Voltage-gated Na⁺ channel activity increases colon cancertranscriptional activity and invasion via persistent MAPK signaling

© 2015 Macmillan Publishers Limited. All rights reserved. Functional expression of voltage-gated Na+ channels (VGSCs) has been demonstrated in multiple cancer cell types where channel activity induces invasive activity. The signaling mechanisms by which VGSCs promote oncogenesis remain poorly understood. We explored the signal transduction process critical to VGSC-mediated invasion on the basis of reports linking channel activity to gene expression changes in excitable cells. Coincidentally, many genes transcriptionally regulated by the SCN5A isoform in colon cancer have an over-representation of cis-acting sites for transcription factors phosphorylated by ERK1/2 MAPK. We hypothesized that VGSC activity promotes MAPK activation to induce transcriptional changes in invasion-related genes. Using pharmacological inhibitors/activators and siRNA-mediated gene knockdowns, we correlated channel activity with Rap1-dependent persistent MAPK activation in the SW620 human colon cancer cell line. We further demonstrated that VGSC activity induces downstream changes in invasion-related gene expression via a PKA/ERK/c-JUN/ELK-1/ETS-1 transcriptional pathway. This is the first study illustrating a molecular mechanism linking functional activity of VGSCs to transcriptional activation of invasion-related genes

Depolarization-induced signaling to Ras, Rap1 and MAPKs in cortical neurons

In neurons, membrane depolarization triggers pleiotropic signaling which includes the activation of the small GTPases, Ras and Rap1, and the mitogen-activated protein kinases (MAPKs) Erk1/2. We have studied the intracellular signaling mechanisms which regulate these events in mouse-cultured cortical neurons. We show that depolarization induces activation of both Ras and Rap1, although with different kinetics: Ras activation is strong and fast while Rap1 activation is slower and weaker. Blockade of calmodulin affects the GTP-loading of Ras and Rap1 and prevents the MAPK response. Moreover, protein kinase A (PKA) activity is required for depolarization-induced Rap1 activation and full Erk stimulation, but is not involved in that of Ras. This PKA-dependent Rap1 activation does not require Src family kinases, but, in contrast to Ras, is sensitive to genistein, indicating the involvement of a tyrosine kinase-dependent mechanism. Our data provide new insights into the regulation of Ras and Rap1 activation in neurons

Chemically controlled formation of a DNA/calcium phosphate coprecipitate: application for transfection of mature hippocampal neurons.

Numerous methods exist for transfecting postmitotic neurons, for example, DNA/calcium phosphate coprecipitation, cationic lipids, viruses, and physical methods such as microinjection, electroporation, and biolistics. Most methods, however, are either toxic to the cell, yield only poor transfection efficiencies, or cells have to be electroporated before plating. In this article, we present a standardized and fast transfection method using DNA/calcium phosphate coprecipitates that efficiently transfer DNA into mature, postmitotic hippocampal neurons. Shifting to CO(2)-independent media with a well-defined pH allows for the tight control of the coprecipitate formation and for adjusting the transfection parameters for the individual DNA plasmid used. The two critical parameters for reproducible and efficient transfections are: the precise pH during crystal formation, and the incubation time of the cells with the coprecipitate. This improved procedure now enables biochemical approaches. By transfecting a dominant-positive Ras mutant, we activate the Erk/MAP kinase signal transduction pathway. Furthermore, using a siRNA plasmid directed against MAP2, the level of an endogenously expressed protein is down-regulated upon transfection. These two approaches demonstrate that the presented transient transfection method can now be used to address questions on a biochemical level in hippocampal neurons

The guanine nucleotide exchange factor RasGRF1 directly binds microtubules via DHPH2-mediated interaction

RasGRF is a family of guanine nucleotide exchange factors with dual specificity for both Ras and Rac GTPases. In this study, using mouse brain exts., we show that both RasGRF1 and RasGRF2 interact with microtubules in an in vitro microtubule assembly system and this binding is very tight. To characterize this assocn., recombinant purified proteins contg. different regions of RasGRF1 were tested for their ability to bind microtubules preassembled from pure tubulin. Only the DHPH2 tandem directly assocs. with microtubules, whereas the isolated DH or PH2 domains do not, indicating that the entire DHPH2 region is required for this assocn. The interaction occurs with high affinity (Kd ~ 2 mM) and with a stoichiometry, at satg. conditions, of one DHPH2 mols. for two tubulin dimers. Competition expts. support the hypothesis that the DHPH2 module is largely responsible for RasGRF1-microtubule interaction. In vivo colocalization of RasGRF1 and microtubules was also obsd. by fluorescence confocal microscopy in nonneuronal cells after stimulation with an oxidative stress agent and in highly differentiated neuron-like cells. Identification of microtubules as new binding partners of RasGRF1 may help to elucidate the signaling network in which RasGRF1 is involved

The neuron specific Ras-exchange factor Ras-GRF1 assembles with polymerized tubulin: microscopy analysis and biochemical studies

5noneForlani G.; Fascio U.; Baldassa S.; Sturani E.; Zippel R.Forlani, Greta; Fascio, U.; Baldassa, S.; Sturani, E.; Zippel, R

N-terminal interaction domain implicates PAK4 in translational regulation and reveals novel cellular localization signals

The serine/threonine kinase PAK4 is a Rho GTPases effector protein implicated in many critical biological processes, including regulation of cell morphology and motility, embryonic development, cell survival, response to infection, and oncogenic transformation. Consistently with its pro-oncogenic features, PAK4 was found to be overexpressed in many cancer cell lines and tissues, and to be necessary to promote activation of survival pathways. PAK4, like other Paks, is now considered a promising target for specific therapy. Little is known on its modes of regulation, molecular partners, and substrates. Because the N-terminal regulatory moiety plays important roles in PAK4 activity and functions, even independently of GTPase interactions, in this study we employed an affinity chromatography approach to identify N-terminal domain binding partners. Within this protein region we identified a novel interaction domain involved in association with ribonucleoprotein (RNP) complexes, suggesting PAK4 implications in translational regulation. Indeed, we found that active PAK4 can affect (cap-independent) translation from specific IRES sequences in vivo, and that the N-terminal domain is critical for this regulation. Further, we could establish that within the RNP interacting sequence PAK4 regulatory domain contains targeting elements that drive cytoplasmic localization and act as nuclear export signal. Functional implication of endogenous PAK4 protein, which was found in both cytoplasmic and nuclear fractions, in IRES-mediated translation further underlines the significance of the reported findings. Our data reveal novel means for PAK4 regulation of gene expression, and provide new elements to understand the molecular mechanisms that determine PAK4 cellular localization and functions