System-Level Analysis of the Effects of RPTPs on Cellular Signaling Networks.

Tyrosine phosphorylation regulates signaling network activity downstream of receptor tyrosine kinase (RTK) activation. Receptor protein tyrosine phosphatases (RPTPs) serve to dephosphorylate RTKs and their proximal adaptor proteins, thus serving to modulate RTK activity. While the general function of RPTPs is well understood, the direct and indirect substrates for each RPTP are poorly characterized. Here we describe a method, quantitative phosphotyrosine phosphoproteomics, that enables the identification of specific phosphorylation sites whose phosphorylation levels are altered by the expression and activity of a given RPTP. In a proof-of-concept application, we use this method to highlight several direct or indirect substrate phosphorylation sites for PTPRJ, also known as DEP1, and show their quantitative phosphorylation in the context of wild-type PTPRJ compared to a mutant form of PTPRJ with increased activity, in EGF-stimulated cells. This method is generally applicable to define the signaling network effects of each RPTP in cells or tissues under different physiological conditions.

Dissecting signaling regulators driving AXL-mediated bypass resistance and associated phenotypes by phosphosite perturbations.

Receptor tyrosine kinase (RTK)-targeted therapies are often effective but invariably limited by drug resistance. A major mechanism of acquired resistance involves "bypass" switching to alternative pathways driven by non-targeted RTKs that restore proliferation. One such RTK is AXL whose overexpression, frequently observed in bypass resistant tumors, drives both cell survival and associated malignant phenotypes such as epithelial-to-mesenchymal (EMT) transition and migration. However, the signaling molecules and pathways eliciting these responses have remained elusive. To explore these coordinated effects, we generated a panel of mutant lung adenocarcinoma PC9 cell lines in which each AXL intracellular tyrosine residue was mutated to phenylalanine. By integrating measurements of phosphorylation signaling and other phenotypic changes associated with resistance through multivariate modeling, we mapped signaling perturbations to specific resistant phenotypes. Our results suggest that AXL signaling can be summarized into two clusters associated with progressive disease and poor clinical outcomes in lung cancer patients. These clusters displayed favorable Abl1 and SFK motifs and their phosphorylation was consistently decreased by dasatinib. High-throughput kinase specificity profiling showed that AXL likely activates the SFK cluster through FAK1 which is known to complex with Src. Moreover, the SFK cluster overlapped with a previously established focal adhesion kinase (FAK1) signature conferring EMT-mediated erlotinib resistance in lung cancer cells. Finally, we show that downstream of this kinase signaling, AXL and YAP form a positive feedback loop that sustains drug tolerant persister cells. Altogether, this work demonstrates an approach for dissecting signaling regulators by which AXL drives erlotinib resistance-associated phenotypic changes.

Predictive data-driven modeling of C-terminal tyrosine function in the EGFR signaling network.

The epidermal growth factor receptor (EGFR) has been studied extensively because of its critical role in cellular signaling and association with disease. Previous models have elucidated interactions between EGFR and downstream adaptor proteins or showed phenotypes affected by EGFR. However, the link between specific EGFR phosphorylation sites and phenotypic outcomes is still poorly understood. Here, we employed a suite of isogenic cell lines expressing site-specific mutations at each of the EGFR C-terminal phosphorylation sites to interrogate their role in the signaling network and cell biological response to stimulation. Our results demonstrate the resilience of the EGFR network, which was largely similar even in the context of multiple Y-to-F mutations in the EGFR C-terminal tail, while also revealing nodes in the network that have not previously been linked to EGFR signaling. Our data-driven model highlights the signaling network nodes associated with distinct EGF-driven cell responses, including migration, proliferation, and receptor trafficking. Application of this same approach to less-studied RTKs should provide a plethora of novel associations that should lead to an improved understanding of these signaling networks.

RNA-binding protein ELAVL4/HuD ameliorates Alzheimer's disease-related molecular changes in human iPSC-derived neurons.

The RNA binding protein ELAVL4/HuD regulates the translation and splicing of multiple Alzheimer's disease (AD) candidate genes. We generated ELAVL4 knockout (KO) human induced pluripotent stem cell-derived neurons to study the effect that ELAVL4 has on AD-related cellular phenotypes. ELAVL4 KO significantly increased the levels of specific APP isoforms and intracellular phosphorylated tau, molecular changes that are related to the pathological hallmarks of AD. Overexpression of ELAVL4 in wild-type neurons and rescue experiments in ELAVL4 KO cells showed opposite effects and also led to a reduction of the extracellular amyloid-beta (Aß)42/40 ratio. All these observations were made in familial AD (fAD) and fAD-corrected neurons. To gain insight into the molecular cascades involved in neuronal ELAVL4 signaling, we conducted pathway and upstream regulator analyses of transcriptomic and proteomic data from the generated neurons. These analyses revealed that ELAVL4 affects multiple biological pathways linked to AD, including those involved in synaptic function, as well as gene expression downstream of APP and tau signaling. The analyses also suggest that ELAVL4 expression is regulated by insulin receptor-FOXO1 signaling in neurons. Taken together, ELAVL4 expression ameliorates AD-related molecular changes in neurons and affects multiple synaptic pathways, making it a promising target for novel drug development.

Phosphoproteomics: a valuable tool for uncovering molecular signaling in cancer cells.

INTRODUCTION: Many pathologies, including cancer, have been associated with aberrant phosphorylation-mediated signaling networks that drive altered cell proliferation, migration, metabolic regulation, and can lead to systemic inflammation. Phosphoproteomics, the large-scale analysis of protein phosphorylation sites, has emerged as a powerful tool to define signaling network regulation and dysregulation in normal and pathological conditions. AREAS COVERED: We provide an overview of methodology for global phosphoproteomics as well as enrichment of specific subsets of the phosphoproteome, including phosphotyrosine and phospho-motif enrichment of kinase substrates. We review quantitative methods, advantages and limitations of different mass spectrometry acquisition formats, and computational approaches to extract biological insight from phosphoproteomics data. Throughout, we discuss various applications and their challenges in implementation. EXPERT OPINION: Over the past 20 years the field of phosphoproteomics has advanced to enable deep biological and clinical insight through the quantitative analysis of signaling networks. Future areas of development include Clinical Laboratory Improvement Amendments (CLIA)-approved methods for analysis of clinical samples, continued improvements in sensitivity to enable analysis of small numbers of rare cells and tissue microarrays, and computational methods to integrate data resulting from multiple systems-level quantitative analytical methods.

[Every physician should know how to take an occupational history: the importance of teaching occupational medicine in medical school]. / Iedere arts moet arbeidsanamnese uitvragen: belang van 'arbeid en gezondheid' in de medische opleiding.

Working and participating in society are important for one's health and wellbeing. This also works the other way around: in order to work or participate, one has to be healthy. Every doctor should learn about this reciprocal relationship during their medical training. In a survey undertaken by the Netherlands Society of Occupational Medicine, the amount of time spent on occupational medicine in the curriculum of eight Dutch medical faculties was examined. The study showed vast differences between faculties in the amount of time spent on occupational medicine, in the learning methods employed and also in the number of students who participate in internships. The situation in the Netherlands compares favourably with that in other European countries. Sufficient specific attention to subjects related to health and work during undergraduate education is important for all doctors and a prerequisite to increase the number of medical students who subsequently choose a career in occupational health medicine.