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)

The epidermal growth factor receptor (EGFR) is integral to basal-like and human epidermal growth factor receptor-2 (Her-2)-overexpressing breast cancers. Such tumors are associated with poor prognosis, the majority of which express high levels of EGFR. We reported that EGFR expression is induced by the oncogenic transcription factor Y-box binding protein-1 (YB-1) that occurs in a manner dependent on phosphorylation by Akt. Herein, we questioned whether blocking Akt with 2-amino-N-[4-[5-(2-phenanthrenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]phenyl]-acetamide (OSU-03012), a phosphoinositide-dependent protein kinase-1 (PDK-1) small-molecule inhibitor, could prevent YB-1 from binding to the EGFR promoter. MDA-MB-468 and SUM 149 are basal-like breast cancer (BLBC) cells that were used for our studies because they express high levels of activated PDK-1, YB-1, and EGFR compared with the immortalized breast epithelial cell line 184htrt. In these cell lines, YB-1 preferentially bound to the -1 kilobase of the EGFR promoter, whereas this did not occur in the 184htrt cells based on chromatin immunoprecipitation. When the cells were exposed to OSU-03012 for 6 h, YB-1/EGFR promoter binding was significantly attenuated. To further confirm this observation, gel-shift assays showed that the drug inhibits YB-1/EGFR promoter binding. The inhibitory effect of OSU-03012 on EGFR was also observed at the mRNA and protein levels. OSU-03012 ultimately inhibited the growth of BLBC in monolayer and soft agar coordinate with the induction of apoptosis using an Array-Scan VTI high-content screening system. Furthermore, OSU-03012 inhibited the expression of EGFR by 48% in tumor xenografts derived from MDA-MB-435/Her-2 cells. This correlated with loss of YB-1 binding to the EGFR promoter. Hence, we find that OSU-03012 inhibits YB-1 resulting in a loss of EGFR expression in vitro and in vivo.
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PMID:The phosphoinositide-dependent kinase-1 inhibitor 2-amino-N-[4-[5-(2-phenanthrenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]phenyl]-acetamide (OSU-03012) prevents Y-box binding protein-1 from inducing epidermal growth factor receptor. 2728 88

Pyruvate dehydrogenase kinase 2 (PDHK2) is a unique mitochondrial protein kinase that regulates the activity of the pyruvate dehydrogenase multienzyme complex (PDC). PDHK2 is an integral component of PDC tightly bound to the inner lipoyl-bearing domains (L2) of the dihydrolipoyl transacetylase component (E2) of PDC. This association has been reported to bring about an up to 10-fold increase in kinase activity. Despite the central role played by E2 in the maintenance of PDHK2 functionality in the PDC-bound state, the molecular mechanisms responsible for the recognition of L2 by PDHK2 and for the E2-dependent PDHK2 activation are largely unknown. In this study, we used a combination of molecular modeling and site-directed mutagenesis to identify the amino acid residues essential for the interaction between PDHK2 and L2 and for the activation of PDHK2 by E2. On the basis of the results of site-directed mutagenesis, it appears that a number of PDHK2 residues located in its R domain (P22, L23, F28, F31, F44, L45, and L160) and in the so-called "cross arm" structure (K368, R372, and K391) are critical in determining the strength of the interaction between PDHK2 and L2. The residues of L2 essential for recognition by PDHK2 include L140, K173, I176, E179, and to a lesser extent D164, D172, and A174. Importantly, certain PDHK2 residues forming interfaces with L2, i.e., K17, P22, F31, F44, R372, and K391, are also critical for the maintenance of enhanced PDHK2 activity in the E2-bound state. Finally, evidence that the blood glucose-lowering compound AZD7545 disrupts the interactions between PDHK2 and L2 and thereby inhibits PDHK2 activity is presented.
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PMID:Recognition of the inner lipoyl-bearing domain of dihydrolipoyl transacetylase and of the blood glucose-lowering compound AZD7545 by pyruvate dehydrogenase kinase 2. 1760 66

Unopposed PI3-kinase activity and 3'-phosphoinositide production in Jurkat T cells, due to a mutation in the PTEN tumour suppressor protein, results in deregulation of PH domain-containing proteins including the serine/threonine kinase PKB/Akt. In Jurkat cells, PKB/Akt is constitutively active and phosphorylated at the activation-loop residue (Thr308). 3'-phosphoinositide-dependent protein kinase-1 (PDK-1), an enzyme that also contains a PH domain, is thought to catalyse Thr308 phosphorylation of PKB/Akt in addition to other kinase families such as PKC isoforms. It is unknown however if the loss of PTEN in Jurkat cells also results in unregulated PDK-1 activity and whether such loss impacts on activation-loop phosphorylation of other putative PDK-1 substrates such as PKC. In this study we have addressed if loss of PTEN in Jurkat T cells affects PDK-1 catalytic activity and intracellular localisation. We demonstrate that reducing the level of 3'-phosphoinositides in Jurkat cells with pharmacological inhibitors of PI3-kinase or expression of PTEN does not affect PDK-1 activity, Ser241 phosphorylation or intracellular localisation. In support of this finding, we show that the levels of PKC activation-loop phosphorylation are unaffected by reductions in the levels of 3'-phosphoinositides. Instead, the dephosphorylation that occurs on PKB/Akt at Thr308 following reductions in 3'-phosphoinositides is dependent on PP2A-like phosphatase activity. Our finding that PDK-1 functions independently of 3'-phosphoinositides in T cells is also confirmed by studies in HuT-78 T cells, a PTEN-expressing cell line with undetectable levels of 3'-phosphoinositides. We conclude therefore that loss of PTEN expression in Jurkat T cells does not impact on the PDK-1/PKC pathway and that only a subset of kinases, such as PKB/Akt, are perturbed as a consequence PTEN loss.
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PMID:Loss of PTEN expression does not contribute to PDK-1 activity and PKC activation-loop phosphorylation in Jurkat leukaemic T cells. 1782 53

Nuclear factor kappaB (NF-kappaB) and activator protein 1 are transcription factors involved in the regulation of cell proliferation that play important roles in tumorigenesis. We investigated whether these two factors cooperate for transcriptional regulation of cyclin D1 (CCND1), a gene whose deregulation is critical during carcinogenesis. We demonstrate that overexpression of JunD in human hepatocarcinoma cells strongly activates transcription mediated by the kappaB2 site of the CCND1 promoter in reporter assays, in a manner strictly dependent on the presence of NF-kappaB proteins. Serum stimulation increased the expression of p65, p50, c-Fos, c-Jun and JunD and induced the recruitment of p65, p50 and JunD to the kappaB2 site of the promoter in DNA pull-down assays. Chromatin immunoprecipitation (ChIP) analysis confirmed the serum-induced recruitment of JunD to the promoter in vivo and showed that the presence of JunD was dependent on the presence of p65 and p50, indicating a protein-protein-dependent mechanism of JunD recruitment. Serum-induced activation of protein binding to kappaB2 correlated with high levels of phosphoinositide-dependent protein kinase-1 (PDK-1) phosphorylation. Both LY294002, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K), and overexpression of a dominant-negative form of PDK-1 inhibited the JunD-stimulating effect in reporter assays. LY294002 also prevented the serum-induced recruitment of JunD, but not p65 or p50 to the promoter in ChIP assay. JunD-p65 complexes, identified in vivo by co-immunoprecipitation, were decreased by LY294002 and by small interfering RNA inhibition of PDK-1. Taken together, our data demonstrate a PI3K/PDK-1-dependent functional cooperation of NF-kappaB and JunD in the transcriptional regulation of CCND1 by serum.
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PMID:Jun D cooperates with p65 to activate the proximal kappaB site of the cyclin D1 promoter: role of PI3K/PDK-1. 1817 38

beta 1-3-Adrenoreceptor (AR)-deficient mice are unable to regulate energy expenditure and develop diet-induced obesity on a high-fat diet. We determined previously that beta2-AR agonist treatment activated expression of the mRNA encoding the orphan nuclear receptor, NOR-1, in muscle cells and plantaris muscle. Here we show that beta2-AR agonist treatment significantly and transiently activated the expression of NOR-1 (and the other members of the NR4A subgroup) in slow-twitch oxidative soleus muscle and fast-twitch glycolytic tibialis anterior muscle. The activation induced by beta-adrenergic signaling is consistent with the involvement of protein kinase A, MAPK, and phosphorylation of cAMP response element-binding protein. Stable cell lines transfected with a silent interfering RNA targeting NOR-1 displayed decreased palmitate oxidation and lactate accumulation. In concordance with these observations, ATP production in the NOR-1 silent interfering RNA (but not control)-transfected cells was resistant to (azide-mediated) inhibition of oxidative metabolism and expressed significantly higher levels of hypoxia inducible factor-1alpha. In addition, we observed the repression of genes that promote fatty acid oxidation (peroxisomal proliferator-activated receptor-gamma coactivator-1alpha/beta and lipin-1alpha) and trichloroacetic acid cycle-mediated carbohydrate (pyruvate) oxidation [pyruvate dehydrogenase phosphatase 1 regulatory and catalytic subunits (pyruvate dehydrogenase phosphatases-1r and -c)]. Furthermore, we observed that beta2-AR agonist administration in mouse skeletal muscle induced the expression of genes that activate fatty acid oxidation and modulate pyruvate use, including PGC-1alpha, lipin-1alpha, FOXO1, and PDK4. Finally, we demonstrate that NOR-1 is recruited to the lipin-1alpha and PDK-4 promoters, and this is consistent with NOR-1-mediated regulation of these genes. In conclusion, NOR-1 is necessary for oxidative metabolism in skeletal muscle.
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PMID:The orphan nuclear receptor, NOR-1, a target of beta-adrenergic signaling, regulates gene expression that controls oxidative metabolism in skeletal muscle. 1832 99

Tamoxifen is a mainstay in the treatment of estrogen receptor (ER)-positive breast cancer patients. Although the efficacy of tamoxifen has been attributed to induction of tumor cell growth arrest and apoptosis by inhibition of ER signaling, recent evidence indicates that tamoxifen possesses ER-independent antitumor activities. Here, we use OSU-03012, a small-molecule inhibitor of phosphoinositide-dependent protein kinase-1 (PDK-1) to address the hypothesis that PDK-1/Akt signaling represents a therapeutically relevant target to sensitize ER-negative breast cancer to tamoxifen. OSU-03012 sensitized both ER-positive MCF-7 and ER-negative MDA-MB-231 cells to the antiproliferative effects of tamoxifen in an ER-independent manner. Flow cytometric analysis of phosphatidylserine externalization revealed that this augmented suppression of cell viability was attributable to a marked enhancement of tamoxifen-induced apoptosis by OSU-03012. Mechanistically, this OSU-03012-mediated sensitization was associated with suppression of a transient tamoxifen-induced elevation of Akt phosphorylation and enhanced modulation of the functional status of multiple Akt downstream effectors, including FOXO3a, GSK3alpha/beta, and p27. The growth of established MDA-MB-231 tumor xenografts was suppressed by 50% after oral treatment with the combination of tamoxifen (60 mg/kg) and OSU-03012 (100 mg/kg), whereas OSU-03012 and tamoxifen alone suppressed growth by 30% and 0%, respectively. These findings indicate that the inhibition of PDK-1/Akt signaling to sensitize ER-negative breast cancer cells to the ER-independent antitumor activities of tamoxifen represents a feasible approach to extending the use of tamoxifen to a broader population of breast cancer patients. Considering the urgent need for novel therapeutic strategies for ER-negative breast cancer patients, this combinatorial approach is worthy of continued investigation.
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PMID:Sensitizing estrogen receptor-negative breast cancer cells to tamoxifen with OSU-03012, a novel celecoxib-derived phosphoinositide-dependent protein kinase-1/Akt signaling inhibitor. 3093 13

cAMP regulates a wide range of processes through its downstream effectors including PKA, and the family of guanine nucleotide exchange factors. Depending on the cell type, cAMP inhibits or stimulates growth and proliferation in a PKA-dependent or independent manner. PKA-independent effects are mediated by PI 3-kinases-Akt signaling and EPAC1 (exchange protein directly activated by cAMP) activation. Recently, we reported PKA-independent activation of the protein kinase Akt as well co-immunoprecipitation of Epac1 with Rap1, p-Akt(Thr-308), and p-Akt(Ser-473) in forskolin-stimulated macrophages. To further probe the role of Epac1 in Akt protein kinase activation and cellular proliferation, we employed the cAMP analog 8-CPT-2-O-Me-cAMP, which selectively binds to Epac1 and triggers Epac1 signaling. We show the association of Epac1 with activated Akt kinases by co-immunoprecipitation and GST-pulldown assays. Silencing Epac1 gene expression by RNA interference significantly reduced levels of Epac1 mRNA, Epac protein, Rap1 GTP, p-ERK1/2, p-B-Raf, p110alpha catalytic subunit of PI 3-kinase, p-PDK, and p-p(70s6k). Silencing Epac1 gene expression by RNA interference also suppressed 8-CPT-2-O-Me-cAMP-upregulated protein and DNA synthesis. Concomitantly, 8-CPT-2-O-Me-cAMP-mediated upregulation of Akt(Thr-308) protein kinase activity and p-Akt(Thr-308) levels was prevented in plasma membranes and nuclei of the cells. In contrast, silencing Epac1 gene expression reduced Akt(Ser-473) kinase activity and p-Akt(Ser-473) levels in plasma membranes, but showed negligible effects on nuclear activity. In conclusion, we show that cAMP-induced Akt kinase activation and cellular proliferation is mediated by Epac1 which appears to function as an accessory protein for Akt activation.
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PMID:The cAMP-activated GTP exchange factor, Epac1 upregulates plasma membrane and nuclear Akt kinase activities in 8-CPT-2-O-Me-cAMP-stimulated macrophages: Gene silencing of the cAMP-activated GTP exchange Epac1 prevents 8-CPT-2-O-Me-cAMP activation of Akt activity in macrophages. 1849 29

Activation of glomerular mesangial cells (MCs) by angiotensin II (Ang II) leads to hypertrophy and extracellular matrix accumulation. Here, we demonstrate that, in MCs, Ang II induces an increase in PDK-1 (3-phosphoinositide-dependent protein kinase-1) kinase activity that required its phosphorylation on tyrosine 9 and 373/376. Introduction into the cells of PDK-1, mutated on these tyrosine residues or kinase-inactive, attenuates Ang II-induced hypertrophy and fibronectin accumulation. Ang II-mediated PDK-1 activation and tyrosine phosphorylation (total and on residues 9 and 373/376) are inhibited in cells transfected with small interfering RNA for Src, indicating that Src is upstream of PDK-1. In cells expressing oxidation-resistant Src mutant C487A, Ang II-induced hypertrophy and fibronectin expression are prevented, suggesting that the pathway is redox-sensitive. Ang II also up-regulates Nox4 protein, and siNox4 abrogates the Ang II-induced increase in intracellular reactive oxygen species (ROS) generation. Small interfering RNA for Nox4 also inhibits Ang II-induced activation of Src and PDK-1 tyrosine phosphorylation (total and on residues 9 and 373/376), demonstrating that Nox4 functions upstream of Src and PDK-1. Importantly, inhibition of Nox4, Src, or PDK-1 prevents the stimulatory effect of Ang II on fibronectin accumulation and cell hypertrophy. This work provides the first evidence that Nox4-derived ROS are responsible for Ang II-induced PDK-1 tyrosine phosphorylation and activation through stimulation of Src. Importantly, this pathway contributes to Ang II-induced MC hypertrophy and fibronectin accumulation. These data shed light on molecular processes underlying the oxidative signaling cascade engaged by Ang II and identify potential targets for intervention to prevent renal hypertrophy and fibrosis.
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PMID:Nox4 NAD(P)H oxidase mediates Src-dependent tyrosine phosphorylation of PDK-1 in response to angiotensin II: role in mesangial cell hypertrophy and fibronectin expression. 1855 49

3'-phosphoinositide-dependent protein kinase-1 (PDK-1) is a crucial serine/threonine kinase in the insulin-like growth factor-I (IGF-I)/AKT signaling pathway, but its function and localization in the nervous system has not been fully characterized. In this study, we compared the localization of PDK-1 in adult neurons and non-neuronal PC-3 cells. We showed that PC-3 cells expressed phosphorylated and nonphosphorylated PDK-1 in the cytoplasm and nucleoplasm. In contrast, neuronal PDK-1 was located in the nucleoplasm and the phosphorylated form was located along the perinuclear region. Furthermore, we found that IGF-I transiently increased phosphorylation of neuronal PDK-1, resulting in its translocation to other cellular compartments. Our findings suggest that IGF-I may regulate neuronal PDK-1 differently than in non-neuronal cells, which may indicate a novel role for PDK-1 in IGF-I-mediated neuroprotective signaling.
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PMID:IGF-I regulated phosphorylation and translocation of PDK-1 in neurons. 1927 99

Deregulation of the 3-phosphoinositide-dependent protein kinase-1 (PDK-1)/Akt signalling pathway is associated with prostate cancer development and progression. Inhibition of PDK-1/Akt signalling can be achieved using structurally optimized celecoxib derivatives such as OSU-03012. In this study, we treated the novel canine prostate cancer cell line, Ace-1, with OSU-03012 or dimethyl sulphoxide in vitro. We found that Akt was constitutively phosphorylated in the canine prostate cancer cell line Ace-1 and that there was a dose-dependent decrease in cell viability, and Akt and glycogen synthase kinase-3beta phosphorylation, in response to OSU-03012 treatment. This was accompanied by a dose-dependent increase in apoptosis. These data suggest that Akt signalling pathway inhibition is a potential strategy for the treatment of dogs with prostate cancer and that canine prostate cancer is a relevant large animal model for evaluating Akt pathway inhibitors such as OSU-03012 for use in people.
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PMID:3-Phosphoinositide-dependent protein kinase-1/Akt signalling and inhibition in a canine prostate carcinoma cell line. 1975 1


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