Journal: Leukemia

Article Title: MAPK/ERK2 phosphorylates ERG at serine 283 in leukemic cells and promotes stem cell signatures and cell proliferation

doi: 10.1038/leu.2016.55

Figure Lengend Snippet: ERG S283 is phosphorylated in human leukemic cells. (a) A schematic of hERG3 scaled to size showing sites of serine phosphorylation in MOLT4 cells relative to its two functional domains. The numbers correspond to the amino acid positions. ETS, E-twenty six; PNT, Pointed. (b) A representative tandem mass spectrum (MS) showing phosphorylated ERG tryptic peptide containing S283. The b- and y-ions are annotated and labeled on the spectrum with the MS1 accurate spectrum shown in the inset. Note that for clarity, some regions of the extracted ion chromatogram have been amplified 10-fold. (c) ERG S283 phosphorylation (pS283) level in MOLT4, KG1 and healthy CD34+ human HSPCs was quantified using MS and expressed as a ratio to the level of pS88 on a log-10 scale. (d) A table summarizing the presence (Y) or absence (N) of ERG phosphorylation. Detection of S55 requires chymotrypsin digestion and was not available for all studies. (e) Lysates of 24 primary leukemic xenografts including 5 ETP ALL, 14 non-ETP T-ALL and 5 primary AML xenografts were probed for pS283 in ERG using a custom anti-pS283 ERG monoclonal antibody. A lysate of healthy CD34+ HSPCs is shown for comparison. Numbers above blots are densitometry reading for pS283 ERG normalized to β-actin. Given variations in exposure time, these numerical values are for within blot interpretation and not for interpretation across individual blots. Expression levels of total ERG and β-actin are also shown. The patient characteristics relating to the xenografts are detailed in previous studies.32–35

Article Snippet: Generation of ERG-pS283 specific antiserum Mouse monoclonal phospho-specific antibody for ERG pS283 was produced by ProMab Biotechnologies, Inc. (Richmond, CA, USA) using the artificially synthesized phosphopeptide PTPQSKAAQP(p)*S*PSTVPKTEDQ, S283 highlighted by asterisk.

Techniques: Phospho-proteomics, Functional Assay, Labeling, Amplification, Comparison, Expressing

Journal: Leukemia

Article Title: MAPK/ERK2 phosphorylates ERG at serine 283 in leukemic cells and promotes stem cell signatures and cell proliferation

doi: 10.1038/leu.2016.55

Figure Lengend Snippet: ERG S283 phosphorylation is mediated by the MAPK/ERK2 pathway. (a) A schematic showing the putative MAPK docking motif, DEF domain, located within ERG exon 12. The amino acid numbers and peptide sequence are shown. (b) A bar graph showing percent phosphorylation of serine residues in WT ERG and in an exon 12 deletion mutant of ERG (ERG-ΔEx12) measured using an in vitro ERK2 kinase assay. Peptide signal intensities of the phosphorylated and unphosphorylated versions were quantified by mass spectrometry (MS). Error bars represent s.d. for all peptide spectral matches quantified in this experiment (n = 3). ****P<0.0001. (c) A bar graph showing changes in phosphorylation in MOLT4 cells in response to treatment with U0126 (10 μM) and/or PMA (100 nM). The levels of phosphorylation were quantified by MS and plotted as a ratio of treated/vehicle on a log-10 scale. (d) Western blots showing the responses of two ETP ALL xenografts with high-level ERG pS283 following treatment with U0126 (10 μM) and/or PMA (100 nM). The levels of ERG pS283, total ERG, active ERK1/2 (pERK1/2), total ERK1/2 and β-actin are shown. Numbers above blots are densitometry readings for pS283 ERG normalized to β-actin. Given variations in exposure time, these numerical values are for within blot interpretation and not for interpretation across individual blots. The upper band in the ERK1/2 panel represents ERK1 and the lower band represents ERK2. (e) Western blots showing levels of endogenous ERG pS283, total ERG and active ERK1/2 (pERK1/2) in a panel of ETP ALL and AML patient-derived xenografts. Levels of each in healthy CD34+ HSPCs are also shown for comparison. Numbers above blots are densitometry reading for pS283 ERG normalized to β-actin. Given variations in exposure time, these numerical values are for within blot interpretation and not for interpretation across individual blots. (f) CFUs (colony-forming units) derived from vehicle-treated (0.1% dimethyl sulfoxide (DMSO)) or U0126-treated (10 μM) KG1, ME1 and CD34+ HSPCs. Error bars represent s.d. from triplicate measurements, *P<0.05 for the relevant comparisons.

Article Snippet: Generation of ERG-pS283 specific antiserum Mouse monoclonal phospho-specific antibody for ERG pS283 was produced by ProMab Biotechnologies, Inc. (Richmond, CA, USA) using the artificially synthesized phosphopeptide PTPQSKAAQP(p)*S*PSTVPKTEDQ, S283 highlighted by asterisk.

Techniques: Phospho-proteomics, Sequencing, Mutagenesis, In Vitro, Kinase Assay, Mass Spectrometry, Western Blot, Derivative Assay, Comparison