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:2.4.2.30 (PARP)
13,611 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

14-3-3 proteins are a family of conserved dimeric molecules that bind to a range of cellular proteins involved in signal transduction and oncogenesis. Our solution of the crystal structure of 14-3-3zeta revealed a conserved amphipathic groove that may allow the association of 14-3-3 with diverse ligands (Liu, D., Bienkowska, J., Petosa, C., Collier, R. J., Fu, H., and Liddington, R. (1995) Nature 376, 191-194). Here, the contributions of three positively charged residues (Lys-49, Arg-56, and Arg-60) that lie in this Raf-binding groove were investigated. Two of the charge-reversal mutations greatly (K49E) or partially (R56E) decreased the interaction of 14-3-3zeta with Raf-1 kinase, whereas R60E showed only subtle effects on the binding. Interestingly, these mutations exhibited similar effects on the functional interaction of 14-3-3zeta with another target protein, exoenzyme S (ExoS), an ADP-ribosyltransferase from Pseudomonas aeruginosa. The EC50 values of 14-3-3zeta required for ExoS activation increased by approximately 110-, 5-, and 2-fold for the K49E, R56E, and R60E mutants, respectively. The drastic reduction of 14-3-3zeta/ligand affinity by the K49E mutation is due to a local electrostatic effect, rather than the result of a gross structural alteration, as evidenced by partial proteolysis and circular dichroism analysis. This work identifies the first point mutation (K49E) that dramatically disrupts 14-3-3zeta/ligand interactions. The parallel effects of this single point mutation on both Raf-1 binding and ExoS activation strongly suggest that diverse associated proteins share a common structural binding determinant on 14-3-3zeta.
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PMID:Raf-1 kinase and exoenzyme S interact with 14-3-3zeta through a common site involving lysine 49. 915 24

Raf-1 activation and Bcl-2 hyperphosphorylation following treatment with paclitaxel (Taxol) or other microtubule-active drugs is associated with mitotic arrest. Here we show that microtubule-active drugs do not activate the mitogen-activated protein kinase (MAPK) pathway in leukemia cells. PD98059, a MEK inhibitor, and SB202190, a p38 MAP kinase inhibitor, do not abrogate Bcl-2 phosphorylation nor apoptosis. Simultaneously with PARP cleavage, paclitaxel induces cleavage of Bcl-2 protein yielding a potentially pro-apoptotic 22 kDa product. In comparison, the stimulation of Raf-1 by phorbol ester (TPA) activates the MAPK pathway, causes MAPK-dependent p21WAF1/CIP1 induction, Rb dephosphorylation and growth arrest without Bcl-2 phosphorylation or apoptosis. Like TPA, cAMP induces p21WAF1/CIP1 but does not cause Bcl-2 phosphorylation. MEKK1 and Ras, upstream activators of JNK and ERK MAPK, also fail to induce Bcl-2 hyperphosphorylation. Although Lck tyrosine kinase has been recently implicated in Raf-1 activation during mitotic arrest, microtubule-active drugs induce Raf-1/Bcl-2 hyperphosphorylation and apoptosis in a Lck-deficient Jurkat cells. Therefore, microtubule-active drugs induce apoptosis which is associated with Raf-1 and Bcl-2 phosphorylation and Bcl-2 cleavage but is independent of the MAPK pathway. In contrast, TPA-activated MAPK pathway causes p21WAF1/CIP1-dependent growth arrest without apoptosis.
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PMID:Mitogen-activated protein kinase pathway is dispensable for microtubule-active drug-induced Raf-1/Bcl-2 phosphorylation and apoptosis in leukemia cells. 1040 Apr 18

Exoenzyme S (ExoS) is a mono-ADP-ribosyltransferase secreted by the opportunistic pathogen Pseudomonas aeruginosa. ExoS requires a eukaryotic factor, the 14-3-3 protein, for enzymatic activity. Here, two aspects of the activation of the ADP-ribosyltransferase activity of ExoS by 14-3-3 proteins are examined. Initial studies showed that several isoforms of 14-3-3, including beta, zeta, eta, sigma, and tau, activated ExoS with similar efficiency. This implicates a conserved structure in 14-3-3 that contributes to the interaction between 14-3-3 and ExoS. One candidate structure is the conserved amphipathic groove that mediates the 14-3-3/Raf-1 interaction. The next series of experiments examined the role of individual amino acids of the amphipathic groove of 14-3-3 zeta in ExoS activation and showed that ExoS activation required the basic residues lining the amphipathic groove of 14-3-3 zeta without extensive involvement of the hydrophobic residues. Strikingly, mutations of Val-176 of 14-3-3 zeta that disrupted its interaction with Raf-1 did not affect the binding and activation of ExoS by 14-3-3. Thus, ExoS selectively employs residues in the Raf-binding groove for its association with 14-3-3 proteins.
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PMID:Residues of 14-3-3 zeta required for activation of exoenzyme S of Pseudomonas aeruginosa. 1050 20

Drug resistance is a well recognized problem in cancer therapy. Despite the current dogma that drug resistance is always an obstacle for treatment, here I show that it provides opportunities for selective protection of non-resistant cells with killing of drug-resistant cancer cells. According to the proposed 'two-drug' strategy, the first drug should be ineffective against a target drug-resistant cell (ie the drug is a substrate of MRP or Pgp pumps). In addition, it must be cytostatic but not cytotoxic. The second drug, which is applied in sequence, must be a cycle-dependent apoptotic drug to which the target cell is not cross-resistant. Thus, low doses of adriamycin, etoposide and actinomycin D, used as the first drugs, were cytostatic to parental HL60 cells. Therefore, these drugs precluded Bcl-2/Raf-1 phosphorylation, PARP cleavage and cell death which are otherwise induced by paclitaxel, a mitosis-selective apoptotic drug for HL60 cells. In contrast, HL60/ADR cells which express MRP, a transporter which pumps out the first drugs from a cell, were insensitive to the first drugs and therefore readily underwent apoptosis following the second drug. This strategy also allowed a selective killing of HL60/TX cells which express MDR-1, with the only difference being that the second drug, paclitaxel, was substituted for epothilones, non-Pgp substrates. Lack of protection by the first drug, a Pgp substrate, resulted in HL60/TX killing by the second drug, whereas parental HL-60 cells were fully protected. Therefore, drug resistant cells can be selectively killed by a combination of drugs not killing sensitive cells. Lack of toxicity against normal cells will be clinically translated in reduction of adverse side-effects of chemotherapy against drug-resistant malignancies.
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PMID:Drug-resistance enables selective killing of resistant leukemia cells: exploiting of drug resistance instead of reversal. 1060 25

Nanomolar concentrations of Taxol, and other antimitotic agents that interact with microtubules, mediate serine phosphorylation of the 66-kDa Shc isoform (p66shc) in A549 human lung carcinoma cells, 9-18 h after drug treatment. This event coincides with the release of PARP cleavage fragments that are early indicators of apoptosis. Taxol-induced serine phosphorylation of p66shc results from a MEK-independent signaling pathway that is activated in A549 cells that have a prolonged or abnormal mitotic phase of the cell cycle [Cancer Res. 60 (2000) 5171]. In contrast, in murine macrophage RAW 264.7 cells, micromolar concentrations of Taxol but not other microtubule-interacting agents induced serine phosphorylation of p66shc that correlated with the phosphorylation of Raf-1 and extracellular signal-regulated kinase (ERK1/2), within 15-30 min after Taxol treatment. This event also was induced by lipopolysaccharide (LPS). The MEK-inhibitor, U0126, that specifically inhibits the activation of ERK also blocked the phosphorylation of p66shc and Raf-1, suggesting that these processes were MEK-dependent, quite different from that which was observed in A549 cells. Taxol also induced phosphorylation of p38 and JNK MAP kinases within 8-15 min after drug treatment. It is known that Taxol, but not other microtubule-interacting agents, induces the production of cytokines, such as tumor necrosis factor alpha (TNF-alpha) in mouse macrophages. The time course of Taxol-induced TNF-alpha expression coincides with that of Taxol-induced p66shc phosphorylation, and U0126 inhibits significantly Taxol-induced TNF-alpha expression in RAW 264.7 cells. Our data indicate that the Taxol-induced serine phosphorylation of p66shc in RAW 264.7 cells is microtubule-independent and may be related to increased TNF-alpha expression after Taxol and LPS treatment. It is concluded that the mechanisms involved in Taxol-induced p66shc phosphorylation are distinct in A549 and RAW 264.7 cells.
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PMID:Distinct mechanisms of taxol-induced serine phosphorylation of the 66-kDa Shc isoform in A549 and RAW 264.7 cells. 1206 70

14-3-3 proteins belong to a family of conserved molecules, which play a regulatory role and participate in signal transduction and checkpoint control pathways. 14-3-3 proteins bind phosphoserine-phosphorylated ligands, such as the Raf-1 kinase and Bad, through recognition of the phosphorylated consensus motif, RSXpSXP (where pS is phosphoserine). Recently, a phosphorylation-independent interaction has been reported to occur between 14-3-3 and a small number of proteins, for example the 43 kDa inositol polyphosphate 5-phosphatase, glycoprotein Ib, p75NTR-associated cell-death executor (NADE) and the bacterial ADP-ribosyltransferase toxin exoenzyme S (ExoS). It has been suggested that specific residues of 14-3-3 proteins are required for activation of the bacterial toxin ExoS. An unphosphorylated peptide derived from a phage display library, known as the R18 peptide, and a synthetic peptide derived from ExoS inhibit the interaction between ExoS and 14-3-3. In this report we identify the amino acid sequence on ExoS which is responsible for its specific interaction with 14-3-3, both in vitro and in vivo. In addition, we believe that this interaction is critical for the ADP-ribosylation of an endogenous target, Ras, by ExoS both in vitro and in vivo. Loss of the 14-3-3-binding site on ExoS results in an ExoS molecule that is unable to efficiently inactivate Ras and shows a reduced capacity to change the morphology of infected cells, together with reduced killing activity.
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PMID:Exoenzyme S binds its cofactor 14-3-3 through a non-phosphorylated motif. 1219 3

By inducing p53-dependent G2 arrest, the pretreatment with low concentrations of DNA damaging drugs (e.g., doxorubicin, DOX) can prevent cell death caused by microtubule-active drugs (e.g., paclitaxel, PTX), thus potentially permitting selective killing of p53-deficient cancer cells. However, DOX still protects a subset of tumor cell lines lacking wt p53 (HL60 and Jurkat leukemia cells), thus limiting the utility of protection of cells with wt p53 (e.g., normal cells). The present work overcomes this obstacle by adding an abrogator of p53-independent checkpoint (e.g., UCN-01) to the DOX-PTX sequence. By inhibiting a p53-independent pathway, UCN-01 overrode DOX-induced G2 arrest and instead induced G1 arrest in HL60 and Jurkat, thus propelling these p53-deficient cells from G2 to G1. Once they entered mitosis, cells were killed by PTX. Induction of G2 arrest with sequential abrogation of a p53-independent checkpoint allows pharmacological manipulation of Raf-1/Bcl-2 hyperphosphorylation, PARP and Rb cleavage and cell death caused by PTX in p53-deficient cells. Unlike previous approaches, this strategy is intended to increase selectivity, not the cytotoxicity of PTX. This rational sequence of agents that induces p53-dependent and abrogates p53-independent arrest represents a cancer-selective strategy for treatment of p53-deficient tumors.
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PMID:Sequential activation and inactivation of G2 checkpoints for selective killing of p53-deficient cells by microtubule-active drugs. 1221 65

Neuroblastomas are the most common extracranial solid tumors of childhood. These tumors are associated with an overall poor prognosis, particularly for advanced stage disease. The benzoquinone ansamycin antibiotic, geldanamycin (GA), exhibits potent antitumor activity in certain cancer cell lines by destabilizing important signal transduction proteins (e.g., Raf-1 and Akt). The purpose of our study was to determine whether GA can alter the expression of Raf-1 and Akt, which have been shown to be critical for neuronal cell survival, and induce apoptosis of neuroblastoma cells. Human neuroblastoma cells (SH-SY5Y, SK-N-SH and LAN-1) were treated with GA for a variable period of time. Cell viability was assessed with MTT assays. Apoptosis was assessed with DNA fragmentation ELISA, TUNEL-flow cytometric assay, Western blot and caspase activities. We found that GA decreases cell viability and induces apoptosis in the SH-SY5Y human neuroblastoma cell line. These effects were mediated through activation of caspase-9 and -3, mitochondrial release of cytochrome c and subsequent PARP cleavage. GA-induced apoptosis was associated with a reduction in the level and activity of Raf-1 and Akt. The importance of these proteins was further demonstrated by induction of apoptosis in SH-SY5Y cells by a combination of U0126 (MEK1/2 inhibitor) and LY294002 (an inhibitor of PI3K). Similar to SH-SY5Y cells, other human neuroblastoma cells (SK-N-SH and LAN-1) were sensitive to the effects of GA-induced apoptosis. Taken together, our findings suggest that GA may be a novel therapeutic agent, which may be effective in the treatment of neuroblastomas.
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PMID:Geldanamycin decreases Raf-1 and Akt levels and induces apoptosis in neuroblastomas. 1247 18

Interactions between the proteasome inhibitor bortezomib and histone deacetylase inhibitors (HDIs) have been examined in Bcr/Abl+ human leukemia cells (K562 and LAMA 84). Coexposure of cells (24-48 hours) to minimally toxic concentrations of bortezomib + either suberoylanilide hydroxamic acid (SAHA) or sodium butyrate (SB) resulted in a striking increase in mitochondrial injury, caspase activation, and apoptosis, reflected by caspases-3 and -8 cleavage and poly(adenosine diphosphate-ribose) polymerase (PARP) degradation. These events were accompanied by down-regulation of the Raf-1/mitogen-induced extracellular kinase (MEK)/extracellular signal-related kinase (ERK) pathway as well as diminished expression of Bcr/Abl and cyclin D1, cleavage of p21CIP1 and phosphorylation of the retinoblastoma protein (pRb), and induction of the stress-related kinases Jun kinase (JNK) and p38 mitogen-activated protein kinase (MAPK). Transient transfection of cells with a constitutively active MEK construct significantly protected them from bortezomib/SAHA-mediated lethality. Coadministration of bortezomib and SAHA resulted in increased reactive oxygen species (ROS) generation and diminished nuclear factor kappa B (NF-kappa B) activation; moreover, the free radical scavenger L-N-acetylcyteine (LNAC) blocked bortezomib/SAHA-related ROS generation, induction of JNK and p21CIP1, and apoptosis. Lastly, this regimen potently induced apoptosis in STI571 (imatinib mesylate)-resistant K562 cells and CD34+ mononuclear cells obtained from a patient with STI571-resistant disease, as well as in Bcr/Abl- leukemia cells (eg, HL-60, U937, Jurkat). Together, these findings raise the possibility that combined proteasome/histone deacetylase inhibition may represent a novel strategy in leukemia, including apoptosis-resistant Bcr/Abl+ hematologic malignancies.
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PMID:The proteasome inhibitor bortezomib interacts synergistically with histone deacetylase inhibitors to induce apoptosis in Bcr/Abl+ cells sensitive and resistant to STI571. 1289 73

Docetaxel (Taxotere, DTX) is a promoter of apoptosis in cancer cells. Since cytotoxic mechanisms of DTX are not yet fully understood, we have investigated the effects of DTX on apoptosis and ras-->Erk-mediated signal transduction in human epidermoid KB, colon HT-29 and breast HCC1937 cancer cells. We have found that the exposure to 0.78 or 1.56 or 2.5 ng/ml DTX for 48 h induced apoptosis and growth inhibition in about 50 % of KB, HCC1937 and HT-29 cell population, respectively. In these experimental conditions, PARP and caspase 3 cleavage was also showed in all cell lines. KB and HCC1937 cells express a wild type p53 while HT-29 display a mutated form. Interestingly, we have found that DTX reduces the expression of mutated p53 in HT-29 and increases the expression of wild type in KB and HCC1937 cells. Moreover, DTX reduces ubiquitination of the wild type p53 in KB and HCC1937 cells and increases the ubiquitin-conjugated form of mutated p53 in HT-29 cells. Furthermore, exposure of cancer cells to DTX for 48 h increases the expression and activity of Ras and up-regulates Raf-1 and the phosphorylated isoforms of Erk-1/2. On the bases of these data, we have hypothesized that the increased activity of the ras-->erk-dependent pathway induced by DTX could be a protective signalling from the apoptosis caused by the drug. Therefore, we have used R115777, a farnesyl transferase inhibitor that inactivates ras, in combination with DTX. The combined treatment with DTX and R115777 resulted in a strong synergism in growth inhibition in the three cell lines. These data suggest the use of the combination in these therapeutic settings even if further experiments are required for the clinical translation.
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PMID:Docetaxel induces p53-dependent apoptosis and synergizes with farnesyl transferase inhibitor r115777 in human epithelial cancer cells. 1597 May 18


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