Systematic Functional Prioritization of Protein Posttranslational Modifications

Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94107, USA.
Cell (Impact Factor: 32.24). 07/2012; 150(2):413-25. DOI: 10.1016/j.cell.2012.05.036
Source: PubMed


Protein function is often regulated by posttranslational modifications (PTMs), and recent advances in mass spectrometry have resulted in an exponential increase in PTM identification. However, the functional significance of the vast majority of these modifications remains unknown. To address this problem, we compiled nearly 200,000 phosphorylation, acetylation, and ubiquitination sites from 11 eukaryotic species, including 2,500 newly identified ubiquitylation sites for Saccharomyces cerevisiae. We developed methods to prioritize the functional relevance of these PTMs by predicting those that likely participate in cross-regulatory events, regulate domain activity, or mediate protein-protein interactions. PTM conservation within domain families identifies regulatory "hot spots" that overlap with functionally important regions, a concept that we experimentally validated on the HSP70 domain family. Finally, our analysis of the evolution of PTM regulation highlights potential routes for neutral drift in regulatory interactions and suggests that only a fraction of modification sites are likely to have a significant biological role.

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Available from: Veronique Albanese, Oct 01, 2015
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    • "Previous studies have suggested that lysine residues found to be targets for ubiquitylation were not more likely to be conserved than non - ub modified lysines residues ( Beltrao et al . , 2012 ) . However , it remains possible that ubiquitylation events that are regulatory in nature are more conserved than ubiquitylation events that target the protein for degrada - tion . Due to the site - specific nature of regulatory 40S ubiquity - lation , we examined if the observed human 40S ubiquitylation was conserved in other species "
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    ABSTRACT: Insults to ER homeostasis activate the unfolded protein response (UPR), which elevates protein folding and degradation capacity and attenuates protein synthesis. While a role for ubiquitin in regulating the degradation of misfolded ER-resident proteins is well described, ubiquitin-dependent regulation of translational reprogramming during the UPR remains uncharacterized. Using global quantitative ubiquitin proteomics, we identify evolutionarily conserved, site-specific regulatory ubiquitylation of 40S ribosomal proteins. We demonstrate that these events occur on assembled cytoplasmic ribosomes and are stimulated by both UPR activation and translation inhibition. We further show that ER stress-stimulated regulatory 40S ribosomal ubiquitylation occurs on a timescale similar to eIF2α phosphorylation, is dependent upon PERK signaling, and is required for optimal cell survival during chronic UPR activation. In total, these results reveal regulatory 40S ribosomal ubiquitylation as an important facet of eukaryotic translational control. Copyright © 2015 Elsevier Inc. All rights reserved.
    Molecular cell 06/2015; 59(1). DOI:10.1016/j.molcel.2015.04.026 · 14.02 Impact Factor
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    • "The seven acetylation sites identified here also align along the dimer interface in the ATP state, implying that they combine with the phosphosite for signal propagation. These hydrogen-bonding interactions provide a rationale for increased dimerization of Hsp70 Sf9 and the conservation of the phosphosite (Beltrao et al., 2012), and the correspondence of the amino acid residues involved in acetylation ( in eukaryotes suggests their functional relevance in vivo. "
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    ABSTRACT: Protein folding in cells is regulated by networks of chaperones, including the heat shock protein 70 (Hsp70) system, which consists of the Hsp40 cochaperone and a nucleotide exchange factor. Hsp40 mediates complex formation between Hsp70 and client proteins prior to interaction with Hsp90. We used mass spectrometry (MS) to monitor assemblies formed between eukaryotic Hsp90/Hsp70/Hsp40, Hop, p23, and a client protein, a fragment of the glucocorticoid receptor (GR). We found that Hsp40 promotes interactions between the client and Hsp70, and facilitates dimerization of monomeric Hsp70. This dimerization is antiparallel, stabilized by post-translational modifications (PTMs), and maintained in the stable heterohexameric client-loading complex Hsp902Hsp702HopGR identified here. Addition of p23 to this client-loading complex induces transfer of GR onto Hsp90 and leads to expulsion of Hop and Hsp70. Based on these results, we propose that Hsp70 antiparallel dimerization, stabilized by PTMs, positions the client for transfer from Hsp70 to Hsp90. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
    Cell Reports 04/2015; 11(5). DOI:10.1016/j.celrep.2015.03.063 · 8.36 Impact Factor
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    • "We determined a significant difference in distribution of rates of evolution (p-value = 0.0.0014) with a Kolmogorov-Smirnov test between the dynamic and static sites. No significant difference in rates of evolution (P-value = 0.3420) was observed between a literature-curated set of functional phosphosites (Beltrao et al., 2012) and our dynamic set of phosphosites. "
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    ABSTRACT: If specific and functional kinase- or phosphatase-substrate interactions are optimized for binding compared to promiscuous interactions, then changes in phosphorylation should occur faster on functional versus promiscuous substrates. To test this hypothesis, we designed a high temporal resolution global phosphoproteomics protocol to study the high-osmolarity glycerol (HOG) response in the budding yeast Saccharomyces cerevisiae. The method provides accurate, stimulus-specific measurement of phosphoproteome changes, quantitative analysis of phosphodynamics at sub-minute temporal resolution, and detection of more phosphosites. Rates of evolution of dynamic phosphosites were comparable to those of known functional phosphosites and significantly lower than static or longer-time-frame dynamic phosphosites. Kinetic profile analyses indicated that putatively functional kinase- or phosphatase-substrate interactions occur more rapidly, within 60 s, than promiscuous interactions. Finally, we report many changes in phosphorylation of proteins implicated in cytoskeletal and mitotic spindle dynamics that may underlie regulation of cell cycle and morphogenesis. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
    Cell Reports 02/2015; 10(7). DOI:10.1016/j.celrep.2015.01.052 · 8.36 Impact Factor
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