Mark L. Grimes

Multiple Levels for Regulation of TrkA in PC12 Cells by Nerve Growth Factor

Abstract: TrkA is a receptor tyrosine kinase for nerve growth factor (NGF). Recent studies indicate that NGF regulates not only activation of trkA kinase but also expression of the trkA gene. To further define NGF actions on trkA, we examined binding and signaling through trkA after both short and long intervals of NGF treatment. Induction of tyrosine phosphorylation on gp140trkA was rapidly followed by down‐regulation of cell surface and total cellular gp140trkA. At later intervals, increased expression of trkA was evident in increased mRNA and protein levels. At 7 days, there was increased binding to gp140trkA and increased signaling through this receptor. NGF appears to regulate trkA at several levels. In neurons persistently exposed to NGF, maintenance of NGF signaling may require increased trkA gene expression.

RCytoscape: tools for exploratory network analysis

BackgroundBiomolecular pathways and networks are dynamic and complex, and the perturbations to them which cause disease are often multiple, heterogeneous and contingent. Pathway and network visualizations, rendered on a computer or published on paper, however, tend to be static, lacking in detail, and ill-equipped to explore the variety and quantities of data available today, and the complex causes we seek to understand.ResultsRCytoscape integrates R (an open-ended programming environment rich in statistical power and data-handling facilities) and Cytoscape (powerful network visualization and analysis software). RCytoscape extends Cytoscapes functionality beyond what is possible with the Cytoscape graphical user interface. To illustrate the power of RCytoscape, a portion of the Glioblastoma multiforme (GBM) data set from the Cancer Genome Atlas (TCGA) is examined. Network visualization reveals previously unreported patterns in the data suggesting heterogeneous signaling mechanisms active in GBM Proneural tumors, with possible clinical relevance.ConclusionsProgress in bioinformatics and computational biology depends upon exploratory and confirmatory data analysis, upon inference, and upon modeling. These activities will eventually permit the prediction and control of complex biological systems. Network visualizations -- molecular maps -- created from an open-ended programming environment rich in statistical power and data-handling facilities, such as RCytoscape, will play an essential role in this progression.

Phosphorylation‐Dependent Akt Cleavage in Neural Cell In Vitro Reconstitution of Apoptosis

Abstract : Neuronal apoptotic execution uses a cytochrome c‐dependent caspase activation mechanism that is conserved in other cell types. Phosphatidylinositol 3‐kinase and its downstream effector, Akt/protein kinase B, appear to control this mechanism and govern the life/death decision. We have developed a cell‐free system using cytosol from human neuroblastoma (SY5Y) cells that reconstitutes biochemical features of neuronal apoptosis. In the presence of cytochrome c and ATP, caspase‐9 and ‐3 were activated, which initiated chromatin condensation and DNA cleavage in rat pheochromocytoma (PC12) nuclei. Akt was cleaved in reactions where caspase‐3 was activated and its cleavage was prevented by the caspase inhibitor DEVD‐aldehyde. The phosphatase inhibitors orthovanadate and okadaic acid prevented catalytic processing and activation of caspase‐3 and digestion of Akt and partially inhibited cleavage of caspase‐9. Caspase‐dependent destruction of Akt irreversibly inactivates this key mediator of survival signaling, ensuring that the execution pathway will prevail.

CREB is cleaved by caspases during neural cell apoptosis

Programmed cell death, or apoptosis, is a tightly regulated process mediated by selective cleavage of proteins by caspases, resulting in ordered destruction of the cell. In addition to structural proteins, proteins that mediate anti‐apoptotic signal transduction are also substrates; their destruction eliminates potential futile attempts to escape execution. We asked whether cAMP response element binding protein (CREB), a transcription factor that mediates nerve growth factor (NGF) survival signals, is a target for caspases during apoptosis. CREB was specifically cleaved by caspases in neuroblastoma extracts, and in cells induced to undergo apoptosis by staurosporine. The destruction of CREB eliminates a key factor that could reverse apoptosis.

SH3 interactome conserves general function over specific form.

Src homology 3 (SH3) domains bind peptides to mediate protein–protein interactions that assemble and regulate dynamic biological processes. We surveyed the repertoire of SH3 binding specificity using peptide phage display in a metazoan, the worm Caenorhabditis elegans, and discovered that it structurally mirrors that of the budding yeast Saccharomyces cerevisiae. We then mapped the worm SH3 interactome using stringent yeast two‐hybrid and compared it with the equivalent map for yeast. We found that the worm SH3 interactome resembles the analogous yeast network because it is significantly enriched for proteins with roles in endocytosis. Nevertheless, orthologous SH3 domain‐mediated interactions are highly rewired. Our results suggest a model of network evolution where general function of the SH3 domain network is conserved over its specific form.

Receptor tyrosine kinase and G-protein coupled receptor signaling and sorting within endosomes

*DivisionofBiologicalSciencesandCenterforStructuralandFunctionalNeuroscience,UniversityofMontana, Missoula, Montana, USA Department of Microbiology, Montana State University, Bozeman, Montana, USAMany neuroscientists desire to understand events that occurat the synapse. We would like to know in exacting detail howreceptors respond to neurotransmitters and neuropeptides.Unfortunately, some mechanisms such as receptors’ mem-brane traffic and signal transduction are not accessible orpractically studied at synapses in vivo, so model systemsmust be used.Data gleaned invitrofrom neuronsor cell linesin culture using the tools ofcell biology has great value infocusing our views about how receptors behave in vivo;nature is conservative and once mechanisms and rules ofbehavior are established they usually hold true in manydifferent contexts. This review examines the hypothesis thatendosomes form organelles that are specialized for signaltransduction, and casts a wide net fishing for clues from cellbiologyabouthowreceptorsmight dothisataxontips.Manyofthese clues are derived from molecular interactions amonggene products with known roles in signal transduction,membrane traffic, and the cytoskeleton.Not so long ago, investigators who studied signaltransduction, membrane traffic, or the cytoskeleton focusedtheir attention mostly within these individual disciplines.Recently these formerly distinct fields have become linkedtogether in surprising and marvelous ways. Endocytosis isregulated by signal transduction and signal transduction isinitiated from endosomes. The first evidence that receptorinternalization is not solely a mechanism for down-regula-tion and termination ofthe receptor’s signal, that receptorscan signal from endosomes, was provided for epidermalgrowth factor (EGF) and insulin receptors by Bergeron andcolleagues (Baass et al. 1995). It is now widely recognizedthat signaling from both receptor tyrosine kinases (RTKs)and G-protein coupled receptors (GPCRs) occurs in intra-cellular organelles derived from endocytosis (Ceresa andSchmid 2000; Di Fiore and De Camilli 2001; Ferguson2001; McPherson et al. 2001; Wiley and Burke 2001).Receptor signal transduction from intracellular organellesactivates distinct pathways and conveys instructions tospecific intracellular locations. In neurons, signaling afterendocytosis is a compelling means to convey neurotrophinsignals from the tips of axons to the cell body.RTK signaling from endocytic organellesand the signaling vesicles hypothesisDuring development ofthe nervous system, more neuronsare born than are needed and in mammals there is a selectionfor those which make functional connections. Neurons thatdo not make proper connections activate programmed celldeath; neurons that do connect survive and differentiate.When projecting axons reach their target they receiveinstructions in the form of polypeptide growth factors,including neurotrophins such as nerve growth factor (NGF),which bind to receptors (Trks) at the axon tip. This distalsignal must traverse down the axon, a great distance in somecases, to direct the cell body towards differentiation andaway from programmed cell death. Defects in initiation,reception, or transport ofneurotrophin signals may lead tomany different diseases of congenital cognitive dysfunctionand neurodegeneration (Cooper et al. 2001; Sofroniew .2001). It is thus ofvital importance to understand inmolecular detail the generation, conveyance, intracellularlocation, and downstream targets ofneurotrophin signals.One indication ofthe perceived importance ofneurotrophinsin the development andmaintenance ofthe nervous system isthe number ofrecent reviews covering this field, no fewer

High‐Resolution Fractionation of Signaling Endosomes Containing Different Receptors

Receptor endocytosis is regulated by ligand binding, and receptors may signal after endocytosis in signaling endosomes. We hypothesized that signaling endosomes containing different types of receptors may be distinct from one another and have different physical characteristics. To test this hypothesis, we developed a high‐resolution organelle fractionation method based on mass and density, optimized to resolve endosomes from other organelles. Three different types of receptors undergoing ligand‐induced endocytosis were localized predominately in endosomes that were resolved from one another using this method. Endosomes containing activated receptor tyrosine kinases (RTKs), TrkA and EGFR, were similar to one another. Endosomes containing p75NTR (in the tumor necrosis receptor superfamily) and PAC1 (a G‐protein‐coupled receptor) were distinct from each other and from RTK endosomes. Receptor‐specific endosomes may direct the intracellular location and duration of signal transduction pathways to dictate response to signals and determine cell fate.

Neuroblastoma Tyrosine Kinase Signaling Networks Involve FYN and LYN in Endosomes and Lipid Rafts

Protein phosphorylation plays a central role in creating a highly dynamic network of interacting proteins that reads and responds to signals from growth factors in the cellular microenvironment. Cells of the neural crest employ multiple signaling mechanisms to control migration and differentiation during development. It is known that defects in these mechanisms cause neuroblastoma, but how multiple signaling pathways interact to govern cell behavior is unknown. In a phosphoproteomic study of neuroblastoma cell lines and cell fractions, including endosomes and detergent-resistant membranes, 1622 phosphorylated proteins were detected, including more than half of the receptor tyrosine kinases in the human genome. Data were analyzed using a combination of graph theory and pattern recognition techniques that resolve data structure into networks that incorporate statistical relationships and protein-protein interaction data. Clusters of proteins in these networks are indicative of functional signaling pathways. The analysis indicates that receptor tyrosine kinases are functionally compartmentalized into distinct collaborative groups distinguished by activation and intracellular localization of SRC-family kinases, especially FYN and LYN. Changes in intracellular localization of activated FYN and LYN were observed in response to stimulation of the receptor tyrosine kinases, ALK and KIT. The results suggest a mechanism to distinguish signaling responses to activation of different receptors, or combinations of receptors, that govern the behavior of the neural crest, which gives rise to neuroblastoma.

NGF Causes TrkA to Specifically Attract Microtubules to Lipid Rafts

Membrane protein sorting is mediated by interactions between proteins and lipids. One mechanism that contributes to sorting involves patches of lipids, termed lipid rafts, which are different from their surroundings in lipid and protein composition. Although the nerve growth factor (NGF) receptors, TrkA and p75NTR collaborate with each other at the plasma membrane to bind NGF, these two receptors are endocytosed separately and activate different cellular responses. We hypothesized that receptor localization in membrane rafts may play a role in endocytic sorting. TrkA and p75NTR both reside in detergent-resistant membranes (DRMs), yet they responded differently to a variety of conditions. The ganglioside, GM1, caused increased association of NGF, TrkA, and microtubules with DRMs, but a decrease in p75NTR. When microtubules were induced to polymerize and attach to DRMs by in vitro reactions, TrkA, but not p75NTR, was bound to microtubules in DRMs and in a detergent-resistant endosomal fraction. NGF enhanced the interaction between TrkA and microtubules in DRMs, yet tyrosine phosphorylated TrkA was entirely absent in DRMs under conditions where activated TrkA was detected in detergent-sensitive membranes and endosomes. These data indicate that TrkA and p75NTR partition into membrane rafts by different mechanisms, and that the fraction of TrkA that associates with DRMs is internalized but does not directly form signaling endosomes. Rather, by attracting microtubules to lipid rafts, TrkA may mediate other processes such as axon guidance.

Neurotrophin signalling in the nervous system

Neurotrophins appear to have important trophic roles in thedeveloping and mature nervous system. The discovery that members of the trk gene family are signalling receptors for the neurotrophins suggests that studies of trk gene expression will be informative with respect to the locus, timing and biological significance of neurotrophin actions. NGF is a target-derived neurotrophin. Localization of trkA expression is predictive of NGF responsiveness in neurons in both the PNS and CNS. The onset of trk expression in the developing PNS appears to allow for NGF signalling as soon as neurites contact NGF in the target. Studies on NGF and trkA raise the possibility that neurotrophins may normally act via internalization of their trk receptors with subsequent retrograde transport of the activated receptor from the neurite tip to the cell body. In the CNS and in PC12 cells, trkA gene expression is induced by NGF. Regulation of trkA by NGF may compensate for receptor internalization and degradation. The localization of expression for trkB and C is more widespread than trkA, and novel trophic relationships are suggested by the complicated, sometimes overlapping patterns for BDNF and NT-3 and their corresponding trk mRNAs. While for some neurons the actions of BDNF, NT-3 and NT-4/5 may be similar to NGF, in other cases there may be autocrine or paracrine modes of action. Understanding where and how activated trks signal is key to elucidating the actions of NGF and the other neurotrophins.