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:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

This is the first report demonstrating that NIH/3T3 fibroblasts utilize the Raf-1/MAPK pathway to sensitize themselves to tumor necrosis factor-alpha (TNF-alpha) cytotoxicity under Ha-rasVal12 oncogene-overexpressed conditions. This paper clearly shows that the sensitivity of NIH/3T3 cells to TNF-alpha cytotoxicity positively correlated with the expression level of activated Ha-ras transgene, which was manipulated either positively by isopropyl-beta-d-thiogalactoside (IPTG) induction or negatively by a ribozyme or a dominant negative Ras suppression. Further analysis revealed that after TNF-alpha treatment, Ha-ras-overexpressed transformants underwent apoptosis. Overexpression of dominant negative Raf-1, Rac1, or RhoA in the Ha-ras transformants clarified that among these factors, only dominant negative Raf-1 could reverse the cell sensitivity to TNF-alpha, indicating that Raf-1, as a proapoptotic factor, indeed participates in TNF-alpha cytotoxicity. The anti-apoptotic roles of Bcl-2 and PI(3) kinase are also demonstrated by the Ha-ras transformants which became more resistant to TNF-alpha while overexpressing Bcl-2 or the activated p110 catalytic subunit. The analyses of the cell cycle and nuclear transcription factor activities revealed that TNF-alpha treatment caused the Ha-ras overexpressed transformants to shift from S to G0/G1 phase and increased the responses of AP-1, c-fos, and c-myc. Taken together, we suggest that the possible action of Ha-ras overexpression to sensitize TNF-alpha-treated fibroblasts is predominantly through the Ras/Raf-1/MAPK pathway to increase the responses of AP-1, c-fos, and c-myc, which are possibly involved in the aberration of cell cycle machinery, and subsequently to turn on the death program.
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PMID:Selective activation of Ha-ras(val12) oncogene increases susceptibilityof NIH/3T3 cells to TNF-alpha. 1022 51

The work of Reddy et al. (S. A. Reddy, J. A. Huang, and W. S. Liao, J. Biol. Chem. 272:29167-29173, 1997) reveals that phosphatidylinositol 3-kinase (PI3K) plays a role in transducing a signal from the occupied interleukin-1 (IL-1) receptor to nuclear factor kappaB (NF-kappaB), but the underlying mechanism remains to be determined. We have found that IL-1 stimulates interaction of the IL-1 receptor accessory protein with the p85 regulatory subunit of PI3K, leading to the activation of the p110 catalytic subunit. Specific PI3K inhibitors strongly inhibit both PI3K activation and NF-kappaB-dependent gene expression but have no effect on the IL-1-stimulated degradation of IkappaBalpha, the nuclear translocation of NF-kappaB, or the ability of NF-kappaB to bind to DNA. In contrast, PI3K inhibitors block the IL-1-stimulated phosphorylation of NF-kappaB itself, especially the p65/RelA subunit. Furthermore, by using a fusion protein containing the p65/RelA transactivation domain, we found that overexpression of the p110 catalytic subunit of PI3K induces p65/RelA-mediated transactivation and that the specific PI3K inhibitor LY294,002 represses this process. Additionally, the expression of a constitutively activated form of either p110 or the PI3K-activated protein kinase Akt also induces p65/RelA-mediated transactivation. Therefore, IL-1 stimulates the PI3K-dependent phosphorylation and transactivation of NF-kappaB, a process quite distinct from the liberation of NF-kappaB from its cytoplasmic inhibitor IkappaB.
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PMID:Activation of phosphatidylinositol 3-kinase in response to interleukin-1 leads to phosphorylation and activation of the NF-kappaB p65/RelA subunit. 1037 29

The effects of cyclic AMP (cAMP) on cell proliferation are cell type specific. Although the growth-inhibitory effects of cAMP have been well studied, much less is known regarding how cAMP stimulates proliferation. We report that cAMP stimulates proliferation through both protein kinase A (PKA)-dependent and PKA-independent signaling pathways and that phosphatidylinositol 3-kinase (PI3K) is required for cAMP-stimulated mitogenesis. In cells where cAMP is a mitogen, cAMP-elevating agents stimulate membrane ruffling, Akt phosphorylation, and p70 ribosomal S6 protein kinase (p70s6k) activity. cAMP effects on ruffle formation and Akt were PKA independent but sensitive to wortmannin. In contrast, cAMP-stimulated p70s6k activity was repressed by PKA inhibitors but not by wortmannin or microinjection of the N-terminal SH2 domain of the p85 regulatory subunit of PI3K, indicating that p70s6k and Akt can be regulated independently. Microinjection of highly specific inhibitors of PI3K or Rac1, or treatment with the p70s6k inhibitor rapamycin, impaired cAMP-stimulated DNA synthesis, demonstrating that PKA-dependent and -independent pathways contribute to cAMP-mediated mitogenesis. Direct elevation of PI3K activity through microinjection of an antibody that stimulates PI3K activity or stable expression of membrane-localized p110 was sufficient to confer hormone-independent DNA synthesis when accompanied by elevations in p70s6k activity. These findings indicate that multiple pathways contribute to cAMP-stimulated mitogenesis, only some of which are PKA dependent. Furthermore, they demonstrate that the ability of cAMP to stimulate both p70s6k- and PI3K-dependent pathways is an important facet of cAMP-regulated cell cycle progression.
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PMID:Protein kinase A-dependent and -independent signaling pathways contribute to cyclic AMP-stimulated proliferation. 1045 35

Phosphoinositide 3-Kinases (PI3-Kinases) are a family of dual specificity enzymes with a unique lipid kinase activity toward the D-3 position of the inositol ring of phosphoinositides and a less well characterized serine/threonine protein kinase activity. Class IA PI3-Kinases comprise a 110-120 kDa catalytic subunit (usually termed p110) and an 85 kDa or 50 to 55 kDa regulatory subunit (often called p85). cDNAs for three mammalian Class IA PI3-Kinase catalytic subunits designated p110alpha, p110beta, and p110delta have been cloned from several species. A YAC clone for the human p110alpha gene has also been cloned and mapped to chromosome 3q26.3. However, structural organization for any of the PI3-Kinase p110alpha genes has not been reported. Here, we report the cloning, structural organization, and chromosomal localization of the mouse PI3-Kinase p110alpha gene. The translated portion of the mouse p110alpha gene is encoded by 19 exons that span at least 24 kb. Dual color fluorescence in situ hybridization (FISH) was performed to determine the chromosomal localization of the mouse PI3-Kinase p110alpha gene. FISH results and DAPI banding demonstrated localization of the p110alpha gene to band B on mouse chromosome 3, a region syntenic with human chromosome 3q26.3.
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PMID:Mouse phosphoinositide 3-kinase p110alpha gene: cloning, structural organization, and localization to chromosome 3 band B. 1046 94

Activation of the protein kinase Raf-1 is a complex process involving association with the GTP-bound form of Ras (Ras-GTP), membrane translocation and both serine/threonine and tyrosine phosphorylation (reviewed in [1]). We have reported previously that p21-activated kinase 3 (Pak3) upregulates Raf-1 through direct phosphorylation on Ser338 [2]. Here, we investigated the origin of the signal for Pak-mediated Raf-1 activation by examining the role of the small GTPase Cdc42, Rac and Ras, and of phosphatidylinositol (PI) 3-kinase. Pak3 acted synergistically with either Cdc42V12 or Rac1V12 to stimulate the activities of Raf-1, Raf-CX, a membrane-localized Raf-1 mutant, and Raf-1 mutants defective in Ras binding. Raf-1 mutants defective in Ras binding were also readily activated by RasV12. This indirect activation of Raf-1 by Ras was blocked by a dominant-negative mutant of Pak, implicating an alternative Ras effector pathway in Pak-mediated Raf-1 activation. Subsequently, we show that Pak-mediated Raf-1 activation is upregulated by both RasV12C40, a selective activator of PI 3-kinase, and p110-CX, a constitutively active PI 3-kinase. In addition, p85Delta, a mutant of the PI 3-kinase regulatory subunit, inhibited the stimulated activity of Raf-1. Pharmacological inhibitors of PI 3-kinase also blocked both activation and Ser338 phosphorylation of Raf-1 induced by epidermal growth factor (EGF). Thus, Raf-1 activation by Ras is achieved through a combination of both physical interaction and indirect mechanisms involving the activation of a second Ras effector, PI 3-kinase, which directs Pak-mediated regulatory phosphorylation of Raf-1.
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PMID:Regulation of the protein kinase Raf-1 by oncogenic Ras through phosphatidylinositol 3-kinase, Cdc42/Rac and Pak. 1071 5

Phosphatidylinositol 3-kinase mediates several actions of insulin including its antilipolytic effect. This effect is elicited by the insulin-stimulated serine phosphorylation and activation of cGMP-inhibited phosphodiesterase (PDE3B). In human adipocytes, we found that insulin differentially stimulated phosphatidylinositol 3-kinase activity; the lipid kinase activity was associated with IRS-1, whereas the serine kinase activity was associated with the insulin receptor and phosphorylated a number of proteins including p85, p110, and a 135-kDa protein identified as PDE3B. PDE3B phosphorylation was associated with enzyme activation, thus initiating the antilipolytic effect of insulin. These results show a novel pathway for intracellular signaling through the insulin receptor leading to the serine phosphorylation of key proteins involved in insulin action.
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PMID:Phosphorylation of PDE3B by phosphatidylinositol 3-kinase associated with the insulin receptor. 1074 89

In our previous study we showed that insulin-like growth factor-I induces a cAMP-response element (CRE) site-containing Bcl-2 promoter through a novel signaling pathway involving mitogen-activated protein kinase kinase 6/p38beta mitogen-activated protein kinase/MAP kinase-activated protein kinase-3/cAMP-response element-binding protein (CREB) (Pugazhenthi, S., Miller, E., Sable, C., Young, P., Heidenreich, K. A., Boxer, L. M., and Reusch, J. E.-B. (1999) J. Biol. Chem. 274, 27529-27535). In the present investigation, we define a second pathway contributing to CREB-dependent up-regulation of Bcl-2 expression as a novel anti-apoptotic function of Akt signaling. To examine the role of Akt on Bcl-2 expression, a series of transient transfections using a luciferase reporter gene driven by the promoter region of Bcl-2 containing a CRE were carried out. Pharmacological inhibition of phosphatidylinositol (PI) 3-kinase, the upstream kinase of Akt, with LY294002 led to a 45% decrease in Bcl-2 promoter activity. The reporter activity was enhanced 2.3-fold by overexpression of active p110 subunit of PI 3-kinase and inhibited 44% by the dominant negative p85 subunit of PI 3-kinase. Cotransfection with 3-phosphoinositide-dependent kinase (PDK1), which is required for the full activation of Akt, resulted in enhanced luciferase activity. Insulin-like growth factor-I-mediated induction of Bcl-2 promoter activity was decreased significantly (p < 0.01) by the dominant negative forms of p85 subunit of PI 3-kinase, PDK1, and Akt. These data indicate that regulation of Bcl-2 expression by IGF-I involves a signaling cascade mediated by PI 3-kinase/PDK1/Akt/CREB. Furthermore, we measured the Bcl-2 mRNA in PC12 cells overexpressing Akt by real-time quantitative reverse transcription-polymerase chain reaction using the TaqMan(TM) fluorogenic probe system. We observed a 2.1-fold increase in Bcl-2 mRNA levels in the Akt cell line compared with control PC12 cells, supporting the observation that enhanced CREB activity by Akt signaling leads to increased Bcl-2 promoter activity and cell survival.
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PMID:Akt/protein kinase B up-regulates Bcl-2 expression through cAMP-response element-binding protein. 1075 67

The discovery that the PTEN tumor suppressor encodes a phosphoinositide 3-phosphatase has raised interest in the effects of constitutive activation of PI 3-kinase. To gain insight into PI 3-kinase function, we have stably expressed a myristoylated form of the catalytic subunit p110alpha (myr-p110) in cells. The myr-p110 associated with the endogenous p85 regulatory subunit and retained lipid and protein kinase activity. Stable lines expressing myr-p110 had 2- to 4-fold more PI 3-kinase activity than controls. Expression of myr-p110 altered cellular morphology and increased the saturation density in culture. These clones were morphologically transformed but Akt and pp70(s6k) were not constitutively activated in contrast to transient assays and from tumor cell lines deficient in PTEN. In addition, the ability of PDGF to induce activation of Akt and pp70(s6k) was diminished. Therefore, expression of a myristoylated PI 3-kinase in murine fibroblasts induces a morphological transformation of the cells.
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PMID:Constitutive cellular expression of PI 3-kinase is distinct from transient expression. 1086 Aug 37

PITSLRE protein kinases are related to the large family of cyclin-dependent kinases. They have been proposed to act as tumor suppressor genes and have been shown to play a role in cell cycle progression. We report that two PITSLRE protein kinase isoforms, namely p11O(PITSLRE) and p58(PITSLRE), are translated from a single transcript by initiation at alternative in-frame AUG codons. p110(PITSLRE) is produced by classical cap-dependent translation, whereas p58(PITSLRE) results from internal initiation of translation controlled by an internal ribosome entry site (IRES) with unique properties. The IRES element is localized to the mRNA coding region, and its activity is cell cycle regulated, which permits translation of p58(PITSLRE) in G2/M.
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PMID:Identification and characterization of a novel cell cycle-regulated internal ribosome entry site. 1088 96

The small G-protein Ras, a critical component in the signalling pathways regulating cell growth, is involved in the tonic upregulation of voltage-dependent calcium channels (VDCCs) in rat sensory neurones. To investigate which downstream effector(s) of Ras is involved in this process, a series of Ras mutant cDNAs were co-expressed with green fluorescent protein (GFP) in primary cultured rat dorsal root ganglion neurones (DRGs). Constitutively active V12Ras (glycine 12 to valine) markedly increased basal calcium current density by 41 % compared with control cells (GFP alone). In contrast, a farnesylation-defective mutant, V12S186Ras (cysteine 186 to serine; activates no downstream effectors), significantly reduced calcium current density by 47 %. Ras effector region mutants V12C40 (tyrosine 40 to cysteine; activates the p110 alpha-subunit of phosphatidylinositol 3-kinase) and V12G37 (glutamic acid 37 to glycine; activates Ral guanine nucleotide dissociation stimulator) had no significant effect on VDCC current. However, V12S35Ras (threonine 35 to serine; activates Raf-1 and the mitogen-activated protein kinase (MAPK) pathway) markedly increased basal calcium current density by 67 %, suggesting that Raf-1 activation is sufficient for Ras enhancement of calcium current in these cells. Raf-1 activates MEK (MAPK kinase) in the MAPK pathway, and the MEK inhibitor U0126 reduced calcium current by 45 % after 10-15 min, whereas the inactive analogue U0124 had no effect. This rapid time course for MEK inhibition suggests direct modulation of VDCCs via the Ras-MAPK pathway rather than gene expression-mediated effects. The relative proportions of omega-conotoxin GVIA- and nicardipine-sensitive N- ( approximately 40 %) and L- ( approximately 40 %) type currents were unaffected by either V12S35Ras expression or U0126 pre-treatment, suggesting that all components of calcium current in DRGs, are enhanced via this pathway.
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PMID:Regulation of voltage-dependent calcium channels in rat sensory neurones involves a Ras-mitogen-activated protein kinase pathway. 1099 May 31


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