Sequence and expression patterns of a human EMAP-related protein-2 (HuEMAP-2)

Lepley, Denise; Palange, Jean; Suprenant, Kathy A.

doi:10.1016/s0378-1119(99)00335-2

Cited by 23 publications

(16 citation statements)

References 17 publications

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“…The more widely expressed isoform 1 of EML2 does not possess a TD region and is therefore unlikely to interact directly with other EMLs but a TD is present in the brain/spinal cord variant isoform 2 studied here [24]. EML3 can also self-associate, and can interact with EML2 isoform 2, but not with EML1.…”

Section: Discussionmentioning

confidence: 76%

Microtubule association of EML proteins and the EML4-ALK variant 3 oncoprotein require an N-terminal trimerization domain

Richards

O’Regan

Röth

et al. 2015

Biochemical Journal

103

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The structural co-ordinates reported for EML2 11-60 and EML4 6-64 I38M appear in the PDB under codes 4CGB and 4CGC respectively. Page heading: EML trimerization domain drives MT-bindingKeywords: EML1, EML2, EML4-ALK, crystal structure, coiled-coil, microtubule 2 SUMMARY STATEMENTWe present crystal structures of a trimeric coiled-coil domain found in EML proteins. This trimerization domain mediates self-association and interactions between a subset of EML proteins. Microtubule-association of EML proteins requires the trimerization domain and an adjacent basic region. ABSTRACTProteins of the echinoderm microtubule associated protein-like (EML) family contribute to formation of the mitotic spindle and interphase microtubule (MT) network. EML1-4 consist of WD40 repeats and an N-terminal region containing a putative coiled-coil. Recurrent gene rearrangements in nonsmall cell lung cancer (NSCLC) fuse EML4 to anaplastic lymphoma kinase (ALK) causing expression of several oncogenic fusion variants. The fusions have constitutive ALK activity due to self-association through the EML4 coiled-coil. We have determined crystal structures of the coiledcoils from EML2 and EML4, which describe the structural basis of both EML self-association and oncogenic EML4-ALK activation. The structures reveal a trimeric oligomerization state directed by a conserved pattern of hydrophobic residues and salt bridges. We show that the trimerization domain (TD) of EML1 is necessary and sufficient for self-association. The TD is also essential for MT binding, however this property requires an adjacent basic region. These observations prompted us to investigate MT association of EML4-ALK and EML1-ABL1 fusions in which variable portions of the EML component are present. Uniquely, EML4-ALK variant 3, which includes the TD and basic region of EML4 but none of the WD40 repeats, was localized to MTs, both when expressed recombinantly and in a patient-derived NSCLC cell line (H2228). This raises the question of whether the mislocalization of ALK activity to MTs might influence downstream signalling and malignant properties of cells. Furthermore, the structure of EML4 TD may enable the development of protein-protein interaction inhibitors targeting the trimerization interface, providing a possible avenue towards therapeutic intervention in EML4-ALK NSCLC.

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Section: Discussionmentioning

confidence: 76%

Microtubule association of EML proteins and the EML4-ALK variant 3 oncoprotein require an N-terminal trimerization domain

Richards

O’Regan

Röth

et al. 2015

Biochemical Journal

103

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“…The 3.2-kb band was also observed in HeLa, colorectal adenocarcinoma, lung carcinoma, and melanoma cells but at much lower levels (data not shown). These results are interesting in light of the fact that, in normal human tissues, the 3.2-kb band is only detected in brain and spinal cord (41). This raises the possibility that ELP70 RNA processing is altered in tumor cells.…”

Section: Conservation Of a Hydrophobic Emap-like Protein (Help)mentioning

confidence: 82%

The Human EMAP-like Protein-70 (ELP70) Is a Microtubule Destabilizer That Localizes to the Mitotic Apparatus

Eichenmüller

Everley

Palange

et al. 2002

Journal of Biological Chemistry

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In this report, we show that the echinoderm microtubule (MT)-associated protein (EMAP) and related EMAP-like proteins (ELPs) share a similar domain organization with a highly conserved hydrophobic ELP (HELP) domain and a large tryptophan-aspartic acid (WD) repeat domain. To determine the function of mammalian ELPs, we generated antibodies against a 70-kDa human ELP and showed that ELP70 coassembled with MTs in HeLa cell extracts and colocalized with MTs in the mitotic apparatus. To determine whether ELP70 bound to MTs directly, human ELP70 was expressed and purified to homogeneity from baculovirus-infected Sf9 cells. Purified ELP70 bound to purified MTs with a stoichiometry of 0.40 ؎ 0.04 mol of ELP70/mol of tubulin dimer and with an intrinsic dissociation constant of 0.44 ؎ 0.13 M. Using a nucleated assembly assay and video-enhanced differential interference contrast microscopy, we demonstrated that ELP70 reduced seeded nucleation, reduced the growth rate, and promoted MT catastrophes in a concentration-dependent manner. As a result, ELP70-containing MTs were significantly shorter than MTs assembled from tubulin alone. These data indicate that ELP70 is a novel MT destabilizer. A lateral destabilization model is presented to describe ELP70's effects on microtubules.

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“…Two additional ELPs are predicted by the human genome sequence. ELP70 protein is predicted to be 57% identical and 77% similar to EMAP at the amino acid level and the gene maps to chromosome 19 [Lepley et al, 1999;Eichenmüller et al, 2001]. ELP120 was previously identified as the restrictively overexpressed proliferation protein 120 (ropp120) and the ELP120 gene maps to chromosome 2 [Heidebrecht et al, 2000].…”

Section: Introductionmentioning

confidence: 99%

Saturable binding of the echinoderm microtubule‐associated protein (EMAP) on microtubules, but not filamentous actin or vimentin filaments

Eichenmüller

Ahrens

et al. 2001

Cell Motil. Cytoskeleton

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The echinoderm microtubule-associated protein (EMAP) is a 75-kDa, WD-repeat protein associated with the mitotic spindle apparatus. To understand EMAP's biological role, it is important to determine its affinity for microtubules (MTs) and other cytoskeletal components. To accomplish this goal, we utilized a low-cost, bubble-column bioreactor to express EMAP as a hexahistidine fusion (6his) protein in baculovirus-infected insect cells. After optimizing cell growth conditions, up to 30 mg of EMAP was obtained in the soluble cell lysate from a 1-liter culture. EMAP was purified to homogeneity in a two-step process that included immobilized metal-affinity chromatography (IMAC) and anion-exchange chromatography. In vitro binding studies on cytoskeletal components were performed with the 6his-EMAP. EMAP bound to MTs, but not actin or vimentin filaments, with an intrinsic dissociation constant of 0.18 microM and binding stoichiometry of 0.7 mol EMAP per mol tubulin heterodimer. In addition, we show that a strong MT binding domain resides in the 137 amino acid, NH(2)-terminus of EMAP and a weaker binding site in the WD-domain. Previous work has shown that the EMAP concentration in the sea urchin egg is over 4 microM. Together, these results show that there is sufficient EMAP in the egg to regulate the assembly of a large pool of maternally stored tubulin.

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Sequence and expression patterns of a human EMAP-related protein-2 (HuEMAP-2)

Cited by 23 publications

References 17 publications

Microtubule association of EML proteins and the EML4-ALK variant 3 oncoprotein require an N-terminal trimerization domain

Microtubule association of EML proteins and the EML4-ALK variant 3 oncoprotein require an N-terminal trimerization domain

The Human EMAP-like Protein-70 (ELP70) Is a Microtubule Destabilizer That Localizes to the Mitotic Apparatus

Saturable binding of the echinoderm microtubule‐associated protein (EMAP) on microtubules, but not filamentous actin or vimentin filaments

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