Trypanosoma brucei: characterization of protein kinases that are capable of autophosphorylation in vitro

Parasitology ◽  
1994 ◽  
Vol 108 (2) ◽  
pp. 161-166 ◽  
Author(s):  
G. Hide ◽  
T. Graham ◽  
N. Buchanan ◽  
A. Tait ◽  
K. Keith
Keyword(s):  
Protein Kinase ◽  
Protein Kinases ◽  
Trypanosoma Brucei ◽  
Mammalian Cells ◽  
Molecular Size ◽  
Regulatory Pathways ◽  
Phosphate Donor ◽  
Epidermal Growth

SUMMARYAutophosphorylation by protein kinases has been implicated as an important control mechanism in signal transduction and growth regulatory pathways in mammalian cells. We have set out to investigate whether any such autophosphorylating protein kinase activities can be found in Trypanosoma brucei. In order to do this, we have developed a system for characterizing such protein kinase activities using an in vitro assay. This assay was carried out by fractionation of trypanosome lysates using isoelectric focusing gel electrophoresis followed by incubation of the gel in γ32P-labelled nucleotide triphosphate and subsequent autoradiography. We have identified two classes of autophosphorylating protein kinase activities. In the first class all were dependent on ATP as the phosphate donor substrate and were all found to have a molecular size of 60 kDa. Differences in the activity of these protein kinases were observed between the bloodstream and procyclic life-cyle stages. Furthermore, the addition of mammalian epidermal growth factor to bloodstream stage lysates stimulated an additional activity. The second class of autophosphorylating protein kinases utilized GTP as the phosphate donor and were all found to be 90 kDa in size. Stage-specific differences were also observed in the activity of these protein kinases.

Characterization of the cellular action of the MSK inhibitor SB-747651A

2011 ◽  
Vol 441 (1) ◽  
pp. 347-357 ◽  
Author(s):  
Shaista Naqvi ◽  
Andrew Macdonald ◽  
Claire E. McCoy ◽  
Joanne Darragh ◽  
Alastair D. Reith ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinases ◽  
Inflammatory Cytokine ◽  
Interleukin 10 ◽  
Camp Response Element Binding ◽  
S6 Kinase ◽  
Ribosomal S6 Kinase ◽  
In Cells

MSK1 (mitogen- and stress-activated kinase 1) and MSK2 are nuclear protein kinases that regulate transcription downstream of the ERK1/2 (extracellular-signal-regulated kinase 1/2) and p38α MAPKs (mitogen-activated protein kinases) via the phosphorylation of CREB (cAMP-response-element-binding protein) and histone H3. Previous studies on the function of MSKs have used two inhibitors, H89 and Ro 31-8220, both of which have multiple off-target effects. In the present study, we report the characterization of the in vitro and cellular properties of an improved MSK1 inhibitor, SB-747651A. In vitro, SB-747651A inhibits MSK1 with an IC50 value of 11 nM. Screening of an in vitro panel of 117 protein kinases revealed that, at 1 μM, SB-747651A inhibited four other kinases, PRK2 (double-stranded-RNA-dependent protein kinase 2), RSK1 (ribosomal S6 kinase 1), p70S6K (S6K is S6 kinase) (p70RSK) and ROCK-II (Rho-associated protein kinase 2), with a similar potency to MSK1. In cells, SB-747651A fully inhibited MSK activity at 5–10 μM. SB-747651A was found to inhibit the production of the anti-inflammatory cytokine IL-10 (interleukin-10) in wild-type, but not MSK1/2-knockout, macrophages following LPS (lipopolysaccharide) stimulation. Both SB-747651A and MSK1/2 knockout resulted in elevated pro-inflammatory cytokine production by macrophages in response to LPS. Comparison of the effects of SB-747651A, both in vitro and in cells, demonstrated that SB-747651A exhibited improved selectivity over H89 and Ro 31-8220 and therefore represents a useful tool to study MSK function in cells.

Characterization of a dual plant protein kinase (FsPK1) up-regulated by abscisic acid and calcium and specifically expressed in dormant seeds ofFagus sylvaticaL.

2003 ◽  
Vol 13 (4) ◽  
pp. 261-271 ◽  
Author(s):  
O. Lorenzo ◽  
C. Nicolás ◽  
G. Nicolás ◽  
D. Rodríguez
Keyword(s):  
Abscisic Acid ◽  
Protein Kinase ◽  
Protein Kinases ◽  
Catalytic Domain ◽  
Consensus Sequence ◽  
Calcium Dependent ◽  
Localization Signal ◽  
Rapid Amplification

An abscisic acid (ABA)-induced cDNA fragment encoding a putative protein kinase (PK) was obtained by differential screening of a cDNA library fromFagus sylvaticaseeds. The full-length clone, named FsPK1, was produced by 5′ rapid amplification of cDNA ends (RACE) extension. This clone contained the 11 catalytic domains present in all protein kinases, but displayed unusual characteristics found only in a few plant PKs. FsPK1 exhibits features of both serine/threonine and tyrosine protein kinases within the catalytic domain, a putative nuclear localization signal within the regulatory domain and the consensus sequence involved in binding of 14-3-3 proteins. The catalytic domain, expressed inEscherichia colias a fusion protein, showed Ca2+-dependentin vitrokinase activity and dual serine/threonine and tyrosine specificity. Transcription of theFsPK1gene was reduced by seed stratification at 4°C, and clearly increased when seeds were treated with 0.1 mM ABA, correlating with the inhibition of germination. Interestingly,FsPK1transcripts were enhanced when ABA (0.1 mM) and calcium (1 mM) were added together, while the addition of EGTA (calcium chelator) and 3,4,5,-trimethoxibenzoic acid 8-(diethylamino) octyl ester (TMB-8, a calcium antagonist) decreased its expression. Furthermore,FsPK1transcript expression was tissue specific and accumulated only in ABA-treated seeds, but not in any ABA-treated vegetative tissues examined. These results suggest that the expression of the corresponding protein could be related to the inhibition of germination mediated by ABA in a calcium-dependent pathway.

scholarly journals Cloning and characterization of mouse extracellular-signal-regulated protein kinase 3 as a unique gene product of 100 kDa

2000 ◽  
Vol 346 (1) ◽  
pp. 169-175 ◽  
Author(s):  
Benjamin TURGEON ◽  
Marc K. SABA-EL-LEIL ◽  
Sylvain MELOCHE
Keyword(s):  
Protein Kinase ◽  
Gene Product ◽  
Mammalian Cells ◽  
Chromosome 9 ◽  
Mouse Tissue ◽  
Mouse Development ◽  
Extracellular Signal ◽  
Mouse Tissues

MAP (mitogen-activated protein) kinases are a family of serine/threonine kinases that have a pivotal role in signal transduction. Here we report the cloning and characterization of a mouse homologue of extracellular-signal-regulated protein kinase (ERK)3. The mouse Erk3 cDNA encodes a predicted protein of 720 residues, which displays 94% identity with human ERK3. Transcription and translation of this cDNA in vitro generates a 100 kDa protein similar to the human gene product ERK3. Immunoblot analysis with an antibody raised against a unique sequence of ERK3 also recognizes a 100 kDa protein in mouse tissues. A single transcript of Erk3 was detected in every adult mouse tissue examined, with the highest expression being found in the brain. Interestingly, expression of Erk3 mRNA is acutely regulated during mouse development, with a peak of expression observed at embryonic day 11. The mouse Erk3 gene was mapped to a single locus on central mouse chromosome 9, adjacent to the dilute mutation locus and in a region syntenic to human chromosome 15q21. Finally, we provide several lines of evidence to support the existence of a unique Erk3 gene product of 100 kDa in mammalian cells.

scholarly journals Characterization of a Cytotoxic Factor in Culture Filtrates of Serratia marcescens

2002 ◽  
Vol 70 (3) ◽  
pp. 1121-1128 ◽  
Author(s):  
Kent B. Marty ◽  
Christopher L. Williams ◽  
Linda J. Guynn ◽  
Michael J. Benedik ◽  
Steven R. Blanke
Keyword(s):  
Cytotoxic Activity ◽  
Serratia Marcescens ◽  
Mammalian Cells ◽  
Divalent Cations ◽  
Recombinant Expression ◽  
Cytotoxic Factor ◽  
Culture Filtrates ◽  
Type Strains

ABSTRACT Serratia marcescens culture filtrates have been reported to be cytotoxic to mammalian cells. Using biochemical and genetic approaches, we have identified a major source of this cytotoxic activity. Both heat and protease treatments abrogated the cytotoxicity of S. marcescens culture filtrates towards HeLa cells, suggesting the involvement of one or more protein factors. A screen for in vitro cytotoxic activity revealed that S. marcescens mutant strains that are deficient in production of a 56-kDa metalloprotease are significantly less cytotoxic to mammalian cells. Cytotoxicity was significantly reduced when culture filtrates prepared from wild-type strains were pretreated with either EDTA or 1,10-phenanthroline, which are potent inhibitors of the 56-kDa metalloprotease. Furthermore, cytotoxic activity was restored when the same culture filtrates were incubated with zinc divalent cations, which are essential for enzymatic activity of the 56-kDa metalloprotease. Finally, recombinant expression of the S. marcescens 56-kDa metalloprotease conferred a cytotoxic phenotype on the culture filtrates of a nonpathogenic Escherichia coli strain. Collectively, these data suggest that the 56-kDa metalloprotease contributes significantly to the in vitro cytotoxic activity commonly observed in S. marcescens culture filtrates.

Identification and characterization of a new mammalian mitogen-activated protein kinase kinase, MKK2

1993 ◽  
Vol 13 (8) ◽  
pp. 4539-4548
Author(s):  
J Wu ◽  
J K Harrison ◽  
P Dent ◽  
K R Lynch ◽  
M J Weber ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Map Kinase ◽  
Protein Kinases ◽  
Map Kinases ◽  
Sodium Dodecyl ◽  
Rat Tissues ◽  
Tyrosine Residues ◽  
Mitogen Activated Protein ◽  
Map Kinase Kinase

Mitogen-activated protein (MAP) kinases are serine/threonine protein kinases activated by dual phosphorylation on threonine and tyrosine residues. A MAP kinase kinase (MKK1 or MEK1) has been identified as a dual-specificity protein kinase that is sufficient to phosphorylate MAP kinases p42mapk and p44mapk on the regulatory threonine and tyrosine residues. Because of the multiplicity of MAP kinase isoforms and the diverse circumstances and agonists leading to their activation, we thought it unlikely that a single MKK could accommodate this complexity. Indeed, two protein bands with MKK activity have previously been identified after renaturation following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We now report the molecular cloning and characterization of a second rat MAP kinase kinase cDNA, MKK2. MKK2 cDNA contains an open reading frame encoding a protein of 400 amino acids, 7 residues longer than MKK1 (MEK1). The amino acid sequence of MKK2 is 81% identical to that of MKK1, but nucleotide sequence differences occur throughout the aligned MKK2 and MKK1 cDNAs, indicating that MKK2 is the product of a distinct gene. MKK1 and MKK2 mRNAs are expressed differently in rat tissues. Both cDNAs when expressed in COS cells displayed the ability to phosphorylate and activate p42mapk and p44mapk, both MKK1 and MKK2 were activated in vivo in response to serum, and both could be phosphorylated and activated by the v-Raf protein in vitro. However, differences between MKK1 and MKK2 in sites of phosphorylation by proline-directed protein kinases predict differences in feedback regulation.

scholarly journals B-1 cell: the precursor of a novel mononuclear phagocyte with immuno-regulatory properties

2009 ◽  
Vol 81 (3) ◽  
pp. 489-496 ◽  
Author(s):  
José Daniel Lopes ◽  
Mario Mariano
Keyword(s):  
Mammalian Cells ◽  
Giant Cells ◽  
Mononuclear Phagocyte ◽  
Different Types ◽  
Series Of Experiments ◽  
B1 Cells ◽  
Regulatory Properties

Characterization of the origin, properties, functions and fate of cells is a fundamental task for the understanding of physiological and pathological phenomena. Despite the bulk of knowledge concerning the diverse characteristics of mammalian cells, some of them, such as B-1 cells, are still poorly understood. Here we report the results obtained in our laboratory on these cells in the last 10 years. After showing that B-1 cells could be cultured and amplified in vitro, a series of experiments were performed with these cells. They showed that B1 cells reside mostly in the peritoneal and pleural cavities, migrate to distant inflammatory foci, coalesce to form giant cells and participate in granuloma formation, both in vitro and in vivo. They are also able to present antigens to immunologically responsive cells and are endowed with regulatory properties. Further, we have also shown that these cells facilitate different types of infection as well as tumor growth and spreading. These data are presently reviewed pointing to a pivotal role that these cells may play in innate and acquired immunity.

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