Lauren Foltz, Mark Grimes, Juan Palacios-Moreno
Photo by Todd Goodrich
Check out the slideshow of 6 items below:
- Slide Title: gradient strategy Slide Caption: Cell Fractionation Strategy
- Slide Title: CoverImage Slide Caption: Science Signaling cover story 22 May 2018
- Slide Title: Separation of endosomes Slide Caption: Separation of signaling endosomes
- Slide Title: Endosome phosphoproteomics Slide Caption: Most prominent phosphorylated proteins in neuroblastoma endosomes
- Slide Title: model Slide Caption: Model for PAG1, LYN, and FYN dynamics
- Slide Title: DRG neuron Slide Caption: Neuroblastoma cells transplanted into chick embryo migrated to DRG
Multiple signals determine cell fates such as cell birth, death, and differentiation during development and in adult multicellular organisms. A major challenge in biology is to understand how signals from different receptors are integrated to determine an appropriate response. This process is particularly complicated in migrating cells such neurons and neural crest cells, and may go awry, resulting in increased cell proliferation or migration in cancer.
Phosphoproteomic analysis has informed much of our current knowledge of cell signaling networks and facilitated drug development. Less appreciated are the inputs of other posttranslational modifications (PTMs). In a paper recently published in Science Signaling and highlighted in Genetic Engineering & Biotechnology News, GenomeWeb, EurekaAlert, Medical press, W3LiveNews, and scoop.it, we integrated genomic, proteomic, phosphoproteomic, acetylomic, and methylomic data from lung cancer cells versus those in normal lung tissue and explored various regulatory patterns. We found that many PTMs are exclusive, in that as phosphorylation increased, acetylation decreased; that the drug geldanamycin broadly alters the PTM landscape and, thus, has effects far beyond its target; and that RNA binding proteins appear to be critical effectors of many signaling paths. These networks may inform new drug development for lung cancer patients and exemplify how cell signaling is regulated by far more extensive PTM networks than was previously appreciated. Lung cancer signaling pathways identified by cluster-filtered networks based on PTMs may be explored here: https://cynetworkbrowser.umt.edu/.
Neuroblastoma cell lines provide a model system to study the molecular mechanisms involved in sorting and transactivation between receptors. Many receptors signal from endosomes: to amplify signals, activate different effectors than those activated at the plasma membrane, or convey signals to different intracellular locations. There is evidence that endosomal signaling from a number of different receptors affects cell fate decisions during development. We hypothesize that multiprotein complexes of activated receptors and their effectors in endosomes play a role in signal integration when more than one receptor is activated.
We have shown that three different types of receptors are localized predominately in endosomes that are resolved from one another using a high-resolution organelle fractionation method based on mass and density (McCaffrey, et al., 2009). The data suggest that receptor sorting into specific signaling endosomes affects the compartmentalization of signaling pathways. The two receptors for nerve growth factor (NGF), TrkA and p75NTR, are rapidly sorted upon ligand binding to distinct endosomes. We have recently shown that sorting of these two receptors away from one another involves dynamic interactions between detergent-insoluble lipid rafts and microtubules (Pryor, et al., 2012). NGF caused TrkA to be attracted to lipid rafts, and p75NTR to sort away from rafts.
To understand tyrosine kinase signaling mechanisms, we undertook a large-scale study of phosphorylated proteins (phosphoproteomics) in neuroblastoma cell lines. We developed new methods to analyze these data with help from collaborators in the fields of pattern recognition, computational biology and bioinformatics, including Gary Bader (University of Toronto), Paul Shannon (Fred Hutchison Cancer Research Institute) and Wan-Jui Lee and Laurens van der Maaten (Delft University of Technology). Our recent paper (Palacios-Moreno, et al., 2015) was highlighted in Science Daily, Science Newsline Biology, EurekaAlert, A-Z News, MedicalNewsToday, and even the Missoulian. The model is that transient networks of multiprotein complexes, whose assembly is governed by interactions between phosphorylated proteins and phospho-specific protein binding domains, convey information that changes cell fate. These complexes assemble at distinct intracellular locations, and contain different components, in response to activation of different receptor tyrosine kinases. A surprising finding was that more than half of the known RTKs in the human genome were detected in neuroblastoma cell lines, and in most cases several RTKs appear to be active in the same cell line. In several cell lines there are multiple RTKs in endosomes. We also detected the SRC-family kinases, FYN and LYN in endosomes as well as lipid rafts. We hypothesize that the dynamic localization of SRC-family kinases in endosomes and lipid rafts plays a role in distinguishing responses to different RTKs.