Article

Human EML4, a novel member of the EMAP family, is essential for microtubule formation. Exp Cell Res

University of Hamburg, Hamburg, Hamburg, Germany
Experimental Cell Research (Impact Factor: 3.37). 11/2006; 312(17):3241-51. DOI: 10.1016/j.yexcr.2006.06.035
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ABSTRACT Human EML4 (EMAP-like protein 4) is a novel microtubule-associated WD-repeat protein of 120 kDa molecular weight, which is classified as belonging to the conserved family of EMAP-like proteins. Cosedimentation assays demonstrated that EML4 associates with in vitro polymerized microtubules. Correspondingly, immunofluorescence stainings and transient expression of EGFP-labeled EML4 revealed a complete colocalization of EML4 with the interphase microtubule array of HeLa cells. We present evidence that the amino-terminal portion of EML4 (amino acids 1-249) is essential for the association with microtubules. Immunoprecipitation experiments revealed that EML4 is hyperphosphorylated on serine/threonine residues during mitosis. In addition, immunofluorescence stainings demonstrated that hyperphosphorylated EML4 is associated with the mitotic spindle, suggesting that the function of EML4 is regulated by phosphorylation. siRNA-mediated knockdown of EML4 in HeLa cells led to a significant decrease in the number of cells. In no case mitotic figures could be observed in EML4 negative HeLa cells. Additionally, we observed a significant reduction of the proliferation rate and the uptake of radioactive [3H]-thymidine as a result of EML4 silencing. Most importantly, EML4 negative cells showed a completely modified microtubule network, indicating that EML4 is necessary for correct microtubule formation.

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    • "The authors were interested in the appearance of the fusion oncogene in human NSCLC patients, thus using antibodies exclusively specific for ALK in the immunohistochemical detection, which explains the conflicting results with the present study. Knowledge about the potential function(s) of wildtype EML4 in general is scarce but the two known publications about this particular protein conclusively demonstrated that EML4 is a microtubule stabilizing protein that is essential for both proliferation and survival of cells [6] [7]. Hence, based on our data related to human tissue, it appears to be likely that EML4 is involved in various immunoreactive processes such as local innate host defense mechanisms in the normal human lung. "
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    ABSTRACT: Despite considerable progress in the development of individualized targeted therapies of tumor diseases, identification of additional reliable target molecules is still mandatory. One of the most recent targets is microtubule-associated human EML4 generating a fusion-type oncogene with ALK demonstrating marked transforming activity in lung cancer. Since EML4 is a poorly characterized protein with regard to expression, function and regulation in human tissue, specimens of human tumor and tumor-free tissues obtained from patients with NSCLC were analyzed to determine the cellular localization. All tissue samples have been previously fixed with the novel HOPE-technique and paraffin embedded. Determination of both gene expression and protein levels of EML4 were performed using RT-PCR, in situ hybridization as well as immunohistochemistry, respectively. In human NSCLC tissue samples, possible regulation of EML4 transcription upon chemotherapy with combinations of most established cytotoxic drugs for NSCLC treatment was also studied employing the recently established ex vivo tissue culture model STST. In normal lung, both marked mRNA and protein levels of EML4 were localized in alveolar macrophages. In contrast, lung tumor tissues always showed consistent transcriptional expression in situ and by RT-PCR. Stimulation of NSCLC tissues with chemotherapeutics revealed heterogeneous effects on EML4 mRNA levels. Based on its expression patterns in both tumor-free lung and NSCLC tissues, human EML4 is likely to be closely associated with processes involved in local inflammation of the lung as well as with tumor behavior. Thus, our results suggest that EML4 may have the potential as a therapeutic target molecule in NSCLC chemotherapy.
    Romanian journal of morphology and embryology = Revue roumaine de morphologie et embryologie 01/2010; 51(4):647-53. · 0.72 Impact Factor
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    • "and a large number of cognate proteins have been found in mammals, plants, Drosophila, C. elegans, yeasts, and in prokaryotes (van Nocker and Ludwig, 2003; Saeki et al., 2006; Li and Roberts, 2001). Members of the WD-40 repeat family have been attributed to biological functions as diverse as RNA transcription and processing (de la Cruz et al., 2005), vesicular trafficking (Fritzius et al., 2007), cytoskeletal assembly (Pollmann et al., 2006), cell signaling (Fritzius et al., 2006) , cell cycle (Yoon et al., 2004) , and cell death (Ogawa et al., 2003). The crystal structures of a number of WD-40 proteins have been solved till date and data suggests that the repeating units form a circular, propeller-like structure with seven blades made up of beta sheets (Sondek et al., 1996; Yu et al., 2000; Cheng et al., 2004). "
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    ABSTRACT: SG2NA is a member of the striatin family of WD-40 repeat proteins with potential scaffolding functions. It was originally identified as a tumor antigen with increased expression during S to G2 phase of cell cycle. We report here that mouse SG2NA has at least five novel splice variants of which two are devoid of the carboxyl terminal WD-40 repeats. The variants of SG2NA are generated by alternative splicing at the exon 7-9 regions and differ in their expression profiles in various tissues tested. While the 83, 78, 38 and 35 kDa variants are present in both brain and heart, the 87 kDa form is brain specific. Also, the expression of 35 kDa variant is more in neonatal than in adult tissues. Western analysis suggests that the SG2NA isoforms differentially respond to growth stimuli. Upon serum stimulation, while the 35 kDa variant is increased, the 78 kDa form is diminished. Splicing variation of SG2NA is conserved in metazoan evolution. In embryonic chicken there are at least four variants of which one is present in brain but absent in heart. Taken together, splicing variation of SG2NA might have some critical roles in differentiation and maturation in metazoan cells.
    Gene 09/2008; 420(1):48-56. DOI:10.1016/j.gene.2008.04.016 · 2.08 Impact Factor
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    • "Eml4 is therefore the first member of the EMAP protein family that stabilizes microtubules. Our results were confirmed in an independent study demonstrating EML4 is essential for microtubule formation (Pollmann et al., 2006). How Eml4 stabilizes microtubules (reduction in catastrophe rate, reduction in shrinkage rate, increase in rescue frequency, increase in growth rate) requires further investigation by e.g. in vitro polymerization assays. "
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    ABSTRACT: Echinoderm microtubule-associated protein (EMAP) is the major microtubule binding protein in dividing sea urchin (Strongylocentrotus purpuratus) eggs. Echinoderm microtubule-associated protein like protein 4 (Eml4, restrictedly overexpressed proliferation-associated protein 120 kDa (Ropp120)) is one of the five mammalian EMAP homologues, the cellular function of which remains to be elucidated. In our first set of experiments we determined the spatio-temporal expression pattern of Eml4 in mouse brain. Our results demonstrate that Eml4 is a highly developmentally regulated gene with high expression levels in the developing nervous system of E11 embryos declining to low levels in adult. Spatially, Eml4 expression becomes restricted to the olfactory bulb, hippocampus and cerebellum. Transient transfection of a fusion construct of full-length mouse Eml4 with green fluorescent protein (GFP-Eml4) into Cos7 and HeLa cells resulted in colocalization of GFP-Eml4 with microtubules. This colocalization was observed both with microtubules of non-dividing cells and with the mitotic spindle of dividing cells. In addition, transient overexpression of GFP-Eml4 in Cos7 cells resulted in microtubules that were resistant to nocodazole treatment suggesting that Eml4 stabilizes microtubules. A consequence of microtubule stabilization is a net reduction in the amount of free tubulin. Microtubule stabilizing proteins therefore are expected to indirectly decrease the microtubule growth rate. Indeed, transient transfection of GFP-Eml4 resulted in a marked decrease in the microtubule growth rate, which is in line with our hypothesis that Eml4 functions as a microtubule stabilizing protein. In summary, our results suggest that Eml4 is a developmentally regulated protein that colocalizes with and stabilizes microtubules.
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