Expanded Cellular Amino Acid Pools Containing Phosphoserine, Phosphothreonine, and Phosphotyrosine

Adding nonstandard amino acids to the genetic code of <i>E. coli</i> expands the chemical and biological functional space for proteins. This is accomplished with engineered, orthogonal aminoacyl-tRNA synthetase and tRNA pairs that require a nonstandard amino acid in sufficient intracellular quantities to support protein synthesis. While cotranslational insertion of phosphoserine into proteins has been accomplished, conditions that modulate intracellular phosphoamino acid concentrations are still poorly understood. Here we used genetic and metabolic engineering to increase the free intracellular levels of phosphoserine in <i>E. coli</i>. We show that deletion of the phosphoserine phosphatase <i>serB</i> elevates the intracellular levels of phosphoserine within ranges comparable to those of standard amino acids. These new conditions improved insertion of phosphoserine into recombinant proteins. Surprisingly, we also observed dramatic increases in intracellular levels of phosphothreonine and phosphotyrosine when WT cells were grown in LB with supplemented phosphothreonine and <i>serB</i> deficient cells were grown in low phosphate media with supplemented phosphotyrosine, respectively. These findings remove a major barrier for further expansion of the genetic code with additional phosphorylated amino acids

Designed Phosphoprotein Recognition in <i>Escherichia coli</i>

Protein phosphorylation is a central biological mechanism for cellular adaptation to environmental changes. Dysregulation of phosphorylation signaling is implicated in a wide variety of diseases. Thus, the ability to detect and quantify protein phosphorylation is highly desirable for both diagnostic and research applications. Here we present a general strategy for detecting phosphopeptide–protein interactions in <i>Escherichia coli</i>. We first redesign a model tetratricopeptide repeat (TPR) protein to recognize phosphoserine in a sequence-specific fashion and characterize the interaction with its target phosphopeptide <i>in vitro</i>. We then combine <i>in vivo</i> site-specific incorporation of phosphoserine with split mCherry assembly to observe the designed phosphopeptide–protein interaction specificity in <i>E. coli</i>. This <i>in vivo</i> strategy for detecting and characterizing phosphopeptide–protein interactions has numerous potential applications for the study of natural interactions and the design of novel ones

Designed Phosphoprotein Recognition in <i>Escherichia coli</i>

Protein phosphorylation is a central biological mechanism for cellular adaptation to environmental changes. Dysregulation of phosphorylation signaling is implicated in a wide variety of diseases. Thus, the ability to detect and quantify protein phosphorylation is highly desirable for both diagnostic and research applications. Here we present a general strategy for detecting phosphopeptide–protein interactions in <i>Escherichia coli</i>. We first redesign a model tetratricopeptide repeat (TPR) protein to recognize phosphoserine in a sequence-specific fashion and characterize the interaction with its target phosphopeptide <i>in vitro</i>. We then combine <i>in vivo</i> site-specific incorporation of phosphoserine with split mCherry assembly to observe the designed phosphopeptide–protein interaction specificity in <i>E. coli</i>. This <i>in vivo</i> strategy for detecting and characterizing phosphopeptide–protein interactions has numerous potential applications for the study of natural interactions and the design of novel ones

Designed Phosphoprotein Recognition in <i>Escherichia coli</i>

Protein phosphorylation is a central biological mechanism for cellular adaptation to environmental changes. Dysregulation of phosphorylation signaling is implicated in a wide variety of diseases. Thus, the ability to detect and quantify protein phosphorylation is highly desirable for both diagnostic and research applications. Here we present a general strategy for detecting phosphopeptide–protein interactions in <i>Escherichia coli</i>. We first redesign a model tetratricopeptide repeat (TPR) protein to recognize phosphoserine in a sequence-specific fashion and characterize the interaction with its target phosphopeptide <i>in vitro</i>. We then combine <i>in vivo</i> site-specific incorporation of phosphoserine with split mCherry assembly to observe the designed phosphopeptide–protein interaction specificity in <i>E. coli</i>. This <i>in vivo</i> strategy for detecting and characterizing phosphopeptide–protein interactions has numerous potential applications for the study of natural interactions and the design of novel ones

The phospho Serine 41 form of DCNL5 does not affect the kinetics of Cullin neddylation reactions.

<p>(A) EcAR7.SEP bacterial strain was transformed with plasmids harboring GST-DCNL5-WT or GST-DCNL5-S41TAG (the S41 codon was replaced by an amber stop codon). Purified proteins were cleaved from the GST tag using precision protease (GE) an analyzed by immunoblot with antibodies against DCNL5 or only the phospho form. <b>(B)</b> Pulse-chase [32P] NEDD8 transfer from UBC12NAc or UBE2FNAc (for Cullin 5 only) to the indicated Cullin C-terminal domain- RBX complexes in the absence or presence of the indicated DCNL5 constructs (PONY domain/ Full length and phosphorylated form. For comparison, all reactions were carried out under the same conditions.</p

Knock down of DCNL5 by siRNA has no effect on the activation of the NF-KB pathway.

<p>(A) RAW264.7 were electroporated with siRNA and stimulated with LPS 24 hours later. Knockdown efficiency was measured by immunoblot. (B) As in (A) RAW264.7 were electroporated with siRNA and stimulated with Poly (I:C) 24 hours later for the indicated times. Total RNA was extracted and the mRNA encoding DCNL5, IFNβ, CXCL10, ISG15 and MX1 were measured by qRT-PCR as described under the “Experimental Procedures.” The results show relative mRNA levels compared with the value of 1.0 measured in unstimulated control cells. The experiment was performed in quadruplicate for each condition. Similar results were obtained in two independent experiments. Adjacent graphs show the means (± s.e.m) of quantified mRNA levels. Statistical significance was determined by two-ways ANOVA. P≤0.05 (C) RAW264.7 were electroporated with siRNA and stimulated with LPS 24 hours later. Knockdown efficiency was measured by immunoblot.</p

DCNL5 is phosphorylated on Serine 41 by IKKα in RAW264.7 macrophages upon TLR stimulation.

<p>(A) HEK293 cell lines were transfected with Flag tagged DCNL5 WT or S41A and HA-tagged IKKα/ β WT (left panel) or kinase dead mutant (right panel). The cell lysates (30 μg) were resolved on SDS-PAGE and analyzed by immunoblotting with indicated antibodies. <b>(B)</b> RAW264.7 macrophages were treated with 100 ng/ml LPS over the indicated time period and subjected to immunoblot analyses. To visualize the phosphorylated form of DCNL5, cell extracts were subjected to immunoprecipitation using the phospho-specific antibody and analyzed by immunoblot with a total DCNL5 antibody. <b>(C)</b> RAW264.7 macrophages were treated with 1 μg/ml Pam3CSK4 over the indicated time period and subjected to immunoblot analyses. To visualize the phosphorylated form of DCNL5, cell extracts were subjected to immunoprecipitation using the phospho-specific antibody and analyzed by immunoblot with a total DCNL5 antibody. <b>(D)</b> RAW264.7 macrophages were treated 1 hour with MRT67307 (1μM), BI605906 (10μM) or NG25 (2μM) prior to LPS stimulation. The cell extracts were analyzed by immunoblots with the indicated antibodies. The phospho-form of DCNL5 was visualized after immunoprecipitation as described in (B). <b>(E)</b> Same as (D), except that RAW264.7 were either mock treated or treated with MLN4924 (3μM) for 3 hours or MRT67307 (1μM) for 1 hour prior to LPS stimulation.</p

DCNL5 is phosphorylated on Serine 41 by IKKα.

<p><b>(A</b>) Immunoblot analysis with the indicated antibodies of cell lysates from different cell lines. DCNL5 is strongly expressed in RAW264.7 (immortalized macrophages), Jurkat (immortalized T lymphocytes), and Raji (derived B lymphocytes) cells, while it is present at low levels in Thp1 cells (a monocyte-derived cell line). <b>(B</b>) Upper panel: Mass spectrum showing phosphopeptide precursor ions corresponding to DCNL5 (amino acids 39–48), which is phosphorylated at Ser41 in RAW264.7 macrophages treated with Poly (I:C) or Pam3CSK4, pre-treatment with MRT67307 (1 μM) for 1 hour enhance the phosphorylation. The results for unstimulated RAW264.7 macrophages are presented in blue (light), results for RAW264.7 macrophages stimulated with Poly (I:C) or Pam3CSK4 are in green (medium) and the results for RAW264.7 macrophages pre-treated with MRT67307 prior to stimulation with Poly (I:C) or Pam3CSK4 are depicted in red (heavy). For the Poly (I:C) treated samples, the ratio of labelled phosphopeptides in the different conditions quantified from MaxQUANT are given. Lower panel: Diagram of DCNL5 depicting the conserved C-ter PONY domain, its specific N-ter domain and the phosphorylated residue (red font; S41) uncovered by MS analysis. <b>(C)</b> Top panel: 1.4 μg of the purified recombinant DCNL5 was incubated with IKKα or IKKβ in the presence of <b>γ</b>32-ATP at 30°C for the times indicated. 1.4 μg of Iκbα was included as a positive control for IKKα and IKKβ. The stopped reactions were run on a gel and exposed to X-ray films. Right panel: same reactions as the top panel using IKKα WT or kinase dead mutant (S176A/S180A). The asterisk depicts degradation products from Iκbα that run at the same position as DCNL5. <b>(D)</b> <i>In vitro</i> phosphorylated DCNL5 was tryptic digested and separated by reverse chromatography followed by radioactivity measurement. Fractions corresponding to DCNL5 phosphopeptide were subjected for Edman sequencing. Flag tagged recombinant DCNL5 WT or S41A mutant was phosphorylated <i>in vitro</i> by IKKα WT in the presence of <b>γ</b>32-ATP at 30 °C for 30 minutes.</p

Kinase Substrate Profiling Using a Proteome-wide Serine-Oriented Human Peptide Library

The human proteome encodes >500 protein kinases and hundreds of thousands of potential phosphorylation sites. However, the identification of kinase–substrate pairs remains an active area of research because the relationships between individual kinases and these phosphorylation sites remain largely unknown. Many techniques have been established to discover kinase substrates but are often technically challenging to perform. Moreover, these methods frequently rely on substrate reagent pools that do not reflect human protein sequences or are biased by human cell line protein expression profiles. Here, we describe a new approach called SERIOHL-KILR (serine-oriented human library–kinase library reactions) to profile kinase substrate specificity and to identify candidate substrates for serine kinases. Using a purified library of >100000 serine-oriented human peptides expressed heterologously in <i>Escherichia coli</i>, we perform <i>in vitro</i> kinase reactions to identify phosphorylated human peptide sequences by liquid chromatography and tandem mass spectrometry. We compare our results for protein kinase A to those of a well-established positional scanning peptide library method, certifying that SERIOHL-KILR can identify the same predominant motif elements as traditional techniques. We then interrogate a small panel of cancer-associated PKCβ mutants using our profiling protocol and observe a shift in substrate specificity likely attributable to the loss of key polar contacts between the kinase and its substrates. Overall, we demonstrate that SERIOHL-KILR can rapidly identify candidate kinase substrates that can be directly mapped to human sequences for pathway analysis. Because this technique can be adapted for various kinase studies, we believe that SERIOHL-KILR will have many new victims in the future

Kinase Substrate Profiling Using a Proteome-wide Serine-Oriented Human Peptide Library

The human proteome encodes >500 protein kinases and hundreds of thousands of potential phosphorylation sites. However, the identification of kinase–substrate pairs remains an active area of research because the relationships between individual kinases and these phosphorylation sites remain largely unknown. Many techniques have been established to discover kinase substrates but are often technically challenging to perform. Moreover, these methods frequently rely on substrate reagent pools that do not reflect human protein sequences or are biased by human cell line protein expression profiles. Here, we describe a new approach called SERIOHL-KILR (serine-oriented human library–kinase library reactions) to profile kinase substrate specificity and to identify candidate substrates for serine kinases. Using a purified library of >100000 serine-oriented human peptides expressed heterologously in <i>Escherichia coli</i>, we perform <i>in vitro</i> kinase reactions to identify phosphorylated human peptide sequences by liquid chromatography and tandem mass spectrometry. We compare our results for protein kinase A to those of a well-established positional scanning peptide library method, certifying that SERIOHL-KILR can identify the same predominant motif elements as traditional techniques. We then interrogate a small panel of cancer-associated PKCβ mutants using our profiling protocol and observe a shift in substrate specificity likely attributable to the loss of key polar contacts between the kinase and its substrates. Overall, we demonstrate that SERIOHL-KILR can rapidly identify candidate kinase substrates that can be directly mapped to human sequences for pathway analysis. Because this technique can be adapted for various kinase studies, we believe that SERIOHL-KILR will have many new victims in the future