A Facile Strategy for Selective Incorporation of Phosphoserine into Histones

Lee, Sangsik; Oh, Shi-Hwan; Yang, Aerin; Kim, Jihyo; Söll, Dieter; Lee, Daeyoup; Park, Hee-Sung

doi:10.1002/ange.201300531

Cited by 19 publications

(23 citation statements)

References 21 publications

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“…To enhance production of different pUb proteins, we introduced 2nd generation pSer system mutations [26] into the pKD-pSer1 plasmid to yield pDS-pSer2. This pSer insertion system is more efficient and able to compete against RF1 [26], so the protein production host was switched to BL21(DE3). Because nearcognate decoding of UAG is minimal in BL21(DE3) ( Fig.…”

Section: Characterization Of Purified Phosphorylated Ubmentioning

confidence: 99%

“…Previous reports have shown it is possible to produce designer phosphoprotein in E. coli [13,26], but the efficient production of pure phosphoprotein has remained challenging. We successfully produced several pUb variants in unambiguously pure form as evidenced by MS/MS and Phos-tag gels.…”

Section: Pure Designer Phosphoproteinmentioning

confidence: 99%

“…Our goal in this study was not to necessarily maximize the yield of phosphoprotein. We [17] (Table S1) and others [11,22,26] have already produced phosphoproteins in sufficient amounts for biochemical and structural studies. Rather, we were concerned with demonstrating unambiguously and in a more rigorous fashion than previous reports that we indeed obtained pure phosphoprotein.…”

Section: Pure Designer Phosphoproteinmentioning

confidence: 99%

“…Previous work relied on MS/MS identification of pSer in the phosphoprotein [11,26], yet mistranslated protein may go undetected with this method. A modified pSer system [11], including mutations in SepRS and the anticodon stem of tRNA Sep , recently reported similar mgÁL À1 culture yields of phosphoprotein as was previously reported for the 2nd generation pSer system [26]. While our yields were somewhat lower, we emphasize that we are reporting pure phosphoprotein yields, which likely reflect some loss of protein during column chromatography.…”

Section: Pure Designer Phosphoproteinmentioning

confidence: 99%

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Generation of phospho‐ubiquitin variants by orthogonal translation reveals codon skipping

et al. 2016

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Edited by Michael IbbaThe activity of the Parkinson's disease-linked E3 ligase parkin is stimulated by phosphorylation at ubiquitin Ser65 (pUb S65 ). The role of other ubiquitin phospho-sites and their kinases are unknown. We produced pUb variants (pS7, pS12, pS20, pS57, pS65) by genetically encoding phosphoserine with the UAG codon. In release factor-deficient Escherichia coli (DRF1), intended to enhance UAG read-through, we discovered ubiquitin variants lacking the UAG-encoded residue, demonstrating previously undocumented +3 frame shifting. We successfully purified each pUb variant from mistranslated products. While pUb S20 failed to stimulate parkin, parkin was partially active with pUb S12 . We observed significant ubiquitination when pUb S65 was the sole substrate.

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Section: Characterization Of Purified Phosphorylated Ubmentioning

confidence: 99%

Section: Pure Designer Phosphoproteinmentioning

confidence: 99%

Section: Pure Designer Phosphoproteinmentioning

confidence: 99%

Section: Pure Designer Phosphoproteinmentioning

confidence: 99%

See 2 more Smart Citations

Generation of phospho‐ubiquitin variants by orthogonal translation reveals codon skipping

et al. 2016

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“…To more directly assess the effects of MSK1-mediated histone phosphorylation on chromatin transcription, we generated histone octamers containing homogenously phosphorylated H3S10 or H3S28 using an amber suppression method 39 and assembled them into chromatin for p53-dependent transcription assays (Fig. 3c ).…”

Section: Resultsmentioning

confidence: 99%

MSK1 functions as a transcriptional coactivator of p53 in the regulation of p21 gene expression

et al. 2018

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Mitogen- and stress-activated kinase 1 (MSK1) is a chromatin kinase that facilitates activator-dependent transcription by altering chromatin structure through histone H3 phosphorylation. The kinase activity of MSK1 is activated by intramolecular autophosphorylation, which is initially triggered by the activation of upstream mitogen-activated protein kinases (MAPKs), such as p38 and ERK1/2. MSK1 has been implicated in the expression of p21, a p53 target gene; however, the precise connection between MSK1 and p53 has not been clearly elucidated. Here, using in vitro and cell-based transcription assays, we show that MSK1 functions as a transcriptional coactivator of p53 in p21 expression, an action associated with MAPK-dependent phosphorylation of MSK1 and elevated kinase activity. Of special significance, we show that MSK1 directly interacts with p53 and is recruited to the p21 promoter, where it phosphorylates histone H3 in a p53-dependent manner. In addition, phosphomimetic mutant analysis demonstrated that negative charges in the hydrophobic motif are critical for serine 212 phosphorylation in the N-terminal kinase domain, which renders MSK1 competent for histone kinase activity. These studies suggest that MSK1 acts through a direct interaction with p53 to function as a transcriptional coactivator and that MSK1 activation by upstream MAPK signaling is important for efficient p21 gene expression.

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Expanding the genetic code of Escherichia coli with phosphotyrosine

Fan

Söll

2016

FEBS Letters

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Protein phosphorylation is one of the most important post-translational modifications in nature. However, the site-specific incorporation of O-phosphotyrosine into proteins in vivo has not yet been reported. Endogenous phosphatases present in cells can dephosphorylate phosphotyrosine as a free amino acid or as a protein residue. Therefore, we deleted the genes of five phosphatases from the genome of Escherichia coli with the aim of stabilizing phosphotyrosine. Together with an engineered aminoacyl-tRNA synthetase (derived from Methanocaldococcus jannaschii tyrosyl-tRNA synthetase) and an elongation factor Tu variant, we were able to co-translationally incorporate O-phosphotyrosine into the super-folder green fluorescent protein at a desired position in vivo. This system will facilitate future studies of tyrosine phosphorylation.

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A Facile Strategy for Selective Incorporation of Phosphoserine into Histones

Cited by 19 publications

References 21 publications

Generation of phospho‐ubiquitin variants by orthogonal translation reveals codon skipping

Generation of phospho‐ubiquitin variants by orthogonal translation reveals codon skipping

MSK1 functions as a transcriptional coactivator of p53 in the regulation of p21 gene expression

Expanding the genetic code of Escherichia coli with phosphotyrosine

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