Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.12.2 (MEK)
18,161 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Raf-1 is a Ser/Thr protein kinase that is involved in regulation of proliferation, differentiation, and apoptosis. Recently, we and others showed that Raf-1 is not only activated in mitogenic pathways leading to cell cycle entry but also during mitosis. Transient expression studies in COS cells now demonstrate that, in contrast to growth factor-dependent activation of Raf-1, mitotic activation of Raf-1 is Ras-independent. Dominant negative RasS17N does not interfere with mitotic activation of Raf-1, whereas epidermal growth factor-dependent stimulation of Raf-1 is inhibited. In addition, the Raf-1 mutant RafR89L, which cannot bind to activated Ras, is still stimulated in mitotic cells. Mitotic activation of Raf-1 seems to be partially dependent on tyrosine phosphorylation since the kinase activity of the Raf mutant RafYY340/341FF, which can no longer be activated by Src, is reduced in mitotic cells. Surprisingly, cell fractionation experiments showed that mitotic-activated Raf-1 is predominantly located in the cytoplasm in contrast to the mitogen-activated Raf-1 that is bound to the plasma membrane. In addition, mitotic activation of Raf-1 does not lead to stimulation of the mitogen-activated protein kinase kinase (MAPKK or MEK) and the extracellular signal-regulated protein kinase (ERK). These data demonstrate that in mitotic cells a Ras-independent mechanism results in a cytoplasmic active Raf-1 kinase which does not signal via the MEK/ERK pathway. These data demonstrate that in mitotic cells a Ras-independent mechanism results in a cytoplasmic active Raf-1 kinase which does not signal via the MEK/ERK pathway.
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PMID:Mitotic Raf-1 is stimulated independently of Ras and is active in the cytoplasm. 972 31

p38 MAPK is a Ser/Thr protein kinase activated by various inflammatory cytokines and a variety of stress stimuli. It is involved in many physiological processes, including the production of inflammatory cytokines. We have previously reported the design and synthesis of a series of pyridinylimidazole compounds that are selective inhibitors of p38 MAPK. These compounds, exemplified by SB 203580, are exceptionally effective in cell-based assays, including the inhibition of inflammatory cytokine production. SB 203580 is widely used as a tool to dissect the role of p38 MAPK in various physiological processes. It has previously been established that SB 203580 acts primarily to block the catalytic activity of p38 MAPK. However, it has been suggested that in cells, the compounds could also inhibit p38 MAPK activation by virtue of their ability to bind to the inactive enzyme. We undertook careful studies to definitively demonstrate that treatment with SB 203580 had no effect on Thr(180) and Tyr(182) phosphorylation, and hence activation of p38 in vivo. SB 203580, however, potently inhibited the activity of p38 MAPK as demonstrated by the inhibition of the activation of MAPKAP K2, a specific physiological substrate of p38 MAPK. This was observed regardless of stimuli or cell type. Identical results were obtained when the p38 MAPK cascade was partially reconstituted in vitro. Thus, our data clearly indicate that SB 203580 specifically inhibits the activity of p38 MAPK but not its activation by upstream MAPKK.
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PMID:Pyridinylimidazole compound SB 203580 inhibits the activity but not the activation of p38 mitogen-activated protein kinase. 1051 65

The c-Jun N-terminal kinase (JNK) group of mitogen-activated protein (MAP) kinases are activated by pleiotropic signals including environmental stresses, growth factors, and hormones. JNK-interacting protein 1 (JIP1) is a scaffold protein that assembles and facilitates the activation of the mixed lineage kinase-dependent JNK module and also establishes an interaction with beta-amyloid precursor protein that has been partially characterized. Here we show that, similarly to other proteins involved in various neurological diseases, JIP1 becomes hyperphosphorylated following activation of stress-activated and MAP kinases. By immobilized metal affinity chromatography and a combined microcapillary LC/MALDI-TOF/ESI-ion trap mass spectrometry approach, we identified 35 sites of mitotic phosphorylation within JIP1, among which eight were present within (Ser/Thr)-Pro sequence. This motif is modified by various kinases in aggregates of the microtubule-associated protein tau, which generates typical intraneuronal lesions occurring in Alzheimer disease. Most of the post-translational modifications found were located within the JNK, MAP kinase kinase, and RAC-alpha Ser/Thr protein kinase binding regions; no modifications occurred in protein Src homology 3 and phosphotyrosine interaction domains, which are essential for binding to kinesin, beta-amyloid precursor protein, and MAP kinase kinase kinase. Protein phosphorylation is known to affect stability and protein-protein interactions. Thus, the findings that JIP1 is extensively phosphorylated after activation of stress-activated and MAP kinases indicate that these signaling pathways might modulate JIP1 signaling by regulating its stability and association with some, but not all, interacting proteins.
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PMID:Hyperphosphorylation of JNK-interacting protein 1, a protein associated with Alzheimer disease. 1619 23

The timely restructuring of the blood-testis barrier (BTB) that facilitates the migration of preleptotene and leptotene spermatocytes from the basal to the adluminal compartment in the seminiferous epithelium of adult rat testes, which occurs at late stage VII through early stage VIII of the epithelial cycle, is a crucial cellular event of spermatogenesis. However, the regulation of BTB dynamics at the biochemical level remains elusive. In this study, tumor necrosis factor alpha (TNFalpha), a secretory product of Sertoli and germ cells in rat testes, was shown to affect junction dynamics in vivo. Following an acute administration of recombinant TNFalpha directly to adult rat testes in vivo at 0.5 and 2 mug/testis (with a body weight ~300 g), this treatment significantly and transiently disrupted the BTB. It also transiently inhibited the steady-state protein levels of occludin, zonula occludens-1, and N-cadherin, but not junction adhesion molecule-A, alpha-, and beta-catenin in testes at the BTB site as illustrated by immunoblottings, immunohistochemistry, electron microscopy, and fluorescent microscopy. This transient disruption of the BTB integrity induced by TNFalpha treatment was further demonstrated by a functional test to assess the passage of a fluorescent dye (e.g. fluorescein-5-isothiocyanate) from the systemic circulation to the adluminal compartment. Additionally, both the phosphorylated-Ser/Thr protein kinase activated by MAP kinase kinase (p-p38) and phosphorylated-externally regulated kinase (p-ERK) mitogen -activated protein kinase-signaling pathways were transiently activated. Collectively, these data coupled with the recently published in vitro studies have illustrated that the BTB is likely utilizing a novel mechanism in which localized production of TNFalpha by Sertoli and germ cells into the microenvironment at the basal compartment facilitates the timely restructuring ('opening'?) of the BTB during spermatogenesis to facilitate germ cell migration.
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PMID:Tumor necrosis factor {alpha} reversibly disrupts the blood-testis barrier and impairs Sertoli-germ cell adhesion in the seminiferous epithelium of adult rat testes. 1689 65

The proteins of the RAF family (A-RAF, B-RAF, and C-RAF) are serine/threonine kinases that play important roles in development, mature cell regulation, and cancer. Although it is widely held that their localization on membranes is an important aspect of their function, there are few data that address this aspect of their mode of action. Here, we report that each member of the RAF family exhibits a specific distribution at the level of cellular membranes and that C-RAF is the only isoform that directly targets mitochondria. We found that the RAF kinases exhibit intrinsic differences in terms of mitochondrial affinity and that C-RAF is the only isoform that binds this organelle efficiently. This affinity is conferred by the C-RAF amino-terminal domain and does not depend on the presence of RAS GTPases on the surface of mitochondria. Finally, we analyzed the consequences of C-RAF activation on mitochondria and observed that this event dramatically changes their morphology and their subcellular distribution. Our observations indicate that: (i) RAF kinases exhibit different localizations at the level of cellular membranes; (ii) C-RAF is the only isoform that directly binds mitochondria; and (iii) through its functional coupling with MEK, C-RAF regulates the shape and the cellular distribution of mitochondria.
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PMID:Isoform-specific interaction of C-RAF with mitochondria. 1835 64

In mammals the RAF family of serine/threonine kinases consists of three members, A-, B-, and C-RAF. Activation of RAF kinases involves a complex series of phosphorylations. Although the most prominent phosphorylation sites of B- and C-RAF are well characterized, little is known about regulatory phosphorylation of A-RAF. Using mass spectrometry, we identified here a number of novel in vivo phosphorylation sites in A-RAF. In particular, we found that Ser-432 participates in MEK binding and is indispensable for A-RAF signaling. On the other hand, phosphorylation within the activation segment does not contribute to epidermal growth factor-mediated activation. Furthermore, we show that the potential 14-3-3 binding domains in A-RAF are phosphorylated independently of its activation status. Of importance, we identified a novel regulatory domain in A-RAF (referred to as IH-segment) positioned between amino acids 248 and 267 that contains seven putative phosphorylation sites. Three of these sites, serines 257, 262, and 264, regulate A-RAF activation in a stimulatory manner. The spatial model of the A-RAF fragment, including residues between Ser-246 and Glu-277, revealed a switch of charge at the molecular surface of the IH-region upon phosphorylation, suggesting a mechanism in which the high accumulation of negative charges may lead to an electrostatic destabilization of protein-membrane interaction resulting in depletion of A-RAF from the plasma membrane. Together, we provide here for the first time a detailed analysis of in vivo A-RAF phosphorylation status and demonstrate that regulation of A-RAF by phosphorylation exhibits unique features compared with B- and C-RAF.
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PMID:Positive regulation of A-RAF by phosphorylation of isoform-specific hinge segment and identification of novel phosphorylation sites. 1866 92

RAF proteins are well known oncoproteins. The B-RAF has been shown to be activated by mutations in a multitude of human cancers. Alterations of C-RAF expression are discussed to play a role in lung cancer. Only for A-RAF no link to tumorigenesis has been published so far. Malignant gliomas are the most prevalent primary brain tumors of adults. They are highly invasive and very difficult to treat, despite of surgery, gamma-irradiation and chemotherapy. Although a role of the mitogenic Ras-RAF-MEK-ERK signalling cascade in brain tumor development is well established, there are only few reports available addressing alterations in RAF sequence or protein expression and function in human gliomas. We analysed the mutational status of A-RAF and B-RAF in human glioblastomas (GBM) by sequencing. Then we checked for RAF gene amplification by dot blot hybridization and examined RAF mRNA and protein expression patterns in human astrocytic gliomas of WHO grade II (LGA) and IV (GBM) by semiquantitative RT-PCR and Western blotting, respectively. The results were correlated with patients prognosis. Finally, we performed functional assays to address a putative function of A-RAF in glioma cell proliferation and migration. We showed that RAF mutations are a rare event in glioblastoma multiforme. A-raf gene amplification was more often detected and overexpression of all three RAF proteins on mRNA and protein level was regularly found in human malignant gliomas. Whereas A-RAF and C-RAF expression was negatively correlated with the patients prognosis, B-RAF expression had a positive effect. Since neither A-RAF, nor C-RAF expression had any influence on proliferation and migration of GBM cells, putative functions of C-RAF in angiogenesis and of A-RAF in regulation of metabolism are discussed. Our data indicate that RAF proteins might be valuable targets for small molecule therapies. However, initially specific functions of RAF during tumorigenesis have to be elucidated.
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PMID:RAF expression in human astrocytic tumors. 1908 3

The Rac1/Cdc42 effector, p21-activated kinase (PAK), is activated by various signaling cascades, including receptor-tyrosine kinases and integrins, and regulates a number of processes such as cell proliferation and motility. PAK activity has been shown to be required for maximal activation of the canonical RAF-MEK-MAPK signaling cascade, possibly because of PAK co-activation of RAF and MEK. Here we have shown that trihydrophobin 1 (TH1), originally identified as a negative regulator of A-RAF kinase, also interacted with PAK1 in cultured cells. Confocal microscopy assay indicated that TH1 colocalized with PAK1 in both the cytoplasm and nucleus, which is consistent with our previous results. GST pulldown and coimmunoprecipitation experiments demonstrated that TH1 interacted directly with PAK1 and bound selectively to the carboxyl-terminal kinase domain of PAK1, and the ability of the binding was enhanced along with activation of PAK1. The binding pattern of PAK1 implies that this interaction was mediated in part by PAK1 kinase activity. As indicated by in vitro kinase activity assays and Western blot detections, TH1 inhibited PAK1 kinase activity and negatively regulated MAPK signal transduction. Interestingly, TH1 bound with MEK1/ERK in cells and in vitro without directly suppressing their kinase activity. Furthermore, we observed that TH1 localized to focal adhesions and filopodia in the leading edge of cells, where TH1 reduced cell migration through affecting actin and adhesion dynamics. Based on these observations, we propose a model in which TH1 interacts with PAK1 and specifically restricts the activation of MAPK modules through the upstream region of the MAPK pathway, thereby influencing cell migration.
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PMID:Trihydrophobin 1 Interacts with PAK1 and Regulates ERK/MAPK Activation and Cell Migration. 1913 54

KSR-1 is a scaffold protein that is essential for Ras-induced activation of the highly conserved RAF-MEK-ERK kinase module. Previously, we identified a close homolog of KSR-1, called KSR-2, through structural homology-based data mining. In order to further understand the role of KSR-2 in MAPK signaling, we undertook a functional proteomics approach to elucidate the dynamic composition of the KSR-2 functional complex in HEK-293 cells under conditions with and without TNF-alpha stimulation. We found nearly 100 proteins that were potentially associated with KSR-2 complex and 43 proteins that were likely recruited to the super molecular complex after TNF-alpha treatment. Our results indicate that KSR-2 may act as a scaffold protein similar as KSR-1 to mediate the MAPK core (RAF-MEK-ERK) signaling but with a distinct RAF isoform specificity, namely KSR-2 may only mediate the A-RAF signaling while KSR-1 is responsible for transducing signals only from c-RAF. In addition, KSR-2 may be involved in the activation of many MAPK downstream signaling molecules such as p38 MAPK, IKAP, AIF, and proteins involved in ubiquitin-proteasome, apoptosis, cell cycle control, and DNA synthesis and repair pathways, as well as mediating crosstalks between MAPK and several other signaling pathways, including PI3K and insulin signaling. While interactions with these molecules are not known for KSR-1, it's reasonable to hypothesize that KSR-1 may also play a similar role in mediating these downstream signaling pathways.
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PMID:Proteomic characterization of the dynamic KSR-2 interactome, a signaling scaffold complex in MAPK pathway. 1956 21

A-RAF, B-RAF, and C-RAF are a family of three protein-serine/threonine kinases that participate in the RAS-RAF-MEK-ERK signal transduction cascade. This cascade participates in the regulation of a large variety of processes including apoptosis, cell cycle progression, differentiation, proliferation, and transformation to the cancerous state. RAS mutations occur in 15-30% of all human cancers, and B-RAF mutations occur in 30-60% of melanomas, 30-50% of thyroid cancers, and 5-20% of colorectal cancers. Activation of the RAF kinases requires their interaction with RAS-GTP along with dephosphorylation and also phosphorylation by SRC family protein-tyrosine kinases and other protein-serine/threonine kinases. The formation of unique side-to-side RAF dimers is required for full kinase activity. RAF kinase inhibitors are effective in blocking MEK1/2 and ERK1/2 activation in cells containing the oncogenic B-RAF Val600Glu activating mutation. RAF kinase inhibitors lead to the paradoxical increase in RAF kinase activity in cells containing wild-type B-RAF and wild-type or activated mutant RAS. C-RAF plays a key role in this paradoxical increase in downstream MEK-ERK activation.
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PMID:RAF protein-serine/threonine kinases: structure and regulation. 2067 47


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