Revealing the Dynamics of the 20 S Proteasome Phosphoproteome: A Combined CID and Electron Transfer Dissociation Approach

Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, 200032 Shanghai, China.
Molecular &amp Cellular Proteomics (Impact Factor: 6.56). 07/2008; 7(11):2073-89. DOI: 10.1074/mcp.M800064-MCP200
Source: PubMed


The 20 S proteasomes play a critical role in intracellular homeostasis and stress response. Their function is tuned by covalent modifications, such as phosphorylation. In this study, we performed a comprehensive characterization of the phosphoproteome for the 20 S proteasome complexes in both the murine heart and liver. A platform combining parallel approaches in differential sample fractionation (SDS-PAGE, IEF, and two-dimensional electrophoresis), enzymatic digestion (trypsin and chymotrypsin), phosphopeptide enrichment (TiO(2)), and peptide fragmentation (CID and electron transfer dissociation (ETD)) has proven to be essential for identifying low abundance phosphopeptides. As a result, a total of 52 phosphorylation identifications were made in mammalian tissues; 44 of them were novel. These identifications include single (serine, threonine, and tyrosine) and dual phosphorylation peptides. 34 phosphopeptides were identified by CID; 10 phosphopeptides, including a key modification on the catalytically essential beta5 subunit, were identified only by ETD; eight phosphopeptides were shared identifications by both CID and ETD. Besides the commonly shared phosphorylation sites, unique sites were detected in the murine heart and liver, documenting variances in phosphorylation between tissues within the proteasome populations. Furthermore the biological significance of these 20 S phosphoproteomes was evaluated. The role of cAMP-dependent protein kinase A (PKA) to modulate these phosphoproteomes was examined. Using a proteomics approach, many of the cardiac and hepatic 20 S subunits were found to be substrate targets of PKA. Incubation of the intact 20 S proteasome complexes with active PKA enhanced phosphorylation in both existing PKA phosphorylation sites as well as novel sites in these 20 S subunits. Furthermore treatment with active PKA significantly elevated all three peptidase activities (beta1 caspase-like, beta2 trypsin-like, and beta5 chymotrypsin-like), demonstrating a functional role of PKA in governing these 20 S phosphoproteomes.

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    • "In the 20S proteasomal phosphoproteome, Ser157 in murine β1 (158 in human) has been suggested to be a PKA (protein kinase A) phosphorylation site [30]. However, the biological significance of this possible phosphorylation is unknown. "
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    ABSTRACT: p27Kip1 is a key cell cycle regulator whose level is primarily regulated by the ubiquitin-proteasome degradation pathway. Its β1 subunit is one of seven β subunits that form the β-ring of the 20S proteasome, which is responsible for degradation of ubiquitinated proteins. We report here that the β1 subunit is up-regulated in esophageal cancer tissues and some ovarian cancer cell lines. It promotes cell growth and migration, as well as colony formation. β1 binds and degrades p27Kip1 directly. Interestingly, the lack of phosphorylation at S158 of the β1 subunit promotes degradation of p27Kip1. We therefore propose that the β1 subunit plays a novel role in tumorigenesis by degrading p27Kip1.
    Bioscience Reports 06/2013; 33(4). DOI:10.1042/BSR20130013 · 2.64 Impact Factor
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    • "The functional consequence of PKCβII-induced 20S proteasome phosphorylation was also demonstrated in isolated neonatal cardiomyocytes since both βIIV5-3, a PKCβII-specific inhibitor (but not selective inhibitors for α, βI or PKCε) and PKCβ knockdown using siRNA abrogated PMA-induced proteasomal dysfunction and the accumulation of damaged proteins (Fig. 2B–C). Since proteasomal activity is regulated by multiple factors, such as intracellular ATP levels [26] and post-translational modification of the proteasome [28], [29], [30], [31], the in vitro findings might not reflect proteasomal regulation in vivo. Thus, we next examined whether PKCβII activation disrupts cardiac PQC related to UPS dysfunction in myocardial infarction- and hypertensive-induced HF rats. "
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    ABSTRACT: Myocardial remodeling and heart failure (HF) are common sequelae of many forms of cardiovascular disease and a leading cause of mortality worldwide. Accumulation of damaged cardiac proteins in heart failure has been described. However, how protein quality control (PQC) is regulated and its contribution to HF development are not known. Here, we describe a novel role for activated protein kinase C isoform βII (PKCβII) in disrupting PQC. We show that active PKCβII directly phosphorylated the proteasome and inhibited proteasomal activity in vitro and in cultured neonatal cardiomyocytes. Importantly, inhibition of PKCβII, using a selective PKCβII peptide inhibitor (βIIV5-3), improved proteasomal activity and conferred protection in cultured neonatal cardiomyocytes. We also show that sustained inhibition of PKCβII increased proteasomal activity, decreased accumulation of damaged and misfolded proteins and increased animal survival in two rat models of HF. Interestingly, βIIV5-3-mediated protection was blunted by sustained proteasomal inhibition in HF. Finally, increased cardiac PKCβII activity and accumulation of misfolded proteins associated with decreased proteasomal function were found also in remodeled and failing human hearts, indicating a potential clinical relevance of our findings. Together, our data highlights PKCβII as a novel inhibitor of proteasomal function. PQC disruption by increased PKCβII activity in vivo appears to contribute to the pathophysiology of heart failure, suggesting that PKCβII inhibition may benefit patients with heart failure. (218 words).
    PLoS ONE 03/2012; 7(3):e33175. DOI:10.1371/journal.pone.0033175 · 3.23 Impact Factor
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    • "Thousands of phosphorylated proteins and their phosphorylation sites have been identified in many model organisms, from bacteria [27], yeast [28] and worms [29] to plants [30] and animals [31]. Over the last few years, many groups have contributed to cardiovascular phosphoproteomic analysis of cardiac myocytes [32], cardiac mitochondria [33] [34], 20S proteasomes [35] and myofilaments [36]. "
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    ABSTRACT: Cardioproteomics (Cardiovascular proteomics) is fast becoming an indispensible technique in deciphering changes in signaling pathways that occur in cardiovascular diseases (CVDs). The quality and availability of the instruments and bioinformatics software used for cardioproteomics continues to improve, and these techniques are now available to most cardiovascular researchers either directly or indirectly via university core centers. The heart and aorta are specialized tissues which present unique challenges to investigate. Currently, the diverse range of proteomic techniques available for cardiovascular research makes the choice of the best method or best combination of methods for the disease parameter(s) being investigated as important as the equipment used. This review focuses on proteomic techniques and their applications which have advanced our understanding of the signaling mechanisms involved in CVDs at the levels of protein complex/protein-protein interaction, post-translational modifications and signaling induced protein changes.
    American Journal of Cardiovascular Disease 01/2011; 1(3):274-92.
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