvate utilization, suppresses reactive oxygen species production, increases lactate production, and promotes brain tumorigenesis. Furthermore, PGK1 S203 and PDHK1 T338 phosphorylation levels correlate with PDH S293 inactivating phosphorylation levels and poor prognosis in glioblastoma patients. This work highlights that PGK1 acts as a protein kinase in coordinating glycolysis and the tricarboxylic acid (TCA) cycle, which is instrumental in cancer metabolism and tumorigenesis. Copyright © 2016 Elsevier Inc. All rights reserved.

Properties of Acetate Kinase Isozymes and a Branched-Chain Fatty Acid Kinase from a Spirochete

PubMed Central

Harwood, Caroline S.; Canale-Parola, Ercole

1982-01-01

Spirochete MA-2, which is anaerobic, ferments glucose, forming acetate as a major product. The spirochete also ferments (but does not utilize as growth substrates) small amounts of l-leucine, l-isoleucine, and l-valine, forming the branched-chain fatty acids isovalerate, 2-methylbutyrate, and isobutyrate, respectively, as end products. Energy generated through the fermentation of these amino acids is utilized to prolong cell survival under conditions of growth substrate starvation. A branched-chain fatty acid kinase and two acetate kinase isozymes were resolved from spirochete MA-2 cell extracts. Kinase activity was followed by measuring the formation of acyl phosphate from fatty acid and ATP. The branched-chain fatty acid kinase was active with isobutyrate, 2-methylbutyrate, isovalerate, butyrate, valerate, or propionate as a substrate but not with acetate as a substrate. The acetate kinase isozymes were active with acetate and propionate as substrates but not with longer-chain fatty acids as substrates. The acetate kinase isozymes and the branched-chain fatty acid kinase differed in nucleoside triphosphate and cation specificities. Each acetate kinase isozyme had an apparent molecular weight of approximately 125,000, whereas the branched-chain fatty acid kinase had a molecular weight of approximately 76,000. These results show that spirochete MA-2 synthesizes a branched-chain fatty acid kinase specific for leucine, isoleucine, and valine fermentation. It is likely that a phosphate branched-chain amino acids is also synthesized by spirochete MA-2. Thus, in spirochete MA-2, physiological mechanisms have evolved which serve specifically to generate maintenance energy from branched-chain amino acids. PMID:6288660

Sunitinib‐Induced Cardiotoxicity Is Mediated by Off‐Target Inhibition of AMP‐Activated Protein Kinase

PubMed Central

Kerkela, Risto; Woulfe, Kathleen C.; Durand, Jean‐Bernard; Vagnozzi, Ronald; Kramer, David; Chu, Tammy F.; Beahm, Cara; Chen, Ming Hui; Force, Thomas

2009-01-01

Abstract Tyrosine kinase inhibitors (TKIs) are transforming the treatment of patients with malignancies. One such agent, sunitinib (Sutent, Pfizer, New York, NY, USA), has demonstrated activity against a variety of solid tumors. Sunitinib is “multitargeted,” inhibiting growth factor receptors that regulate both tumor angiogenesis and tumor cell survival. However, cardiac dysfunction has been associated with its use. Identification of the target of sunitinib‐associated cardiac dysfunction could guide future drug design to reduce toxicity while preserving anticancer activity. Herein we identify severe mitochondrial structural abnormalities in the heart of a patient with sunitinib‐induced heart failure. In cultured cardiomyocytes, sunitinib induces loss of mitochondrial membrane potential and energy rundown. Despite the latter, 5′ adenosine monophosphate‐activated protein kinase (AMPK) activity, which should be increased in the setting of energy compromise, is reduced in hearts of sunitinib‐treated mice and cardiomyocytes in culture, and this is due to direct inhibition of AMPK by sunitinib. Critically, we find that adenovirus‐mediated gene transfer of an activated mutant of AMPK reduces sunitinib‐induced cell death. Our findings suggest AMPK inhibition plays a central role in sunitinib cardiomyocyte toxicity, highlighting the potential of off‐target effects of TKIs contributing to cardiotoxicity. While multitargeting can enhance tumor cell killing, this must be balanced against the potential increased risk of cardiac dysfunction. PMID:20376335

Activation of multiple mitogen-activated protein kinases by recombinant calcitonin gene-related peptide receptor.

PubMed

Parameswaran, N; Disa, J; Spielman, W S; Brooks, D P; Nambi, P; Aiyar, N

2000-02-18

Calcitonin gene-related peptide is a 37-amino-acid neuropeptide and a potent vasodilator. Although calcitonin gene-related peptide has been shown to have a number of effects in a variety of systems, the mechanisms of action and the intracellular signaling pathways, especially the regulation of mitogen-activated protien kinase (MAPK) pathway, is not known. In the present study we investigated the role of calcitonin gene-related peptide in the regulation of MAPKs in human embryonic kidney (HEK) 293 cells stably transfected with a recombinant porcine calcitonin gene-related peptide-1 receptor. Calcitonin gene-related peptide caused a significant dose-dependent increase in cAMP response and the effect was inhibited by calcitonin gene-related peptide(8-37), the calcitonin gene-related peptide-receptor antagonist. Calcitonin gene-related peptide also caused a time- and concentration-dependent increase in extracellular signal-regulated kinase (ERK) and P38 mitogen-activated protein kinase (P38 MAPK) activities, with apparently no significant change in cjun-N-terminal kinase (JNK) activity. Forskolin, a direct activator of adenylyl cyclase also stimulated ERK and P38 activities in these cells suggesting the invovement of cAMP in this process. Calcitonin gene-related peptide-stimulated ERK and P38 MAPK activities were inhibited significantly by calcitonin gene-related peptide receptor antagonist, calcitonin gene-related peptide-(8-37) suggesting the involvement of calcitonin gene-related peptide-1 receptor. Preincubation of the cells with the cAMP-dependent protein kinase inhibitor, H89 [¿N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, hydrochloride¿] inhibited calcitonin gene-related peptide-mediated activation of ERK and p38 kinases. On the other hand, preincubation of the cells with wortmannin ¿[1S-(1alpha,6balpha,9abeta,11alpha, 11bbeta)]-11-(acetyloxy)-1,6b,7,8,9a,10,11, 11b-octahydro-1-(methoxymethyl)-9a,11b-dimethyl-3H-furo[4,3, 2-de]indeno[4,5-h]-2

Perfluorooctanoic acid exposure for 28 days affects glucose homeostasis and induces insulin hypersensitivity in mice

NASA Astrophysics Data System (ADS)

Yan, Shengmin; Zhang, Hongxia; Zheng, Fei; Sheng, Nan; Guo, Xuejiang; Dai, Jiayin

2015-06-01

Perfluoroalkyl acids (PFAAs) are widely used in many applications due to their unique physical and chemical characteristics. Because of the increasing prevalence of metabolic syndromes, including obesity, dyslipidemia and insulin resistance, concern has arisen about the roles of environmental pollutants in such diseases. Earlier epidemiologic studies showed a potential association between perfluorooctanoic acid (PFOA) and glucose metabolism, but how PFOA influences glucose homeostasis is still unknown. Here, we report on the modulation of the phosphatidylinositol 3-kinase-serine/threonine protein kinase (PI3K-AKT) signaling pathway in the livers of mice after 28 d of exposure to PFOA. Compared with normal mice, PFOA exposure significantly decreased the expression of the phosphatase and tensin homologue (PTEN) protein and affected the PI3K-AKT signaling pathway in the liver. Tolerance tests further indicated that PFOA exposure induced higher insulin sensitivity and glucose tolerance in mice. Biochemical analysis revealed that PFOA exposure reduced hepatic glycogen synthesis, which might be attributed to gluconeogenesis inhibition. The levels of several circulating proteins were altered after PFOA exposure, including proteins potentially related to diabetes and liver disease. Our results suggest that PFOA affected glucose metabolism and induced insulin hypersensitivity in mice.

Purine inhibitors of protein kinases, G proteins and polymerases

DOEpatents

Gray, Nathanael S.; Schultz, Peter; Kim, Sung-Hou; Meijer, Laurent

2004-10-12

The present invention relates to 2-N-substituted 6-(4-methoxybenzylamino)-9-isopropylpurines that inhibit, inter alia, protein kinases, G-proteins and polymerases. In addition, the present invention relates to methods of using such 2-N-substituted 6-(4-methoxybenzylamino)-9-isopropylpurines to inhibit protein kinases, G-proteins, polymerases and other cellular processes and to treat cellular proliferative diseases.

Calcium Hydroxide-induced Proliferation, Migration, Osteogenic Differentiation, and Mineralization via the Mitogen-activated Protein Kinase Pathway in Human Dental Pulp Stem Cells.

PubMed

Chen, Luoping; Zheng, Lisha; Jiang, Jingyi; Gui, Jinpeng; Zhang, Lingyu; Huang, Yan; Chen, Xiaofang; Ji, Jing; Fan, Yubo

2016-09-01

Calcium hydroxide has been extensively used as the gold standard for direct pulp capping in clinical dentistry. It induces proliferation, migration, and mineralization in dental pulp stem cells (DPSCs), but the underlying mechanisms are still unclear. The aim of this study was to investigate the role of the mitogen-activated protein (MAP) kinase pathway in calcium hydroxide-induced proliferation, migration, osteogenic differentiation, and mineralization in human DPSCs. Human DPSCs between passages 3 and 6 were used. DPSCs were preincubated with inhibitors of MAP kinases and cultured with calcium hydroxide. The phosphorylated MAP kinases were detected by Western blot analysis. Cell viability was analyzed via the methylthiazol tetrazolium assay. Cell migration was estimated using the wound healing assay. Alkaline phosphatase (ALP) expression was analyzed using the ALP staining assay. Mineralization was studied by alizarin red staining analysis. Calcium hydroxide significantly promoted the phosphorylation of the c-Jun N-terminal kinase (JNK), p38, and extracellular signal-regulated kinase. The inhibition of JNK and p38 signaling abolished calcium hydroxide-induced proliferation of DPSCs. The inhibition of JNK, p38, and extracellular signal-regulated kinase signaling suppressed the migration, ALP expression, and mineralization of DPSCs. Our study showed that the MAP kinase pathway was involved in calcium hydroxide-induced proliferation, migration, osteogenic differentiation, and mineralization in human DPSCs. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

n-Propyl gallate suppresses lipopolysaccharide-induced inducible nitric oxide synthase activation through protein kinase Cδ-mediated up-regulation of heme oxygenase-1 in RAW264.7 macrophages.

PubMed

Jeon, Wookwang; Park, Seong Ji; Kim, Byung-Chul

2017-04-15

n-Propyl gallate is a synthetic phenolic antioxidant with potential anti-inflammatory effects. However, the underlying mechanism remains largely unknown. In the present study, we showed that n-propyl gallate increases the expression and activity of the heme oxygenase-1 (HO-1), a stress-inducible protein with potent anti-inflammatory activity, in RAW264.7 macrophages. The inhibition of the HO-1 activity by treatment with zinc (II) protoporphyrin IX (ZnPP) or by knockdown of the HO-1 expression with small interference RNA significantly reversed the inhibitory effect of n-Propyl gallate on activations of nuclear factor-κB (NF-κB) and inducible nitric oxide synthase (iNOS) induced by lipopolysaccharide (LPS). An additional mechanism study using inhibitors of signaling kinases revealed the involvement of protein kinase Cδ (PKCδ) in the expression of HO-1 induced by n-Propyl gallate. Consistent with these results, n-Propyl gallate increased the intracellular levels of phosphorylated PKCδ in concentration- and time-dependent manners. The inhibitory effects of n-Propyl gallate on LPS-induced iNOS expression and nitric oxide production were also significantly attenuated by pretreatment with the PKCδ inhibitor, rottlerin, or by transfection with PKCδ (K376R), a kinase-inactive form of PKCδ. Taken together, these findings provide the first evidence that n-Propyl gallate exerts its anti-inflammatory effect through PKCδ-mediated up-regulation of HO-1 in macrophages. Copyright © 2017 Elsevier B.V. All rights reserved.

A framework for classification of prokaryotic protein kinases.

PubMed

Tyagi, Nidhi; Anamika, Krishanpal; Srinivasan, Narayanaswamy

2010-05-26

Overwhelming majority of the Serine/Threonine protein kinases identified by gleaning archaeal and eubacterial genomes could not be classified into any of the well known Hanks and Hunter subfamilies of protein kinases. This is owing to the development of Hanks and Hunter classification scheme based on eukaryotic protein kinases which are highly divergent from their prokaryotic homologues. A large dataset of prokaryotic Serine/Threonine protein kinases recognized from genomes of prokaryotes have been used to develop a classification framework for prokaryotic Ser/Thr protein kinases. We have used traditional sequence alignment and phylogenetic approaches and clustered the prokaryotic kinases which represent 72 subfamilies with at least 4 members in each. Such a clustering enables classification of prokaryotic Ser/Thr kinases and it can be used as a framework to classify newly identified prokaryotic Ser/Thr kinases. After series of searches in a comprehensive sequence database we recognized that 38 subfamilies of prokaryotic protein kinases are associated to a specific taxonomic level. For example 4, 6 and 3 subfamilies have been identified that are currently specific to phylum proteobacteria, cyanobacteria and actinobacteria respectively. Similarly subfamilies which are specific to an order, sub-order, class, family and genus have also been identified. In addition to these, we also identify organism-diverse subfamilies. Members of these clusters are from organisms of different taxonomic levels, such as archaea, bacteria, eukaryotes and viruses. Interestingly, occurrence of several taxonomic level specific subfamilies of prokaryotic kinases contrasts with classification of eukaryotic protein kinases in which most of the popular subfamilies of eukaryotic protein kinases occur diversely in several eukaryotes. Many prokaryotic Ser/Thr kinases exhibit a wide variety of modular organization which indicates a degree of complexity and protein-protein interactions in the

AMP-Activated Protein Kinase Deficiency Rescues Paraquat-Induced Cardiac Contractile Dysfunction Through an Autophagy-Dependent Mechanism

PubMed Central

Wang, Qiurong; Yang, Lifang; Hua, Yinan; Nair, Sreejayan; Xu, Xihui; Ren, Jun

2014-01-01

Aim: Paraquat, a quaternary nitrogen herbicide, is a highly toxic prooxidant resulting in multi-organ failure including the heart although the underlying mechanism still remains elusive. This study was designed to examine the role of the cellular fuel sensor AMP-activated protein kinase (AMPK) in paraquat-induced cardiac contractile and mitochondrial injury. Results: Wild-type and transgenic mice with overexpression of a mutant AMPK α2 subunit (kinase dead, KD), with reduced activity in both α1 and α2 subunits, were administered with paraquat (45 mg/kg) for 48 h. Paraquat elicited cardiac mechanical anomalies including compromised echocardiographic parameters (elevated left ventricular end-systolic diameter and reduced factional shortening), suppressed cardiomyocyte contractile function, intracellular Ca2+ handling, reduced cell survival, and overt mitochondrial damage (loss in mitochondrial membrane potential). In addition, paraquat treatment promoted phosphorylation of AMPK and autophagy. Interestingly, deficiency in AMPK attenuated paraquat-induced cardiac contractile and intracellular Ca2+ derangement. The beneficial effect of AMPK inhibition was associated with inhibition of the AMPK-TSC-mTOR-ULK1 signaling cascade. In vitro study revealed that inhibitors for AMPK and autophagy attenuated paraquat-induced cardiomyocyte contractile dysfunction. Conclusion: Taken together, our findings revealed that AMPK may mediate paraquat-induced myocardial anomalies possibly by regulating the AMPK/mTOR-dependent autophagy. PMID:25092649

High-fat diet induces protein kinase A and G-protein receptor kinase phosphorylation of β2 -adrenergic receptor and impairs cardiac adrenergic reserve in animal hearts.

PubMed

Fu, Qin; Hu, Yuting; Wang, Qingtong; Liu, Yongming; Li, Ning; Xu, Bing; Kim, Sungjin; Chiamvimonvat, Nipavan; Xiang, Yang K

2017-03-15

Patients with diabetes show a blunted cardiac inotropic response to β-adrenergic stimulation despite normal cardiac contractile reserve. Acute insulin stimulation impairs β-adrenergically induced contractile function in isolated cardiomyocytes and Langendorff-perfused hearts. In this study, we aimed to examine the potential effects of hyperinsulinaemia associated with high-fat diet (HFD) feeding on the cardiac β 2 -adrenergic receptor signalling and the impacts on cardiac contractile function. We showed that 8 weeks of HFD feeding leads to reductions in cardiac functional reserve in response to β-adrenergic stimulation without significant alteration of cardiac structure and function, which is associated with significant changes in β 2 -adrenergic receptor phosphorylation at protein kinase A and G-protein receptor kinase sites in the myocardium. The results suggest that clinical intervention might be applied to subjects in early diabetes without cardiac symptoms to prevent further cardiac complications. Patients with diabetes display reduced exercise capability and impaired cardiac contractile reserve in response to adrenergic stimulation. We have recently uncovered an insulin receptor and adrenergic receptor signal network in the heart. The aim of this study was to understand the impacts of high-fat diet (HFD) on the insulin-adrenergic receptor signal network in hearts. After 8 weeks of HFD feeding, mice exhibited diabetes, with elevated insulin and glucose concentrations associated with body weight gain. Mice fed an HFD had normal cardiac structure and function. However, the HFD-fed mice displayed a significant elevation of phosphorylation of the β 2 -adrenergic receptor (β 2 AR) at both the protein kinase A site serine 261/262 and the G-protein-coupled receptor kinase site serine 355/356 and impaired adrenergic reserve when compared with mice fed on normal chow. Isolated myocytes from HFD-fed mice also displayed a reduced contractile response to

Mechanisms of aldehyde-induced adenosinetriphosphatase activities of kinases.

PubMed

Rendina, A R; Cleland, W W

1984-10-23

Aldehyde analogues of the normal alcohol substrates induce ATPase activities by glycerokinase (D-glyceraldehyde), fructose-6-phosphate kinase (2,5-anhydromannose 6-phosphate), fructokinase (2,5-anhydromannose or 2,5-anhydrotalose), hexokinase (D-gluco-hexodialdose), choline kinase (betaine aldehyde), and pyruvate kinase (glyoxylate). Since purified deuterated aldehydes give V and V/K isotope effects near 1.0 for glycerokinase, fructokinase with 2,5-anhydro[1-2H]talose, hexokinase, choline kinase, and pyruvate kinase, the hydrates of these almost fully hydrated aldehydes are the activators of the ATPase reactions. Fructose-6-phosphate kinase and fructokinase with 2,5-anhydro[1-2H]mannose show V/K deuterium isotope effects of 1.10 and 1.22, respectively, suggesting either that both hydrate and free aldehyde may be activators (predicted values are 1.37 if only the free aldehyde activates the ATPase) or, more likely, that the phosphorylated hydrate breaks down in a rate-limiting step on the enzyme while MgADP is still present and the back-reaction to yield free hydrate in solution is still possible. 18O was transferred from the aldehyde hydrate to phosphate during the ATPase reactions of glycerokinase, fructose-6-phosphate kinase, fructokinase, and hexokinase but not with choline kinase or pyruvate kinase. Thus, direct phosphorylation of the hydrates by the first four enzymes gives the phosphate adduct of the aldehyde, which decomposes nonenzymatically, while with choline kinase and pyruvate kinase the hydrates induce transfer to water (metal-bound hydroxide or water with pyruvate kinase on the basis of pH profiles). Observation of a lag in the release of phosphate from the glycerokinase ATPase reaction at 15 degrees C supports the existence of a phosphorylated hydrate intermediate with a rate constant for breakdown of 0.035-0.043 s-1 at this temperature. Kinases that phosphorylate creatine, 3-phosphoglycerate, and acetate did not exhibit ATPase activities in the

Aspirin-induced AMP-activated protein kinase activation regulates the proliferation of vascular smooth muscle cells from spontaneously hypertensive rats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sung, Jin Young; Choi, Hyoung Chul, E-mail: hcchoi@med.yu.ac.kr

Highlights: {yields} Aspirin-induced AMPK phosphorylation was greater in VSMC from SHR than WKY. {yields} Aspirin-induced AMPK phosphorylation inhibited proliferation of VSMC from SHR. {yields} Low basal AMPK phosphorylation in SHR elicits increased VSMC proliferation. {yields} Inhibition of AMPK restored decreased VSMC proliferation by aspirin in SHR. {yields} Aspirin exerts anti-proliferative effect through AMPK activation in VSMC from SHR. -- Abstract: Acetylsalicylic acid (aspirin), used to reduce risk of cardiovascular disease, plays an important role in the regulation of cellular proliferation. However, mechanisms responsible for aspirin-induced growth inhibition are not fully understood. Here, we investigated whether aspirin may exert therapeutic effectsmore » via AMP-activated protein kinase (AMPK) activation in vascular smooth muscle cells (VSMC) from wistar kyoto rats (WKY) and spontaneously hypertensive rats (SHR). Aspirin increased AMPK and acetyl-CoA carboxylase phosphorylation in a time- and dose-dependent manner in VSMCs from WKY and SHR, but with greater efficacy in SHR. In SHR, a low basal phosphorylation status of AMPK resulted in increased VSMC proliferation and aspirin-induced AMPK phosphorylation inhibited proliferation of VSMCs. Compound C, an AMPK inhibitor, and AMPK siRNA reduced the aspirin-mediated inhibition of VSMC proliferation, this effect was more pronounced in SHR than in WKY. In VSMCs from SHR, aspirin increased p53 and p21 expression and inhibited the expression of cell cycle associated proteins, such as p-Rb, cyclin D, and cyclin E. These results indicate that in SHR VSMCs aspirin exerts anti-proliferative effects through the induction of AMPK phosphorylation.« less

Auto-phosphorylation Represses Protein Kinase R Activity.

PubMed

Wang, Die; de Weerd, Nicole A; Willard, Belinda; Polekhina, Galina; Williams, Bryan R G; Sadler, Anthony J

2017-03-10

The central role of protein kinases in controlling disease processes has spurred efforts to develop pharmaceutical regulators of their activity. A rational strategy to achieve this end is to determine intrinsic auto-regulatory processes, then selectively target these different states of kinases to repress their activation. Here we investigate auto-regulation of the innate immune effector protein kinase R, which phosphorylates the eukaryotic initiation factor 2α to inhibit global protein translation. We demonstrate that protein kinase R activity is controlled by auto-inhibition via an intra-molecular interaction. Part of this mechanism of control had previously been reported, but was then controverted. We account for the discrepancy and extend our understanding of the auto-inhibitory mechanism by identifying that auto-inhibition is paradoxically instigated by incipient auto-phosphorylation. Phosphor-residues at the amino-terminus instigate an intra-molecular interaction that enlists both of the N-terminal RNA-binding motifs of the protein with separate surfaces of the C-terminal kinase domain, to co-operatively inhibit kinase activation. These findings identify an innovative mechanism to control kinase activity, providing insight for strategies to better regulate kinase activity.

Ethanol increases affinity of protein kinase C for phosphatidylserine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chin, J.H.

1986-03-01

Protein kinase C is a calcium-dependent enzyme that requires phospholipid for its activation. It is present in relatively high concentration in the brain and may be involved in neuronal function. The present experiments test whether the membrane disorder induced by ethanol affects the activity of kinase C by changing its interaction with membrane lipid. Fractions rich in kinase C were purified from rat brain cytosol by DEAE-cellulose chromatography and Sephadex G-200 gel filtration. Enzyme activity was assayed by measuring the phosphorylation of histone H1. As expected, phosphatidylserine activated the enzyme, and the stimulation was further increased by the addition ofmore » calcium and/or diacylglycerol. At low concentration of free calcium (0.5-1..mu..M), ethanol (800 mM0 enhanced kinase C activity if the presence of phospholipid. similar results were observed in the absence of calcium. Double reciprocal plots of the data showed that ethanol increased the affinity of the enzyme for phosphatidylserine without affecting the V/sub max. The stimulation of kinase C activity by ethanol was not observed at high calcium concentrations. These experiments suggest that ethanol may activated protein kinase C at physiological levels of calcium by facilitating its transfer into the hydrophobic membrane environment.« less

Protein Kinase C Regulates Human Pluripotent Stem Cell Self-Renewal

PubMed Central

Kinehara, Masaki; Kawamura, Suguru; Tateyama, Daiki; Suga, Mika; Matsumura, Hiroko; Mimura, Sumiyo; Hirayama, Noriko; Hirata, Mitsuhi; Uchio-Yamada, Kozue; Kohara, Arihiro; Yanagihara, Kana; Furue, Miho K.

2013-01-01

Background The self-renewal of human pluripotent stem (hPS) cells including embryonic stem and induced pluripotent stem cells have been reported to be supported by various signal pathways. Among them, fibroblast growth factor-2 (FGF-2) appears indispensable to maintain self-renewal of hPS cells. However, downstream signaling of FGF-2 has not yet been clearly understood in hPS cells. Methodology/Principal Findings In this study, we screened a kinase inhibitor library using a high-throughput alkaline phosphatase (ALP) activity-based assay in a minimal growth factor-defined medium to understand FGF-2-related molecular mechanisms regulating self-renewal of hPS cells. We found that in the presence of FGF-2, an inhibitor of protein kinase C (PKC), GF109203X (GFX), increased ALP activity. GFX inhibited FGF-2-induced phosphorylation of glycogen synthase kinase-3β (GSK-3β), suggesting that FGF-2 induced PKC and then PKC inhibited the activity of GSK-3β. Addition of activin A increased phosphorylation of GSK-3β and extracellular signal-regulated kinase-1/2 (ERK-1/2) synergistically with FGF-2 whereas activin A alone did not. GFX negated differentiation of hPS cells induced by the PKC activator, phorbol 12-myristate 13-acetate whereas Gö6976, a selective inhibitor of PKCα, β, and γ isoforms could not counteract the effect of PMA. Intriguingly, functional gene analysis by RNA interference revealed that the phosphorylation of GSK-3β was reduced by siRNA of PKCδ, PKCε, and ζ, the phosphorylation of ERK-1/2 was reduced by siRNA of PKCε and ζ, and the phosphorylation of AKT was reduced by PKCε in hPS cells. Conclusions/Significance Our study suggested complicated cross-talk in hPS cells that FGF-2 induced the phosphorylation of phosphatidylinositol-3 kinase (PI3K)/AKT, mitogen-activated protein kinase/ERK-1/2 kinase (MEK), PKC/ERK-1/2 kinase, and PKC/GSK-3β. Addition of GFX with a MEK inhibitor, U0126, in the presence of FGF-2 and activin A provided a long

AMP-Activated Protein Kinase Interacts with the Peroxisome Proliferator-Activated Receptor Delta to Induce Genes Affecting Fatty Acid Oxidation in Human Macrophages.

PubMed

Kemmerer, Marina; Finkernagel, Florian; Cavalcante, Marcela Frota; Abdalla, Dulcineia Saes Parra; Müller, Rolf; Brüne, Bernhard; Namgaladze, Dmitry

2015-01-01

AMP-activated protein kinase (AMPK) maintains energy homeostasis by suppressing cellular ATP-consuming processes and activating catabolic, ATP-producing pathways such as fatty acid oxidation (FAO). The transcription factor peroxisome proliferator-activated receptor δ (PPARδ) also affects fatty acid metabolism, stimulating the expression of genes involved in FAO. To question the interplay of AMPK and PPARδ in human macrophages we transduced primary human macrophages with lentiviral particles encoding for the constitutively active AMPKα1 catalytic subunit, followed by microarray expression analysis after treatment with the PPARδ agonist GW501516. Microarray analysis showed that co-activation of AMPK and PPARδ increased expression of FAO genes, which were validated by quantitative PCR. Induction of these FAO-associated genes was also observed upon infecting macrophages with an adenovirus coding for AMPKγ1 regulatory subunit carrying an activating R70Q mutation. The pharmacological AMPK activator A-769662 increased expression of several FAO genes in a PPARδ- and AMPK-dependent manner. Although GW501516 significantly increased FAO and reduced the triglyceride amount in very low density lipoproteins (VLDL)-loaded foam cells, AMPK activation failed to potentiate this effect, suggesting that increased expression of fatty acid catabolic genes alone may be not sufficient to prevent macrophage lipid overload.

AMP-Activated Protein Kinase Interacts with the Peroxisome Proliferator-Activated Receptor Delta to Induce Genes Affecting Fatty Acid Oxidation in Human Macrophages

PubMed Central

Kemmerer, Marina; Finkernagel, Florian; Cavalcante, Marcela Frota; Abdalla, Dulcineia Saes Parra; Müller, Rolf; Brüne, Bernhard; Namgaladze, Dmitry

2015-01-01

AMP-activated protein kinase (AMPK) maintains energy homeostasis by suppressing cellular ATP-consuming processes and activating catabolic, ATP-producing pathways such as fatty acid oxidation (FAO). The transcription factor peroxisome proliferator-activated receptor δ (PPARδ) also affects fatty acid metabolism, stimulating the expression of genes involved in FAO. To question the interplay of AMPK and PPARδ in human macrophages we transduced primary human macrophages with lentiviral particles encoding for the constitutively active AMPKα1 catalytic subunit, followed by microarray expression analysis after treatment with the PPARδ agonist GW501516. Microarray analysis showed that co-activation of AMPK and PPARδ increased expression of FAO genes, which were validated by quantitative PCR. Induction of these FAO-associated genes was also observed upon infecting macrophages with an adenovirus coding for AMPKγ1 regulatory subunit carrying an activating R70Q mutation. The pharmacological AMPK activator A-769662 increased expression of several FAO genes in a PPARδ- and AMPK-dependent manner. Although GW501516 significantly increased FAO and reduced the triglyceride amount in very low density lipoproteins (VLDL)-loaded foam cells, AMPK activation failed to potentiate this effect, suggesting that increased expression of fatty acid catabolic genes alone may be not sufficient to prevent macrophage lipid overload. PMID:26098914

2,5-hexanedione (HD) treatment alters calmodulin, Ca{sup 2+}/calmodulin-dependent protein kinase II, and protein kinase C in rats' nerve tissues

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang Qingshan; Hou Liyan; National Research Institute for Family Planning, Beijing 100081

2008-10-01

Calcium-dependent mechanisms, particularly those mediated by Ca{sup 2+}/calmodulin (CaM)-dependent protein kinase II (CaMKII), have been implicated in neurotoxicant-induced neuropathy. However, it is unknown whether similar mechanisms exist in 2,5-hexanedione (HD)-induced neuropathy. For that, we investigated the changes of CaM, CaMKII, protein kinase C (PKC) and polymerization ratios (PRs) of NF-L, NF-M and NF-H in cerebral cortex (CC, including total cortex and some gray), spinal cord (SC) and sciatic nerve (SN) of rats treated with HD at a dosage of 1.75 or 3.50 mmol/kg for 8 weeks (five times per week). The results showed that CaM contents in CC, SC andmore » SN were significantly increased, which indicated elevation of Ca{sup 2+} concentrations in nerve tissues. CaMKII contents and activities were also increased in CC and were positively correlated with gait abnormality, but it could not be found in SC and SN. The increases of PKC contents and activities were also observed in SN and were positively correlated with gait abnormality. Except for that of NF-M in CC, the PRs of NF-L, NF-M and NF-H were also elevated in nerve tissues, which was consistent with the activation of protein kinases. The results suggested that CaMKII might be partly (in CC but not in SC and SN) involved in HD-induced neuropathy. CaMKII and PKC might mediate the HD neurotoxicity by altering the NF phosphorylation status and PRs.« less

Pyruvate kinase M knockdown-induced signaling via AMP-activated protein kinase promotes mitochondrial biogenesis, autophagy, and cancer cell survival.

PubMed

Prakasam, Gopinath; Singh, Rajnish Kumar; Iqbal, Mohammad Askandar; Saini, Sunil Kumar; Tiku, Ashu Bhan; Bamezai, Rameshwar N K

2017-09-15

Preferential expression of the low-activity (dimeric) M2 isoform of pyruvate kinase (PK) over its constitutively active splice variant M1 isoform is considered critical for aerobic glycolysis in cancer cells. However, our results reported here indicate co-expression of PKM1 and PKM2 and their possible physical interaction in cancer cells. We show that knockdown of either PKM1 or PKM2 differentially affects net PK activity, viability, and cellular ATP levels of the lung carcinoma cell lines H1299 and A549. The stable knockdown of PK isoforms in A549 cells significantly reduced the cellular ATP level, whereas in H1299 cells the level of ATP was unaltered. Interestingly, the PKM1/2 knockdown in H1299 cells activated AMP-activated protein kinase (AMPK) signaling and stimulated mitochondrial biogenesis and autophagy to maintain energy homeostasis. In contrast, knocking down either of the PKM isoforms in A549 cells lacking LKB1, a serine/threonine protein kinase upstream of AMPK, failed to activate AMPK and sustain energy homeostasis and resulted in apoptosis. Moreover, in a similar genetic background of silenced PKM1 or PKM2, the knocking down of AMPKα1/2 catalytic subunit in H1299 cells induced apoptosis. Our findings help explain why previous targeting of PKM2 in cancer cells to control tumor growth has not met with the expected success. We suggest that this lack of success is because of AMPK-mediated energy metabolism rewiring, protecting cancer cell viability. On the basis of our observations, we propose an alternative therapeutic strategy of silencing either of the PKM isoforms along with AMPK in tumors. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

3-Phosphoinositide-dependent PDK1 negatively regulates transforming growth factor-beta-induced signaling in a kinase-dependent manner through physical interaction with Smad proteins.

PubMed

Seong, Hyun-A; Jung, Haiyoung; Kim, Kyong-Tai; Ha, Hyunjung

2007-04-20

We have reported previously that PDK1 physically interacts with STRAP, a transforming growth factor-beta (TGF-beta) receptor-interacting protein, and enhances STRAP-induced inhibition of TGF-beta signaling. In this study we show that PDK1 coimmunoprecipitates with Smad proteins, including Smad2, Smad3, Smad4, and Smad7, and that this association is mediated by the pleckstrin homology domain of PDK1. The association between PDK1 and Smad proteins is increased by insulin treatment but decreased by TGF-beta treatment. Analysis of the interacting proteins shows that Smad proteins enhance PDK1 kinase activity by removing 14-3-3, a negative regulator of PDK1, from the PDK1-14-3-3 complex. Knockdown of endogenous Smad proteins, including Smad3 and Smad7, by transfection with small interfering RNA produced the opposite trend and decreased PDK1 activity, protein kinase B/Akt phosphorylation, and Bad phosphorylation. Moreover, coexpression of Smad proteins and wild-type PDK1 inhibits TGF-beta-induced transcription, as well as TGF-beta-mediated biological functions, such as apoptosis and cell growth arrest. Inhibition was dose-dependent on PDK1, but no inhibition was observed in the presence of an inactive kinase-dead PDK1 mutant. In addition, confocal microscopy showed that wild-type PDK1 prevents translocation of Smad3 and Smad4 from the cytoplasm to the nucleus, as well as the redistribution of Smad7 from the nucleus to the cytoplasm in response to TGF-beta. Taken together, our results suggest that PDK1 negatively regulates TGF-beta-mediated signaling in a PDK1 kinase-dependent manner via a direct physical interaction with Smad proteins and that Smad proteins can act as potential positive regulators of PDK1.

Identification of ATM Protein Kinase Phosphorylation Sites by Mass Spectrometry.

PubMed

Graham, Mark E; Lavin, Martin F; Kozlov, Sergei V

2017-01-01

ATM (ataxia-telangiectasia mutated) protein kinase is a key regulator of cellular responses to DNA damage and oxidative stress. DNA damage triggers complex cascade of signaling events leading to numerous posttranslational modification on multitude of proteins. Understanding the regulation of ATM kinase is therefore critical not only for understanding the human genetic disorder ataxia-telangiectasia and potential treatment strategies, but essential for deciphering physiological responses of cells to stress. These responses play an important role in carcinogenesis, neurodegeneration, and aging. We focus here on the identification of DNA damage inducible ATM phosphorylation sites to understand the importance of autophosphorylation in the mechanism of ATM kinase activation. We demonstrate the utility of using immunoprecipitated ATM in quantitative LC-MS/MS workflow with stable isotope dimethyl labeling of ATM peptides for identification of phosphorylation sites.

Immunoprecipitation of PDE2 phosphorylated and inactivated by an associated protein kinase.

PubMed

Bentley, J Kelley

2005-01-01

A PDE2A2-associated protein kinase phosphorylates PDE2A2 in vivo and in vitro to inhibit its catalytic activity. Rat brain PDE2A2 may be solubilized using nona (ethylene glycol) mono dodecyl ether (Lubrol 12A9). PDE2A2 exists in a complex with a protein kinase regulating its activity in an adenosine triphosphate-dependent manner. When native or recombinant PDE2 is immunoprecipitated from PC12 cells using an antibody to the amino terminus in a buffer containing Lubrol 12A9, protease inhibitors, and phosphatase inhibitors, a coimmunoprecipitating nerve growth factor-stimulated protein kinase acts to phosphorylate it. PDE2A2 phosphoryla-tion occurs optimally at pH 6.5 in a sodium 2-(4-morpholino)-ethane sulfonate buffer with 5 mM MgCl2 and 1 mM Na3VO4. I describe protocols for producing an antibody to an amino-terminal bacterial fusion protein encoding amino acids 1-251 of PDE2A2 as well as the use of this antibody in immunoprecipitating a PDE2: tyrosine protein-kinase complex from rat brain or PC12 cells.

Odor-induced phosphorylation of olfactory cilia proteins.

PubMed Central

Boekhoff, I; Schleicher, S; Strotmann, J; Breer, H

1992-01-01

Stimulation of isolated rat olfactory cilia in the presence of [gamma-32P]ATP leads to a significantly enhanced incorporation of [32P]phosphate. Depending on the type of odorants applied, the induced phosphorylation is completely blocked by specific inhibitors of either protein kinase A or protein kinase C. Time-course experiments indicate that the odor-induced modification of ciliary proteins is transient; the intensity of labeling decayed over time (1-10 sec). Separation of ciliary proteins by SDS/polyacrylamide gel electrophoresis followed by autoradiography demonstrated that upon stimulation with lilial, a single polypeptide (50,000 Da) was phosphorylated; the size of the modified protein is in line with the hypothesis that odorant receptors are phosphorylated subsequent to activation by specific odors. Images PMID:1334554

The regulation of smooth muscle contractility by zipper-interacting protein kinase.

PubMed

Ihara, Eikichi; MacDonald, Justin A

2007-01-01

Smooth muscle contractility is mainly regulated by phosphorylation of the 20 kDa myosin light chains (LC20), a process that is controlled by the opposing activities of myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP). Recently, intensive research has revealed that various protein kinase networks including Rho-kinase, integrin-linked kinase, zipper-interacting protein kinase (ZIPK), and protein kinase C (PKC) are involved in the regulation of LC20 phosphorylation and have important roles in modulating smooth muscle contractile responses to Ca2+ (i.e., Ca2+ sensitization and Ca2+ desensitization). Here, we review the general background and structure of ZIPK and summarize our current understanding of its involvement in a number of cell processes including cell death (apoptosis), cell motility, and smooth muscle contraction. ZIPK has been found to induce the diphosphorylation of LC20 at Ser-19 and Thr-18 in a Ca2+-independent manner and to regulate MLCP activity directly through its phosphorylation of the myosin-targeting subunit of MLCP or indirectly through its phosphorylation of the PKC-potentiated inhibitory protein of MLCP. Future investigations of ZIPK function in smooth muscle will undoubtably focus on determining the mechanisms that regulate its cellular activity, including the identification of upstream signaling pathways, the characterization of autoinhibitory domains and regulatory phosphorylation sites, and the development of specific inhibitor compounds.

Drug-activated multiple pathways of defensin mRNA regulation in HL-60 cells are defined by reversed roles of participating protein kinases.

PubMed

Herwig, S; Su, Q; Tempst, P

1998-10-01

Defensin transcription in HL-60 promyelocytic leukemia cells is greatly enhanced during retinoic acid (RA)-induced differentiation. We have probed this regulatory pathway by selective modulation of various kinase activities. Induction was potentiated by elevated cAMP and attenuated by protein kinase C inhibition, entirely correlated to enhanced or blocked morphological differentiation, respectively. Yet, defensin mRNA was also induced in undifferentiated HL-60 cells, but not in others, by cAMP alone. By contrast, modulators that cooperated with RA had adverse effects on the normal capacity of dimethyl sulfoxide to up regulate these transcripts as well. Thus, defensin mRNA accumulation can be selectively uncoupled from maturation stage; and transcript levels may be regulated by multiple pathways, each independently acted upon by different chemical inducers.

Cooperative roles of fish protein kinase containing Z-DNA binding domains and double-stranded RNA-dependent protein kinase in interferon-mediated antiviral response.

PubMed

Liu, Ting-Kai; Zhang, Yi-Bing; Liu, Ying; Sun, Fan; Gui, Jian-Fang

2011-12-01

The double-stranded RNA (dsRNA)-dependent protein kinase (PKR) inhibits protein synthesis by phosphorylating eukaryotic translation initiation factor 2α (eIF2α). In fish species, in addition to PKR, there exists a PKR-like protein kinase containing Z-DNA binding domains (PKZ). However, the antiviral role of fish PKZ and the functional relationship between fish PKZ and PKR remain unknown. Here we confirmed the coexpression of fish PKZ and PKR proteins in Carassius auratus blastula embryonic (CAB) cells and identified them as two typical interferon (IFN)-inducible eIF2α kinases, both of which displayed an ability to inhibit virus replication. Strikingly, fish IFN or all kinds of IFN stimuli activated PKZ and PKR to phosphorylated eIF2α. Overexpression of both fish kinases together conferred much more significant inhibition of virus replication than overexpression of either protein, whereas morpholino knockdown of both made fish cells more vulnerable to virus infection than knockdown of either. The antiviral ability of fish PKZ was weaker than fish PKR, which correlated with its lower ability to phosphorylate eIF2α than PKR. Moreover, the independent association of fish PKZ or PKR reveals that each of them formed homodimers and that fish PKZ phosphorylated eIF2α independently on fish PKR and vice versa. These results suggest that fish PKZ and PKR play a nonredundant but cooperative role in IFN antiviral response.

Expression and characterization of a brain-specific protein kinase BSK146 from zebrafish.

PubMed

Chou, Chih-Ming; Chen, Yi-Chung; Lee, Ming-Ting; Chen, Gen-Der; Lu, I-Ching; Chen, Shui-Tsung; Huang, Chang-Jen

2006-02-17

We have previously identified a novel protein kinase, pk146, in the brain of Tetraodon. In the present study, we cloned the homologous protein kinase gene encoding a protein of 385 amino acid residues from zebrafish. The overall amino acid sequence and the kinase domain of zebrafish BSK146 shows 48% and 69% identity to that of rat sbk, a SH3-containing serine/threonine protein kinase. By whole-mount in situ hybridization and RT-PCR, the expression of bsk146 mRNA was mainly in the brain. To explore the in vivo function of BSK146 during zebrafish development, we used morpholino knockdown approach and found that BSK146 morphants displayed enlarged hindbrain ventricle and smaller eyes. Whole-mount in situ hybridization was further performed to analyze the brain defects in BSK146-MO-injected embryos. The expression of brain-specific markers, such as otx2, pax2.1, and krox20, was found normal in morphant embryos at 24hpf, while expression of pax2.1 exerted changes in midbrain-hindbrain boundary and hindbrain in morphant embryos at 48hpf. These data suggest that BSK146 may play an important role in later ventricle expansion in zebrafish brain development. Although the recombinant BSK146 protein produced in insect cells was active and could phosphorylate both histone H1 and histone 2B, the endogenous substrate of BSK146 in the embryonic brain of zebrafish is not clear at the present time and needs further investigation.

Contribution of RhoA kinase and protein kinase C to weak relaxant effect of pinacidil on carbachol-induced contractions in sensitized guinea-pig trachealis.

PubMed

Gok, Sule; Izanli-Paksoy, Ahenk; Vural, Kamil

2009-02-01

The exact mechanisms underlying the weak bronchodilator effect of K(ATP) channel openers on cholinergic stimulations is unknown. The present study was designed to examine the relaxant efect of pinacidil in guinea-pig trachea stimulated with carbachol by the presence of calcium sensitizer inhibitors; HA 1077, a rhoA kinase inhibitor, and chelerythrine, a protein kinase C inhibitor. Adenosine (10 microM) was used as other contractile agent for comparison. Tracheal tissues were isolated from ovalbumin sensitized guineapigs and changes in tension were recorded isometrically. Pinacidil (1-100 muM, cumulatively) and HA 1077 (0.01-30 microM, cumulatively) produced concentration-dependent relaxations in unstimulated tisues. The relaxant response to pinacidil decreased in carbachol contracted tissues, but increased in adenosine-stimulated tissues. Pretreatment of the tissues with HA 1077 (0.1 microM) and chelerythrine (10 microM) increased the pinacidil-induced relaxations by approximately %100 and %40, respectively. Glibenclamide, a KATP channel blocker, partially antagonized the pinacidil response in contracted tissues. Glibenclamide also inhibited the carbachol and adenosine induced contractions. These results suggest that diminish effect of pinacidil may have related to the enhanced calcium sensitization by cholinergic stimulation. Rho kinase inhibitors appear more effective than PKC inhibitors to achieve of this failure.

ERK1/2 and p38 MAP kinases control prion protein fragment 90-231-induced astrocyte proliferation and microglia activation.

PubMed

Thellung, Stefano; Villa, Valentina; Corsaro, Alessandro; Pellistri, Francesca; Venezia, Valentina; Russo, Claudio; Aceto, Antonio; Robello, Mauro; Florio, Tullio

2007-11-01

Astrogliosis and microglial activation are a common feature during prion diseases, causing the release of chemoattractant and proinflammatory factors as well as reactive free radicals, involved in neuronal degeneration. The recombinant protease-resistant domain of the prion protein (PrP90-231) displays in vitro neurotoxic properties when refolded in a beta-sheet-rich conformer. Here, we report that PrP90-231 induces the secretion of several cytokines, chemokines, and nitric oxide (NO) release, in both type I astrocytes and microglial cells. PrP90-231 elicited in both cell types the activation of ERK1/2 MAP kinase that displays, in astrocytes, a rapid kinetics and a proliferative response. Conversely, in microglia, PrP90-231-dependent MAP kinase activation was delayed and long lasting, inducing functional activation and growth arrest. In microglial cells, NO release, dependent on the expression of the inducible NO synthase (iNOS), and the secretion of the chemokine CCL5 were Ca(2+) dependent and under the control of the MAP kinases ERK1/2 and p38: ERK1/2 inhibition, using PD98059, reduced iNOS expression, while p38 blockade by PD169316 inhibited CCL5 release. In summary, we demonstrate that glial cells are activated by extracellular misfolded PrP90-231 resulting in a proliferative/secretive response of astrocytes and functional activation of microglia, both dependent on MAP kinase activation. In particular, in microglia, PrP90-231 activated a complex signalling cascade involved in the regulation of NO and chemokine release. These data argue in favor of a causal role for misfolded prion protein in sustaining glial activation and, possibly, glia-mediated neuronal death.

TEC protein tyrosine kinase is involved in the Erk signaling pathway induced by HGF

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Feifei; Jiang, Yinan; Zheng, Qiping

Research highlights: {yields} TEC is rapidly tyrosine-phosphorylated and activated by HGF-stimulation in vivo or after partial hepatectomy in mice. {yields} TEC enhances the activity of Elk and serum response element (SRE) in HGF signaling pathway in hepatocyte. {yields} TEC promotes hepatocyte proliferation through the Erk-MAPK pathway. -- Abstract: Background/aims: TEC, a member of the TEC family of non-receptor type protein tyrosine kinases, has recently been suggested to play a role in hepatocyte proliferation and liver regeneration. This study aims to investigate the putative mechanisms of TEC kinase regulation of hepatocyte differentiation, i.e. to explore which signaling pathway TEC is involvedmore » in, and how TEC is activated in hepatocyte after hepatectomy and hepatocyte growth factor (HGF) stimulation. Methods: We performed immunoprecipitation (IP) and immunoblotting (IB) to examine TEC tyrosine phosphorylation after partial hepatectomy in mice and HGF stimulation in WB F-344 hepatic cells. The TEC kinase activity was determined by in vitro kinase assay. Reporter gene assay, antisense oligonucleotide and TEC dominant negative mutant (TEC{sup KM}) were used to examine the possible signaling pathways in which TEC is involved. The cell proliferation rate was evaluated by {sup 3}H-TdR incorporation. Results: TEC phosphorylation and kinase activity were increased in 1 h after hepatectomy or HGF treatment. TEC enhanced the activity of Elk and serum response element (SRE). Inhibition of MEK1 suppressed TEC phosphorylation. Blocking TEC activity dramatically decreased the activation of Erk. Reduced TEC kinase activity also suppressed the proliferation of WB F-344 cells. These results suggest TEC is involved in the Ras-MAPK pathway and acts between MEK1 and Erk. Conclusions: TEC promotes hepatocyte proliferation and regeneration and is involved in HGF-induced Erk signaling pathway.« less

Indomethacin promotes apoptosis in gastric cancer cells through concomitant degradation of Survivin and Aurora B kinase proteins.

PubMed

Chiou, Shiun-Kwei; Hoa, Neil; Hodges, Amy; Ge, Lishen; Jadus, Martin R

2014-09-01

Regular usage of nonsteroidal anti-inflammatory drugs (NSAIDs) is associated with reduced incidence of a variety of cancers. The molecular mechanisms underlying these chemopreventive effects remain poorly understood. This current investigation showed that in gastric cancer cells: (1) Indomethacin treatment enhanced the degradation of chromosomal passenger proteins, Survivin and Aurora B kinase; (2) Indomethacin treatment down-regulated Aurora B kinase activity in a cell cycle-independent fashion; (3) siRNA knockdown of Survivin level promoted Aurora B kinase protein degradation, and vice versa; (4) ectopic overexpression of Survivin blocked reduction of Aurora B kinase level and activity by indomethacin treatment, and vice versa; (5) siRNA knockdown of Aurora B kinase level and AZD1152 inhibition of its activity induced apoptosis, and overexpression of Aurora B kinase inhibited indomethacin-induced apoptosis; (6) indomethacin treatment reduced Aurora B kinase level, coinciding with reduction of Survivin level and induction of apoptosis, in KATO III and HT-29 cells, and in mouse gastric mucosa. A role for Aurora B kinase function in NSAID-induced apoptosis was not previously explored. Thus this report provides better understanding of the molecular mechanisms underlying the anti-cancer effect of NSAIDs by elucidating a significant role for Aurora B kinase in indomethacin-induced apoptosis.

Mitogen-activated protein kinase pathway mediates DBP-maf-induced apoptosis in RAW 264.7 macrophages.

PubMed

Gumireddy, Kiranmai; Reddy, C Damodar; Swamy, Narasimha

2003-09-01

Vitamin D-binding protein-macrophage-activating factor (DBP-maf) is derived from serum vitamin D binding protein (DBP) by selective deglycosylation during inflammation. In the present study, we investigated the effect of DBP-maf on RAW 264.7 macrophages and the underlying intracellular signal transduction pathways. DBP-maf increased proapoptotic caspase-3, -8, and -9 activities and induced apoptosis in RAW 264.7 cells. However, DBP, the precursor to DBP-maf did not induce apoptosis in these cells. Cell cycle analysis of DBP-maf-treated RAW 264.7 cells revealed growth arrest with accumulation of cells in sub-G(0)/G(1) phase. We also investigated the role of mitogen-activated protein kinase (MAPK) pathways in the DBP-maf-induced apoptosis of RAW264.7 cells. DBP-maf increased the phosphorylation of p38 and JNK1/2, while it decreased the ERK1/2 phosphorylation. Treatment with the p38 MAPK inhibitor, SB202190, attenuated DBP-maf-induced apoptosis. PD98059, a MEK specific inhibitor, did not show a significant inhibition of apoptosis induced by DBP-maf. Taken together, these results suggest that the p38 MAPK pathway plays a crucial role in DBP-maf-mediated apoptosis of macrophages. Our studies indicate that, during inflammation DBP-maf may function positively by causing death of the macrophages when activated macrophages are no longer needed at the site of inflammation. In summary, we report for the first time that DBP-maf induces apoptosis in macrophages via p38 and JNK1/2 pathway. Copyright 2003 Wiley-Liss, Inc.

Eleostearic acid induces RIP1-mediated atypical apoptosis in a kinase-independent manner via ERK phosphorylation, ROS generation and mitochondrial dysfunction

PubMed Central

Obitsu, S; Sakata, K; Teshima, R; Kondo, K

2013-01-01

RIP1 is a serine/threonine kinase, which is involved in apoptosis and necroptosis. In apoptosis, caspase-8 and FADD have an important role. On the other hand, RIP3 is a key molecule in necroptosis. Recently, we reported that eleostearic acid (ESA) elicits caspase-3- and PARP-1-independent cell death, although ESA-treated cells mediate typical apoptotic morphology such as chromatin condensation, plasma membrane blebbing and apoptotic body formation. The activation of caspases, Bax and PARP-1, the cleavage of AIF and the phosphorylation of histone H2AX, all of which are characteristics of typical apoptosis, do not occur in ESA-treated cells. However, the underlying mechanism remains unclear. To clarify the signaling pathways in ESA-mediated apoptosis, we investigated the functions of RIP1, MEK, ERK, as well as AIF. Using an extensive study based on molecular biology, we identified the alternative role of RIP1 in ESA-mediated apoptosis. ESA mediates RIP1-dependent apoptosis in a kinase independent manner. ESA activates serine/threonine phosphatases such as calcineurin, which induces RIP1 dephosphorylation, thereby ERK pathway is activated. Consequently, localization of AIF and ERK in the nucleus, ROS generation and ATP reduction in mitochondria are induced to disrupt mitochondrial cristae, which leads to cell death. Necrostatin (Nec)-1 blocked MEK/ERK phosphorylation and ESA-mediated apoptosis. Nec-1 inactive form (Nec1i) also impaired ESA-mediated apoptosis. Nec1 blocked the interaction of MEK with ERK upon ESA stimulation. Together, these findings provide a new finding that ERK and kinase-independent RIP1 proteins are implicated in atypical ESA-mediated apoptosis. PMID:23788031

Protein Kinase D-dependent Phosphorylation and Nuclear Export of Histone Deacetylase 5 Mediates Vascular Endothelial Growth Factor-induced Gene Expression and Angiogenesis*S⃞

PubMed Central

Ha, Chang Hoon; Wang, Weiye; Jhun, Bong Sook; Wong, Chelsea; Hausser, Angelika; Pfizenmaier, Klaus; McKinsey, Timothy A.; Olson, Eric N.; Jin, Zheng-Gen

2008-01-01

Vascular endothelial growth factor (VEGF) is essential for normal and pathological angiogenesis. However, the signaling pathways linked to gene regulation in VEGF-induced angiogenesis are not fully understood. Here we demonstrate a critical role of protein kinase D (PKD) and histone deacetylase 5 (HDAC5) in VEGF-induced gene expression and angiogenesis. We found that VEGF stimulated HDAC5 phosphorylation and nuclear export in endothelial cells through a VEGF receptor 2-phospholipase Cγ-protein kinase C-PKD-dependent pathway. We further showed that the PKD-HDAC5 pathway mediated myocyte enhancer factor-2 transcriptional activation and a specific subset of gene expression in response to VEGF, including NR4A1, an orphan nuclear receptor involved in angiogenesis. Specifically, inhibition of PKD by overexpression of the PKD kinase-negative mutant prevents VEGF-induced HDAC5 phosphorylation and nuclear export as well as NR4A1 induction. Moreover, a mutant of HDAC5 specifically deficient in PKD-dependent phosphorylation inhibited VEGF-mediated NR4A1 expression, endothelial cell migration, and in vitro angiogenesis. These findings suggest that the PKD-HDAC5 pathway plays an important role in VEGF regulation of gene transcription and angiogenesis. PMID:18332134

p56Lck and p59Fyn Regulate CD28 Binding to Phosphatidylinositol 3-Kinase, Growth Factor Receptor-Bound Protein GRB-2, and T Cell-Specific Protein-Tyrosine Kinase ITK: Implications for T-Cell Costimulation

NASA Astrophysics Data System (ADS)

Raab, Monika; Cai, Yun-Cai; Bunnell, Stephen C.; Heyeck, Stephanie D.; Berg, Leslie J.; Rudd, Christopher E.

1995-09-01

T-cell activation requires cooperative signals generated by the T-cell antigen receptor ξ-chain complex (TCRξ-CD3) and the costimulatory antigen CD28. CD28 interacts with three intracellular proteins-phosphatidylinositol 3-kinase (PI 3-kinase), T cell-specific protein-tyrosine kinase ITK (formerly TSK or EMT), and the complex between growth factor receptor-bound protein 2 and son of sevenless guanine nucleotide exchange protein (GRB-2-SOS). PI 3-kinase and GRB-2 bind to the CD28 phosphotyrosine-based Tyr-Met-Asn-Met motif by means of intrinsic Src-homology 2 (SH2) domains. The requirement for tyrosine phosphorylation of the Tyr-Met-Asn-Met motif for SH2 domain binding implicates an intervening protein-tyrosine kinase in the recruitment of PI 3-kinase and GRB-2 by CD28. Candidate kinases include p56Lck, p59Fyn, ξ-chain-associated 70-kDa protein (ZAP-70), and ITK. In this study, we demonstrate in coexpression studies that p56Lck and p59Fyn phosphorylate CD28 primarily at Tyr-191 of the Tyr-Met-Asn-Met motif, inducing a 3- to 8-fold increase in p85 (subunit of PI 3-kinase) and GRB-2 SH2 binding to CD28. Phosphatase digestion of CD28 eliminated binding. In contrast to Src kinases, ZAP-70 and ITK failed to induce these events. Further, ITK binding to CD28 was dependent on the presence of p56Lck and is thus likely to act downstream of p56Lck/p59Fyn in a signaling cascade. p56Lck is therefore likely to be a central switch in T-cell activation, with the dual function of regulating CD28-mediated costimulation as well as TCR-CD3-CD4 signaling.

Identification of the regulatory autophosphorylation site of autophosphorylation-dependent protein kinase (auto-kinase). Evidence that auto-kinase belongs to a member of the p21-activated kinase family.

PubMed

Yu, J S; Chen, W J; Ni, M H; Chan, W H; Yang, S D

1998-08-15

Autophosphorylation-dependent protein kinase (auto-kinase) was identified from pig brain and liver on the basis of its unique autophosphorylation/activation property [Yang, Fong, Yu and Liu (1987) J. Biol. Chem. 262, 7034-7040; Yang, Chang and Soderling (1987) J. Biol. Chem. 262, 9421-9427]. Its substrate consensus sequence motif was determined as being -R-X-(X)-S*/T*-X3-S/T-. To characterize auto-kinase further, we partly sequenced the kinase purified from pig liver. The N-terminal sequence (VDGGAKTSDKQKKKAXMTDE) and two internal peptide sequences (EKLRTIV and LQNPEK/ILTP/FI) of auto-kinase were obtained. These sequences identify auto-kinase as a C-terminal catalytic fragment of p21-activated protein kinase 2 (PAK2 or gamma-PAK) lacking its N-terminal regulatory region. Auto-kinase can be recognized by an antibody raised against the C-terminal peptide of human PAK2 by immunoblotting. Furthermore the autophosphorylation site sequence of auto-kinase was successfully predicted on the basis of its substrate consensus sequence motif and the known PAK2 sequence, and was further demonstrated to be RST(P)MVGTPYWMAPEVVTR by phosphoamino acid analysis, manual Edman degradation and phosphopeptide mapping via the help of phosphorylation site analysis of a synthetic peptide corresponding to the sequence of PAK2 from residues 396 to 418. During the activation process, auto-kinase autophosphorylates mainly on a single threonine residue Thr402 (according to the sequence numbering of human PAK2). In addition, a phospho-specific antibody against a synthetic phosphopeptide containing this identified sequence was generated and shown to be able to differentially recognize the activated auto-kinase autophosphorylated at Thr402 but not the non-phosphorylated/inactive auto-kinase. Immunoblot analysis with this phospho-specific antibody further revealed that the change in phosphorylation level of Thr402 of auto-kinase was well correlated with the activity change of the kinase during both

Identification of the regulatory autophosphorylation site of autophosphorylation-dependent protein kinase (auto-kinase). Evidence that auto-kinase belongs to a member of the p21-activated kinase family.

PubMed Central

Yu, J S; Chen, W J; Ni, M H; Chan, W H; Yang, S D

1998-01-01

Autophosphorylation-dependent protein kinase (auto-kinase) was identified from pig brain and liver on the basis of its unique autophosphorylation/activation property [Yang, Fong, Yu and Liu (1987) J. Biol. Chem. 262, 7034-7040; Yang, Chang and Soderling (1987) J. Biol. Chem. 262, 9421-9427]. Its substrate consensus sequence motif was determined as being -R-X-(X)-S*/T*-X3-S/T-. To characterize auto-kinase further, we partly sequenced the kinase purified from pig liver. The N-terminal sequence (VDGGAKTSDKQKKKAXMTDE) and two internal peptide sequences (EKLRTIV and LQNPEK/ILTP/FI) of auto-kinase were obtained. These sequences identify auto-kinase as a C-terminal catalytic fragment of p21-activated protein kinase 2 (PAK2 or gamma-PAK) lacking its N-terminal regulatory region. Auto-kinase can be recognized by an antibody raised against the C-terminal peptide of human PAK2 by immunoblotting. Furthermore the autophosphorylation site sequence of auto-kinase was successfully predicted on the basis of its substrate consensus sequence motif and the known PAK2 sequence, and was further demonstrated to be RST(P)MVGTPYWMAPEVVTR by phosphoamino acid analysis, manual Edman degradation and phosphopeptide mapping via the help of phosphorylation site analysis of a synthetic peptide corresponding to the sequence of PAK2 from residues 396 to 418. During the activation process, auto-kinase autophosphorylates mainly on a single threonine residue Thr402 (according to the sequence numbering of human PAK2). In addition, a phospho-specific antibody against a synthetic phosphopeptide containing this identified sequence was generated and shown to be able to differentially recognize the activated auto-kinase autophosphorylated at Thr402 but not the non-phosphorylated/inactive auto-kinase. Immunoblot analysis with this phospho-specific antibody further revealed that the change in phosphorylation level of Thr402 of auto-kinase was well correlated with the activity change of the kinase during both

Overexpression of protein kinase FA/GSK-3 alpha (a proline-directed protein kinase) correlates with human hepatoma dedifferentiation/progression.

PubMed

Yang, S D; Yu, J S; Yang, C C; Lee, S C; Lee, T T; Ni, M H; Kuan, C Y; Chen, H C

1996-05-01

Computer analysis of protein phosphorylation sites sequence revealed that transcriptional factors and viral oncoproteins are prime targets for regulation of proline-directed protein phosphorylation, suggesting an association of the proline-directed protein kinase (PDPK) family with neoplastic transformation and tumorigenesis. In this report, an immunoprecipitate activity assay of protein kinase FA/glycogen synthase kinase-3 alpha (kinase F(A)/GSK-3 alpha) (a member of PDPK family) has been optimized for human hepatoma and used to demonstrate for the first time significantly increased (P < 0.01) activity in poorly differentiated SK-Hep-1 hepatoma (24.2 +/- 2.8 units/mg) and moderately differentiated Mahlavu hepatoma (14.5 +/- 2.2 units/mg) when compared to well differentiated Hep 3B hepatoma (8.0 +/- 2.4 units/mg). Immunoblotting analysis revealed that increased activity of kinase FA/GSK-3 alpha is due to overexpression of the protein. Elevated kinase FA/GSK-3 alpha expression in human hepatoma biopsies relative to normal liver tissue was found to be even more profound. This kinase appeared to be fivefold overexpressed in well differentiated hepatoma and 13-fold overexpressed in poorly differentiated hepatoma when compared to normal liver tissue. Taken together, the results provide initial evidence that overexpression of kinase FA/GSK-3 alpha is involved in human hepatoma dedifferentiation/progression. Since kinase FA/GSK-3 alpha is a PDPK, the results further support a potential role of this kinase in human liver tumorigenesis, especially in its dedifferentiation/progression.

ERK and p38 MAPK-Activated Protein Kinases: a Family of Protein Kinases with Diverse Biological Functions

PubMed Central

Roux, Philippe P.; Blenis, John

2004-01-01

Conserved signaling pathways that activate the mitogen-activated protein kinases (MAPKs) are involved in relaying extracellular stimulations to intracellular responses. The MAPKs coordinately regulate cell proliferation, differentiation, motility, and survival, which are functions also known to be mediated by members of a growing family of MAPK-activated protein kinases (MKs; formerly known as MAPKAP kinases). The MKs are related serine/threonine kinases that respond to mitogenic and stress stimuli through proline-directed phosphorylation and activation of the kinase domain by extracellular signal-regulated kinases 1 and 2 and p38 MAPKs. There are currently 11 vertebrate MKs in five subfamilies based on primary sequence homology: the ribosomal S6 kinases, the mitogen- and stress-activated kinases, the MAPK-interacting kinases, MAPK-activated protein kinases 2 and 3, and MK5. In the last 5 years, several MK substrates have been identified, which has helped tremendously to identify the biological role of the members of this family. Together with data from the study of MK-knockout mice, the identities of the MK substrates indicate that they play important roles in diverse biological processes, including mRNA translation, cell proliferation and survival, and the nuclear genomic response to mitogens and cellular stresses. In this article, we review the existing data on the MKs and discuss their physiological functions based on recent discoveries. PMID:15187187

Abscisic acid-regulated protein degradation causes osmotic stress-induced accumulation of branched-chain amino acids in Arabidopsis thaliana.

PubMed

Huang, Tengfang; Jander, Georg

2017-10-01

Whereas proline accumulates through de novo biosynthesis in plants subjected to osmotic stress, leucine, isoleucine, and valine accumulation in drought-stressed Arabidopsis thaliana is caused by abscisic acid-regulated protein degradation. In response to several kinds of abiotic stress, plants greatly increase their accumulation of free amino acids. Although stress-induced proline increases have been studied the most extensively, the fold-increase of other amino acids, in particular branched-chain amino acids (BCAAs; leucine, isoleucine, and valine), is often higher than that of proline. In Arabidopsis thaliana (Arabidopsis), BCAAs accumulate in response to drought, salt, mannitol, polyethylene glycol, herbicide treatment, and nitrogen starvation. Plants that are deficient in abscisic acid signaling accumulate lower amounts of BCAAs, but not proline and most other amino acids. Previous bioinformatic studies had suggested that amino acid synthesis, rather than protein degradation, is responsible for the observed BCAA increase in osmotically stressed Arabidopsis. However, whereas treatment with the protease inhibitor MG132 decreased drought-induced BCAA accumulation, inhibition of BCAA biosynthesis with the acetolactate synthase inhibitors chlorsulfuron and imazapyr did not. Additionally, overexpression of BRANCHED-CHAIN AMINO ACID TRANSFERASE2 (BCAT2), which is upregulated in response to osmotic stress and functions in BCAA degradation, decreased drought-induced BCAA accumulation. Together, these results demonstrate that BCAA accumulation in osmotically stressed Arabidopsis is primarily the result of protein degradation. After relief of the osmotic stress, BCAA homeostasis is restored over time by amino acid degradation involving BCAT2. Thus, drought-induced BCAA accumulation is different from that of proline, which is accumulated due to de novo synthesis in an abscisic acid-independent manner and remains elevated for a more prolonged period of time after removal of

Differential expression of poplar sucrose nonfermenting1-related protein kinase 2 genes in response to abiotic stress and abscisic acid.

PubMed

Yu, Xiang; Takebayashi, Arika; Demura, Taku; Ohtani, Misato

2017-09-01

Knowledge on the responses of woody plants to abiotic stress can inform strategies to breed improved tree varieties and to manage tree species for environmental conservation and the production of lignocellulosic biomass. In this study, we examined the expression patterns of poplar (Populus trichocarpa) genes encoding members of the sucrose nonfermenting1-related protein kinase 2 (SnRK2) family, which are core components of the abiotic stress response. The P. trichocarpa genome contains twelve SnRK2 genes (PtSnRK2.1- PtSnRK2.12) that can be divided into three subclasses (I-III) based on the structures of their encoded kinase domains. We found that PtSnRK2s are differentially expressed in various organs. In MS medium-grown plants, all of the PtSnRK2 genes were significantly upregulated in response to abscisic acid (ABA) treatment, whereas osmotic and salt stress treatments induced only some (four and seven, respectively) of the PtSnRK2 genes. By contrast, soil-grown plants showed increased expression of most PtSnRK2 genes under drought and salt treatments, but not under ABA treatment. In soil-grown plants, drought stress induced SnRK2 subclass II genes in all tested organs (leaves, stems, and roots), whereas subclass III genes tended to be upregulated in leaves only. These results suggest that the PtSnRK2 genes are involved in abiotic stress responses, are at least partially activated by ABA, and show organ-specific responses.

Casein kinase II protein kinase is bound to lamina-matrix and phosphorylates lamin-like protein in isolated pea nuclei

NASA Technical Reports Server (NTRS)

Li, H.; Roux, S. J.

1992-01-01

A casein kinase II (CK II)-like protein kinase was identified and partially isolated from a purified envelope-matrix fraction of pea (Pisum sativum L.) nuclei. When [gamma-32P]ATP was directly added to the envelope-matrix preparation, the three most heavily labeled protein bands had molecular masses near 71, 48, and 46 kDa. Protein kinases were removed from the preparation by sequential extraction with Triton X-100, EGTA, 0.3 M NaCl, and a pH 10.5 buffer, but an active kinase still remained bound to the remaining lamina-matrix fraction after these treatments. This kinase had properties resembling CK II kinases previously characterized from animal and plant sources: it preferred casein as an artificial substrate, could use GTP as efficiently as ATP as the phosphoryl donor, was stimulated by spermine, was calcium independent, and had a catalytic subunit of 36 kDa. Some animal and plant CK II kinases have regulatory subunits near 29 kDa, and a lamina-matrix-bound protein of this molecular mass was recognized on immunoblot by anti-Drosophila CK II polyclonal antibodies. Also found associated with the envelope-matrix fraction of pea nuclei were p34cdc2-like and Ca(2+)-dependent protein kinases, but their properties could not account for the protein kinase activity bound to the lamina. The 71-kDa substrate of the CK II-like kinase was lamin A-like, both in its molecular mass and in its cross-reactivity with anti-intermediate filament antibodies. Lamin phosphorylation is considered a crucial early step in the entry of cells into mitosis, so lamina-bound CK II kinases may be important control points for cellular proliferation.

Proteolytic Inhibition of Salmonella enterica Serovar Typhimurium-Induced Activation of the Mitogen-Activated Protein Kinases ERK and JNK in Cultured Human Intestinal Cells

PubMed Central

Mynott, Tracey L.; Crossett, Ben; Prathalingam, S. Radhika

2002-01-01

Bromelain, a mixture of cysteine proteases from pineapple stems, blocks signaling by the mitogen-activated protein (MAP) kinases extracellular regulated kinase 1 (ERK-1) and ERK-2, inhibits inflammation, and protects against enterotoxigenic Escherichia coli infection. In this study, we examined the effect of bromelain on Salmonella enterica serovar Typhimurium infection, since an important feature of its pathogenesis is its ability to induce activation of ERK-1 and ERK-2, which leads to internalization of bacteria and induction of inflammatory responses. Our results show that bromelain dose dependently blocks serovar Typhimurium-induced ERK-1, ERK-2, and c-Jun NH2-terminal kinase (JNK) activation in Caco-2 cells. Bromelain also blocked signaling induced by carbachol and anisomycin, pharmacological MAP kinase agonists. Despite bromelain inhibition of serovar Typhimurium-induced MAP kinase signaling, it did not prevent subsequent invasion of the Caco-2 cells by serovar Typhimurium or alter serovar Typhimurium -induced decreases in resistance across Caco-2 monolayers. Surprisingly, bromelain also did not block serovar Typhimurium-induced interleukin-8 (IL-8) secretion but synergized with serovar Typhimurium to enhance IL-8 production. We also found that serovar Typhimurium does not induce ERK phosphorylation in Caco-2 cells in the absence of serum but that serovar Typhimurium-induced invasion and decreases in monolayer resistance are unaffected. Collectively, these data indicate that serovar Typhimurium-induced invasion of Caco-2 cells, changes in the resistance of epithelial cell monolayers, and IL-8 production can occur independently of the ERK and JNK signaling pathways. Data also confirm that bromelain is a novel inhibitor of MAP kinase signaling pathways and suggest a novel role for proteases as inhibitors of signal transduction pathways in intestinal epithelial cells. PMID:11748167

Mitogen activated protein kinase (MAPK) pathway regulates heme oxygenase-1 gene expression by hypoxia in vascular cells.

PubMed

Ryter, Stefan W; Xi, Sichuan; Hartsfield, Cynthia L; Choi, Augustine M K

2002-08-01

Hypoxia induces the stress protein heme oxygenase-1 (HO-1), which participates in cellular adaptation. The molecular pathways that regulate ho-1 gene expression under hypoxia may involve mitogen activated protein kinase (MAPK) signaling and reactive oxygen. Hypoxia (8 h) increased HO-1 mRNA in rat pulmonary aortic endothelial cells (PAEC), and also activated both extracellular signal-regulated kinase 1 (ERK1)/ERK2 and p38 MAPK pathways. The role of these kinases in hypoxia-induced ho-1 gene expression was examined using chemical inhibitors of these pathways. Surprisingly, SB203580, an inhibitor of p38 MAPK, and PD98059, an inhibitor of mitogen-activated protein kinase kinase (MEK1), strongly enhanced hypoxia-induced HO-1 mRNA expression in PAEC. UO126, a MEK1/2 inhibitor, enhanced HO-1 expression in PAEC under normoxia, but not hypoxia. Diphenylene iodonium, an inhibitor of NADPH oxidase, also induced the expression of HO-1 in PAEC under both normoxia and hypoxia. Similar results were observed in aortic vascular smooth muscle cells. Furthermore, hypoxia induced activator protein (AP-1) DNA-binding activity in PAEC. Pretreatment with SB203580 and PD98059 enhanced AP-1 binding activity under hypoxia in PAEC; UO126 stimulated AP-1 binding under normoxia, whereas diphenylene iodonium stimulated AP-1 binding under normoxia and hypoxia. These results suggest a relationship between MAPK and hypoxic regulation of ho-1 in vascular cells, involving AP-1.

Non-degradative Ubiquitination of Protein Kinases

PubMed Central

Ball, K. Aurelia; Johnson, Jeffrey R.; Lewinski, Mary K.; Guatelli, John; Verschueren, Erik; Krogan, Nevan J.; Jacobson, Matthew P.

2016-01-01

Growing evidence supports other regulatory roles for protein ubiquitination in addition to serving as a tag for proteasomal degradation. In contrast to other common post-translational modifications, such as phosphorylation, little is known about how non-degradative ubiquitination modulates protein structure, dynamics, and function. Due to the wealth of knowledge concerning protein kinase structure and regulation, we examined kinase ubiquitination using ubiquitin remnant immunoaffinity enrichment and quantitative mass spectrometry to identify ubiquitinated kinases and the sites of ubiquitination in Jurkat and HEK293 cells. We find that, unlike phosphorylation, ubiquitination most commonly occurs in structured domains, and on the kinase domain, ubiquitination is concentrated in regions known to be important for regulating activity. We hypothesized that ubiquitination, like other post-translational modifications, may alter the conformational equilibrium of the modified protein. We chose one human kinase, ZAP-70, to simulate using molecular dynamics with and without a monoubiquitin modification. In Jurkat cells, ZAP-70 is ubiquitinated at several sites that are not sensitive to proteasome inhibition and thus may have other regulatory roles. Our simulations show that ubiquitination influences the conformational ensemble of ZAP-70 in a site-dependent manner. When monoubiquitinated at K377, near the C-helix, the active conformation of the ZAP-70 C-helix is disrupted. In contrast, when monoubiquitinated at K476, near the kinase hinge region, an active-like ZAP-70 C-helix conformation is stabilized. These results lead to testable hypotheses that ubiquitination directly modulates kinase activity, and that ubiquitination is likely to alter structure, dynamics, and function in other protein classes as well. PMID:27253329

Mitogen-activated protein kinase cascades in Vitis vinifera

PubMed Central

Çakır, Birsen; Kılıçkaya, Ozan

2015-01-01

Protein phosphorylation is one of the most important mechanisms to control cellular functions in response to external and endogenous signals. Mitogen-activated protein kinases (MAPK) are universal signaling molecules in eukaryotes that mediate the intracellular transmission of extracellular signals resulting in the induction of appropriate cellular responses. MAPK cascades are composed of four protein kinase modules: MAPKKK kinases (MAPKKKKs), MAPKK kinases (MAPKKKs), MAPK kinases (MAPKKs), and MAPKs. In plants, MAPKs are activated in response to abiotic stresses, wounding, and hormones, and during plant pathogen interactions and cell division. In this report, we performed a complete inventory of MAPK cascades genes in Vitis vinifera, the whole genome of which has been sequenced. By comparison with MAPK, MAPK kinases, MAPK kinase kinases and MAPK kinase kinase kinase kinase members of Arabidopsis thaliana, we revealed the existence of 14 MAPKs, 5 MAPKKs, 62 MAPKKKs, and 7 MAPKKKKs in Vitis vinifera. We identified orthologs of V. vinifera putative MAPKs in different species, and ESTs corresponding to members of MAPK cascades in various tissues. This work represents the first complete inventory of MAPK cascades in V. vinifera and could help elucidate the biological and physiological functions of these proteins in V. vinifera. PMID:26257761

Protective effect of naringin on 3-nitropropionic acid-induced neurodegeneration through the modulation of matrix metalloproteinases and glial fibrillary acidic protein.

PubMed

Gopinath, Kulasekaran; Sudhandiran, Ganapasam

2016-01-01

Naringin (4',5,7-trihydroxy-flavonone-7-rhamnoglucoside), a flavonone present in grapefruit, has recently been reported to protect against neurodegeration, induced with 3-nitropropionic acid (3-NP), through its antioxidant, anti-inflammatory, and antiapoptotic properties. This study used a rat model of 3-NP-induced neurodegeneration to investigate the neuroprotective effects of naringin exerted by modulating the expression of matrix metalloproteinases and glial fibrillary acidic protein. Neurodegeneration was induced with 3-NP (10 mg/kg body mass, by intraperitoneal injection) once a day for 2 weeks, and induced rats were treated with naringin (80 mg/kg body mass, by oral gavage, once a day for 2 weeks). Naringin ameliorated the motor abnormalities caused by 3-NP, and reduced blood-brain barrier dysfunction by decreasing the expression of matrix metalloproteinases 2 and 9, along with increasing the expression of the tissue inhibitors of metalloproteinases 1 and 2 in 3-NP-induced rats. Further, naringin reduced 3-NP-induced neuroinflammation by decreasing the expression of nuclear factor-kappa B and glial fibrillary acidic protein. Thus, naringin exerts protective effects against 3-NP-induced neurodegeneration by ameliorating the expressions of matrix metalloproteinases and glial fibrillary acidic protein.

Calcium-Dependent Protein Kinase Genes in Corn Roots

NASA Technical Reports Server (NTRS)

Takezawa, D.; Patil, S.; Bhatia, A.; Poovaiah, B. W.

1996-01-01

Two cDNAs encoding Ca-2(+) - Dependent Protein Kinases (CDPKs), Corn Root Protein Kinase 1 and 2 (CRPK 1, CRPK 2) were isolated from the root tip library of corn (Zea mays L., cv. Merit) and their nucleotide sequences were determined. Deduced amino acid sequences of both the clones have features characteristic of plant CDPKS, including all 11 conserved serine/threonine kinase subdomains, a junction domain and a calmodulin-like domain with four Ca-2(+), -binding sites. Northern analysis revealed that CRPKI mRNA is preferentially expressed in roots, especially in the root tip; whereas, the expression of CRPK2 mRNA was very low in all the tissues tested. In situ hybridization experiments revealed that CRPKI mRNA is highly expressed in the root apex, as compared to other parts of the root. Partially purified CDPK from the root tip phosphorylates syntide-2, a common peptide substrate for plant CDPKs, and the phosphorylation was stimulated 7-fold by the addition of Ca-2(+). Our results show that two CDPK isoforms are expressed in corn roots and they may be involved in the Ca-2(+)-dependent signal transduction process.

A rho-binding protein kinase C-like activity is required for the function of protein kinase N in Drosophila development.

PubMed

Betson, Martha; Settleman, Jeffrey

2007-08-01

The Rho GTPases interact with multiple downstream effectors to exert their biological functions, which include important roles in tissue morphogenesis during the development of multicellular organisms. Among the Rho effectors are the protein kinase N (PKN) proteins, which are protein kinase C (PKC)-like kinases that bind activated Rho GTPases. The PKN proteins are well conserved evolutionarily, but their biological role in any organism is poorly understood. We previously determined that the single Drosophila ortholog of mammalian PKN proteins, Pkn, is a Rho/Rac-binding kinase essential for Drosophila development. By performing "rescue" studies with various Pkn mutant constructs, we have defined the domains of Pkn required for its role during Drosophila development. These studies suggested that Rho, but not Rac binding is important for Pkn function in development. In addition, we determined that the kinase domain of PKC53E, a PKC family kinase, can functionally substitute for the kinase domain of Pkn during development, thereby exemplifying the evolutionary strategy of "combining" functional domains to produce proteins with distinct biological activities. Interestingly, we also identified a requirement for Pkn in wing morphogenesis, thereby revealing the first postembryonic function for Pkn.

Phosphorylation of spore coat proteins by a family of atypical protein kinases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nguyen, Kim B.; Sreelatha, Anju; Durrant, Eric S.

The modification of proteins by phosphorylation occurs in all life forms and is catalyzed by a large superfamily of enzymes known as protein kinases. We recently discovered a family of secretory pathway kinases that phosphorylate extracellular proteins. One member, family with sequence similarity 20C (Fam20C), is the physiological Golgi casein kinase. While examining distantly related protein sequences, we observed low levels of identity between the spore coat protein H (CotH), and the Fam20C-related secretory pathway kinases. CotH is a component of the spore in many bacterial and eukaryotic species, and is required for efficient germination of spores in Bacillus subtilis;more » however, the mechanism by which CotH affects germination is unclear. In this paper, we show that CotH is a protein kinase. The crystal structure of CotH reveals an atypical protein kinase-like fold with a unique mode of ATP binding. Examination of the genes neighboring cotH in B. subtilis led us to identify two spore coat proteins, CotB and CotG, as CotH substrates. Furthermore, we show that CotH-dependent phosphorylation of CotB and CotG is required for the efficient germination of B. subtilis spores. Finally and collectively, our results define a family of atypical protein kinases and reveal an unexpected role for protein phosphorylation in spore biology.« less

Phosphorylation of spore coat proteins by a family of atypical protein kinases

DOE PAGES

Nguyen, Kim B.; Sreelatha, Anju; Durrant, Eric S.; ...

2016-05-16

The modification of proteins by phosphorylation occurs in all life forms and is catalyzed by a large superfamily of enzymes known as protein kinases. We recently discovered a family of secretory pathway kinases that phosphorylate extracellular proteins. One member, family with sequence similarity 20C (Fam20C), is the physiological Golgi casein kinase. While examining distantly related protein sequences, we observed low levels of identity between the spore coat protein H (CotH), and the Fam20C-related secretory pathway kinases. CotH is a component of the spore in many bacterial and eukaryotic species, and is required for efficient germination of spores in Bacillus subtilis;more » however, the mechanism by which CotH affects germination is unclear. In this paper, we show that CotH is a protein kinase. The crystal structure of CotH reveals an atypical protein kinase-like fold with a unique mode of ATP binding. Examination of the genes neighboring cotH in B. subtilis led us to identify two spore coat proteins, CotB and CotG, as CotH substrates. Furthermore, we show that CotH-dependent phosphorylation of CotB and CotG is required for the efficient germination of B. subtilis spores. Finally and collectively, our results define a family of atypical protein kinases and reveal an unexpected role for protein phosphorylation in spore biology.« less

Inhibition of AMP-Activated Protein Kinase Signaling Alleviates Impairments in Hippocampal Synaptic Plasticity Induced by Amyloid β

PubMed Central

Ma, Tao; Chen, Yiran; Vingtdeux, Valerie; Zhao, Haitian; Viollet, Benoit; Marambaud, Philippe

2014-01-01

The AMP-activated protein kinase (AMPK) is a Ser/Thr kinase that is activated in response to low-energy states to coordinate multiple signaling pathways to maintain cellular energy homeostasis. Dysregulation of AMPK signaling has been observed in Alzheimer's disease (AD), which is associated with abnormal neuronal energy metabolism. In the current study we tested the hypothesis that aberrant AMPK signaling underlies AD-associated synaptic plasticity impairments by using pharmacological and genetic approaches. We found that amyloid β (Aβ)-induced inhibition of long-term potentiation (LTP) and enhancement of long-term depression were corrected by the AMPK inhibitor compound C (CC). Similarly, LTP impairments in APP/PS1 transgenic mice that model AD were improved by CC treatment. In addition, Aβ-induced LTP failure was prevented in mice with genetic deletion of the AMPK α2-subunit, the predominant AMPK catalytic subunit in the brain. Furthermore, we found that eukaryotic elongation factor 2 (eEF2) and its kinase eEF2K are key downstream effectors that mediate the detrimental effects of hyperactive AMPK in AD pathophysiology. Our findings describe a previously unrecognized role of aberrant AMPK signaling in AD-related synaptic pathophysiology and reveal a potential therapeutic target for AD. PMID:25186765

Never in mitosis gene A related kinase-6 attenuates pressure overload-induced activation of the protein kinase B pathway and cardiac hypertrophy.

PubMed

Bian, Zhouyan; Liao, Haihan; Zhang, Yan; Wu, Qingqing; Zhou, Heng; Yang, Zheng; Fu, Jinrong; Wang, Teng; Yan, Ling; Shen, Difei; Li, Hongliang; Tang, Qizhu

2014-01-01

Cardiac hypertrophy appears to be a specialized form of cellular growth that involves the proliferation control and cell cycle regulation. NIMA (never in mitosis, gene A)-related kinase-6 (Nek6) is a cell cycle regulatory gene that could induce centriole duplication, and control cell proliferation and survival. However, the exact effect of Nek6 on cardiac hypertrophy has not yet been reported. In the present study, the loss- and gain-of-function experiments were performed in Nek6 gene-deficient (Nek6-/-) mice and Nek6 overexpressing H9c2 cells to clarify whether Nek6 which promotes the cell cycle also mediates cardiac hypertrophy. Cardiac hypertrophy was induced by transthoracic aorta constriction (TAC) and then evaluated by echocardiography, pathological and molecular analyses in vivo. We got novel findings that the absence of Nek6 promoted cardiac hypertrophy, fibrosis and cardiac dysfunction, which were accompanied by a significant activation of the protein kinase B (Akt) signaling in an experimental model of TAC. Consistent with this, the overexpression of Nek6 prevented hypertrophy in H9c2 cells induced by angiotonin II and inhibited Akt signaling in vitro. In conclusion, our results demonstrate that the cell cycle regulatory gene Nek6 is also a critical signaling molecule that helps prevent cardiac hypertrophy and inhibits the Akt signaling pathway.

Effects of simulated weightlessness on the kinase activity of MEK1 induced by bone morphogenetic protein-2 in rat osteosarcoma cells

NASA Astrophysics Data System (ADS)

Zhang, S.; Wang, B.; Cao, X. S.; Yang, Z.

Objective The mRNA expression of alpha 1 chain of type I collagen COL-I alpha 1 in rat osteosarcoma ROS17 2 8 cells induced by bone morphogenetic protein-2 BMP-2 was reduced under simulated microgravity The protein kinase MEK1 of MAPK signal pathway plays an important role in the expression of COL-I alpha 1 mRNA The purpose of this study is to investigate the effects of simulated weightlessness on the activity of MEK1 induced by BMP-2 in ROS17 2 8 cells Methods ROS17 2 8 cells were cultured in 1G control and rotating clinostat simulated weightlessness for 24 h 48 h and 72 h BMP-2 500 ng ml was added into the medium 1 h before the culture ended There was a control group in which ROS17 2 8 cells were cultured in 1G condition without BMP-2 Then the total protein of cells was extracted and the expression of phosphated-ERK1 2 p-ERK1 2 protein was detected by means of Western Blotting to show the kinase activity of MEK1 Results There were no significant differences in the expression of total ERK1 2 among all groups The expression of p-ERK1 2 was unconspicuous in the control group without BMP-2 but increased significantly when BMP-2 was added P 0 01 The level of p-ERK1 2 in simulated weightlessness group was much more lower than that in 1G group in every time point P 0 01 The expression of p-ERK1 2 gradually decreased along with the time of weightlessness simulation P 0 01 Conclusions The kinase activity of MEK1 induced by BMP-2 in rat osteosarcoma cells was reduced under simulated weightlessness

G protein-coupled receptor kinase 2 promotes cardiac hypertrophy

PubMed Central

Tscheschner, Henrike; Gao, Erhe; Schumacher, Sarah M.; Yuan, Ancai; Backs, Johannes; Most, Patrick; Wieland, Thomas; Koch, Walter J.; Katus, Hugo A.; Raake, Philip W.

2017-01-01

The increase in protein activity and upregulation of G-protein coupled receptor kinase 2 (GRK2) is a hallmark of cardiac stress and heart failure. Inhibition of GRK2 improved cardiac function and survival and diminished cardiac remodeling in various animal heart failure models. The aim of the present study was to investigate the effects of GRK2 on cardiac hypertrophy and dissect potential molecular mechanisms. In mice we observed increased GRK2 mRNA and protein levels following transverse aortic constriction (TAC). Conditional GRK2 knockout mice showed attenuated hypertrophic response with preserved ventricular geometry 6 weeks after TAC operation compared to wild-type animals. In isolated neonatal rat ventricular cardiac myocytes stimulation with angiotensin II and phenylephrine enhanced GRK2 expression leading to enhanced signaling via protein kinase B (PKB or Akt), consecutively inhibiting glycogen synthase kinase 3 beta (GSK3β), such promoting nuclear accumulation and activation of nuclear factor of activated T-cells (NFAT). Cardiac myocyte hypertrophy induced by in vitro GRK2 overexpression increased the cytosolic interaction of GRK2 and phosphoinositide 3-kinase γ (PI3Kγ). Moreover, inhibition of PI3Kγ as well as GRK2 knock down prevented Akt activation resulting in halted NFAT activity and reduced cardiac myocyte hypertrophy. Our data show that enhanced GRK2 expression triggers cardiac hypertrophy by GRK2-PI3Kγ mediated Akt phosphorylation and subsequent inactivation of GSK3β, resulting in enhanced NFAT activity. PMID:28759639

Okadaic acid-induced, naringin-sensitive phosphorylation of glycine N-methyltransferase in isolated rat hepatocytes.

PubMed Central

Møller, Michael T N; Samari, Hamid R; Fengsrud, Monica; Strømhaug, Per E; øStvold, Anne C; Seglen, Per O

2003-01-01

Glycine N-methyltransferase (GNMT) is an abundant cytosolic enzyme that catalyses the methylation of glycine into sarcosine, coupled with conversion of the methyl donor, S -adenosylmethionine (AdoMet), into S -adenosylhomocysteine (AdoHcy). GNMT is believed to play a role in monitoring the AdoMet/AdoHcy ratio, and hence the cellular methylation capacity, but regulation of the enzyme itself is not well understood. In the present study, treatment of isolated rat hepatocytes with the protein phosphatase inhibitor okadaic acid, was found to induce an overphosphorylation of GNMT, as shown by proteomic analysis. The analysis comprised two-dimensional gel electrophoretic separation of (32)P-labelled phosphoproteins and identification of individual protein spots by matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry. The identity of GNMT was verified by N-terminal Edman sequencing of tryptic peptides. Chromatographic separation of proteolytic peptides and (32)P-labelled amino acids suggested that GNMT was phosphorylated within a limited region, and only at serine residues. GNMT phosphorylation could be suppressed by naringin, an okadaic acid-antagonistic flavonoid. To assess the possible functional role of GNMT phosphorylation, the effect of okadaic acid on hepatocytic AdoMet and AdoHcy levels was examined, using HPLC separation for metabolite analysis. Surprisingly, okadaic acid was found to have no effect on the basal levels of AdoMet or AdoHcy. An accelerated AdoMet-AdoHcy flux, induced by the addition of methionine (1 mM), was likewise unaffected by okadaic acid. 5-Aminoimidazole-4-carboxamide riboside, an activator of the hepatocytic AMP-activated protein kinase, similarly induced GNMT phosphorylation without affecting AdoMet and AdoHcy levels. Activation of cAMP-dependent protein kinase by dibutyryl-cAMP, reported to cause GNMT phosphorylation under cell-free conditions, also had little effect on hepatocytic AdoMet and AdoHcy levels

High-throughput kinase assays with protein substrates using fluorescent polymer superquenching.

PubMed

Rininsland, Frauke; Stankewicz, Casey; Weatherford, Wendy; McBranch, Duncan

2005-05-31

High-throughput screening is used by the pharmaceutical industry for identifying lead compounds that interact with targets of pharmacological interest. Because of the key role that aberrant regulation of protein phosphorylation plays in diseases such as cancer, diabetes and hypertension, kinases have become one of the main drug targets. With the exception of antibody-based assays, methods to screen for specific kinase activity are generally restricted to the use of small synthetic peptides as substrates. However, the use of natural protein substrates has the advantage that potential inhibitors can be detected that affect enzyme activity by binding to a site other than the catalytic site. We have previously reported a non-radioactive and non-antibody-based fluorescence quench assay for detection of phosphorylation or dephosphorylation using synthetic peptide substrates. The aim of this work is to develop an assay for detection of phosphorylation of chemically unmodified proteins based on this polymer superquenching platform. Using a modified QTL Lightspeed assay, phosphorylation of native protein was quantified by the interaction of the phosphorylated proteins with metal-ion coordinating groups co-located with fluorescent polymer deposited onto microspheres. The binding of phospho-protein inhibits a dye-labeled "tracer" peptide from associating to the phosphate-binding sites present on the fluorescent microspheres. The resulting inhibition of quench generates a "turn on" assay, in which the signal correlates with the phosphorylation of the substrate. The assay was tested on three different proteins: Myelin Basic Protein (MBP), Histone H1 and Phosphorylated heat- and acid-stable protein (PHAS-1). Phosphorylation of the proteins was detected by Protein Kinase Calpha (PKCalpha) and by the Interleukin -1 Receptor-associated Kinase 4 (IRAK4). Enzyme inhibition yielded IC50 values that were comparable to those obtained using peptide substrates. Statistical parameters that

Sensitization of TRPA1 by Protein Kinase A

PubMed Central

Meents, Jannis E.; Fischer, Michael J. M.; McNaughton, Peter A.

2017-01-01

The TRPA1 ion channel is expressed in nociceptive (pain-sensitive) somatosensory neurons and is activated by a wide variety of chemical irritants, such as acrolein in smoke or isothiocyanates in mustard. Here, we investigate the enhancement of TRPA1 function caused by inflammatory mediators, which is thought to be important in lung conditions such as asthma and COPD. Protein kinase A is an important kinase acting downstream of inflammatory mediators to cause sensitization of TRPA1. By using site-directed mutagenesis, patch-clamp electrophysiology and calcium imaging we identify four amino acid residues, S86, S317, S428, and S972, as the principal targets of PKA-mediated phosphorylation and sensitization of TRPA1. PMID:28076424

AMP-activated protein kinase confers protection against TNF-{alpha}-induced cardiac cell death.

PubMed

Kewalramani, Girish; Puthanveetil, Prasanth; Wang, Fang; Kim, Min Suk; Deppe, Sylvia; Abrahani, Ashraf; Luciani, Dan S; Johnson, James D; Rodrigues, Brian

2009-10-01

Although a substantial role for 5' adenosine monophosphate-activated protein kinase (AMPK) has been established in regulating cardiac metabolism, a less studied action of AMPK is its ability to prevent cardiac cell death. Using established AMPK activators like dexamethasone (DEX) or metformin (MET), the objective of the present study was to determine whether AMPK activation prevents tumour necrosis factor-alpha (TNF-alpha) induced apoptosis in adult rat ventricular cardiomyocytes. Cardiomyocytes were incubated with DEX, MET, or TNF-alpha for varying durations (0-12 h). TNF-alpha-induced cell damage was evaluated by measuring caspase-3 activity and Hoechst staining. Protein and gene estimation techniques were employed to determine the mechanisms mediating the effects of AMPK activators on TNF-alpha-induced cardiomyocyte apoptosis. Incubation of myocytes with TNF-alpha for 8 h has increased caspase-3 activation and apoptotic cell death, an effect that was abrogated by DEX and MET. The beneficial effect of DEX and MET was associated with stimulation of AMPK, which led to a rapid and sustained increase in Bad phosphorylation. This event reduced the interaction between Bad and Bcl-xL, limiting cytochrome c release and caspase-3 activation. Addition of Compound C to inhibit AMPK reduced Bad phosphorylation and prevented the beneficial effects of AMPK against TNF-alpha-induced cytotoxicity. Our data demonstrate that although DEX and MET are used as anti-inflammatory agents or insulin sensitizers, respectively, their common property to phosphorylate AMPK promotes cardiomyocyte cell survival through its regulation of Bad and the mitochondrial apoptotic mechanism.

Phosphoinositide 3-kinase-dependent Ras activation by tauroursodesoxycholate in rat liver.

PubMed Central

Kurz, A K; Block, C; Graf, D; Dahl, S V; Schliess, F; Häussinger, D

2000-01-01

Ursodesoxycholic acid, widely used for the treatment of cholestatic liver disease, causes choleretic, anti-apoptotic and immunomodulatory effects. Here the effects on choleresis of its taurine conjugate tauroursodesoxycholate (TUDC), which is present in the enterohepatic circulation, were correlated with the activation of important elements of intracellular signal transduction in cultured rat hepatocytes and perfused rat liver. TUDC induced a time- and concentration-dependent activation of the small GTP-binding protein Ras and of phosphoinositide 3-kinase (PI 3-kinase) in cultured hepatocytes. Ras activation was dependent on PI 3-kinase activity, without the involvement of protein kinase C- and genistein-sensitive tyrosine kinases. Ras activation by TUDC was followed by an activation of the mitogen-activated protein kinases extracellular-signal-regulated kinase-1 (Erk-1) and Erk-2. In perfused rat liver, PI 3-kinase inhibitors largely abolished the stimulatory effect of TUDC on taurocholate excretion, suggesting an important role for a PI 3-kinase/Ras/Erk pathway in the choleretic effect of TUDC. PMID:10926845

A Screen for Novel Phosphoinositide 3-kinase Effector Proteins*

PubMed Central

Dixon, Miles J.; Gray, Alexander; Boisvert, François-Michel; Agacan, Mark; Morrice, Nicholas A.; Gourlay, Robert; Leslie, Nicholas R.; Downes, C. Peter; Batty, Ian H.

2011-01-01

Class I phosphoinositide 3-kinases exert important cellular effects through their two primary lipid products, phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2). As few molecular targets for PtdIns(3,4)P2 have yet been identified, a screen for PI 3-kinase-responsive proteins that is selective for these is described. This features a tertiary approach incorporating a unique, primary recruitment of target proteins in intact cells to membranes selectively enriched in PtdIns(3,4)P2. A secondary purification of these proteins, optimized using tandem pleckstrin homology domain containing protein-1 (TAPP-1), an established PtdIns(3,4)P2 selective ligand, yields a fraction enriched in proteins of potentially similar lipid binding character that are identified by liquid chromatography-tandem MS. Thirdly, this approach is coupled to stable isotope labeling with amino acids in cell culture using differential isotope labeling of cells stimulated in the absence and presence of the PI 3-kinase inhibitor wortmannin. This provides a ratio-metric readout that distinguishes authentically responsive components from copurifying background proteins. Enriched fractions thus obtained from astrocytoma cells revealed a subset of proteins that exhibited ratios indicative of their initial, cellular responsiveness to PI 3-kinase activation. The inclusion among these of tandem pleckstrin homology domain containing protein-1, three isoforms of Akt, switch associated protein-70, early endosome antigen-1 and of additional proteins expressing recognized lipid binding domains demonstrates the utility of this strategy and lends credibility to the novel candidate proteins identified. The latter encompass a broad set of proteins that include the gene product of TBC1D2A, a putative Rab guanine nucleotide triphosphatase activating protein (GAP) and IQ motif containing GAP1, a potential tumor promoter. A sequence comparison of the former protein indicates