scholarly journals Synapsins as major neuronal Ca2+/S100A1-interacting proteins

1999 ◽  
Vol 344 (2) ◽  
pp. 577-583 ◽  
Author(s):  
Jörg HEIERHORST ◽  
Ken I. MITCHELHILL ◽  
Richard J. MANN ◽  
Tony TIGANIS ◽  
Andrew J. CZERNIK ◽  
...  
Keyword(s):  
Protein Kinases ◽  
Double Labelling ◽  
Dependent Manner ◽  
Interacting Proteins ◽  
Confocal Immunofluorescence Microscopy ◽  
Confocal Immunofluorescence ◽  
Brain Extracts ◽  
Dependent Protein

The mammalian S100A1 protein can activate the invertebrate myosin-associated giant protein kinase twitchin in a Ca2+-dependent manner by more than 1000-fold in vitro; however, no mammalian S100-dependent protein kinases are known. In an attempt to identify novel mammalian Ca2+/S100A1-regulated protein kinases, brain extracts were subjected to combined Ca2+-dependent affinity chromatography with S100A1 and an ATP analogue. This resulted in the purification to near-homogeneity of the four major synapsin isoforms Ia, Ib, IIa and IIb. All four synapsins were specifically affinity-labelled with the ATP analogue 5′-p-fluorosulphonylbenzoyladenosine. S100A1 bound to immobilized synapsin IIa in BIAcore experiments in a Ca2+-dependent and Zn2+-enhanced manner with submicromolar affinity; this interaction could be competed for with synthetic peptides of the proposed S100A1-binding sites of synapsin. Double-labelling confocal immunofluorescence microscopy demonstrated that synapsins and S100A1 are both present in the soma and neurites of PC12 cells, indicating their potential to interact in neurons in vivo.

Roscovitine, a specific inhibitor of cyclin-dependent protein kinases, reversibly inhibits chromatin condensation during in vitro maturation of porcine oocytes

Zygote ◽  
2001 ◽  
Vol 9 (4) ◽  
pp. 309-316 ◽  
Author(s):  
Carsten Krischek ◽  
Burkhard Meinecke
Keyword(s):  
Map Kinase ◽  
Protein Kinases ◽  
Chromatin Condensation ◽  
Specific Inhibitor ◽  
Histone H1 ◽  
Dependent Manner ◽  
Free Medium ◽  
Concentration Dependent Manner ◽  
Dependent Protein

In the present study the effects of roscovitine on the in vitro nuclear maturation of porcine oocytes were investigated. Roscovitine, a specific inhibitor of cyclin-dependent protein kinases, prevented chromatin condensation in a concentration-dependent manner. This inhibition was reversible and was accompanied by non-activation of p34cdc2/histone H1 kinase. It also decreased enzyme activity of MAP kinase, suggesting a correlation between histone H1 kinase activation and the onset of chromatin condensation. The addition of roscovitine (50 μM) to extracts of metaphase II oocytes revealed that the MAP kinase activity was not directly affected by roscovitine, which indicates a possible link between histone H1 and MAP kinase. Chromatin condensation occurred between 20 and 28 h of culture of cumulus-oocyte complexes (COCs) in inhibitor-free medium (germinal vesicle stage I, GV1: 74.6% and 13.7%, respectively). Nearly the same proportion of chromatin condensation was detected in COCs incubated initially in inhibitor-free medium for 20-28 h and subsequently in roscovitine-supplemented medium (50 μM) for a further 2-10 h (GV I: 76.2% and 18.8%, respectively). This observation indicates that roscovitine prevents chromatin condensation even after an initial inhibitor-free cultivation for 20 h. Extending this initial incubation period to ≥22 h led to an activation of histone H1 and MAP kinase and increasing proportions of oocytes exhibiting chromatin condensation in the presence of roscovitine. It is concluded that histone H1 kinase is involved in the induction of chromatin condensation during in vitro maturation of porcine oocytes.

scholarly journals Cyclic AMP-Dependent Kinase Regulates Raf-1 Kinase Mainly by Phosphorylation of Serine 259

2002 ◽  
Vol 22 (10) ◽  
pp. 3237-3246 ◽  
Author(s):  
Amardeep S. Dhillon ◽  
Claire Pollock ◽  
Helge Steen ◽  
Peter E. Shaw ◽  
Harald Mischak ◽  
...  
Keyword(s):  
Cyclic Amp ◽  
Kinase Activity ◽  
Dependent Manner ◽  
Dependent Protein Kinase ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal ◽  
Dependent Protein ◽  
Kinase Pathway

ABSTRACT The Raf-1 kinase activates the ERK (extracellular-signal-regulated kinase) pathway. The cyclic AMP (cAMP)-dependent protein kinase (PKA) can inhibit Raf-1 by direct phosphorylation. We have mapped all cAMP-induced phosphorylation sites in Raf-1, showing that serines 43, 259, and 621 are phosphorylated by PKA in vitro and induced by cAMP in vivo. Serine 43 phosphorylation decreased the binding to Ras in serum-starved but not in mitogen-stimulated cells. However, the kinase activity of a RafS43A mutant was fully inhibited by PKA. Mutation of serine 259 increased the basal Raf-1 activity and rendered it largely resistant to inhibition by PKA. cAMP increased Raf-1 serine 259 phosphorylation in a PKA-dependent manner with kinetics that correlated with ERK deactivation. PKA also decreased Raf-1 serine 338 phosphorylation of Raf-1, previously shown to be required for Raf-1 activation. Serine 338 phosphorylation of a RafS259A mutant was unaffected by PKA. Using RafS259 mutants we also demonstrate that Raf-1 is the sole target for PKA inhibition of ERK and ERK-induced gene expression, and that Raf-1 inhibition is mediated mainly through serine 259 phosphorylation.

scholarly journals Reconstitution of Calcium-Regulated Parathyroid Hormone Secretion from Streptolysin-O-Permeabilized Parathyroid Cells by Guanosine 5′-O-(Thio)Triphosphate*

Endocrinology ◽  
1997 ◽  
Vol 138 (3) ◽  
pp. 1170-1179 ◽  
Author(s):  
Lisa M. Matovcik ◽  
Steven S. Rhee ◽  
Jean F. Schaefer ◽  
Barbara K. Kinder
Keyword(s):  
Hormone Secretion ◽  
Dependent Manner ◽  
Intact Cells ◽  
Permeabilized Cells ◽  
Calcium Calmodulin Dependent ◽  
Streptolysin O ◽  
Dependent Protein ◽  
Vitro Model

Abstract Intracellular Ca2+ levels determine the amount of PTH secretion from parathyroid cells. Dissociated calf parathyroid cells were permeabilized with streptolysin-O (SLO) to provide an in vitro model system to examine Ca2+-dependent regulation of hormone secretion. PTH release from these cells was energy dependent and increased by cytosolic cofactors. Guanosine 5′-O-(thio)triphosphate (GTPγS) increased PTH secretion from SLO-permeabilized cells in a dose-dependent manner from 0.1–100 μm. In the absence of GTPγS there was no relationship between the ambient Ca2+ concentration and the rate of PTH secretion. However, in the presence of GTPγS, intracellular Ca2+ inhibited PTH secretion with an EC50 of approximately 0.1 μm, corresponding to physiological intracellular Ca2+ levels. Thus, the addition of GTPγS to SLO-permeabilized parathyroid cells reconstituted the inverse relationship between extracellular Ca2+ and PTH secretion that is observed in vivo and in intact cells. The data indicate that this effect is mediated at least in part by heterotrimeric guanosine triphosphatases. In addition, calcium/calmodulin-dependent protein kinase II appears to mediate low Ca2+-dependent PTH secretion from these cells.

scholarly journals Phosphodiesterase 3A binds to 14-3-3 proteins in response to PMA-induced phosphorylation of Ser428

2005 ◽  
Vol 392 (1) ◽  
pp. 163-172 ◽  
Author(s):  
Mercedes Pozuelo Rubio ◽  
David G. Campbell ◽  
Nicholas A. Morrice ◽  
Carol Mackintosh
Keyword(s):  
Protein Kinase ◽  
Metal Ion ◽  
Mitogen Activated Protein Kinase ◽  
Dependent Manner ◽  
Cell Extracts ◽  
Mitogen Activated Protein ◽  
Dependent Protein ◽  
Pkc Protein Kinase C

PDE3A (phosphodiesterase 3A) was identified as a phosphoprotein that co-immunoprecipitates with endogenous 14-3-3 proteins from HeLa cell extracts, and binds directly to 14-3-3 proteins in a phosphorylation-dependent manner. Among cellular stimuli tested, PMA promoted maximal binding of PDE3A to 14-3-3 proteins. While p42/p44 MAPK (mitogen-activated protein kinase), SAPK2 (stress-activated protein kinase 2)/p38 and PKC (protein kinase C) were all activated by PMA in HeLa cells, the PMA-induced binding of PDE3A to 14-3-3 proteins was inhibited by the non-specific PKC inhibitors Ro 318220 and H-7, but not by PD 184352, which inhibits MAPK activation, nor by SB 203580 and BIRB0796, which inhibit SAPK2 activation. Binding of PDE3A to 14-3-3 proteins was also blocked by the DNA replication inhibitors aphidicolin and mimosine, but the PDE3A–14-3-3 interaction was not cell-cycle-regulated. PDE3A isolated from cells was able to bind to 14-3-3 proteins after in vitro phosphorylation with PKC isoforms. Using MS/MS of IMAC (immobilized metal ion affinity chromatography)-enriched tryptic phosphopeptides and phosphospecific antibodies, at least five sites on PDE3A were found to be phosphorylated in vivo, of which Ser428 was selectively phosphorylated in response to PMA and dephosphorylated in cells treated with aphidicolin and mimosine. Phosphorylation of Ser428 therefore correlated with 14-3-3 binding to PDE3A. Ser312 of PDE3A was phosphorylated in an H-89-sensitive response to forskolin, indicative of phosphorylation by PKA (cAMP-dependent protein kinase), but phosphorylation at this site did not stimulate 14-3-3 binding. Thus 14-3-3 proteins can discriminate between sites in a region of multisite phosphorylation on PDE3A. An additional observation was that the cytoskeletal cross-linker protein plectin-1 coimmunoprecipitated with PDE3A independently of 14-3-3 binding.

Scutellarin Suppresses RPMI7951 Melanoma Cell Proliferation by Targeting TOPK

2020 ◽  
Vol 20 ◽  
Author(s):  
Xin Mu ◽  
Lijuan Wang ◽  
Zixi Zhang ◽  
Rui Ge ◽  
Jian Zhang ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinases ◽  
Histone H3 ◽  
Soft Agar ◽  
Kinase Assay ◽  
Dependent Manner ◽  
Dose Dependent ◽  
Extracellular Regulated Protein Kinases

Background: T-LAK cell-originated protein kinase (TOPK) belongs to the serine/threonine protein kinase family. It is highly expressed in RPMI7951 melanoma cells. Scutellarin (SCU) is an active ingredient extracted from Erigeron breviscapus (Vant.) Hand.–Mazz. Its main physiological functions are related to its anti-inflammatory and antitumour activities. Methods: The relationship between SCU and TOPK was assessed by molecular docking, an in vitro binding assay and an in vitro kinase assay. The effect of SCU on RPMI7951 cells was detected by MTS and soft agar assays. TOPK knockdown was induced by lentiviral infection. The TOPK downstream signalling pathway was detected by western blot and immunohistochemical analyses in vitro and in vivo. Results: SCU was found to directly bind with TOPK and inhibit TOPK activity in vitro. SCU inhibited the proliferation and colony formation of RPMI7951 cells in a dose-dependent manner. Silencing TOPK decreased the sensitivity of colon cancer cells to SCU. SCU inhibited the phosphorylation levels of extracellular regulated protein kinases 1/2 (ERK1/2) and histone H3 in a time- and dose-dependent manner in RPMI7951 cells. In addition, SCU inhibited the growth of xenograft tumours of RPMI7951 cells and decreased the phosphorylation levels of extracellular regulated protein kinases 1/2 and histone H3 in vivo. Conclusion: The results showed that SCU exerts promising antitumour effects on human RPMI7951 cells by inhibiting the activity of TOPK.

scholarly journals Rhubarb Tannins Extract Inhibits the Expression of Aquaporins 2 and 3 in Magnesium Sulphate-Induced Diarrhoea Model

2014 ◽  
Vol 2014 ◽  
pp. 1-14 ◽  
Author(s):  
Chunfang Liu ◽  
Yanfang Zheng ◽  
Wen Xu ◽  
Hui Wang ◽  
Na Lin
Keyword(s):  
Magnesium Sulphate ◽  
Camp Response Element Binding ◽  
Dependent Manner ◽  
Active Components ◽  
Camp Response Element ◽  
Element Binding Protein ◽  
Dependent Protein ◽  
Ht 29

Tannins, a group of major active components of Chinese rhubarb and widely distributed in nature, have a significant antidiarrhoeal activity. Aquaporins (AQPs) 2 and 3 play important roles in regulating water transfer during diarrhoea. The present study aims to determine the effect of the total tannins extract of rhubarb on aquaporins (AQPs) 2 and 3 in diarrhoea mice and HT-29 cells both induced by magnesium sulphate (MgSO4). Our results showed that rhubarb tannins extract (RTE) significantly decreased the faecal water content in colon and evaluation index of defecation of diarrhoea mice. Interestingly, RTE could markedly reduce the mRNA and protein expression levels of AQPs 2 and 3 in apical and lateral mucosal epithelial cells in the colons of diarrhoea mice and HT-29 cells both induced by MgSO4in a dose-dependent manner. Furthermore, RTE suppressed the production of cyclic monophosphate- (cAMP-) dependent protein kinase A catalytic subunitsα(PKA C-α) and phosphorylated cAMP response element-binding protein (p-CREB, Ser133) in MgSO4-induced HT-29 cells. Our data showed for the first time that RTE inhibit AQPs 2 and 3 expressionin vivoandin vitrovia downregulating PKA/p-CREB signal pathway, which accounts for the antidiarrhoeal effect of RTE.

scholarly journals Induction of a cytoplasmic activator of DNA synthesis in lymphocytes is mediated through a membrane-associated protein kinase.

1990 ◽  
Vol 1 (13) ◽  
pp. 1015-1025 ◽  
Author(s):  
M V Autieri ◽  
K L Fresa ◽  
F D Coffman ◽  
M E Katz ◽  
S Cohen
Keyword(s):  
Protein Kinase ◽  
Dna Synthesis ◽  
Protein Kinases ◽  
Phorbol Esters ◽  
Adenosine Monophosphate ◽  
Peripheral Blood Lymphocytes ◽  
Lymphoid Cells ◽  
Dependent Manner ◽  
Dependent Protein

We have shown previously that cytoplasmic extracts from actively dividing lymphoid cells are capable of inducing DNA synthesis in isolated nuclei. One of the factors involved in this activity, ADR, appears to be a greater than 90 kDa heat-labile protease. Cytoplasmic extracts prepared from nonproliferating lymphocytes express little to no ADR activity. However, ADR activity can be generated in these extracts by brief exposure to a membrane-enriched fraction of spontaneously proliferating, leukemic human T lymphoblastoid (MOLT-4) cells. This suggests that ADR activity is present in the resting cytoplasm in an inactive or precursor form. This in vitro generation of ADR activity can be inhibited in a dose-dependent manner by the isoquinolinesulfonamide derivative, H-7 (1-(5-isoquinoline-sulfonyl)-2-methylpiperazine dihydrochloride), an inhibitor of both cyclic adenosine monophosphate (cAMP)-dependent protein kinases and protein kinase C (PKC). However, more specific inhibitors of cAMP-dependent protein kinases, including N-[( 2-methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H8) and N-(2-gua-nidinoethyl)-5-isoquinolinesulfonamide (HA-1004), had little to no effect on the in vitro generation of ADR activity. Furthermore, membranes from MOLT-4 cells depleted of PKC by long-term exposure (24 h) to phorbol esters and calcium ionophores were unable to induce ADR activity in resting peripheral blood lymphocytes extracts. The results of these studies suggest 1) ADR activity is present in resting cell cytoplasm in an inactive or precursor form; and 2) ADR activity can be induced in this resting cytoplasm through a mechanism involving a membrane-associated protein kinase, possibly PKC. The ability of alkaline phosphatase to deplete the activity of preformed ADR suggests the possibility that ADR itself is phosphoprotein.

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