Recombinant Human Phosphoserine Aminotransferase 1, His-tagged

• Cat.No.: PSAT1-1328H
• Product Overview: Recombinant human PSAT1 protein, fused to His-tag at N-terminus, was expressed in E.coli and purified by using conventional chromatography.

Specification

• Species: Human
• Source: E.coli
• Tag: His
• Protein Length: 1-370 a.a.
• Description: PSAT1, also known as phosphoserine aminotransferase, catalyses the conversion of 3-phosphohydroxypyruvate into 3-phosphoserine which is dephosphorylated subsequently by phosphoserine phosphatase to form L-serine.
• Form: Liquid. In 20 mM Tris-HCl buffer (pH 8.0) containing 1 mM DTT, 20% glycerol.
• Molecular Weight: 42.9 kDa (394 aa), confirmed by MALDI-TOF
• Purity: > 90 % by SDS-PAGE
• Concentration: 1 mg/ml (determined by Bradford)
• Sequences of amino acids: MGSSHHHHHH SSGLVPRGSH MGSHMDAPRQ VVNFGPGPAK LPHSVLLEIQ KELLDYKGVG ISVLEMSHRS SDFAKIINNT ENLVRELLAV PDNYKVIFLQ GGGCGQFSAV PLNLIGLKAG RCADYVVTGA WSAKAAEEAK KFGTINIVHP KLGSYTKIPD PSTWNLNPDA SYVYYCANET VHGVEFDFIP DVKGAVLVCD MSSNFLSKPV DVSKFGVIFA GAQKNVGSAG VTVVIVRDDL LGFALRECPS VLEYKVQAGN SSLYNTPPCF SIYVMGLVLE WIKNNGGAAA MEKLSSIKSQ TIYEIIDNSQ GFYVCPVEPQ NRSKMNIPFR IGNAKGDDAL EKRFLDKALE LNMLSLKGHR SVGGIRASLY NAVTIEDVQK LAAFMKKFLE MHQL
• Storage: Can be stored at +4°C short term (1-2 weeks). For long term storage, aliquot and store at -20°C or -70°C. Avoid repeated freezing and thawing cycles.
• Pathways: Glycine, serine and threonine metabolism; Metabolic pathways; Vitamin B6 metabolism; Serine biosynthesis

Gene Information

• Gene Name: PSAT1 phosphoserine aminotransferase 1 [ Homo sapiens ]
• Official Symbol: PSAT1
• Synonyms: PSAT1; phosphoserine aminotransferase 1; PSA; EPIP; PSAT; MGC1460; endometrial progesterone-induced protein; EC 2.6.1.52
• Gene ID: 29968
• mRNA Refseq: NM_021154
• Protein Refseq: NP_066977
• MIM: 610936
• UniProt ID: Q9Y617
• Chromosome Location: 9q21.2
• Function: O-phospho-L-serine:2-oxoglutarate minotransferase activity; pyridoxal phosphate binding; transferase activity

Citation

The breast cancer oncogene IKKε coordinates mitochondrial function and serine metabolism

Journal: bioRxiv    Data: 2019/11/26

Authors: Xu Ruoyan, Jones William, Bianchi Katiuscia

Article Snippet:PrePrint: HA-tagged IKKε was isolated and purified from 1mg of whole cell lysate by pull-down using Anti-HA Agarose beads (Sigma Aldrich) for 1hr at 4°C with continuous mixing.HA-tagged IKKε was isolated and purified from 1mg of whole cell lysate by pull-down using Anti-HA Agarose beads (Sigma Aldrich) for 1hr at 4°C with continuous mixing.. 5 pmol of purified recombinant PSAT1 protein (PSAT1-1328H, Creative BioMart) was added to the pelleted and washed agarose beads along with kinase buffer (25mM Tris HCl (pH 7.8), 10mM MgCl 2 , 0.5 mM EGTA, 1mM DTT, 1mM Na 3 VO 4 , 2.5mM Na 2 P 2 H 2 O 7 and 20mM β-glycerol phosphate) and 10mM ATP (New England Biolabs) to a total volume of 50μl.. The reaction was incubated at room temperature for 20 minutes and halted by the addition of solid urea to a final concentration of 8M.The reaction was incubated at room temperature for 20 minutes and halted by the addition of solid urea to a final concentration of 8M.

(A) Principal component analysis of differentially phosphorylated substrates in three independent single cell clones of Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (100 ng/ml, 16 hours). The phosphoproteomes in the three clones were analysed by mass spectrometry as described in material and methods. (B) Level of IKKε in three independent single cell clones of Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells following treatment with doxycycline (100 ng/ml, 16 hours). (C) Pathway enrichment analysis of phosphoproteomic data identifying modification of proteins in key metabolic and signalling pathways as a result of IKKε expression. Enriched pathways from differentially phosphorylated substrates were determined using the David pathway enrichment analysis tool (see material and methods). (D) Volcano plot representation of statistically significant differentially phosphorylated substrates in indicated Flp-In 293 HA-IKKε expressing clones relative to HA-GFP expressing controls (n=4 independent replicates). P values were calculated by unpaired Student’s t-test of log transformed normalised phosphopeptide intensity values (peak areas of extracted ion chromatograms). Highlighted dots represent IKKε auto-phosphorylation sites indicative of kinase activity, and PSAT1 S331 phosphosite. (E) Level of endogenous PSAT1 and HA-tagged wild-type (WT) or mutant (S>A, S>E) PSAT1 in Flp-In 293 HA-PSAT1 wt, Flp-In 293 HA-PSAT1 S>A and Flp-In 293 HA-PSAT1 S>E cells treated with doxycycline (50 ng/ml, 16 hours). Endogenous PSAT1 was silenced in indicated samples prior to doxycycline treatment. (F) Levels of HA-tagged PSAT1 variants in endogenous PSAT1 -silenced Flp-In 293 HA-PSAT1 wt, Flp-In 293 HA-PSAT1 S>A and Flp-In 293 HA-PSAT1 S>E cells treated with doxycycline (50 ng/ml, 16 hours). Densitometry analysis quantified single sample density as a percentage of total blot densitometry prior to vinculin normalisation (n=9, mean ± SEM, *p<0.05, one-way ANOVA with Bonferroni post-hoc tests). (G) Quantitative Real-Time PCR (qRT-PCR) analysis of PSAT1 mRNA level in Flp-In 293 HA-PSAT1 wt, Flp-In 293 HA-PSAT1 S>A and Flp-In 293 HA-PSAT1 S>E cells treated with doxycycline (50 ng/ml, 16 hours). Data are expressed as relative to levels in non-treated cells and normalised to β-Actin (n=4, mean ± SEM) (H-K) 15 N 2 -glutamine flux analysis of endogenous PSAT1 -silenced Flp-In 293 HA-PSAT1 wt, Flp-In 293 HA-PSAT1 S>A and Flp-In 293 HA-PSAT1 S>E cells treated with doxycycline (50 ng/ml, 16 hours). Fractional enrichment of (H) glutamate, (I) glutamine, (J) glycine and (K) serine 15 N-isotopologues. m+1 shows the naturally occurring 13 C isotopologue (n=10). Data Information: In (E) and (H-K), endogenous PSAT1 was silenced by transfection with a pool of 3 siRNA oligos targeting the non-coding sequence of PSAT1 mRNA. In (H-K), data are presented as mean ± SD, *p<0.05 (one-way ANOVA with Bonferroni post-hoc tests).

(A) Levels of IKKε, IRF3, phosphorylated IRF3 (S396) and PSAT1 in Flp-In 293 HA-IKKε cells treated with doxycycline (100 ng/ml) for the indicated number of hours. (B) Levels of IKKε, PHGDH, PSAT1 and PSPH in IKBKE -silenced ZR-75-1, T47D, MDA-MB-468 and MCF7 breast cancer cell lines. (C-E) Levels of the SBP enzymes in a panel of IKBKE -silenced breast cancer cell lines. (C) PHGDH, (D) PSAT1 and (E) PSPH level normalised to Vinculin. Densitometry analysis quantified single sample density as a percentage of total blot density per cell line prior to vinculin normalisation (n≥3, mean ± SEM, *p<0.05, two-tailed Student’s t-test). (F) qRT-PCR analysis of PHGDH, PSAT1 and PSPH mRNA levels in Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (100 ng/ml, 16 hours). Data are expressed as relative to levels in non-treated Flp-In 293 HA-GFP cells and normalised to β-Actin (n=4, mean ± SEM, *p<0.05, two-way ANOVA with Bonferroni post-hoc tests) (G) qRT-PCR analysis of PHGDH, PSAT1 and PSPH mRNA levels in a panel of IKBKE -silenced breast cancer cell lines. Data are expressed as relative to levels in a non-silenced control of each cell line and normalised to β-Actin (n≥3, mean ± SEM, *p < 0.05, as measured by one sample t-test).

(A) Basal oxygen consumption in Flp-In 293 HA-IKKε wt, Flp-In 293 HA-IKKε KD-m and Flp-In 293 HA-IKKε UbLD-m cells following treatment with doxycycline (16 hours), measured using Oroboros high resolution respirometry. Data are normalised to non-treated control cells. (B) Average TMRM staining intensity in Flp-In 293 HA-IKKε wt, Flp-In 293 HA-IKKε KD-m and Flp-In 293 HA-IKKε UbLD-m expressing cells induced by doxycycline (16 hours). Data are normalised to non-treated controls cells. (C) Basal mitochondrial oxygen consumption rate (OCR) in a panel of IKBKE -silenced breast cancer cell lines, transfected with a pool of 4 targeting siRNA oligos, measured using Seahorse XF96 analysis (n ≥ 3, mean ± SEM, *p < 0.05, paired). (D) Basal oxygen consumption in PSAT1- silenced Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (16 hours). Cells were transfected with a pool of 4 targeting siRNA oligos. Measured using Oroboros high resolution respirometry. Data are normalised to non-treated Flp-In 293 HA-GFP control cells. (E) Basal OCR in a panel of PSAT1- silenced breast cancer cell lines, transfected with a pool of 4 targeting siRNA oligos, measured using Seahorse XF96 analysis. (n=3, mean ± SEM). (F) Relative pyruvate dehydrogenase (PDH) activity in Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (50 ng/ml, 16 hours) (n=3, mean ± SEM, *p < 0.05 by Student’s t-test). (G) Average TMRM staining intensity in Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline and DCA (both for 16 hours). Data are normalised to non-treated Flp-In 293 HA-IKKε cells (n=3, mean ± SEM, *p < 0.05 as measured by two-way ANOVA with Bonferroni post hoc test). (H) Basal OCR in Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (50 ng/ml) in combination with dichloroacetate (DCA) for 16 hours, measured using Oroboros high resolution respirometry (n=8). (I) Basal OCR in Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (50 ng/ml) in combination with pyruvate deprivation for 16 hours, measured using Oroboros high resolution respirometry (n=8) Data Information: All data are n≥3 and presented as mean ± SEM, *p<0.05. In (A-D, H, I), data were normalised to total sample protein concentration. In (A, B) two-way ANOVA, in (G, H) one-way ANOVA with Fischer’s LSD post-hoc tests, in (C, E, F) two tailed paired Student’s t-tests for each cell line (C, E), and in (D, I) one-way ANOVA with Tukey’s multiple comparison tests were applied.