Hyperphosphorylation of rat liver proteasome subunits: the effects of ethanol and okadaic acid are compared

In experimental alcoholic liver disease, protein degradation by the ATP-ubiquitin-proteasome pathway is inhibited. Failure of the proteasome to eliminate cytoplasmic proteins leads to the accumulation of oxidized and otherwise modified proteins. One possible explanation for the inhibition of the proteasome is hyperphosphorylation of proteasome subunits. To examine this possibility, the 26S proteasomes from the liver of rats fed ethanol and a pair-fed control were studied by isolating the proteasomes in a purified fraction. The effect of ethanol on the phosphorylation of proteasomal subunits was compared with the hyperphosphorylation of the proteasomes caused by okadaic acid given to rats in vivo. Ethanol ingestion caused an inhibition of the chymotrypsin-like activity of the purified proteasome. The 2D electrophoresis and Western blot analysis of the purified 20S and 26S proteasomes from the ethanol-fed rats indicated that hyperphosphorylation of proteasomal subunits had occured. The proteasomal alpha type subunits C9/alpha3 and C8/alpha7 were hyperphosphorylated compared to the controls. Chymotrypsin-like activity was also inhibited by okadaic acid treatment similar to ethanol feeding. The 26S proteasome fraction examined by isoelectric focusing gel revealed many hyperphosphorylated bands in the proteasomes from the okadaic acid treated and the ethanol fed rat livers compared with the controls. In conclusion hyperphosphorylation of the proteasome subunits occurs in the ethanol treated proteasomal subunits which could be one mechanism of the inhibition of the 26S proteasome caused by ethanol feeding.     

In vivo and in vitro phosphorylation of Candida albicans 20S proteasome

In this paper we demonstrate that the Candida albicans 20S proteasome is in vivo phosphorylated and is a good in vitro substrate (S(0.5) 14nM) of homologous protein kinase CK2 (CK2). We identify alpha6/C2, alpha3/C9, and alpha5/Pup2 proteasome subunits as the main in vivo phosphorylated and in vitro CK2-phosphorylatable proteasome components. In vitro phosphorylation by homologous CK2 holoenzyme occurs only in the presence of polylysine, a characteristic that distinguishes the yeast proteasomes from mammalian proteasomes which are phosphorylated by CK2 in the absence of polycations. The major in vivo phosphate acceptor is the alpha3/C9 subunit, being phosphorylated in serine, both in vivo and in vitro. The phosphopeptides generated by endoproteinase Glu-C digestion from in vivo labeled alpha3/C9 subunit, from in vitro phosphorylation by homologous CK2 holoenzyme, and from the recombinant alpha3/C9 subunit phosphorylated by recombinant human CK2-alpha subunit are identical, suggesting that CK2 is likely responsible for in vivo phosphorylation of this subunit. Direct mutational analysis shows that serine 248 is the residue of the alpha3/C9 subunit phosphorylated by CK2. The in vitro stoichiometry of phosphorylation of the alpha6/C2 and alpha3/C9 proteasome subunits by CK2 can be estimated as 0.7-0.8 and 0.4-0.5 mol of phosphate per mole of subunit, respectively. These results are consistent with the relative abundance of the unphosphorylated and phosphorylated isoforms of these subunits present in the purified 20S proteasome preparation. Our demonstration of phosphorylation of C. albicans proteasome suggests that phosphorylation might be a general mechanism of regulation of proteasome activity.     

Phosphorylation of the C2 subunit of the proteasome in rice (Oryza sativa L.)

Proteasomes function mainly in the ATP-dependent degradation of proteins that have been conjugated with ubiquitin. To demonstrate the phosphorylation of proteasomes in plants, we conducted an enzymatic dephosphorylation experiment with a crude extract of rice cultured cells. The results indicated that the C2 subunit of the 20S proteasome is phosphorylated in vivo in cultured cells. An in-gel kinase assay and analysis of phosphoamino acids revealed that the C2 subunit is phosphorylated by a 40-kDa serine/threonine protein kinase, the activity of which is inhibited by heparin, a specific inhibitor of casein kinase II. The catalytic subunit of casein kinase II from Arabidopsis was also able to phosphorylate the C2 subunit. These results suggest that the C2 subunit in rice is probably phosphorylated by casein kinase II. Our demonstration of the phosphorylation of proteasomes in plants suggests that phosphorylation might be involved in the general regulation of the functions of proteasomes.© 1997 Federation of European Biochemical Societies.     

Regulation of proteasome complexes by gamma-interferon and phosphorylation

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.     

Analysis of Drosophila 26 S proteasome using RNA interference.

We have utilized double-stranded RNA interference (RNAi) to examine the effects of reduced expression of individual subunits of the 26 S proteasome in Drosophila S2 cells. RNAi significantly decreased mRNA and protein levels of targeted subunits of both the core 20 S proteasome and the PA700 regulatory complex. Cells deficient in any of several 26 S proteasome subunits (e.g. d beta 5, dRpt1, dRpt2, dRpt5, dRpn2, and dRpn12) displayed decreased proteasome activity (as judged by hydrolysis of succinyl-Leu-Leu-Val-Tyr-aminomethylcoumarin), increased apoptosis, decreased cell proliferation without a specific block of the cell cycle, and accumulation of ubiquitinated cellular proteins. RNAi of many individual 26 S proteasome subunits promoted increased expression of many non-targeted subunits. This effect was not mimicked by chemical proteasome inhibitors such as lactacystin. Reduced expression of most targeted subunits disrupted the assembly of the 26 S proteasome. RNAi of six of eight targeted PA700 subunits disrupted that structure and caused accumulation of increased levels of uncapped 20 S proteasome. Notable exceptions included RNAi of dRpn10, a polyubiquitin binding subunit, and dUCH37, a ubiquitin isopeptidase. dRpn10-deficient cells showed a significant increase in succinyl-Leu-Leu-Val-Tyr-aminomethylcoumarin hydrolyzing activity of the 26 S proteasomes but accumulated polyubiquitinated proteins. d beta 5-Deficient cells had a phenotype similar to that of most PA700-deficient cells but also accumulated low molecular mass complexes containing subunits of the 20 S proteasome, probably representing unassembled precursors of the 20 S proteasomes. Cells deficient in several of the 26 S proteasome subunits were more resistant to otherwise toxic concentrations of various proteasome inhibitors. Our data suggest that those cells adapted to grow in conditions of impaired ubiquitin and proteasome-dependent protein degradation.     

Proteasome structures affected by ionizing radiation

Exposure of cells to ionizing radiation slows the rate of degradation of substrates through the proteasome. Because the 26S proteasome degrades most short-lived cellular proteins, changes in its activity might significantly, and selectively, alter the life span of many signaling proteins and play a role in promoting the biological consequences of radiation exposure, such as cell cycle arrest, DNA repair, and apoptosis. Experiments were therefore undertaken to identify the radiation target that is associated with the proteasome. Regardless of whether they were irradiated before or after extraction and purification from human prostate cancer PC3 cells, 26S proteasomes remained intact but showed a rapid 30% to 50% dose-independent decrease in their three major enzymatic activities following exposure to 1 to 20 Gy. There was no effect on 20S proteasomes, suggesting that the radiation-sensitive target is located in the 19S cap of the 26S proteasome, rather than in the enzymatically active core. Because the base of the 19S cap contains an ATPase ring that mediates substrate unfolding, pore opening, and translocation of substrates into the catalytic chamber, we examined whether the ATPase activity of purified 26S proteasomes was affected. In fact, in vitro irradiation of proteasomes enhanced their ATPase activity. Furthermore, pretreatment with low concentrations of the free radical scavenger tempol was able to prevent both the radiation-induced decrease in proteolytic activity and the increase in ATP utilization, indicating that free radicals are mediators of these radiation-induced phenomena. Finally, we have shown that cell irradiation results in the accumulation of proteasome substrates: polyubiquitinated proteins and ornithine decarboxylase, indicating that the observed decrease in proteasome function is physiologically relevant.     

Interaction between alphaB-crystallin and the human 20S proteasomal subunit C8/alpha7

alphaB-Crystallin, a member of the small heat shock protein (sHsp) family, can bind unfolding proteins, but is unable to refold them. To fulfil its protective function in vivo it is therefore likely to interact with other cellular proteins. Here we report that alphaB-crystallin binds very specifically both in vitro and in vivo to C8/alpha7, one of the 14 subunits of the 20S proteasome. The C8/alpha7 protein forms heterogeneous complexes with alphaB-crystallin of about 540 kDa. However, no strong interaction between alphaB-crystallin and 20S proteasomes was observed. Since both proteins are localized in the cytoplasm, the interaction between alphaB-crystallin and C8/alpha7 subunit might affect the assembly of the proteasome complex or facilitate the degradation of unfolded proteins bound to alphaB-crystallin.     

Specificity of changes in proteasome properties in diethylmaleate-induced apoptosis of K562 cells

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.     

Interaction between αB-crystallin and the human 20S proteasomal subunit C8/α7

αB-Crystallin, a member of the small heat shock protein (sHsp) family, can bind unfolding proteins, but is unable to refold them. To fulfil its protective function in vivo it is therefore likely to interact with other cellular proteins. Here we report that αB-crystallin binds very specifically both in vitro and in vivo to C8/α7, one of the 14 subunits of the 20S proteasome. The C8/α7 protein forms heterogeneous complexes with αB-crystallin of about 540 kDa. However, no strong interaction between αB-crystallin and 20S proteasomes was observed. Since both proteins are localized in the cytoplasm, the interaction between αB-crystallin and C8/α7 subunit might affect the assembly of the proteasome complex or facilitate the degradation of unfolded proteins bound to αB-crystallin.     

Affinity labeling of the proteasome by a belactosin A derived inhibitor

Belactosin A is a potent proteasome inhibitor isolated from Streptomyces metabolites. Here we show that a hydrophobic belactosin A derivative, dansyl-KF33955, can covalently, and specifically, affinity label the catalytic subunits of the 26S proteasome, which consists of the 20S protein degrading core particle and the 19S regulatory particles. The labeling of catalytic subunits proceeds faster in intact proteasomes in vivo than in isolated 20S core particles. These data suggest that the 19S regulatory particle may facilitate entry of the inhibitor into the 20S core particle. This cell-permeable chemical probe is an excellent tool with which to study the interactions of this proteasome inhibitor with proteasomes in intact cells.     

A quantitative proteomic approach using two-dimensional gel electrophoresis and isotope-coded affinity tag labeling for studying human 20S proteasome heterogeneity

Mammalian proteasomes are macromolecular complexes formed of a catalytic 20S core associated to two regulatory complexes. The 20S core complex consists of four stacked rings of seven alpha or beta subunits. Three beta subunits contain a catalytic site and can be replaced by three interferon gamma-inducible counterparts to form the immunoproteasome. Cells may constitutively possess a mixture of both 20S proteasome types leading to a heterogeneous proteasome population. Purified rat 20S proteasome has been separated in several chromatographic fractions indicating an even higher degree of complexity in 20S proteasome subunit composition. This complexity may arise from the presence of subunit isoforms, as previously detected in purified human erythrocyte 20S proteasome. In this study, we have used a quantitative proteomic approach based on two-dimensional gel electrophoresis and isotope-coded affinity tag (ICAT) labeling to quantify the variations in subunit composition, including subunit isoforms, of 20S proteasomes purified from different cells. The protocol has been adapted to the analysis of low quantities of 20S proteasome complexes. The strategy has then been validated using standard proteins and has been applied to the comparison of 20S proteasomes from erythrocytes and U937 cancer cells. The results obtained show that this approach represents a valuable tool for the study of 20S proteasome heterogeneity.     

Phosphorylation of ATPase subunits of the 26S proteasome

The 26S proteasome complex plays a major role in the non-lysosomal degradation of intracellular proteins. Purified 26S proteasomes give a pattern of more than 40 spots on 2D-PAGE gels. The positions of subunits have been identified by mass spectrometry of tryptic peptides and by immunoblotting with subunit-specific antipeptide antibodies. Two-dimensional polyacrylamide gel electrophoresis of proteasomes immunoprecipitated from [³²P]phosphate-labelled human embryo lung L-132 cells revealed the presence of at least three major phosphorylated polypeptides among the regulatory subunits as well as the C8 and C9 components of the core 20S proteasome. Comparison with the positions of the regulatory polypeptides revealed a minor phosphorylated form to be S7 (MSS1). Antibodies against S4, S6 (TBP7) and S12 (MOV34) all cross-reacted at the position of major phosphorylated polypeptides suggesting that several of the ATPase subunits may be phosphorylated. The phosphorylation of S4 was confirmed by double immunoprecipitation experiments in which 26S proteasomes were immunoprecipitated as above and dissociated and then S4 was immunoprecipitated with subunit-specific antibodies. Antibodies against the non-ATPase subunit S10, which has been suggested by others to be phosphorylated, did not coincide with the position of a phosphorylated polypeptide. Some differences were observed in the 2D-PAGE pattern of proteasomes immunoprecipitated from cultured cells compared to purified rat liver 26S proteasomes suggesting possible differences in subunit compositions of 26S proteasomes.     

Proteasome disassembly and downregulation is correlated with viability during stationary phase

During prolonged starvation, yeast cells enter a stationary phase (SP) during which the synthesis of many proteins is dramatically decreased. We show that a parallel decrease in proteasome-dependent proteolysis also occurs. The reduction in proteolysis is correlated with disassembly of 26S proteasome holoenzymes into their 20S core particle (CP) and 19S regulatory particle (RP) components. Proteasomes are reassembled, and proteolysis resumes prior to cell cycle reentry. Free 20S CPs are found in an autoinhibited state in which the N-terminal tails from neighboring alpha subunits are anchored by an intricate lattice of interactions blocking the channel that leads into the 20S CPs. By deleting channel gating residues of CP alpha subunits, we generated an "open channel" proteasome that exhibits faster rates of protein degradation both in vivo and in vitro, indicating that gating contributes to regulation of proteasome activity. This open channel mutant is delayed in outgrowth from SP and cannot survive following prolonged starvation. In summary, we have found that the ubiquitin-proteasome pathway can be subjected to global downregulation, that the proteasome is a target of this regulation, and that proteasome downregulation is linked to survival of SP cells. Maintaining high viability during SP is essential for evolutionary fitness, which may explain the extreme conservation of channel gating residues in eukaryotic proteasomes.     

Changes in composition and activities of 26S proteasomes under the action of doxorubicin--apoptosis inductor of erythroleukemic K562 cells

Changes in the subunit composition, phosphorylation of the subunits, and regulation of the activities of 26S proteasomes in proliferating cells undergoing programmed cell death have not been studied so far. Moreover, there are no reports on phosphorylation of proteasome subunits both in normal and in neoplastic cells during apoptosis. The data of the present study show for the first time that apoptosis inductor doxorubicin regulates subunit composition, enzymatic activities, and phosphorylation state of 26S proteasomes in neoplastic (proerythroleukemic K562) cells or, in other words, induces reprogramming of proteasome population. Furthermore, the phosphorylation state of proteasomes is found to be the mechanism controlling specificity of proteasomal proteolytic and endoribonuclease activities.     

Reprogramming of nuclear proteasomes in K562 cells undergoing apoptosis. I. Effect of glutathione-depleting agent, diethylmaleate

Here we have studied changes in the subunit composition, phosphorylation state and enzymatic activities of 26S proteasomes in cells undergoing the programmed cell death. Apoptosis in proerythroleukemic K562 cells was induced by glutathione-depleting agent, diethylmaleate (DEM). We have shown for the first time that proteasomes isolated from the nuclei of control and induces K562 cells differ in their subunit patterns, as well as in the phosphorylation state of subunits on threonine and tyrosine residues. We observed trypsin- and chymotrypsin-like activities on nuclear proteasomes and the specificity of proteasomal nucleolysis of several individual messenger RNAs (c-fos and c-myc) to be changed under effect of DEM on K562 cells. Treatment of K562 cells with DEM leads to modification of zeta/alpha5 and iota/alpha6 proteasomal subunits associated with RNAse activity of proteasomes. These findings confirm our hypothesis about so-called reprogramming of nuclear proteasome population in undergoing apoptosis K562 cells which is manifested by the changes in proteasomal composition, phosphorylation state, and enzymatic activities during the programmed cell death.     

20S proteasome biogenesis

26S proteasomes are multi-subunit protease complexes responsible for the turnover of short-lived proteins. Proteasomal degradation starts with the autocatalytic maturation of the 20S core particle. Here, we summarize different models of proteasome assembly. 20S proteasomes are assembled as precursor complexes containing alpha and unprocessed beta subunits. The propeptides of the beta subunits are thought to prevent premature conversion of the precursor complexes into matured particles and are needed for efficient beta subunit incorporation. The complex biogenesis is tightly regulated which requires additional components such as the maturation factor Ump1/POMP, an ubiquitous protein in eukaryotic cells. Ump1/POMP is associated with precursor intermediates and degraded upon final maturation. Mammalian proteasomes are localized all over the cell, while yeast proteasomes mainly localize to the nuclear envelope/endoplasmic reticulum (ER) membrane network. The major localization of yeast proteasomes may point to the subcellular place of proteasome biogenesis.     

Identification of the Immunoproteasome as a Novel Regulator of Skeletal Muscle Differentiation

While many of the molecular details of myogenesis have been investigated extensively, the function of immunoproteasomes (i-proteasomes) in myogenic differentiation remains unknown. We show here that the mRNA of i-proteasome subunits, the protein levels of constitutive and inducible proteasome subunits, and the proteolytic activities of the 20S and 26S proteasomes were significantly upregulated during differentiation of skeletal muscle C2C12 cells. Knockdown of the i-proteasome catalytic subunit PSMB9 by short hairpin RNA (shRNA) decreased the expression of both PSMB9 and PSMB8 without affecting other catalytic subunits of the proteasome. PSMB9 knockdown and the use of i-proteasome-specific inhibitors both decreased 26S proteasome activities and prevented C2C12 differentiation. Inhibition of the i-proteasome also impaired human skeletal myoblast differentiation. Suppression of the i-proteasome increased protein oxidation, and these oxidized proteins were found to be more susceptible to degradation by exogenous i-proteasomes. Downregulation of the i-proteasome also increased proapoptotic proteins, including Bax, as well as cleaved caspase 3, cleaved caspase 9, and cleaved poly(ADP-ribose) polymerase (PARP), suggesting that impaired differentiation is likely to occur because of significantly increased apoptosis. These results demonstrate for the first time that i-proteasomes, independent of constitutive proteasomes, are critical for skeletal muscle differentiation of mouse C2C12 cells.