scholarly journals Tyrosine phosphorylation and activation of a new mitogen-activated protein (MAP)-kinase cascade in human neutrophils stimulated with various agonists

1996 ◽  
Vol 318 (1) ◽  
pp. 247-253 ◽  
Author(s):  
Nabeel NAHAS ◽  
Thaddeus F. P. MOLSKI ◽  
Gustavo A FERNANDEZ ◽  
Ramadan I. SHA'AFI
Keyword(s):  
Protein Kinase ◽  
Map Kinase ◽  
Tyrosine Phosphorylation ◽  
Map Kinases ◽  
P38 Map Kinase ◽  
Human Neutrophils ◽  
Low Concentrations ◽  
Mitogen Activated Protein ◽  
Experimental Findings ◽  
Stimulation Of

The presence of a novel 38 kDa protein that is tyrosine phosphorylated in human neutrophils, a terminally differentiated cell, upon stimulation of these cells with low concentrations of lipopolysaccharide (LPS) in combination with serum has been demonstrated. This 38 kDa protein was identified as the mammalian homologue of HOG1 in yeast, the p38 mitogen-activated protein (MAP) kinase. This conclusion is based on the experimental findings that anti-phosphotyrosine (anti-PY) antibody immunoprecipitates a 38 kDa protein that is recognized by anti-p38 MAP kinase antibody, and conversely, anti-p38 MAP kinase antibody immunoprecipitates a 38 kDa protein that can be recognized by anti-PY antibody. Moreover, this tyrosine phosphorylated protein is found associated entirely with the cytosol. It was also found that this p38 MAP kinase is activated following stimulation of these cells with low concentrations of LPS in combination with serum. This conclusion is based on three experimental findings. First, soluble fractions isolated from LPS-stimulated cells phosphorylate heat shock protein 27 (hsp27) in an in vitro assay, and this effect is not inhibited by protein kinase C and protein kinase A inhibitor peptides. This effect is similar to the effect produced by the commercially available phosphorylated and activated MAPKAP kinase-2 (MAP kinase activated protein kinase-2). Secondly, a 27 kDa protein that aligns with a protein recognized by anti-hsp27 antibody is phosphorylated upon LPS stimulation of intact human neutrophils prelabelled with radioactive phosphate. Lastly, immune complex protein kinase assays, using [γ-32P]ATP and activating transcription factor 2 (ATF2) as substrates, showed increased p38 MAP kinase activity from LPS-stimulated human neutrophils. The phosphorylation and activation of this p38 MAP kinase can be affected by both G-protein-coupled receptors such as platelet-activating factor (PAF) and non-G-protein-coupled receptors such as the cytokine-coupled receptors for granulocyte–macrophage colony-stimulating factor (GM-CSF) and tumour necrosis factor α (TNF-α). The effect of low concentrations of PAF is greatly increased in cells pretreated with LPS. The tyrosine phosphorylation of the p38 MAP kinase is not restricted to stimuli that mediate their actions through membrane-associated receptors, but it can be affected by agents that bypass membrane-associated receptors such as the protein translation blocker anisomycin. While anisomycin is known to increase the tyrosine phosphorylation of the 54 kDa SAPK (stress-activated protein kinase), this is the first report that shows that anisomycin also tyrosine phosphorylates the p38 MAP kinase. Cytokine receptors that increase the tyrosine phosphorylation and activation of the erk1 and erk2 MAP kinases have less effect on this p38 MAP kinase than those that do not affect the erk1 and erk2 MAP kinases. The possible role of the p38 MAP kinase in the phosphorylation of cytosolic phospholipase A2 is discussed.

Ionomycin causes activation of p38 and p42/44 mitogen-activated protein kinases in human neutrophils

2001 ◽  
Vol 281 (1) ◽  
pp. C350-C360 ◽  
Author(s):  
David J. Elzi ◽  
A. Jason Bjornsen ◽  
Todd MacKenzie ◽  
Travis H. Wyman ◽  
Christopher C. Silliman
Keyword(s):  
Map Kinase ◽  
Map Kinases ◽  
P38 Map Kinase ◽  
Polymorphonuclear Neutrophils ◽  
Human Neutrophils ◽  
Kinase Activation ◽  
Extracellular Signal ◽  
Mitogen Activated Protein ◽  
Activating Transcription Factor

Many receptor-linked agents that prime or activate the NADPH oxidase in polymorphonuclear neutrophils (PMNs) elicit changes in cytosolic Ca2+concentration and activate mitogen-activated protein (MAP) kinases. To investigate the role of Ca2+in the activation of p38 and p42/44 MAP kinases, we examined the effects of the Ca2+-selective ionophore ionomycin on priming and activation of the PMN oxidase. Ionomycin caused a rapid rise in cytosolic Ca2+that was due to both a release of cytosolic Ca2+stores and Ca2+influx. Ionomycin also activated (2 μM) and primed (20–200 nM) the PMN oxidase. Dual phosphorylation of p38 MAP kinase and phosphorylation of its substrate activating transcription factor-2 were detected at ionomycin concentrations that prime or activate the PMN oxidase, while dual phosphorylation of p42/44 MAP kinase and phosphorylation of its substrate Elk-1 were elicited at 0.2–2 μM. SB-203580, a p38 MAP kinase antagonist, inhibited ionomycin-induced activation of the oxidase (68 ± 8%, P < 0.05) and tyrosine phosphorylation of 105- and 72-kDa proteins; conversely, PD-98059, an inhibitor of MAP/extracellular signal-related kinase 1, had no effect. Treatment of PMNs with thapsigargin resulted in priming of the oxidase and activation of p38 MAP kinase. Chelation of cytosolic but not extracellular Ca2+completely inhibited ionomycin activation of p38 MAP kinase, whereas chelation of extracellular Ca2+abrogated activation of p42/44 MAP kinase. These results demonstrate the importance of changes in cytosolic Ca2+for MAP kinase activation in PMNs.

scholarly journals Inhibition of p38 Mitogen-Activated Protein Kinase Augments Lipopolysaccharide-Induced Cell Proliferation in CD14-Expressing Chinese Hamster Ovary Cells

2001 ◽  
Vol 69 (2) ◽  
pp. 931-936 ◽  
Author(s):  
Dipshikha Chakravortty ◽  
Yutaka Kato ◽  
Tsuyoshi Sugiyama ◽  
Naoki Koide ◽  
Mya Mya Mu ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Protein Kinase ◽  
Dna Synthesis ◽  
Map Kinase ◽  
Cho Cells ◽  
Map Kinases ◽  
Chinese Hamster ◽  
P38 Map Kinase ◽  
Mitogen Activated Protein ◽  
Membrane Bound

ABSTRACT CD14-expressing Chinese hamster ovary (CD14-CHO) cells, established by transfection of human CD14 DNA, acquired high responsiveness to lipopolysaccharide (LPS) through membrane-bound CD14 expression. LPS induced DNA synthesis and activated a series of mitogen-activated protein (MAP) kinases, extracellular signal-regulated kinase 1/2 (Erk1/2), p38, and c-Jun N-terminal kinase/stress-activated protein kinase, in CD14-CHO cells but not in mock-transfected CHO cells. Anti-CD14 antibody completely abrogated both LPS-induced DNA synthesis and LPS-induced phosphorylation of those MAP kinases, suggesting a critical role of membrane-bound CD14 in LPS signaling. A p38 MAP kinase inhibitor, SB203580, markedly augmented LPS-induced DNA synthesis in CD14-CHO cells, whereas an Erk1/2 inhibitor, PD98059, had no affect. On the other hand, SB203580 exhibited no effect on epidermal growth factor-induced DNA synthesis in CD14-CHO cells, although PD98059 inhibited it significantly. The activation and inactivation of p38 MAP kinase with dominant negative and dominant positive mutants also suggested the participation of p38 MAP kinase in LPS-induced DNA synthesis. It was therefore suggested that the activation of p38 MAP kinase can negatively regulate LPS-induced cell proliferation in CD14-CHO cells.

scholarly journals Effect of lipopolysaccharide on mitogen-activated protein kinases and cytosolic phospholipase A2

1995 ◽  
Vol 308 (3) ◽  
pp. 815-822 ◽  
Author(s):  
S I Fouda ◽  
T F P Molski ◽  
M S E Ashour ◽  
R I Sha′afi
Keyword(s):  
Phospholipase A2 ◽  
Map Kinase ◽  
Map Kinases ◽  
Kinase Inhibitor ◽  
Cytosolic Phospholipase A2 ◽  
Mitogen Activated Protein Kinase ◽  
Human Neutrophils ◽  
Cytosolic Phospholipase ◽  
Low Concentrations ◽  
Mitogen Activated Protein

The addition of platelet-activating factor (PAF) to human neutrophils increases phosphorylation on tyrosine residues and stimulates the activity of p42erk2 mitogen-activated protein kinase (MAP kinase). This action is rapid and transient. In contrast, p42erk2, p44erk1 and the p40hera MAP kinase isoforms are all not tyrosine phosphorylated or activated in human neutrophils stimulated with low concentrations of lipopolysaccharide (LPS) in combination with serum. In spite of this, the PAF-induced tyrosine phosphorylation and activation of the p42erk2 MAP kinase are greatly potentiated in cells pretreated with LPS. More interestingly, although low concentrations of LPS do not affect MAP kinase isoforms in these cells, they cause the phosphorylation of cytosolic phospholipase A2 (cPLA2), as evidenced by a decrease in the electrophoretic mobility of the enzyme. In addition, this stimulus-induced upward shift in the mobility of the enzyme is not inhibited by the tyrosine kinase inhibitor, genistein. Furthermore, LPS increases the release of arachidonic acid in control and PAF-stimulated human neutrophils. These observations clearly show that cPLA2 can be phosphorylated and activated by kinases other than the currently known MAP kinases. It is proposed that there are MAP kinase-dependent and -independent mechanisms for the phosphorylation of cPLA2.

scholarly journals Regulation of cyclin-dependent kinase (Cdk) 2 Thr-160 phosphorylation and activity by mitogen-activated protein kinase in late G1 phase

2000 ◽  
Vol 349 (3) ◽  
pp. 869-876 ◽  
Author(s):  
Mario CHIARIELLO ◽  
Eliana GOMEZ ◽  
J. Silvio GUTKIND
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Dna Synthesis ◽  
Map Kinase ◽  
Map Kinases ◽  
Mitogen Activated Protein Kinase ◽  
G1 Phase ◽  
Cyclin Dependent Kinase ◽  
Mitogen Activated Protein ◽  
Stimulation Of

Mitogen-activated protein (MAP) kinases, p42MAPK and p44MAPK, are central components of growth-promoting signalling pathways. However, how stimulation of MAP kinases culminates in cell-cycle progression is still poorly understood. Here we show that mitogenic stimulation of NIH 3T3 cells causes a sustained activation of MAP kinases, which lasts until cells begin progressing through the G1/S boundary. Furthermore, we observed that disruption of the MAP-kinase pathway with a selective MEK (MAP kinase/extracellular-signal-regulated protein kinase kinase) inhibitor, PD98059, prevents the activation of cyclin-dependent kinase (Cdk) 2 and DNA synthesis, even when added during late G1 phase, once the known mechanisms by which MAP kinase controls G1 progression, accumulation of G1 cyclins and degradation of Cdk inhibitors have already taken place. Moreover, we provide evidence indicating that MAP kinases control Cdk2 Thr-160 activating phosphorylation and function, possibly by regulating the activity of a Cdk-activating kinase, thus promoting the re-initiation of DNA synthesis. These findings suggest the existence of a novel mechanism whereby signal-transducing pathways converging on MAP kinases can affect the cell-cycle machinery and, ultimately, participate in cell-growth control.

A yeast mitogen-activated protein kinase homolog (Mpk1p) mediates signalling by protein kinase C

1993 ◽  
Vol 13 (5) ◽  
pp. 3067-3075 ◽  
Author(s):  
K S Lee ◽  
K Irie ◽  
Y Gotoh ◽  
Y Watanabe ◽  
H Araki ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Map Kinase ◽  
Protein Kinases ◽  
Map Kinases ◽  
Pheromone Response ◽  
Pheromone Response Pathway ◽  
Kinase C ◽  
Mitogen Activated Protein ◽  
Kinase Cascade ◽  
Protein Kinase Cascade

Mitogen-activated protein (MAP) kinases are activated in response to a variety of stimuli through a protein kinase cascade that results in their phosphorylation on tyrosine and threonine residues. The molecular nature of this cascade is just beginning to emerge. Here we report the isolation of a Saccharomyces cerevisiae gene encoding a functional analog of mammalian MAP kinases, designated MPK1 (for MAP kinase). The MPK1 gene was isolated as a dosage-dependent suppressor of the cell lysis defect associated with deletion of the BCK1 gene. The BCK1 gene is also predicted to encode a protein kinase which has been proposed to function downstream of the protein kinase C isozyme encoded by PKC1. The MPK1 gene possesses a 1.5-kb uninterrupted open reading frame predicted to encode a 53-kDa protein. The predicted Mpk1 protein (Mpk1p) shares 48 to 50% sequence identity with Xenopus MAP kinase and with the yeast mating pheromone response pathway components, Fus3p and Kss1p. Deletion of MPK1 resulted in a temperature-dependent cell lysis defect that was virtually indistinguishable from that resulting from deletion of BCK1, suggesting that the protein kinases encoded by these genes function in a common pathway. Expression of Xenopus MAP kinase suppressed the defect associated with loss of MPK1 but not the mating-related defects associated with loss of FUS3 or KSS1, indicating functional conservation between the former two protein kinases. Mutation of the presumptive phosphorylated tyrosine and threonine residues of Mpk1p individually to phenylalanine and alanine, respectively, severely impaired Mpk1p function. Additional epistasis experiments, and the overall architectural similarity between the PKC1-mediated pathway and the pheromone response pathway, suggest that Pkc1p regulates a protein kinase cascade in which Bck1p activates a pair of protein kinases, designated Mkk1p and Mkk2p (for MAP kinase-kinase), which in turn activate Mpk1p.

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 Insulin signal transduction in rat small intestine: role of MAP kinases in expression of mucosal hydrolases

2001 ◽  
Vol 280 (2) ◽  
pp. G229-G240 ◽  
Author(s):  
Soheila Marandi ◽  
Nadine De Keyser ◽  
Alain Saliez ◽  
Anne-Sophie Maernoudt ◽  
Etienne Marc Sokal ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Map Kinase ◽  
Insulin Receptor ◽  
Map Kinases ◽  
Kinase Inhibitor ◽  
Protein Kinase B ◽  
Insulin Signal Transduction ◽  
Mitogen Activated Protein ◽  
Src Homology ◽  
Mucosal Mass

The postreceptor events regulating the signal of insulin downstream in rat intestinal cells have not yet been analyzed. Our objectives were to identify the nature of receptor substrates and phosphorylated proteins involved in the signaling of insulin and to investigate the mechanism(s) by which insulin enhances intestinal hydrolases. In response to insulin, the following proteins were rapidly phosphorylated on tyrosine residues: 1) insulin receptor substrates-1 (IRS-1), -2, and -4; 2) phospholipase C-isoenzyme-γ; 3) the Ras-GTPase-activating protein (GAP) associated with Rho GAP and p62Src; 4) the insulin receptor β-subunit; 5) the p85 subunits of phosphatidylinositol 3-kinase (PI 3-kinase); 6) the Src homology 2 α-collagen protein; 7) protein kinase B; 8) mitogen-activated protein (MAP) kinase-1 and -2; and 9) growth receptor-bound protein-2. Compared with controls, insulin enhanced the intestinal activity of MAP kinase-2 and protein kinase B by two- and fivefold, respectively, but did not enhance p70/S6 ribosomal kinase. Administration of an antireceptor antibody or MAP-kinase inhibitor PD-98059 but not a PI 3-kinase inhibitor (wortmannin) to sucklings inhibited the effects of insulin on mucosal mass and enzyme expression. We conclude that normal rat enterocytes express all of the receptor substrates and mediators involved in different insulin signaling pathways and that receptor binding initiates a signal enhancing brush-border membrane hydrolase, which appears to be regulated by the cascade of MAP kinases but not by PI 3-kinase.

scholarly journals Conditional transformation of cells and rapid activation of the mitogen-activated protein kinase cascade by an estradiol-dependent human raf-1 protein kinase.

1993 ◽  
Vol 13 (10) ◽  
pp. 6241-6252 ◽  
Author(s):  
M L Samuels ◽  
M J Weber ◽  
J M Bishop ◽  
M McMahon
Keyword(s):  
Protein Kinase ◽  
Map Kinase ◽  
Map Kinases ◽  
Growth Media ◽  
Hormone Binding ◽  
Map Kinase Cascade ◽  
Mitogen Activated Protein ◽  
Kinase Cascade ◽  
Hormone Binding Domain ◽  
Map Kinase Kinase

We report a strategy for regulating the activity of a cytoplasmic signaling molecule, the protein kinase encoded by raf-1. Retroviruses encoding a gene fusion between an oncogenic form of human p74raf-1 and the hormone-binding domain of the human estrogen receptor (hrafER) were constructed. The fusion protein was nontransforming in the absence of estradiol but could be reversibly activated by the addition or removal of estradiol from the growth media. Activation of hrafER was accompanied in C7 3T3 cells by the rapid, protein synthesis-independent activation of both mitogen-activated protein (MAP) kinase kinase and p42/p44 MAP kinase and by phosphorylation of the resident p74raf-1 protein as demonstrated by decreased electrophoretic mobility. The phosphorylation of p74raf-1 had no effect on the kinase activity of the protein, indicating that mobility shift is an unreliable indicator of p74raf-1 enzymatic activity. Removal of estradiol from the growth media led to a rapid inactivation of the MAP kinase cascade. These results demonstrate that Raf-1 can activate the MAP kinase cascade in vivo, independent of other "upstream" signaling components. Parallel experiments performed with rat1a cells conditionally transformed by hrafER demonstrated activation of MAP kinase kinase in response to estradiol but no subsequent activation of p42/p44 MAP kinases or phosphorylation of p74raf-1. This result suggests that in rat1a cells, p42/p44 MAP kinase activation is not required for Raf-1-mediated oncogenic transformation. Estradiol-dependent activation of p42/p44 MAP kinases and phosphorylation of p74raf-1 was, however, observed in rat1a cells expressing hrafER when the cells were pretreated with okadaic acid. This result suggests that the level of protein phosphatase activity may play a crucial role in the regulation of the MAP kinase cascade. Our results provide the first example of a cytosolic signal transducer being harnessed by fusion to the hormone-binding domain of the estrogen receptor. This conditional system not only will aid the elucidation of the function of Raf-1 but also may be more broadly useful for the construction of conditional forms of other kinases and signaling molecules.

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