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Molecular & Cellular Proteomics 7:2073-2089, 2008.
© 2008 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

Revealing the Dynamics of the 20 S Proteasome Phosphoproteome

A Combined CID and Electron Transfer Dissociation Approach^*,S

Haojie Lu^,, Chenggong Zong^,¶, Yueju Wang^¶, Glen W. Young^¶, Ning Deng^¶, Pete Souda^¶, Xiaohai Li^¶, Julian Whitelegge^¶, Oliver Drews^¶, Peng-Yuan Yang and Peipei Ping^¶,||

From the Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, 200032 Shanghai, China and ^¶ Departments of Physiology and Medicine, Division of Cardiology, UCLA School of Medicine, University of California at Los Angeles, Los Angeles, California 90095

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 (TiO₂), 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 β5 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 (β1 caspase-like, β2 trypsin-like, and β5 chymotrypsin-like), demonstrating a functional role of PKA in governing these 20 S phosphoproteomes.

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