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1.
The 26S proteasome is an ATP-dependent proteolytic complex found in all eukaryotes, archaebacteria, and some eubacteria. Inhibition
of the 26S proteasome causes pleiotropic effects in cells, including cellular apoptosis, a fact that has led to the use of
the 26S proteasome inhibitor, bortezomib, for treatment of the multiple myeloma cancer. We previously showed that in addition
to the effects of proteolysis, inhibition of the 26S proteasome causes a rapid decrease in the protein synthesis rate due
to phosphorylating alfa subunit of the eukaryotic translation initiation factor 2 (eIF2α) by the heme-regulated inhibitor
kinase (HRI). In order to test whether inhibition of the 26S proteasome causes the same effect in cancer cells, we have investigated
the influence of two commonly used proteasome inhibitors, bortezomib and MG132, on the phosphorylation status of eIF2α in
B16F10 melanoma and 4T1 breast cancer cells. It was found that both of the inhibitors caused rapid phosphorylation of eIF2α.
Taking into account that the Hsp70 is a critical component needed for the HRI activation and enzymatic activity, we have tested
a possible participation of this protein in the eIF2α phosphorylation event. However, treatment of the cells with two structurally
different Hsp70 inhibitors, quercetin and KNK437, in the presence of the proteasome inhibitors did not affect the eIF2α phosphorylation.
In addition, neither protein kinase C (PKC) nor p38 mitogen-activated protein kinase (MAPK) was required for the proteasome
inhibitor-induced eIF2α phosphorylation; furthermore, both the PKC inhibitor staurosporine and the p38 MAPK inhibitor SB203580
caused enchanced phosphorylation of eIF2α. Zinc(II) protoporphyrine IX (ZnPP), an inhibitor of the heme-oxygenase-1 (HO-1),
which has also been previously reported to be involved in HRI activation, also failed to prevent the induction of eIF2α phosphorylation
in the presence of the proteasome inhibitor bortezomib or MG132. 相似文献
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3.
DSG (15-deoxyspergualin), an immunosuppressant with tumoricidal properties, binds potently to the regulatory C-terminal 'EEVD' motif of Hsps (heat-shock proteins). In the present study we demonstrate that DSG inhibits eukaryotic protein synthesis by sequestering Hsp70 which is required for maintaining HRI (haem-regulated inhibitor), a kinase of the eIF2alpha (eukaryotic initiation factor 2alpha), inactive. DSG stalled initiation of protein synthesis through phosphorylation of HRI and eIF2alpha. Addition of a recombinant eIF2alpha (S51A) protein, which lacks the phosphorylation site, lowered the inhibitory potential of DSG in reticulocyte lysate. The inhibitory effect of DSG was also attenuated in HRI knockdown cells. Moreover, exogenous addition of Hsp70 or the peptide 'EEVD' reversed the inhibitory effect of DSG. Interestingly, the inhibitory effect of DSG in different mammalian cancer cells was found to negatively correlate with the amount of Hsp70 expressed in the cells, emphasizing the link with Hsp70 in DSG inhibition of eukaryotic translation. 相似文献
4.
Raven JF Baltzis D Wang S Mounir Z Papadakis AI Gao HQ Koromilas AE 《The Journal of biological chemistry》2008,283(6):3097-3108
Cyclin D1 plays a critical role in controlling the G(1)/S transition via the regulation of cyclin-dependent kinase activity. Several studies have indicated that cyclin D1 translation is decreased upon activation of the eukaryotic initiation factor 2alpha (eIF2alpha) kinases. We examined the effect of activation of the eIF2alpha kinases PKR and PKR-like endoplasmic reticulum kinase (PERK) on cyclin D1 protein levels and translation and determined that cyclin D1 protein levels decrease upon the induction of PKR and PERK catalytic activity but that this decrease is not due to translation. Inhibition of the 26 S proteasome with MG132 rescued cyclin D1 protein levels, indicating that rather than inhibiting translation, PKR and PERK act to increase cyclin D1 degradation. Interestingly, this effect still requires eIF2alpha phosphorylation at serine 51, as cyclin D1 remains unaffected in cells containing a non-phosphorylatable form of the protein. This proteasome-dependent degradation of cyclin D1 requires an intact ubiquitination pathway, although the ubiquitination of cyclin D1 is not itself affected. Furthermore, this degradation is independent of phosphorylation of cyclin D1 at threonine 286, which is mediated by the glycogen synthase kinase 3beta and mitogen-activated protein kinase pathways as described in previous studies. Our study reveals a novel functional cross-talk between eIF2alpha phosphorylation and the proteasomal degradation of cyclin D1 and that this degradation is dependent upon eIF2alpha phosphorylation during short, but not prolonged, periods of stress. 相似文献
5.
Translation Initiation Control by Heme-Regulated Eukaryotic Initiation Factor 2α Kinase in Erythroid Cells under Cytoplasmic Stresses
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Cytoplasmic stresses, including heat shock, osmotic stress, and oxidative stress, cause rapid inhibition of protein synthesis in cells through phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha) by eIF2alpha kinases. We have investigated the role of heme-regulated inhibitor (HRI), a heme-regulated eIF2alpha kinase, in stress responses of erythroid cells. We have demonstrated that HRI in reticulocytes and fetal liver nucleated erythroid progenitors is activated by oxidative stress induced by arsenite, heat shock, and osmotic stress but not by endoplasmic reticulum stress or nutrient starvation. While autophosphorylation is essential for the activation of HRI, the phosphorylation status of HRI activated by different stresses is different. The contributions of HRI in various stress responses were assessed with the aid of HRI-null reticulocytes and fetal liver erythroid cells. HRI is the only eIF2alpha kinase activated by arsenite in erythroid cells, since HRI-null cells do not induce eIF2alpha phosphorylation upon arsenite treatment. HRI is also the major eIF2alpha kinase responsible for the increased eIF2alpha phosphorylation upon heat shock in erythroid cells. Activation of HRI by these stresses is independent of heme and requires the presence of intact cells. Both hsp90 and hsc70 are necessary for all stress-induced HRI activation. However, reactive oxygen species are involved only in HRI activation by arsenite. Our results provide evidence for a novel function of HRI in stress responses other than heme deficiency. 相似文献
6.
Phosphorylation of eukaryotic initiation factor 2 by heme-regulated inhibitor kinase-related protein kinases in Schizosaccharomyces pombe is important for fesistance to environmental stresses
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Zhan K Vattem KM Bauer BN Dever TE Chen JJ Wek RC 《Molecular and cellular biology》2002,22(20):7134-7146
Protein synthesis is regulated by the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha) in response to different environmental stresses. One member of the eIF2alpha kinase family, heme-regulated inhibitor kinase (HRI), is activated under heme-deficient conditions and blocks protein synthesis, principally globin, in mammalian erythroid cells. We identified two HRI-related kinases from Schizosaccharomyces pombe which have full-length homology with mammalian HRI. The two HRI-related kinases, named Hri1p and Hri2p, exhibit autokinase and kinase activity specific for Ser-51 of eIF2alpha, and both activities were inhibited in vitro by hemin, as previously described for mammalian HRI. Overexpression of Hri1p, Hri2p, or the human eIF2alpha kinase, double-stranded-RNA-dependent protein kinase (PKR), impeded growth of S. pombe due to elevated phosphorylation of eIF2alpha. Cells from strains with deletions of the hri1(+) and hri2(+) genes, individually or in combination, exhibited a reduced growth rate when exposed to heat shock or to arsenic compounds. Measurements of in vivo phosphorylation of eIF2alpha suggest that Hri1p and Hri2p differentially phosphorylate eIF2alpha in response to these stress conditions. These results demonstrate that HRI-related enzymes are not unique to vertebrates and suggest that these eIF2alpha kinases are important participants in diverse stress response pathways in some lower eukaryotes. 相似文献
7.
Shibuya Y Hirasawa N Sakai T Togashi Y Muramatsu R Ishii K Yamashita M Takayanagi M Ohuchi K 《Life sciences》2004,75(4):435-446
The role of p44/42 mitogen-activated protein kinase (MAPK) in the expression of intercellular adhesion molecule-1 (ICAM-1) in NCI-H292 cells, a human bronchial epithelial cell line, was analyzed. Treatment with the protein kinase C (PKC) activator 12-O-tetradecanoylphorbol 13-acetate (TPA) (16.2 nM) or interferon-gamma (IFN-gamma) (100 U/ml) induced phosphorylation of p44/42 MAPK. The MEK inhibitor U0126 (0.1 to 10 microM) enhanced the TPA-induced ICAM-1 expression but not the IFN-gamma-induced one. U0126 also enhanced the ICAM-1 expression induced by two other PKC activators teleocidin (22.5 nM) and aplysiatoxin (14.9 nM). Furthermore, PD98059 (0.5 to 50 microM), another MEK inhibitor, enhanced the TPA-induced ICAM-1 expression as well. The inhibitor of p38 MAPK SB203580 did not affect the TPA-induced ICAM-1 expression. BAY11-7082, an inhibitor of nuclear factor kappaB (NF-kappaB) activation, and MG132, a 26S proteasome inhibitor, reduced the TPA-induced ICAM-1 expression but not the IFN-gamma-induced one. TPA partially decreased the level of IkappaB-alpha and the reduction was further augmented by U0126 in a concentration-dependent manner. These findings suggested that, in NCI-H292 cells, p44/42 MAPK suppresses PKC activator-induced NF-kappaB activation, thus negatively regulating the PKC activator-induced ICAM-1 expression but not the IFN-gamma-induced one. 相似文献
8.
Background
Cytoplasmic stress granules (SGs) are specialized storage sites of untranslated mRNAs whose formation occurs under different stress conditions and is often associated with cell survival. SGs-inducing stresses include radiations, hypoxia, viral infections, and chemical inhibitors of specific translation initiation factors. The FDA-approved drug bortezomib (Velcade®) is a peptide boronate inhibitor of the 26S proteasome that is very efficient for the treatment of myelomas and other hematological tumors. Solid tumors are largely refractory to bortezomib. In the present study, we investigated the formation of SGs following bortezomib treatment.Results
We show that bortezomib efficiently induces the formation of SGs in cancer cells. This process involves the phosphorylation of translation initiation factor eIF2α by heme-regulated inhibitor kinase (HRI). Depletion of HRI prevents bortezomib-induced formation of SGs and promotes apoptosis.Conclusions
This is the first study describing the formation of SGs by a chemotherapeutic compound. We speculate that the activation of HRI and the formation of SGs might constitute a mechanism by which cancer cells resist bortezomib-mediated apoptosis.9.
10.
Ito H Iwamoto I Inaguma Y Takizawa T Nagata K Asano T Kato K 《Journal of cellular biochemistry》2005,95(5):932-941
There are several reports describing participation of small heat shock proteins (sHsps) in cellular protein quality control. In this study, we estimated the endoplasmic reticulum (ER) stress-induced response of Hsp27 and alphaB-crystallin in mammalian cells. Treatment targeting the ER with tunicamycin or thapsigargin induced the phosphorylation of Hsp27 but not of alphaB-crystallin in U373 MG cells, increase being observed after 2-10 h and decline at 24 h. Similar phosphorylation of Hsp27 by ER stress was also observed with U251 MG and HeLa but not in COS cells and could be blocked using SB203580, an inhibitor of p38 MAP kinase. Other protein kinase inhibitors, like G?6983, PD98059, and SP600125, inhibitors of protein kinase C (PKC), p44/42 MAP kinase, and JNK, respectively, were without major influence. Prolonged treatment with tunicamycin but not thapsigargin for 48 h caused the second induction of the phosphorylation of Hsp27 in U251 MG cells. Under these conditions, the intense perinuclear staining of Hsp27, with some features of aggresomes, was observed in 10%-20% of the cells. 相似文献
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12.
Neznanov N Dragunsky EM Chumakov KM Neznanova L Wek RC Gudkov AV Banerjee AK 《PloS one》2008,3(4):e1887
Proteasome activity is an important part of viral replication. In this study, we examined the effect of proteasome inhibitors on the replication of vesicular stomatitis virus (VSV) and poliovirus. We found that the proteasome inhibitors significantly suppressed VSV protein synthesis, virus accumulation, and protected infected cells from toxic effect of VSV replication. In contrast, poliovirus replication was delayed, but not diminished in the presence of the proteasome inhibitors MG132 and Bortezomib. We also found that inhibition of proteasomes stimulated stress-related processes, such as accumulation of chaperone hsp70, phosphorylation of eIF2alpha, and overall inhibition of translation. VSV replication was sensitive to this stress with significant decline in replication process. Poliovirus growth was less sensitive with only delay in replication. Inhibition of proteasome activity suppressed cellular and VSV protein synthesis, but did not reduce poliovirus protein synthesis. Protein kinase GCN2 supported the ability of proteasome inhibitors to attenuate general translation and to suppress VSV replication. We propose that different mechanisms of translational initiation by VSV and poliovirus determine their sensitivity to stress induced by the inhibition of proteasomes. To our knowledge, this is the first study that connects the effect of stress induced by proteasome inhibition with the efficiency of viral infection. 相似文献
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14.
Nakayama K Furusu A Xu Q Konta T Kitamura M 《Journal of immunology (Baltimore, Md. : 1950)》2001,167(3):1145-1150
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16.
Exogenous stimuli such as nerve growth factor (NGF) exert their effects on neurite outgrowth via Trk neurotrophin receptors. TrkA receptors are known to be ubiquitinated via proteasome inhibition in the presence of NGF. However, the effect of proteasome inhibition on neurite outgrowth has not been studied extensively. To clarify these issues, we investigated signaling events in PC12 cells treated with NGF and the proteasome inhibitor MG132. We found that MG132 facilitated NGF-induced neurite outgrowth and potentiated the phosphorylation of the extracellular signal-regulated kinase/mitogen- activated protein kinase (ERK/MAPK) and phosphatidylinositol- 3-kinase (PI3K)/AKT pathways and TrkA receptors. MG132 stimulated internalization of surface TrkA receptor and stabilized intracellular TrkA receptor, and the Ub(K63) chain was found to be essential for stability. These results indicate that the ubiquitin-proteasome system potentiated neurite formation by regulating the stability of TrkA receptors. 相似文献
17.
Activation of protein kinase C (PKC) prevents apoptosis in certain cells; however, the mechanisms are largely unknown. Inhibitors of apoptosis (IAP) family members, including NAIP, cIAP-1, cIAP-2, XIAP/hILP, survivin, and BRUCE, block apoptosis by binding and potently inhibiting caspases. Activation of NF-kappa B contributes to cIAP-2 induction; however, the cellular mechanisms regulating cIAP-2 expression have not been entirely defined. In this study, we examined the role of the PKC and NF-kappa B pathways in the regulation of cIAP-2 in human colon cancers. We found that cIAP-2 mRNA levels were markedly increased in human colon cancer cells by treatment with the phorbol ester, phorbol-12-myristate-13-acetate (PMA), or bryostatin 1. Inhibitors of the Ca2+-independent, novel PKC isoforms, but not inhibitors of MAPK, PI3-kinase, or PKA, blocked PMA-stimulated cIAP-2 mRNA expression, suggesting a role of PKC in PMA-mediated cIAP-2 induction. Pretreatment with the PKC delta-selective inhibitor rottlerin or transfection with an antisense PKC delta oligonucleotide inhibited PMA-induced cIAP-2 expression, whereas cotransfection with a PKC delta plasmid induced cIAP-2 promoter activity, which, taken together, identifies a role for PKC delta in cIAP-2 induction. Treatment with the proteasome inhibitor, MG132 or inhibitors of NF-kappa B (e.g. PDTC and gliotoxin), decreased PMA-induced up-regulation of cIAP-2. PMA-induced NF-kappa B activation was blocked by either GF109203x, MG132, PDTC, or gliotoxin. Moreover, overexpression of PKC delta-induced cIAP-2 promoter activity and increased NF-kappa B transactivation, suggesting regulation of cIAP-2 expression by a PKC delta/NF-kappa B pathway. In conclusion, our findings demonstrate a role for a PKC/NF-kappa B-dependent pathway in the regulation of cIAP-2 expression in human colon cancer cells. These data suggest a novel mechanism for the anti-apoptotic function mediated by the PKC delta/NF-kappa B/cIAP-2 pathway in certain cancers. 相似文献
18.
Yoshiko Iida Keiko Sawabe Masayo Kojima Kazuya Oguro Nobuo Nakanishi Hiroyuki Hasegawa 《European journal of biochemistry》2002,269(19):4780-4788
We previously demonstrated in mast cell lines RBL2H3 and FMA3 that tryptophan hydroxylase (TPH) undergoes very fast turnover driven by 26S-proteasomes [Kojima, M., Oguro, K., Sawabe, K., Iida, Y., Ikeda, R., Yamashita, A., Nakanishi, N. & Hasegawa, H. (2000) J. Biochem (Tokyo) 2000, 127, 121-127]. In the present study, we have examined an involvement of TPH phosphorylation in the rapid turnover, using non-neural TPH. The proteasome-driven degradation of TPH in living cells was accelerated by okadaic acid, a protein phosphatase inhibitor. Incorporation of 32P into a 53-kDa protein, which was judged to be TPH based on autoradiography and Western blot analysis using anti-TPH serum and purified TPH as the size marker, was observed in FMA3 cells only in the presence of both okadaic acid and MG132, inhibitors of protein phosphatase and proteasome, respectively. In a cell-free proteasome system constituted mainly of RBL2H3 cell extracts, degradation of exogenous TPH isolated from mastocytoma P-815 cells was inhibited by protein kinase inhibitors KN-62 and K252a but not by H89. Consistent with the inhibitor specificity, the same TPH was phosphorylated by exogenous Ca2+/calmodulin-dependent protein kinase II in the presence of Ca2+ and calmodulin but not by protein kinase A (catalytic subunit). TPH protein thus phosphorylated by Ca2+/calmodulin-dependent protein kinase II was digested more rapidly in the cell-free proteasome system than was the nonphosphorylated enzyme. These results indicated that the phosphorylation of TPH was a prerequisite for proteasome-driven TPH degradation. 相似文献
19.
Choi CH Lee BH Ahn SG Oh SH 《Biochemical and biophysical research communications》2012,418(4):759-764
Proteasome inhibition is a promising approach for cancer treatment; however, the underlying mechanisms involved have not been fully elucidated. Here, we show that proteasome inhibition-induced p38 mitogen-activated protein kinase regulates autophagy and apoptosis by modulating the phosphorylation status of glycogen synthase kinase 3β (GSK3β) and 70kDa ribosomal S6 kinase (p70S6K). The treatment of MDA-MB-231 cells with MG132 induced endoplasmic reticulum stress through the induction of ATF6a, PERK phosphorylation, and CHOP, and apoptosis through the cleavage of Bax and procaspase-3. MG132 caused the phosphorylation of GSK3β at Ser(9) and, to a lesser extent, Thr(390), the dephosphorylation of p70S6K at Thr(389), and the phosphorylation of p70S6K at Thr(421) and Ser(424). The specific p38 inhibitor SB203080 reduced the p-GSK3β(Ser9) and autophagy through the phosphorylation of p70S6K(Thr389); however, it augmented the levels of p-ERK, p-GSK3β(Thr390), and p-70S6K(Thr421/Ser424) induced by MG132, and increased apoptotic cell death. The GSK inhibitor SB216763, but not lithium, inhibited the MG132-induced phosphorylation of p38, and the downstream signaling pathway was consistent with that in SB203580-treated cells. Taken together, our data show that proteasome inhibition regulates p38/GSK(Ser9)/p70S6K(Thr380) and ERK/GSK3β(Thr390)/p70S6K(Thr421/Ser424) kinase signaling, which is involved in cell survival and cell death. 相似文献
20.
Previously, we have shown that translation eukaryotic initiation factor (eIF) 4GI is cleaved during anti-Fas-mediated apoptosis. Here, we have investigated the effects of the proteasome inhibitors, MG132 and lactacystin, and the immunosuppressants, 2-amino-2[2-(4-octylphenyl)ethyl]-1,3,propane diol (FTY720) and cyclosporin A, on the integrity of eIF4GI and eIF4GII in T cells. Using wild-type Jurkat T cells, we show that the proteasome inhibitors MG132 and lactacystin promote the cleavage of eIF4G, activate caspase-8 and caspase-3-like activities and decrease cell viability. Furthermore, MG132 also promotes the cleavage of eIF4G and the activation of caspase-3-like activity in a caspase-8-deficient Jurkat cell line which is resistant to anti-Fas-mediated apoptosis. Using specific anti-peptide antisera, we show that both eIF4GI and eIF4GII are cleaved in either cell line in response to MG132 and lactacystin. In response to such treatments, we demonstrate that the fragments of eIF4GI generated include those previously observed with anti-Fas antiserum together with a novel product which lacks the ability to interact with eIF4E. In contrast, cells treated with the immunosuppressants FTY720 and cyclosporin A appear to contain only the novel cleavage fragment of eIF4GI and to lack those characteristic of cells treated with anti-Fas antiserum. These data suggest that caspase-8 activation is not required for apoptosis and eIF4G cleavage mediated by proteasome inhibitors and immunosuppressants in human T cells. 相似文献