Assembly Dynamics and Stoichiometry of the Apoptosis Signal-regulating Kinase (ASK) Signalosome in Response to Electrophile Stress.

Apoptosis signal-regulating kinase 1 (ASK1) is a key sensor kinase in the mitogen-activated protein kinase pathway that transduces cellular responses to oxidants and electrophiles. ASK1 is regulated by a large, dynamic multiprotein signalosome complex, potentially including over 90 reported ASK1-interacting proteins. We employed both shotgun and targeted mass spectrometry assays to catalogue the ASK1 protein-protein interactions in HEK-293 cells treated with the prototypical lipid electrophile 4-hydroxy-2-nonenal (HNE). Using both epitope-tagged overexpression and endogenous expression cell systems, we verified most of the previously reported ASK1 protein-protein interactions and identified 14 proteins that exhibited dynamic shifts in association with ASK1 in response to HNE stress. We used precise stable isotope dilution assays to quantify protein stoichiometry in the ASK signalosome complex and identified ASK2 at a 1:1 stoichiometric ratio with ASK1 and 14-3-3 proteins (YWHAQ, YWHAB, YWHAH, and YWHAE) collectively at a 0.5:1 ratio with ASK1 as the main components. Several other proteins, including ASK3, PARK7, PRDX1, and USP9X were detected with stoichiometries of 0.1:1 or less. These data support an ASK signalosome comprising a multimeric core complex of ASK1, ASK2, and 14-3-3 proteins, which dynamically engages other binding partners needed to mediate diverse stress-response signaling events. This study further demonstrates the value of combining global and targeted MS approaches to interrogate multiprotein complex composition and dynamics.

Facile labeling of lipoglycans with quantum dots.

Bacterial endotoxins or lipopolysaccharides (LPS) are among the most potent activators of the innate immune system, yet mechanisms of their action and in particular the role of glycans remain elusive. Efficient non-invasive labeling strategies are necessary for studying interactions of LPS glycans with biological systems. Here we report a new method for labeling LPS and other lipoglycans with luminescent quantum dots. The labeling is achieved by partitioning of hydrophobic quantum dots into the core of various LPS aggregates without disturbing the native LPS structure. The biofunctionality of the LPS-Qdot conjugates is demonstrated by the labeling of mouse monocytes. This simple method should find broad applicability in studies concerned with visualization of LPS biodistribution and identification of LPS binding agents.

Ibrutinib inhibition of ERBB4 reduces cell growth in a WNT5A-dependent manner.

Alterations in ERBB family members have been associated with many tumor malignancies. EGFR and ERBB2 have been extensively explored in clinical oncology and several drugs currently target them therapeutically. However, the significance of ERBB4 as a potential therapeutic target remains mostly unexplored, even though ERBB4 is overexpressed or mutated in many solid tumors. Using a unique functional protein microarray platform, we found that ibrutinib inhibits ERBB4 activity in the same nM range as its canonical target, BTK. Cell-based assays revealed that ibrutinib treatment inhibited cell growth and decreased phosphorylation of ERBB4 and downstream targets MEK and ERK in cancer cell lines with high levels of endogenous ERBB4. In vivo, ibrutinib-responsive mouse xenograft tumors showed decreased tumor volumes with ibrutinib treatment. Interestingly, global gene expression comparisons between responsive and non-responsive cells identified a signature featuring the WNT pathway that predicts growth responsiveness to ibrutinib. Non-responsive ERBB4-expressing cell lines featured elevated activity of the WNT pathway, through the overexpression of WNT5A. Moreover, inhibition of WNT5A expression led to an ibrutinib response in non-responsive cell lines. Our data show that inhibiting ERBB4 reduces cell growth in cells that have low WNT5A expression and reveal a link between the ERBB4 and WNT pathways.