scholarly journals 3pK, a novel mitogen-activated protein (MAP) kinase-activated protein kinase, is targeted by three MAP kinase pathways.

1996 ◽  
Vol 16 (12) ◽  
pp. 6687-6697 ◽  
Author(s):  
S Ludwig ◽  
K Engel ◽  
A Hoffmeyer ◽  
G Sithanandam ◽  
B Neufeld ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Map Kinase ◽  
Stress Responses ◽  
Specific Inhibitor ◽  
Mitogen Activated Protein Kinase ◽  
Mapk Cascades ◽  
Mitogen Activated Protein ◽  
Stimulation Of

Recently we have identified a mitogen-activated protein kinase (MAPK)-activated protein kinase, named 3pK (G. Sithanandam, F. Latif, U. Smola, R. A. Bernal, F.-M. Duh, H. Li, I. Kuzmin, V. Wixler, L. Geil, S. Shresta, P. A. Lloyd, S. Bader, Y. Sekido, K. D. Tartof, V. I. Kashuba, E. R. Zabarovsky, M. Dean, G. Klein, B. Zbar, M. I. Lerman, J. D. Minna, U. R. Rapp, and A. Allikmets, Mol. Cell. Biol. 16:868-876, 1996). In vitro characterization of the kinase revealed that 3pK is activated by ERK. It was further shown that 3pK is phosphorylated in vivo after stimulation of cells with serum. However, the in vivo relevance of this observation in terms of involvement of the Raf/MEK/ERK cascade has not been established. Here we show that 3pK is activated in vivo by the growth inducers serum and tetradecanoyl phorbol acetate in promyelocytic HL60 cells and transiently transfected embryonic kidney 293 cells. Activation of 3pK was Raf dependent and was mediated by the Raf/MEK/ERK kinase cascade. 3pK was also shown to be activated after stress stimulation of cells. In vitro studies with recombinant proteins demonstrate that in addition to ERK, members of other subgroups of the MAPK family, namely, p38RK and Jun-N-terminal kinases/stress-activated protein kinases, were also able to phosphorylate and activate 3pK. Cotransfection experiments as well as the use of a specific inhibitor of p38RK showed that these in vitro upstream activators also function in vivo, identifying 3pK as the first kinase to be activated through all three MAPK cascades. Thus, 3pK is a novel convergence point of different MAPK pathways and could function as an integrative element of signaling in both mitogen and stress responses.

scholarly journals Phosphorylation of the c-Fos transrepression domain by mitogen-activated protein kinase and 90-kDa ribosomal S6 kinase

1993 ◽  
Vol 90 (23) ◽  
pp. 10952-10956 ◽  
Author(s):  
R H Chen ◽  
C Abate ◽  
J Blenis
Keyword(s):  
Protein Kinase ◽  
Map Kinase ◽  
Mitogen Activated Protein Kinase ◽  
S6 Kinase ◽  
Downstream Signaling ◽  
C Terminus ◽  
Mitogen Activated Protein ◽  
Ribosomal S6 Kinase

Phosphorylation of the C terminus of c-Fos has been implicated in serum response element-mediated repression of c-fos transcription after its induction by serum growth factors. The growth-regulated enzymes responsible for this phosphorylation in early G1 phase of the cell cycle and the sites of phosphorylation have not been identified. We now provide evidence that two growth-regulated, nucleus- and cytoplasm-localized protein kinases, 90-kDa ribosomal S6 kinase (RSK) and mitogen-activated protein kinase (MAP kinase), contribute to the serum-induced phosphorylation of c-Fos. The major phosphopeptides derived from biosynthetically labeled c-Fos correspond to phosphopeptides generated after phosphorylation of c-Fos in vitro with both RSK and MAP kinase. The phosphorylation sites identified for RSK (Ser-362) and MAP kinase (Ser-374) are in the transrepression domain. Cooperative phosphorylation at these sites by both enzymes was observed in vitro and reflected in vivo by the predominance of the peptide phosphorylated on both sites, as opposed to singly phosphorylated peptides. This study suggests a role for nuclear RSK and MAP kinase in modulating newly synthesized c-Fos phosphorylation and downstream signaling.

scholarly journals Human mitogen-activated protein kinase kinase kinase mediates the stress-induced activation of mitogen-activated protein kinase cascades

1998 ◽  
Vol 336 (3) ◽  
pp. 599-609 ◽  
Author(s):  
Po-Ying CHAN-HUI ◽  
Robert WEAVER
Keyword(s):  
Protein Kinase ◽  
Sequence Similarity ◽  
K562 Cells ◽  
Mitogen Activated Protein Kinase ◽  
Hek293 Cells ◽  
Mapk Cascades ◽  
Mitogen Activated Protein ◽  
Factor Α

The mitogen-activated protein kinase (MAPK) cascades represent one of the important signalling mechanisms in response to environmental stimuli. We report the identification of a human MAPK kinase kinase, MAPKKK4, via sequence similarity with other MAPKKKs. When truncated MAPKKK4 (ΔMAPKKK4) was overexpressed in HEK293 cells, it was constitutively active and induced the activation of endogenous p38α, c-Jun N-terminal kinase (JNK)1/2 and extracellular signal-regulated kinase (ERK)2 in vivo. Kinase-inactive ΔMAPKKK4 partly inhibited the activation of p38α, JNK1/2 and ERK2 induced by stress, tumour necrosis factor α or epidermal growth factor, suggesting that MAPKKK4 might be physiologically involved in all three MAPK cascades. Co-expressed MAP kinase kinase (MKK)-1, MKK-4, MKK-3 and MKK-6 were activated in vivo by ΔMAPKKK4. All of the above MKKs purified from Escherichia coli were phosphorylated and activated by ΔMAPKKK4 immunoprecipitates in vitro. When expressed by lower plasmid doses, ΔMAPKKK4 preferentially activated MKK-3 and p38α in vivo. Overexpression of ΔMAPKKK4 did not activate the NF-κB pathway. Immunoprecipitation of endogenous MAPKKK4 by specific antibodies showed that MAPKKK4 was activated after the treatment of K562 cells with various stress conditions. As a broadly distributed kinase, MAPKKK4 might serve as a stress responder. MAPKKK4 is 91% identical with the recently described murine MEKK-4β and might be its human homologue. It is also identical with the recently cloned human MAP three kinase 1 except for the lack of an internal sequence homologous to the murine MEKK-4α isoform. Differences in the reported functional activities of the three kinases are discussed.

An Inhibitor of p38 Mitogen-Activated Protein Kinase Abrogates Procoagulant and Proinflammatory Effects of Antiphospholipid Antibodies In Vivo.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 136-136
Author(s):  
Silvia S. Pierangeli ◽  
Mariano E. Vega-Ostertag ◽  
Xiaowei Liu
Keyword(s):  
Protein Kinase ◽  
Carotid Artery ◽  
P38 Mapk ◽  
Specific Inhibitor ◽  
Mitogen Activated Protein Kinase ◽  
Peritoneal Cells ◽  
Mitogen Activated Protein ◽  
Pre Treatment

Abstract Background: Activation of p38 mitogen-activated protein kinase (p38 MAPK) has been shown to play a fundamental role in antiphospholipid-induced up regulation of tissue factor (TF) expression and function in monocytes and in endothelial cells (ECs) and increased expression of intercellular adhesion molecule -1 (ICAM-1) in vitro. Those effects correlate with the thrombogenic and pro-inflammatory effects of aPL in vivo. However, It is not clear whether aPL-induceTF in vivo. Methods: To examine this question, we treated CD1 male mice, in groups of 4, with IgG from 3 patients with Antiphospholipid Syndrome (IgG-APS) or with control IgG from healthy controls (IgG-NHS), twice. Seventy-two hours after the first injection, the adhesion of leukocytes per capillary venule (#WBC) to EC in cremaster muscle (as an indication of EC activation in vivo), as well the size of an induced thrombus in the femoral vein of the mice were examined. Some mice were infused i.p. with 25 mg/kg of SB203580 (a p38 MAPK-specific inhibitor) 30 minutes prior to the each IgG-APS injection. TF activity was determined using a chromogenic assay that measures the conversion of factor X into Factor Xa, in homogenates of carotid artery, and in peritoneal cells of mice treated with IgG-APS or with IgG-NHS. Expression of TF and ICAM-1 was determined by cyto-ELISA on cultured HUVECs after treatment of the cells with IgG-APS or with IgG-NHS. Results: At the time of the surgical procedures, the mean aCL titer in the sera of the mice injected with IgG-APS was 73 ± 34 GPL. In vivo, IgG-APS increased significantly the #WBC adhering to EC, when compared to control mice (5.25 ± 0.96 vs 1.85 ± 0.72) and these effects were significantly reduced (2.1 ± 0.74), when mice were pre-treated with SB203580. IgG-APS increased significantly the thrombus size when compared to IgG-NHS-treated mice (3189 ± 558 μm2 vs 1468 ± 401 μm2) and SB203580 inhibited this effect by 65%. Treatment of the mice with IgG-APS also induced significantly increased TF function in peritoneal cells and in homogenates of carotid artery when compared to IgG-NHS-treated mice (17.5 ±11.1 pM vs. 0.8 ±0.2 pM and 8.31 ± 1.59 vs 0.69 ± 0.03, respectively). Pre-treatment of the mice with SB203580 abrogated completely those effects (0.61 ± 0.06 pM in peritoneal cells and 0.75 ± 0.28 pM in carotid artery preparations of mice treated with IgG-APS). Significant expression of TF and ICAM-1 was observed in vitro when HUVECs were treated with any of the three IgG-APS. TF upregulation and ICAM-1 expression were significantly reduced by pre-treatment of the cells with SB203580 (49–97% for TF and 25–69% for ICAM-1). Conclusions: The data show that IgG-APS up regulates TF function in vivo, and this correlates with an in vivo pro-inflammatory and pro-thrombotic effect. Importantly, those effects were abrogated in vivo by a p38 MAPK specific inhibitor. These findings may be important in designing new modalities of targeted therapies to treat thrombosis in patients with APS.

scholarly journals Mitotic Phosphorylation of Golgi Reassembly Stacking Protein 55 by Mitogen-activated Protein Kinase ERK2

2001 ◽  
Vol 12 (6) ◽  
pp. 1811-1817 ◽  
Author(s):  
Stephen A. Jesch ◽  
Timothy S. Lewis ◽  
Natalie G. Ahn ◽  
Adam D. Linstedt
Keyword(s):  
Protein Kinase ◽  
Specific Inhibitor ◽  
Mitogen Activated Protein Kinase ◽  
Erk Pathway ◽  
Erk Activation ◽  
Mitogen Activated Protein ◽  
Mitotic Phosphorylation

The role of the mitogen-activated protein kinase kinase (MKK)/extracellular-activated protein kinase (ERK) pathway in mitotic Golgi disassembly is controversial, in part because Golgi-localized targets have not been identified. We observed that Golgi reassembly stacking protein 55 (GRASP55) was phosphorylated in mitotic cells and extracts, generating a mitosis-specific phospho-epitope recognized by the MPM2 mAb. This phosphorylation was prevented by mutation of ERK consensus sites in GRASP55. GRASP55 mitotic phosphorylation was significantly reduced, both in vitro and in vivo, by treatment with U0126, a potent and specific inhibitor of MKK and thus ERK activation. Furthermore, ERK2 directly phosphorylated GRASP55 on the same residues that generated the MPM2 phospho-epitope. These results are the first demonstration of GRASP55 mitotic phosphorylation and indicate that the MKK/ERK pathway directly phosphorylates the Golgi during mitosis.

scholarly journals Identification of Functional MKK3/6 and MEK1/2 Homologs from Echinococcus granulosus and Investigation of Protoscolecidal Activity of Mitogen-Activated Protein Kinase Signaling Pathway Inhibitors In Vitro and In Vivo

2018 ◽  
Vol 63 (1) ◽  
Author(s):  
Chuanshan Zhang ◽  
Jing Li ◽  
Tuerganaili Aji ◽  
Liang Li ◽  
Xiaojuan Bi ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Echinococcus Granulosus ◽  
Sensu Stricto ◽  
Mitogen Activated Protein Kinase ◽  
Content Type ◽  
Mapk Cascades ◽  
Significant Difference ◽  
Mitogen Activated Protein

ABSTRACT Cystic echinococcosis is a zoonosis caused by the larval stage of Echinococcus granulosus sensu lato. There is an urgent need to develop new drugs for the treatment of this disease. In this study, we identified two new members of mitogen-activated protein kinase (MAPK) cascades, MKK3/6 and MEK1/2 homologs (termed EgMKK1 and EgMKK2, respectively), from E. granulosus sensu stricto. Both EgMKK1 and EgMKK2 were expressed at the larval stages. As shown by yeast two-hybrid and coimmunoprecipitation analyses, EgMKK1 interacted with the previously identified Egp38 protein but not with EgERK. EgMKK2, on the other hand, interacted with EgERK. In addition, EgMKK1 and EgMKK2 displayed kinase activity toward the substrate myelin basic protein. When sorafenib tosylate, PD184352, or U0126-ethanol (EtOH) was added to the medium for in vitro culture of E. granulosus protoscoleces (PSCs) or cysts, an inhibitory and cytolytic effect was observed via suppressed phosphorylation of EgMKKs and EgERK. Nonviability of PSCs treated with sorafenib tosylate or U0126-EtOH, and not with PD184352, was confirmed through bioassays, i.e., inoculation of treated and untreated protoscoleces into mice. In vivo treatment of E. granulosus sensu stricto-infected mice with sorafenib tosylate or U0126-EtOH for 4 weeks demonstrated a reduction in parasite weight, but the results did not show a significant difference. In conclusion, the MAPK cascades were identified as new targets for drug development, and E. granulosus was efficiently inhibited by their inhibitors in vitro. The translation of these findings into in vivo efficacy requires further adjustment of treatment regimens using sorafenib tosylate or, possibly, other kinase inhibitors.

Role of IRS-1-GRB-2 complexes in insulin signaling

1994 ◽  
Vol 14 (6) ◽  
pp. 3577-3587
Author(s):  
M G Myers ◽  
L M Wang ◽  
X J Sun ◽  
Y Zhang ◽  
L Yenush ◽  
...  
Keyword(s):  
Map Kinase ◽  
Insulin Receptor ◽  
Insulin Receptors ◽  
Growth Factor Signaling ◽  
Insulin Stimulation ◽  
Mitogen Activated Protein ◽  
D Cells ◽  
Stimulation Of

GRB-2 is a small SH2- and SH3 domain-containing adapter protein that associates with the mammalian SOS homolog to regulate p21ras during growth factor signaling. During insulin stimulation, GRB-2 binds to the phosphorylated Y895VNI motif of IRS-1. Substitution of Tyr-895 with phenylalanine (IRS-1F-895) prevented the IRS-1-GRB-2 association in vivo and in vitro. The myeloid progenitor cell line, 32-D, is insensitive to insulin because it contains few insulin receptors and no IRS-1. Coexpression of IRS-1 or IRS-1F-895 with the insulin receptor was required for insulin-stimulated mitogenesis in 32-D cells, while expression of the insulin receptor alone was sufficient to mediate insulin-stimulated tyrosine phosphorylation of Shc and activation of p21ras and mitogen-activated protein (MAP) kinase. The Shc-GRB-2 complex formed during insulin stimulation is a possible mediator of p21ras and MAP kinase activation in IRS-1-deficient 32-D cells. Interestingly, IRS-1, but not IRS-1F-895, enhanced the stimulation of MAP kinase by insulin in 32-D cells expressing insulin receptors. Thus, IRS-1 contributes to the stimulation of MAP kinase by insulin, probably through formation of the IRS-1-GRB-2 complex at Tyr-895. Our results suggest that the Shc-GRB-2 complex and the activation of p21ras-dependent signaling pathways, including MAP kinase, are insufficient for insulin-stimulated mitogenesis and that the essential function(s) of IRS-1 in proliferative signaling is largely unrelated to IRS-1-GRB-2 complex formation.

scholarly journals FGIN-1-27 Inhibits Melanogenesis by Regulating Protein Kinase A/cAMP-Responsive Element-Binding, Protein Kinase C-β, and Mitogen-Activated Protein Kinase Pathways

2020 ◽  
Vol 11 ◽  
Author(s):  
Jinpeng Lv ◽  
Songzhou Jiang ◽  
Ying Yang ◽  
Ximei Zhang ◽  
Rongyin Gao ◽  
...  
Keyword(s):  
Protein Kinase ◽  
The Body ◽  
Tyrosinase Activity ◽  
Mitogen Activated Protein Kinase ◽  
Mushroom Tyrosinase ◽  
Mitogen Activated Protein ◽  
Element Binding Protein ◽  
Responsive Element

FGIN-1-27 is a synthetic mitochondrial diazepam binding inhibitor receptor (MDR) agonist that has demonstrated pro-apoptotic, anti-anxiety, and steroidogenic activity in various studies. Here we report, for the first time, the anti-melanogenic efficacy of FGIN-1-27 in vitro and in vivo. FGIN-1-27 significantly inhibited basal and α-melanocyte-stimulating hormone (α-MSH)-, 1-Oleoyl-2-acetyl-sn-glycerol (OAG)- and Endothelin-1 (ET-1)-induced melanogenesis without cellular toxicity. Mushroom tyrosinase activity assay showed that FGIN-1-27 did not directly inhibit tyrosinase activity, which suggested that FGIN-1-27 was not a direct inhibitor of tyrosinase. Although it was not capable of modulating the catalytic activity of mushroom tyrosinase in vitro, FGIN-1-27 downregulated the expression levels of key proteins that function in melanogenesis. FGIN-1-27 played these functions mainly by suppressing the PKA/CREB, PKC-β, and MAPK pathways. Once inactivated, it decreased the expression of MITF, tyrosinase, TRP-1, TRP-2, and inhibited the tyrosinase activity, finally inhibiting melanogenesis. During in vivo experiments, FGIN-1-27 inhibited the body pigmentation of zebrafish and reduced UVB-induced hyperpigmentation in guinea pig skin, but not a reduction of numbers of melanocytes. Our findings indicated that FGIN-1-27 exhibited no cytotoxicity and inhibited melanogenesis in both in vitro and in vivo models. It may prove quite useful as a safer skin-whitening agent.

scholarly journals A Redox-Sensitive Thiol in Wis1 Modulates the Fission Yeast Mitogen-Activated Protein Kinase Response to H2O2 and Is the Target of a Small Molecule

2020 ◽  
Vol 40 (7) ◽  
Author(s):  
Johanna J. Sjölander ◽  
Agata Tarczykowska ◽  
Cecilia Picazo ◽  
Itziar Cossio ◽  
Itedale Namro Redwan ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Fission Yeast ◽  
Inhibitory Effect ◽  
Mitogen Activated Protein Kinase ◽  
Activation Loop ◽  
Content Type ◽  
Kinase Activation ◽  
Mitogen Activated Protein

ABSTRACT Oxidation of a highly conserved cysteine (Cys) residue located in the kinase activation loop of mitogen-activated protein kinase kinases (MAPKK) inactivates mammalian MKK6. This residue is conserved in the fission yeast Schizosaccharomyces pombe MAPKK Wis1, which belongs to the H2O2-responsive MAPK Sty1 pathway. Here, we show that H2O2 reversibly inactivates Wis1 through this residue (C458) in vitro. We found that C458 is oxidized in vivo and that serine replacement of this residue significantly enhances Wis1 activation upon addition of H2O2. The allosteric MAPKK inhibitor INR119, which binds in a pocket next to the activation loop and C458, prevented the inhibition of Wis1 by H2O2 in vitro and significantly increased Wis1 activation by low levels of H2O2 in vivo. We propose that oxidation of C458 inhibits Wis1 and that INR119 cancels out this inhibitory effect by binding close to this residue. Kinase inhibition through the oxidation of a conserved Cys residue in MKK6 (C196) is thus conserved in the S. pombe MAPKK Wis1.

Sign in / Sign up

Export Citation Format

Share Document