Publications by Type: Journal Article
2008
2007
2005
2003
Protein ubiquitination has been implicated in the regulation of axonal growth and synaptic plasticity as well as in the pathogenesis of neurodegenerative diseases. Here we show that depolarization-dependent Ca2+ influx into synaptosomes produces a global, rapid (range of seconds), and reversible decrease of the ubiquitinated state of proteins, which correlates with the Ca2+-dependent dephosphorylation of several synaptic proteins. A similar general decrease in protein ubiquitination was observed in nonneuronal cells on Ca2+ entry induced by ionomycin. Both in synaptosomes and in nonneuronal cells, this decrease was blocked by FK506 (a calcineurin antagonist). Proteins whose ubiquitinated state was decreased include epsin 1, a substrate for the deubiquitinating enzyme fat facets/FAM, which we show here to be concentrated at synapses. These results reveal a fast regulated turnover of protein ubiquitination. In nerve terminals, protein ubiquitination may play a role both in the regulation of synaptic function, including vesicle traffic, and in the coordination of protein turnover with synaptic use.
2002
2000
Epsin (Eps15 interactor) is a cytosolic protein involved in clathrin-mediated endocytosis via its direct interactions with clathrin, the clathrin adaptor AP-2, and Eps15. The NH(2)-terminal portion of epsin contains a phylogenetically conserved module of unknown function, known as the ENTH domain (epsin NH(2)-terminal homology domain). We have now solved the crystal structure of rat epsin 1 ENTH domain to 1.8 A resolution. This domain is structurally similar to armadillo and Heat repeats of beta-catenin and karyopherin-beta, respectively. We have also identified and characterized the interaction of epsin 1, via the ENTH domain, with the transcription factor promyelocytic leukemia Zn(2)+ finger protein (PLZF). Leptomycin B, an antifungal antibiotic, which inhibits the Crm1- dependent nuclear export pathway, induces an accumulation of epsin 1 in the nucleus. These findings suggest that epsin 1 may function in a signaling pathway connecting the endocytic machinery to the regulation of nuclear function.
The Saccharomyces cerevisiae SAC1 gene was identified via independent analyses of mutations that modulate yeast actin function and alleviate the essential requirement for phosphatidylinositol transfer protein (Sec14p) activity in Golgi secretory function. The SAC1 gene product (Sac1p) is an integral membrane protein of the endoplasmic reticulum and the Golgi complex. Sac1p shares primary sequence homology with a subfamily of cytosolic/peripheral membrane phosphoinositide phosphatases, the synaptojanins, and these Sac1 domains define novel phosphoinositide phosphatase modules. We now report the characterization of a rat counterpart of Sac1p. Rat Sac1 is a ubiquitously expressed 65-kDa integral membrane protein of the endoplasmic reticulum that is found at particularly high levels in cerebellar Purkinje cells. Like Sac1p, rat Sac1 exhibits intrinsic phosphoinositide phosphatase activity directed toward phosphatidylinositol 3-phosphate, phosphatidylinositol 4-phosphate, and phosphatidylinositol 3,5-bisphosphate substrates, and we identify mutant rat sac1 alleles that evoke substrate-specific defects in this enzymatic activity. Finally, rat Sac1 expression in Deltasac1 yeast strains complements a wide phenotypes associated with Sac1p insufficiency. Biochemical and in vivo data indicate that rat Sac1 phosphatidylinositol-4-phosphate phosphatase activity, but not its phosphatidylinositol-3-phosphate or phosphatidylinositol-3, 5-bisphosphate phosphatase activities, is essential for the heterologous complementation of Sac1p defects in vivo. Thus, yeast Sac1p and rat Sac1 are integral membrane lipid phosphatases that play evolutionary conserved roles in eukaryotic cell physiology.
1999
Clathrin-mediated endocytosis was shown to be arrested in mitosis due to a block in the invagination of clathrin-coated pits. A Xenopus mitotic phosphoprotein, MP90, is very similar to an abundant mammalian nerve terminal protein, epsin, which binds the Eps15 homology (EH) domain of Eps15 and the alpha-adaptin subunit of the clathrin adaptor AP-2. We show here that both rat epsin and Eps15 are mitotic phosphoproteins and that their mitotic phosphorylation inhibits binding to the appendage domain of alpha-adaptin. Both epsin and Eps15, like other cytosolic components of the synaptic vesicle endocytic machinery, undergo constitutive phosphorylation and depolarization-dependent dephosphorylation in nerve terminals. Furthermore, their binding to AP-2 in brain extracts is enhanced by dephosphorylation. Epsin together with Eps15 was proposed to assist the clathrin coat in its dynamic rearrangements during the invagination/fission reactions. Their mitotic phosphorylation may be one of the mechanisms by which the invagination of clathrin-coated pits is blocked in mitosis and their stimulation-dependent dephosphorylation at synapses may contribute to the compensatory burst of endocytosis after a secretory stimulus.
Eps15 was originally identified as a substrate for the kinase activity of the epidermal growth factor receptor (EGFR). Eps15 has a tripartite structure comprising a NH2-terminal portion, which contains three EH domains, a central putative coiled-coil region, and a COOH-terminal domain containing multiple copies of the amino acid triplet Aspartate-Proline-Phenylalanine. A pool of Eps15 is localized at clathrin coated pits where it interacts with the clathrin assembly complex AP-2 and a novel AP-2 binding protein, Epsin. Perturbation of Eps15 and Epsin function inhibits receptor-mediated endocytosis of EGF and transferrin, demonstrating that both proteins are components of the endocytic machinery. Since the family of EH-containing proteins is implicated in various aspects of intracellular sorting, biomolecular strategies aimed at interfering with these processes can now be envisioned. These strategies have potentially far reaching implications extending to the control of cell proliferation. In this regard, it is of note that Eps15 has the potential of transforming NIH-3T3 cells and that the eps15 gene is rearranged with the HRX/ALL/MLL gene in acute myelogeneous leukemias, thus implicating this protein in the subversion of cell proliferation in neoplasia.