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J. Biol. Chem., Vol. 275, Issue 27, 20295-20301, July 7, 2000

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Ca²⁺-free Calmodulin and Calmodulin Damaged by in Vitro Aging Are Selectively Degraded by 26 S Proteasomes without Ubiquitination^*

Edit Tarcsa^§, Grazyna Szymanska^§¶, Stewart Lecker, Clare M. O'Connor^¶, and Alfred L. Goldberg^§

From the  Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115 and the ^¶ Department of Biology, Boston College, Chestnut Hill, Massachusetts 02467

Received for publication, February 24, 2000, and in revised form, April 27, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The ubiquitin-proteasome pathway is believed to selectively degrade post-synthetically damaged proteins in eukaryotic cells. To study this process we used calmodulin (CaM) as a substrate because of its importance in cell regulation and because it acquires isoaspartyl residues in its Ca²⁺-binding regions both in vivo and after in vitro "aging" (incubation for 2 weeks without Ca²⁺). When microinjected into Xenopus oocytes, in vitro aged CaM was degraded much faster than native CaM by a proteasome-dependent process. Similarly, in HeLa cell extracts aged CaM was degraded at a higher rate, even though it was not conjugated to ubiquitin more rapidly than the native species. Ca²⁺ stimulated the ubiquitination of both species, but inhibited their degradation. Thus, for CaM, ubiquitination and proteolysis appear to be dissociated. Accordingly, purified muscle 26 S proteasomes could degrade aged CaM and native Ca²⁺-free (apo) CaM without ubiquitination. Addition of Ca²⁺ dramatically reduced degradation of the native molecules but only slightly reduced the breakdown of the aged species. Thus, upon Ca²⁺ binding, native CaM assumes a non-degradable conformation, which most of the age-damaged species cannot assume. Thus, flexible conformations, as may arise from age-induced damage or the absence of ligands, can promote degradation directly by the proteasome without ubiquitination.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

One function of the ubiquitin (Ub)¹-proteasome pathway is the selective degradation of abnormal proteins, as may arise by mutation or post-synthetic damage (1, 2). Although proteins can undergo various types of chemical modifications with time, the intracellular fate of such damaged proteins has not been systematically studied. We chose to use CaM to investigate this process, because it plays an important role in cell regulation (3), therefore factors influencing its function and degradation can have important physiological consequences. Moreover, its tertiary structure is known in detail (4, 5) and spontaneous chemical modifications that occur in CaM upon aging (6, 7) have been shown to promote its degradation (8).

CaM has been reported to have a half-life of 18-25 h (1-2%/h) (9, 10), which resembles the half-life of the bulk of cell proteins (11). Over time, such long-lived proteins are susceptible to a variety of spontaneous chemical modifications, including the deamidation of asparaginyl residues and the isomerization of aspartyl residues (7, 12). These modifications arise through slow, non-enzymatic rearrangements that yield isoaspartyl residues and also some racemized aspartyl residues (13). The appearance of these residues has been detected by enzymatic carboxyl methylation using protein-L-isoaspartate (D-aspartate) O-methyltransferase (14), and has been demonstrated to occur in several proteins in vivo as well as in vitro upon prolonged incubation at physiological pH and temperatures (15, 16).

When CaM was incubated for 2 weeks at pH 7.4 and 37 °C in the presence of Ca²⁺, small amounts of isoaspartyl residues were formed only from two aspartates at flexible regions of CaM, which do not participate in Ca²⁺ binding (6). Isoaspartates were found at these same positions in CaM isolated from bovine brain (17). When Ca²⁺ was not present during the 2-week incubation, there was a dramatic increase in the isoaspartyl content of CaM, due to the deamidation of several asparaginyl residues and the isomerization of additional aspartates (6, 12). Because these isoaspartates were in positions implicated in Ca²⁺ binding, their appearance correlated with a significant loss of Ca²⁺-dependent regulatory activity (18). Interestingly, in CaM isolated from human erythrocytes, the principal sites of isoaspartyl residues were also in these Ca²⁺-binding regions (7). Thus, preincubation of CaM in the absence of Ca²⁺ may serve as an in vitro model for protein "aging" in intact cells (12).

Age-induced chemical modifications, damage by oxygen-free radicals and other post-translational damage are believed to trigger rapid degradation of proteins in vivo (19). In previous studies, we demonstrated that in vitro aged (deamidated) CaM was rapidly degraded following microinjection into Xenopus oocytes (8). By contrast recently isolated CaM, as well as CaM preincubated for 2 weeks in the presence of Ca²⁺, were stable for several hours. In the present studies we sought to uncover the molecular basis for the differential degradation of age-damaged CaM and to define the role of the Ub-proteasome pathway in this process.

In the Ub-proteasome pathway, regulatory proteins or proteins with abnormal conformation are marked for rapid degradation by a series of enzymatic reactions leading to the attachment of a chain of ubiquitin molecules to lysine residues on the substrate (2). These Ub-conjugated proteins are then digested rapidly to small peptides by the 26 S proteasome, a 2.4-MDa complex containing multiple proteolytic sites within its 20 S core particle (20, 21). Recognition of short-lived regulatory proteins by the ubiquitin-protein ligases (E3s) can depend on the presence of particular sequences (e.g. the destruction box of mitotic cyclins or substrate phosphorylation) (2), but the mode of recognition of substrates with abnormal conformations is unknown. It has been suggested that the appearance of modified amino acids leads directly to the proteins recognition and rapid degradation (19). Alternatively, these modified residues may have an indirect effect by disrupting the proteins normal conformation, thus leading to its selective degradation. It is assumed that the Ub-protein ligases (E3s) are the recognition elements, or that ubiquitination of abnormal proteins may follow their selective binding to molecular chaperones (22). Although Ub conjugation is essential for the rapid elimination of many regulatory and mutant polypeptides (2, 23) and for accelerated proteolysis under certain physiological conditions (24-26), proteasome-mediated degradation of some proteins can occur without ubiquitination (27-29). While ubiquitination of proteins generally enhances their breakdown (30, 31), in vitro certain unfolded proteins and the short lived enzyme, ornithine decarboxylase, can be hydrolyzed by proteasomes rapidly in an ATP-dependent manner in the absence of ubiquitination (27, 32). It remains uncertain to what extent this Ub-independent process occurs in vivo.

In this study we have used oocytes, HeLa cell lysates, and purified 26 S proteasomes to explore the biochemical mechanisms leading to the rapid destruction of the in vitro aged CaM. We demonstrate here that aged CaM and the apo-form of native CaM are degraded rapidly by 26 S proteasomes, apparently without ubiquitination. Interestingly, upon Ca²⁺ binding, native CaM assumes a conformation that prevents its degradation by the proteasome, even though Ca²⁺ binding promotes CaM ubiquitination. Furthermore, the chemical modifications that occur during aging, which make CaM less able to bind Ca²⁺, lead to its rapid degradation directly by the proteasomes, even in the presence of Ca²⁺.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Purification of CaM-- Recombinant chicken hemagglutinin-tagged CaM (HA-CaM) and metabolically labeled CaM ([³⁵S]CaM) were purified from P4830 Escherichia coli cells that had been transformed with the pCaM_pL plasmid containing the chicken CaM gene under the control of the  phage left promoter. The P4830 host E. coli (Amersham Pharmacia Biotech) contains a temperature-sensitive variant of the cI repressor. For HA-CaM purification the E. coli cells were grown in LB medium, and for [³⁵S]CaM purification in M9 medium (33) supplemented with 0.1% glucose, 1 µg/ml thiamine, and 0.2 mM each of the amino acids, excluding methionine. Both cultures were grown at 30 °C until the culture density reached an A₆₀₀ = 0.4 and CaM synthesis was induced by shifting the cultures to 41 °C. For [³⁵S]CaM preparation, [³⁵S]Express ³⁵S Tag labeling solution (1175 Ci/mmol, NEN Life Science Products Inc.) was added to the cultures at the time of the temperature shift to give a concentration of 10 µCi/ml. After 2 h the cells were harvested, and CaM was purified as described (34, 35). Approximately 80 µg of [³⁵S]CaM was obtained from a 100-ml culture (specific activity: 200,000 cpm/µg).

Preparation and Analysis of Aged CaM-- HA-CaM and [³⁵S]CaM (100 µM) were incubated for 2 weeks at 37 °C in 50 mM Na-HEPES or K-HEPES, pH 7.4, containing 1 mM EGTA as described (18). Control ("native") samples were incubated for the same time in 50 mM Na-HEPES or K-HEPES, pH 7.4, containing 5 mM CaCl₂. Native and aged CaM has the same mobility on SDS-PAGE (36); therefore they were routinely analyzed by nondenaturing PAGE, in the presence of 2 mM EDTA (18). For two-dimensional separations, strips containing the different CaMs were cut from nondenaturing gels containing EDTA and equilibrated for 10 min in sample buffer containing 2 mM EDTA or 5 mM CaCl₂. These strips were then layered across the top of a second native gel that contained either 2 mM EDTA or 5 mM CaCl₂ in the gel solutions and running buffer.

Microinjection of HA-CaM into Xenopus Oocytes-- Xenopus oocytes were first injected with 47 nl of a solution containing 10 pmol of lactacystin in 10 mM sodium phosphate, pH 6.8, using a Nanoject injection apparatus. The lactacystin was diluted from a 10 mM stock in dimethyl sulfoxide immediately before injection. A control group was injected with a similar dilution of dimethyl sulfoxide alone. Thirty min later, oocytes were injected with 2.5 ng of aged HA-CaM in 47 nl. The oocytes were incubated at 20 °C, and at indicated times, HA-CaM levels were measured in extracts prepared from groups of 10 oocytes using the immunoblotting method described previously (8).

Preparation of HeLa Cell Extract-- HeLa cells were harvested by centrifugation and osmotically lysed by the addition of 5 volumes of hypotonic buffer (10 mM Tris-Cl, pH 7.9, 1.5 mM MgCl₂, 10 mM KCl, 0.2 mM phenylmethylsulfonyl fluoride, 0.5 mM DTT). The mixture was incubated on ice for 10 min, then homogenized with 12 strokes of a Dounce homogenizer. After centrifugation for 15 min at 10,000 × g, the supernatant was ultracentrifuged at 100,000 × g for 30 min. This supernatant was concentrated by the addition of solid (NH₄)₂SO₄ to 80% saturation, stirred at 4 °C for 30 min, and centrifuged for 15 min at 20,000 × g. The precipitate was resuspended in volume of 20 mM Tris-Cl, pH 7.6, 20 mM KCl, 5 mM MgCl₂, 1 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride, 50 µM chymostatin, and 20 µM E64, and dialyzed against 20 mM Tris-Cl, pH 7.6, 20 mM KCl, 5 mM MgCl₂, 1 mM DTT, 10% glycerol at 4 °C overnight and stored at 70 °C until use.

Degradation of CaM in HeLa Extract-- Native or aged [³⁵S]CaM (~2 µg) was added to a 50-µl reaction containing 500 µg of HeLa cell extract supplemented with an ATP-regenerating system (10 mM creatine phosphate, 0.2 mg/ml creatine kinase) and protease inhibitors (50 µM E64, 1 mM phenylmethylsulfonyl fluoride, 50 µM chymostatin) in Buffer H (Buffer H: 50 mM Na-HEPES, pH 7.5, 5 mM MgCl₂, 1 mM ATP, 1 mM DTT) in the presence of 5 mM CaCl₂ or EGTA. The samples were incubated at 37 °C for 3 h, precipitated with trichloroacetic acid (at a final concentration of 12%), and the acid soluble radioactivity was measured in a liquid scintillation counter. (To determine the proteasome-mediated degradation of [³⁵S]CaM, parallel reactions were carried out in the presence of the proteasome inhibitor, MG-132 (50 µM), and the difference between total degradation and that in the presence of MG-132 was calculated.) Degradation was linear during the time period studied and proportional to the protein concentration of the extract.

Ubiquitination Assay-- Native or aged [³⁵S]CaM (~2 µg in 50 mM Na-HEPES, pH 7.5, 1 mM EGTA) was added to a 20-µl reaction containing 2 mM AMP-PNP, 30 µM MG-132, 1.5 µM ubiquitin aldehyde, 25 µM ubiquitin, and 50 µg of HeLa cell extract (37) in 20 mM Tris-Cl, pH 7.6, 20 mM KCl, 10 mM MgCl₂, 1 mM DTT. Additional Ca²⁺ or EGTA was added to the individual reactions to reach final concentrations of 5 mM. In the control experiment in Fig. 3a, 10 µg of Me-Ub was also added to the reaction (38). The samples were incubated at 37 °C for 60 min and analyzed by SDS-PAGE on 15% acrylamide gels. After electrophoresis, the gels were incubated for 20 min in 30% methanol, 10% acetic acid with gentle stirring, dried, and analyzed using a Fuji PhosphorImager. Densitometric analysis of the gels was carried out with Image Gauge software; the results are presented in arbitrary units.

Degradation of Proteins by 26 S Proteasomes-- 26 S proteasomes were prepared from young rabbit muscles as described previously (32). Native or aged [³⁵S]CaM (13.0 µM) were incubated with the proteasomes (28 nM) in Buffer H in the presence of 5 mM CaCl₂ or EGTA. Aliquots were removed at the indicated time points, mixed with 100 µl of 1 mg/ml bovine serum albumin, and precipitated with trichloroacetic acid (final concentration 12%). Acid-soluble counts were measured as above. In certain experiments ¹⁴C--casein and ¹⁴C--lactalbumin (reduced and carboxymethylated (32)), specific activity 40,000 cpm/µg) were incubated with the proteasomes under the same conditions.

Peptidase activity of the 26 S proteasomes (33 pM) was determined by measuring the hydrolysis of succinyl-LLVY-aminomethyl coumarin (100 µM, Bachem, Bubendorf, Switzerland) in Buffer H containing either 5 mM CaCl₂ or EGTA. The release of the fluorescent product, 7-amino-4-methylcoumarin, was measured in an Aminco-Bowman luminescence spectrometer by continuous assay (wavelength of excitation: 380 nm, emission 460 nm).

To assay ATP-dependent proteolysis, the ATP present in the 26 S proteasome preparations was removed by gel filtration using a NICK spin column (Amersham Pharmacia Biotech), equilibrated with 50 mM Na-HEPES, pH 7.5, 50 mM KCl, 5 mM MgCl₂, 1 mM DTT, 20% glycerol. After spinning, the proteasomes were divided into two parts and used immediately for the degradation reactions: one part was supplemented with ATP (1 mM) and the other with buffer only.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Aged and Native CaM Differ in Conformation and Ca²⁺ Binding-- Conformational changes accompanying Ca²⁺ binding and deamidation of CaM have been demonstrated using nondenaturing polyacrylamide gels (18, 36). To compare the conformational changes that occur upon Ca²⁺ binding in native CaM and in the in vitro aged species (incubated for 2 weeks at 37 °C and pH 7.4 in the absence of Ca²⁺), we extended this approach, using two-dimensional gels that were run in the presence of EDTA in the first dimension and in the presence of either EDTA or Ca²⁺ in the second dimension. In the first dimension, in the presence of EDTA, native CaM migrated as one predominant species plus two minor, slower variants. By contrast, aged CaM was resolved into at least four major and two minor variants, most of which showed a lower electrophoretic mobility than the predominant form of native CaM (data not shown). These differences were most clear when electrophoresis was carried out in the second dimension in the presence of EDTA, when the different aged species were distributed along the diagonal (Fig. 1). The reduced electrophoretic mobility of aged CaM variants on native gels has been attributed to their having a more extended conformation (18), since on the basis of charge alone, the deamidation occurring during aging would be expected to increase the proteins negative charge and electrophoretic mobility. The low amounts of slowly migrating variants in native CaM are due to isomerization of aspartyl residues at positions outside the Ca²⁺-binding regions (6, 17).

View larger version (57K): [in this window] [in a new window]   Fig. 1.   Ca2+-induced alterations in the conformation of aged and native CaM. Native and aged CaM preparations (10 µg each) were analyzed on two-dimensional nondenaturing gel electrophoresis (12, 18) in the presence of 2 mM EDTA in the first dimension, and either 2 mM EDTA or 5 mM CaCl2 in the second dimension. Proteins were visualized by Coomassie Blue staining.

When electrophoresis was performed in the presence of Ca²⁺ (5 mM) in the second dimension, the native CaM (including the minor variants) showed a large decrease in electrophoretic mobility, which is characteristic of Ca²⁺-liganded CaM. By contrast, most of the species present in the aged CaM preparation, when separated in the presence of Ca²⁺, showed a smaller mobility shift away from the diagonal than was seen with the native CaM. This mobility difference may reflect decreased Ca²⁺ binding by the aged species and/or a reduced ability of the bound Ca²⁺ to induce the same conformational changes in the aged CaM as in the native form. In addition, a small amount of the aged CaM showed an electrophoretic mobility shift, which was similar to that observed for the native species. Thus, some unmodified CaM appears to persist even after 2 weeks of in vitro aging. These findings support the prior observations that the presence of isoaspartyl residues in the Ca²⁺-binding regions of aged CaM alters its conformation and may interfere with Ca²⁺-binding or Ca²⁺-induced conformational changes (6).

Proteasomes Mediate the Degradation of Aged CaM in Oocytes-- Following in vitro aging, hemagglutinin-tagged CaM (HA-CaM) is rapidly degraded upon microinjection into Xenopus oocytes (8). By contrast, HA-CaM that was freshly isolated or was preincubated for 2 weeks in the presence of Ca²⁺ was stable for several hours after microinjection (8). To determine if proteasomes catalyze this selective destruction of the aged CaM, the oocytes were injected with the specific proteasome inhibitor, lactacystin (39), 30 min prior to CaM injection. In the untreated oocytes, the aged HA-CaM had a half-life of approximately 40 min (Fig. 2, upper panel). However, when lactacystin was injected in amounts that should give a final intracellular concentration found maximally effective in cultured cells (~10 µM) (39), it prevented the degradation of the aged HA-CaM (Fig. 2, lower panel). These results clearly implicate the proteasomes in the degradation of aged HA-CaM in oocytes. However, no accumulation of ubiquitinated derivatives of HA-CaM (i.e. higher molecular weight forms) could be detected in either the presence or absence of lactacystin (see below).

View larger version (57K): [in this window] [in a new window]   Fig. 2.   The proteasome inhibitor, lactacystin, increases the stability of in vitro aged HA-CaM upon microinjection into Xenopus oocytes. After in vitro aging (i.e. incubation for 2 weeks at pH 7.4 and 37 °C in the presence of EGTA), HA-CaM was microinjected into Xenopus oocytes that had been previously microinjected with the proteasome inhibitor lactacystin or with an equal volume of buffer alone. Following incubation at 20 °C for the indicated times, extracts were prepared, and HA-CaM levels were analyzed by immunoblotting.

Aged CaM Is Degraded More Rapidly Than Native CaM in Cell Extracts-- To study the mechanisms responsible for the rapid degradation of aged CaM, we investigated this process in HeLa cell extracts, which have previously been shown to carry out both the ubiquitination and the degradation of many intracellular proteins (37). Degradation and ubiquitination of native and aged [³⁵S]CaM were studied both in the presence and absence of Ca²⁺, because the data in Fig. 1 suggested major structural differences between native and aged CaM upon Ca²⁺ binding. In extracts containing EGTA to chelate Ca²⁺, aged [³⁵S]CaM was degraded 8 times more rapidly than native [³⁵S]CaM (Table I). The addition of high concentrations of Ca²⁺ (5 mM) reduced the rate of hydrolysis of native [³⁵S]CaM to almost half of that observed in the absence of Ca²⁺. The rate of degradation of aged [³⁵S]CaM was also reduced by the addition of Ca²⁺, but the relative effect was smaller (26%). Thus, in the presence of Ca²⁺, the difference in the rate of degradation between aged and native [³⁵S]CaM was even more pronounced; the aged species was degraded 11 times faster. These results are in good agreement with the findings in oocytes (Fig. 2 and Ref. 8) demonstrating that aged CaM is degraded much more rapidly than the native molecule.

To analyze how Ca²⁺ and aging influence degradation by the proteasomes, we focused on the component of the proteolysis that is sensitive to the proteasome inhibitor, MG-132 (Table I). In the absence of Ca²⁺, the proteasome-mediated degradation of aged [³⁵S]CaM (i.e. the MG-132 sensitive component) was 11 times larger than that of native [³⁵S]CaM. The addition of Ca²⁺ reduced the proteasomal degradation of native [³⁵S]CaM by over 50% and that of aged CaM by about 30%. Therefore, in the presence of Ca²⁺ the proteasome-mediated degradation of aged [³⁵S]CaM was actually 16 times that of native CaM. MG-132 reduced total proteolysis in these crude extracts by 50-60%, however, these measurements most probably underestimate the contribution of proteasomes to CaM degradation, since these peptide aldehyde inhibitors are less effective in crude extracts than in intact cells (40). Although additional proteases may also contribute to the degradation of CaM in these extracts, the application of inhibitors of serine and cysteine proteases (chymostatin, phenylmethylsulfonyl fluoride, E64) and incubation at pH 7.4 should have minimized the contribution of lysosomal proteases or calpains. (In fact, the addition of Ca²⁺, which usually activates calpains, inhibited the degradation of CaM.)

The results obtained in the extracts are consistent with the in vivo data indicating a major role for the proteasomes in the degradation of aged CaM. In addition, the degradation of native CaM by proteasomes appears to be very sensitive to Ca²⁺-induced conformational changes, and these effects appear less pronounced with the aged species.

Aging Does Not Promote CaM Ubiquitination-- Ubiquitin conjugation to [³⁵S]CaM was studied in these same HeLa extracts by following the ATP-dependent appearance of radioactive species with higher apparent molecular weights, which represent conjugates of [³⁵S]CaM with multiple ubiquitin molecules. To prevent the hydrolysis of any conjugates formed during the assay, experiments were performed in the presence of ubiquitin aldehyde, which inhibits disassembly of ubiquitin chains by ubiquitin peptide hydrolases (41), the proteasome inhibitor MG-132, and the ATP analog AMP-PNP, which allows ubiquitination, but does not support protein degradation by the proteasome. In these extracts the extent of ubiquitin conjugation to CaM was low (Fig. 3a), which is consistent with its being a relatively stable protein in vivo (9, 10). The addition of Ca²⁺ to the HeLa extracts stimulated the formation of high molecular weight adducts of [³⁵S]CaM (Fig. 3a, lane 2 versus 3), as was also found in reticulocyte lysates (42). To prove that these adducts were indeed ubiquitin-CaM conjugates, Me-Ub was added to the reactions to inhibit the formation of poly-Ub chains (38). As expected, the addition of Me-Ub resulted in the disappearance of the high molecular weight [³⁵S]CaM adducts from the top of the gel, and in an increase in the amount of the monoubiquitinated form (Fig. 3a, lane 4), indicating that the high molecular weight species were [³⁵S]CaM conjugates with Ub chains of different lengths. The effect of Ca²⁺ was specific for the ubiquitination of CaM, since overall ubiquitination in the extracts, as determined by ¹²⁵I-Ub conjugation to endogenous substrates, was not influenced by the presence of Ca²⁺ (Fig. 3b).

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Aged and native CaM are ubiquitinated at similar rates in HeLa cell extracts. Native (a and c) and aged (c) [35S]CaM were ubiquitinated with Ub and ATP in HeLa extracts in the presence of Ca2+ or EGTA as described under "Experimental Procedures." After 60 min, proteins were separated on 15% SDS-PAGE and visualized by autoradiography. a, native CaM was incubated without (lane 1) or with HeLa extract (lanes 2-4), in the presence of EGTA (lane 2) or Ca2+ (lanes 3 and 4) and Me-Ub (lane 4). b, ubiquitination of HeLa cell proteins was assayed by incubation of the crude extracts (50 µg of protein) with 125I-Ub in the presence of Ca2+ (lane 2) or EGTA (lane 3). Lane 1, 125I-Ub incubated without HeLa extract. c, native CaM (lanes 2, 4, and 6) and aged CaM (lanes 1, 3, and 5) were incubated without (lanes 1 and 2) or with HeLa extract (lanes 3-6) in the presence of Ca2+ (lanes 5 and 6) or EGTA (lanes 1, 2, 5, and 6). (In most experiments, aged and native CaM were ubiquitinated to a similar extent. But in some cases, as shown here, the ubiquitin conjugation to the aged CaM was less than to native CaM.)

When the ubiquitination of aged CaM was compared with that of the native molecule, no major differences were found in the extent of Ub conjugation (Fig. 3c), despite the much faster degradation of the aged species. In the absence of Ca²⁺ (i.e. with EGTA present), only a small amount of monoubiquitinated [³⁵S]CaM was observed with both the native and aged substrates, even though the degradation of both proteins was rapid under these conditions. The presence of Ca²⁺, while inhibiting CaM degradation, stimulated the ubiquitination of both aged and native forms. In some experiments, as shown in Fig. 3c, the extent of ubiquitination of the aged molecule was actually lower than that of native [³⁵S]CaM. Thus, the ubiquitination of the aged and native CaM does not correlate with its rate of degradation (Table I).

Because we had anticipated greater ubiquitination of the more rapidly degraded aged CaM, the rates of ubiquitination of these proteins were also compared in extracts prepared from Xenopus eggs, rabbit reticulocytes, and rabbit skeletal muscle. In all extracts, Ub conjugation to [³⁵S]CaM was stimulated by Ca²⁺, and the degree of Ub conjugation to aged and native [³⁵S]CaM was similar (data not shown). Clearly, while aged [³⁵S]CaM is degraded much more rapidly than the native form, it is ubiquitinated to a similar or lesser extent. These various observations suggested that the age-damaged CaM may be degraded by the proteasome in a primarily Ub-independent manner.

Native and Aged [³⁵S]CaM Are Degraded by Pure 26 S Proteasomes and Ca²⁺ Inhibits this Process-- Using 26 S proteasomes purified from rabbit skeletal muscles (32), we tested the possibility that the aged [³⁵S]CaM was degraded directly by the proteasome without prior ubiquitination. No ubiquitin or ubiquitinating enzymes were present in these reactions. As expected from the above experiments, aged [³⁵S]CaM was rapidly degraded by the 26 S proteasomes in the absence of ubiquitination (Fig. 4). Surprisingly, however, native apo-CaM was also degraded at a significant rate by the 26 S proteasomes, but not as rapidly as the aged species (Fig. 4, left panel). The degradation of both native and aged [³⁵S]CaM was stimulated by ATP (1 mM) by about 3-fold (Table II), indicating that the degradation was catalyzed primarily (or exclusively) by the 26 S form of the proteasome.

View larger version (15K): [in this window] [in a new window]   Fig. 4.   26 S proteasomes degrade native and aged CaM without prior ubiquitination. Native and aged [35S]CaM (13.0 µM) were incubated at 37 °C with purified rabbit muscle 26 S proteasomes (28 nM) in the presence of ATP (1 mM) in buffer containing 5 mM CaCl2 or EGTA. Aliquots were removed at the indicated time points, treated with trichloroacetic acid, and the amount of acid soluble radioactivity was determined by liquid scintillation counting.

In the absence of Ca²⁺, both aged and native [³⁵S]CaM were rapidly degraded, and the rate of degradation of the aged species was about twice that observed for the native molecule. The addition of Ca²⁺ was found to reduce the degradation of both native and aged [³⁵S]CaM by the 26 S proteasome, but the magnitude of this inhibition was very different for the two species. In the presence of Ca²⁺, native [³⁵S]CaM degradation was almost completely blocked, while the breakdown of aged [³⁵S]CaM was decreased by only about 15%. This dramatic effect of Ca²⁺ on the degradation of the native protein is most likely due to the change in CaM's conformation and cannot be attributed to an effect of Ca²⁺ on the functional properties of the 26 S proteasome, because the degradation of other proteins was not inhibited by the addition of Ca²⁺, but instead appeared to be stimulated (Table III). Similarly, Ca²⁺ enhanced (by 21%) the cleavage of the specific tetrapeptide substrate, succinyl-LLVY-aminomethyl coumarin (100 µM). These observations indicate that upon Ca²⁺ binding, native CaM assumes a conformation that cannot be degraded by the 26 S proteasome. However, the chemical modifications, which occur during aging, appears to interfere with Ca²⁺-binding or with Ca²⁺-induced conformational changes, allowing the aged CaM to be rapidly degraded by the 26 S proteasome regardless of the Ca²⁺ levels.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Ubiquitin-independent Degradation of Native CaM in Vitro-- Although our initial goal was to identify the mechanism for the selective degradation of age-damaged proteins using CaM as a model substrate, these studies have uncovered unexpected findings regarding the pathway for the degradation of the native molecule. Rapid degradation of proteins in vivo by the 26 S proteasome usually requires their conjugation with a chain of ubiquitin molecules (2). The generality of this ubiquitin requirement, however, has been very difficult to establish in vivo due to lack of specific inhibitors of ubiquitination, and because of the transient nature of the Ub-protein conjugates. The only clear exception to this Ub requirement for proteolysis in vivo is the enzyme ornithine decarboxylase, which is targeted for degradation by the 26 S proteasome through its association with the specific regulatory protein, antizyme (27). Although both native and aged CaM can be conjugated with ubiquitin (Fig. 3c), this step did not correlate at all with proteolysis and was not required for degradation by the purified 26 S proteasomes (Fig. 4). Ubiquitination was markedly stimulated by Ca²⁺ binding, while proteolysis was greatly reduced (Table I). Thus, for CaM, and presumably some other proteins, ubiquitination and degradation can be uncoupled (31, 43). CaM is one of the few clear examples of an intracellular protein that can be degraded rapidly by the 26 S proteasome in the absence of ubiquitination (28-30). Recently, the Cdk inhibitor p21 has also been shown to be degraded in vivo by proteasomes without ubiquitination, and it was proposed that the lack of a "well-defined tertiary structure" triggers its recognition by the proteasomes (44). CaM without bound Ca²⁺, however, has a specific tertiary structure although it differs from that of the Ca²⁺-bound form (5). Therefore other factors, e.g. increased flexibility, are more likely to contribute to recognition and degradation by the proteasomes. Indeed, NMR studies have shown that native CaM, with four Ca²⁺ ions bound, is significantly less flexible than the Ca²⁺-free, apo-form (5). In the Ca²⁺-liganded form, the Ca²⁺-binding loops are highly structured, and the only flexible regions are at the N terminus and in the middle region between the two globular domains (4). In the absence of Ca²⁺, there is a major change in helix packing leading to a large increase in the mobility of the Ca²⁺-binding loops (5). Presumably, the rigidity of the Ca²⁺-liganded form accounts for its resistance to degradation by the 26 S proteasome, while the greater flexibility of the apo-form allows it to be readily unfolded and translocated into the 20 S core particle.

Ubiquitin-independent Degradation of Aged CaM-- In vitro aging results in the formation of isoaspartyl residues predominantly in the Ca²⁺-binding loops of CaM (12), which presumably alters its conformation, increases its flexibility, and reduces its Ca²⁺ binding, as suggested by Fig. 1. Although aged CaM was selectively degraded in intact oocytes and cell lysates by proteasomes, the altered conformation of the aged CaM did not increase its rate of ubiquitination. On the contrary, in several experiments, Ub conjugation to the aged CaM was actually reduced (e.g. Fig. 3c), even though it was degraded many times faster than the native molecule (Table I). Since purified 26 S proteasomes were consistently able to degrade the aged species without ubiquitination, the structural modifications during aging must directly target the damaged protein to this degradative complex. These findings do not support the widespread belief that Ub-protein ligases (E3s) are responsible for the recognition of substrates with abnormal conformation and their channeling to the proteasomes via ubiquitination. Instead, components of the 26 S proteasome must be able to selectively bind the aged molecules. Recognition is most probably mediated by the 19 S regulatory particle, which has recently been found to preferentially bind denatured proteins (45). More specifically, substrate binding might occur through the six ATPase subunits of the 19 S particle, since the homologous protease-regulating ATPase complexes in prokaryotes (HslV, ClpA, and PAN) are all critical in substrate recognition and preferentially bind unfolded polypeptides (46, 47). Alternatively, it is also possible that the native and aged forms both bind to the proteasome, but only the more flexible one can be unfolded and degraded at an appreciable rate.

It seems quite unlikely that the proteasomes directly recognize the isoaspartyl residues or other amino acid modifications in the aged protein, since after Ca²⁺ removal, the native CaM was degraded almost as fast as the aged molecule. More likely the increased conformational flexibility arising from chemical damage or ligand-removal enhances a generally Ub-independent degradation by the 26 S proteasome. It is noteworthy that a number of unfolded proteins have been shown both to be degraded in extracts without ubiquitination (48) and also to be digested by isolated proteasomes in an ATP-dependent manner (32). It remains unclear to what extent other polypeptides may be degraded by the proteasome without ubiquitin conjugation in vivo.

The Effect of Ligand (Ca²⁺) Binding on CaM Ubiquitination and Degradation-- As noted, the ubiquitination and degradation of CaM paradoxically show opposite sensitivities to Ca²⁺ (Table I). In the absence of Ca²⁺, some monoubiquitination of CaM occurred (Fig. 3c), although most data suggest that the attachment of one Ub to a substrate does not generally promote its degradation (31, 49). The monoubiquitination of CaM probably involves Lys-115, a residue only available for ubiquitination in bacterially expressed CaMs (as used here) and in Dictyostelium discoideum (50). In other species, Lys-115 is blocked by post-translational trimethylation, therefore the functional importance, if any, of CaM monoubiquitination is unclear. On the other hand, the formation of multiubiquitin conjugates in these extracts required the presence of Ca²⁺ (Fig. 3a), as was also found in reticulocyte lysates by Laub and co-workers (51) who showed that Ub is conjugated to Lys-21 in the first Ca²⁺-binding loop of CaM. Although the formation of multiubiquitin conjugates would be expected to promote proteolysis (31, 52), the degradation of CaM is clearly decreased in the extract in the presence of Ca²⁺ (Table I) (53). It is possible that the multiubiquitinated CaM retains sufficient Ca²⁺ binding ability to maintain a structure too rigid to be unfolded and translocated into the proteasome. Whether ligand binding can prevent the degradation of other substrates by the 26 S proteasome remains to be investigated. Another example in which ligand binding prevented proteasomal degradation is dihydrofolate reductase, whose degradation, after ubiquitination, was inhibited by binding of its ligands folic acid or methotrexate (31, 43).

CaM Degradation in Vivo-- Our data clearly demonstrate that aged and native apo-CaM can be degraded by purified proteasomes without prior ubiquitination in vitro. Since CaM in cells is generally in the Ca²⁺-free form, Ub-independent degradation may also represent a major pathway for CaM degradation in vivo. However, we cannot exclude the possibility that ubiquitination may still influence CaM degradation. Perhaps when cytosolic Ca²⁺ levels rise (e.g. in contracting muscle), Ub conjugation to CaM is temporarily stimulated, and subsequently, when Ca²⁺ levels return to normal, and Ca²⁺ dissociates from CaM, these ubiquitinated apo-species are degraded by the proteasomes.

In addition to influencing proteasomal degradation directly, the greater flexibility of the apo-CaM also makes it more susceptible to the age-related reactions that generate isoaspartyl residues (12), thereby further enhancing its proteasomal degradation. Since the degradation of the aged CaM was much less inhibited by the presence of Ca²⁺, these damaged species are probably eliminated continuously, without ubiquitination, regardless of the Ca²⁺ level in the cell. However, these damaged species can have alternative fates. Enzymatic carboxyl methylation of isoaspartyl residues in aged CaM was shown to reduce the degradation of the aged molecule in Xenopus oocytes (8). This protective effect of methylation may be due to enzymatic repair and refolding of the damaged molecule. In fact the enzyme protein-isoaspartyl methyltransferase has been shown to catalyze the formation of a new electrophoretic variant from aged CaM with a partial recovery of its activity (18). Thus selective hydrolysis by the proteasome and enzymatic repair may constitute alternative mechanisms for preventing the intracellular accumulation of damaged proteins.

	ACKNOWLEDGEMENTS

We thank to Drs. Olga Kandror, Nadia Benaroudj, Alexei Kisselev, Tomo Saric, and Thomas Jagoe for critical reading of the manuscript.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants GM46147 (to A. L. G.), AG08109 (to C. M. O.), and DK02707 (to S. H. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Contributed equally to the results of this paper.

To whom correspondence should be addressed: Dept. of Cell Biology, Harvard Medical School, 240 Longwood Ave., Bldg. C1-415, Boston, MA 02115. Tel.: 617-432-1855; Fax: 617-232-0173; E-mail: alfred_goldberg@hms.harvard.edu.

Published, JBC Papers in Press, April 28, 2000, DOI 10.1074/jbc.M001555200

	ABBREVIATIONS

The abbreviations used are: Ub, ubiquitin; CaM, calmodulin; PAGE, polyacrylamide gel electrophoresis; DTT, dithiothreitol; Me-, methyl-; HA, hemagglutinin; AMP-PNP, 5'-adenylyl-,-imidodiphosphate.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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