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

George, Susanna; Aguirre, Jacob D.; Spratt, Donald E.; Bi, Yumin; Jeffery, Madeline; Shaw, Gary S.; O’Donoghue, Patrick

doi:10.1002/1873-3468.12182

Cited by 34 publications

(43 citation statements)

References 52 publications

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“…In our current study, and previous work [11,[17][18][19], we demonstrated that genetic code expansion with pSer in E. coli provides a simple route to produce designer phosphoproteins. Here, we have produced differentially phosphorylated Akt1 variants with and without the PH domain in E. coli using genetic code expansion and enzymatic phosphorylation in E. coli.…”

Section: Synthetic Biology Approach To Generate Active Akt1supporting

confidence: 70%

“…Full length pAkt1 variants were produced from pCDF-Duet1 plasmids as described previously [11]. The genetic code expansion system for phosphoserine (pSer) is encoded on the pDS-pSer2 plasmid [11,17,18], which contains 5 copies of tRNA Sep [19], phosphoseryl-tRNA synthetase (SepRS9), and elongation factor Tu mutant (EFSep21) [20]. Incorporation of pSer also required site-directed mutagenesis of the Ser473 codon to TAG in the ΔPH-akt1 constructs.…”

Section: Bacterial Strains and Plasmidsmentioning

confidence: 99%

“…The pDS-pSer2 plasmid [18] was used as before [11] to incorporate pSer in response to a UAG codon at position 473 in ΔPHAkt1 and full length Akt1 variants. To produce both full length and PH domain deficient Akt1 variants containing pSer473 (Table S1), the pCDFDuet-1 Akt1 bearing plasmid was co-transformed with pDS-pSer2 into E. coli Bl21(DE3) and plated on LB agar plates with 25 μg/ml kanamycin and 50 μg/ml streptomycin.…”

Section: Protein and Phosphoprotein Productionmentioning

confidence: 99%

“…Using genetic code expansion [11,18], we incorporated pSer in response to a UAG stop codon at position 473 in the relevant Akt1 constructs. Thr308 was site-specifically phosphorylated by the co-expression of the upstream kinase PDK1 in E. coli.…”

Section: Production Of Recombinant Akt1 Variantsmentioning

confidence: 99%

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Phosphorylation-Dependent Inhibition of Akt1

Balasuriya¹,

McKenna²,

Liu³

et al. 2018

Preprint

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Akt1 is a proto-oncogene that is over active in most cancers. Akt1 activation requires phosphorylation at Thr308; phosphorylation at Ser473 further enhances catalytic activity. Akt1 activity is also regulated via interactions between the kinase domain and the N-terminal autoinhibitory pleckstrin homology (PH) domain. As it was previously difficult to produce Akt1 in sitespecifically phosphorylated forms, the contribution of each activating phosphorylation site to autoinhibition was unknown. Using a combination of genetic code expansion and in vivo enzymatic phosphorylation, we produced Akt1 variants containing programmed phosphorylation to probe the interplay between Akt1 phosphorylation status and the auto-inhibitory function of the PH domain. Deletion of the PH domain increased the enzyme activity for all three phosphorylated Akt1 variants. For the doubly phosphorylated enzyme, deletion of the PH domain relieved auto-inhibition by 295-fold. We next found that phosphorylation at Ser473 provided resistance to chemical inhibition by Akti-1/2 inhibitor VIII. The Akti-1/2 inhibitor was most effective against pAkt1 T308 and showed 4-fold decreased potency with Akt1 variants phosphorylated at Ser473. The data highlight the need to design more potent Akt1 inhibitors that are effective against the doubly phosphorylated and most pathogenic form of Akt1.

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Section: Synthetic Biology Approach To Generate Active Akt1supporting

confidence: 70%

Section: Bacterial Strains and Plasmidsmentioning

confidence: 99%

Section: Protein and Phosphoprotein Productionmentioning

confidence: 99%

Section: Production Of Recombinant Akt1 Variantsmentioning

confidence: 99%

See 2 more Smart Citations

Phosphorylation-Dependent Inhibition of Akt1

Balasuriya¹,

McKenna²,

Liu³

et al. 2018

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…This preparation is thus not of the same homogeneous quality as TrxR1 produced above in the presence of a SECIS element or as expected of native GPx1 purified from mammalian tissue. UAG-directed Gln suppression has also been reported earlier (45)(46)(47). The hereby enabled recombinant production methodology nonetheless gives the possibilities for new types of studies, such as evaluating the effects of point mutations within a selenoprotein scaffold.…”

Section: Resultsmentioning

confidence: 74%

Selenocysteine Insertion at a Predefined UAG Codon in a Release Factor 1 (RF1)-depleted Escherichia coli Host Strain Bypasses Species Barriers in Recombinant Selenoprotein Translation

Cheng

Arnér

2017

Journal of Biological Chemistry

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Edited by Ronald C. WekSelenoproteins contain the amino acid selenocysteine (Sec), co-translationally inserted at a predefined UGA opal codon by means of Sec-specific translation machineries. In Escherichia coli, this process is dependent upon binding of the Sec-dedicated elongation factor SelB to a Sec insertion sequence (SECIS) element in the selenoprotein-encoding mRNA and competes with UGA-directed translational termination. Here, we found that Sec can also be efficiently incorporated at a predefined UAG amber codon, thereby competing with RF1 rather than RF2. Subsequently, utilizing the RF1-depleted E. coli strain C321.⌬A, we could produce mammalian selenoprotein thioredoxin reductases with unsurpassed purity and yield. We also found that a SECIS element was no longer absolutely required in such a system. Human glutathione peroxidase 1 could thereby also be produced, and we could confirm a previously proposed catalytic tetrad in this selenoprotein. We believe that the versatility of this new UAG-directed production methodology should enable many further studies of diverse selenoproteins.

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Delivery of AKT1 phospho‐forms to human cells reveals differential substrate selectivity

Siddika,

Shao,

Heinemann

et al. 2024

IUBMB Life

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Protein kinase B (AKT1) is a serine/threonine kinase that regulates fundamental cellular processes, including cell survival, proliferation, and metabolism. AKT1 activity is controlled by two regulatory phosphorylation sites (Thr308, Ser473) that stimulate a downstream signaling cascade through phosphorylation of many target proteins. At either or both regulatory sites, hyperphosphorylation is associated with poor survival outcomes in many human cancers. Our previous biochemical and chemoproteomic studies showed that the phosphorylated forms of AKT1 have differential selectivity toward peptide substrates. Here, we investigated AKT1‐dependent activity in human cells, using a cell‐penetrating peptide (transactivator of transcription, TAT) to deliver inactive AKT1 or active phospho‐variants to cells. We used enzyme engineering and genetic code expansion relying on a phosphoseryl‐transfer RNA (tRNA) synthetase (SepRS) and tRNASep pair to produce TAT‐tagged AKT1 with programmed phosphorylation at one or both key regulatory sites. We found that all TAT‐tagged AKT1 variants were efficiently delivered into human embryonic kidney (HEK 293T) cells and that only the phosphorylated AKT1 (pAKT1) variants stimulated downstream signaling. All TAT‐pAKT1 variants induced glycogen synthase kinase (GSK)‐3α phosphorylation, as well as phosphorylation of ribosomal protein S6 at Ser240/244, demonstrating stimulation of downstream AKT1 signaling. Fascinatingly, only the AKT1 variants phosphorylated at S473 (TAT‐pAKT1S473 or TAT‐pAKT1T308,S473) were able to increase phospho‐GSK‐3β levels. Although each TAT‐pAKT1 variant significantly stimulated cell proliferation, cells transduced with TAT‐pAKT1T308 grew significantly faster than with the other pAKT1 variants. The data demonstrate differential activity of the AKT1 phospho‐forms in modulating downstream signaling and proliferation in human cells.

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

Cited by 34 publications

References 52 publications

Phosphorylation-Dependent Inhibition of Akt1

Phosphorylation-Dependent Inhibition of Akt1

Selenocysteine Insertion at a Predefined UAG Codon in a Release Factor 1 (RF1)-depleted Escherichia coli Host Strain Bypasses Species Barriers in Recombinant Selenoprotein Translation

Delivery of AKT1 phospho‐forms to human cells reveals differential substrate selectivity

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