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)

Okadaic acid (OA) causes meiotic progression and chromosome condensation in cultured pachytene spermatocytes and an increase in maturation promoting factor (cyclin B1/cdc2 kinase) activity, as evaluated by H1 phosphorylative activity in anti-cyclin B1 immunoprecipitates. OA also induces a strong increase of phosphorylative activity toward the mitogen-activated protein kinase substrate myelin basic protein (MBP). Immunoprecipitation experiments with anti-extracellular signal-regulated kinase 1 (ERK1) or anti-ERK2 antibodies followed by MBP kinase assays, and direct in-gel kinase assays for MBP, show that p44/ERK1 but not p42/ERK2 is stimulated in OA-treated spermatocytes. OA treatment stimulates phosphorylation of ERK1, but not of ERK2, on a tyrosine residue involved in activation of the enzyme. ERK1 immunoprecipitated from extracts of OA-stimulated spermatocytes induces a stimulation of H1 kinase activity in extracts from control pachytene spermatocytes, whereas immunoprecipitated ERK2 is uneffective. We also show that natural G(2)/M transition in spermatocytes is associated to intracellular redistribution of ERKs, and their association with microtubules of the metaphase spindle. Preincubation of cultured pachytene spermatocytes with PD98059 (a selective inhibitor of ERK-activating kinases MEK1/2) completely blocks the ability of OA to induce chromosome condensation and progression to meiotic metaphases. These results suggest that ERK1 is specifically activated during G(2)/M transition in mouse spermatocytes, that it contributes to the mechanisms of maturation promoting factor activation, and that it is essential for chromosome condensation associated with progression to meiotic metaphases.
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PMID:Activation of the mitogen-activated protein kinase ERK1 during meiotic progression of mouse pachytene spermatocytes. 1055 44

An ERK2-binding site at the N terminus of MEK1 was reported to mediate their stable association. We examined the importance of this binding site in the feedback phosphorylation of MEK1 on Thr(292) and Thr(386) by ERK2, the phosphorylation and activation of ERK2 by MEK1, and the interaction of MEK1 with ERK2 and Raf-1. Deletion of the binding site from MEK1 reduced its phosphorylation by ERK2, but had no effect on its phosphorylation by p21-activated protein kinase-1 (PAK1). A MEK1 N-terminal peptide containing the binding site inhibited MEK1 phosphorylation by ERK2. However, it did not affect MEK1 phosphorylation by p21-activated protein kinase or myelin basic protein phosphorylation by ERK2. Deletion of the N-terminal ERK-binding domain of MEK1 also reduced its ability to phosphorylate ERK2 in vitro, to co-immunoprecipitate with ERK2, and to stimulate ERK2 activation in transfected cells, but it did not alter the association with endogenous Raf-1. Using ERK2-p38 chimeras and an ERK2 deletion mutant, a MEK1-binding site of ERK2 was localized to its N terminus.
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PMID:The N-terminal ERK-binding site of MEK1 is required for efficient feedback phosphorylation by ERK2 in vitro and ERK activation in vivo. 1056 69

Transforming growth factor-beta1 (TGF-beta1) stimulates articular chondrocyte cell proliferation and extracellular matrix formation. We reported previously that immediate and transient expression of c-fos mRNA through protein kinase C activation is required for the mitogenic effect of TGF-beta1 on cultured rat articular chondrocytes (CRAC). In gel kinase assays using myelin basic protein (MBP) showed that total cell lysates from cells treated with TGF-beta1 caused rapid phosphorylation of MBP, which suggests the involvement of mitogen-activated protein kinase (MAPK) activation. To identify specific MAPK pathways activated by TGF-beta1, we performed in vitro kinase assays using specific substrates. TGF-beta1 induced a rapid activation of extracellular signal regulated kinase (ERK) with a peak at 5 min, which decreased to basal levels within 240 min after TGF-beta1 stimulation. In contrast, the c-jun N-terminal kinase activity increased only about 2.5-fold after 240 min of stimulation and p38 MAPK activity did not change significantly. ERK activation by TGF-beta1 was also confirmed by in vivo phosphorylation assays of Elk1. However, a specific MEK1 inhibitor, PD98059, significantly decreased TGF-beta1 induced Elk1 phosphorylation in a dose-dependent manner. Furthermore, PD98059 reduced the TGF-beta1-induced cell growth by 40%. These results indicate that TGF-beta1 specifically activates MEK1 and subsequent ERK pathways in CRAC, and that the activation of this MAPK pathway plays a role in the mitogenic response to TGF-beta1.
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PMID:Transforming growth factor-beta stimulates articular chondrocyte cell growth through p44/42 MAP kinase (ERK) activation. 1058 Jul 47

A tobacco MAP kinase termed SIPK (Salicylic acid-Induced Protein Kinase) is activated in response to a variety of stress signals, including pathogen attack and wounding (S. Zhang and D.F. Klessig, Proc. Natl. Acad. Sci. USA 95:7225-7230, 1998; S. Zhang and D.F. Klessig, Proc. Natl. Acad. Sci. USA 95:7433-7438, 1998). Using the yeast two-hybrid system, we have identified a gene encoding a protein that interacts with SIPK but not the wounding induced protein kinase (WIPK), which is another tobacco MAP kinase. Sequence analysis indicated that this SIPK-interacting protein is a member of the MAP kinase kinase family; thus, it was named SIPK kinase (SIPKK). Co-immunoprecipitation experiments demonstrated that SIPKK and SIPK interact in vitro. Consistent with its putative function as a kinase, SIPKK phosphorylated myelin basic protein in vitro. Interestingly, SIPKK was induced at the mRNA level after Tobacco mosaic virus (TMV) infection or wounding, albeit with kinetics that are too slow to account for the activation of SIPK following these stimuli.
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PMID:Molecular cloning and characterization of a tobacco MAP kinase kinase that interacts with SIPK. 1065 93

Degenerate polymerase chain reaction against conserved kinase catalytic subdomains identified 15 tyrosine and serine-threonine kinases expressed in surgically removed prostatic carcinoma tissues, including six receptor kinases (PDGFBR, IGF1-R, VEGFR2, MET, RYK, and EPH-A1), six non-receptor kinases (ABL, JAK1, JAK2, TYK2, PLK-1, and EMK), and three novel kinases. Several of these kinases are oncogenic, and may function in the development of prostate cancer. One of the novel kinases is a new member of the sterile 20 (STE20) family of serine-threonine kinases which we have called prostate-derived STE20-like kinase (PSK) and characterized functionally. PSK encodes an open reading frame of 3705 nucleotides and contains an N-terminal kinase domain. Immunoprecipitated PSK phosphorylates myelin basic protein and transfected PSK stimulates MKK4 and MKK7 and activates the c-Jun N-terminal kinase mitogen-activated protein kinase pathway. Microinjection of PSK into cells results in localization of PSK to a vesicular compartment and causes a marked reduction in actin stress fibers. In contrast, C-terminally truncated PSK (1-349) did not localize to this compartment or induce a decrease in stress fibers demonstrating a requirement for the C terminus. Kinase-defective PSK (K57A) was unable to reduce stress fibers. PSK is the first member of the STE20 family lacking a Cdc42/Rac binding domain that has been shown to regulate both the c-Jun N-terminal kinase mitogen-activated protein kinase pathway and the actin cytoskeleton.
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PMID:PSK, a novel STE20-like kinase derived from prostatic carcinoma that activates the c-Jun N-terminal kinase mitogen-activated protein kinase pathway and regulates actin cytoskeletal organization. 1066 Jun

Parathyroid hormone (PTH), a major physiologic regulator of proximal renal tubule cell sodium-phosphate cotransport, stimulates several signal transduction pathways including extracellular signal-regulated kinases (ERK). The physiologic role of PTH-stimulated ERK is unknown. The purpose of the present study was to identify signaling components involved in PTH-stimulated ERK activity and to determine the role of PTH-stimulated ERK activity in regulation of phosphate transport. PTH-stimulated ERK activity was measured in opossum kidney (OK) cell lysates as phosphorylation of myelin basic protein by an in vitro kinase assay. PTH stimulated a dose-dependent increase in ERK activity with a peak at 10(-7) M. The time course was biphasic with an early peak at 10 min and a later peak at 20 min. Pretreatment of OK cells with the nonreceptor tyrosine kinase inhibitors genistein and herbimycin A or with the phosphatidylinositol 3-kinase (PI-3K) inhibitors wortmannin and LY294002 blocked the early and late peaks of PTH-stimulated ERK activity. Pretreatment with the protein kinase C inhibitor calphostin C blocked only the later phase of PTH-stimulated ERK. To determine the role of ERK in regulation of phosphate transport, PTH inhibition of phosphate uptake and PTH regulation of sodium-phosphate cotransporter (NaPi-4) expression were measured in OK cells pretreated with the MEK inhibitor PD098059. PD098059 significantly attenuated PTH inhibition of phosphate uptake but did not prevent PTH downregulation of NaPi-4. It is concluded that PTH stimulates ERK through two signal transduction pathways: an early pathway dependent on tyrosine kinase and PI-3K and a late pathway dependent on protein kinase C. PTH-stimulated ERK regulates phosphate transport by a mechanism other than downregulation of NaPi-4 expression.
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PMID:Parathyroid hormone stimulates extracellular signal-regulated kinase (ERK) activity through two independent signal transduction pathways: role of ERK in sodium-phosphate cotransport. 1066 29

Raf-1 is a serine-threonine protein kinase that functions as a central component of the mitogen-activated protein kinase signal transduction pathway. Raf-1 activity is currently assayed in vitro by either measuring 32P incorporation into MEK, Raf-1's only characterized substrate, or by using the phosphorylated MEK to initiate a coupled assay culminating in the phosphorylation of myelin basic protein by MAP kinase. These assays are plagued by a potential lack of specificity in the case of the former, and the time consuming and error-prone nature of the later indirect assay. In this report, we demonstrate a novel single step assay for Raf-1 kinase activity based on phosphorylation of recombinant MEK-1, detected using an activation-specific MEK antibody that recognizes MEK only when specifically phosphorylated by Raf-1 on Ser 217 and Ser 221. The assay readily detected stem cell factor-mediated Raf-1 activation. MEK phosphorylation by immunoprecipitated Raf-1 plateaued at 10 min following initiation of the kinase reaction and was completely dependent on the inclusion of Raf-1. There was a linear correlation between the degree of MEK phosphorylation and the amount of Raf-1 protein immunoprecipitated. In addition to detecting growth factor-mediated activation, the assay was also able to detect paclitaxel-mediated Raf-1 activation. This assay is rapid, sensitive, and specific and therefore is a marked improvement over currently utilized techniques.
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PMID:A novel assay for the measurement of Raf-1 kinase activity. 1104 90

We describe a versatile intracellular reporter of ERK/MAP kinase activity: a cDNA construct, pGFP.MBP, encoding amino acids 85-144 of the human myelin basic protein fused to the C-terminus of an enhanced green fluorescent protein (GFP). The fused fragment of myelin basic protein contains a single consensus ERK/MAP kinase phosphorylation motif (PRTP, where the threonine is phosphorylated). Phosphorylation of the specific motif can be detected via immunoblotting or immunofluorescence with a commercially available phospho-specific monoclonal antibody. When expressed in mammalian cells by either transient or stable transfection, the fusion protein acts as a bona fide kinase substrate, as demonstrated by rapid serum-induced phosphorylation that is blocked by a specific MEK inhibitor. Moreover, the localization of the total substrate pool is easily visualized by GFP autofluorescence and the extent of its phosphorylation simultaneously detected within intact fixed cells by immunofluorescence using the commercially available phospho-specific antibody. The approach described should be generally applicable to the intracellular analysis of many specific protein kinase substrates for which phospho-specific antibodies have been produced.
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PMID:A green fluorescent protein kinase substrate allowing detection and localization of intracellular ERK/MAP kinase activity. 1139 42

Hypertrophy is an adaptive response of the heart to myocardial injury or hemodynamic overload that may progress and contribute to cardiac decompensation and eventually to heart failure. The signaling pathways controlling this response in the cardiac myocyte are poorly understood. A data mining effort of a human failed heart cDNA library was undertaken in an effort to identify novel signaling molecules involved in cardiac hypertrophy. This effort identified a novel kinase (MLK7) homologous to the mixed lineage kinase family of proteins. The mixed lineage kinases are mitogen-activated protein kinase kinase kinases (MAPKKKs) which activate stress activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) and p38 kinase pathways. They contain a catalytic domain with homology to both serine/threonine and tyrosine-specific kinases and a dual leucine zipper. MLK7 is identical to leucine zipper and sterile-alpha motif protein kinase (ZAK) through the leucine zipper domain but has a completely divergent COOH-terminus and shares approximately 40% homology with the other MLKs overall. Expression of MLK7 mRNA is most abundant in skeletal muscle and heart, with expression restricted to the cardiac myocyte. The recombinant histidine tagged MLK7 expressed and purified from insect cells exhibited serine/threonine kinase activity in vitro with myelin basic protein as substrate. When expressed in cardiac myocytes, MLK7 activated SAPK/JNK1, and ERK and p38 to a lesser extent. Additionally, MLK7 altered fetal gene expression and increased protein synthesis in cardiac myocytes. These data suggest that MLK7 is a new member of the mixed lineage kinase family that modulates cardiac SAPK/JNK pathway and may play a role in cardiac hypertrophy and progression to heart failure.
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PMID:Tissue distribution and functional expression of a cDNA encoding a novel mixed lineage kinase. 1154 52

The involvement of mitogen-activated protein (MAP) kinases in the mitogenic effect of thyrotropin (TSH) is not fully elucidated. In FRTL-5 cells, we found that the MAP kinase kinase (MEK) inhibitors UO126 and PD98059 substantially decreased TSH-induced DNA synthesis, indicating that MAP kinases are involved in the TSH-stimulated proliferative response. Accordingly, TSH, forskolin (FSK) and 8-bromo-cAMP induced a rapid (3 min) and transient activation of ERK1/2, as assessed by phosphorylation of myelin basic protein and ERK1/2. This effect was cAMP-dependent and protein kinase A (PKA)-independent. The activation of Rap1 and B-Raf was involved in the mechanism of MAP kinase stimulation by TSH. TSH induced rapid (3 min) GDP/GTP exchange and activation of Rap1. After a 3-min exposure to FSK, B-Raf was recruited to a vesicular compartment, where it colocalized with Rap1. Both activation of Rap1 and translocation of B-Raf were PKA-independent. The Rap1 dominant negative Rap1N17 significantly reduced TSH-stimulated but not insulin-like growth factor 1-stimulated ERK1/2 phosphorylation, whereas the Ras dominant negative RasN17 inhibited the effect of both agonists. In conclusion, our results document that TSH increases intracellular cAMP, which rapidly stimulates MAP kinase cascade independent of PKA. This novel mechanism could integrate other pathways involved in TSH-stimulated proliferative response.
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PMID:Thyrotropin activates mitogen-activated protein kinase pathway in FRTL-5 by a cAMP-dependent protein kinase A-independent mechanism. 1164 20


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