Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.2 (PDK1)
 2,238 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We explored three approaches to create tissue-specific knock-in mice by generating knock-in mice in which a substrate-docking site of the PDK1 protein kinase was ablated in Cre-expressing tissues in a way that prevented activation of one of its substrates, p70 ribosomal S6 kinase (S6K), but not another (protein kinase B (PKB)). Employing two of the approaches, termed the "heterozygous" and "minigene" methods, we generated mice in which Cre-expressing skeletal and cardiac muscle produced the mutant rather than wild type PDK1. Consistent with this, injection of these mice with insulin only induced activation of PKB but not S6K in muscle tissues. We have also demonstrated that insulin-stimulated glucose uptake proceeds normally in knock-in mice, consistent with the notion that PKB mediates this process. In contrast to conditional knock-out of PDK1 in muscle, the knock-in mice did not develop dilated cardiomyopathy, suggesting that PKB plays a key role in protecting mice from heart failure. The third knock-in strategy that was evaluated, termed the "inversion" method, did not proceed with high efficiency. We discuss the merits and disadvantages of each of the conditional knock-in approaches, along with the applications for which they may be most suited, and suggest how they could be further refined.
J Biol Chem 2006 Sep 29
PMID:Evaluation of approaches to generation of tissue-specific knock-in mice. 1688 94

To elucidate the role of 3-phosphoinositide-dependent protein kinase-1 (PDK1) in cellular signaling, we constructed and expressed a pseudosubstrate of PDK1, designated as deltaAL-PIF, and characterized its properties in cultured cells. deltaAL-PIF consists of two fused proteins of the protein kinase Cdelta (deltaPKC) activation loop (deltaAL) and PDK1-interacting fragment (PIF). The phosphorylation of deltaAL-PIF was detected with anti-deltaPKC phospho-Thr505-specific antibody and was increased in proportion to the expression level of co-expressed GST-PDK1, indicating that it acts as a pseudosubstrate of PDK1. In cells expressing deltaAL-PIF, basal phosphorylation level at the activation loop of PKBalpha, deltaPKC and gammaPKC was reduced, compared with that in control cells, suggesting that deltaAL-PIF functions as an inhibitory molecule for PDK1. deltaAL-PIF affected the stability, translocation and endogenous activity of PKCs. These effects of deltaAL-PIF on gammaPKC properties were confirmed by investigation using conditioned PDK1 knockout cells. Furthermore, apoptosis frequently occurred in cells expressing deltaAL-PIF for 3 days. These findings revealed that deltaAL-PIF served as an effective pseudosubstrate and an inhibitory molecule for PDK1, suggesting that this molecule can be used as a tool for investigating PDK-mediated cellular functions as well as being applicable for anti-cancer therapy.
Genes Cells 2006 Sep
PMID:Fused protein of deltaPKC activation loop and PDK1-interacting fragment (deltaAL-PIF) functions as a pseudosubstrate and an inhibitory molecule for PDK1 when expressed in cells. 1692 25

Our previous studies have demonstrated that hypoxic precondition (HPC) increased membrane translocation of protein kinase C isoforms and decreased phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) in the brain of mice. The goal of this study was to determine the involvement of p90 KD ribosomal S6 kinase (RSK) in cerebral HPC of mice. Using Western-blot analysis, we found that the levels of membrane/nuclear translocation, but not protein expression of RSK increased significantly in the frontal cortex and hippocampus of HPC mice. In addition, we found that the phosphorylation levels of RSK at the Ser227 site (a PDK1 phosphorylation site), but not at the Thr359/Ser363 sites (ERK1/2 phosphorylated sites) increased significantly in the brain of HPC mice. Similar results were confirmed by an immunostaining study of total RSK and phospho-Ser227 RSK. To further define the cellular populations to express phospho-Ser227 RSK, we found that the expression of phospho-Ser227 RSK co-localized with neurogranin, a neuron-specific marker, in cortex and hippocampus of HPC mice by using double-labeled immunofluorescent staining method. These results suggest that increased RSK membrane/nuclear translocation and PDK1 mediated neuron-specific phosphorylation of RSK at Ser227 might be involved in the development of cerebral HPC of mice.
Neurochem Res 2007 Sep
PMID:Increased membrane/nuclear translocation and phosphorylation of p90 KD ribosomal S6 kinase in the brain of hypoxic preconditioned mice. 1740 33

Rituximab (chimeric anti-CD20 monoclonal antibody) is currently being used, alone or in combination with chemotherapy, in the treatment of B-non-Hodgkin's lymphoma (B-NHL). We have reported that rituximab treatment of B-NHL cell lines sensitizes the drug-resistant tumor cells to apoptosis by various chemotherapeutic drugs and chemosensitization was, in large part, owing to the selective inhibition of the anti-apoptotic Bcl-(XL) gene product. The constitutive activation of the Akt pathway in B-NHL results in overexpression and functional activation of Bcl-(xL). Hence, we hypothesized that rituximab-induced inhibition of Bcl-(xL) expression and chemosensitization may result, in part, from its inhibitory activity of the Akt pathway. This hypothesis was tested using the drug-resistant Ramos and Daudi B-NHL cell lines. Time kinetic analysis revealed that treatment with rituximab inhibited phosphorylation of Akt, but not unphosphorylated Akt, and the inhibition was first detected at 6 h post-rituximab treatment. Similar time kinetics revealed rituximab-induced inhibition of p-PDK1, p-Bad, p-IKKalpha/beta and p-Ikappabetaalpha and no inhibition of unphosphorylated proteins. In addition, rituximab treatment resulted in significant increase of Bcl-(xL)-Bad heterodimeric complexes as compared to untreated cells. The role of the Akt pathway in the regulation of resistance was corroborated by the use of the Akt inhibitor, LY294002, and by transfection with siRNA Akt. Treatment of tumor cells with LY294002 or with Akt siRNA, but not control siRNA, resulted in inhibition of Bcl-(xL) expression and sensitization to drug-induced apoptosis. Although rituximab did not inhibit the Akt pathway nor sensitized the rituximab-resistant Ramos RR1 clone, treatment with LY294002 or Akt siRNA sensitized the clone to drug-induced apoptosis. The present findings demonstrate for the first time that rituximab inhibits the constitutively activated Akt pathway in B-NHL cell lines, and this inhibition contributes to sensitization of drug-resistant cells to apoptosis by chemotherapeutic drugs. The findings also identify the Akt pathway as target for therapeutic intervention in the reversal of rituximab and drug-resistant B-NHL.
Oncogene 2007 Sep 13
PMID:Rituximab inhibits the constitutively activated PI3K-Akt pathway in B-NHL cell lines: involvement in chemosensitization to drug-induced apoptosis. 2734 1

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.
Mol Pharmacol 2007 Sep
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

Statins activate phosphatidylinositol-3-kinase, which activates ecto-5'-nucleotidase and phosphorylates 3-phosphoinositide-dependent kinase-1 (PDK-1). Phosphorylated (P-)PDK-1 phosphorylates Akt, which phosphorylates endothelial nitric oxide synthase (eNOS). We asked if the blockade of adenosine receptors (A(1), A(2A), A(2B), or A(3) receptors) could attenuate the induction of Akt and eNOS by atorvastatin (ATV) and whether ERK1/2 is involved in the ATV regulation of Akt and eNOS. In protocol 1, mice received intraperitoneal ATV, theophylline (TH), ATV + TH, or vehicle. In protocol 2, mice received intraperitoneal injections of ATV, U0126 (an ERK1/2 inhibitor), ATV + U0126, or vehicle; 8 h later, hearts were assessed by immunoblot analysis. In protocol 3, mice received intraperitoneal ATV alone or with 8-sulfophenyltheophylline (SPT); 1, 3, and 6 h after injection, hearts were assessed by immunoblot analysis. In protocol 4, mice received intraperitoneal ATV alone or with SPT, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), 1,3,7-trimethyl-8-(3-chlorostyryl)xanthine (CSC), alloxazine, or MRS-1523; 3 h after injection, hearts were assessed by immunoblot analysis. ATV increased P-ERK, P-PDK-1, Ser(473) P-Akt, Thr(308) P-Akt, and P-eNOS levels. TH blocked ATV-induced increases in P-ERK, Ser(473) P-Akt, Thr(308) P-Akt, and P-eNOS levels without affecting the induction of P-PDK-1 by ATV. U0126 blocked the ATV induction of Ser(473) P-Akt and Thr(308) P-Akt while attenuating the induction of P-eNOS. A detectable increase in P-ERK, Ser(473) P-Akt and P-eNOS was seen 3 and 6 h after injection but not at 1 h. DPCPX, CSC, and alloxazine partially blocked the ATV induction of P-ERK, Ser(473) P-Akt, and P-eNOS. In conclusion, blockade of adenosine A(1), A(2A), and A(2B) receptors but not A(3) receptors inhibited the induction of Akt and eNOS by statins. Adenosine was required for ERK1/2 activation by statins, which resulted in Akt and eNOS phosphorylation.
Am J Physiol Heart Circ Physiol 2007 Sep
PMID:The central role of adenosine in statin-induced ERK1/2, Akt, and eNOS phosphorylation. 1761 49

The purpose of this study was to determine whether OE treatment affects the expression of genes related to lipid metabolism under two physiological conditions: late pregnancy and mid-lactation, both characterized by lipid mobilization. Samples of periovarian and retroperitoneal adipose tissue from 21-day pregnant or 15-day lactating dams were used. The expression of LPL, FATP1, FABP4, HSL, ACC1, FAS, PEPCK, GLUT4, PDK4, SREBP1c, adiponutrin and leptin, were compared with their expression in virgin rats. In pregnant rats, FABP4, HSL, PEPCK and PDK4 were over expressed in the periovarian site compared to virgin rats, whereas adiponutrin, FAS, GLUT4 and SREBP1c were underexpressed; the retroperitoneal fat depot showed a similar pattern but ACC1 and leptin were also underexpressed. OE treatment caused a generalized decrease in gene expression in both adipose depots. In lactating dams, the gene expression profile at the periovarian depot was similar to that observed in pregnant rats. OE treatment mimicked the trend observed in pregnant rats, although the intensity of the gene expression changes was lower. After OE treatment, the retroperitoneal adipose depot showed a completely different pattern since the values were close to those of virgin rats. These results corroborate that OE effects in adipose tissue, lowering lipids and depressing their metabolism, already described under other physiological situations, can be also found in late pregnancy and lactation.
Lipids 2007 Sep
PMID:Oleoyl-estrone treatment to late pregnant and mid-lactating rats affects the expression of lipid metabolism genes. 1762 18

The nuclear receptors peroxisome proliferator-activated receptors (PPARs) are known for their critical role in the metabolic syndrome. Here, we show that they are direct regulators of the family of pyruvate dehydrogenase kinase (PDK) genes, whose products act as metabolic homeostats in sensing hunger and satiety levels in key metabolic tissues by modulating the activity of the pyruvate dehydrogenase complex. Mis-regulation of this tightly controlled network may lead to hyperglycemia. In human embryonal kidney cells we found the mRNA expression of PDK2, PDK3 and PDK4 to be under direct primary control of PPAR ligands, and in normal mouse kidney tissue Pdk2 and Pdk4 are PPAR targets. Both, treatment of HEK cells with PPARbeta/delta-specific siRNA and the genetic disruption of the Pparbeta/delta gene in mouse fibroblasts resulted in reduced expression of Pdk genes and abolition of induction by PPARbeta/delta ligands. These findings suggest that PPARbeta/delta is a key regulator of PDK genes, in particular the PDK4/Pdk4 gene. In silico analysis of the human PDK genes revealed two candidate PPAR response elements in the PDK2 gene, five in the PDK3 gene and two in the PDK4 gene, but none in the PDK1 gene. For seven of these sites we could demonstrate both PPARbeta/delta ligand responsiveness in context of their chromatin region and simultaneous association of PPARbeta/delta with its functional partner proteins, such as retinoidXreceptor, co-activator and mediator proteins and phosphorylated RNA polymerase II. In conclusion, PDK2, PDK3 and PDK4 are primary PPARbeta/delta target genes in humans underlining the importance of the receptor in the control of metabolism.
J Mol Biol 2007 Sep 14
PMID:Three members of the human pyruvate dehydrogenase kinase gene family are direct targets of the peroxisome proliferator-activated receptor beta/delta. 1766 20

Rhabdomyosarcoma (RMS) is the most common paediatric soft-tissue sarcoma including two major subtypes, alveolar rhabdomyosarcoma (ARMS) and embryonal rhabdomyosarcoma (ERMS). Increasing evidence suggests that oncogenesis of RMS involves multiple stages of signalling protein dysregulation which may include prolonged activation of serine/threonine kinases such as phosphoinositide-dependent kinase-1 (PDK-1) and AKT. To date, whether PDK-1/AKT pathway is activated in RMS is unknown. This study was to examine phosphorylation status of AKT and to evaluate a novel small molecular inhibitor, OSU-03012 targeting PDK-1 in RMS. We examined phosphorylation levels of AKT using ARMS and ERMS tissue microarray and immunohistochemistry staining. Our results showed phospho-AKT(Thr308) level is elevated 42 and 35% in ARMS and ERMS, respectively. Phospho-AKT(Ser473) level is also increased 43% in ARMS and 55% in ERMS. Furthermore, we showed that OSU-03012 inhibits cell viability and induces apoptosis in ARMS and ERMS cell lines (RH30, SMS-CTR), which express elevated phospho-AKT levels. Normal cells are much less sensitive to OSU-03012 and in which no detectable apoptosis was observed. This study showed, for the first time, that PDK-1/AKT pathway is activated in RMS and may play an important role in survival of RMS. PDK-1/AKT pathway may be an attractive therapeutic target for cancer intervention in RMS using OSU-03012.
Br J Cancer 2007 Sep 17
PMID:PDK-1/AKT pathway as a novel therapeutic target in rhabdomyosarcoma cells using OSU-03012 compound. 1784 13

Immunohistochemical evaluation of lung carcinomas for key enzymes involved in cellular metabolism (lactate dehydrogenase LDH 1 and 5, pyruvate dehydrogenase PDH, pyruvate dehydrogenase kinase PDHK-1, monocarboxylate transporters MCT 1, 2 and 4, glucose transporter GLUT1, hypoxia inducible factors HIF1alpha and 2alpha) show a complementary metabolic profile between cancer cells and tumor-associated stroma. Cancer cells share enzyme/transporter activities suggestive of an anaerobic metabolism with high affinity for glucose absorption, anaerobic glycolysis and lactate extrusion. On the other hand, the tumour-associated fibroblasts express patterns involved in aerobic pathways and lactate oxidation. These findings bring forward the hypothesis that tumor associated stroma is an accomplice in tumor growth and survival sustaining an independent cellular and metabolic tumor domain. The development of agents exploiting such cancer specific metabolic pathways may prove of importance in the treatment of lung cancer.
Cancer Biol Ther 2007 Sep
PMID:Lung cancer: a comparative study of metabolism related protein expression in cancer cells and tumor associated stroma. 1788 95


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