A Role for Epsin N-terminal Homology/AP180 N-terminal Homology (ENTH/ANTH) Domains in Tubulin Binding
Bibliographic record
Abstract
The epsin N-terminal homology (ENTH) domain is a protein module of ∼150 amino acids found at the N terminus of a variety of proteins identified in yeast, plants, nematode, frog, and mammals. ENTH domains comprise multiple α-helices folded upon each other to form a compact globular structure that has been implicated in interactions with lipids and proteins. In characterizing this evolutionarily conserved domain, we isolated and identified tubulin as an ENTH domain-binding partner. The interaction, which is direct and has a dissociation constant of ∼1 μm, was observed with ENTH domains of proteins present in various species. Tubulin is co-immunoprecipitated from rat brain extracts with the ENTH domain-containing proteins, epsins 1 and 2, and punctate epsin staining is observed along the microtubule cytoskeleton of dissociated cortical neurons. Consistent with a role in microtubule processes, the over-expression of epsin ENTH domain in PC12 cells stimulates neurite outgrowth. These data demonstrate an evolutionarily conserved property of ENTH domains to interact with tubulin and microtubules. The epsin N-terminal homology (ENTH) domain is a protein module of ∼150 amino acids found at the N terminus of a variety of proteins identified in yeast, plants, nematode, frog, and mammals. ENTH domains comprise multiple α-helices folded upon each other to form a compact globular structure that has been implicated in interactions with lipids and proteins. In characterizing this evolutionarily conserved domain, we isolated and identified tubulin as an ENTH domain-binding partner. The interaction, which is direct and has a dissociation constant of ∼1 μm, was observed with ENTH domains of proteins present in various species. Tubulin is co-immunoprecipitated from rat brain extracts with the ENTH domain-containing proteins, epsins 1 and 2, and punctate epsin staining is observed along the microtubule cytoskeleton of dissociated cortical neurons. Consistent with a role in microtubule processes, the over-expression of epsin ENTH domain in PC12 cells stimulates neurite outgrowth. These data demonstrate an evolutionarily conserved property of ENTH domains to interact with tubulin and microtubules. The epsin N-terminal homology (ENTH) 1The abbreviations used are: ENTH, epsin N-terminal homology; ANTH, AP180 N-terminal homology; E/ANTH, epsin/AP180 N-terminal homology; DH, Dbl homology; GFP, green fluorescent protein; GST, glutathione S-transferase; HIP, Huntingtin-interacting protein; MAP, microtubule-associated protein; MES, 4-morpholineethanesulfonic acid; MP90, mitotic phosphoprotein of 90 kDa; PH, pleckstrin homology; PIPES, 1,4-piperazinediethanesulfonic acid; PtdIns(4,5)P2, phosphatidylinositol 4,5-bisphosphate; SH3, Src homology 3; TRITC, tetramethylrhodamine isothiocyanate. domain is an evolutionarily conserved globular module of ∼150 amino acids that occurs at the amino terminus of a variety of proteins (1Kay B.K. Yamabhai M. Wendland B. Emr S.D. Protein Sci. 1999; 8: 435-438Crossref PubMed Scopus (102) Google Scholar, 2Chen H. Fre S. Slepnev V.I. Capua M.R. Takei K. Butler M.H. Di Fiore P.P. De Camilli P. Nature. 1998; 394: 793-797Crossref PubMed Scopus (273) Google Scholar). Originally noted in the plant protein, Af10 (3Jones H.D. Smith S.J. Desikan R. Plakidou-Dymock S. Lovegrove A. Hooley R. Plant Cell. 1998; 10: 245-254Crossref PubMed Scopus (113) Google Scholar), the ENTH domain has subsequently been characterized in epsins (2Chen H. Fre S. Slepnev V.I. Capua M.R. Takei K. Butler M.H. Di Fiore P.P. De Camilli P. Nature. 1998; 394: 793-797Crossref PubMed Scopus (273) Google Scholar) and enthoprotin (4Wasiak S. Legendre-Guillemin V. Puertollano R. Blondeau F. Girard M. de Heuvel E. Boismenu D. Bell A.W. Bonifacino J.S. McPherson P.S. J. Cell Biol. 2002; 158: 855-862Crossref PubMed Scopus (167) Google Scholar) (also termed epsinR (5Hirst J. Motley A. Harasaki K. Peak Chew S.Y. Robinson M.S. 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Adaptor protein 180 (AP180), clathrin assembly lymphoid myeloid leukemia protein (CALM), Huntingtin-interacting protein-1 (HIP1) and HIP12, and the yeast proteins yAP180 and Sla2p contain a module that is so similar in structure to the epsin ENTH domain that they were initially denoted ENTH-bearing proteins (11Hyman J. Chen H. Di Fiore P.P. De Camilli P. Brunger A.T. J. Cell Biol. 2000; 149: 537-546Crossref PubMed Scopus (146) Google Scholar, 12Itoh T. Koshiba S. Kigawa T. Kikuchi A. Yokoyama S. Takenawa T. Science. 2001; 291: 1047-1051Crossref PubMed Scopus (392) Google Scholar, 13Ford M.G. Pearse B.M. Higgins M.K. Vallis Y. Owen D.J. Gibson A. Hopkins C.R. Evans P.R. McMahon H.T. Science. 2001; 291: 1051-1055Crossref PubMed Scopus (611) Google Scholar). However, recent structural studies have refined our understanding such that ENTH-like domains from these proteins have been re-designated ANTH domain-containing proteins in accordance with their higher structural similarity to AP180 rather than epsin (14Ford M.G. Mills I.G. Peter B.J. Vallis Y. Praefcke G.J. Evans P.R. McMahon H.T. Nature. 2002; 419: 361-366Crossref PubMed Scopus (811) Google Scholar). In an effort to simplify the nomenclature applied in this study, we refer to these homologous structures as E/ANTH domains when collectively discussing proteins bearing either domain, but maintain the ENTH or ANTH nomenclature when discussing individual proteins. A common feature among many E/ANTH domain-bearing proteins is that their C termini contain peptide motifs, indicative of a functional role in clathrin-mediated membrane budding including clathrin and clathrin adaptor protein-binding elements (1Kay B.K. Yamabhai M. Wendland B. Emr S.D. 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Cell Biol. 2003; 160: 213-222Crossref PubMed Scopus (204) Google Scholar, 7Kalthoff C. Groos S. Kohl R. Mahrhold S. Ungewickell E.J. Mol. Biol. Cell. 2002; 13: 4060-4073Crossref PubMed Scopus (106) Google Scholar, 10Duncan M.C. Costaguta G. Payne G.S. Nat. Cell Biol. 2003; 5: 77-81Crossref PubMed Scopus (83) Google Scholar). Recent studies have demonstrated that the E/ANTH domains of epsin and AP180 can mediate lipid binding, particularly to phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), and that this interaction is required for efficient clathrin-mediated endocytosis i
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.001 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".