Amphiphysin I Is Associated with Coated Endocytic Intermediates and Undergoes Stimulation-dependent Dephosphorylation in Nerve Terminals

Bauerfeind, Rudolf; Takei, Kohji; Camilli, Pietro De

doi:10.1074/jbc.272.49.30984

Cited by 150 publications

(172 citation statements)

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“…A downward shift of epsin in depolarized synaptosomes can be seen. This shift correlates with the concomitant downwards shift of amphiphysin 1 previously shown to reflect its Ca 2ϩ -dependent dephosphorylation (23). In control synaptosomes, Eps15 migrated as a doublet.…”

Section: Regulated Phosphorylation Of Epsin and Eps15 3258mentioning

confidence: 77%

“…We report here that both epsin and Eps15 are phosphorylated in mitosis and that their phosphorylation inhibits binding to the clathrin adaptor AP-2. We also report that both epsin and Eps15, like other accessory proteins of clathrin-mediated endocytosis, undergo stimulation-dependent dephosphorylation in nerve terminals (22)(23)(24)(25), with a resulting increase in their binding to each other and to AP-2. Their dephosphorylation may facilitate endocytosis of synaptic vesicle membranes following an exocytotic burst.…”

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confidence: 99%

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The Interaction of Epsin and Eps15 with the Clathrin Adaptor AP-2 Is Inhibited by Mitotic Phosphorylation and Enhanced by Stimulation-dependent Dephosphorylation in Nerve Terminals

Chen¹,

Slepnev²,

Fiore³

et al. 1999

Journal of Biological Chemistry

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130

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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 ␣-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 ␣-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.Recent studies have implicated several cytosolic proteins besides clathrin and the clathrin adaptor AP-2 in clathrinmediated endocytosis, including the endocytosis of synaptic vesicles in nerve terminals (1-5). Two such proteins are Eps15 and epsin (6, 7). Eps15 was first identified as an endogenous substrate for EGF 1 receptor kinase (8) and was subsequently found to be an interacting partner for the "appendage domain" of the AP-2 subunit ␣-adaptin. Binding of Eps15 to AP-2 is mediated by its COOH-terminal region (9, 10), whereas the NH 2 -terminal region of Eps15 includes three Eps15 homology (EH) domains (11). Via these modules, Eps15 binds proteins with the consensus amino sequence NPF (12). Epsin, which contains three NPF motifs in its COOH-terminal region (NPF domain), is a major binding partner for Eps15 (7). Its NH 2 -terminal portion comprises an evolutionary conserved domain of unknown function, the ENTH domain (epsin NH 2 terminal homology domain), whereas its central region, which contains eight DPW repeats (DPW domain), binds the appendage domain of ␣-adaptin at a site that overlaps with the Eps15-binding site (7). Perturbation of the interactions of both Eps15 and epsin with AP-2, as well as disruption of the function of both proteins by antibody injection, block clathrin-mediated endocytosis (7, 13-16). It was proposed that Eps15 and epsin play an important role in clathrin-mediated endocytosis, possibly by participating in dynamic rearrangements of the clathrin coat during bud invagination and fission (7,17,18).Clathrin-mediated endocytosis is blocked during mitosis. In mitotic cells clathrin coats assemble, but their invagination is impaired (19,20). The identification of substrates of mitotic kinases responsible for this effect may therefore shed new light on the still elusive mechanisms underlying th...

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Section: Regulated Phosphorylation Of Epsin and Eps15 3258mentioning

confidence: 77%

mentioning

confidence: 99%

The Interaction of Epsin and Eps15 with the Clathrin Adaptor AP-2 Is Inhibited by Mitotic Phosphorylation and Enhanced by Stimulation-dependent Dephosphorylation in Nerve Terminals

Chen¹,

Slepnev²,

Fiore³

et al. 1999

Journal of Biological Chemistry

Self Cite

130

101

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“…Dephosphorylation of components of endocytic machinery also appears to be necessary in vivo. When calcium enters the nerve terminal, calcineurin triggers the dephosphorylation of dynamin, amphiphysin, synaptojanin, epsin, and eps15, allowing them to assemble with AP-2 into a macromolecular complex, which presumably is required for the internalization of plasma membrane (Bauerfeind et al, 1997;Slepnev et al, 1998;Chen et al, 1999). The dephosphorylation of ␤-arrestin also is required for its association with clathrin (Lin et al, 1997).…”

Section: Ap-3 Adaptor Phosphorylation By Casein Kinasementioning

confidence: 99%

The AP-3 Complex Required for Endosomal Synaptic Vesicle Biogenesis is Associated with a Casein Kinase Ια-Like Isoform

Faúndez

Kelly

2000

MBoC

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The formation of small vesicles is mediated by cytoplasmic coats the assembly of which is regulated by the activity of GTPases, kinases, and phosphatases. A heterotetrameric AP-3 adaptor complex has been implicated in the formation of synaptic vesicles from PC12 endosomes . When the small GTPase ARF1 is prevented from hydrolyzing GTP, we can reconstitute AP-3 recruitment to synaptic vesicle membranes in an assembly reaction that requires temperatures above 15°C and the presence of ATP suggesting that an enzymatic step is involved in the coat assembly. We have now found an enzymatic reaction, the phosphorylation of the AP-3 adaptor complex, that is linked with synaptic vesicle coating. Phosphorylation occurs in the ␤3 subunit of the complex by a kinase similar to casein kinase 1␣. The kinase copurifies with neuronal-specific AP-3. In vitro, purified casein kinase I selectively phosphorylates the ␤3A and ␤3B subunit at its hinge domain. Inhibiting the kinase hinders the recruitment of AP-3 to synaptic vesicles. The same inhibitors that prevent coat assembly in vitro also inhibit the formation of synaptic vesicles in PC12 cells. The data suggest, therefore, that the mechanism of AP-3-mediated vesiculation from neuroendocrine endosomes requires the phosphorylation of the adaptor complex at a step during or after AP-3 recruitment to membranes. INTRODUCTIONMembrane proteins are carried from donor membranes to acceptor membranes by two steps, the vesiculation of a carrier vesicle from the donor and the fusion of the carrier vesicle with the acceptor. Vesiculation of the donor membrane can itself be divided into five steps: recognition of cargo proteins; recruitment of coating molecules; the imposition of curvature on the forming vesicle membrane; fission of the carrier vesicle from the donor membrane; and, finally, the loss of the vesicle coat (Springer et al., 1999). To discover the molecular events that take place at each step, several laboratories have reconstituted either the entire process of vesiculation or individual steps in vitro.The first two steps of vesiculation, cargo recognition and coating, can be regulated by phosphorylation. Membrane proteins are recognized as cargo because they contain sorting domains. Adaptors interact with a tyrosine or a dileucine motif (Schmid, 1997;Kirchhausen et al., 1997;Bonifacino and Dell'Angelica, 1999). The recognition of cargo molecules can be regulated by the phosphorylation of sites close to the sorting domains; for example, in the trafficking of furin (Wan et al., 1998;Teuchert et al., 1999;Molloy et al., 1999), CD4 (Pitcher et al., 1999), CTLA-4 (Shiratori et al., 1997), MHC-II-Ii complex (Anderson and Roche, 1998), and pIgAR (Casanova et al., 1990;Okamoto et al., 1994;Luton et al., 1998). The cargo molecules are recruited into a newly forming carrier vesicle by cytoplasmic coat molecules (Matsuoka et al., 1998;Bremser et al., 1999). The recruitment of two of the known coats, COPI and COPII, is regulated by small ARF-like GTPases and appears to be a relatively simple p...

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“…Each protein can independently promote tubulation or vesiculation of membranes (26,27), and both proteins are predicted to coordinate membrane deformation with actin cytoskeleton dynamics (28,29). Endophilin and amphiphysin are localized to the highly curved necks that link a deeply invaginated vesicle to the donor membrane (30,31), and they are thought to function at a late step in vesicle budding together with dynamin to promote vesicle scission. Like CIN85, endophilin is implicated in regulation of the actin cytoskeleton.…”

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confidence: 99%

The Rsp5 Ubiquitin Ligase Binds to and Ubiquitinates Members of the Yeast CIN85-Endophilin Complex, Sla1-Rvs167

Stamenova¹,

Dunn²,

Adler

et al. 2004

Journal of Biological Chemistry

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Sla1 and Rvs167 are yeast proteins required for receptor internalization and organization of the actin cytoskeleton. Here we provide evidence that Sla1 and Rvs167 are orthologues of the mammalian CIN85 and endophilin proteins, respectively, which are required for ligand-stimulated growth factor receptor internalization. Sla1 is similar in domain structure to CIN85 and binds directly to the endophilin-like Rvs167. Akin to CIN85, Sla1 interacts with synaptojanins and a ubiquitin ligase that regulates endocytosis. This ubiquitin ligase, Rsp5, binds directly to both Sla1 and Rvs167. The interaction between Rsp5 and Rvs167 is mediated through Rsp5 WW domains and PXY motifs in the central Gly-Pro-Ala-rich domain of Rvs167. Rvs167 PXY motifs are required for Rsp5-dependent monoubiquitination of Rvs167 on Lys 481 in the Src homology 3 (SH3) domain. Mutation of Lys 481 3 Arg causes cells to grow slowly on medium containing 1 M NaCl, although this phenotype is not due to the defect in ubiquitination caused by the K481R mutation. We propose that Rsp5 interaction with Sla1-Rvs167 promotes Rvs167 ubiquitination and regulates activity of this protein complex. Rvs167 ubiquitination is not required for general function of Rvs167, but may control specific Rvs167 SH3 domain-protein interactions or negatively regulate SH3 domain activity.

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Amphiphysin I Is Associated with Coated Endocytic Intermediates and Undergoes Stimulation-dependent Dephosphorylation in Nerve Terminals

Cited by 150 publications

References 63 publications

The Interaction of Epsin and Eps15 with the Clathrin Adaptor AP-2 Is Inhibited by Mitotic Phosphorylation and Enhanced by Stimulation-dependent Dephosphorylation in Nerve Terminals

The Interaction of Epsin and Eps15 with the Clathrin Adaptor AP-2 Is Inhibited by Mitotic Phosphorylation and Enhanced by Stimulation-dependent Dephosphorylation in Nerve Terminals

The AP-3 Complex Required for Endosomal Synaptic Vesicle Biogenesis is Associated with a Casein Kinase Ια-Like Isoform

The Rsp5 Ubiquitin Ligase Binds to and Ubiquitinates Members of the Yeast CIN85-Endophilin Complex, Sla1-Rvs167

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