PKCε-dependent H-Ras activation encompasses the recruitment of the RasGEF SOS1 and of the RasGAP neurofibromin in the lipid rafts of embryonic neurons

Karouzaki, Sophia; Peta, Charoula; Tsirimonaki, Emmanouella; Mangoura, Dimitra

doi:10.1016/j.neuint.2019.104582

Cited by 5 publications

(10 citation statements)

References 73 publications

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“…Because protein conformation in cell lysates differs and may closer resemble endogenous states than those attained after fixation or exposure to SDS, we reasonably presume that inclusion, or not, of the 41 amino acids of exon 51 may indeed alter the conformation of the molecule. With an estimated molecular mass of ~320 kDa, neurofibromin has been repeatedly shown to migrate as a 220–250 kDa protein in SDS polyacrylamide gels [ 32 , 48 , 49 , 59 , 60 , 75 ], most likely due to the known intramolecular interactions between its domains and specific protein folding. At any case, the expected differential conformation of the ΔNLS or NLS neurofibromins would also impact its well established intermolecular interactions [ 18 , 35 , 60 , 75 ].…”

Section: Discussionmentioning

confidence: 99%

“…Western blot analysis was essentially done as we have previously described [ 18 , 49 , 57 , 59 , 60 ]: equal amounts (60 μg) of total cell homogenates in radioimmunoprecipitation (RIPA) buffer, supplemented with protease and phosphatase inhibitors, were resuspended in 5× Laemmli buffer and proteins were separated with Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, proteins were transferred into nitrocellulose membranes and probed with primary antibodies.…”

Section: Methodsmentioning

confidence: 99%

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Nuclear Isoforms of Neurofibromin Are Required for Proper Spindle Organization and Chromosome Segregation

Peta

Tsirimonaki

Samouil

et al. 2020

Cells

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Mitotic spindles are highly organized, microtubule (MT)-based, transient structures that serve the fundamental function of unerring chromosome segregation during cell division and thus of genomic stability during tissue morphogenesis and homeostasis. Hence, a multitude of MT-associated proteins (MAPs) regulates the dynamic assembly of MTs in preparation for mitosis. Some tumor suppressors, normally functioning to prevent tumor development, have now emerged as significant MAPs. Among those, neurofibromin, the product of the Neurofibromatosis-1 gene (NF1), a major Ras GTPase activating protein (RasGAP) in neural cells, controls also the critical function of chromosome congression in astrocytic cellular contexts. Cell type- and development-regulated splicings may lead to the inclusion or exclusion of NF1exon51, which bears a nuclear localization sequence (NLS) for nuclear import at G2; yet the functions of the produced NLS and ΔNLS neurofibromin isoforms have not been previously addressed. By using a lentiviral shRNA system, we have generated glioblastoma SF268 cell lines with conditional knockdown of NLS or ΔNLS transcripts. In dissecting the roles of NLS or ΔNLS neurofibromins, we found that NLS-neurofibromin knockdown led to increased density of cytosolic MTs but loss of MT intersections, anastral spindles featuring large hollows and abnormal chromosome positioning, and finally abnormal chromosome segregation and increased micronuclei frequency. Therefore, we propose that NLS neurofibromin isoforms exert prominent mitotic functions.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Nuclear Isoforms of Neurofibromin Are Required for Proper Spindle Organization and Chromosome Segregation

Peta

Tsirimonaki

Samouil

et al. 2020

Cells

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“…In addition, immunofluorescence showed that the co-localization of PKCε and TRPV1 increased in the model rats. Additionally, the intradermal injection of ψεRACK, a PKCε-specific activator peptide ( Karouzaki et al, 2019 ), instead of PGE2 produced an increase in the expression of TRPV1 in L4–L6 DRGs and induced chronic pain. All these results indicate that PGE2 produced long-lasting hyperalgesia by activating the PKCε–TRPV1 pathway in the peripheral nervous system in a Car-induced priming model.…”

Section: Discussionmentioning

confidence: 99%

Electroacupuncture Regulates Pain Transition Through Inhibiting PKCε and TRPV1 Expression in Dorsal Root Ganglion

et al. 2021

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Many cases of acute pain can be resolved with few side effects. However, some cases of acute pain may persist beyond the time required for tissue injury recovery and transit to chronic pain, which is hard to treat. The mechanisms underlying pain transition are not entirely understood, and treatment strategies are lacking. In this study, the hyperalgesic priming model was established on rats to study pain transition by injection of carrageenan (Car) and prostaglandin E2 (PGE2). The expression levels of protein kinase C epsilon (PKCε) and transient receptor potential vanilloid 1 (TRPV1) in the L4–L6 dorsal root ganglion (DRG) were investigated. Electroacupuncture (EA) is a form of acupuncture in which a small electric current is passed between a pair of acupuncture needles. EA was administrated, and its effect on hyperalgesia and PKCε and TRPV1 expression was investigated. The PKCε–TRPV1 signaling pathway in DRG was implicated in the pain transition. EA increased the pain threshold of model animals and regulated the high expression of PKCε and TRPV1. Moreover, EA also regulated hyperalgesia and high TRPV1 expression induced by selective PKCε activation. We also found that EA partly increased chronic pain threshold, even though it was only administered between the Car and PGE2 injections. These findings suggested that EA could prevent the transition from acute to chronic pain by inhibiting the PKCε and TRPV1 expression in the peripheral nervous system.

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“…Accumulated experimental evidence has pointed out the importance of coalescing lipid rafts as signalling platforms, formed within constrains applied by the underlying cortical filamentous actin (Factin) cytoskeleton and its associated proteins (Gupta et al 2006;Morone et al 2006;Chichili and Rodgers 2007;Goswami et al 2008;Simons and Gerl 2010;Asimaki et al 2011;Asimaki and Mangoura 2011;Karouzaki et al 2019). Lipid rafts represent preordained, liquid-ordered, cholesterol-rich microdomains of the plasma membrane bilayer, with glycosphingolipids and glycosyl phosphatidylinositol-anchored proteins in their outer leaflet, integral transmembrane, palmitoylated proteins (mostly receptors and transmembrane kinases, like JAKs) (Ren et al 2013;Lorent et al 2017), and high concentrations of major lipidated signalling molecules, such as the lipid raft prototype resident H-Ras, Src-family kinases, G proteins, and adaptor molecules in their inner leaflet and cytoplasmic face (e.g., (Asimaki et al 2011;Karouzaki et al 2019)). These specialized concentrations contribute, through lipid-lipid and lipid-protein interactions (Raghupathy et al, 2015), to the generation and dynamic behavior of lipid raft domains (reviewed in (Harayama and Riezman 2018;Kusumi et al 2020)).…”

Section: Introductionmentioning

confidence: 99%

“…Indeed, the significance of biomolecular condensates, that is concentrations of macromolecules not surrounded by a membrane, in the biological outcomes of membrane receptor signalling has now become evident (Asimaki and Mangoura 2011;Mangoura 2016;Banani et al 2017;Shin and Brangwynne 2017;Karouzaki et al 2019;Jaqaman and Ditlev 2021;Schuster et al 2021). Such condensates, formed at the plasma membrane upon receptor -agonist binding, regulate the activity of signalling proteins, by regulating the times that such molecules spend in the membrane (Huang et al, 2019).…”

Section: Introductionmentioning

confidence: 99%

Lipid rafts integrity is essential for prolactin-induced mitogenesis in mouse embryonic stem cells

Karouzaki¹,

Peta²,

Tsirimonaki³

et al. 2022

Int. J. Dev. Biol.

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Embryonic stem cells, ESCs, retain the capacity to self-renew, yet, the protein machinery essential in maintaining this undifferentiated status remains largely undefined. Signalling interactions are initiated and enhanced at the plasma membrane lipid rafts, within constrains and regulation applied by the actin and tubulin cytoskeleton systems. First, we undertook a comprehensive approach using twodimensional gel electrophoresis and mass spectrometry analysis combined with Western blotting and immunofluorescence analyses at the single cell level to compile the proteome profile of detergentfree preparations of lipid rafts of E14 mouse embryonic stem cells. In comparison with the proteomic profiles of other membrane fractions, recovery of actin and tubulin network proteins, including folding chaperones, was impressively high. At equally high frequency we detected annexins, pleiotropic proteins that may bind membrane lipids and actin filaments to regulate important membrane processes, and we validated their expression in lipid rafts. Next, we tested whether lipid raft integrity is required for completion of mitogenic signalling pathways. Disruption of the rafts with the cholesterol sequestering methyl-β-cyclodextrin (MCD) greatly downregulated the mitotic index of ESCs, in a dose- and time of exposure-dependent manner. Moreover, MCD greatly reduced the mitogenic actions of prolactin, a hormone known to stimulate proliferation in a great variety of stem and progenitor cells. Taken together, our data postulate that lipid rafts in ESCs are in close association with the actin and tubulin cytoskeletons to support signal compartmentalization, especially for signalling pathways pertinent to symmetric divisions for self-renewal.

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PKCε-dependent H-Ras activation encompasses the recruitment of the RasGEF SOS1 and of the RasGAP neurofibromin in the lipid rafts of embryonic neurons

Cited by 5 publications

References 73 publications

Nuclear Isoforms of Neurofibromin Are Required for Proper Spindle Organization and Chromosome Segregation

Nuclear Isoforms of Neurofibromin Are Required for Proper Spindle Organization and Chromosome Segregation

Electroacupuncture Regulates Pain Transition Through Inhibiting PKCε and TRPV1 Expression in Dorsal Root Ganglion

Lipid rafts integrity is essential for prolactin-induced mitogenesis in mouse embryonic stem cells

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