Global Identification and Characterization of Both O-GlcNAcylation and Phosphorylation at the Murine Synapse

Trinidad, Jonathan C.; Barkan, David T.; Gulledge, Brittany F.; Thalhammer, Agnes; Šali, Andrej; Schoepfer, Ralf; Burlingame, Alma L.

doi:10.1074/mcp.o112.018366

Cited by 375 publications

(460 citation statements)

References 65 publications

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“…We believe that this constitutes the first broad proteomic analysis of O-GlcNAc-modified proteins in primary human T cells. Many of the proteins that we identified appeared in prior proteomics studies (55,(76)(77)(78), supporting the validity of our results. However, we also identified novel O-GlcNAc substrates, including ARNT, HCLS1, ZAP-70, SHIP1, LCK, and PI3K.…”

Section: Discussionsupporting

confidence: 87%

Global Analysis of O-GlcNAc Glycoproteins in Activated Human T Cells

Lund

Elias

Davis

2016

The Journal of Immunology

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T cell activation in response to Ag is largely regulated by protein posttranslational modifications. Although phosphorylation has been extensively characterized in T cells, much less is known about the glycosylation of serine/threonine residues by O-linked N-acetylglucosamine (O-GlcNAc). Given that O-GlcNAc appears to regulate cell signaling pathways and protein activity similarly to phosphorylation, we performed a comprehensive analysis of O-GlcNAc during T cell activation to address the functional importance of this modification and to identify the modified proteins. Activation of T cells through the TCR resulted in a global elevation of O-GlcNAc levels and in the absence of O-GlcNAc, IL-2 production and proliferation were compromised. T cell activation also led to changes in the relative expression of O-GlcNAc transferase (OGT) isoforms and accumulation of OGT at the immunological synapse of murine T cells. Using a glycoproteomics approach, we identified >200 O-GlcNAc proteins in human T cells. Many of the identified proteins had a functional relationship to RNA metabolism, and consistent with a connection between O-GlcNAc and RNA, inhibition of OGT impaired nascent RNA synthesis upon T cell activation. Overall, our studies provide a global analysis of O-GlcNAc dynamics during T cell activation and the first characterization, to our knowledge, of the O-GlcNAc glycoproteome in human T cells.

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Section: Discussionsupporting

confidence: 87%

Global Analysis of O-GlcNAc Glycoproteins in Activated Human T Cells

Lund

Elias

Davis

2016

The Journal of Immunology

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“…S18), or in other words when the residue affected is followed by a Pro. Since in all vertebrates more than 40% of Ser/Thr residues that are phosphorylated are followed by Pro at P+1 (28), reciprocal phosphorylation/O-GlcNAcylation crosstalk, as suggested previously (12), is much more restricted and less frequent than initially proposed.…”

Section: Discussionmentioning

confidence: 79%

“…Thus, doubt still exists as to whether on a specific protein molecule O-GlcNAcylation directly affects phosphorylation or vice versa. Work by Trinidad et al (12) suggested that phosphorylation and O-GlcNAcylation occur randomly with respect to each another and that negative crosstalk is likely not a common phenomenon. From that work it can be hypothesized that phosphorylation/O-GlcNAcylation crosstalk is not really widespread.…”

mentioning

confidence: 99%

Elucidating crosstalk mechanisms between phosphorylation and O-GlcNAcylation

Leney

Atmioui

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

138

127

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Proteins can be modified by multiple posttranslational modifications (PTMs), creating a PTM code that controls the function of proteins in space and time. Unraveling this complex PTM code is one of the great challenges in molecular biology. Here, using mass spectrometry-based assays, we focus on the most common PTMs-phosphorylation and O-GlcNAcylation-and investigate how they affect each other. We demonstrate two generic crosstalk mechanisms. First, we define a frequently occurring, very specific and stringent phosphorylation/ O-GlcNAcylation interplay motif, (pSp/T)P(V/A/T)(gS/gT), whereby phosphorylation strongly inhibits O-GlcNAcylation. Strikingly, this stringent motif is substantially enriched in the human (phospho)proteome, allowing us to predict hundreds of putative O-GlcNAc transferase (OGT) substrates. A set of these we investigate further and show them to be decent substrates of OGT, exhibiting a negative feedback loop when phosphorylated at the P-3 site. Second, we demonstrate that reciprocal crosstalk does not occur at PX(S/T)P sites, i.e., at sites phosphorylated by proline-directed kinases, which represent 40% of all sites in the vertebrate phosphoproteomes.O-GlcNAcylation | phosphorylation | crosstalk | signaling | regulation

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“…However, the recent development of lectin weak affinity chromatography (LWAC) and chemical/enzymatic tagging strategies has facilitated enrichment of these modified peptides from complex proteolytic digest mixtures (9,10), and the advent of electron transfer dissociation (ETD) mass spectrometry has facilitated the robust assignment of modification sites (11). These advances have been used effectively for studies in mammalian systems, and over 1,000 O-GlcNAc-modified proteins have been identified (9)(10)(11)(12)(13)(14). However, no similar study has been reported in plants, and thus it's unclear whether O-GlcNAc modification controls similar cellular process in plants and animals.…”

mentioning

confidence: 99%

Proteomic analysis reveals O-GlcNAc modification on proteins with key regulatory functions in Arabidopsis

Chalkley

Maynard

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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113

161

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Genetic studies have shown essential functions of O-linked N-acetylglucosamine (O-GlcNAc) modification in plants. However, the proteins and sites subject to this posttranslational modification are largely unknown. Here, we report a large-scale proteomic identification of O-GlcNAc-modified proteins and sites in the model plant Arabidopsis thaliana. Using lectin weak affinity chromatography to enrich modified peptides, followed by mass spectrometry, we identified 971 O-GlcNAc-modified peptides belonging to 262 proteins. The modified proteins are involved in cellular regulatory processes, including transcription, translation, epigenetic gene regulation, and signal transduction. Many proteins have functions in developmental and physiological processes specific to plants, such as hormone responses and flower development. Mass spectrometric analysis of phosphopeptides from the same samples showed that a large number of peptides could be modified by either O-GlcNAcylation or phosphorylation, but cooccurrence of the two modifications in the same peptide molecule was rare. Our study generates a snapshot of the O-GlcNAc modification landscape in plants, indicating functions in many cellular regulation pathways and providing a powerful resource for further dissecting these functions at the molecular level.of proteins consisting of a single O-linked N-acetylglucosamine attached to serine and threonine residues. It has been extensively studied in animals, where it regulates a wide range of developmental and metabolic processes. O-GlcNAcylation is dynamically controlled by two enzymes: an O-GlcNAc transferase (OGT) and an O-GlcNAcase (OGA), which add and remove O-GlcNAc, respectively. O-GlcNAcylation occurs in the cytoplasm, nucleus, and mitochondria and has been implicated in cellular processes, including transcription, translation, signal transduction, nuclear pore function, epigenetic regulation and proteasomal degradation (1). Altered levels of protein O-GlcNAcylation in animals have been associated with neurodegeneration, diabetes, cardiovascular diseases, and cancer (2) whereas knock out of OGT is embryonically lethal (3).The model plant Arabidopsis has two putative OGTs: SPINDLY (SPY) and SECRET AGENT (SEC). The spy mutant was identified based on its phenotypes that mimic gibberellin-treated plants, with elongated stems (4). The spy plants also show defects in light and cytokinin responses, leaf morphology and phyllotaxy, root growth, meristem activity, and circadian rhythms (5). The sec mutant displays defects in flower development (6). Although OGT enzymatic activity has been demonstrated in SEC, similar activity in SPY has not been confirmed (7). However, the spy;sec double mutants show severe defects in the development of gametes and are embryonically lethal (7), similar to the OGT knockout mutant in animals. Thus, genetic evidence indicates that O-GlcNAc modification is as important in plants as in animals. But little is known about its specific functions because few O-GlcNAc-modified proteins have been identified...

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Global Identification and Characterization of Both O-GlcNAcylation and Phosphorylation at the Murine Synapse

Cited by 375 publications

References 65 publications

Global Analysis of O-GlcNAc Glycoproteins in Activated Human T Cells

Global Analysis of O-GlcNAc Glycoproteins in Activated Human T Cells

Elucidating crosstalk mechanisms between phosphorylation and O-GlcNAcylation

Proteomic analysis reveals O-GlcNAc modification on proteins with key regulatory functions in Arabidopsis

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