Mu, F.-T. et al. EEA1, an early endosome-associated protein. EEA1 is a conserved -helical peripheral membrane protein flanked by cysteine 'fingers' and contains a calmodulin-binding IQ motif. J. Biol. Chem. 270, 13503-13511

Department of Pathology, National University of Singapore.
Journal of Biological Chemistry (Impact Factor: 4.57). 07/1995; 270(22):13503-11. DOI: 10.1074/jbc.270.22.13503
Source: PubMed


Early endosomes are cellular compartments receiving endocytosed material and sorting them for vesicular transport to late endosomes and lysosomes or for recycling to the plasma membrane. We have cloned a human cDNA encoding an evolutionarily conserved 180-kDa protein on early endosomes named EEA1 (Early Endosome Antigen1). EEA1 is associated with early endosomes since it co-localizes by immunofluorescence with the transferrin receptor and with Rab5 but not with Rab7. Immunoelectron microscopy shows that it is associated with tubulovesicular early endosomes containing internalized bovine serum albumin-gold. EEA1 is a hydrophilic peripheral membrane protein present in cytosol and membrane fractions. It partitions in the aqueous phase after Triton X-114 solubilization and is extracted from membranes by 0.3 M NaCl. It is a predominantly alpha-helical protein sharing 17-20% sequence identity with the myosins and contains a calmodulin-binding IQ motif. It is flanked by metal-binding, cysteine "finger" motifs. The COOH-terminal fingers, Cys-X2-Cys-X12-Cys-X2-Cys and Cys-X2-Cys-X16-Cys-X2-Cys, are present within a region that is strikingly homologous with Saccharomyces cerevisiae FAB1 protein required for endocytosis and with Caenorhabditis elegans ZK632. These fingers also show limited conservation with S. cerevisiae VAC1, Vps11, and Vps18p proteins implicated in vacuolar transport. We propose that EEA1 is required for vesicular transport of proteins through early endosomes and that its finger motifs are required for this activity.

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    • "Inhibition of dynein-dynactin function, in addition to Golgi fragmentation also causes relocalization of other organelles, in particular endosomes that in conditions of impaired dynein-dynactin function redistribute to the cell periphery in non-polarized cells [53-55]. We therefore studied the distribution of endosomes in motor neurons of BN1 and G1del mice using antibodies against early endosome antigen 1 (EEA1) [57] and Rab4 binding protein GRASP-1 [58,59]. Strikingly, instead of a redistribution of endosomes to the cell periphery, BICD2-N-expressing motor neurons in BN1 mice showed an overall reduction of endosomal labeling in both EAA1 and GRASP1 stained sections (Figure 6A-C). "
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    ABSTRACT: Fragmentation of stacked cisterns of the Golgi apparatus into dispersed smaller elements is a feature associated with degeneration of neurons in amyotrophic lateral sclerosis (ALS) and some other neurodegenerative disorders. However, the role of Golgi fragmentation in motor neuron degeneration is not well understood. Here we use a SOD1-ALS mouse model (low-copy Gurney G93A-SOD1 mouse) to show that motor neurons with Golgi fragmentation are retrogradely labeled by intramuscularly injected CTB (beta subunit of cholera toxin), indicating that Golgi fragmentation precedes neuromuscular denervation and axon retraction. We further show that Golgi fragmentation may occur in the absence of and precede two other pathological markers, i.e. somatodendritic SOD1 inclusions, and the induction of ATF3 expression. In addition, we show that Golgi fragmentation is associated with an altered dendritic organization of the Golgi apparatus, does not depend on intact apoptotic machinery, and is facilitated in transgenic mice with impaired retrograde dynein-dependent transport (BICD2-N mice). A connection to altered dynein-dependent transport also is suggested by reduced expression of endosomal markers in neurons with Golgi fragmentation, which also occurs in neurons with impaired dynein function. Together the data indicate that Golgi fragmentation is a very early event in the pathological cascade in ALS that is associated with altered organization of intracellular trafficking.
    04/2014; 2(1):38. DOI:10.1186/2051-5960-2-38
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    • "GAPDH is also associated with the GTPase Rab2 at the ER and Golgi apparatus [31]. The early endosome antigen 1 (EEA1) was used as endosomal marker to show that endosomes primarily exist in the cytoplasm and light membrane fractions [32]. NUP62 as a marker for the nuclear membrane, as well as lamin B1 and histone H3 as nuclear markers were employed and were also detected in the nucleolar fraction and the nuclear membrane as well as in the nucleoplasm in the case of lamin B1 [33]. "
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    ABSTRACT: Fragile X mental Retardation Protein (FMRP) is a well-known regulator of local translation of its mRNA targets in neurons. However, despite its ubiquitous expression, the role of FMRP remains ill-defined in other cell types. In this study we investigated the subcellular distribution of FMRP and its protein complexes in HeLa cells using confocal imaging as well as detergent-free fractionation and size exclusion protocols. We found FMRP localized exclusively to solid compartments, including cytosolic heavy and light membranes, mitochondria, nuclear membrane and nucleoli. Interestingly, FMRP was associated with nucleolin in both a high molecular weight ribosomal and translation-associated complex (≥6 MDa) in the cytosol, and a low molecular weight complex (∼200 kDa) in the nucleoli. Consistently, we identified two functional nucleolar localization signals (NoLSs) in FMRP that are responsible for a strong nucleolar colocalization of the C-terminus of FMRP with nucleolin, and a direct interaction of the N-terminus of FMRP with the arginine-glycine-glycine (RGG) domain of nucleolin. Taken together, we propose a novel mechanism by which a transient nucleolar localization of FMRP underlies a strong nucleocytoplasmic translocation, most likely in a complex with nucleolin and possibly ribosomes, in order to regulate translation of its target mRNAs.
    PLoS ONE 03/2014; 9(3):e91465. DOI:10.1371/journal.pone.0091465 · 3.23 Impact Factor
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    • "EEA-1 plays a key role in the clathrin-dependent pathway and contains an FYVE finger, which interacts with PI3 K [45, 46]. PI3 K phosphorylates Rab5, which helps EEA-1 to localize to early-endocytic compartments [47]. "
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    ABSTRACT: Chlorotoxin (CTX) is a 36-amino acid peptide derived from Leiurus quinquestriatus (scorpion) venom, which inhibits low-conductance chloride channels in colonic epithelial cells. It has been reported that CTX also binds to matrix metalloproteinase-2 (MMP-2), membrane type-1 MMP, and tissue inhibitor of metalloproteinase-2, as well as CLC-3 chloride ion channels and other proteins. Pancreatic cancer cells require the activation of MMP-2 during invasion and migration. In this study, the fusion protein was generated by joining the CTX peptide to the amino terminus of the human IgG-Fc domain without a hinge domain, the monomeric form of chlorotoxin (M-CTX-Fc). The resulting fusion protein was then used to target pancreatic cancer cells (PANC-1) in vitro. M-CTX-Fc decreased MMP-2 release into the media of PANC-1 cells in a dose-dependent manner. M-CTX-Fc internalization into PANC-1 cells was observed. When the cells were treated with chlorpromazine (CPZ), the internalization of the fusion protein was reduced, implicating a clathrin-dependent internalization mechanism of M-CTX-Fc in PANC-1 cells. Furthermore, M-CTX-Fc clearly exhibited the inhibition of the migration depending on the concentration, but human IgG, as negative control of Fc, was not affected. The M-CTX-Fc may be an effective instrument for targeting pancreatic cancer.
    01/2014; 2014(1):152659. DOI:10.1155/2014/152659
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