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)

In this study, the abilities of constitutive and conditional forms of the three Raf kinases to abrogate the cytokine dependency of FDC-P1 cells were examined. The constitutively active forms (delta) of all three Raf kinases were fused to the hormone-binding domain of the estrogen receptor (ER), rendering their activities conditionally dependent upon exogenous beta-estradiol. The vast majority of deltaRaf:ER-infected FDC-P1 cells remained cytokine-dependent; however, cells were obtained at low frequency in which expression of deltaRaf:ER abrogated cytokine dependency. Isoform specific differences between the Raf kinases were observed as cytokine-independent cells were obtained more frequently from deltaA-Raf:ER than either deltaRaf-1:ER or deltaB-Raf:ER infected cells. To determine whether the regulatory phosphorylation sites in the Raf proteins were necessary for abrogation of cytokine dependency, they were changed by site-directed mutagenesis. Substitution with phenylalanine eliminated the transforming ability of the deltaB-Raf:ER and deltaRaf-1:ER kinases. However, a similar substitution in A-Raf did not extinguish its transforming activity. The activated Raf proteins induced essential downstream MEK1 activity as treatment with the MEK1 inhibitor, PD98059, suppressed Raf-mediated growth. Activated MAP kinases (ERK1 and ERK2) were detected in deltaRaf:ER-transformed cells, and their presence was dependent upon a functional MEK1 protein. The cytokine-independent phenotype required the continued activity of the deltaRaf:ER proteins as removal of beta-estradiol caused the cells to stop growing and undergo apoptosis. The Raf-responsive cells were found to express autocrine growth factors, which promoted their growth. Constitutive activation of the Raf-1 oncogene resulted in malignant transformation as cytokine-independent FDC-P1 cells infected with a retrovirus encoding an activated Raf-1 protein formed tumors upon injection of immunocompromised mice. In summary, Raf kinases can abrogate cytokine dependency, prevent apoptosis and induce the tumorigenicity of a certain subpopulation of FDC-P1 cells by a MEK1-dependent mechanism.
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PMID:Differential abilities of the Raf family of protein kinases to abrogate cytokine dependency and prevent apoptosis in murine hematopoietic cells by a MEK1-dependent mechanism. 1076 50

Isoform-specific expression of endothelin-converting enzyme (ECE)-1, the major big endothelin-processing enzyme, is controlled by alternative promoters. Signaling pathways and transcriptional mechanisms of ECE-1 mRNA expression are largely unknown. To investigate ECE-1 isoform expression after protein kinase C (PKC) activation, we used phorbol 12-myristate 13-acetate (PMA) to stimulate primary cultured human umbilical vein endothelial cells and the related EA.hy926 cell line. ECE-1a mRNA was up-regulated (approximately 3-fold), whereas mRNA of alternative isoforms (b, c, and d) was unchanged, which was confirmed on the protein level. PMA effects on mRNA expression were suppressed by the PKC inhibitors H-7 and Calphostin C. Because increased ECE-1a expression was preceded by induction of the transcription factor Ets-1, we performed gel shift assays and demonstrated specific DNA/protein interactions involving the ETS binding motif GGAA. Luciferase reporter assays showed that PMA induced ECE-1a promoter activity about 2.5-fold in EA.hy926 cells. Similarly, coexpression of Ets-1 protein resulted in a dose-dependent increase in ECE-1a promoter activity (more than 8-fold). Using gel shift assays and mutation analysis, we identified two tandemly arranged Ets-1 binding sites (EBS) at -638 and -658, respectively, that are involved in transcriptional activation of the ECE-1a promoter by PMA or Ets-1. Moreover, we also found evidence for binding of a transcriptional repressor to EBS -638. The inhibitor of mitogen-activated protein kinase kinase, PD98059, inhibited PMA effects on ECE-1a mRNA expression and promoter activity, respectively. Our results provide the first detailed analysis of signaling pathways and transcriptional mechanisms involved in isoform-specific ECE-1 gene expression.
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PMID:Transcriptional mechanism of protein kinase C-induced isoform-specific expression of the gene for endothelin-converting enzyme-1 in human endothelial cells. 1172 40

Transforming growth factor (TGF)-beta, bone morphogenetic protein (BMP), and interleukin-1beta activate TGF-beta-activated kinase 1 (TAK1), which lies upstream of the p38 MAPK, JNK, and NF-kappaB pathways. Our knowledge remains incomplete of TAK1 target genes, requirement for cooperative signaling, and capacity for shared or segregated ligand-dependent responses. We show that adenoviral overexpression of TAK1a in articular chondrocytes stimulated type II collagen protein synthesis 3-6-fold and mimicked the response to TGF-beta1 and BMP2. Both factors activated endogenous TAK1 and its activating protein, TAB1, and the collagen response was inhibited by dominant-negative TAK1a. Isoform-specific antibodies to TGF-beta blocked the response to endogenous and exogenous TGF-beta but not the response to TAK1a. Expression of Smad3 did not stimulate type II collagen synthesis or enhance that caused by TGF-beta1 or TAK1a, in contrast to its effects on its endogenous targets, CTGF and plasminogen-activated inhibitor-1. TAK1a, overexpressed alone and immunoprecipitated, phosphorylated MKK6 and stimulated the plasminogen-activated inhibitor-1 promoter following transient transfection; both effects were enhanced by TAB1 coexpression, but type II collagen synthesis was not. Stimulation by TAK1a or TGF-beta did not require increased Col2a1 mRNA, and TAK1 actually reduced Col2a1 mRNA in parallel with the cartilage markers, SRY-type HMG box 9 (Sox9) and aggrecan. Thus, TAK1 increased target gene expression (Col2a1) by translational or posttranslational mechanisms as a Smad3-independent response shared by TGF-beta1 and BMP2.
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PMID:Transforming growth factor (TGF)-beta-activated kinase 1 mimics and mediates TGF-beta-induced stimulation of type II collagen synthesis in chondrocytes independent of Col2a1 transcription and Smad3 signaling. 1574 58

The fully executed epidermal growth factor receptor (EGFR)/Ras/MEK/ERK pathway serves a pro-survival role in renal epithelia under moderate oxidative stress. We and others have demonstrated that during severe oxidative stress, however, the activated EGFR is disconnected from ERK activation in cultured renal proximal tubule cells and also in renal proximal tubules after ischemia/reperfusion injury, resulting in necrotic death. Studies have shown that the tyrosine-phosphorylated p46/52 isoforms of the ShcA family of adaptor proteins connect the activated EGFR to activation of Ras and ERK, whereas the p66(shc) isoform can inhibit this p46/52(shc) function. Here, we determined that severe oxidative stress (after a brief period of activation) terminates activation of the Ras/MEK/ERK pathway, which coincides with ERK/JNK-dependent Ser(36) phosphorylation of p66(shc). Isoform-specific knockdown of p66(shc) or mutation of Ser(36) to Ala, but not to Asp, attenuated severe oxidative stress-mediated ERK inhibition and cell death in vitro. Also, severe oxidative stress (unlike ligand stimulation and moderate oxidative stress, both of which support survival) increased binding of p66(shc) to the activated EGFR and Grb2. This binding dissociated the SOS1 adaptor protein from the EGFR-recruited signaling complex, leading to termination of Ras/MEK/ERK activation. Notably, Ser(36) phosphorylation of p66(shc) and its increased binding to the EGFR also occurred in the kidney after ischemia/reperfusion injury in vivo. At the same time, SOS1 binding to the EGFR declined, similar to the in vitro findings. Thus, the mechanism we propose in vitro offers a means to ameliorate oxidative stress-induced cell injury by either inhibiting Ser(36) phosphorylation of p66(shc) or knocking down p66(shc) expression in vivo.
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PMID:p66shc inhibits pro-survival epidermal growth factor receptor/ERK signaling during severe oxidative stress in mouse renal proximal tubule cells. 1817 62