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  1. Article ; Online: Applying imaging flow cytometry and immunofluorescence in studying the dynamic Golgi structure in cultured cells

    Inbal Wortzel / Ziv Porat / Rony Seger / Galia Maik-Rachline

    STAR Protocols, Vol 3, Iss 2, Pp 101278- (2022)

    2022  

    Abstract: Summary: The Golgi apparatus is subjected to fragmentation under several cellular processes such as mitosis. Here we describe two complementary approaches to analyze different Golgi morphological changes during its mitotic fragmentation, using classical ... ...

    Abstract Summary: The Golgi apparatus is subjected to fragmentation under several cellular processes such as mitosis. Here we describe two complementary approaches to analyze different Golgi morphological changes during its mitotic fragmentation, using classical immunofluorescence and imaging flow cytometry. Although fluorescent microscopy provides information on the exact Golgi architecture in distinct cells, the imaging flow cytometry combines the morphological data with the high-throughput quantification of flow cytometry. Taken together, both approaches provide robust and significant unbiased data analysis.For complete details on the use and execution of this protocol, please refer to Wortzel et al. (2021).
    Keywords Cell Biology ; Flow Cytometry/Mass Cytometry ; Microscopy ; Science (General) ; Q1-390
    Language English
    Publishing date 2022-06-01T00:00:00Z
    Publisher Elsevier
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  2. Article ; Online: Alternative Splicing of MAPKs in the Regulation of Signaling Specificity

    Galia Maik-Rachline / Inbal Wortzel / Rony Seger

    Cells, Vol 10, Iss 3466, p

    2021  Volume 3466

    Abstract: The mitogen-activated protein kinase (MAPK) cascades transmit signals from extracellular stimuli to a variety of distinct cellular processes. The MAPKKs in each cascade specifically phosphorylate and activate their cognate MAPKs, indicating that this ... ...

    Abstract The mitogen-activated protein kinase (MAPK) cascades transmit signals from extracellular stimuli to a variety of distinct cellular processes. The MAPKKs in each cascade specifically phosphorylate and activate their cognate MAPKs, indicating that this step funnels various signals into a seemingly linear pathway. Still, the effects of these cascades vary significantly, depending on the identity of the extracellular signals, which gives rise to proper outcomes. Therefore, it is clear that the specificity of the signals transmitted through the cascades is tightly regulated in order to secure the desired cell fate. Indeed, many regulatory components or processes that extend the specificity of the cascades have been identified. Here, we focus on a less discussed mechanism, that is, the role of distinct components in each tier of the cascade in extending the signaling specificity. We cover the role of distinct genes, and the alternatively spliced isoforms of MAPKKs and MAPKs, in the signaling specificity. The alternatively spliced MEK1b and ERK1c, which form an independent signaling route, are used as the main example. Unlike MEK1/2 and ERK1/2, this route’s functions are limited, including mainly the regulation of mitotic Golgi fragmentation. The unique roles of the alternatively spliced isoforms indicate that these components play an essential role in determining the proper cell fate in response to distinct stimulations.
    Keywords MAPK ; alternative splicing ; ERK ; JNK ; p38 ; ERK1c ; Biology (General) ; QH301-705.5
    Subject code 570 ; 572
    Language English
    Publishing date 2021-12-01T00:00:00Z
    Publisher MDPI AG
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  3. Article ; Online: Mitotic HOOK3 phosphorylation by ERK1c drives microtubule-dependent Golgi destabilization and fragmentation

    Inbal Wortzel / Galia Maik-Rachline / Suresh Singh Yadav / Tamar Hanoch / Rony Seger

    iScience, Vol 24, Iss 6, Pp 102670- (2021)

    2021  

    Abstract: Summary: ERK1c is an alternatively spliced isoform of ERK1 that specifically regulates mitotic Golgi fragmentation, which allows division of the Golgi during mitosis. We have previously shown that ERK1c translocates to the Golgi during mitosis where it ... ...

    Abstract Summary: ERK1c is an alternatively spliced isoform of ERK1 that specifically regulates mitotic Golgi fragmentation, which allows division of the Golgi during mitosis. We have previously shown that ERK1c translocates to the Golgi during mitosis where it is activated by a resident MEK1b to induce Golgi fragmentation. However, the mechanism of ERK1c functions in the Golgi remained obscure. Here, we searched for ERK1c substrates and identified HOOK3 as a mediator of ERK1c-induced mitotic Golgi fragmentation, which requires a second phosphorylation by AuroraA for its function. In cycling cells, HOOK3 interacts with microtubules (MTs) and links them to the Golgi. Early in mitosis, HOOK3 is phosphorylated by ERK1c and later by AuroraA, resulting in HOOK3 detachment from the MTs, and elevated interaction with GM130. This detachment modulates Golgi stability and allows fragmentation of the Golgi. This study demonstrates a novel mechanism of Golgi apparatus destabilization early in mitosis to allow mitotic progression.
    Keywords cell biology ; functional aspects of cell biology ; Science ; Q
    Subject code 571
    Language English
    Publishing date 2021-06-01T00:00:00Z
    Publisher Elsevier
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  4. Article ; Online: Nuclear ERK

    Galia Maik-Rachline / Avital Hacohen-Lev-Ran / Rony Seger

    International Journal of Molecular Sciences, Vol 20, Iss 5, p

    Mechanism of Translocation, Substrates, and Role in Cancer

    2019  Volume 1194

    Abstract: The extracellular signal-regulated kinases 1/2 (ERK) are central signaling components that regulate stimulated cellular processes such as proliferation and differentiation. When dysregulated, these kinases participate in the induction and maintenance of ... ...

    Abstract The extracellular signal-regulated kinases 1/2 (ERK) are central signaling components that regulate stimulated cellular processes such as proliferation and differentiation. When dysregulated, these kinases participate in the induction and maintenance of various pathologies, primarily cancer. While ERK is localized in the cytoplasm of resting cells, many of its substrates are nuclear, and indeed, extracellular stimulation induces a rapid and robust nuclear translocation of ERK. Similarly to other signaling components that shuttle to the nucleus upon stimulation, ERK does not use the canonical importinmechanism of nuclear translocation. Rather, it has its own unique nuclear translocation signal (NTS) that interacts with importin7 to allow stimulated shuttling via the nuclear pores. Prevention of the nuclear translocation inhibits proliferation of B-Raf- and N/K-Ras-transformed cancers. This effect is distinct from the one achieved by catalytic Raf and MEK inhibitors used clinically, as cells treated with the translocation inhibitors develop resistance much more slowly. In this review, we describe the mechanism of ERK translocation, present all its nuclear substrates, discuss its role in cancer and compare its translocation to the translocation of other signaling components. We also present proof of principle data for the use of nuclear ERK translocation as an anti-cancer target. It is likely that the prevention of nuclear ERK translocation will eventually serve as a way to combat Ras and Raf transformed cancers with less side-effects than the currently used drugs.
    Keywords mitogen-activated protein kinase (MAPK) ; Beta-like importins ; nuclear translocation ; nuclear substrates ; negative feedback loop ; Biology (General) ; QH301-705.5 ; Chemistry ; QD1-999
    Subject code 570
    Language English
    Publishing date 2019-03-01T00:00:00Z
    Publisher MDPI AG
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  5. Article ; Online: GqPCR-stimulated dephosphorylation of AKT is induced by an IGBP1-mediated PP2A switch

    Guy Nadel / Zhong Yao / Ehud Wainstein / Izel Cohen / Ido Ben-Ami / Amir Schajnovitz / Galia Maik-Rachline / Zvi Naor / Benjamin A. Horwitz / Rony Seger

    Cell Communication and Signaling, Vol 20, Iss 1, Pp 1-

    2022  Volume 18

    Abstract: Abstract Background G protein-coupled receptors (GPCRs) usually regulate cellular processes via activation of intracellular signaling pathways. However, we have previously shown that in several cell lines, GqPCRs induce immediate inactivation of the AKT ... ...

    Abstract Abstract Background G protein-coupled receptors (GPCRs) usually regulate cellular processes via activation of intracellular signaling pathways. However, we have previously shown that in several cell lines, GqPCRs induce immediate inactivation of the AKT pathway, which leads to JNK-dependent apoptosis. This apoptosis-inducing AKT inactivation is essential for physiological functions of several GqPCRs, including those for PGF2α and GnRH. Methods Here we used kinase activity assays of PI3K and followed phosphorylation state of proteins using specific antibodies. In addition, we used coimmunoprecipitation and proximity ligation assays to follow protein–protein interactions. Apoptosis was detected by TUNEL assay and PARP1 cleavage. Results We identified the mechanism that allows the unique stimulated inactivation of AKT and show that the main regulator of this process is the phosphatase PP2A, operating with the non-canonical regulatory subunit IGBP1. In resting cells, an IGBP1-PP2Ac dimer binds to PI3K, dephosphorylates the inhibitory pSer608-p85 of PI3K and thus maintains its high basal activity. Upon GqPCR activation, the PP2Ac-IGBP1 dimer detaches from PI3K and thus allows the inhibitory dephosphorylation. At this stage, the free PP2Ac together with IGBP1 and PP2Aa binds to AKT, causing its dephosphorylation and inactivation. Conclusion Our results show a stimulated shift of PP2Ac from PI3K to AKT termed “PP2A switch” that represses the PI3K/AKT pathway, providing a unique mechanism of GPCR-stimulated dephosphorylation. Video Abstract
    Keywords AKT ; PI3K ; PP2A ; IGBP1 ; PKC ; Medicine ; R ; Cytology ; QH573-671
    Language English
    Publishing date 2022-01-01T00:00:00Z
    Publisher BMC
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  6. Article ; Online: G protein-coupled receptor 37 is a negative regulator of oligodendrocyte differentiation and myelination

    Hyun-Jeong Yang / Anna Vainshtein / Galia Maik-Rachline / Elior Peles

    Nature Communications, Vol 7, Iss 1, Pp 1-

    2016  Volume 11

    Abstract: The molecular mechanism controlling oligodendrocyte differentiation is not fully understood. Here the authors show that G protein coupled receptor 37 acts as a negative regulator of CNS myelination, and this effect is mediated by suppression of ERK ... ...

    Abstract The molecular mechanism controlling oligodendrocyte differentiation is not fully understood. Here the authors show that G protein coupled receptor 37 acts as a negative regulator of CNS myelination, and this effect is mediated by suppression of ERK signalling.
    Keywords Science ; Q
    Language English
    Publishing date 2016-03-01T00:00:00Z
    Publisher Nature Portfolio
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  7. Article ; Online: Author Correction

    Rand Arafeh / Karen Flores / Alona Keren-Paz / Galia Maik-Rachline / Naomi Gutkind / Steven Rosenberg / Rony Seger / Yardena Samuels

    Scientific Reports, Vol 9, Iss 1, Pp 1-

    Combined inhibition of MEK and nuclear ERK translocation has synergistic antitumor activity in melanoma cells

    2019  Volume 1

    Abstract: A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper. ...

    Abstract A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.
    Keywords Medicine ; R ; Science ; Q
    Language English
    Publishing date 2019-03-01T00:00:00Z
    Publisher Nature Publishing Group
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  8. Article ; Online: Intrinsically active MEK variants are differentially regulated by proteinases and phosphatases

    Merav Ordan / Chiara Pallara / Galia Maik-Rachline / Tamar Hanoch / Francesco Luigi Gervasio / Fabian Glaser / Juan Fernandez-Recio / Rony Seger

    Scientific Reports, Vol 8, Iss 1, Pp 1-

    2018  Volume 16

    Abstract: Abstract MAPK/ERK kinase (MEK) 1/2 are central signaling proteins that serve as specificity determinants of the MAPK/ERK cascade. More than twenty activating mutations have been reported for MEK1/2, and many of them are known to cause diseases such as ... ...

    Abstract Abstract MAPK/ERK kinase (MEK) 1/2 are central signaling proteins that serve as specificity determinants of the MAPK/ERK cascade. More than twenty activating mutations have been reported for MEK1/2, and many of them are known to cause diseases such as cancers, arteriovenous malformation and RASopathies. Changes in their intrinsic activity do not seem to correlate with the severity of the diseases. Here we studied four MEK1/2 mutations using biochemical and molecular dynamic methods. Although the studied mutants elevated the activating phosphorylation of MEK they had no effect on the stimulated ERK1/2 phosphorylation. Studying the regulatory mechanism that may explain this lack of effect, we found that one type of mutation affects MEK stability and two types of mutations demonstrate a reduced sensitivity to PP2A. Together, our results indicate that some MEK mutations exert their function not only by their elevated intrinsic activity, but also by modulation of regulatory elements such as protein stability or dephosphorylation.
    Keywords Medicine ; R ; Science ; Q
    Subject code 570
    Language English
    Publishing date 2018-08-01T00:00:00Z
    Publisher Nature Publishing Group
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  9. Article ; Online: Combined inhibition of MEK and nuclear ERK translocation has synergistic antitumor activity in melanoma cells

    Rand Arafeh / Karen Flores / Alona Keren-Paz / Galia Maik-Rachline / Naomi Gutkind / Steven Rosenberg / Rony Seger / Yardena Samuels

    Scientific Reports, Vol 7, Iss 1, Pp 1-

    2017  Volume 9

    Abstract: Abstract Genetic alterations in BRAF, NRAS and NF1 that activate the ERK cascade, account for over 80% of metastatic melanomas. However, ERK cascade inhibitors have been proven beneficial almost exclusively for BRAF mutant melanomas. One of the hallmarks ...

    Abstract Abstract Genetic alterations in BRAF, NRAS and NF1 that activate the ERK cascade, account for over 80% of metastatic melanomas. However, ERK cascade inhibitors have been proven beneficial almost exclusively for BRAF mutant melanomas. One of the hallmarks of the ERK cascade is the nuclear translocation of ERK1/2, which is important mainly for the induction of proliferation. This translocation can be inhibited by the NTS-derived peptide (EPE) that blocks the ERK1/2-importin7 interaction, inhibits the nuclear translocation of ERK1/2, and arrests active ERK1/2 in the cytoplasm. In this study, we found that the EPE peptide significantly reduced the viability of not only BRAF, but also several NRAS and NF1 mutant melanomas. Importantly, combination of the EPE peptide and trametinib showed synergy in reducing the viability of some NRAS mutant melanomas, an effect driven by the partial preservation of negative feedback loops. The same combination significantly reduced the viability of other melanoma cells, including those resistant to mono-treatment with EPE peptide and ERK cascade inhibitors. Our study indicates that targeting the nuclear translocation of ERK1/2, in combination with MEK inhibitors can be used for the treatment of different mutant melanomas.
    Keywords Medicine ; R ; Science ; Q
    Subject code 570
    Language English
    Publishing date 2017-11-01T00:00:00Z
    Publisher Nature Publishing Group
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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