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1.
Proteasomes function as the main nonlysosomal machinery of intracellular proteolysis and are involved in the regulation of the majority of important cellular processes. Despite the considerable progress that has been made in understanding the functioning of proteasomes, some issues (in particular, the RNase activity of these ribonucleoprotein complexes and its regulation) remain poorly investigated. In this study, we found to several proteins with electrophoretic mobility that corresponds to that of 20S subunits of the core proteasome complex exhibit endoribonuclease activity with respect to the sense and antisense sequences of the c-myc mRNA 3′-UTR. Mass-spectrometric analysis of tryptic hydrolysates of these proteins showed that the samples contained 20S proteasome subunits—α1 (PSMA6), α5 (PSMA5), α6 (PSMA1), and α7 (PSMA3). A number of new phosphorylation sites of α1 (PSMA6) and α7 (PSMA3) subunits were found, and a form of α5 (PSMA5) subunit with a deletion of 20 N-terminal amino-acid residues was identified. The observed differences in the manifestation of endonuclease activity by individual subunits are apparently due to posttranslational modifications of these proteins (in particular, phosphorylation). It was shown that the specificity of RNase activity changes upon proteasome dephosphorylation and under the influence of Ca2+ and Mg2+ cations. It is concluded that posttranslational modifications of proteasome subunits affect the specificity of their RNase activity.  相似文献   

2.
3.
The 26S proteasome is a large multi-subunit protein complex that exerts specific degradation of proteins in the cell. The 26S proteasome consists of the 20S proteolytic particle and the 19S regulator. In order to be targeted for proteasomal degradation most of the proteins must undergo the post-translational modification of poly-ubiquitination. However, a number of proteins can also be degraded by the proteasome via a ubiquitin-independent pathway. Such degradation is exercised largely through the binding of substrate proteins to the PSMA3 (alpha 7) subunit of the 20S complex. However, a systematic analysis of proteins interacting with PSMA3 has not yet been carried out. In this report, we describe the identification of proteins associated with PSMA3 both in the cytoplasm and nucleus. A combination of two-dimensional gel electrophoresis (2D-GE) and tandem mass-spectrometry revealed a large number of PSMA3-bound proteins that are involved in various aspects of mRNA metabolism, including splicing. In vitro biochemical studies confirmed the interactions between PSMA3 and splicing factors. Moreover, we show that 20S proteasome is involved in the regulation of splicing in vitro of SMN2 (survival motor neuron 2) gene, whose product controls apoptosis of neurons.  相似文献   

4.
26S proteasome is a large multi-subunit protein complex involved in proteolytic degradation of proteins. In addition to its canonical proteolytic activity, the proteasome is also associated with recently characterized endoribonuclease (endo-RNAse) activity. However, neither functional significance, nor the mechanisms of its regulation are currently known. In this report, we show that 26S proteasome is able to hydrolyze various cellular RNAs, including AU-rich mRNA of c-myc and c-fos. The endonucleolytic degradation of these mRNAs is exerted by one of the 26S proteasome subunits, PSMA5 (α5). The RNAse activity of 26S proteasome is differentially affected by various extra-cellular signals. Moreover, this activity contributes to the process of degradation of c-myc mRNA during induced differentiation of K562 cells, and may be controlled by phosphorylation of the adjacent subunits, PSMA1 (α6) and PSMA3 (α7). Collectively, the data presented in this report suggest a causal link between cell signalling pathways, endo-RNAse activity of the 26S proteasome complex and metabolism of cellular RNAs.  相似文献   

5.
6.
Regulated proteolysis plays important roles in cell biology and pathological conditions. A crosstalk exists between apoptosis and the ubiquitin?Cproteasome system, two pathways responsible for regulated proteolysis executed by different proteases. To investigate whether the apoptotic process also affects the 20S proteasome, we performed three independent SILAC-based quantitative proteome approaches: 1-DE/MALDI-MS, small 2-DE/MALDI-MS and large 2-DE/nano-LC?CESI?CMS. Taking the results of all experiments together, no quantitative changes were observed for the ??- and ??-subunits of the 20S proteasome except for subunit ??7. This protein was identified in two protein spots with a down-regulation of the more acidic protein species (??7a) and up-regulation of the more basic protein species (??7b) during apoptosis. The difference in these two ??7 protein species could be attributed to oxidation of cysteine-41 to cysteine sulfonic acid and phosphorylation at serine-250 near the C terminus in ??7a, whereas these modifications were missing in ??7b. These results pointed to the biological significance of posttranslational modifications of proteasome subunit ??7 after induction of apoptosis.  相似文献   

7.
The participation of proteasome in the programmed cells death is now extensively investigated. Studies using selective inhibitors of proteasomes have provided a direct evidence of both pro- and anti-apoptotic functions of proteasomes. Such opposite roles of 26S proteasomes in regulation of apoptosis may be defined by the proliferative state of cell. The induction of apoptosis in K562 cells by diethylmaleate was used as a model to investigate changes in the subunit composition, phosphorylation state and enzymatic activities of 26S proteasomes undergoing the programmed cell death. Here we have shown that proteasomes isolated from the cytoplasm of control and diethylmaleate treated K562 cells differ in their subunit patterns, as well as in the phosphorylation state of subunits on threonine and tyrosine residues. It has been shown for the first time that proteolytic activity of 26S proteasomes is decreased, and endoribonuclease activity of 26S proteasomes is affected under diethylmaleate action on K562 cells. Treatment of K562 cells with an inductor of apoptosis--diethylmaleate--leads to modification of a proteasomal subunit (zeta/alpha5) associated with RNase activity of proteasomes. These data suggest the subunit composition and enzymatic activities of 26S proteasomes to be changed in K562 cells undergoing apoptosis, and that specific subtypes of 26S proteasomes participate in execution of programmed death of these cells.  相似文献   

8.
Mutations in the parkin gene encoding an E3 ligase are responsible for autosomal recessive Parkinson's disease. Putative parkin substrates and interacting partners have been identified, but the molecular mechanism underlying parkin-related neurodegeneration is still unclear. We have identified the 20S proteasomal subunit alpha4 (synonyms: PSMA7, XAPC7, subunit alpha type 7) as a new interacting partner of parkin. The C-terminal IBR-RING domain of parkin and the C-terminal part of alpha4 were essential for the interaction. Biochemical studies revealed that alpha4 was not a substrate for parkin-dependent ubiquitylation. Putative functions of the interaction might therefore be substrate presentation to the proteasome or regulation of proteasomal activity. Full-length parkin and parkin lacking the N-terminal ubiquitin-like domain slightly increased the proteasomal activity in HEK 293T cells, in line with the latter hypothesis.  相似文献   

9.
Regulation of proteasome complexes by gamma-interferon and phosphorylation   总被引:7,自引:0,他引:7  
Rivett AJ  Bose S  Brooks P  Broadfoot KI 《Biochimie》2001,83(3-4):363-366
Proteasomes play a major role in non-lysosomal proteolysis and also in the processing of proteins for presentation by the MHC class I pathway. In animal cells they exist in several distinct molecular forms which contribute to the different functions. 26S proteasomes contain the core 20S proteasome together with two 19S regulatory complexes. Alternatively, PA28 complexes can bind to the ends of the 20S proteasome to form PA28-proteasome complexes and PA28-proteasome-19S hybrid complexes have also been described. Immunoproteasome subunits occur in 26S proteasomes as well as in PA28-proteasome complexes. We have found differences in the subcellular distribution of the different forms of proteasomes. The gamma-interferon inducible PA28 alpha and beta subunits are predominantly located in the cytoplasm, while 19S regulatory complexes (present at significant levels only in 26S complexes) are present in the nucleus as well as in the cytoplasm. Immunoproteasomes are greatly enriched at the endoplasmic reticulum (ER) where they may facilitate the generation of peptides for transport into the lumen of the ER. We have also investigated the effects of gamma-interferon on the levels and subcellular distribution of inducible subunits and regulator subunits. In each case gamma-interferon was found to increase the level but not to alter the distribution. Several subunits of proteasomes are phosphorylated including alpha subunits C8 (alpha7) and C9 (alpha3), and ATPase subunit S4 (rpt2). Our studies have shown that gamma-interferon treatment decreases the level of phosphorylation of proteasomes. We have investigated the role of phosphorylation of C8 by casein kinase II by site directed mutagenesis. The results demonstrate that phosphorylation at either one of the two sites is essential for the association of 19S regulatory complexes and that the ability to undergo phosphorylation at both sites gives the most efficient incorporation of C8 into the 26S proteasome.  相似文献   

10.
The proteasome is the main intracellular proteolytic machine involved in the regulation of numerous cellular processes, including gene expression. In addition to their proteolytic activity, proteasomes also exhibit ATPase/helicase (the 19S particle) and RNAse (the 20S particle) activities, which are regulated by post-translational modifications. In this report we uncovered that several 20S particle subunits: α1 (PSMA6), α2 (PSMA2), α4 (PSMA7), α5 (PSMA5), α6 (PSMA1) and α7 (PSMA3) possess RNAse activity against the p53 mRNA in vitro. Furthermore, we found that the RNAse activity of PSMA1 and PSMA3 was regulated upon hemin-induced differentiation of K562 proerythroleukemia cells. The decrease in RNAse activity of PSMA1 and PSMA3 was paralleled by changes in their status of phosphorylation and ubiquitylation. Collectively, our data support the notion that proteasomal RNAse activity may be functionally important and provide insights into the potential mechanism of p53 repression in erythroleukemia cells by RNAse activity of the 20S α-type subunits.  相似文献   

11.
The 26S proteasome complex, which consists of a 20S proteasome and a pair of 19S regulatory particles, plays important roles in the degradation of ubiquitinated proteins in eukaryotic cells. The alpha7 subunit of the budding yeast 20S proteasome is a major phosphorylatable subunit; serine residue(s) in its C-terminal region are phosphorylated in vitro by CKII. However, the exact in vivo phosphorylation sites have not been identified. In this study, using electrospray ionization quadrupole time-of-flight mass spectrometry analysis, we detected a mixture of singly, doubly, and triply phosphorylated C-terminal peptides isolated from a His-tagged construct of the alpha7 subunit by nickel-immobilized metal affinity chromatography. In addition, we identified three phosphorylation sites in the C-terminal region using MS/MS analysis and site-directed mutagenesis: Ser258, Ser263, and Ser264 residues. The MS/MS analysis of singly phosphorylated peptides showed that phosphorylation at these sites did not occur successively.  相似文献   

12.
Cyclin B targets p34cdc2 for tyrosine phosphorylation.   总被引:28,自引:7,他引:21       下载免费PDF全文
L Meijer  L Azzi    J Y Wang 《The EMBO journal》1991,10(6):1545-1554
A universal intracellular factor, the 'M phase-promoting factor' (MPF), triggers the G2/M transition of the cell cycle in all organisms. In late G2, it is present as an inactive complex of tyrosine-phosphorylated p34cdc2 and unphosphorylated cyclin Bcdc13. In M phase, its activation as an active MPF displaying histone H1 kinase (H1K) originates from the concomitant tyrosine dephosphorylation of the p34cdc2 subunit and the phosphorylation of the cylin Bcdc13 subunit. We have investigated the role of cyclin in the formation of this complex and the tyrosine phosphorylation of p34cdc2, using highly synchronous mitotic sea urchin eggs as a model. As cells leave the S phase and enter the G2 phase, a massive tyrosine phosphorylation of p34cdc2 occurs. This large p34cdc2 tyrosine phosphorylation burst does not arise from a massive increase in p34cdc2 concentration. It even appears to affect only a fraction (non-immunoprecipitable by anti-PSTAIR antibodies) of the total p34cdc2 present in the cell. Several observations point to an extremely close association between accumulation of unphosphorylated cyclin and p34cdc2 tyrosine phosphorylation: (i) both events coincide perfectly during the G2 phase; (ii) both tyrosine-phosphorylated p34cdc2 and cyclin are not immunoprecipitated by anti-PSTAIR antibodies; (iii) accumulation of unphosphorylated cyclin by aphidicolin treatment of the cells, triggers a dramatic accumulation of tyrosine-phosphorylated p34cdc2; and (iv) inhibition of cyclin synthesis by emetine inhibits p34cdc2 tyrosine phosphorylation without affecting the p34cdc2 concentration. These results show that, as it is synthesized, cyclin B binds and recruits p34cdc2 for tyrosine phosphorylation; this inactive complex then requires the completion of DNA replication before it can be turned into fully active MPF. These results fully confirm recent data obtained in vitro with exogenous cyclin added to cycloheximide-treated Xenopus egg extracts.  相似文献   

13.
14.
Sphingosine 1-phosphate (S1P), a ligand for endothelial differentiation gene family proteins, is one of the most potent signal mediators released from activated platelets. Here, we report that S1P induces membrane ruffling of human umbilical vein endothelial cells (HUVECs) via the vascular endothelial growth factor receptor (VEGFR), Src family tyrosine kinase(s), and the CrkII adaptor protein. S1P induced prominent phosphorylation of CrkII in HUVECs, indicating that CrkII was involved in the S1P-induced signaling pathway. S1P-induced CrkII phosphorylation was blocked by pertussis toxin and overexpression of the carboxyl terminus of beta-adrenergic receptor kinase, indicating that the betagamma subunit of G(i) was required for the phosphorylation. Notably, the S1P-induced CrkII phosphorylation was also abolished by inhibitors of VEGFR or Src family tyrosine kinases. By using Picchu, a real time monitoring protein for CrkII phosphorylation, we found that S1P induced rapid CrkII phosphorylation at membrane ruffles. Finally, we observed that expression of a dominant negative mutant of CrkII inhibited the S1P-induced membrane ruffling and cell migration. These results delineated a novel S1P signaling pathway that involves sequential activation of G(i)-coupled receptor(s), VEGFR, Src family tyrosine kinase(s), and the CrkII adaptor protein, and which is responsible for both the induction of membrane ruffling and the increase in cell motility.  相似文献   

15.
The proteasome, a proteolytic complex present in all eukaryotic cells, is part of the ATP-dependent ubiquitin/proteasome pathway. It plays a critical role in the regulation of many physiological processes. The 20 S proteasome, the catalytic core of the 26 S proteasome, is made of four stacked rings of seven subunits each (alpha7beta7beta7alpha7). Here we studied the human 20 S proteasome using proteomics. This led to the establishment of a fine subunit reference map and to the identification of post-translational modifications. We found that the human 20 S proteasome, purified from erythrocytes, exhibited a high degree of structural heterogeneity, characterized by the presence of multiple isoforms for most of the alpha and beta subunits, including the catalytic ones, resulting in a total of at least 32 visible spots after Coomassie Blue staining. The different isoforms of a given subunit displayed shifted pI values, suggesting that they likely resulted from post-translational modifications. We then took advantage of the efficiency of complementary mass spectrometric approaches to investigate further these protein modifications at the structural level. In particular, we focused our efforts on the alpha7 subunit and characterized its N-acetylation and its phosphorylation site localized on Ser(250).  相似文献   

16.
Stimulation of receptors coupled to G(q)/G(11) protein may induce phosphorylation on a tyrosine residue of the alpha subunit of this G protein, which is an essential event for G(q)/G(11) activation. Here we observed that in HEK293 cells stably expressing high levels of thyrotropin-releasing hormone (TRH) receptors and G(11)alpha protein the maximal tyrosine phosphorylation of G(q)/G(11)alpha was reached within 10 min of TRH stimulation and then it faded away at longer time periods of agonist exposure. The G(q)/G(11)alpha protein levels did not change during this treatment. Incubation of intact cells with beta-cyclodextrin (beta CD) for 40 min prior to hormone exposure significantly decreased the rapid transient tyrosine phosphorylation. Subsequent replenishment of cholesterol levels reversed the former negative effect of beta CD. Isolation of caveolin-enriched, detergent-resistant membrane domains indicated destruction of these structures in beta CD-treated cells. These data indicate that the preserved integrity of plasma membrane domains/caveolae is required for complete agonist-induced phosphorylation of G(q)/G(11)alpha.  相似文献   

17.
Despite the central role of the 26 S proteasome in eukaryotic cells, many facets of its structural organization and functioning are still poorly understood. To learn more about the interactions between its different subunits, as well as its possible functional partners in cells, we performed, with Marc Vidal's laboratory (Dana-Farber Cancer Institute, Boston, MA, U.S.A.), a systematic two-hybrid analysis using Caenorhaditis elegans 26 S proteasome subunits as baits [Davy, Bello, Thierry-Mieg, Vaglio, Hitti, Doucette-Stamm, Thierry-Mieg, Reboul, Boulton, Walhout et al. (2001) EMBO Rep. 2, 821-828]. A pair-wise matrix of all subunit combinations allowed us to detect numerous possible intra-complex interactions, among which some had already been reported by others and eight were novel. Interestingly, four new interactions were detected between two ATPases of the 19 S regulatory complex and three alpha-subunits of the 20 S proteolytic core. Possibly, these interactions participate in the association of these two complexes to form the 26 S proteasome. Proteasome subunit sequences were also used to screen a cDNA library to identify new interactors of the complex. Among the interactors found, most (58) have no clear connection to the proteasome, and could be either substrates or potential cofactors of this complex. Few interactors (7) could be directly or indirectly linked to proteolysis. The others (12) interacted with more than one proteasome subunit, forming 'interaction clusters' of potential biological interest.  相似文献   

18.
Abstract

Hydrogen sulfide (H2S) shows antioxidative, anti-inflammatory, antiapoptotic, and cytoprotective effects in kidneys. Recently, H2S has been reported to alleviate uranium-induced rat nephrotoxicity through oxidative stress and inflammatory response via Nrf2-NF-κB pathways. Here, the protective effect and molecular mechanism of H2S on uranium-induced apoptosis were examined in normal rat kidney proximal cells (NRK-52E) in vitro. The results indicate that NaHS (an H2S donor) administration in uranium-intoxicated kidney cells ameliorated uranium-induced reactive oxygen species generation, caspase-3-dependent apoptosis, and endoplasmic reticulum (ER) stress identified through several key markers including GRP78, C/EBP homologous protein (CHOP), and caspase-12. NaHS treatment in uranium-intoxicated kidney cells abolished the effects of uranium on Akt phosphorylation, GSK-3β activation, increased Fyn nuclear expression, and concomitantly decreased Nrf2 nuclear expression. NaHS administration in uranium-treated kidney cells resorted uranium-decreased the expression of two key subunit PSMA6 and PSMB7 in 20S proteasome. But, DRB (an Nrf2 inhibitor) administration abrogated the effects of NaHS on PSMA6 and PSMB7 expression in uranium-contaminated kidney cells. Bortezomib (a proteasome inhibitor) treatment in NaHS pulsing uranium cotreated kidney cells reversed the effects of NaHS on not only PSMA6 and PSMB7 but also GRP78 and CHOP. Taken together, all data suggest that H2S can attenuate uranium-induced kidney cell apoptosis mediated by ER stress via 20S proteasome involving in Akt/GSK-3β/Fyn-Nrf2 signaling axis.  相似文献   

19.
Huang YT  Chen SU  Chou CH  Lee H 《Cellular signalling》2008,20(8):1521-1527
Sphingosine 1-phosphate (S1P) is a multifunctional phospholipid which acts through a specific family of G protein-coupled receptors. Platelet/endothelial cell adhesion molecule-1 (PECAM-1) form trans-homophilic binding at lateral cell border. Upon stimulation, its cytoplasmic tyrosine residues could be phosphorylated and interact with various downstream signaling molecules. In this study, we demonstrated that S1P induced PECAM-1 tyrosine phosphorylation in human umbilical cord vein cells (HUVECs). By pharmacological inhibitors, it was suggested that G(i) and Src family kinases were involved in PECAM-1 phosphorylation. Moreover, cSrc and Fyn siRNA significantly suppressed S1P-induced PECAM-1 phosphorylation. These results suggested that S1P-induced PECAM-1 phosphorylation through G(i) and subsequent cSrc and Fyn. Our findings provide further understanding of S1P and PECAM-1 signaling as well as their functions in endothelial cells.  相似文献   

20.
The nin1-1 mutant of Saccharomyces cerevisiae cannot perform the G1/S and G2/M transitions at restrictive temperatures. At such temperatures, nin1-1 strains fail to activate histone H1 kinase after release from alpha factor-imposed G1 block and after release from hydroxyurea-imposed S block. The nin1-1 mutation shows synthetic lethality with certain cdc28 mutant alleles such as cdc28-IN. Two lines of evidence indicate that Nin1p is a component of the 26S proteasome complex: (i) Nin1p, as well as the known component of the 26S proteasome, shifted to the 26S proteasome peak in the glycerol density gradient after preincubation of crude extract with ATP-Mg2+, and (ii) nin1-1 cells accumulated polyubiquitinated proteins under restrictive conditions. These results suggest that activation of Cdc28p kinase requires proteolysis. We have cloned a human cDNA encoding a regulatory subunit of the 26S proteasome, p31, which was found to be a homolog of Nin1p.  相似文献   

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