Identification of significantly different phosphorylation sites.

<p>(A) Volcano plot of NRG1-regulated phosphorylation in Ba/F3 cells expressing ERBB3 and ERBB4. (B) Volcano plot comparison of phosphorylation sites in NRG1-treated ERBB3/ERBB4 <i>versus</i> NRG1-treated ERBB4 expressing Ba/F3 cells. In both comparisons, log₁₀-transformed, average phosphosite ratios are plotted against their standard deviations determined from mTRAQ replicate quantifications. Significantly regulated class I sites according to the Global Mean Rank test are depicted in red, all other sites in blue. The dashed grey lines indicate two-fold regulation.</p

Contribution of ERBB3 to phosphosite regulation.

<p>Scatter plot of the mean ERBB3/ERBB4 ± NRG1 ratios with mean ERBB3/ERBB4 <i>versus</i> ERBB4 ratios from NRG1-treated cells. Reproducibly quantified ERBB3 phosphosites are encircled.</p

NRG1-induced proliferation and signaling of Ba/F3 cells expressing ERBB3 and/or ERBB4.

<p>(A) Cells expressing either ERBB4 (upper panel), ERBB4 and ERBB3 (middle panel) or ERBB3 receptors (lower panel) were cultured in medium without ligand, in the presence of 10 ng/ml IL3, or in the presence of 10 or 100 ng/ml NRG1. Cell numbers were counted at the indicated time points. (B) Parental Ba/F3 or Ba/F3 cells expressing ERBB3, ERBB4 or both receptors were incubated in the absence of IL3 and treated with 100 ng/mL NRG1 where indicated. Total cell lysate were then prepared and immunoblotted with phosphoepitope- and protein-specific antibodies for ERBB3, ERBB4, Akt and MEK.</p

Quantitative phosphoproteomics workflow and experimental design.

<p>Ba/F3 ERBB3/ERBB4 or ERBB4 cells were treated in the three replicate experiments as indicated. Upon lysis and proteolytic digestion, peptides were differentially labeled with the three isotopic variant of mTRAQ and then pooled prior to peptide separation by high pH reversed phase chromatography and IMAC phosphopeptide enrichment. Phosphopeptide fractions were then analyzed by quantitative LC-MS on a LTQ Orbitrap Velos instrument. Lower panel: Characteristic mTRAQ patterns shown for a peptide harboring a NRG1-induced phosphosite in ERBB3/ERBB4 cells, which less strongly up-regulated in the absence of ERBB3 in ERBB4-expressing Ba/F3 cells. </p

Fragment-Based Discovery of Type I Inhibitors of Maternal Embryonic Leucine Zipper Kinase

Fragment-based drug design was successfully applied to maternal embryonic leucine zipper kinase (MELK). A low affinity (160 μM) fragment hit was identified, which bound to the hinge region with an atypical binding mode, and this was optimized using structure-based design into a low-nanomolar and cell-penetrant inhibitor, with a good selectivity profile, suitable for use as a chemical probe for elucidation of MELK biology