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(Circulation Research. 2006;99:372.)
© 2006 American Heart Association, Inc.

Molecular Medicine

Regulation of Murine Cardiac 20S Proteasomes

Role of Associating Partners

Chenggong Zong, Aldrin V. Gomes, Oliver Drews, Xiaohai Li, Glen W. Young, Beniam Berhane, Xin Qiao, Samuel W. French, Fawzia Bardag-Gorce, Peipei Ping

From the Departments of Physiology and Medicine (C.Z., A.V.G., O.D., X.L., G.W.Y., B.B., X.Q., P.P.), School of Medicine, University of California at Los Angeles; and the Department of Pathology and Medicine (S.W.F., F.B.-G.), Harbor-UCLA Medical Center, Torrance, Calif.

Correspondence to Peipei Ping, Department of Physiology, UCLA School of Medicine, MRL Building, Suite 1609 CVRL, 675 CE Young Dr, Los Angeles, CA 90095. E-mail pping{at}mednet.ucla.edu

See related article, pages 362–371

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Our recent studies have provided a proteomic blueprint of the 26S proteasome complexes in the heart, among which 20S proteasomes were found to contain cylinder-shaped structures consisting of both and ß subunits. These proteasomes exhibit a number of features unique to the myocardium, including striking differences in post-translational modifications (PTMs) of individual subunits and novel PTMs that have not been previously reported. To date, mechanisms contributing to the regulation of this myocardial proteolytic core system remain largely undefined; in particular, little is known regarding PTM-dependent regulation of cardiac proteasomes. In this investigation, we seek to elucidate the function and regulation of 20S proteasome complexes in the heart. Functionally viable murine cardiac 20S proteasomes were purified. Tandem mass spectrometry analyses, combined with native gel electrophoresis, immunoprecipitation, and immunoblotting, revealed the identification of 2 previously unrecognized functional partners in the endogenous intact cardiac 20S complexes: protein phosphatase 2A (PP2A), and protein kinase A (PKA). Furthermore, our results demonstrated that PP2A and PKA profoundly impact the proteolytic function of 20S proteasomes: phosphorylation of 20S complexes enhances the peptidase activity of individual subunits in a substrate-specific fashion. Moreover, inhibition of PP2A or the addition of PKA significantly modified both the serine- and threonine-phosphorylation profile of proteasomes; multiple individual subunits of 20S (eg, 1 and ß2) were targets of PP2A and PKA. Taken together, these studies provide the first demonstration that the function of cardiac 20S proteasomes is modulated by associating partners and that phosphorylation may serve as a key mechanism for regulation.

Key Words: 20S proteasomes • mass spectrometry • phosphorylation • associating partners

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The 20S proteasomes are a collection of multimeric protease complexes that consist of 2 groups of homologous proteins, namely 7 and 7 ß subunits, forming 4 stacked rings (ßß).^1,2 In eukaryotes, 3 ß subunits (ß1, ß2, and ß5) are catalytically active, whereas other subunits are catalytically silent but may participate in functions such as controlling substrates access. The 26S proteasomes, which are composed of one 20S core particle flanked by one or two 19S regulatory particles, are responsible for degradation of polyubiquitin-tagged proteins.² In contrast, 20S proteasomes by themselves can degrade oxidatively damaged proteins^3,4 and certain nontagged proteins.⁵

The cardiac 26S proteasome complexes have been recently mapped.⁶ Recent investigations identified deficits in the ubiquitin-proteasome pathway and offered significant insights in protein quality control of diseased myocardium,^7–11 suggesting important roles of the protein degradation process in the heart. However, the functionality of individual cardiac 20S proteasome subunits and their regulatory mechanisms remain largely unknown.

Regulation of 20S proteasome is thought to be critical to maintain homeostasis.^1,2,12 In mammalian cells, the inducible ß subunits (ß1i, ß2i, and ß5i) can replace their constitutively expressed counterparts (ß1, ß2, and ß5) to form different species of proteasome complexes, which are shown to be important in the immune response.¹³ In 26S proteasomes of noncardiac tissues, regulatory particles, including 19S and 11S, and PA200 positively regulate protein degradation, whereas PI31 and PR39 have inhibitory effects.^2,14–16 Most members of the regulatory particles function via modulation of substrate entry. Post-translational modification of 20S subunits (eg, phosphorylation of subunits) has been reported,^17–23 however, a comprehensive phosphorylation profile on cardiac 20S proteasomes is lacking. It remains unknown whether phosphorylation plays a regulatory role in cardiac proteasome function and, if so, which molecules conduct the phosphorylation and which molecules are targeted by the phosphorylation.

In this investigation, we used an integrated proteomic and biochemical approach to establish regulatory mechanisms of cardiac 20S proteasomes. We characterized peptidase activities for ß1, ß2, and ß5 in the heart, and we defined endogenous phosphorylation patterns of individual 20S subunits. Importantly, protein kinase A (PKA) and a phosphatase, protein phosphatase 2A (PP2A), were identified as novel functional associating partners of the native cardiac 20S complexes. Phosphorylation of 20S subunits enhanced proteasome function in the heart. These results provide fundamental information pertaining to regulation of 20S proteasomes in the heart.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
All procedures were performed in accordance with the Animal Research Committee guidelines at the University of California-Los Angeles and the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health. All methods are abbreviated and details are provided in the online data supplement available at http://circres.ahajournals.org.

Materials
Z-LLE-AMC, Boc-LSTR-AMC, and PKA were acquired from Sigma and Suc-LLVY-AMC from Bachem. Okadaic acid and phospho-antibodies were from Calbiochem; anti-3 antibodies, Biosource; anti-rabbit IgG and anti-mouse IgG, Rockland Immunochemicals; anti-mouse IgM, SYPRO Ruby, and Pro-Q diamond stains, Invitrogen; and PKA and PP2A antibodies, BD Biosciences.

Purification of Murine Cardiac 20S Proteasomes
Highly purified, 98% pure, murine cardiac 20S proteasomes were obtained via numerous trial-and-error pilot studies leading to a protocol effective to isolate proteasomes from cardiac muscle. Briefly, hearts were homogenized. Cytosolic fraction was collected and fractionated by 40% to 60% of ammonium sulfate precipitation to yield a pellet. A stepwise separation with preparative-scale ion-exchange chromatography (Q-FastFlow) enriched 20S proteasomes in the 75% buffer B eluted fraction (Figure 1A). This fraction was centrifuged to collect high-molecular-weight complexes; the 20S complexes were recovered at 60% of buffer B with an analytical-scale ion-exchange chromatography (Mono-Q) gradient elution (Figure 1B).

View larger version (36K): [in this window] [in a new window]   Figure 1. Purification of murine cardiac 20S proteasomes. A combined proteomic and biochemical approach was used. Hearts were homogenized and enriched 20S proteasomes were obtained in the 40% to 60% ammonium sulfate pellet. A, Subsequent to removing excess salts, the 20S-enriched sample was eluted by 75% of buffer B during Q Fast Flow preparative-scale ion-exchange chromatography and recovered by ultracentrifugation. The 3-step salt elution protocol, as well as the ultracentrifugation, were created to gain adequate separation and yield of cardiac 20S proteasomes. B, 20S Proteasomes were further resolved by analytical-scale Mono-Q ion-exchanged chromatography with a linear salt concentration gradient from 0% buffer B (0 mmol/L KCl) to 100% buffer B (600 mmol/L KCl). Purified 20S proteasomes were collected at 60% buffer B (360 mmol/L KCl). The 20S proteasomes eluted in the major peak of UV280, which corresponded to fractions (D9–E2) and were well separated from contaminants. C, Coomassie-stained SDS-PAGE documented high purity of 20S proteasome subunits in fractions D9–E2 in the correct MW range (20 to 30 kDa), whereas fractions D6–D7 contained residual contaminants. D, Immunoblotting using antibodies against the core 20S subunits (recognizing six subunits, 1, 2, 3, 5, 6, 7) confirmed the presence of 20S proteasomes in fractions D9–E2. E, The functional integrity of purified 20S proteasomes in fractions D9–E2 was determined by their proteolytic activities, which were consistent with their protein expression in Panel D. F, The structural integrity of purified 20S proteasomes (fraction D10) was examined by electron microscopy as shown in the left; the bottom view and side view of a murine cardiac proteasome illustrated the structure of 4 stacking rings (pictures in the middle); a wireframe model of bovine liver 20S proteasome as retrieved from PDB (http://www.pdb.org/36).

Characterization of 20S Proteasomes
Endogenous cardiac 20S proteasomes were separated by 2-dimensional electrophoresis (2-DE) and stained with SYPRO Ruby or immunoblotted with antibodies against phospho-serine, phospho-threonine, or phospho-tyrosine. Images acquired from fluorescently labeled immunoblots (Odyssey) were superimposed with SYPRO Ruby-stained images, from which the protein spots were identified by liquid chromatography/tandem mass spectroscopy (LC/MS/MS). In a separate study,⁶ we verified the cardiomyocyte-specific expression of 20S subunits. In parallel experiments, blue-native gel electrophoresis (BN-PAGE) was used to resolve the intact native cardiac 20S complexes, which contain all subunits and associating partners. The BN-PAGEs were immunoblotted simultaneously with antibodies specific to proteasome subunits, associating proteins, or both.²⁴ A dual-laser fluorescent scanner (Odyssey) was used to detect both signals simultaneously.

The electron microscopic study of cardiac 20S proteasome complexes is detailed in the online data supplement.

LC/MS/MS Identification of 20S Proteasome Complexes
20S Proteasome complexes separated by 1-dimensional electrophoresis (1-DE), 2-DE, or BN-PAGE were subjected to in-gel digestion. The resulting peptides were resolved with reverse phase high-performance liquid chromatography (PicoFrit) coupled to LC/MS/MS, as detailed in the online data supplement.

Measurement of 20S Proteasome Peptidase Activities
The 3 enzymatically active subunits (ß1, ß2, and ß5) were assayed using fluorescently tagged substrates (Z-LLE-AMC for ß1, Boc-LSTR-AMC for ß2, Suc-LLVY-AMC for ß5). In particular, a significant effort was made to optimize buffer cocktails that unmasked the proteolytic activities of cardiac proteasomes. The buffers contained distinct concentrations of unique detergents as inducers. To determine roles of PP2A and PKA in proteasome function, we incubated PP2A inhibitor or PKA in cocktail buffers with purified cardiac proteasomes and examined the peptidase activities specific for the ß1, ß2, and ß5 subunits as detailed in the online data supplement.

Defining Phosphorylation of 20S Proteasome Subunits
Cardiac 20S proteasomes were incubated with either PKA or PP2A. Phosphatase- or kinase-treated proteasomes were precipitated, resolved by 2-DE, and probed with phospho-specific antibodies to detect changes in phosphorylation state. The Odyssey dual-laser scanner enabled the use of 3 as an internal loading control. The anti-phospho-antibody cocktail (Calbiochem) was used.

Statistical Analyses
All data are presented as mean±SE. Student t test was used for unpaired data. P<0.05 was considered statistically significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Characterization of Murine Cardiac 20S Proteasomes
Significant effort was made to optimize the isolation procedures to gain high- purity cardiac 20S proteasomes (see the online data supplement).⁶ Using our protocol, 10 g of cardiac tissue yielded 860 µg of highly purified 20S proteasomes (>98% purity as determined by Coomassie staining). Briefly, the proteasome complexes were purified using a 3-step preparative ion-exchange chromatography (Figure 1A). A fraction from each elution step was collected and analyzed. The chromatogram documents ratios of UV260 nm/280 nm, indicating that the first 2 fractions were enriched in proteins (Figure 1A). Subsequently, a linear gradient elution of an analytical ion-exchange column (Figure 1B) was applied to gain highly enriched 20S proteasomes (Figure 1C and 1D). The proteolytic function of purified 20S proteasomes was determined (Figure 1E), and the structure integrity of the purified cardiac 20S proteasomes was examined by electron microscopy (EM) analyses (Figure 1F), which consistently showed a population of cardiac 20S proteasomes exhibiting a barrel-like structure. Multiple EM micrographs were reviewed; an individual 20S proteasome complex is shown at a magnification of 100 000x.

The murine cardiac 20S proteasomes have been recently mapped by our group.⁶ All 17 subunits of 20S proteasomes were identified with high-sequence coverage (this information is shown in online Table I). The high sequence coverage facilitated the investigation of post-translational modifications (PTMs) on multiple 20S subunits. Mass spectra analyses revealed N-terminal acetylation on subunits 2, 5, 7, ß3, and ß4 (online Table I). 2-DE analysis of cardiac 20S proteasomes documented the expression of 7 and 7 ß subunits (Figure 2A).

View larger version (26K): [in this window] [in a new window]   Figure 2. Phosphorylation profile of the cardiac 20S proteasome subunits. A, Purified cardiac 20S proteasomes were resolved by 4 sets of 2-DEs. The first set was stained with SYPRO Ruby, imaged, and proteasome subunit identifications made (LC/MS/MS); the remaining 3 sets were immunoblotted with phospho-specific antibodies. The signals of immunoblots were aligned with proteasome subunits identified. The 1, 2, 3, 6, ß2 subunits were detected by phospho-serine antibodies; 1 and 6 recognized by phospho-threonine and phospho-tyrosine antibodies. B, LC/MS/MS identified a C-terminal serine-250 phosphorylation of 7, which was not detected by phospho-serine antibodies.

Endogenous Phosphorylation of 20S Proteasomes
Using 2-DE, immunoblotting, and LC/MS/MS, we delineated the endogenous phosphorylation profile of individual 20S subunits (Figure 2A). As shown in Figure 2A, 1, 2, 3, 6, and ß2 subunits were detected by phospho-serine antibodies; 1 and 6 subunits were detected by both phospho-threonine and phospho-tyrosine antibodies (Figure 2A). In separate studies, 7 subunit was stained by Pro-Q (a phospho dye), and phosphorylation of its C-terminal serine-250 residue was confirmed by LC/MS/MS (Figure 2B). The lack of phospho-serine antibody staining of the 7 subunit is likely because of bias against its sequence motif.

Optimization of Peptidase Activity Assays
ß1, ß2, and ß5 are proteolytically active subunits with distinct substrates bias, which can be experimentally differentiated with specific substrates.² However, we met challenges in demonstrating the proteolytic activities of ß1, ß2, and ß5 subunits in the murine cardiac 20S proteasomes using published protocols. In particular, the enzymatic activities of ß1 and ß2 were less demonstrable, thus giving the false impression that these 2 subunits were attenuated in the normal heart. Traditionally, 0.03% sodium dodecyl sulfate (SDS) was used to reveal the latency of the 20S proteasomes for activity assays,^25,26 but this was ineffective for the cardiac ß1 and ß2. To address this issue, we conducted experiments with a variety of detergent choices to optimize assay conditions (online Figure I). Optimal detergents and their concentrations for the cardiac 20S proteasome activities were determined: 0.05% NP-40 and 0.001% SDS cocktails were identified to be optimal for both ß1 and ß2 activities (Figure 3A and 3B), whereas 0.03% SDS was found to be the best for the ß5 activity (Figure 3C).

View larger version (32K): [in this window] [in a new window]   Figure 3. Buffer conditions for the determination of cardiac 20S proteolytic activities. Assay conditions were optimized to gain demonstration of proteolytic activities of purified cardiac ß1 (A), ß2 (B), and ß5 (C). 20S Proteasomes remain latent in the absence of activator complexes (eg, 19S); detergents were required to release the latency. The effect of various detergents was carefully examined, with the proteolytic activity of both ß1 and ß2 best optimized in 0.05% NP-40 and 0.001% SDS, whereas that of the ß5 was best optimized in 0.03% SDS. Additional information is available in the online data supplement (online Figures I and II).

Identification of Endogenous Associating Partners of 20S Proteasomes
We reasoned that if a molecule is a functional associating partner, it must satisfy the following criteria. First, it resides within the native and enzymatically active 20S complexes. Second, it impacts the functionality of 20S proteasome complexes. Accordingly, 4 sets of experiments were conducted to characterize functional associating partners of murine cardiac 20S proteasomes: (1) identification of candidate proteins that are associated with the purified 20S proteasomes; (2) confirmation of candidate proteins that reside within the native 20S proteasome complexes; (3) determination of their impact on 20S proteasome function; and (4) characterization of molecular targets of functional associating partners.

Identification of Candidate Proteins That May Serve as Associating Partners
Purified cardiac 20S proteasomes were separated on 1-DE, and gel plugs were sequentially excised. LC/MS/MS analysis identified all 20S proteasome subunits. In addition, potential associating partners were found in the 20S complexes, among which are , ß, and c subunits of PP2A (Figure 4A and Table), as well as the catalytic subunit of PKA (Table).

View larger version (31K): [in this window] [in a new window]   Figure 4. Identification of PP2A and PKA as cardiac 20S associating partners. 1-DE, BN-PAGE, immunoblotting, immunoprecipitation, and LC/MS/MS were used to identify associating partners that coexist with the intact 20S proteasome complexes. A, An example of LC/MS/MS identification of PP2A catalytic subunit from the purified cardiac 20S proteasomes; a representative MS spectrum illustrates 1 peptide sequence from a total of 15 unique peptides identified for the PP2A catalytic subunit (see Table 1). B, The intact 20S complexes were displayed by native electrophoresis; the expression of PP2A (upper blot) and PKA (lower blot) in the intact 20S complexes were detected. The 3 subunit (illustrated by a Cy5.5 labeled secondary antibody) was used to document the expression of 20S proteasomes.

View this table: [in this window] [in a new window]   Table 1. Identification of Associating Partners of Cardiac 20S Proteasomes

Confirmation of Candidate Proteins Within the Native 20S Complexes
To determine whether PP2A and PKA reside within the native cardiac 20S proteasomes, purified cardiac 20S proteasomes were immunoprecipitated with 3 antibodies. The bound protein complexes were resolved by 1-DE and analyzed by LC/MS/MS; an example of the PP2A catalytic c subunit is shown (Figure 4A). Subsequently, purified 20S complexes were displayed on a nondenaturing gel (under which conditions, intact 20S proteasomes are maintained in their native forms) and immunoblotted with antibodies against PKA or PP2A (Figure 4B).

Regulation of Cardiac 20S Proteasomes by Associating Partners
The evidence that various 20S subunits show endogenous phosphorylation, combined with the identification of phosphatase and kinase as associating partners, served as the basis of our hypotheses that phosphorylation may be a mechanism for regulation and that PP2A and PKA may impact the proteolytic function of 20S proteasomes. Because current technologies do not permit separation of the associating partners from the proteasomes without impacting the integrity of 20S complexes, we choose to take a pharmacological approach. The effect of associating partners was examined against an estimated range of substrate concentrations (0, 2, 10, 20, 50, 100, 200, and 500 µmol/L) in a subunit specific fashion for all 3 subunits (ß1, ß2, and ß5). All experiments were repeated 6 times, and statistic significance was evaluated (P<0.05).

The role of PP2A was tested using its inhibitor, okadaic acid. Incubation of cardiac 20S proteasomes with 10 or 50 nmol/L okadaic acid affected all 3 proteolytic activities in a similar fashion. The concentrations of okadaic acid were chosen based on their half-maximum inhibition concentration (IC50) and their previously documented effects in cardiac cells (see the online data supplement). Compared with vehicle treated (control), the inhibition of proteasome associating PP2A enhanced peptidase activities. The activity curve was shifted upward, and a maximum change of up to 20% was observed (Figure 5A-I, 5A-II, and 5A-III). Selective inhibition of PKA in 20S proteasomes is a challenge, because the presence of PKA in 20S (molar ratio to /ß subunits) has not been quantitatively determined. To circumvent this issue, we used various amounts of enzymatically active recombinant PKA together with the inactive PKA and vehicle treated as negative controls. The molar amount of PKA versus 20S was selected based on their enzymatic activity. Our data showed that PKA regulates cardiac 20S proteasome function. Specifically, PKA treatment drastically enhanced 20S peptidase activities in a dose-dependent fashion. Both ß1 and ß5 activities were elevated significantly; the maximal change was up by 2-fold. The PKA effect on the ß2 activity was less but statistically significant, the maximal change was up by 20% (Figure 5B).

View larger version (30K): [in this window] [in a new window]   Figure 5. PP2A and PKA contribute to functional regulation of cardiac 20S proteasomes. A, Purified murine cardiac 20S proteasomes were treated with various concentration of okadaic acid, a PP2A inhibitor, at 35°C for 30 minutes. Inhibition of PP2A with 10 nmol/L okadaic acid augmented proteolytic activities of ß1 (panels A-I), ß2 (A-II), and ß5 (A-III) subunits in a substrate concentration dependent fashion. The overall effect was modest (15% to 20% changes), however, the changes are statistically significant and reproducible. A higher concentration of okadaic acid (50 nmol/L) did not produce additional effect. B, Catalytically active recombinant PKA had a significant effect on the proteolytic function of cardiac 20S proteasomes. PKA treatment induced significant increases in the proteolytic activities under all substrate concentrations tested for ß1, ß2, and ß5 subunits. Vehicle and kinase dead PKA were not significantly different; both were used as negative controls. The changes in proteolytic activities of either ß1, ß2, or ß5, were profound, statistically significant, and consistent for all 3 subunits tested.

Molecular Targets of Cardiac 20S Associating Partners
To determine whether 20S subunits are molecular targets for PP2A and PKA, we examined whether these associating partners impact the phosphorylation profiles of the individual 20S subunits. Purified cardiac 20S proteasomes were either treated with vehicle, PP2A, or PKA, and their phosphorylation profiles were gained by 2-DE immunoblotting with phospho-antibodies (Figure 6A through 6C); importantly, the 3 subunit expression (by antibodies) documented internal controls for 2-DE analyses (Figure 6D). Our data showed that PP2A and PKA were independently effective to alter phosphorylation profiles of individual 20S subunits. Specifically, PP2A attenuated serine phosphorylation signals on multiple subunits, most strikingly, on the 1 and ß2 subunit; PP2A also attenuated threonine signals on the 1 (Figure 6A through 6C). Recombinant PKA markedly changed the phosphorylation profile of 20S proteasomes. It enhanced serine phosphorylation of the 1, 2, and 3 subunits as well as the ß2, ß3, and ß7 subunits; PKA also augmented threonine phosphorylation on 3, ß3, and ß7 subunits (Figure 6A through 6C). Neither PP2A nor PKA had a significant impact on the tyrosine phosphorylation profile (Figure 6A through 6C). These data provide the first evidence demonstrating that cardiac 20S proteasome subunits are regulatory targets of PP2A and PKA. In separate studies, the potential phosphorylation sites on 2 (Figure 7) and 3 (data not shown) were characterized by LC/MS/MS on PKA-treated recombinant proteins.

View larger version (19K): [in this window] [in a new window]   Figure 6. 20S Subunits are targets of PKA and PP2A. Purified murine cardiac 20S proteasomes were analyzed in activation conditions of either PP2A or PKA. The effect of PP2A and PKA on 20S proteasome subunit specific serine, threonine, and tyrosine phosphorylation was characterized by 2-DE followed by immunoblotting. Immunoblotting of total 3 was used as internal standard for loading control and was differentially recognized from the monoclonal antibodies using different fluorescently labeled secondary antibodies and the Odyssey scanner. A, Ser-phosphorylation. Compared with the control sample, PP2A attenuated phosphorylation of 1, 3, 6, and ß2 subunits; PKA significantly enhanced phosphorylations of 1, 2, 3, ß2, ß3, and ß7 subunits. B, Threonine-phosphorylation. Compared with the control, PP2A attenuated phosphorylation of the 1 subunit and the 6 subunit, whereas PKA significantly enhanced phosphorylation of 1, 3, ß3, and ß7 subunits. C, Tyrosine-phosphorylation. Compared with the control, neither PP2A nor PKA, had any recognizable effect on tyrosine phosphorylation. D, 3 as an internal control. An example of serine-phosphorylation profile of 20S subunits, where 3 protein expression was used as an internal control.

View larger version (22K): [in this window] [in a new window]   Figure 7. Identification of PKA phosphorylation sites on the 2 subunit. PKA phosphorylation site was investigated by examining PKA-treated recombinant 2 subunit of 20S using LC/MS/MS running at neutral loss mode. The product ions of N-terminal peptide with neutral loss in a MS/MS scan were isolated and further fragmented in the MS3 scan for sequence information. Based on database search results of MS/MS and MS3 spectra, phosphorylation of the N-terminal serine-8 residue of the 2 subunit was identified.

In summary, using this experimental approach, PP2A and PKA were found to meet the criteria established for the identification of functional associating partners, that is, they reside within the native 20S complexes, and they impact the proteolytic function of cardiac 20S proteasomes. The presence of both phosphatase and kinase as associating partners of 20S proteasomes has never been reported in any tissue or cell type, to the best our knowledge. This is a significant finding potentially unique to cardiac 20S proteasomes; our data suggest an elegant interplay between the phosphatases and kinases in the regulation of murine cardiac 20S proteasome function, supporting the hypothesis that phosphorylation serves as a key regulatory mechanism for cardiac 20S proteasome function.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The 20S proteasomes in the murine heart exhibit complex features with respect to both their structural assembly and their molecular compositions,⁶ contrasting to the proteasome system reported for yeast²⁷ and the limited information documented for proteasomes in the skeletal muscle.^28,29 In this study, we begin a long-term effort to comprehensively explore regulatory mechanisms of proteasome function in the heart. Our results showed that the cardiac 20S proteasomes display distinct patterns of endogenous phosphorylation, and these phosphorylation patterns were modulated by phosphatase PP2A and PKA, which coexisted within the 20S proteasome complexes. Furthermore, PP2A and PKA play dichotomous roles in the regulation of myocardial proteasome function: PKA or inhibition of PP2A significantly augmented proteolytic function of cardiac proteasomes, supporting the concept that phosphorylation serves as a key regulatory mechanism of cardiac 20S proteasome function.

Purification, Isolation, and Identification of Cardiac 20S Proteasomes
Several investigations have purified 20S proteasomes from yeast and mammalian tissues^{7,25,27,29,30} yet a full characterization of cardiac 20S proteasome has never been reported, largely because of the technical challenges involved in its purification procedures. Thus, optimization of the purification process became a key step. Three principles of purification strategies had been previously applied: classic biochemical purification, immunoprecipitation, and affinity purification protocols²⁴; biochemical strategies were documented to achieve high yield. The 20S barrel-like structure enables each subunit to interact with multiple neighboring subunits, forming stable complexes²⁷ that are soluble in high ionic-strength solution, bearing high molecular masses and displaying a unique surface charge ratio. In view of these considerations, we selected our purification strategies using a protocol adapted from rat liver proteasomes purification³⁰ with key modifications detailed below. First, a hand-held homogenizer instead of a tissue tearer or polytron homogenizer was used to avoid mitochondrial contamination of cytosol. Second, a high throughput preparative scale ion-exchange chromatography with a 3-step salt elution combined with subsequent ultracentrifugation was created to eliminate contaminants and to improve yield. Third, in the final step, a shallow salt gradient around 20S proteasome containing fractions enabled a high resolution of 20S proteasome complexes without any overlapping peaks from other proteins (Figure 1A and 1B). This approach yielded 20S proteasome samples with high purity (>98%), structural integrity (EM analysis), and proteolytic activities.

Associating Partners of Intact 20S Proteasome Complexes
19S and 11S are known regulatory particles of 20S proteasomes.^2,14–16 They positively impact 20S proteasome function by controlling substrate entry. Polyubiquitin receptors and deubiquitinating enzymes regulate activities of 20S.^24,31 Although PTMs of 20S proteasomes have been reported,^17–23 little data exist regarding the molecular nature or the subcellular location of these post-translational modifying enzymes. Furthermore, it remains unknown if these modifications have a functional impact. Previous studies implicated the association of kinases with proteasomes, including PKA in the pituitary gland²¹ and CKII in erythrocytes,²⁰ although their coexpression within the intact and functionally active 20S proteasome complexes was never examined.

In this study, PKA and PP2A have been consistently identified as associating partners of murine cardiac 20S proteasomes. The experiments were repeated using different sample preparations of the cardiac 20S proteasomes; PKA and PP2A were identified in 2 independent sets of high-sensitivity ion-trap mass spectrometry and were further confirmed by native electrophoresis with immunoblotting. In parallel experiments, their association with proteasome complexes was examined by LC/MS/MS. The identification of a phosphatase together with a kinase as associating members of 20S proteasome complexes suggests dynamic molecular interplay for the phosphorylation-dependent regulation of 20S proteasome function in the heart. At this juncture, it remains to be determined whether the kinase and phosphatase coexist in the same 20S proteasome complex or if they reside in separate species of 20S proteasome complexes.

Phosphorylation as a Regulatory Mechanism of Cardiac 20S Proteasome Function
Multiple subunits of proteasomes have been found to bear phosphorylation sites via both mutagenesis and mass spectrometry.^17–19,21 The known phosphorylated subunits of 20S proteasomes include subunits 2, 3, 4, 5, 6, 7, and ß6 in both yeast and mammalian tissues. The 6 subunit was phosphorylated in rice²²; 2, 3, 5, 7, and ß6 in liver²³; 3, 5, and 6 in Candida albicans¹⁸; and 2, 4, and 7 in Saccharomyces cerevisiae.¹⁹ We characterized the phosphorylation profile of individual 20S proteasome subunits in the murine heart. A phosphorylation profile was determined by 2-DE and immunoblotting analyses with phospho-serine, phospho-threonine, and phospho-tyrosine specific antibodies. The phosphorylation of 4 subunits, including 1 (serine, threonine, and tyrosine), ß2 (threonine), ß3 (serine and threonine), and ß7 (serine and threonine), are unique to murine cardiac 20S proteasomes, as they have not been previously reported in the yeast or in other mammalian cells.

Importantly, the use of a large range of substrate concentrations and an optimized proteolytic assay for cardiac 20S proteasomes enabled us to uncover the regulatory effect of PKA on ß1, ß2, and ß5 for the first time. Phosphorylation of 20S proteasomes with the active PKA enhanced 20S proteolytic activities, and inhibiting the 20S proteasome associating PP2A augmented 20S proteolytic activities. Our results showed that both and ß subunits are targets of PKA and PP2A. Future investigations to characterize specific phosphorylation sites and the role of each in the altered proteolytic activities will be important.

Implications of Proteasome Function in Cardiovascular Diseases
Increased ubiquitination and protein aggregates are observed in failing hearts and may involve proteasome dysfunction.^10,11 Ischemia reperfusion injury has been shown to parallel a decline in proteasome activity.^7,9,32 However, proteasome inhibition was shown to be protective against ischemia/reperfusion injury in other models.^32–34 These conflicting results are likely because of the lack of a fundamental understanding of molecular mechanisms involved in the regulation of proteasome function. The current study offers a first glimpse on how this important organelle is regulated in the heart. Given the high complexity of cardiac 26S proteasomes in their structure and molecular compositions, we envision that there may exist multiple sites for functional regulation, including modifications of individual subunits, modulations of distinct complex assembly, and regulation by associating partners.³⁵ Future studies focusing on delineating the structure, function, and regulation of cardiac 20S proteasomes will aid to advance our understanding of this organelle in the myocardium and its contributions to cardiac phenotypes.

	Acknowledgments

 
Sources of Funding

This work is supported by NIH grants HL63901, HL65431 and HL-80111 (P.P.), a Laubisch Endowment at the University of California-Los Angeles, and AHA 0315086B (C.Z.).

Disclosures

None.

	Footnotes

 
Original received February 25, 2006; revision received June 12, 2006; accepted July 7, 2006.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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