Large-scale determination of absolute phosphorylation stoichiometries in human cells by motif-targeting quantitative proteomics

Tsai, Chia Feng; Wang, Yi Ting; Yen, Hsin Yung; Tsou, Chih-Chiang; Ku, Wei-Chi; Lin, Pei; Chen, Hsuan Yu; Nesvizhskii, Alexey I.; Ishihama, Yasushi; Chen, Yu‐Ju

doi:10.1038/ncomms7622

Cited by 153 publications

(134 citation statements)

References 36 publications

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“…Thus, it has been demonstrated that His and Asp phosphorylation regulate the functions of pathogenesis, such as adherence, motility, enhancement of toxicity, quorum sensing, capsule formation, and drug resistance (100). Recently, in cancer drug development studies, it has been suggested that changes in phosphorylation stoichiometry are helpful for characterizing the kinase-dependent pathway in gefitinib-resistant lung cancer cells (101). Our study showed that Ser-90 phosphorylation of AmpC may be part of the kinase/ phosphatase machinery conferring imipenem resistance, ␤-lactamase activity, and the ability to neutralize imipenem in A. baumannii.…”

Section: Discussionmentioning

confidence: 72%

Comparative Phosphoproteomics Reveals the Role of AmpC β-lactamase Phosphorylation in the Clinical Imipenem-resistant Strain Acinetobacter baumannii SK17

Lai

Yang

Chern

et al. 2016

Molecular & Cellular Proteomics

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Nosocomial infectious outbreaks caused by multidrugresistant Acinetobacter baumannii have emerged as a serious threat to human health. Phosphoproteomics of pathogenic bacteria has been used to identify the mechanisms of bacterial virulence and antimicrobial resistance. In this study, we used a shotgun strategy combined with high-accuracy mass spectrometry to analyze the phosphoproteomics of the imipenem-susceptible strain SK17-S and -resistant strain SK17-R. We identified 410 phosphosites on 248 unique phosphoproteins in SK17-S and 285 phosphosites on 211 unique phosphoproteins in SK17-R. The distributions of the Ser/Thr/Tyr/Asp/His phosphosites in SK17-S and SK17-R were 47.0%/27.6%/ 12.4%/8.0%/4.9% versus 41.4%/29.5%/17.5%/6.7%/ 4.9%, respectively. The Ser-90 phosphosite, located on the catalytic motif S 88 VS 90 K of the AmpC ␤-lactamase, was first identified in SK17-S. Based on site-directed mutagenesis, the nonphosphorylatable mutant S90A was found to be more resistant to imipenem, whereas the phosphorylation-simulated mutant S90D was sensitive to imipenem. Additionally, the S90A mutant protein exhibited higher ␤-lactamase activity and conferred greater bacterial protection against imipenem in SK17-S compared with the wild-type. In sum, our results revealed that in A. baumannii, Ser-90 phosphorylation of AmpC negatively regulates both ␤-lactamase activity and the ability to counteract the antibiotic effects of imipenem. These findings highlight the impact of phosphorylation-mediated regulation in antibiotic-resistant bacteria on future drug design and new therapies.

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Section: Discussionmentioning

confidence: 72%

Comparative Phosphoproteomics Reveals the Role of AmpC β-lactamase Phosphorylation in the Clinical Imipenem-resistant Strain Acinetobacter baumannii SK17

Lai

Yang

Chern

et al. 2016

Molecular & Cellular Proteomics

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“…Therefore, ZNF687 res373-382 was synthesized, in which we phosphorylated Ser374 and Thr377 (at P−6 and P−3, respectively, with respect to the O-GlcNAc site), and the percentage O-GlcNAcylation on the phosphopeptides was compared with that of the unmodified peptide ( Table 2). The phosphosites were chosen based on their reported detection (32). Consistent with our previous data on CRMP2 (SI Appendix, Fig.…”

Section: Reciprocal Interplay Does Not Occur On Ser/thr Sites Targetementioning

confidence: 96%

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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“…In addition, NQO1 can undergo post-translational modifications such as phosphorylation [42] and ubiquitination [43]. It has been shown previously that phosphorylation and other post-translational modifications of RBPs including HuR, BRF1, TTP, KSRP, TIAR/TIA-1 have been linked to changes in their subcellular localization and their association with target mRNAs [44].…”

Section: Discussionmentioning

confidence: 99%

Novel RNA-binding activity of NQO1 promotes SERPINA1 mRNA translation

Francesco

Germanio

Panda

et al. 2016

Free Radical Biology and Medicine

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NAD(P)H:quinone oxidoreductase (NQO1) is essential for cell defense against reactive oxidative species, cancer, and metabolic stress. Recently, NQO1 was found in ribonucleoprotein (RNP) complexes, but NQO1-interacting mRNAs and the functional impact of such interactions are not known. Here, we used ribonucleoprotein immunoprecipitation (RIP) and microarray analysis to identify comprehensively the subset of NQO1 target mRNAs in human hepatoma HepG2 cells. One of its main targets, SERPINA1 mRNA, encodes the serine protease inhibitor α-1-antitrypsin, A1AT, which is associated with disorders including obesity-related metabolic inflammation, chronic obstructive pulmonary disease (COPD), liver cirrhosis and hepatocellular carcinoma. Biotin pulldown analysis indicated that NQO1 can bind the 3′ untranslated region (UTR) and the coding region (CR) of SERPINA1 mRNA. NQO1 did not affect SERPINA1 mRNA levels; instead, it enhanced the translation of SERPINA1 mRNA, as NQO1 silencing decreased the size of polysomes forming on SERPINA1 mRNA and lowered the abundance of A1AT. Luciferase reporter analysis further indicated that NQO1 regulates SERPINA1 mRNA translation through the SERPINA1 3′UTR. Accordingly, NQO1-KO mice had reduced hepatic and serum levels of A1AT and increased activity of neutrophil elastase (NE), one of the main targets of A1AT. We propose that this novel mechanism of action of NQO1 as RNA-binding protein may help to explain its pleiotropic biological effects.

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Large-scale determination of absolute phosphorylation stoichiometries in human cells by motif-targeting quantitative proteomics

Cited by 153 publications

References 36 publications

Comparative Phosphoproteomics Reveals the Role of AmpC β-lactamase Phosphorylation in the Clinical Imipenem-resistant Strain Acinetobacter baumannii SK17

Comparative Phosphoproteomics Reveals the Role of AmpC β-lactamase Phosphorylation in the Clinical Imipenem-resistant Strain Acinetobacter baumannii SK17

Elucidating crosstalk mechanisms between phosphorylation and O-GlcNAcylation

Novel RNA-binding activity of NQO1 promotes SERPINA1 mRNA translation

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