Characterization of membrane-associated proteasomes in WB rat liver epithelial cells

Although proteasomes are mainly located in the cytosol, it is known that significant amounts are also associated with endoplasmic reticulum (ER) membranes where they may play a role in the degradation of specific ER membrane proteins. The present studies were undertaken to compare ER and cytosolic proteasomal activities in WB rat liver cells. N-Heptyl-beta-thioglucopyranoside (HTG) extracts of membrane or cytosol fractions were chromatographed in glycerol/ATP buffers on size-exclusion and ion-exchange columns and the elution profiles of proteasomal peptidase activity and immunoreactive components of the 20S complex, 19S complex, and PA28 were compared. Cytosol fractions showed a single peak of chymotrypsin-like peptidase activity (Cht-L), which was inhibited completely by 5 microM lactacystin (LC) or SDS (0.03%) and corresponded to 26S proteasomes based upon the presence of both 20S and 19S components. By comparison, membrane fractions contained two major peaks of Cht-L activity. The first peak shared the same properties as the peak activity observed in cytosol fractions. However, the second peak was stimulated by SDS and was LC-insensitive (5 microM) and contained trypsin-like (T-L) and peptide-glutamyl peptidase (PGPH) but no cathepsin or calcium-activated protease activities. PA28 activator protein was present in both membrane and cytosol fractions. Thus, the principal difference between cytosolic and membrane activity was that the latter fractions contained a novel membrane-associated LC-insensitive protease(s) catalyzing three of the major peptidase activities of the proteasome.     

Selective effect of inductors of apoptosis on the endoribonuclease activity of 26S proteasomes and alpha-RNP particles in K562 cells: possible involvement of 26S proteasomes and alpha-RNP in the regulation of RNA stability

It has been shown that endoribonuclease activity of alpha-RNP particles and 26S proteasomes are changed under the action of inductors of programmed cell death. Treatment of K562 cells with inductors of apoptosis--doxorubicin (adriamycin) and diethylmaleate--lead to a significant stimulation of RNAse activity of alpha-RNP and to reduction of proteasome RNase activity. The enzymatic activity under study has been shown to be specifically and selectively dependent on phosphorylation of subunits of alpha-RNP particles and 26S proteasomes. The characteristics of RNAse activity of different subpopulations of proteasomes differ. The specificity of a subpopulation of proteasomes exported from the cell has been demonstrated. Proteasome and alpha-RNP involvement in the coordinated control of stability of various specific messenger RNA molecules is suggested, and one of the mechanisms of this control might be the export of specific subpopulation of proteasomes from the cell.     

Native structure of rat liver immune proteasomes

Native structure of active forms of rat liver immune proteasomes has been studied by two-dimensional electrophoresis method modified for analysis of unpurified protein fractions. The developed method allowed revealing the proteasome immune subunits LMP7 and LMP2 in 20S subparticles and in the structures bound to one or two PA28αβ activators, but not to the PA700 activator, which is involved in the hydrolysis of ubiquitinated proteins. The results obtained indicate the participation of the immune proteasomes in delicate regulatory mechanisms based on the production of biologically active peptides and exclude their participation in processes of crude degradation of “rotated” ubiquitinated proteins.     

Phosphorylation of rat liver glucocorticoid receptor

Rat liver glucocorticoid-receptor complex (GRc) was purified 2000-fold by a combination of methods including (NH4)2SO4-fractionation and phosphocellulose and DNA-cellulose chromatography. The purified glucocorticoid receptor preparation contained a major peptide of Mr = 90,000 and the GRc sedimented as 4 S in 5-20% sucrose gradients. An additional peptide of Mr = 45,000 (45K) was also observed. Some preparations yielded only the Mr = 90,000 (90K) peptide suggesting that the 45K peptide may be a proteolyzed portion of the 90K protein. The purified GRc was incubated with [gamma-32P]ATP in the presence of cAMP-dependent kinase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the above preparation revealed the presence of two 32P-containing bands with apparent Mr = 90,000 and 45,000. The 32P incorporation was dependent on the availability of divalent cation (Mg2+). GRc in cytosol labeled with [3H]dexamethasone mesylate and purified as above co-migrated with 32P-containing bands. GRc was also purified from cytosol obtained from livers of rats injected with [32P]orthophosphate. Both 32P and 3H bands were associated with 90K and 45K peptides. Our results indicate that rat liver glucocorticoid receptor is a phosphoprotein and that both the phosphorylated peptides 90K and 45K also contain the steroid and the DNA binding regions of the glucocorticoid receptor.     

cDNA cloning and sequencing of component C9 of proteasomes from rat hepatoma cells

The nucleotide sequence of component C9 of rat proteasomes (multicatalytic proteinase complexes) has been determined from a recombinant cDNA clone isolated by screening a Reuber H4TG hepatoma cell cDNA library using synthetic oligodeoxynucleotide probes corresponding to partial amino acid sequences of the protein. The predicted sequence of C9 consists of 261 amino acid residues with a calculated molecular weight of 29,496. The C9 component is a novel protein, differing from known proteins, but its primary structure resembles those of other proteasome components, including C2, C3 and C5, although its similarity to C5 is relatively low, suggesting that proteasomes consist of a family of proteins that have evolved from a common ancestor.     

cDNA cloning and sequencing of component C8 of proteasomes from rat hepatoma cells

The primary structure of component C8 of rat proteasomes (multicatalytic proteinase complexes) has been determined by sequencing on isolated cDNA clone. C8 consists of 255 amino acid residues with a calculated molecular weight of 28,417. These values are consistent with those obtained by protein chemical analyses. Computer-assisted homology comparison showed that C8 is a new protein, differing from all proteins reported so far. The overall amino acid sequence of C8 resembles those of most other components of proteasomes reported, such as components C2, C3 and C9 of rat proteasomes and certain components of other eukaryotic proteasomes, such as those of Drosophila and yeast, but shows little similarity with component C5 of rat proteasomes. C8 showed particularly close structural similarity to component YC1 of yeast proteasomes, suggesting that C8 has been highly conserved during evolution and functions ubiquitously in all eukaryotes.     

Autodegradation of rat liver proteasomes (large multicatalytic proteinase complexes)

Purified proteasomes (large multicatalytic proteinase complexes) were found to be very stable, showing no change in activities or structures during prolonged incubation in medium of pH 7.5 at 37 degrees C. However, on addition of urea they were degraded autocatalytically in a time- and dose-dependent manner, suggesting that destruction of the proteasomal complexes acts as a signal for their autolysis. ATP at a physiological concentration greatly stimulated the urea-dependent breakdown of proteasomes. The autolysis induced by urea was almost completely inhibited by hemin, but not by other protease inhibitors tested, such as leupeptin, chymostation and Ep-475. Thus, autolytic degradation of proteasomes appears to be important for the regulation of enzyme levels in eukaryotic cells.     

Isolation and characterization of two 20S proteasomes from the endoplasmic reticulum of rat liver microsomes.

Two new forms of proteasomes, designated as the endoplasmic reticulum (ER) membrane-associated proteasome (ERa proteasome) and ER membrane-bound proteasome (ERb proteasome), were purified to homogeneity from 0.0125 and 2.5% sodium cholate extracts, respectively, of a rat liver microsomal fraction. SDS-PAGE analysis revealed that the purified ERa and ERb proteasomes were composed of multiple subunits similar to the cytosolic 20S proteasome. However, electrophoretic, structural and immunochemical differences between the ERa, ERb and cytosolic 20S proteasomes were observed on native PAGE, two-dimensional (2D) PAGE, and immunoblot analyses. Purification of ERb from a 2.5% sodium cholate extract of the trypsin-treated microsomal fraction yielded a trypsin-modified form of ERb (tERb), which lacked the C2 subunit at least. On the other hand, no ERa proteasome was obtained from the 0.0125% sodium cholate extract of the trypsin-treated microsomes, suggesting that ERa and ERb are ER membrane-associated and -bound proteasomes, respectively. The ERa, ERb, and cytosolic 20S proteasomes exhibited similar specificities as to peptide hydrolyzing activity, although differences in their activities were noted in the presence of SDS and phospholipid. With respect to the proteolysis of protein substrates, only the ERb proteasome cleaved beta-casein, and it also degraded reduced and carboxymethylated lysozyme considerably faster than the cytosolic 20S and ERa proteasomes. Collectively our results suggest that the ERa and ERb proteasomes may play roles in intracellular proteolysis distinct from that of the cytosolic 20S proteasome.     

Phosphorylation of D-glucosamine by rat liver glucokinase.

D-Glucosamine was found to be phosphorylated by a rat liver extract in the presence of a high concentration of glucose, which was formerly believed to be a strong competitive inhibitor of this reaction. Results suggested that glucosamine may be phosphorylated by high Km hexokinase, i.e. glucokinase [EC 2.7.1.2]. The enzyme involved was separated from specific N-acetyl-D-glucosamine kinase [EC 2.7.1.59]. The phosphorylation was not inhibited by a physiological level of glucose or glucose 6-phosphate, which strongly inhibited low Km hexokinase. The apparent Km of glucokinase for glucosamine was estimated as 8 mM, which is ten times that of low Km hexokinase.     

Phosphorylation of purified glucocorticoid receptor from rat liver by an endogenous protein kinase

Glucocorticoid receptor was purified from rat liver cytosol using a dexamethasone affinity column. The receptor thus purified displayed a single protein band when subjected to SDS-polyacrylamide gel electrophoresis. It had a molecular weight of 90,000 which was consistent with the reported value for other glucocorticoid receptor preparations. Incubation of the purified preparation with [gamma 32P] ATP and Mg2+ resulted in transfer of [32P] to the receptor protein indicating the presence of an endogeneous protein kinase activity capable of phosphorylating the receptor molecule. Phosphorylation of the glucocorticoid receptor by the endogenous protein kinase might serve as a direct mechanism for the activation of the receptor.     

Properties of endoribonuclease activity of proteasomes from K562 cells. III. The specific endonuclease activity of ribonucleoprotein particles (alpha-RNP) tightly bound to chromatin and containing 20S proteosomes

The present work demonstrates the ability of 20S proteasome-containing, tightly bound to chromatin RNP-particles (alpha-RNP) to endonucleolyse specific messenger RNAs (in particular, human mRNA for p53 gene and mRNA for luciferase from Renilla sp.). The dependence of individual mRNA endonucleolysis by alpha-RNP particles on both the substrate and enzyme was found.     

Phosphorylation of rat liver glycogen synthase by phosphorylase kinase

Phosphorylation of rat liver glycogen synthase by rabbit skeletal muscle phosphorylase kinase results in the incorporation of approximately 0.8-1.2 mol of PO4/subunit. Analyses of the tryptic peptides by isoelectric focusing and thin layer chromatography reveal the presence of two major 32P-labeled peptides. Similar results were obtained when the synthase was phosphorylated by rat liver phosphorylase kinase. This extent of phosphorylation does not result in a significant change in the synthase activity ratio. In contrast, rabbit muscle glycogen synthase is readily inactivated by rabbit muscle phosphorylase kinase; this inactivation is further augmented by the addition of rabbit muscle cAMP-dependent protein kinase or cAMP-independent synthase (casein) kinase-1. Addition of cAMP-dependent protein kinase after initial phosphorylation of liver synthase with phosphorylase kinase, however, does not result in an inactivation or additional phosphorylation. The lack of additive phosphorylation under this condition appears to result from the phosphorylation of a common site by these two kinases. Partial inactivation of liver synthase can be achieved by sequential phosphorylation with phosphorylase kinase followed by synthase (casein) kinase-1. Under this assay condition, the phosphate incorporation into the synthase is additively increased and the synthase activity ratio (-glucose-6-P/+glucose-6-P) is reduced from 0.95 to 0.6. Nevertheless, if the order of the addition of these two kinases is reversed, neither additive phosphorylation nor inactivation of the synthase is observed. Prior phosphorylation of the synthase by phosphorylase kinase transforms the synthase such that it becomes a better substrate for synthase (casein) kinase-1 as evidenced by a 2- to 4-fold increase in the rate of phosphorylation. This increased rate of phosphorylation of the synthase appears to result from the rapid phosphorylation of a site neighboring that previously phosphorylated by phosphorylase kinase.     

Phosphorylation of nuclear proteins in rat regenerating liver.

In studies of the phosphorylated proteins in rat liver and Walker-256, it was established that the ratio of various fractions of P-N linkages to P-O linkages varies from 0.6 to 3.1. In rat regenerating liver nuclei, the ratio of P-N and P-O varies with time after partial hepatectomy. Using [3H]-lysine and 32Pi, it is shown that phosphoryllysine forms in some new and, presumably, some preexisting H1 molecules. Using [3H]histidine and 32Pi, it is shown that phosphohistidine forms exclusively in preexisting H4. The half-life of H4 phosphohistidine appears to be about 2 h.