scholarly journals Mos stimulates MAP kinase in Xenopus oocytes and activates a MAP kinase kinase in vitro.

1993 ◽  
Vol 13 (4) ◽  
pp. 2546-2553 ◽  
Author(s):  
J Posada ◽  
N Yew ◽  
N G Ahn ◽  
G F Vande Woude ◽  
J A Cooper
Keyword(s):  
Fusion Protein ◽  
Map Kinase ◽  
Xenopus Oocytes ◽  
De Novo ◽  
Rabbit Muscle ◽  
Wild Type ◽  
Kinase Activation ◽  
Mitogen Activated Protein ◽  
Map Kinase Kinase

Several protein kinases, including Mos, maturation-promoting factor (MPF), mitogen-activated protein (MAP) kinase, and MAP kinase kinase (MAPKK), are activated when Xenopus oocytes enter meiosis. De novo synthesis of the Mos protein is required for progesterone-induced meiotic maturation. Recently, bacterially synthesized maltose-binding protein (MBP)-Mos fusion protein was shown to be sufficient to initiate meiosis I and MPF activation in fully grown oocytes in the absence of protein synthesis. Here we show that MAP kinase is rapidly phosphorylated and activated following injection of wild-type, but not kinase-inactive mutant, MBP-Mos into fully grown oocytes. MAP kinase activation by MBP-Mos occurs within 20 min, much more rapidly than in progesterone-treated oocytes. The MBP-Mos fusion protein also activates MPF, but MPF activation does not occur until approximately 2 h after injection. Extracts from oocytes injected with wild-type but not kinase-inactive MBP-Mos contain an activity that can phosphorylate MAP kinase, suggesting that Mos directly or indirectly activates a MAPKK. Furthermore, activated MBP-Mos fusion protein is able to phosphorylate and activate a purified, phosphatase-treated, rabbit muscle MAPKK in vitro. Thus, in oocytes, Mos is an upstream activator of MAP kinase which may function through direct phosphorylation of MAPKK.

Mos stimulates MAP kinase in Xenopus oocytes and activates a MAP kinase kinase in vitro

1993 ◽  
Vol 13 (4) ◽  
pp. 2546-2553
Author(s):  
J Posada ◽  
N Yew ◽  
N G Ahn ◽  
G F Vande Woude ◽  
J A Cooper
Keyword(s):  
Fusion Protein ◽  
Map Kinase ◽  
Xenopus Oocytes ◽  
De Novo ◽  
Rabbit Muscle ◽  
Wild Type ◽  
Kinase Activation ◽  
Mitogen Activated Protein ◽  
Map Kinase Kinase

Several protein kinases, including Mos, maturation-promoting factor (MPF), mitogen-activated protein (MAP) kinase, and MAP kinase kinase (MAPKK), are activated when Xenopus oocytes enter meiosis. De novo synthesis of the Mos protein is required for progesterone-induced meiotic maturation. Recently, bacterially synthesized maltose-binding protein (MBP)-Mos fusion protein was shown to be sufficient to initiate meiosis I and MPF activation in fully grown oocytes in the absence of protein synthesis. Here we show that MAP kinase is rapidly phosphorylated and activated following injection of wild-type, but not kinase-inactive mutant, MBP-Mos into fully grown oocytes. MAP kinase activation by MBP-Mos occurs within 20 min, much more rapidly than in progesterone-treated oocytes. The MBP-Mos fusion protein also activates MPF, but MPF activation does not occur until approximately 2 h after injection. Extracts from oocytes injected with wild-type but not kinase-inactive MBP-Mos contain an activity that can phosphorylate MAP kinase, suggesting that Mos directly or indirectly activates a MAPKK. Furthermore, activated MBP-Mos fusion protein is able to phosphorylate and activate a purified, phosphatase-treated, rabbit muscle MAPKK in vitro. Thus, in oocytes, Mos is an upstream activator of MAP kinase which may function through direct phosphorylation of MAPKK.

Characterization of MAP kinase and PKC isoform and effect of ACE inhibition in hypertrophy in vivo

1999 ◽  
Vol 277 (5) ◽  
pp. H1808-H1816 ◽  
Author(s):  
L. Kim ◽  
T. Lee ◽  
J. Fu ◽  
M. E. Ritchie
Keyword(s):  
Map Kinase ◽  
Gene Activation ◽  
Pressure Overload ◽  
Ace Inhibition ◽  
Binding Activity ◽  
Sham Operation ◽  
Kinase Activation ◽  
Mitogen Activated Protein

Protein kinase C (PKC) and mitogen-activated protein (MAP) kinase activation appear important in conferring hypertrophy in vitro. However, the response of PKC and MAP kinase to stimuli known to induce hypertrophy in vivo has not been determined. We recently demonstrated that pressure-overload hypertrophy induced a transiently transfected gene driven by an hypertrophy responsive enhancer (HRE) through a marked increase in binding activity of its interacting nuclear factor (HRF). These data suggested that the HRE/HRF could serve as a target for evaluating the signal transduction events responsible for hypertrophy in vivo. Accordingly, we characterized MAP kinase and PKC isoform activation, injected HRE driven reporter gene expression, and HRF binding activity in rat hearts subjected to ascending aortic clipping or sham operation in the presence of the angiotensin-converting enzyme (ACE) inhibitor fosinopril, hydralazine, or no treatment. Analyses showed that PKC-ε and MAP kinase were acutely activated following ascending aortic ligature and that fosinopril significantly inhibited but did not completely abrogate PKC-ε and MAP kinase activation. However, fosinopril completely prevented pressure overload-mediated induction of HRE containing constructs and obviated increased HRF binding activity. These results suggest a direct relationship between ACE activity and HRE/HRF-mediated gene activation and imply that PKC-ε and MAP kinase may be involved in transducing this signal.

scholarly journals Identification of a Novel Mitogen-Activated Protein Kinase Kinase Activation Domain Recognized by the Inhibitor PD 184352

2002 ◽  
Vol 22 (21) ◽  
pp. 7593-7602 ◽  
Author(s):  
Amy M. Delaney ◽  
John A. Printen ◽  
Huifen Chen ◽  
Eric B. Fauman ◽  
David T. Dudley
Keyword(s):  
Protein Kinase ◽  
Map Kinase ◽  
Genetic Screen ◽  
Mitogen Activated Protein Kinase ◽  
Erk Signaling ◽  
Signaling Cascade ◽  
Kinase Activation ◽  
Basal Activity ◽  
Mitogen Activated Protein

ABSTRACT Utilizing a genetic screen in the yeast Saccharomyces cerevisiae, we identified a novel autoactivation region in mammalian MEK1 that is involved in binding the specific MEK inhibitor, PD 184352. The genetic screen is possible due to the homology between components of the yeast pheromone response pathway and the eukaryotic Raf-MEK-ERK signaling cascade. Using the FUS1::HIS3 reporter as a functional readout for activation of a reconstituted Raf-MEK-ERK signaling cascade, randomly mutagenized MEK variants that were insensitive to PD 184352 were obtained. Seven single-base-change mutations were identified, five of which mapped to kinase subdomains III and IV of MEK. Of the seven variants, only one, a leucine-to-proline substitution at amino acid 115 (Leu115Pro), was completely insensitive to PD 184352 in vitro (50% inhibitory concentration >10 μM). However, all seven mutants displayed strikingly high basal activity compared to wild-type MEK. Overexpression of the MEK variants in HEK293T cells resulted in an increase in mitogen-activated protein (MAP) kinase phosphorylation, a finding consistent with the elevated basal activity of these constructs. Further, treatment with PD 184352 failed to inhibit Leu115Pro-stimulated MAP kinase activation in HEK293T cells, whereas all other variants had some reduction in phospho-MAP kinase levels. By using cyclic AMP-dependent protein kinase (1CDK) as a template, an MEK homology model was generated, with five of the seven identified residues clustered together, forming a potential hydrophobic binding pocket for PD 184352. Additionally, the model allowed identification of other potential residues that would interact with the inhibitor. Directed mutation of these residues supported this region's involvement with inhibitor binding.

MAP Kinase Activation Is Essential for the Development of Acute Leukemia by MLL Fusion Protein and FLT3 Tyrosine Kinase Mutation.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2067-2067
Author(s):  
Ryoichi Ono ◽  
Hidetoshi Kumgai ◽  
Hideaki Nakajima ◽  
Yukio Tonozuka ◽  
Ai Hishiya ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Acute Leukemia ◽  
Tyrosine Kinase ◽  
Map Kinase ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Oncogenic Potential ◽  
Kinase Activation

Abstract MLL (mixed lineage leukemia)-fusion-mediated acute leukemia in infants has frequently been found to have FLT3 overexpression or tyrosine kinase domain (TKD) mutation. We have recently developed both in vitro and in vivo leukemogenesis models where MLL fusion proteins cooperate with another FLT3 mutant, internal tandem duplication (ITD). However, little has been clear about molecular mechanism of the cooperativity between MLL fusion protein and FLT3 mutants, not only FLT3-ITD but also FLT3-TKD. The present study demonstrates that MLL-SEPT6 fusion protein associated with infantile acute leukemia cooperates in vitro with FLT3-TKD mainly through activated MAP kinase pathway, while MLL-SEPT6 cooperates with FLT3-ITD mainly through activated STAT5 pathway. We first found that the interleukin (IL) -3 dependent murine hematopoietic cell line immortalized by MLL-SEPT6, named HF6, was transformed to grow without IL-3 by forced expression of FLT3 mutants which activated MAP kinase and STAT5, as shown in IL-3 dependent murine pro-B Ba/F3 cells. A dominant negative mutant of STAT5A suppressed the proliferation of the HF6 cells transformed by FLT3-ITD more effectively than that by FLT3-TKD, similarly to the transformed Ba/F3 cells. However, unlike the transformed Ba/F3 cells, the proliferation of transformed HF6 cells was suppressed with an MEK inhibitor more effectively in the HF6 cells transformed by FLT3-TKD than by FLT3-ITD. These results suggested that, in the transformation of HF6 cells, MAP kinase activation is more critical for FLT3-TKD than STAT5, while STAT5 activation is more critical for FLT3-ITD than MAP kinase. Furthermore, HF6 cells became IL-3 independent by direct activation of Raf-MAP kinase, while Ba/F3 cells did not. In contrast, a constitutively active mutant of STAT5 enabled, not HF6, but Ba/F3 cells to grow without IL-3, thus suggesting the essential role of activation of the Raf-MAP kinase cascade in the growth of the cells expressing MLL fusion protein. We next examined the oncogenic potential of MLL-SEPT6 and either of the FLT3 mutants by leukemogenesis assays in vivo using bone marrow transplantation. Interestingly, FLT3-TKD cooperated with MLL-SEPT6 in vivo to induce acute leukemia in mice rapidly (26±5.5 days), similarly to FLT3-ITD (27±5.1 days), although the individual oncogenic potential of FLT3-TKD leading to T-cell lymphoma (119±11 days), was much weaker than that of FLT3-ITD leading to myeloproliferative disease (56±16 days). Taken together, these results suggest that MLL fusion protein can induce human acute leukemia in concert with MAP kinase activation through secondary genetic events including FLT3-TKD mutation or other mechanisms which activate MAP kinase.

scholarly journals Role of receptor desensitization, phosphatase induction and intracellular cyclic AMP in the termination of mitogen-activated protein kinase activity in UTP-stimulated EAhy 926 endothelial cells

1996 ◽  
Vol 315 (2) ◽  
pp. 563-569 ◽  
Author(s):  
Anne GRAHAM ◽  
Angela McLEES ◽  
Kevin MALARKEY ◽  
Gwyn W. GOULD ◽  
Robin PLEVIN
Keyword(s):  
Endothelial Cells ◽  
Protein Synthesis ◽  
Map Kinase ◽  
Kinase Activity ◽  
Mitogen Activated Protein Kinase ◽  
Receptor Desensitization ◽  
Kinase Activation ◽  
Map Kinase Phosphatase ◽  
Mitogen Activated Protein ◽  
Map Kinase Kinase

We have investigated the mechanisms that bring about the termination of mitogen-activated protein kinase (MAP kinase) activation in response to UTP in EAhy 926 endothelial cells. UTP-stimulated MAP kinase activity was transient, returning to basal values by 60 min. At this time MAP kinase activation was desensitized; re-application of UTP did not further activate MAP kinase, full re-activation of MAP kinase being only apparent after a 1–2 h wash period. However, activation of MAP kinase by UTP could be sustained beyond 60 min by preincubation of the cells with the protein synthesis inhibitor cycloheximide. UTP also stimulated expression of MAP kinase phosphatase-1 and this was abolished after pretreatment with cycloheximide. Pretreatment of cells with forskolin abolished the initial activation of MAP kinase kinase or c-Raf-1 by UTP, but only affected MAP kinase activity during prolonged stimulation. The effect of forskolin on prolonged MAP kinase activation was also prevented by cycloheximide. These results suggest that the termination of MAP kinase activity in response to UTP involves a number of interacting mechanisms including receptor desensitization and the induction of a phosphatase. However, several pieces of evidence do not support a major role for MAP kinase phosphatase-1 in termination of the MAP kinase signal. Raising intracellular cyclic AMP may also be involved but only after an initial protein-synthesis step and by a mechanism that does not involve the inactivation of c-Raf-1 or MAP kinase kinase.

scholarly journals Spred-1 Negatively Regulates Interleukin-3-mediated ERK/Mitogen-activated Protein (MAP) Kinase Activation in Hematopoietic Cells

2004 ◽  
Vol 279 (50) ◽  
pp. 52543-52551 ◽  
Author(s):  
Atsushi Nonami ◽  
Reiko Kato ◽  
Koji Taniguchi ◽  
Daigo Yoshiga ◽  
Takaharu Taketomi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mast Cells ◽  
Map Kinase ◽  
Cell Lines ◽  
Colony Formation ◽  
Wild Type ◽  
Kinase Activation ◽  
Erk Activation ◽  
Protein Map ◽  
Mitogen Activated Protein

Sprouty/Spred family proteins have been identified as negative regulators of growth factor-induced ERK/mitogen-activated protein (MAP) kinase activation. However, it has not been clarified whether these proteins regulate cytokine-induced ERK activity. We found that Spred-1 is highly expressed in interleukin-3 (IL-3)-dependent hematopoietic cell lines and bone marrow-derived mast cells. To investigate the roles of Spred-1 in hematopoiesis, we expressed wild-type Spred-1 and a dominant negative form of Spred-1, ΔC-Spred, in IL-3- and stem cell factor (SCF)-dependent cell lines as well as hematopoietic progenitor cells from mouse bone marrow by retrovirus gene transfer. In IL-3-dependent Ba/F3 cells expressing c-kit, forced expression of Spred-1 resulted in a reduced proliferation rate and ERK activation in response to not only SCF but also IL-3. In contrast, ΔC-Spred augmented IL-3-induced cell proliferation and ERK activation. Wild-type Spred-1 inhibited colony formation of bone marrow cells in the presence of cytokines, whereas ΔC-Spred-1 expression enhanced colony formation. Augmentation of ERK activation and proliferation in response to IL-3 was also observed in Spred-1-deficient bone marrow-derived mast cells. These data suggest that Spred-1 negatively regulates hematopoiesis by suppressing not only SCF-induced but also IL-3-induced ERK activation.

Activation of MAP kinase kinase (MEK) and Ras by cholecystokinin in rat pancreatic acini

1995 ◽  
Vol 268 (6) ◽  
pp. G1060-G1065 ◽  
Author(s):  
R. D. Duan ◽  
C. F. Zheng ◽  
K. L. Guan ◽  
J. A. Williams
Keyword(s):  
Map Kinase ◽  
Threshold Concentration ◽  
Maximal Effect ◽  
Glutathione S Transferase ◽  
Pancreatic Acini ◽  
Kinase Activation ◽  
Slow Decline ◽  
Significant Stimulation ◽  
Mitogen Activated Protein ◽  
Map Kinase Kinase

Cholecystokinin (CCK) has recently been shown to activate mitogen-activated protein (MAP) kinase in rat pancreatic acini [Duan and Williams, Am. J. Physiol. 267 (Gastrointest. Liver Physiol. 30): G401-G408, 1994]. To evaluate the mechanism of MAP kinase activation, we studied the effects of CCK on MAP kinase kinase (MEK) in rat pancreatic acini. Two forms of MEK were identified by immunoblotting, using antibodies specific to MEK1 and MEK2. MEK activity in acinar extracts and after immunoprecipitation with anti-MEK was detected using a recombinant fusion protein, glutathione S-transferase-MAP kinase, as a substrate. MEK activity rapidly increased after stimulation of acini by CCK, with significant stimulation at 1 min and a maximal effect at 5 min, followed by a slow decline to slightly above control levels after 30 min. The threshold concentration of CCK was approximately 10 pM, and the maximal effect was induced by 1 nM CCK, which increased MEK activity by 120%. In addition to CCK, bombesin and carbachol, but not secretin or vasoactive intestinal peptide, enhanced MEK activity. Phorbol ester mimicked the effect of CCK, whereas ionomycin and thapsigargin failed to activate MEK. We further studied the activation of Ras, an important component leading to activation of MEK by growth factors. Ras in acini was immunoprecipitated and identified by Western blotting. CCK and 12-O-tetradecanoylphorbol-13-acetate stimulated the incorporation of GTP into Ras, a requirement for its activation, reaching a maximum at 10 min of approximately 120% over control. In conclusion, the activation of MAP kinase by CCK can be explained by activation of MEK and may involve the activation of Ras by a protein kinase C-dependent mechanism.

scholarly journals Phosphorylation of the Human Cytomegalovirus 86-Kilodalton Immediate-Early Protein IE2

1998 ◽  
Vol 72 (7) ◽  
pp. 5481-5492 ◽  
Author(s):  
Noam Y. Harel ◽  
James C. Alwine
Keyword(s):  
Map Kinase ◽  
Human Cytomegalovirus ◽  
Transcriptional Activation ◽  
Immediate Early ◽  
Wild Type ◽  
Independent Manner ◽  
Early Protein ◽  
Mitogen Activated Protein

ABSTRACT We have investigated the phosphorylation state of the human cytomegalovirus 86-kDa immediate-early (IE) protein IEP86 from transfected and infected cells. We show that multiple domains of IEP86 are phosphorylated by cellular kinases, both in vitro and in vivo. Our data suggest that serum-inducible kinases play a significant role in cell-mediated IE protein phosphorylation and that a member of the mitogen-activated protein (MAP) kinase (MAPK) family, extracellular regulated kinase 2 (ERK2), phosphorylates several domains of IEP86 in vitro. Alanine substitution mutagenesis was performed on specific serines or threonines (T27, S144, T233/S234, and T555) found in consensus MAP kinase motifs. Analysis of these mutations showed that T27 and T233/S234 are the major sites for serum-inducible kinases and are the major ERK2 sites in vitro. S144 appeared to be phosphorylated in a serum-independent manner in vitro. All of the mutations except T555 eliminated specific phosphorylation in vivo. In transient transfection analyses, IEP86 isoforms containing mutations in S144 and, especially, T233/S234displayed increased transcriptional activation relative to the wild type, suggesting that phosphorylation at these sites in wild-type IEP86 may result in reduction of its transcriptional activation ability.

scholarly journals Mitogen-activated protein kinase (MAP kinase), MAP kinase kinase and c-Mos stimulate glucose transport in Xenopus oocytes

1993 ◽  
Vol 295 (2) ◽  
pp. 351-355 ◽  
Author(s):  
N W Merrall ◽  
R J Plevin ◽  
D Stokoe ◽  
P Cohen ◽  
A R Nebreda ◽  
...  
Keyword(s):  
Growth Factors ◽  
Protein Kinase ◽  
Map Kinase ◽  
Glucose Transport ◽  
Xenopus Oocytes ◽  
Mitogen Activated Protein Kinase ◽  
Sugar Uptake ◽  
Mitogen Activated Protein ◽  
General Response ◽  
Map Kinase Kinase

Mitogens and growth factors acutely stimulate glucose transport in all cells to supply energy for their growth and division, but little is known about the signalling mechanism by which these agonists promote sugar uptake. Here we show that the transport of deoxyglucose and 3-O-methylglucose into Xenopus laevis oocytes is stimulated about 2.5-fold when mitogen-activated protein kinase (MAP kinase) is microinjected into these oocytes. We also demonstrate that microinjection of the proto-oncogene product c-Mos (an activator of MAP kinase kinase, which activates MAP kinase in Xenopus oocytes), and purified MAP kinase kinase produce similar increases in deoxyglucose transport. Since the activation of MAP kinase is a general response to almost all mitogens and growth factors, we propose that one of its downstream effects is the stimulation of glucose-transport activity.

scholarly journals Mitogen-activated protein kinase activation down-regulates a mechanism that inactivates cyclin B-cdc2 kinase in G2-arrested oocytes.

1997 ◽  
Vol 8 (2) ◽  
pp. 249-261 ◽  
Author(s):  
A Abrieu ◽  
M Dorée ◽  
A Picard
Keyword(s):  
Map Kinase ◽  
Xenopus Oocytes ◽  
Cyclin B ◽  
Mitogen Activated Protein Kinase ◽  
Cdc2 Kinase ◽  
Hormonal Stimulation ◽  
Germinal Vesicle Breakdown ◽  
Kinase Activation ◽  
Map Kinase Phosphatase ◽  
Mitogen Activated Protein

The G2 arrest of oocytes from frogs, clams, and starfish requires that preformed cyclin B-cdc2 complexes [prematuration-promoting factor (MPF)] be kept in an inactive form that is largely due to inhibitory phosphorylation of this pre-MPF. We have investigated the role of mitogen-activated protein (MAP) kinase in the activation of this pre-MPF. The cytoplasm of both frog and starfish oocytes contains an activity that can rapidly inactivate injected MPF. When the MAP kinase of G2-arrested starfish or Xenopus oocytes was prematurely activated by microinjection of c-mos or Ste-11 delta N fusion proteins, the rate and extent of MPF inactivation was much reduced. Both effects were suppressed by expression of the specific MAP kinase phosphatase Pyst 1. These results show that MAP kinase down-regulates a mechanism that inactivates cyclin B-cdc2 kinase in Xenopus oocytes. In starfish oocytes, however, MAP kinase activation occurs only after germinal vesicle breakdown, much after MPF activation. In this case, down-regulation of the cyclin B-cdc2 inhibiting pathway is a sensitive response to hormonal stimulation that does not require MAP kinase activation.

Sign in / Sign up

Export Citation Format

Share Document